It: IL-Lt 1' .u'i ’g-‘cww Y K ‘ \ 4 y .‘ S w; W? an; "F .‘ i.- “35.. m:- an “" US“. X l “L"? . 93A H a“... I J‘éG K YO ' «N d. I: .‘mwfizt “33.91.; ~ r- fix. mfg I i Elllg‘b‘w‘.‘“" 3.. 7": 'f~ J...‘ ‘-)"‘ a - .- I o .2 . ¥! ~ ,‘ In 1L A1‘5')&“ U niv CE; W er «a. .2»; v» ‘kfl" tun-w This is to certify that the thesis entitled THE RELATIONSHIP OF ULTRASTRUCTURAL AND BIOCHEMICAL PARAMETERS TO TENDERNESS IN YOUNG BULLS presented by Gary Lee Gann has been accepted towards fulfillment Ph.D. of the requirements for Food Science & Date May 29, 1974 degree in Human Nutfit ion Major professor 0-7639 Mi. ‘7 30- l" u ' -I I A‘ 1m We 1‘ I. ‘9? twnd- l“. ' 1- - f;t0 ntwd 5‘ 'CIMJCIH‘» -l| | yfid‘iO? “ o4; Qf) ABSTRACT (I THE RELATIONSHIP OF ULTRASTRUCTURAL AND BIOCHEMICAL PARAMETERS T0 TENDERNESS IN YOUNG BULLS by Gary Lee Gann Two genetic lines of Hereford bulls, consisting of 16 animals, 1 selected for tenderness and the other an unselected tenderness control line, were used to study the effects of postmortem aging on sarcomere length, muscle ultrastructure, protein solubility, ATPase and ITPase activity and superprecipitation of longissimus muscle. Longissimus muscle samples were removed from the 12th rib area 1, 48 and 216 hr. postmortem. Samples were prepared for electron microscopy with the remainder of the sample being qrr=»4 frozen in liquid N and powdered in a Waring Blendor. Warner-Bratzler shear r flu‘-‘ and taste panel data were obtained from the 216 hr. postmortem samples. Sarcomere length was measured on the 48 and 216 hr. samples and the ATPase, ITPase and superprecipitation assays were run on the 5 most tender and 5 toughest samples. Two groups of fibers, type I and type II, classified according to Z- line width and density and mitochondrial location and number, were found to differ in susceptibility to postmortem Z-line degradation. Type I fibers did not lose Z-line material during postmortem aging, whereas type II fibers consistently lost Z-material in all samples although the amount lost varied between fibers. Some Z-line degradation was apparent at 1 hr. postmortem, however, most of the Z-line degradation occurred between 1 and 48 hr. post- mortem- Little additional degradation occurred between 48 and 216 hr. post- mortem. Tender and control sample type II fibers were equally susceptible to Z-line degradation during all postmortem aging intervals measured. Myo- fibril fragmentation at the 1-2 junction occurred only in the 48 and 216 hr. '. Gary Lee Gann postmortem samples. The amount of myofibril fragmentation at 48 hr. post- mortem was limited to a few isolated sarcomeres, however, at 216 hr. post- mortem, the entire fibers of several samples, were found to be transversely broken. Type I and Type II fibers appeared to be equally susceptible to fragmentation at the 1-2 junction. The most tender sample (Warner-Bratzler shear) had more fiber breakage than the toughest sample, although, among all samples, fragmentation and shear score were not highly related. There were no statistically significant differences between the 48 and 216 hr. postmortem sarcomere lengths nor were there sarcomere length differences between tender and control line samples. The amount of myo- fibrillar N extracted by KCL at 48 hr. postmortem was significantly (P < .05) correlated with 48 hr. sarcomere length, however, no significant correlations were found between 216 hr. KCL or KI extracted myofibrillar N and 216 hr. sarcomere length. Sarcomere length at 216 hr. postmortem was significantly and negatively (P < .05) correlated with Warner-Bratzler shear but not with taste panel score. Sarcoplasmic N increased significantly (P < .05) between 1 and 48 hr; poatmortem and then decreased to the 1 hr. postmortem level. There were no significant sarcoplasmic N differences between control and tender lines. Shear and taste panel data were not significantly correlated with sarco- plasmic N at any postmortem time interval measured. No significant dif- ference was obtained for 1.1M K1 extracted myofibrillar N among the 3 postmortem time periods or between tender and control lines. Myofibrillar N extracted by 1.1M KCL at 216 hr. postmortem was significantly (P < .05) greater than that at 1 hr. postmortem, but not from that at 48 hr. postmortem. r. ,7, . . . . Gary Lee Gann No significant differences were obtained between tender and control lines. The KCL extracted myofibrillar N was significantly (P < .05) correlated with 1 hr. muscle temperature at all postmortem time intervals measured. There were no significant alterations in NPN as the result of postmortem aging, however, the tender line NPN at 216 hr. postmortem was signifi- cantly (P < .05) greater than all but the 1 hr. mean of the tender samples. NPN at 216 hr. postmortem was significantly (P < .05) negatively correlated with taste panel score, but only approached significance with Warner- Bratzler shear. Stroma N was not significantly different among the 3 postmortem time intervals on control or tender lines determined after ex- traction of the myofibrillar N by 1.1M KI. Hawever, the 1 hr. postmortem KCL determined stroma N was statistically different (P < .05) from the 48 hr. sample but not the 216 hr. postmortem sample. In the present study, it was concluded that protein solubility was not an adequate criterion for categorizing bovine longissimus muscle samples into tough or tender grOups. C82+;, Mg2+;, EDTA- and EGTA-modified ATPase activities did not change significantly during the postmortem time intervals measured or between tender and control samples. Ca2+;modified ITPase activity decreased be- tween 1 and 48 hr. pastmortem, however, Mg2+-modified ITPase activity changed very little among the 3 postmortem time intervals measured. No significant differences were observed between tender and control line sam- ples at any postmortem time interval measured for the ITPase activities. The superprecipitation assay (Mgz+ + EGTA) of the most tender and toughest samples showed that the 1 hr. postmortem samples had the fastest Gary Lee Gann ‘W Knit! onset. The tender sample low Ca2+ (0.05 mM; 100 mM KCL) ‘éi’iahlifisay' had the fastest rate of turbidity onset as com- ‘cafi‘lihph, however, in both tender and tough samples the H in sample had the fastest rate of turbidity onset. '>vilLATIONSHIP 0F ULTRASTRUCTURAL AND BIOCHEMICAL .IERAMETERS T0 TENDERNESS IN YOUNG BULLS BY Gary Lee Gann A THESIS Submitted to Michigan State university 1.1! épartial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY -nttofvrood-Science and Human Nutrition 1974 ACKNOWLEDGEMENTS The author wishes to express appreciation to his major professor, Dr. R. A. Merkel, for his support and guidance during the course of this study. The author also expresses gratitude to Drs. R. E. Carrow, J. F. Price, J. R. Brunner and G. C. Spink for serving as members of the guidance committee. Special thanks are extended to Dr. G. C. Spink and Dr. G. R. Hooper for making electron microscope facilities available and for helpful discussions concerning electron microscopy. The author is grateful to Dr. w. T. Magee for furnishing the experi- mental animals in this study and for assistance With the statistical analysis. Special thanks go to Mrs. Dora Spooner for her contribution to the biochemical analyses and to Mrs. Beatrice Eichelberger for typing this manuscript. The author would also like to thank Dr. A. M. Pearson for the use of the electron microscope preparation facilities and Dr. T. R. Dutson for helpful discussions and the use of his microscopy facilities. The author is appreciative of the many intellectual discussions with fellow graduate students in the Meat Laboratory. The author would particularly like to express appreciation to his parents, Mr. and Mrs. Gordon Gann, for their continued support throughout his academic career. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . 3 Tenderness Studies Prior to 1960 . . . . . . . . . . . . . 3 Tenderness Studies After 1960 . . . . . . . . . . . . . . . 17 Connective Tissue Proteins . . . . . . . . . . . 17 Proteolysis . . . . . . . . . . . . . . . . 27 Muscle Protein Solubility . . . . . . . . . . . . . . 36 Water-Holding Capacity . . . . . . . . . . . . . . . . 51 Rigor Mortis . . . . . . . . . . . . . . . . . . 53 Chemical Changes . . . . . . . . . . . . . . . . 53 Structural Changes . . . . . . . . . . . . . . . 57 Postmortem Shortening of Myofibrils . . . . . . . . . . . . 59 Factors Affecting Shortening . . . . . . . . . . . . . 59 Rigor Shortening . . . . . . . . . . . . . . . . 59 Temperature Effects . . . . . . . . . . . . . . . 60 Prerigor Muscle Excision . . . . . . . . . . 61 Effect of Shortening on Tenderness . . . . . . . 61 Restraints on Shortening . . . . . . . . . . . . 68 Resolution of Rigor Mortis . . . . . . . . . . . . . . . . 71 Concept of Rigor Resolution . . . . . . . . . . . . . 71 Rigor Resolution and Tenderness . . . . . . . . . . . . . . 74 Morphological Changes . . . . . . . . . . . . . . . . . . . 76 Z-line Structure . . . . . . . . . . . . . . . . 76 ATPase Activity . . . . . . . . . . . . . . . . . . . . . . 83 Regulatory Proteins . . . . . . . . . . . . . . . . . . . . 89 Superprecipitation . . . . . . . . . . 89 Other Indicators of Regulatory Changes . . . . . . . . 94 Sulfhydryl Groups . . . . . . . . . . . . . . . . . . . . . 96 Proteolytic Probes . . . . . . . . . . . . . . . . . . . . 99 iii Page EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . 105 Experimental Animals . . . . . . . . . . . . . . 105 Sample Preparation . . . . . . . . . . . . . . . . . . . . 105 Electron Microscopy . . . . . . . . . . . . . . . . . . . . 106 Sampling . . . . . . . . . . . . . . . . . . . 106 Fixation and Embedding . . . . . . . . . . . . . 106 Section Preparation and Staining . . . . . . . . . . . 108 Specimen Observation and Photography . . . . . . . . . 108 Sarcomere Measurement . . . . . . . . . . . . . . . . . . . 109 Sample Preparation . . . . . . . . . . . . . . . . . . 109 Protein Extraction . . . . . . . . . . . . . . . . . . . . 110 Sarcoplasmic Protein . . . . . . . . . . . . . . . . . 111 Myofibrillar Protein . . . . . . . . . . . . . . . . . 111 Non-Protein Nitrogen . . . . . . . . . . . . . . . . . 111 Total Nitrogen . . . . . . . . . . . . . . . . . . . . 112 Stroma Nitrogen . . . . . . . . . . . . . . . . . . . 112 ATPase and ITPase Activity . . . . . . . . . . . . . . . . 112 Preparation of Myofibrils . . . . . . . . . . . . . . 112 CaZ+-Activated ATPase Activity . . . . . . . . . . . . 113 Mg2+-Activated ATPase Activity . . . . . . . . . . . 113 EGTA + Mg2+-Activated ATPase Activity . . . . . . . . 113 EDTA-Activated ATPase Activity . . . . . . . . . . 114 Mg2+- and Ca2+-Activated ITPase Activity . . . . . . . 114 Phosphate Determination . . . . . . . . . . 114 Myofibril Protein Determination . . . . . . . . . . . 114 Superprecipitation . . . . . . . . . . . . . . 115 Natural Actomyosin Preparation . . . . . . . . . . . . 115 Superprecipitation Assay . . . . . . . . . . . . . . . 115 Low Ca + Superprecipitation . . . . . . . . . . . . . 116 ug2+ + Ca2+ Superprecipitation . . . . . . . . . . . . 116 Statistical Analyses . . . . . . . . . . . . . . . . . . . 116 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . 117 Electron Microscopy . . . . . . . . . . . . . . . . . . . . 117 1 Hr. Postmortem . . . . . . . . . . . . . . . . . . . 117 Mitochondria . . . . . . . . . . . . . . . . . . . . . 117 iv Z— —Line . . Contractile State . M-Line Glycogen Treatment Effects . 48 Hr. Samples . . . . 216 Mitochondria Z-Line I- Z Breakage M-Line Contractile State . Glycogen Hr. Samples . Mitochondria . Z-Line I—Z Breakage Fiber Breakage M—Line Contractile State . Glycogen Ultrastructure and Tenderness . Sarcomere Length . . . Protein Fractionation . Sarcoplasmic Proteins NPN Myofibrillar Proteins Stroma . . . Total N . Myofibrillar N to Sarcoplasmic N Ratio (MF—N Protein Solubility and Ultrastructure . Adenosine Triphosphatase Activity . Ca2 -Modified ATPase . Mg2+-Modified ATPase . EDTA-Modified ATPase . EGTA+ Mg 2+-Modified ATPase Inosine Triphosphatase Activity . Ca2+-Modified ITPase . Mg2+-Modified ITPase Activity ATPase, ITPase and Ultrastructure Superprecipitation M82+ + EGTA Superprecipitation . Low Ca2+ Superprecipitation c :SP-N) Page 119 124 127 127 128 128 128 130 134 141 141 143 145 145 147 150 157 161 161 161 163 167 170 170 170 174 175 176 177 178 179 179 181 183 184 184 184 187 188 189 189 191 .1 I . _4 a . t— - IT'S . n .‘ . vi 1 Page 194 . } an; :Pm ‘ I -‘lmd .315". ‘1 I l I I I I I I I I I I I I I I I I I I I o I 198 ' la cn :‘r ~ 220 hlgcl‘rg" I I I I I I I I I I I I I I I I I I I I I I M :img. 'lktracrxm " .1 4-. “In I‘ltlr’ : LIST OF TABLES Mean sarcomere length of the longissimus muscle of each line and aging period . . . . Simple correlation coefficients between sarcomere length and biochemical and tenderness data . . . . . . . . Mean protein fraction N of the longissimus muscle of each line and aging period . . . . . . . . Some simple correlation coefficients between protein fraction N and palatability, muscle pH and temperature Mean adenosine triphosphatase activities by line and aging period . . . . . . . . . . . . . . . . . . . . Simple correlation coefficients for ATPase, ITPase and NPN Mean inosine triphosphatase activities by line and aging period . . . . . . . . . . . . . . . . . . . . . vii Page 167 169 171 174 180 185 Figure 10 11 12 13 14 15 16 17 18 19 LIST OF FIGURES 1 hr. postmortem bovine longissimus muscle fiber . 1 hr. postmortem bovine longissimus muscle subsarcolemmal mitochondria . . . . . . . . . . . . . 1 hr. postmortem bovine longissimus muscle fiber . 1 hr. postmortem bovine longissimus muscle type I fiber. 1 hr. postmortem bovine lgngissimgs muscle type I fiber. 1 hr. postmortem bovine longissimus muscle type II fiber Type II fiber in 1 hr. bovine longissimus muscle . Type II fiber in 1 hr. bovine longissimus muscle . 1 hr. postmortem bovine longissimus muscle type II fiber illustrating partially relaxed muscle sarcomeres . 1 hr. postmortem bovine longissimus muscle type I fiber illustrating contracted muscle sarcomeres . . . Type II fiber, 1 hr. postmortem, illustrating relaxed condition . . . . . . . . . . . . . . . . . . . . . Type I fiber, 48 hr. postmortem Two type II fibers, 48 hr. postmortem . . . . . . . . . Type I fiber from 48 hr. postmortem bovine longissimus muscle . . . . . . . Type I fiber from 48 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . Type I fiber from 48 hr. postmortem bovine longissimus muscle . . . . . . . . . Type II fiber, 48 hr. postmortem, showing Z— line degradation . . . . . . . . . Higher magnification of Z-line area from figure 17 . . . Two type II fibers, A and B . . . . . . . . . . . Viii Page 118 118 120 120 122 122 123 123 125 125 126 129 129 131 131 132 132 133 133 Figure 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Page 48 hr. postmortem type II fiber . . . . . . . . . . . . 135 48 hr. postmortem type II fiber . . . . . . . . . . . . 135 Type II fiber in 48 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . . . . 136 Type II fiber in 48 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . . . . 136 Type I fiber in 48 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . . . . 137 48 hr. postmortem bovine longissimus type I fiber . . . 137 Low magnification electron micrograph of a portion of 2 fibers in 48 hr. postmortem bovine longissimus muscle . 139 Higher magnification of figure 26 showing I- Z junction breakage . . . . . . . . 139 Type I fiber in 48 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . . . . 140 Type II fiber in 48 hr. postmortem bovine longissimus muscle . . . . . . . . 140 Low magnification electron micrograph of a portion of 2 fibers, A and B . . . . . . . . . . . . . . . . . . 142 Higher magnification electron micrograph of the area within the square in figure 30 . . . . . . . . . . . 142 Mitochondria in 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . 146 Two fiber portions of 216 hr. bovine longissimus muscle 146 216 hr. postmortem bovine longissimus muscle fiber . . . 148 216 hr. postmortem bovine longissimus muscle type I fiber showing essentially no change . . . . . 148 Higher magnification of a portion of a 216 hr. postmortem bovine longissimus muscle type I fiber showing essentially no change . . . . . . . . . . . 149 Figure l 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Page Type I 216 hr. postmortem bovine longissimus muscle fiber showing essentially no change . . . . 149 216 hr. postmortem bovine longissimus muscle . . . . . . 151 Type II fiber in 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . . . 151 216 hr. postmortem bovine longissimus muscle type II fiber . . . . . . . . . 152 Type II fiber in 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . 152 Two type II fibers, A and B, in 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . 153 Type I 216 hr. postmortem bovine longissimus muscle fiber 153 216 hr. postmortem bovine longissimus muscle type I fiber 155 216 hr. postmortem bovine longissimus type I fiber . . . 155 216 hr. postmortem bovine longissimus muscle type II fiber . . . . . . . 156 Type II fiber in 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . . . . 156 216 hr. postmortem bovine longissimus muscle type 11 fiber . . . . . . 158 Portions of 3 fibers, A, B and C, from 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . 158 Low magnification electron micrograph of an area of oblique fiber breakage in 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . 159 Low magnification electron micrograph of a portion of 6 fibers, A, B, C, D, E and F, of 216 hr. postmortem bovine longissimus muscle . . . . . . . . . 159 Portion of a broken fiber from 216 hr. postmortem bovine longissimus muscle . . . . . . . . . . . . . . . . . . . 160 Page I magnification electron micrograph of a trans- . ,7 divided 216 hr. postmortem bovine longissimus fair. C O I I I I I O O I l I O I I O I O I I I I 160 Ll c . lfi§+ + 0.1 mM EGTA Superprecipitation Assay of Most ‘1 ' Jtoughest saWJ-es I o s o o u I I I o I o o I 190 +110. 05 mM) Superprecipitation Assay of Most r and Toughest Samples . . . . . . . . . . 192 11*”?! rmll?“ Metih‘ - I19 “Icy...» Appendix II III IV XVIII XIX liiiiEiIIV LIST OF APPENDIX TABLES Schedule For Preparation of 1.25% Glutaraldehyde Fixative . . . . . . . . . . . . . . . . Schedule for Preparation of Washing Buffer . . . Osmium Tetroxide Fixation (1%) . . . . . . Epon Embedding Media . . . . . . . . . . . . . . . Procedure for Embedding Muscle Fibers . . . . . . Stain Preparation Procedure . . . . . . . . . . . Staining Procedure for Thin Sections Reagent Preparation for Protein Fractionation . Reagents for Kjeldahl Nitrogen Determination . . Procedure for Myofibril Preparation . ATPase and ITPase Procedure . . . . . . . . . . . . . . Phosphate Determination . . . . . . Biuret Reagent . . . . . . Myosin B (Natural Actomyosin) Preparation . . . . . . Definition of Variable Names . . . . . . . . Simple Correlation Coefficients Between Shear and Sensory, Biochemical and Histological Parameters Simple Correlation Coefficients Between Taste Panel and Shear, Biochemical and Histological Parameters . Simple Correlation Coefficients . . . . . . . . Simple Correlation Coefficients . . . . . . . . . . . . Page 220 220 221 222 223 224 227 228 229 230 232 233 234 235 236 237 238 239 2:1”— 7 INTRODUCTION As society becomes increasingly more affluent, the consumer not only insists that adequate quantity of foodstuffs be available but also that these foodstuffs be of a consistent and predictable quality. With such a consumer directance the animal scientist has had to become increasingly more sophisticated not only in techniques, but also in innovativeness. Not only has affluence given rise to a discriminating consumer, it has also fueled the arrival of competitive products that are offered and will, in the future, increase both the quality and quantity of alternatives available to the consumer. Economic inflation pressures even the strong— est willed consumer to use some of the many substitutes for animal products and, as such, the animal scientist must be continually improving the economic and qualitative position of his products. In addition to the scruntiny of the consumer, the animal scientist must be cognizant of the restrictions placed on his manipulative procedures by governmental and consumer protective agencies. The questionable status of diethylstilbestrol and sodium nitrite is indicative of the impact that the "potential carcinogen" label can impart to any agent used in the production system. The animal scientist must have foresight and the ability to economically manipulate the animal system within regulatory restraints. Tenderness has been at the forefront of meat investigations, based on its importance, for many years. The beneficial effects of aging on meat tenderness have been recognized for many years, however, the specific » 7 i I mechanism by which aging affects tenderness has been elusive. In contem— porary marketing methods, the meat packers must efficiently utilize facilities which has prevented the majority of carcasses from receiving adequate storage time to achieve maximum tenderization. Although aging of carcasses has not been a panacea for all tenderness deficiencies, it is sufficient for the animal scientist to search for the components regu— lating postmortem tenderness changes. Considerable progress has been made on the effects of temperature, pH and histological modifications, however, the animal scientists are still searching for answers to most of the questions. With these ideas in mind, this project was designed to meaSure ultra- structural and biochemical parameters of postmortem bovine longissimus muscle. The animals used in this project were young Hereford bulls that had been selected for tenderness or leanness for several generations. Most postmortem studies involve study of biochemical and histological parameters, however, few of these studies have attempted to relate the postmortem observations to tenderness data. This study was designed to not only observe postmortem changes, but to determine if these changes were related to objective and subjective tenderness scores. REVIEW OF LITERATURE Tenderness Studies Prior to 1960 For thousands of years the primary concern of man in the handling of meat has been its storage and preservation and only within the last century with the advent of mechanical refrigeration has he been able to preserve meat in the fresh state during all seasons of the year. He has subsequently became concerned with the palatability aspects of fresh meat. With this concern, meat scientists began to investigate the properties of postmortem muscle in an effort to control and manipulate those factors deemed important to the consumer. 0f the several palatability characteristics of meat, tenderness has generally been considered to be the single most important component. With the importance placed on this component, considerable work has been expended on determining the factors which are not only responsible for the inherent tenderness of a muscle, but also in what way these factors promote in- creases in tenderization during postmortem aging or ripening of meat. It is not clear when the first ideas concerning tenderization were formulated, however, when histologists first identified the components of a muscle, it became apparent that the connective tissue proteins and the proteins of the muscle fibers would certainly play an important role based solely on their dominance within the system. The early investigators in the field were rather equally divided in belief as to whether the connect— ive tissue proteins (primarily collagen) or the muscle fiber proteins underwent changes during aging that would account for the tenderization observed. The first observations that meat hanging for several days improved in tenderness apparently has been lost in antiquity, however, Hoppe-Seyler (1871) made the general observation that dead tissues liquefied without accompanying putrefaction which prompted him to suggest a similarity to digestive fermentation. Whether this observation was the spark that caused many to subscribe to the belief that autolysis was the mechanism of postmortem tenderization is unknown, however, the concept developed a following among many eminent scientists. Lehman (1907) and associates concluded that the toughness of meat, determined by mechanical measures, was closely related to the connective tissue content. Hoagland, McBryde and Powick (1917) observed that the primary change during postmortem aging was a marked increase in tenderness. This same group concluded that the chemical changes occurring during storage, including the increase in non-protein nitrogen, could be attri- buted to enzyme activity. Apparently these changes did not manifest them- selves microscopically even after 77 days of storage. Mitchell, Zimmerman and Hamilton (1927) chemically determined the amount of connective tissue in various cuts of meat, but due to low sen- sitivity of the technique the results were inconclusive. However, they were able to observe relative differences such as that observed between the wholesale shank and the rib of cattle. It was observations such as this that prompted and encouraged some scientists to believe that the .aauafigu;15. ’ connective tissue proteins were the primary components of tenderness. It was readily ascertained that mere quantity was Sufficient to cause differ- ences between anatomical location of muscles and a direct extrapolation to explain the tenderness between the same muscle from two different animals was considered to be a valid inference. Mitchell 35 El' (1927) didn't make this direct extrapolation, however, they attributed collagen and elastin differences between similar wholesale cuts to condition differences among animals. In a subsequent paper, Mitchell, Hamilton and Haines (1928) correlated collagen and elastin nitrogen in various muscles of the beef carcass with such parameters as age, sex, grade, texture and firmness of steers and heifers fed from 0 to 266 days. A ranking of the various muscles of the carcass was possible utilizing collagen and elastin nitrogen, however, no significant correlations were obtained with age, grade, sex, texture or firmness. While maintaining that connective tissue content was the major factor in meat tenderness, these authors concluded that the parameters used were not a reliable method to determine tender- ness of lean meat. The concept of connective tissue quantity dictating tenderness was not universally accepted. Some scientists believed that Ithe aging period was responsible for chemical changes in stroma proteins (connective tissue proteins) that resulted in increased tenderness. Ewell (1940) reported that some German scientists believed that coarse textured (high in connective tissue) cuts of meat which showed a greater relative increase in tenderness than cuts initially more tender supported the con- cept that postmortem aging altered connective tissue proteins. .1: }mrked that the general consensus among the butcher trade was that Ernest for a length of time improved the palatability aspects parti- V}V»tenderness. In a consideration of the various components of ten— ‘.U.f5 the authors reported that muscle fibers, connective tissue and gtiected tenderness. They suggested that much of the tenderness ",r.:,:,. between muscles in different anatomical locations could be jfi3 myosin A > actin > myosin B. The sarcoplasmic protein de- gradation is not surprising since previous work by Bodwell and Pearson (1964) showed no major activity on the salt soluble proteins. Addition- aIIJVSuzuki g; _l, (1969b) observed that cathepsin D decreased the Mg2+- erflianced adenosinetriphosphatase (ATPase) activity of muscle at pH 5.0 but not at pH 5.5. The Ca2+4enhanced ATPase activity was not changed irrespective of the pH used. 33 The concept of the lysosome has elicited considerable interest since the presence of acid pH optima, hydrolytic enzymes seems to fit the mold for a ready reservoir of postmortem autolytic enzymes. However, the con- cept at first fails since some authors have reported that histochemical enzyme markers suggest that normal muscle exhibits activity only as a part of the vascular system (Smith, 1964), or as the result of dystrophy or atrophy (Pellegrino and Franzini, 1963). However, Bird (1971) reported that by utilizing ultracentrifugation he and his coworkers have been able to define two distinct sources of lysosomal enzymes. By determining relative quantitives of several of the enzymes known to be present in lysosomes, Canonico and Bird (1970) have demonstrated that some of the lysosomal enzymes are present as the result of phagocytic cells but that enzymes are also present in endogenous muscle lysosomes. However clear the concept of intracellular lysosomes may be, the overriding fact that needs to be clarified is the association of these enzymes with postmortem tenderization. Martins and Whitaker (1968) and Caldwell (1970) suggested that even though difficulty has been encountered in showing catheptic activity towards salt soluble proteins it is possible that a combination of the various cathepsins (A, B, C, D) might be necess- ary for degradation. Ono (1970) biochemically substantiated the presence of lysosomes in bovine longissimus muscle, however, he did not investigate the capacity of these enzymes to degrade salt soluble protein. Parrish et al. (1969a) attempted to put the catheptic enzyme-tenderi- zation question in proper perspective. By measuring NPN, free amino groups a .o I; 34 and shear values, these authors concluded that postmortem proteolysis did occur, but tenderization did not parallel the proteolytic products. Parrish _£‘_l. (19699 suggested that even if proven that the amino acids come from connective or myofibrillar tissue proteins it would be an assumption that this is related directly to tenderness. These authors proposed that a few highly specific points of myofibrillar protein cleav- age, such as those at the I-Z junction, would be more likely to improve tenderness rather than N or C-terminal degradation of actin or myosin. Eino and Stanley (1973) followed the development of proteolytic activity in psoas major muscles aged at O to 5 C up to 10 to 14 days. By utilizing a crude catheptic enzyme preparation and maintaining an optimum pH of 3.8 these individuals found that the activity (cathepsin D) on several natural substrates generally followed the shape of the aging curve. Little additional lysosome release with Triton X could be observed after 8 to 14 days aging which suggested to them that maximal release and activity coincided with tenderization. Even though enzyme assays were conducted in an environment (pH = 3.8) dissimilar to postmortem muscle these authors suggested that the observed maximum release of cathepsins coincident with tenderization should be adequate to implicate these enzymes in tenderization. Additional textural measurements were made which coin- cided with observations of maximum cathepsin release. Most investigations have concluded that probably some type of proteo- lysis is involved in postmortem tenderization, but the proper measurements and conditions have not been found that unequivocably demonstrate this fact. 35 With this in mind, G011 _£‘_1. (1971a, b) attempted to demonstrate that proteolysis was indeed a functioning part of postmortem muscle tenderiza- tion. They utilized the concept that the myofibril has certain areas which are sensitive to proteolysis by trypsin. They argued that a mild proteo- lytic attack on the sensitive myosin and tropomyosin-troponin (TM-TN) complex might be a fruitful indirect approach to determining the role of proteoly- tic enzymes in postmortem muscle. Their results showed that a mild pro- teolysis by trypsin effectively mimicked nearly all of the postmortem changes observed in muscle. These changes apparently are prevalent and measurable before the proteolytic activity cleaves myosin into heavy and light mero- myosin fractions or destruction of the TM-TN complex. A more in depth review of this work will be made later, however, it appears that work such as this certainly suggests that a mild specific proteolysis probably occurs in postmortem muscle. The most apparent problem that exists is determining what endogenous (if it is such) substance is responsible and how one can properly measure all components of the system. Although it is not appropriate at this point of this literature review to consider in depth the work reported by Busch _£__ls (l972b),they reported on the isolation of a endogenous component of muscle capable of removing the Z-line from myofibrils. Although complete characterization has not been reported, preliminary results suggest that it is an enzyme which re- quires activation by greater than O.l mM calcium. All other ultrastructural components appear to be unaltered by this enzyme, but until adequate char- acterization is obtained one can only speculate as to whether this is the elusive proteolytic agent sought after for so many years. 36 Muscle Protein Solubility Changes in the myofibrillar proteins during postmortem storage re- ceived considerable attention for a number of years. Saxl (1907) observed decreased protein solubility as the result of rigor mortis which he attri- buted to protein denaturation. Deuticke (1930) found that both postmortem muscle and prerigor muscle that received repeated stimulations yielded about 30% less protein upon extraction with 0.09M potassium phosphate pH 7.2 containing 0.03M potassium iodide (KI). This solubility differ- ential of the stimulated muscle can be associated with early onset of rigor mortis (Helander, 1957). Any discussion of postmortem muscle protein solubility is intimately involved with rigor mortis. The interpretation of rigor mortis relies solely on the structural aspects of muscle which Huxley (1958) clearly described. The sliding filament model proposes the presence of inter- digitating thick and thin filaments. The overlap of these filaments pro- vide not only the striated appearance, but also performs the shortening necessary for force generation. The presence or absence of several ions and specific nucleotide phosphates dictate whether the system is contract- ing or relaxed. The overall picture of rigor mortis and resolution of rigor will be considered in depth at a later point, however, suffice it to say at this point that in postmortem muscle the absence of the plasti- cizing effect of Mg adenosine triphosphatase (Mg ATPZ') allows permanent bonds to be formed between actin and myosin. The postmortem contraction and actomyosin formation results in stiffness and is characteristic of the rigor mortis phenomenon. 37 As mentioned previously, the observation that muscle solubility de- creased postmortem prompted the investigation of what caused "resolution of rigor" and the corresponding increases in tenderness. One of the earl- iest observations in this area was that of Wierbicki _£.él- (1954) who suggested that initial toughening might be due to formation of actomyosin (rigor) and the observed tenderization could be due to dissociation of actomyosin. Additional results from the same laboratory (Wierbicki t a1., 1956) supported the concept that rigor mortis was associated with the formation of actomyosin, however, these authors failed to find support for tenderization to be due to its dissociation. Helander (1957) investigated solubility of contracted and non-contracted muscle, but he was unable to find decreased solubility. However, he assumed that the extracting system, KI which depolymerizes F-actin to G-actin, could be responsible for the apparent discrepancy in extractability. Zender _£‘_1. (1958) in their studies involving aseptic autolysis of muscle found no diminution of the fibril proteins extraction during rigor mortis such as was observed by Wierbicki _£_al, (1956). However, they used a buffer system consisting of l M glycine. Whether or not this buffer can be classified with strong dissociating agents such as KI or act simi- larly to pyrophosphate in dissociating the actomyosin complex is unknown. These authors did find decreasing extractability of protein from post- rnortem muscle, but they attributed this to proteolysis since the quantity caf free amino acids increased. Locker (1960) in studying proteolysis in beef noted that the extraction (Df PCstmortem muscle with Weber-Edsall solution (W-E) was highly variable 38 but the trend was for a decline during rigor with an increase following rigor. This increase in many cases equaled or exceeded that extracted from (3 hour samples. Since it is rather well accepted that W-E solution extracts the actin-myosin A (myosin B) complex (Fujimaki gt al., 1965b), it would appear that Locker's results support the postulate of Wierbicki .E£.§£' (1956) that no actomyosin dissociation occurs postrigor. Whitaker (1959) in a review thought it would be reasonable to assume that the pro- cess of tenderization followed a reversal of rigor mortis, however, he concluded that the data do not suggest that this was actually occurring. Weinberg and Rose (1960) investigated changes in protein extracta- bility during postrigor storage of chicken breast muscle. These authors used several extraction methods based on differential solubility that allowed the quantitation of actomyosin and myosin in prerigor and post- rigor muscle. They determined that the increase in extractability post- rigor was due to the increase in the actomyosin fraction. These individuals, however, postulated that since the free myosin content decreased postrigor and actomyosin increased that it could have been possible that dissocia- tion of actomyosin occurred but it reassociated during extraction. In concluding their work, Weinberg and Rose (1960) suggested that contrary to the popular autolysis theory it appeared that tenderization was the result of specific cleavage of an actin association responsible for main- tenance of the muscle matrix. Hill (1962) studied the distribution of the various nitrogen fractions in two muscles, longissimus and semitendinosus, among lambs, swine and 39 cattle. He reasoned that by using two muscles that are considerably diver- gent in tenderness, information regarding tenderness differences might be determined. The results showed that beef muscle contained a greater quantity of stroma and myofibrillar nitrogen with the latter quantity determined on a stroma nitrogen free basis. Hill (1962) stated that it was assumed that the greater toughness of the semitendinosus versus the longissimus muscle was primarily associated with the stroma content. However, on a stroma free basis, he observed that the semitendinosus had a greater quan— tity of myofibrillar nitrogen and less sarcoplasmic nitrogen and suggested that in some way an association between these parameters and tenderness existed. Additionally, he suggested that a similar argument could be ex— tended to the observed differences among the species since tenderness ranking appears to coincide with the distribution of myofibrillar and sarcoplasmic nitrogen. Partmann (1963) in discussing rigor mortis and tenderization concluded that the resolution of rigor mortis is not a simple reversal of those events leading to rigor. However, he observed that if fully aged beef muscle was gently homogenized in isotonic KCl a mixture of fiber fragments was obtained that contracted upon addition of 10‘2 M ATP. He concluded that this supplied sufficient information to believe that the actomyosin complex has either been dissociated somewhat or became easily dissociated as the result of the aging period. This information and other reported data suggested to him that little change takes place in the fibrillar pro- teins during aging. 40 Hegarty, Bratzler and Pearson (1963) investigated the distribution of the various nitrogen fractions in muscle from twenty yearling bulls that had been selected for tenderness. Tenderness was highly correlated ‘with fibrillar solubility when using shear data or taste panel compari- sons. Although studying anabberant muscle situation rather than effects of aging, Sayre and Briskey (1963) found that a combination of pH and temperature could affect the solubility of the myofibrillar proteins. As muscle was exposed to a medium (5.7 to 5.9) or low pH (5.3 to 5.6) in com- bination with a high temperature (>35 C), myofibrillar protein solubility decreased considerably. This situation is dramatically represented by the condition known as pale, soft exudative pork, however, whether the pH and temperature relationship alters the solubility of bovine muscle is certainly speculative. Scopes (1964) investigated various temperature and pH combinations and their affect on the solubilities of bovine longissimus muscle proteins. He found a situation paralleling that reported by Sayre and Briskey (1963). Bovine muscle protein extractability dramatically decreased if the muscle preparation was exposed to 37 C temperature for 4 hr. then cooled to O Centigrade. Similar samples that were either extracted at O ' hour or allowed to "age" 20 hr. at O C and then extracted showed virtually no changes in extracta- bility. They speculated that the heat denatured sarcoplasmic proteins in some way inhibited high ionic strength KCl from extracting the myofibrillar proteins. 0. 41 In a somewhat similar study, 0011, Henderson and Kline (1964a) investi- gated postmortem tenderness and solubility changes in the bovine semiten- dinosus. Samples were obtained from paired muscles, 1 excised immediately postmortem and the other left on the skeleton. After storage for l of 6 time periods, (at 4 C) 0, 6, 12, 24, 72 and 312 hr., the samples were tested for tenderness and protein solubility. It was found that the 2 sample groups, excised and nonexcised, responded differently to the tests performed on them. A negative correlation coefficient (r = -0.32) existed between shear and myofibrillar protein solubility of the excised muscle, but a positive correlation (r = 0.44) was determined for unexcised muscle. They also observed that the 0 hour excised muscle yielded more protein and was less tender than the unexcised muscle which appears to contradict earlier results (Hegarty gg_al., 1963). These authors suggested that pro- tein solubility and tenderness were related only casually and that the excised muscles might have an opportunity to cool faster and thus not be— come denatured during postmortem storage. Aberle and Merkel (1966) investigated the solubility and electrophoretic behavior of proteins from aged bovine muscle. They found an expected decline in fibrillar protein solubility during rigor mortis, however, contrary to Goll gt 2;, (1964althey also found a significantly higher solu- bility at 168 and 336 hr. postmortem. The increase in solubility was reported to be positively correlated with the decrease in Warner-Bratzler shear values at 168 and 336 hours. These authors also found a decrease in sarcoplasmic protein solubility from the semitendinosus muscle post— mortem, but no change was observed in the longissimus. A few changes were 42 also noted in the electrophoretic pattern of the sarcoplasmic proteins as the result of aging. Maier and Fischer (1966) also observed changes in certain bands and increased intensity of others when water soluble proteins were submitted to gel electrophoresis. Fibrillar protein solu— bility was essentially static during postmortem storage, however, as reported in previous work the latter authors found that the water soluble proteins became less extractable as storage time increased. McIntosh (1967) reported that bovine semitendinosus fibrillar protein including actomyosin became more extractable as aging time increased through 2 weeks. Similar responses were reported for pork and chicken fibrillar proteins except the time scale necessary for chicken was less than that for bovine or porcine muscle. Cook (1967) studied the effect of certain physical treatments on the solubility of postmortem bovine muscle. He reported that stretching pre- rigor muscle and allowing rigor to occur, prevented the occurrence of fibrillar solubility changes. This observation led him to suggest that the contractile state played a large part in the solubility of the proteins of the myofibers. The contractile state was only significant for postrigor samples which led him to suggest that the formation of actomyosin was an important aspect of decreased solubility during the rigor state and if acto- uwosin formation could be inhibited it would improve tenderness attributes (fl muscle. Buck, Stanley and Commissiong (1970) investigated similar aspects Ofnmscle as that of Cook (1967) and found that stretching significantly increased the extractability of protein from postrigor muscles in all but 43 one trial. They reported that the increased solubility was positively correlated with tenderness. However, investigation of the properties of the protein extracted revealed that more actomyosin was extracted from the stretched muscle in all trials. These authors were unable to clarify the enigmatic results, however, some evidence for the individual extraction of myosin and actin and subsequent combination of the two in the extract- ing buffer has been postulated previously by Weinberg and Rose (1960). The data presented by Buck gt El' (1970) doesn't suggest that this would account for all the observed discrepancies, however, it is possible that improved resolution of the extracted proteins might clarify this point. Davey and Gilbert (1968a) investigated the changes in extractability of bovine and rabbit muscle during aging. By using the Hasselbach-Schneider (H-S) buffer which preferentially extracts myosin and the W-E buffer which extracts only actomyosin, these authors determined that fibrillar solubi- lity increased with aging. It was found that less actomyosin was extracted from unaged than aged meat when short extraction periods (10 min.) were used. A time necessary for solubilization and period of aging was described with the aged samples requiring considerably less time. A similar time response was reported when the H-S buffer was used for extraction. The authors concluded that the more extensive and increased ease of extraction Cf fibrillar proteins was consistent with weakening of the linkages of the ruoteins and in some way the ultimate pH helped determine the extent of extractability of the proteins. In a subsequent report, Davey and Gilbert (1968b) reported moving boundary electrophoresis data suggesting that actin 44 was released in increasing amounts into the H-S buffer during aging. They reported that the actin recombined with myosin during dialysis in prepara- tion for electrophoresis and the increased peak area attributed to acto— myosin was in essence due to more solubilized actin which had increased during aging. This was consistent with the observation that myosin extrac- tion did not fluctuate as a result of the aging process. From their observations, Davey and Gilbert (1968b) concluded that in some unknown manner the tropomyosin and actin association could be weakened and as a result actin became more extractable with aging time. Some support for dissolution of the Z—line during aging (Davey and Gilbert, 1967b) apparently lends credence to the concept of Davey and Gilbert (1968b). Penny (1968) utilized different extracting buffers and other chemical tests to determine the properties of prerigor and aged rabbit muscle. He found an increasing extraction of proteins during aging when using 1 M KCl. Pyrophosphate extraction yielded similar results, however, by coupling extraction proce- dures with measurement of Ca2+ ATPase activities it was possible to charac- terize the properties of the increase in extraction. The initial extraction with pyrophosphate yielded all the Ca2+ ATPase activity as evidenced by the lack of any further increase with aging. Extraction with KCl yielded less of the Ca2+ ATPase activity from prerigor muscle than with the pyrophosphate extraction, however, the KCl Ca2+ ATPase activity increased with aging time. Penny (1968) suggested that the differences observed between the two buffered extractants was an indication that more actin and tropomyosin was solubilized as the result of aging. Reduced viscosity data suggested 45 that no changes occurred in the myosin molecule since a probable component, acto-heavy meromyosin would have revealed results inconsistent with those observed. All of these results allowed Penny (1968) to conclude that a breakdown of bonds had occurred within the myofibrillar component. The probable site of this bond breakage was the complex binding area of thin filament with the Z-line (Davey and Gilbert, 1967i)- The weakening of attachments in this area would allow more actin to be solubilized which did occur as was shown by KCl extraction. Results obtained using myofibril suspensions suggested that the component or agent responsible for weakening of the thin filament-Z lattice structure was easily removed by buffered washes of the suspensions. In two subsequent papers, Penny (1970a, b) sought to more thoroughly investigate and characterize the components being solubilized as the result of aging. In the first paper, Penny (1970a) set out to characterize and quantify the specific proteins extractable with H-S solution and 5 mM tris (pH 8.2) buffer. The composition of the muscle extract was determined to be 47% myosin, 12% tropomyosin and 3% troponin. He stated that actin could not be quantified due to it being complexed with a-actinin, troponin and an insoluble residue. The second paper (Penny, 1970b) verified some of his earlier work that aging was responsible for allowing more myosin, actin, tropomyosin and troponin to be extracted. None of the work firmly suggested that these proteins were degraded during storage. In evaluation of his data and after supplementation with work from other authors, he concluded that a-actinin, as a component of the Z-band, loses its affinity for the thin 46 filament during aging and as such the thin filament proteins are more readily released upon extraction. H383.2£.§l° (1966) reported that pre- ceding solubilization of actin, a break occurs between the thin filament and the Z-line, Goll'gg'al. (1970) suggested that the strength of the actin-Z-line bonds contributes substantially not only to the extraction of actin but also affects myosin indirectly since actin influences the solu- bility of myosin. Sayre (1968) determined the change in extractability of the myofi- brillar proteins of chicken pectoralis muscles during 24 hr. of storage. He found that myosin extractability dropped considerably during the first 3 hr. and showed a continuous decline through 24 hours. The loss of myosin extractability was paralleled by an increase in extractability of actomyosin. The constancy of the other measurable protein fractions, NPN, sarcoplasmic and stroma, led Sayre to eliminate the probability that major proteolysis was involved in promoting the changes in extractability. Instead, Sayre (1968) suggested that the combination of actin and myosin during rigor was responsible for the decrease in myosin extractability observed postmortem. The increase in actomyosin extraction that occurred was suggested to be due to either a breakage of the thin filament or in some way a detachment from the Z-line which then allowed the actomyosin to become extracted. These observations support the view of several of the previous studies in that extractability is affected by the actin-myosin combination and by thin filament bonding to the Z-disk. Valin (1968) used bovine longissimus muscle stored for either 1 or 8 days at 4 C to prepare both actomyosin (W—E) and metin (TM-TN) which was 47 extracted either directly from muscle or from the isolated actomyosin. Valin (1968) found that more myofibrillar protein was solubilized as post- mortem storage time increased and associated with this increase was an increase in extraction of metin from the isolated actomyosin. The impor- tance of the extraction of metin was the lack of extractability after one day of aging and the observation that 8 day aged muscle yielded a consi- derable quantity. Valin (1968) suggested that an association exists be- tween metin extraction and tenderness since the apparent intimacy of TM- TN with the thin filament and the Z-line coincides with the previous report by Davey and Gilbert (1968b) concerning aging and the disappearance of Z-component. Chaudhry, Parrish and G011 (1969) investigated the effect of temper- ature and extracting buffer on the solubility of rabbit and bovine muscle. The sarcoplasmic proteins showed little change in extractability at 2 C, however, these proteins increased in solubility during postmortem storage at 25, 28 and 31 centigrade. At 37 C solubility increased up to 24 hr., however, a decrease of 18 to 25% was observed thereafter. The myofibrillar solubility in 0.5M KCl increased considerably when determined either 48 or 312 hr. postmortem. This increased extraction was apparent in both rabbit and bovine muscle and increased with increasing temperature up to IN centigrade. Myofibrillar extraction in 1.1M KI generally paralleled Hfl.extraction up to 25 C, however, KI extractions were not as great as that of KCl. At 28 and 31 C little change was noted and at 37 C a decrease afmnr6 to 12 hr. was noted. These authors observed that they could not 48 find any evidence for any of the myofibrillar extracts to contain actomyo- sin. Chaudhry E£.él° (1969) could find no association between the solu- bility of myofibrillar proteins and tenderness. These authors reported a decrease in solubility at 37 C which does not coincide with previous work showing muscle to be more tender at this temperature (Busch, Parrish and G011, 1967). The role of the water soluble (sarcoplasmic) proteins in postmortem tenderization has been investigated and in many cases the solubility patterns are similar to those of the myofibrillar proteins, in that a slow decrease occurs postrigor (Goll t 1., 1964a; Sayre and Briskey, 1963; Wierbicki _£._l-a 1956). Generally the sarcoplasmic proteins have been shown to change little quantitatively and qualitatively as shown by elec- trophoresis (Aberle and Merkel, 1966). Fukazawa _£.§l- (1970) found little change in low (< 0.2) and high (> 0.57) ionic strength extracts from pre- and postrigor chicken muscle. However, by extracting the residue of both the low and high ionic strength fractions with 0.03M KCl and H 0 these 2 latter authors found considerable increases occurring during postmortem storage. It was noted that it could be more than fortuitous that this increase is congruent with the loss of Z-line materials. The water soluble extract was fractionated further with NH4804 and the properties of each of two fractions were tested. They found that considerable differences existed between these fractions from pre- and postrigor muscle on their effect on SuPerprecipitation and gelation of F-actin. The 2 components were identi- fied as a-actinin and native tropomyosin which have been postulated to be associated with the Z-line and thin filament, respectively. 49 Landes, Dawson and Price (1971) extracted the various protein frac- tions from treated (sodium pentobarbital) and control turkey- breast muscle during pre- and postrigor time periods. They found that fibrillar protein solubility (control) increased rapidly and then leveled off after 3 hr. postmortem. The treated samples revealed a delayed response but increased gradually before leveling off at 12 hr. postmortem. Initially the control birds had the most extractable fibrillar protein, but after aging more protein was extracted from the treated birds. Wu and Sayre (1971) investigated the effect of aging on myosin from red and white chicken muscle. They found an increase in protein extraction with time postmortem. They also isolated a new component (T) that increased as aging progressed, however, this component was found only in white muscle. Hay, Currie and Wolfe (1972) reported the observation of a peak of unknown composition in sedimentation diagrams of chicken fibrillar protein that corresponded closely to the component T of Wu and Sayre (1971). These authors concluded that myosin becomes more dissociated during long (7 days) periods of aging as compared to that from muscle aged less time. deFremery (1971, 1972) in two closely related papers isolated actin from chicken breast and leg muscle. In the initial paper, he found no change in actin extractability during aging nor could an association between removal of C-terminal amino acids from actin and tenderization be found. In the sub- sequent paper, some increase in polymerizable actin with aging was reported, however, no correlation with tenderness could be found. Much of the in- formation available concerning aging of meat is contradictory, however, 50 the breakage or rupture of myofibrils has been relatively well documented by Davey and Gilbert (1967b, 1969). Since considerable evidence exists for the presence of a—actinin in the Z-band (Goll‘_£‘_l., 1969), Penny (1972) regarded the changes of this protein during postmortem aging as of primary importance in myofibrillar disruption. Penny (1972) extracted and quantified a-actinin from 1, l4 and 21 day postmortem bovine longissimus. It was found that the quantity of a-actinin remained the same (1.4%) ir- respective of the aging time. When comparing aged to unaged muscle, a small difference was found in the amount of a-actinin that could bind F- actin, however, the author felt that the differences were insufficient to account for all the myofibrillar changes in postmortem muscle. Hay _£ _1. (1972) and Hay, Currie and Wolfe (1973b) investigated the effect of postmortem aging on the physicochemical changes in chicken acto- myosin and fibrils prepared from breast and leg muscle. In the initial paper, Hay t al. (1972) found only minor differences in Ca2+ ATPase acti- vities during aging, however, in the latter paper, Hay _£._1. (1973b) found aging affected electrophoretic patterns. Electrophoresis of sodium dodecylsulfate (SDS) incubated myofibrils quite clearly revealed the dis- appearance of some and appearance of several new components during a 168 hr. aging period. The authors suggested that the disappearance of a 44,000 Dalton component at 48 hr. in breast muscle could possibly be more than coincidental with the early reported loss of the M-line in electron micro- scope studies (Hay gg al., 1973a). The appearance of a 30,000 Dalton com- ponent in both breast and leg muscle was attributed to a possible degradation of myosin. 51 In summary, only general conclusions can be made concerning protein solubility postmortem and during subsequent aging. It is generally be- lieved that muscle reaches a low point in extractability during rigor and slowly increases during aging. It has been observed that prerigor muscle is tender, however, considerable discrepancy exists in the data as to its protein solubility. Most observations suggest increases in tenderness with aging up to a point, however, not all solubility data reported coin- cides directly with the increased tenderness. Much of the recent work has focused not only on quantitative changes but also on qualitative changes of postmortem muscles. Biochemical data corresponding to observed histo- logical changes have been promising, but additional sophistication and experimentation will be necessary to validate the observations. A general trend has been seen from the older method of mere quantitation of the various nitrogen fractions postmortem to both quantitative and qualitative evaluation of all protein fractions in an effort to determine if the specu- lated minor proteolysis actually occurs in muscle (Coll t 1., 1971b) and if these changes are associated with tenderization differences. Water-Holding Capacity The relationship between water-holding capacity (WHC) and tenderness is well documented (Hamm, 1959, 1960). According to Hamm (1960) there exists a complicated relationship between glycolysis and pH that affects the ultimate hydration of the muscle proteins and therefore the tenderness Ofnmat. Tenderness tends to be at a low point coinciding with the least 52 hydration of the proteins. Hamm (1959) determined that WHC was more related to the degradation of ATP postmortem than to pH. This relationship was suggested to be involved with the association of ATP with the alkaline earth metals and when ATP is degraded the cations are free to bind to the proteins and cause a tighter structure of less hydration. Hamm (1959) presented a graph of bound water versus pH and it showed a minimum of hy- dration at approximately pH 5.0 or near the isoelectric point of muscle proteins. The association of WHC with tenderness suggested to several groups that a modification of rigor mortis might prove fruitful to increase tenderness and improve other quality attributes of muscle (Khan and Nakamura,l97l; Weiner and Pearson, 1966; Weiner, Pearson and Schweigert, 1969; deFremery, 1966; Radcnro-Thomas _£__1,, 1959). As a general rule, the attenuation of pH fall postmortem has been obtained and as a result increased hydration has been observed. The injecti0t1 of several of the chemicals used to impede pH fall[1,2-bis-(2-dicarboxymethylaminoethoxy)- ethane (EGTA), ethylenediaminetetracetic acid (EDTA), etc.] have proven to be lethal and the practical application appears to be limited at this time. Another possible limitation could be the probable increased bacterial proliferation if the pH is allowed to remain near neutrality which is the pH optimum for most bacteria (Lechowich, 1971). Hamm (1960) very adequately summarized the relationship between WHC and tenderness when he said that a correlation is apparent and important (Hay if the difference between samples is relatively great. 53 Rigor Mortis Chemical Changes The literal translation of rigor mortis is "stiffness of death". Much of the early work on rigor mortis involved the association of the observed rigidity with precipitation of the muscle proteins by lactic acid. An early observation by Bernard (1877) suggested that glycogen was the forerunner of the acid production in postmortem muscle and without much difficulty an animal could be depleted of glycogen and alkaline rigor would be established. This appeared to be the first reference that contradicts the necessity for lactic acid for the obtainment of rigor. According to Needham (1971) researchers failed to recognize the implications of Bernard's observation and as such considerable time passed with the lactic acid theory still tenable. This is readily apparent in the report by Moran and Smith (1929) who attributed rigor to coagulation of the muscle proteins. Clarification of the enigmas of rigor mortis appeared to be related to the interpretation and elucidation of the biochemical energy pathways in muscle. The discovery of creatine phosphate (CP) in 1929 was apparently unnoticed in its association with rigor, however, the discovery and demonstration of the ATPase activity of myosin accelerated the work on contraction and rigor (Needham, 1971). Additionally, the discovery (Erdos, 1942) that a close association existed between the rigidity of muscle during rigor and the disappearance of ATP prompted a more thorough investigation of the adenosine triphosphate (ATP) concept (Needham, 1971). 54 Although earlier workers had reported the association of ATP and rigor, Bate-Smith and Bendall (1947) confirmed and extended this concept. Additionally they determined that animals with low glycogen reserves followed the same pattern except that the pH did not fall to normal levels. Earlier, Bate-Smith (1939) found a rapid change in the modulus of elas- ticity at pH 6.2. Bate-Smith and Bendall (1947) observed the same occurrence, but they considered that some other mechanism must be involved "alkaline rigor" could occur. In a subsequent paper, Bate- since a rapid Smith and Bendall (1949) reported the effect of animal condition on the onset of rigor, duration of rigor delay, initial pH and ultimate pH. They suggested that the struggle of an animal at death (as verified by muscle relaxants) affected the initial pH, but the glycogen reserves were the only factor that affected the ultimate pH. Bate-Smith (1948) in a classical review on rigor mortis discussed the overall knowledge of rigor mortis which included pH, ATP and CP, however, it was 3 years later before Bendall (1951) reported the precise relationship between CP, ATP and pH. He suggested that no ATP was broken down until approximately 70% of the CP had disappeared. All of this agrees with the observations by Bate- Smith and Bendall (1947, 1949) on the condition of the animal at slaughter. Well rested animals not only have considerable quantities of glycogen re- serves, but also the CP reserve is adequate to maintain the ATP supply until it is almost exhausted. Additionally, Bendall (1951) suggested that the shortening observed during rigor, even though differing greatly in time sequence must be similar in mechanism to physiological contraction. 55 Marsh (1952a, 1954) extended the rigor mortis and ATP dephosphoryla- tion concept to bovine and whale muscle. Although the investigation of postmortem whale muscle extended and verified earlier observations on other species, it also provided information on previously undescribed states of rigor. Marsh (19523)reported that fresh whale muscle existed either in a dry and firm, wet and dull or dry, hard and rubbery state. He found an important relationship between pH, ATP dephosphorylation and the physical state of the muscle. If the pH was greater than 6.3, the muscle appeared dry and firm, however, at pH values below 6.1, the muscle was invariably wet and dull. Within the transitional range of pH 6.1 to 6.3 a mixture of wet and dry muscles were observed. In concluding the study on whale muscle, Marsh (1952a)suggested that the transition from the dry to wet states represented the onset of rigor mortis. His observations on beef muscle verified the earlier observations on rabbit muscle (Bate-Smith and Bendall, 1947, 1949). Certain variations in temperature effects were observed between rabbit and bovine muscle but the dependence of rigor onset on ATP dephosphorylation unequivocably demonstrated that ATP content dictated the time course of rigor mortis. Marsh (1954) also reported the verifi- cation of the report by Bendall (1951) that shortening during rigor was essentially a slow and irreversible physiological contraction. He suggested that the resolution of rigor was in no way a reversal of the inextensibility obtained during rigor since the modulus of elasticity did not change after 7 days of storage at 7 C in a nitrogen atmosphere. 56 Since the shortening aspect of rigor mortis has been suggested to be essentially the same as contraction except for the time sequence (Bendall, 1951), the accumulation of information concerning contraction certainly should give an insight to the mechanism of rigor shortening. Marsh (1952b) discovered the method of how muscle maintained the relaxed state without splitting ATP. The presence of a relaxing factor in the super- natant and additional experimental evidence allowed him to speculate that the relaxing property was due to the binding of Ca2+ ions. Porter and Palade (1957) described a three-dimensional membrane and tubular struc- ture surrounding muscle which has been implicated in conduction of nerve impulses resulting in Ca2+ release (Huxley, 1965). Subsequent work has verified and extended this concept and showed that the relaxing factor is particulate in nature and is composed of components of the sarcoplasmic reticulum (SR) (Marsh, 1966). The clarification of the role of the SR in contraction and the suggestion that rigor shortening is essentially the same as contraction (Bendall, 1951) has led to the development of the theory that the increase in free sarcoplasm Ca2+ causes rigor shortening (Schmidt, Cassens and Briskey, 1970). Goll‘_£‘_1. (1971b) proposed that the loss 0f C32+ accumulating ability was the direct and immediate cause of rigor onset. They also proposed that limited proteolysis of the SR might be the direct cause of the loss of Ca2+ accumulating ability and the subsequent onset of rigor mortis. Needham (1971) succinctly summarized the sequence in the development of rigor mortis. The loss of circulation prevents 57 oxidative phosphorylation which necessitates the replenishment of ATP by PC and glycolysis. The usage of glycogen stores for anaerobic glycolysis causes the production of lactic acid which accumulates during the delay period. The pH drop is dependent on the quantity of glycogen reserves with exhausted animals showing little or no pH fall. Rapid ATP hydrolysis ensues when a critical pH is reached and the actomyosin ATPase appears to be activated when the Ca2+ pump fails to maintain the concentration gra- dient. The actomyosin ATPase initiates hydrolysis if ATP is available and fiber contraction may occur. The muscle fibers become inextensible when ATP is unavailable for plasticizing myosin and actin. Certain modi- fications of the sequence of events are seen if elevated temperatures are used or if insulin and muscle relaxants alter the normal situation. Structural Changes Before the introduction of such sophisticated research tools as the electron microscope, it had been difficult to explain physical measure- ments taken on prerigor and rigor muscle (Bendall, 1960). The increased knowledge of the muscle proteins and the knowledge that actin and myosin will combine when ATP is absent led to a clarification of the contractile mechanism (Szent-Gyorgyi, 1944). The explanation of the mechanism of shortening by the sliding of thin and thick filaments past each other contributed substantially to the interpretation of contraction and rigor (Huxley, 1958). Earlier structural models were unable to account for 58 certain observations made on muscle, however, the Hanson and Huxley model of filament sliding fulfilled nearly all the requirements necessary to explain rigor mortis changes (Bendall, 1960). Earlier proposals that suggested similarity between physiological contraction and rigor shorten- ing seemed to fit nicely into the sliding filament hypothesis. The iso- lation and characterization of several new proteins of the myofibril (Bailey, 1948; Ebashi and Ebashi, 1964; Ebashi and Kodama, 1966) has more definitively defined the contractile process whether it be physiological or postmortem. The previously mentioned relaxing factor described by Marsh (1952b) and the regulatory proteins discovered by Ebashi and co- workers (1964, 1966) in addition to the biochemical explanation of rigor mortis by a host of workers (Bate-Smith, 1939; Bate-Smith and Bendall, 1947, 1949; Bendall, 1951) has allowed considerable insight into mechan- isms which govern muscle activity. Although this description of rigor mortis is at best superficial, it does report the empirical observations necessary to account for most of the changes responsible for its development. The interpretation of postrigor changes which are responsible for the observed tenderness changes are no less difficult than that encountered in explaining the onset of rigor mortis. A detailed account of these postrigor changes implicated as tenderness factors follows under the title of "resolution 0f rigor mortis." 59 Postmortem Shortening of Myofibrils Factors Affecting Shortening Rigor Shortening. The observation that muscle shortens as it passes into rigor has long been known, but one of the earliest observations on rigor shortening in meat producing animals was reported by Bate-Smith (1939). He reported that rigor shortening appeared to be an unusual situation rather than a normal occurrence. He suggested that shortening appeared to be related to a rapid onset of rigor. Bate-Smith and Bendall (1947) concluded that shortening occurred only when the stiffening of rigor was associated with a pH higher than 6.2 and is not a normal con- comitancy of rigor. Bate-Smith and Bendall (1949) investigated the time course of rigor and reported a temperature dependent relationship for rigor shortening. Bendall (1951) suggested that rigor shortening, although involving a different time sequence, was similar to physiological con- traction in that the contractile elements were involved in both cases. Marsh (1954) reported results that supported the observation by Bendall (1951) that rigor shortening was similar to physiological contraction. In addition, he also suggested that this shortening was a slow and irre- versible contraction. All of the early work reported by Bate-Smith (1939), Bendall (1951) and co-workers defined all aspects of rigor mortis including shortening based on isotonic measurements. Jungk t 1. (1967), Goll (1968) and Busch gt a}. (1972£)developed an isometer that measured tension produced 60 by postmortem muscle and this apparatus definitively showed that muscle shortened during rigor onset. The overall implication of this type of measurement will be discussed in the section concerning rigor resolution, however, at this point it is sufficient to note that shortening, irrespect- ive of amount, plays an important role in the overall concept of rigor mortis. Temperature Effects. Although the discovery of cold shortening has been attributed to Locker and Hagyard (1963), the foundation for this work has a much longer history. Although Lowe and Stewart (1946) did not asso- ciate their observed toughness of prerigor excised chicken breast muscle with myofibril shortening, it was one of the earliest observations of this phenomena. Bate-Smith and Bendall (1949) and Bendall (1951) reported that muscle stored at 17 C shortened considerably less than muscle stored at 37 centigrade. Thus the early work implied a linear association between storage temperature and muscle shortening. Locker and Hagyard (1963) investigated the effects of temperature on shortening using a range of temperatures between 0 and 37 centigrade. They observed that maximum shortening occurred at 0 C and a minimum was reached between 14 to 19 centigrade. Shortening increased between 19 and 37 C al- though the extent of shortening at 37 C was not as great as that observed at 0 centigrade. The authors commented on the complexity of this enigma by reporting that rabbit muscle did not cold shorten. Jungk _5 a1. (1967) and Henderson, Goll and Stromer (1970) extended the cold shortening phe- nomena by clearly demonstrating that cold shortening was a temperature and 61 species dependent phenomenon. They reported the similar minimum and maxi- mum temperature for bovine muscle as reported by Locker and Hagyard (1963). Henderson _£._l, (1970) reported that rabbit muscle shortened, however, the maximum occurred at 37 C and shortening was minimal at 0 to 2 centigrade. Rabbit muscle shortened slightly between 2 to 16 C, remained constant be- tween 16 to 25 C and then shortened dramatically above 25 C and reached a maximum at 37 centigrade. Henderson _£__l. (1970) also reported that the shortening of porcine muscle was temperature dependent. Temperature de- pendent shortening of porcine muscle was reported to be intermediate com- pared to that of bovine and rabbit muscle but more nearly approximating rabbit muscle. Porcine muscle shortened more at 2 C than 16 or 25 C, however, the maximum occurred at 37 centigrade. Busch _5 al. (1972a) verified and extended all the previous reports on temperature dependent muscle shortening. Preriggr Muscle Excision. Locker and Hagyard (1963) reported that the stimulus of excision caused a small quantity of the contraction ulti- mately obtained. The overall implications of prerigor excision is its effect on tenderness which will be discussed subsequently. Effect of Shortening on Tenderness. As mentioned previously, Lowe and Stewart (1946) observed that prerigor excised breast muscle from chicken was considerably tougher than muscle that was left attached to the skeleton. Although these authors did not suggest that shortening was responsible for the tenderness differences, it was one of the earlier reports that described a tenderness relationship between excised and non- 62 excised samples. Ramsbottom and Strandine (1949) removed the longissimus from beef carcasses before chilling and reported that these samples were considerably less tender than paired non-excised muscles even after 12 days of storage. The authors attributed the tenderness differences to the change in the chemical and physical state of the muscle possibly due to the stimulation of muscle and nerve cells by cutting. The classical papers in the literature concerning shortening and tenderness are those of Locker (1960) and Locker and Hagyard (1963). In the initial paper, Locker suggested that the muscles of the ox go into rigor in various states of contraction. Locker (1960) suggested that the restraint a muscle has imposed on it as a result of hanging the carcass determines the final contractile condition of each muscle. The removal of a muscle either completely or by severing one attachment allows shortening to occur, and more importantly, the shortened muscle is tougher than a less contracted muscle. In the subsequent paper, Locker and Hagyard (1963) expanded the original observation to include temperature dependent shorten- ing. The latter observation becomes important since ig_§i£g muscles exposed to low temperatures may shorten and as a result be tough. Goll _£.§l° (1964a) verified the tenderness differences between excised and non-excised muscles and concluded that aging improved the tenderness of excised muscles but they were still less tender than controls after 312 hours. In a series of papers, Herring g; _l. (1965a, b, 1967b) investigated the influence of sarcomere length on the organoleptic qualities of bovine muscle. They found that prerigor excised muscle was considerably tougher 63 than prerigor excised but stretched-restrained muscle. This phenomenon existed for both pso E major and semitendinosus muscles which were consi- derably divergent in initial sarcomere length and original shear scores. These authors concluded that it was more important to prevent shortening than to stretch the muscle. Additionally, in light of previous observa- t a1., 1953; Tuma t 1., 1962), Herring tions on fiber diameter (Hiner _£.§l. (1965b) found a positive relationship between shear force and fiber diameter. However, the relationship became complicated since they were unable to ascertain the contribution of shortening to fiber diameter versus that due to inherent differences in diameter of the fibers. Gothard t l. (1966) measured sarcomere lengths of semimembranosus and longissimus muscles at slaughter through 7 days of aging. They found considerable shortening in both muscles, and the results suggested a strong association between final contractile state and tenderness. They also reported the tendency for sarcomeres to lengthen once maximum con- traction was obtained. These authors concluded that final contractile state played a role in overall tenderness although it apparently was not the major contributing factor. Cook and Langsworth (1966a, b) reported that several pre- and post- mortem treatments affected both the shortening and shear values of ovine muscle. They found cold shortening to occur maximally at 0 C, a minimum occurred at 5 C followed by an insignificant rise at 20 C and another maximum at 40 C that approached the overall maximum that appeared at 0 centigrade. The shear values did not correspond to the amount of shorten- ing in all cases. Maximum shear values were obtained from the 0 C samples, 64 however, the minimum value was obtained from the 40 C samples which were shortened to approximately the same degree as those at 0 centigrade. Marsh and Leet (1966) reported results that conflicted somewhat with Cook and Langsworth (1966a, b). Marsh and Leet (1966) reported shortening to highly influence shear values, however, certain limits were defined that govern the amount of shear resistance of a sample. Up to 20% short- ening did not appear to affect shear resistance, however, shear values increased from a low at 20% shortening to a maximum at 40 percent. Be- tween 40 to 60% shortening, shear values began to decrease and at 60% the amount of shear resistance approached the minimum obtained at 20 percent. Although all measurements were obtained from excised muscles, Marsh and Leet (1966) suggested that certain muscles in the carcass such as the longissimus can shorten ig_§i£u_due to lack of bOny attachments on both ends of the fibers. Indeed, Marsh, Woodhams and Leet (1968) found that ovine longissimus in situ increased significantly in toughness if exposed to low temperatures within 16 hr. of slaughter. These authors suggested that the toughness was probably due to the shortening of the muscle fibers as previously shown to occur in excised muscle strips (Marsh and Leet, 1966). Parrish t l. (1969b) reported work on Choice beef carcasses that contradicted the observations of Marsh _£_gl, (1968) on lamb carcasses. Several different temperature combinations were used to allow the phenomedfll of cold shortening to manifest itself if indeed it was a problem of i§_ situ muscles. These authors found that shear values were essentially the same for all time-temperature treatments. Thus this work suggested that 65 2 or 15 C temperatures did not influence the tenderness of bovine muscle that remained attached to the carcass. Parrish _£_al. (1969b) suggested that the rate of cooling could have been attenuated enough by the larger muscle mass and fat covering of the beef carcass which prevented fiber shortening. Davey, Kuttel and Gilbert (1967a) investigated the relationship be- tween cold shortening and meat aging. They found a similar association between the percentage of shortening and tenderness as reported by Marsh and Leet (1966). In addition, they reported that aging (shear decrease) was dramatically affected by the extent of shortening. Below 20% shorten- ing, shear Values were lowest and the effects of aging were maximized, however, beyond 20% shortening, the effects of aging decreased and they were minimal at 40% shortening. They surmised that at 40% shortening, or peak toughness, the sarcomere had decreased in length to the point that no I band was visible, i.e., the sarcomere was the same length as the A band (1.5 gm). Jungk _£‘_1. (1967),uti1izing isometric tension measurements,suggested that the increase and decrease in tension postmortem, probably corresponds to similar phases of decreasing and increasing tenderness. However, Busch _£‘_1. (1967) reported that isometric tension measurements are not necessarily a valid method for determination of shear values. They con- cluded similarly to previous reports (Gothard _£._l,, 1965; Marsh and Leet, 1966) that shortening contributes to muscle tenderness, but probably is not the main contributor. 66 Buck E; El. (1970) determined the force necessary to shear rabbit muscle that was excised and allowed to shorten and on a similar sample that was stretched zuui both stored at l to 2 C for 24 hours. The muscle that was allowed to shorten required more force to shear than the stretched sample in all cases. Bouton _£._l- (1973b)reported similar results using bovine muscle. The contractile state influenced the shear values signi- ficantly, and in addition these authors found an interaction between pH and sarcomere length. Most of the evidence overwhelmingly suggests that the contractile state of a muscle influences the eating quality of the final cooked pro- duct. Voyle (1969) recognized the amount of literature that supports the phenomena of toughening and shortening; however, he considered the situa- tion to be considerably more complex than a mere shortening of the sarco- mere. Additionally, he suggested that the compression of the myosin filaments against the Z-discs might be the most important aspect of muscle shortening. Weidemann, Kaess and Carruthers (1967) reported that prerigor unre- strained (cooked without restraint) bovine semitendinosus muscle was always tender, however, prerigor muscle that was restrained during cooking was always tough. Muscle stored unrestrained at 0 C remained tough even during 1 to 5 days storage. Stretched muscle underwent tenderization during one day of storage and remained tender throughout storage. Klose, Luyet and Menz (1970) utilized prerigor restrained and contracted chicken muscle to determine if shear values were related to the contractile condi- tion. They reported that the restrained samples had shear values 67 approximately twice as large as the contracted muscle samples. The authors concluded that the difference between samples could be obviated if the shear values were reported on a per filament basis. Hegarty and Allen (1972) stretched rigor turkey leg muscle and com- pared the shear values to a paired group of folded muscles on the opposite leg. Unexpectedly, the authors reported a significantly lower shear value for the folded muscles versus the stretched muscles even though before cooking the stretched muscles had a significantly greater sarcomere length. The authors could not explain these discrepancies of their data in light of those in the literature. Considering all the reports involving the effect of cold shortening on tenderness, Schmidt and Gilbert (1970) and Parrish _£._l' (1973) stated that they could possibly circumvent this problem. Schmidt and Gilbert (1970) reported that muscles excised from prerigor beef carcasses could be maintained in a tender state if stored for 24 hr. at 15 centigrade. An additional 24 hr. of storage produced a considerable aging effect in several of the excised muscles. Parrish _£._l. (1973) examined both pre- rigor excised muscle stored at 2 and 16 C and similar samples that were left ifl situ at the same temperature. They found that one day of aging at 16 C vastly improved the tenderness of bovine longissimus muscle. These authors suggested that 2 C chilling of muscles allowed to remain on the carcass apparently was not a deterrent to tenderness possibly due to the slowed rate of cooling as effected by muscle mass and fat cover. Smith, Arango and Carpenter (1971) reported similar results in that bovine 68 longissimus muscle increased significantly in tenderness by holding car- casses at 16 C for 16 hr. postmortem and then placed in a 2 C cooler. McCrae _£._l- (1971) found similar relationships when they reported that a 16 hr. delay at 18 C before freezing lamb carcasses considerably im- proved tenderness of several muscles. The original work on cold shortening (Locker and Hagyard, 1963) cer- tainly pointed out the hazard involved with exposing excised muscles to near freezing temperatures. Subsequent work has verified this early ob- servation, however, others have failed to find a one to one association between shortening and tenderness. Some researchers have suggested that cold shortening probably doesn't affect those muscles normally restrained on the carcass. Marsh (1972) suggested that shortening ig_§i£g_must be considered on an individual muscle basis since carcass restraint will minimize shortening in some but will not prevent it in others. Restraints on Shortening. Marsh and Thompson (1958) observed that muscle which was restrained during rigor did not shorten once the restraint was removed. Locker (1960) suggested that the final contraction state of a muscle depended on the restraint put on it in the hung carcass and it could be modified by excision or partial detachment. Herring _£_§l, (1965a) investigated muscle shortening and alteration of the normal carcass suspension method. The right sides of fraternal twins were hung normally by the achilles tendon and the left sides were Placed horizontally, bone down, on a flat surface. The limbs of the left side were tied perpendicular to the long axis which approximated the 69 standing position. The vertically suspended carcasses had sarcomere lengths ranging from 1.8 um to 3.6 um with the horizontal carcasses vary- ing from 2.0 gm to 2.7 um. They suggested that the long sarcomeres of a few muscles from the vertically suspended sides in particular the psoas EQlEE could be attributed to stretching. Due to the anatomy of the car- cass, other muscles were not under tension and could shorten appreciably. Horizontal suspension prevented several muscles from shortening that were free to do so in vertically suspended sides and conversely several muscles had tension removed and could shorten. In view of earlier work (Locker, 1960) these authors found that shortened muscles had larger fiber diameters and higher shear values. It was suggested that practical application might be possible since horizontal placement improves the tenderness of several important muscles. Indeed, Eisenhut t l. (1965) reported that horizon- tal placement markedly changed fiber angles of the longissimus muscle relative to the spinous and transverse processes and the sarcomere length. Hostetler t al. (1970) compared 5 muscles from vertically suspended (leg suspension) carcasses and carcasses suspended from the ischium (hip suspension). One muscle, triceps brachii (TB) (long head), from the thoracic limb, two from the lumbar region, longissimus (LD) and psoas major (PM), and two from the pelvic limb, semimembranosus (SM) and semi- tendinosus (ST) were studied. The SM, ST and LD had significantly greater sarcomere lengths due to the hip suspension method and the SM and LD had lower shear scores and higher taste panel scores. The ST shear and taste panel scores were not significantly different from leg suspended carcasses. 70 The PM had shorter sarcomeres and higher but nonsignificant shear values. The TB was not significantly different for sarcomere length, taste panel or shear scores. Hostetler _£._l- (1972) extended these observations to include modifications of the hip suspension (hip tied) and new methods, neck tied and horizontal. The observations revealed that the hip free method as used previously was the most beneficial in improving tenderness of the major muscles and in particular the muscles of the loin and round. Bouton t al. (1973a) used some variation of the techniques reported by Hostetler _£.él- (1972), however, they also concluded that the aitch bone suspension (hip free) yielded shear values at 2 to 3 days postmortem that were comparable to muscles aged 2 to 3 weeks in the carcass form suspended by the achilles tendon. Similar conclusions were made by Quarrier _£._l, (1972) and Bouton and Harris (1972) concerning ovine muscles. Again with the phenomena of shortening and sarcomere length as with connective tissue proteins and all other tenderness associated phenomena, there appears to be no unanimity among the researchers in the field as to the contribution of these factors to tenderness. Although conclusive evidence has been reported by numerous reseachers concerning sarcomere length and tenderness, Hostetler _£‘_l. (1972) reported that sarcomere length could account for only 12% of the variation in tenderness among animals. 80 it appears that shortening and sarcomere length appear along with the other variables in the complex equation involving tenderness and not as the only variable. 71 Resolution of Rigor Mortis Concept of Rigor Resolution The usage of "rigor resolution" has been loosely implied to describe the postrigor changes in muscle which apparently influences the eating quality of meat. Prior to the work of Bendall (1951) and Bate-Smith (1939) at Cambridge and Marsh (1954) in New Zealand, it was assumed that tenderness increases were in some way related to a reversal of the events causing the stiffness of muscle. The observations by Moran and Smith (1929) attributed the onset of rigor mortis to protein coagulation and the subsequent "resolution of rigor mortis" to the change of the proteins back to a soluble form. However, Bate—Smith (1939) and Bendall (1951) began using the measurement of extensibility to define phases of rigor mortis. This type of observation allowed them to conclude that little or no change or "resolution of rigor" occurred as could be measured iso- tonically. Accordingly, the elucidation of many of the secrets of the contractile process has contributed to the apparent demise of the concept in its literal interpretation. The argument for nonexistence of resolu— tion has firm roots both biochemically and physiologically. The concept of proteolysis doesn't stem necessarily from the philosophy of rigor resolution, however, the maintenance and proliferation of this concept is based considerably on the feeling that "rigor resolution" does not occur in the form of a reversal of rigor onset. Even though the majority of reported data do not support the possibility of proteolysis occurring 72 (Locker, 1960), as measured by conventional methods, the concept has been attractive to help explain the increased tenderness apparent in aged muscle. Increased extensibility during postrigor storage has been ruled out by the observations of Bate-Smith (1939), Bendall (1951) and Marsh (1954) who could not find any evidence of changes during storage. Many other reports support the theory that inextensibility is maintained during aging and that proteolysis of the myofibrillar proteins or alterations in stroma proteins as measured by changes in content or presence of degraded products apparently cannot be found, at least with the present level of instrumental sophistication. G011 and co-workers (1968) have added a new technique to postmortem muscle measurements that apparently does not contradict the originally de- fined philosophy of rigor resolution. Jungk gt 31. (1967) developed an isometer in an effort to describe rigor and postrigor changes in terms of isometric tension. The observations were made using both rabbit and bovine skeletal muscle. Temperature played a role in the time of onset and ten- sion developed, however, in bovine muscle isometric tension developed at all temperatures and reached a peak which subsequently declined to levels much lower than the maximum levels reached. Goll (1968) redefined "resolu- ' in terms of isometric tension which did not refute tion of rigor mortis' earlier insistence that rigor resolution was non—existent. He divided rigor mortis into a shortening or contraction aspect and loss of extensi- bility. The extensibility aspect was divided into a macroscopic and mole- cular phase to facilitate the extension of the concept of "resolution of rigor mortis". The macroscopic aspect of extensibility is manifested as 73 inextensibility as measured and reported by Bate-Smith and Bendall (1949). The molecular extensibility occurs at the sarcomere level when myosin and actin combine and are unable to freely slide past each other as is apparent in physiologically contracted muscle. Goll (1968) attributed macroscopic inextensibility to be the result of numerous sarcomeres having lost mole- cular extensibility. The stiffness or rigidity aspect of rigor mortis, although not necessarily a prerequisite for rigor, was a result of the shortening or contraction phase mentioned previously. All the aspects of rigor were directly influenced by the ATP loss during rigor development. The presence of tension development in postmortem muscle in itself is not surprising since some shortening has been observed by Bendall (1951) and by various other individuals. The point of most interest is the observed inability of a muscle to maintain the isometric tension obtained at the height of rigor mortis (Jungk 33 21" 1967). 6011 (1968) suggested that the inability of muscle to maintain isometric tension corresponded to "resolution of rigor mortis." Additionally, he attributed isometric tension loss to inability of the muscle to maintain the contracted or shortened state. The concept of rigor resolution appears to be a viable topic when considered from the perspective presented by Goll (1968). The validation of the concept rests on determining the causes and effects of rigor reso- lution which will be presented by discussing the various instruments and criteria that are utilized to visualize postmortem muscle changes. 74 Rigor Resolution and Tenderness The overall concept and definition of "rigor resolution" is dependent totally on the methodology used for measurement. As alluded to previously, Bendall (1951) and others have decried usage of the term "rigor resolution" since it implicitly requires that rigor onset must be a reversible reaction. Marsh (1952r)had shown previously that muscle stored in an inert atmosphere does not become freely extensible as seen for prerigor muscle. The concept of rigor resolution has been reported earlier in this review only as a definition by Goll (1968). The overall implication is not its existence since by definition it has been shown to exist (Coll, 1968), but what affect it has on muscle structure and tenderness. Since the dimensions of rigor resolution are microscopic, measurements of this change must, at best, be indirect and to be reported later, alteration of ATPase activities, supercontraction, sulfhydryl content and histological evidence are used to describe these changes. As with its inception, by far the majority of the work on rigor reso— lution has emanated from the laboratories of Cell (1968) and co-workers. The association of tenderness and rigor resolution has been an inference from this group and their concepts will be the basis for the review of this topic. Goll (1968) and G011 35 El. (1971b) have proposed two main causes that might explain the inability of postmortem muscle to maintain isometric tension. Criteria used to characterize the loss of isometric tension are a modification of the actin—myosin interaction measured as a change in the 75 nucleoside triphosphatase (NTPase) activity, in_yi££g contractile activity (superprecipitation) modification of actomyosin, lengthening of rigor- shortened sarcomeres and modification of dissociability of actomyosin by ATP and secondly, the loss of the integrity of the Z—disk resulting in fragmentation. Goll (1971b) based much of the causes of rigor resolution on the in- formation gained from observing the characteristics of trypsin treated muscle. Not only will trypsin initiate rigor onset, it also produces similar changes in NTPase activities as seen during postmortem storage and it induces the normally seen reduction in time for turbidity response. Additionally, trypsin lengthens rigor shortened sarcomeres similar to that described for postrigor muscle (Gothard $5.31., 1966) Trypsin also selectively degrades the Z-line of muscle which has been implicated by several researchers as a primary characteristic of aged muscle. Although earlier workers (Davey and Gilbert, 1969; Davey and Dick- son, 1970) have reported fiber breakage at the I—Z junction more frequently than mere loss of fibrillar structure, the effects of trypsin on this par- ticular loss of Z—disk continuity has been left unexplained. The interrelationship of the above phenomena and tenderness merely shows that during the aging or postrigor period many of these conditions accompany the simultaneous increase in tenderness. Based on previous ob- servations that sarcomere length and tenderness are associated (Marsh, 1972) and if the modification of the actin-myosin interaction does result in a slippage or lengthening of the sarcomere (6011, 1968) the association 76 with tenderness is more than mere conjecture. The weakening and degradation of the Z-disk appears to have strong theoretical grounds for diminishing shear values, however, explicit data supporting this concept have not been reported. In summary, the character of postmortem muscle has been measured in- numerable ways and many of these characteristics appear to have an intimate association with tenderness. However, the contribution of each component to tenderness still is unresolved and if a "most important component” does exist it has not been elucidated with respect to the myofibrillar proteins during postmortem aging. Morphological Changes Z—line Structure Most of the early workers studying the histological changes associated with postmortem storage reported that some degree of fiber breakage occurred and apparently in the region of the I—Z junction (Hanson 32.31., 1942; Paul 35 21., 1944). The Z-line has been recognized as a distinctive component of the sarcomere for many years, however, its structure and composition has been and still is somewhat unclear. Knappeis and Carlsen (1962) suggested that the actin (I) filaments terminated on each side of the Z-line as rod-like projections. The thin filaments as such do not extend through the Z-line, but one I filament is positioned between two I filaments from the adjacent sarcomere. This pattern in effect imparts a zig-zag appearance to the Z-band. 77 Other structures have been suggested to account for the morphology of the Z-band, however, they do not explain the ultrastructural properties and biochemical data. Kelly (1966) investigating desmosomes and related structures found that intracellular filaments which attach at the desmosome sites actually loop past the area of attachment and return into the cell interior. Kelly (1967) proposed two models based upon electron microscopic observations of skeletal muscle Z-disks. In the first model, the actin double helix separates into its two component strands adjacent to the Z-line and each of the separated strands enters into the Z-band and loops.over an actin filament from the adjacent sarcomere. After looping over, the actin filament returns to its original sarcomere and becomes incorporated into a double helix of an adjacent actin filament. Certain inconsistencies with known biochemical data exist for this model, however, Kelly (1967) in describing the second model suggested that the filament looping through the Z-band could be the tropomyosin molecule which would follow the same pattern as suggested for the actin filament. Rowe (1971) expanded Kellys (1967) observations to include a model that would satisfy certain inconsis- tencies with reported lattice dimensions. Rowe (1973) included the looping filament model to explain Z-disk dimension differences observed for the red, white and intermediate fibers ‘with a modification of the looping fila- ment model. Certainly the looping filament model doesn't account for all the known observations either biochemically or ultrastructurally' and the rePOrt that a-actinin resides in the Z-line (Goll 33 a1., 1969) raises the question as 78 to its role in the looping filament model. Whatever the discrepancies, the looping filament model fulfills many of the ultrastructural observa- tions on skeletal muscle and as additional information concerning the Z- disk becomes available the model of the actual Z-line ultrastructure will become more definitive. In an earlier section of this review, Hanson g£_al. (1942), Paul 33 31, (1944), Ramsbottom and Strandine (1949) and others reported that post- mortem aging was accompanied by fiber splitting and transverse breakage. Sharp (1963) found that storage at 5 C for 19 days produced fibers that broke transversely and eventually produced short sections of fibrils. Paul (1963) described the effect of heating on fiber breaks as possibly occurring at the angles of the Z-Z contractions and these breaks apparently increase in number with heating. This implies aninherent weakness at this point in the myofibril which is expressed upon heating. Cook and Wright (1966) came to similar conclusions when they suggested that the Z-line may be the first structural component degraded by heat treatment. In a subse- quent paper, Paul (1965) observed fiber breakage beginning in the I band which she suggested was a greater ability for the actin filament to break as compared to the thick filament. Gothard gt 31. (1966) attempted to section samples that were aged 6 to 7 days and generally the fibers frag- mented very easily. The fibers were reported to always shatter at the level of the I band. This was interpreted as a possible degradation of the protein actin as the result of postmortem aging. Fukazawa and Yasui (1967) and Takahashi, Fukazawa and Yasui (1967) studied the change in the zig—zag configuration of the Z-line and fragmentation . 79 of myofibrils of chicken pectoral muscle during postmortem storage. They determined that myofibrils isolated immediately postmortem were in the form of intact myofibrils, however, as aging progressed the isolated myofibrils were obtained in progressively smaller fragments. Accordingly, they attri- buted the fragmentation to either a Z-line loss or a disruption of the bands between tropomyosin and actin. Fukazawa _£._l~ (1969) recognized 2 types of Z-line distruction when 24 hr. stored chicken pectoral muscle samples were blendorized. First, the Z-line was degraded or disappeared completely and secondly, the sarcomeres broke at the Z-line and I-filament junction. Fukazawa _£‘_l. (1970) reported that protein extractability increased at the same time that the changes occurred in the Z-line. Davey and Gilbert (1967b) found that bovine sternomadibularis muscle removed from the carcass underwent 2 primary changes during aging. First, they observed a complete loss of the Z-line and a lengthening of the A-band at the expense of the I-zone, and secondly, they suggested that the disin- tegration of the Z-line allowed the actin filaments to collapse onto the thick or myosin filaments. These changes parallel closely the objective and subjective increases in tenderness which occur during aging. In a subsequent paper, Davey and Gilbert (1969) found that aged myofibrils dis- rupted both laterally and transversely (Z-line dissolution) during brief homogenization as compared to the refractory unaged myofibrils. These authors also reported that the disappearance of Z-lines and loss of lateral association was completely inhibited by EDTA. Davey and Dickson (1970) extended the previous observations to include stretched and cold shortened 80 muscle. Ultrastructural observations of these preparations revealed fiber breakage when aged muscle was placed under light extension loads. The breaks were indicative of a weakness in the myofibril structures. Unaged meat only responded after much greater extension loads and generally by a withdrawal of thin filaments from beteen the thick filaments. The short- ened fibers broke in the shortened I-zone, however, the slightly stretched myofibrils broke near the Z-disk. The authors concluded that the weaken- ing of the fiber at the level of the I-filament-Z-disk junction contributed more to meat aging than the loss of the lateral linkages. Weidemann _£._l- (1967) concurred when they stated that the degree of tenderness was related to the degree of disruption of the myofilaments. Sayre (1970) investigated the relationship between fragmentation and tenderness in chicken pectoralis major muscle. He observed that the myo- fibrils always broke in the I-band region and the A-band never broke which supports the early observations of Fukazawa, Hashimoto and Tonomura (1963). Sayre (1970) found that the Z-line didn't disappear during 24 hr. storage, but rather fragments of it were attached to the I-band and generally no I- filaments were found to protrude from the fragmented Z-line. This contra- dicts observations by Davey and Gilbert (1967b) that the Z-line completely disappears in 4-day aged bovine muscle. Sayre (1970) observed that frag- mentation takes place at the same time as tenderization, however, he did not find evidence that would conclusively link fragmentation with tender- ness. Henderson t l. (1970) compared ultrastructural changes in bovine, porcine and rabbit muscle stored at 2, 16, 25 and 37 C for 4, 8 or 24 hr. 81 postmortem with at death muscle in each of the species. Z-line degradation occurred much sooner postmortem and to a greater extent when the incubation temperature was 25 or 37 C as compared to lower temperatures. Bovine muscle was reported to be considerably more resistant than rabbit or porcine muscle, however, many of the same changes were evident in bovine muscle if incuba- tion time was extended. The M-lines of rabbit and porcine muscle were fre- quently lost at higher temperatures, however, after aging up to 24 hr. bovine M-lines were not degraded. Some fragmentation of myofibrils was observed to occur mainly at the level of the Z-line, however, no particular significance was attributed to this phenomenon (Henderson gg 31., 1970). Bovine muscle myofibrils exhibited little change during postmortem storage at 2 or 16 centigrade. It was suggested that Z-line degradation might be the result of disruption of the bond between the Z-line and the thin fila- ment rather than a disruption of the Z-line itself. Parrish _£‘_l. (1973) concurred with the observation that Z-line fragmentation occurs primarily at or near the Z-line and that fragmentation seems to result in greater tenderness. Moller, Westergaard and Wismer-Pedersen (1973) reported that measurements of fragmentation occurring after homogenization of raw muscle tissue was an important measure of tenderness in heated bull longissimus muscle. Several observations in the literature suggest the presence of endogen- ous proteolytic enzymes or other agents that control the degradation of the Z-line in postmortem muscle. Davey and Gilbert (1969), as reported earlier, were able to inhibit Z-line degradation with EDTA and Fukazawa t 1. (1969) found the extraction of sarcoplasmic proteins from muscle to influence the 82 degree of fragmentation during storage. Using these observations and others concerning cathepsins and the observation that trypsin can remove the Z-line (Goll.g£-§l., 1971a) Busch g£_§1, (19720 isolated a sarcoplasmic factor that removed Z-lines exclusively from rabbit muscle. These authors reported that the sarcoplasmic factor was protein in nature and required Ca2+ for activation. Incubation of muscle samples with a Ca2+ chelator (EGTA) prevented Z-line removal. The isolation of this factor fulfills the search for a cathepsin or other enzyme that will effect degradation of Z-lines, however, greater than known physiological levels of Caz+'are required for optimum activation. The clarification of the role of this component in postmortem muscle and its relationship to tenderness must be awaited, however, the implications are of major importance. The alteration of skeletal muscle Z-lines has been demonstrated quite clearly, however, the existence of several fiber types differing not only morphologically (Gauthier, 1970) but also biochemically and physiologically (Brooke, 1970) suggests possible differing effects of postmortem storage. Few data have been reported supporting this concept, however, Goll__£,_1. (1970) reported that the Z-line of red muscle fibers might be less labile than those of white muscle. The ultrastructure of chicken breast muscle has been reported previously, however, Hay g£.§l. (1973a) reported that chicken leg muscle Z-lines were virtually resistant to degradation even after 168 hr. of storage, whereas, at the same time period, breast muscle Z-lines were virtually absent. The authors suggested that Z-line degradation might not totally reflect changes in tenderness in light of the red muscle Z-lines 83 not being degraded during postmortem storage. Dutson g£_al. (1974) reported data on normal and low quality porcine muscle that supports the observa- tion that white muscle fiber Z—lines are more labile than red muscle fiber Z-lines. ATPase Activity 0f the studies concerned with research on muscle, few can rival the discovery that myosin has the capability to hydrolyze the terminal phosphate from ATP. This remarkable discovery was first reported by Engelhardt and Lyubimova (1939). This report was subsequently verified by Needham (1942) and Bailey (1942). Bailey (1942) suggested that the enzyme was activated by Ca2+ and an+ and that no inconsistencies were found to rule out the fact that the enzyme and myosin were one and the same entity. Some compli- cations exist on interpretation of early work concerning activators of the enzyme since no information was available concerning the presence of other proteins in the system and much was done unknowingly on either myosin A or myosin B and sometimes both (Needham, 1971). Suffice it to say at this point that highly purified myosin A is activated by Ca2+ and inhibited by Mgz+ and myosin B is activated by Mg+2 and traces of Ca2+ (Gergely, 1970; Seidel, 1969a). Early observations suggested that tenderization could proceed by dis- sociation into separate actin and myosin components, however, the extensi- bility concept virtually refutes this speculation (Bendall, 1960). Fujimaki 35.31. (19653) utilized the previously mentioned properties of actin, 84 myosin and actomyosin to ascertain postmortem changes in the muscle pro- teins. By utilizing the activities of Ca2+ and Mg2+ on the ATPase proper- ties of rabbit fibrillar proteins, they determined that postrigor muscle had 1.2 to 1.3 times the Mg2+-activated ATPase activity of prerigor muscle. Aged muscle (7 days) Mg2+ ATPase activity decreased from postrigor values, but never below prerigor levels. The Ca2+ activated ATPase activity in— creased during rigor, however, the aged muscle activity decreased below that observed for prerigor muscle. They also found that sensitivity to dissociation increased as postmortem time increased. Other physico- chemical measurements supported their suggestion that the contractile pro- teins and in particular the actin-myosin interaction was undergoing some modification during the transition from prerigor to aged condition. Okitani, Takagi and Fujimaki (1967) reported that the interaction between actin and myosin was weakened by storage at low temperatures, low pH or high ionic strength conditions. They speculated that myosin B became denatured either by spontaneous aggregation or irreversible dissociation into myosin A and actin. Scharpf, Marion and Forsythe (1966) reported some evidence utilizing gradient ultracentrifugation and Mg2t and Ca2+Lmodified ATPase activity that they considered as some evidence for dissociation of myosin B into its components actin and myosin. In a report that bears some importance to the measurement of ATPase activity is that of Hayashi and Tonomura (1966) who found that ATPase acti- vity was related to the sarcomere length of the muscle fibers. A peak of activity was noted at 2.57 um with a fairly sharp drop of activity on each side of the maximum. 85 In a series of reports, Goll and Robson (1967), Robson, 0011 and Main (1967), Chaudhry 35 31. (1969) and Arakawa, G011 and Temple (1970a, b) in- vestigated the ATPase and ITPase activities of bovine and rabbit myofibrils and myosin B. The initial work involved activities of sucrose prepared myofibrils incubated at 2 and 16 C from 0 to 312 hr. postmortem. The Ca2+ and Mg2+ ATPase activities showed a variable response, however, at 24 hr. both values were 20 to 50% higher than at death values. Little change was observed in the EDTA-modified ATPase activity, but the EGTA-modified re- sponse increased slightly at 24 hours. All except the Mg2+-modified enzyme remained higher than the 0 hr. samples. The ITPase activities were less responsive with the Mg2+ -modified enzyme showing some increase by 24 hr., but the Ca2+-modified enzyme showed no response during postmortem storage. Some temperature and ionic strength interactions were involved, but at > 0.5 ionic strength the actomyosin complex appeared to be dissociated and only the myosin enzyme could be assayed. In a subsequent paper, Robson gt El. (1967) utilized myosin B preparations to determine ATPase activities. Little difference could be found between at death preparation and samples stored at 2 or 16 C. It was hypothesized that possibly the lack of a-actinin might be responsible for the results. Chaudhry E£.§l: (1969) reported a complete lack of Ca2+ and Mg2+ ATPase activity, but the authors suggested exhaustive dialysis or KI extraction as possible reasons rather than post- mortem changes. Although the role of the minor protein components will be considered in depth later, Arakawa 35.31. (1970a) reported that a-actinin and the TMrTN complex are not the primary causative agents for the variable NTPase activities. 86 Penny (1967, 1968) investigated the solubility and ATPase activities of aged rabbit muscle and model systems to determine the mechanism of the reported ATPase fluctuations. He concluded in the initial report that loss of ATPase activity was primarily due to denaturation of fibrillar protein. The loss of the Ca2+;activated enzyme occurred earlier since myosin not bound to actin is denatured easier than the Mg2+-activated en- zyme associated with the more resistant actomyosin. Penny (1967) also found a parallel between loss of extractability and ATPase activity and considered that uncoiling of the myosin molecule might be responsible. In the subsequent paper, he found no effects of aging on Ca2+;activated ATPase activity, but the Mg2+-activated enzyme showed a definite decline after 4 days at 15 to 18 and 4 centigrade. He reported that denaturation of myosin would be inconsistent with the Ca2+-activated stability and con- cluded that the myosin-actin linkage must be weakened in some way. Herring t al. (1969a, c) studied the physiochemical properties of natural actomyosin from tough and tender bovine longissimus aged 0 to 10 days. The highest ATPase values (1.1 to 1.2 times the 0 hour sample) were obtained from the 12 and 24 hr. aged samples. The Mg2+- and Ca2+;modified activities of both tough and tender samples responded similarly and as such little relationship between tenderness and ATPase activity was reported. Yang, Okitani, Fujimaki (1970) and Okitani and Fujimaki (1970a, b) investigated the postmortem changes in rabbit myofibril and actomyosin ATPase activity. In the initial report, Yang g£_ 31, + (1970) observed that the Mg2 -activated ATPase activity increased 87 with increased aging time. At the same time, they found that aging in- creased the dependence of this activity on ionic strength. Due to some similarities between isolated actomyosin and 8 day aged myofibrils the authors suggested that Mg2+ ATPase activities appeared to be related to the structural continuity of the myofibril. The aging of muscle was reported to produce similar Mg2+-activated ATPase activities as seen in high ionic strength extractions of muscle. Since aging has been reported to alter myofibril structure, the authors suggested an association between myofibril structural alterations and ATPase activities. In the second paper, Okitani and Fujimaki (1970a) reported that the loss in CaZ+-enhanced ATPase activity when stored in 0.6 M KCl can be attributed to inactivation of myosin A ATPase activity whereas Mg2+-enhanced ATPase (low ionic strength) activity loss was due to a loss of the activating ability of F—actin. In the subse- quent paper, Okitani and Fujimaki (1970b) expanded on the loss of activating influence of myosin A and F-actin. They found that if the ATPase activity of myosin A.was lost, it still retained the ability to combine with F-actin. However, if F-actin lost ATPase activating ability it also lost the ability to combine with myosin A. These same authors also concluded that the actin— myosin interaction became insensitive to dissociation by ATP during storage. Earlier reports on ATPase activities of bovine and rabbit actomyosin 2"Land Mg2+-modified ATPase activity during showed an increase in both Ca rigor mortis (Fujimaki 3£_31,, 1965a, b; G011 and Robson, 1967; Robson 33 31., 1967). Hay 33 31. (1972) and Jones (1972) reported that chicken muscle actomyosin ATPase activities responded somewhat differently than that re- ported for rabbit and bovine muscle. Hay 33.31, (1972) reported values for 88 chicken breast and leg muscle actomyosin and observed that Ca2+-activated ATPase did not change from 0 to 168 hr. of storage except for chicken breast muscle actomyosin during rigor. The Mg2+-modified ATPase activities increased during rigor and after 168 hr. the values were lower than rigor but not as low as 0 hr. samples. The Mg2+-modified values paralleled those reported by Goll 33 31. (1971b) for bovine myofibrils, but the Ca2+ mediated values are considerably different than those reported by these same authors. Hay 33 31. (1972) suggested that the results might reflect the actin con- tent of the actomyosin preparation. Jones (1972) used chicken breast actomyosin and reported values similar to Hay 3£_31, (1972), however, he suggested that rather than containing less actin, the rigor actomyosin possibly contained more actin that was enzymatically inactive than that of 0 hr. samples. Jones (1972) additionally suggested that stored chicken actomyosin underwent some unknown modification of the actin-myosin inter- action. Most of the data reportedtD date involves changes in ATPase activities of postmortem muscle as related to the reported phenomena of "resolution of rigor mortis", however, few data relate these activities to actual sen- sory and objective evaluations of tenderness. Parrish 33 31. (1973) utilized Choice grade steers in an effort to interrelate most of the known measurements of postmortem muscle changes with both taste panel and Warner— Bratzler shear values. The carcasses utilized were stored at either 2 or 16 C and samples were obtained at 4 postmortem time periods varying from 0 hr. to 7 days. The Mg2*3 Ca2t-and EGTA-modified ATPase activities increased 89 with postmortem storage at 2 and 16 C with the 16 C values generally greater but not significantly greater. The association between observed ATPase values and tenderness was not discussed, however, the authors utilized these measurements in an effort to clarify the changes occurring in post- mortem.muscle. The relationship between the histological observations and tenderness will be included in a later section of this review. In summary, the ATPase data reported are not consistent, however, the procedures used and techniques utilized were almost as varied as the re- sults. The concept of muscle fiber type and ATPase activity was not in- cluded in this discussion. The differences in biochemical, physiological and histological structure has been well documented (Dubowitz, 1970; Gauthier, 1970); the overall implication of these differences was consi- dered earlier. Generally, it can be concluded that the ATPase activities vary during postmortem storage which must reflect rigor and postrigor changes in the state of the muscle proteins. Even though the reported values vary with laboratories and experiments, it is important to use this type of technique in an effort to determine the molecular mechanism of the myofibrillar changes. Regulatory Proteins Superprecipitation The phenomenon of superprecipitation of actomyosin was first reported by Szent-Gyorgyi (1944). Under 13_vitro situations of low ionic strength 90 which resembles that in living muscle, the interaction of actin and myosin forms a fine suspension which does not settle readily (Szent-Gyorgyi, 1947). In the presence of Ca2+, Mg2+ and ATP, the fine suspension of the combined actin and myosin contracts with the comcomitant splitting of ATP (Briskey and Fukazawa, 1971). Szent—Gyorgyi (1947) described the transformation of the loose flocculi of the actomyosin in the gel state to that of a granular precipitate of reduced volume upon the addition of ATP. The use of the word superprecipitation was destined to separate it from low KCl precipita- tion and was classified as an 12.31532 model of contraction (Briskey and Fukazawa, 1971). The response of an 13_y1££3_model to ATP in the presence of Mg2+ and absence of Ca2+ has been called clearing which is analogous to EEHXEXQ relaxation (Spicer, 1952). Since superprecipitation has been described as an EEHXEEEQ model of muscle contraction, it has been used to describe various parameters of the contractile process. Difficulty in quantitatively measuring superprecipi- tation existed at first, but Ebashi (1961) gained considerable success by using turbidity changes as an index of superprecipitation. The scattering of incident light by actomyosin after ATP induced syneresis has been attri- buted to an increase in refractive index of the actomyosin gel which appears to more than compensate for the decreased volume reducing scattering (Endo, 1964). Considerable work has been reported on the mechanism of superprecipita- tion (Weber and Winicur, 1961; Watanabe and Yasui, 1965; Yasui and Watanabe, 1965; Tada and Tonomura, 1966; Matsunaga and Noda, 1966; and Sekine and Yamaguchi, 1966), however, superprecipitation has also been used to determine 91 the effects of aging on the postmortem muscle system. Although some questions concerning the exact comparisons of superprecipitation to 13H31£3 contraction have been issued, the system has been used to characterize the changes in the postmortem muscle system as compared to at death muscle. This section of the review will be primarily concerned with the work re- porting changes in the postmortem system that might clarify the mechanism of tenderization. The recent discovery of the presence of several new proteins in the myofibril has certainly complicated measurement of postmortem protein alter- ations. The regulatory proteins account for 25% or less of the total myofibrillar protein, however, they exert profound influence on the total system. This regulatory influence coupled with superprecipitation has given the meat scientist a tool to gain insight into the molecular altera- tions. Ebashi and Ebashi (198;) reported the isolation and characterization of a protein from muscle which they called a-actinin. The protein was iso- lated from native tropomyosin preparations and was found to markedly influ- ence the superprecipitation of synthetic actomyosin. The authors reported that a-actinin accelerated the turbidity response of an actomyosin prepara- tion. From additional research by Maruyama and Ebashi (1970), they deter- mined that o-actinin was composed of a 68 and 108 component with the 6S component being the active part of the system. It has been recognized for several years that myofibrillar ATPase acti- vity increased after a short time period of storage postmortem (Fujimaki 33 ‘31., 1965a). Herring 33 31. (1969b) measured the turbidity changes in 92 postmortem bovine muscle to determine if differences between tender and tough muscles could be ascertained. Aged muscle (12 to 24 hr.) had an increased rate of turbidity rise when using natural actomyosin in 100 mM KCl. The authors suggested that 12 to 24 hr. postmortem muscle had a stronger actin-myosin interaction than prerigor samples. They also observed that when the assay was conducted in 100 mM KCl, 5 days aging was adequate to observe a decrease in rate of superprecipitation of tender muscles, however, tough muscles had to be aged 10 days to elicit a comparable change. The rate of turbidity response for tender and tough muscle in 50 mM KCl showed a more rapid response for tough muscle, however, in 100 mM KCl this relationship was reversed. The authors suggested that a possible inter- action between ionic strength and a—actinin enhanced superprecipitation. Additionally, they surmised that a possible difference in either a-actinin or another regulatory protein existed between tough and tender muscle. The earlier mentioned discovery of a—actinin and the previously des- cribed regulatory protein troponin by Ebashi and Ebashi (1964) led Arakawa .33 31. (19703, b) to speculate that postmortem modification of either of these proteins could be responsible for the observed changes. They con— sidered that proteolysis of troponin would in effect derepress the inhibi- tory activity that this protein exhibited towards the interaction of actin and myosin. a-actinin has been isolated from the Z-line (Goll 33 31., 1967) and they suggested that Z-line disintegration might release this protein to induce an increase in Mg2+-modified ATPase activity (Arakawa 3£_31., 1970c). By utilizing rabbit muscle stored at several temperatures and pH the authors prepared a-actinin and troponin and measured the response these 93 2 proteins had. on superprecipitation. The authors concluded that even though postmortem storage appeared to decrease the time necessary to observe the turbidity response, all of the difference could not be attributed to a-actinin and troponin modification. Their results showed that a-actinin slowly lost its capability to accelerate turbidity and ATPase activity. This was certainly contrary to the results which would be necessary to account for the increased Mg2+-modified ATPase activity and decreased time for tur- bidity to occur. The (TMrTN) system also appeared to have considerable resistance to proteolysis and appeared to not be involved in modifying the activities of postmortem muscle. Some change was noted in both systems, however, most could be attributed to abnormal pH and temperatures not gen- erally encountered in normal postmortem muscle environment. The use of superprecipitation as an indicator of molecular alterations has been demonstrated quite adequately and in particular has been a very essential tool to measure the modifications of some of the minor components of the myofibril. Jones (1972) observed much the same modifications of the time course of superprecipitation in chicken muscle as that observed by Arakawa 33_31. (19703) for rabbit muscle and suggested that some protein in the regulatory group was being modified with postmortem storage. Removal of low ionic strength proteins was accomplished by exhaustive dialysis and centrifugation, however, the results suggested that regulatory protein modification was not primarily responsible for the observed changes. Fukazawa 33 31. (1970) ob- tained somewhat different results from chicken pectoralis muscle which they suggested was the result of an increased release of a-actinin occurring in 94 conjunction with Z-line dissolution. These authors isolated a fraction (Fr.2) which originally contained superprecipitation depressing activity on trypsin-treated myosin B. The activity of Fr.2 decreased with postmortem storage and Fukazawa 33 31. (1970) concluded that it was troponin. Another fraction (Fr.1) was considered to be a-actinin, specifically the 108 or inactive component and not the active 68 component. Other Indicators of Regulatory Changes Penny (19703, b) extracted the proteins from bovine longissimus muscle after aging for 8 and 15 days at 4 centigrade. He found no evidence that would suggest that any protein fraction had been degraded or disappeared during aging. However, his data suggested that the actin-a-actinin complex had been altered in some unknown manner during storage. In a subsequent paper, Penny (1972) prepared a—actinin from 7, l4 and 21 day aged bovine muscle. No quantitative differences were obtained as the result of aging, however, a small and insignificant effect on a-actinin binding properties was obtained. Penny (1972) concluded similarly to that of Arakawa_3£_31. (19703, b) that some small changes were occurring in properties of a-actinin, however, he felt that these changes were not primarily responsible for ob- served postmortem alterations. Hay 33 31, (1973b) investigated the effects of aging chicken breast and leg muscle on the SDS disc gel electrophoresis patterns. The appearance and disappearance of several bands, particularly in the breast muscle, was considered to be rather consistent with ultrastructural observations on similar muscles (Hay,gt.31,, 1973a). Although several band changes were 95 discussed, the authors considered the disappearance of 3 44,000 Dalton component and the appearance of a 30,000 Dalton component during aging to be the most important of the changes. It was suggested that the 44,000 Dalton component could possibly be the result of the disappearance of the Mkline as reported earlier (Hay 33_31., 1973a). The leg muscle did not show a similar loss, however, the 0 hr. gels did not contain a band corres- ponding to 44,000 Daltons. The authors also reported the appearance of a 30,000 Dalton component which was apparent at 48 hr. in breast muscle, but did not appear until 168 hr. of aging in the leg muscle. Hay 33 31. (1973b) interpreted this to be either a degradation product of myosin or possible one from troponin-B although they considered the latter to be un- likely. Other aspects of the investigation support the contention that¢y- actinin and actin are not being degraded. TheSe latter authors felt that since the myosin band was broad and relatively diffuse it probably masked subtle alterations if they occurred in this fraction. The physiological and biochemical properties of slow (red) and fast (white) muscle have been known for a number of years (Dubowitz, 1970). Interspecies differences in ATPase activity and superprecipitation have been reported (Barany 33 31., 1967) along with subunit variations between red and white myosin (Locker and Hagyard, 1968). Suzuki 33 31. (1973) reported little apparent difference in the biochemical properties of a- actinin prepared from red and white portions of the semitendinosus muscle. The biochemical properties are divergent enough to possibly impart some importance to some of the parameters measured in muscles, however, the use 96 of mixed fiber muscles precludes accurate biochemical determination of the contributions of each type to the observations. Sulflydryl Groups The ATPase activity of myosin preparations is a universally known phenomenon. However, the molecular architecture of the active site has not been characterized as clearly, but it is known that myosin contains two sulfhydryl groups (SH) that affect the ATPase activity differently (Blum, 19623). Blum (1962b) reported that the two SH groups can be blocked with N-ethylmaleimide(NEM), however, the amount of blocking action is dependent on the reaction time. One SH group can be blocked that activates C32+- modified ATPase activity without inhibiting superprecipitation or the effect of relaxing factor grana. Increased reaction time will effectively block both ATPase activity and superprecipitation. Yamaguchi and Sekine (1966) called the SH group responsible for activating the C32+-activ3ted ATPase $1 and the group that inhibits ATPase activity 82. They also deter- mined that the C32+'activated ATPase SH group is present as one group per myosin subunit. Seidel (1969b) selectively blocked the slower reacting 52 group and determined that by blocking only 32 does not inhibit 032+ ATPase activity. He concluded that both groups lead to a conformational change in the regulatory site and it necessitates blockage of both groups to lose superprecipitation and ATPase activity. Daniel and Hartshorne (1972) 2+ reported that the SH groups that are responsible for Ca sensitivity of natural actomyosin apparently are located on the heavy subunits of myosin 97 (subfragment 1). Other additional data suggested to them that the Ca2+ sensitivity of myofibrils may be affected more by the myosin molecule than earlier suspected. This could change the complexity of the relationship of the TM-TN complex to ATPase activity. The measurement of postmortem changes in SH content in relation to tenderness has been rather limited. One of the earlier attempts was con- ducted by McCarthy and King (1942) in conjunction with work on the "Tenderay" process of aging beef. They reported that the number of titratable SH groups increased during aging at both normal cooler temperatures and at elevated "Tenderay" process temperatures. No definite conclusions were' made except for the observation that the increase in tenderness was parallel by the increase in titratable SH groups. Much of the work associating the role of SH groups with tenderization has been done with poultry muscle. Chajuss and Spencer (19623, b) reported work that suggested that SH groups may play a role in the onset and "resolu- tion of rigor mortis". They hypothesized that the formation, cleavage or reorientation of disulfide bonds may be intimately associated with the rigor state in muscle. The relaxation of the rigor state or "resolution of rigor" by an exchange reaction involving disulfide-sulfhydryl groups was 3 suggested mechanism. Gawronski, Spencer and Pubols (1967) concurred with this hypothe- sis when they observed a modification of rigor and tenderization with NEM modified muscle preparations. NEM was reported to ultimately increase the shear resistance of muscle, however, they concluded that no firm role for SH groups could be stated until the nature of NEM.alterations are fully understood. Caldwell and Lineweaver (1969) investigated similar parameters 98 with chicken muscle, however, they were unable to substantiate the role of the SH group as a rigor initiator or tenderization mechanism. Wu and Sayre (1971) concurred with the observation of Caldwell and Lineweaver (1969) that aged chicken muscle did not differ from fresh muscle in its SH content. Hay 33H31. (1972) measured the SH groups in chicken red and white muscle by several methods and except in one instance all were similar to that re- ported by wu and Sayre (1971). In the one exception the quantity of SH groups exposed by 20 mM KCl in breast muscle was increased during rigor mortis. These authors suggested a possible association between this obser- vation and the reduction of Ca2+ ATPase activity during the same time period. Stranberg 33 31. (1973) investigated the effect of various SH protecting reagents and postmortem storage on ATPase and superprecipitation activities of rabbit muscle myosin B. A variety of results were obtained particularly as modified by the ionic strength of the assay medium. They reported that quantitative differences in SH groups were apparent only during storage at elevated temperatures (3 days, 25 C). Based on quantitation of SH groups, the authors concluded that the postmortem change in ATPase activity and superprecipitation could not solely result from SH group alteration. Iodo- acetamide (IAA) and NEM modified myosin B reacted similarly to postmortem myosin B preparations. Stranberg 35 31. (1973) observed that 50% blockage of total SH groups of at death myosin B accelerated the rate of turbidity response and that of the Mg2+ + C32+-3nd Mg2+ + EGTA-modified ATPase activi- ties. This is similar to previous reports (Coll, 1968) that postmortem storage modifies ATPase activity and superprecipitation. Some protection 99 against increased ATPase activity could be conferred to myosin B by dialyzing it against 2-mercaptoethanol (MCE). However, at low ionic strength (0.052) dialysis against MCE did not prevent an increase in Mg2+ + EGTA ATPase activity. The authors were cautious in interpreting the results, however, they concluded that modification of SH groups should be credited with par- tial responsibility for superprecipitation and ATPase changes in postmortem myosin B. However, Strandberg 3£_31, (1973) reported that the increase in Mgz+ + EGTA.ATPase activity was indicative of loss of Ca2+ sensitivity which suggested that proteolysis might play a role in postmortem muscle. Previous work by Arakawa 3£_31, (19703) showed that a-actinin and the TM-TN complex was not changed in sufficient magnitude to account for observed postmortem changes. With Ca2+ sensitivity attributed to the TM-TN complex, the source 2+ responsible for the increased Mg EGTA activity is presently unknown. Proteolytic Probes The size of the myosin molecule has contributed to the difficulty of determining molecular weight, a-helix content and dimension.(Needham, 1971). Earlier attempts had been made to disrupt secondary structure linkages by urea or other agents, however, the use of trypsin to cleave the molecule into smaller fragments has clarified many aspects of myosin structure (Gergely, 1950; Perry, 1950). Subsequent work by Szent-Gyorgyi (1953) helped clarify earlier observations when he obtained two sub-units from trypsin proteolysis and named them light meromyosin (LMM) and heavy meromyosin (HMM). The important results of this and previous work showed that LMM maintained 100 much of the solubility characteristics of the parent molecule, whereas HMM was water soluble but possessed most of the ATPase activity. The structure of myosin known to date has been determined from the use of proteolytic tools such as trypsin, papain, chymotrypsin and others together with X-ray diffraction data. The present day evidence suggests that the myosin molecule is composed of two major polypeptide chains running the length of the molecule (Lowey 33_31., 1969). The myosin molecule can be split into smaller fragments by proteolytic enzymes as reported earlier by Szent-Gyorgyi (1953). These smaller fragments are called LMM and HMM, however, the HMM appears to be able to be subdivided into two additional fractions called heavy meromyosin subfragment l (HMMS-l) and heavy meromyo- sin subfragment a (HMMS—Z) (Lowey 3£_31,, 1969). The junction between HMM and LMM, as previously mentioned, is susceptible to trypsin hydrolysis, however, with the use of insoluble complexes of papain (Nihei and Kay, 1968; Lowey 33 31., 1969) it was determined that the globular head containing the ATPase activity is connected to LMM by 3 highly helical fraction, HMMS-2 (Huxley, 1969). Although the description of the myosin molecule has been considerably superficial, the clarification of molecular structure has allowed the pur- suit of postmortem perspectives that allow an insight into the changes occurring during the transition of muscle to meat. Proteolysis has been covered in a previous section of this review, however, most measures of proteolysis that have been used are of the type that measure degraded products such as peptides or amino acids. Suzuki 33 .31. (19693, b) and Okitani 33 31. (1972) utilized cathepsin D as a proteolytic 101 agent to determine postmortem changes in rabbit muscle. Suzuki 3£_31, (1969b) reported that cathepsin D does not degrade native tropomyosin in myosin B which indicates a specificity difference between it and other commonly used proteolytic agents. Cathepsin D lowered Mg2+-enhanced ATPase activity when the assay was run at pH 5.0, but when pH 5.5 or what has been considered to be approximately the ultimate pH of meat was used, no lowering of the ATPase activity was observed. Cathepsin D showed no effect on Caz+-activated ATP- ase irrespective of pH which suggests that no activity occurs against myosin A. Suzuki 33 31. (19693) reported similar specificities for pepsin and cathepsin D when using the oxidized B-chain of insulin as substrate. Pepsin was reported to significantly decrease the Mg2+-activated ATPase activity of myosin B which is in contrast with the effect of cathepsin D. Okitani ‘33 31. (1972) observed the same ATPase activities under conditions reflect- ing normal postmortem situations. They concluded that cathepsin D does not play a major role in postmortem degradation of muscle proteins of the myofibril. Yang 33 31. (1972) incubated actomyosin and myofibrils from rabbit ske- letal muscle with trypsin and measured the alteration of the ATPase activi- ties. They found that the Mg2+-enhanced ATPase activity underwent an alter- ation that was dependent on ionic strength and time of aging. Actomyosin and myofibril preparations underwent qualitatively similar modifications with the changes of the former being considerably larger than the latter. These authors concluded that aging of myofibrils results in some alteration of the structural components with the Z-line and tropomyosin the most probable sites of change. 102 G011 33 31. (19713) reported data that support the concept that a brief incubation of myofibrillar protein with trypsin modifies the actin-myosin interaction before the cleavage into HMM and LMM fractions occurs. These authors listed three specific modifications that support this concept. First, 30 min. of tryptic digestion causes an 8-fold increase in the Ca2t modified inosine triphosphatase (ITPase) activity of actomyosin without causing a similar response in myosin preparations. In addition to the ITP- ase activity trypsin causes a 10 to 30% increase in the Mgz+ + C32+-modified ATPase activities during the first 1 to 2 min. but 60 min. of digestion caused a drop in activity to only 20% of the original value. Secondly, trypsin caused an increase in the rate of turbidity response of actomyosin suspensions initially, but after 60 min. the rate of response was much less than control samples. The final line of evidence presented was the obser- vation that supercontracted myofibrils (ATP contracted to 50% of initial length) incubated for 4 min. with trypsin lengthen to 70% of resting length. This lengthening was accompanied by cleavage of myosin into HMM and LMM, but the authors concluded that lengthening was primarily due to the sliding of thick and thin filaments rather than by cleavage of the myosin molecule into HMM and LMM. This latter observation supports the earlier data reported by Stromer, 0011 and Roth (1967) that trypsin lengthened rigor shortened myofibrils. The culmination of all work on muscle utilizing proteolytic probes must be either as a method to investigate and relate to 13_y133_molecular struc~ ture or as a tool to help determine causes of postmortem changes. The former method has been demonstrated quite adequately by Gergely (1950), Perry (1950) 103 and Lowey 35 31. (1969), however, only a few researchers utilized proteoly— tic enzymes to study postmortem changes. Goll_33 31. (1971b) reported the striking similarities between normal postmortem muscle parameters and those produced by limited tryptic proteolysis. Greaser 33 31. (1969) attributed rigor mortis onset to the loss of Ca2+ accumulating ability of the SR. G011 33 31. (19713) concurred with this observation, however, information gained from limited tryptic incubation suggests that proteolysis of the SR causes the loss of Ca2+ accumulating ability of these membranes. The resolution of rigor mortis apparently is more complex in origin than rigor onset, but many of the known parameters of aged muscle can be duplicated by mild tryptic proteolysis (Golll33 31., 1971b). Trypsin incubation mimics 3+ 2+ the changes in the Mg. and Ca -activated ATPase activities that have been reported earlier (Arakawa 3£_31., 19703). The Mg2+-activated ATPase was elevated in postmortem muscle (24 to 72 hr.) and also in trypsin treated (2 min.) at death myofibrils, however, longer incubations (4 min.) caused the decline in Mg2+ ATPase activity which paralleled the activity in 312 hr. postmortem muscle. The C32+-activated ATPase activity increased in a simi- lar trial and remained elevated in both cases even after 312 hr. of storage. A second line of evidence reported by these same authors (Goll 3E 31., 1971b) is the increased rate of turbidity response in both normal postmortem muscle and trypsin treated muscle. Some inconsistencies in this comparison were reported between 8—day aged muscle and greater than 5 min. trypsin treatment. The authors concluded that proteolysis may be only a partial explanation for postmortem changes. 104 Two final lines of evidence presented by Goll 33 31. (1971b) to sup- port the similarities between postmortem changes and trypsin incubation are the lengthening of shortened myofibrils and Z-disk alterations. The former observation was reported in a earlier paper (Coll 3£_31,, 19713) concerning the effects of trypsin on mimicking postmortem changes. Normal rigor shortened muscle lengthens during storage much the same as trypsin treated muscle. The latter observation was considered in detail earlier, however, these authors again cite the similarities between normal untreated aged muscle and trypsin treated at death myofibrils. The Z-disk in both cases was reported to undergo a gradual disintegration and eventual loss of continuity. Call 33 31. (1971b) concluded that it appears that very limited pro- teolysis plays an important role in determining not only the onset of rigor but also the controversial "resolution of rigor mortis." They also reported that other factors probably contribute to the observed changes and cautioned that changes in postmortem muscle are probably affected by other alterations in the muscle system. EXPERIMENTAL METHODS Experimental Animals Sixteen Hereford bulls, 14 to 16 months of age, were used in this study. The bulls were obtained during the last year of a 12 year study that was designed to genetically select for tenderness. One-half of the bulls were unselected for tenderness, but selected for leanness. The latter line served as the tenderness control line. Semen was collected from each bull prior to slaughter and frozen. The semen from the 2 most tender bulls was used to inseminate the females in the original herd designated as the tender group; likewise, semen from the 2 leannest bulls was used to inseminate the females in the lean group to obtain the next years“ calf crop. The 2 most tender and leanest bulls were identified by a tenderness or leanness index as follows: tenderness index = 10 + 1.4 X taste panel score — shear value; index of leanness = weight of the round, rump and loin divided by carcass weight and the percentage obtained was adjusted to a carcass weight of 500 pounds by linear regression. Sample Preparation Approximately 1 hr. after exsanguination, a 300 to 400 g sample of the longissimus muscle was removed adjacent to the 12th rib. The sample was trimmed free of epimysium and a pH determination was made on the muscle Surface using a Corning model 12 pH meter. 105 106 A 1 hr. postmortem temperature was obtained by inserting a thermo- meter in the loggissimus muscle anterior to the point where the sample for electron microscopy and protein fractionation was removed. Several small bundles of fibers were removed from different locations within the muscle sample for electron microscopy preparation. The remainder of the sample was cut into smaller portions and frozen in 2—methoxybutane cooled with dry ice. The frozen samples were placed in Whirl-Pak bags (Nasco, Ft. Atkinson, Wisconsin) and stored at —29 C until used- Electron Microscopy Sampling. The prerigor muscle samples were removed from the right longissimus muscle approximately adjacent to the 12th rib at 1 hr. post— mortem. Muscle samples were removed after 48 and 216 hr. of postmortem storage from the same area of the left longissimus. An attempt was made to prevent shortening of the prerigor samples, however, no mechanical re- straining device was used. The samples were trimmed to approximately 2 mm in cross sectional area and 1 cm in length and placed in glutaralde- hyde fixative. Fixation and Embeddigg. The glutaraldehyde fixation was a modified procedure of Karlson and Schultz (1965) described by Sjostrand (1967). The samples were fixed for 2 hr. in a 1.25% glutaraldehyde solution pH 7.4 in sodium phosphate (P04) buffer as described in Appendix I. The fixative and P04 buffer also contained N3Cl to provide an approximately 13_situ osmotic condition to help prevent osmotic damage. 107 After the samples were fixed for 2 hr., the fixative was removed and the samples were washed 3 times for 20 min. each using only the P04 buffer containing NaCl (Appendix II). After rinsing, the samples were transferred to a 1% osmium tetroxide solution made up in veronal acetate buffer pH 7.4. The tonicity of the veronal acetate buffer was adjusted to that of blood (300 milliosmolar) by adding NaCl, KCl and C3C12 (Appendix III). The procedure for preparing the 1% osmium tetroxide solution was reported by Sjostrand (1967). The samples were postfixed in osmium tetroxide with gentle agitation for 1 hour. The samples were dehydrated with a graded series of ethanol, 25, 50, 75 and 95% for 10 min. each. The samples were then allowed two 15 min. changes in absolute (100%) ethanol for final dehydration. The samples were placed into propylene oxide for two 30 min. changes and then into a 1:1 (v/v) mixture of Epon 812 and propylene oxide for 12 hr. in a dessicator. After the 12 hr. period in the propylene oxide : epon mixture, the samples were removed and trimmed to approximately 1.0 mm x 0.5 millimeter. These trimmed samples were then transferred to 00 size gelatin capsules containing 100% epon (Appendix IV). Three replicates were embedded for each sample. The capsules were placed in a dessicator under slight vacuum for 12 hours. After the 12 hr. settling period, the samples were oriented for proper longitudinal sectioning and placed in a 60 C oven and allowed to polymerize for 48 hours. The polymerized epon blocks were removed from the oven and stored in a dessicator until used (Appendix V). 108 Section Preparation and Staining. The epon embedded tissue blocks were hand trimmed, using razor blades, into the shape of a truncated pyra- ‘mid with no dimension greater than 0.5 millimeters. The trimmed tissue block was then sectioned on a LKB 4801A ultramicrotome using either glass or diamond knives. Sixty to 100 nm (silver to gold) sections were picked up on 300-mesh uncoated copper grids or 75 to 200 mesh formvar (0.25%) coated copper grids. Three to 5 grids were collected for each replicate which gave a total of 9 to 15 grids per muscle sample. The copper grids containing the tissue sections were stained by floating on a saturated solution of aqueous uranyl acetate (Sjostrand, 1967) for 30 min. or for 5 min. on a 3% alcoholic (ethanol : methanol, 3:1; Appendix VI, VII) phosphotungstic acid (PTA) solution. After the required staining period, the grids were rinsed thoroughly with a jet of glass distilled water for the uranyl acetate stained grids or 3 ethanol methanol (3:1) solution for PTA stained grids (Appendix VI, VII). In the case of the uranyl acetate stained grids, a second staining was accomplished by floating the grids on a lead citrate stain, proposed by Reynolds (1963), for 5 min. or a modification of this method for 10 sec. (personal communi- cation, Richard Ruffing)(Appendix VI). After staining, a jet of 0.02N NaOH followed by glass distilled water was used for rinsing and then the grids were allowed to dry before use. The PTA stained sections were not doubly stained and were ready for use after drying. §pecimen Observation and Photography. Specimen containing grids pre- viously stained were placed in a Philips EM2300 electron microscope and 109 observed at an accelerating voltage of 60 kilovolts. At least 9 grids were observed for each sample and representative photographs were taken using Kodak 8.25 cm x 10.16 cm sheet film. The film was developed for 4 min. in Kodak D-19 developer, washed for 1 1/2 min. in running water, fixed for 8 to 10 min. in Kodak Fixer, washed in running water for l min., rinsed in Kodak Hypo-Cleaning Agent and washed for 10 min. in running water. The washed negatives were dipped in Kodak Photo-Flo solution and dried for 45 min. with warmed air. All processing from the latent image to the final negative was performed on an Arkay nitrogen burst machine. The 8.25 cm x 10.16 cm negatives were placed in a Durst S'45-EM point light source enlarger and Ilford Ilfoprint rapid stabilization pro- cess paper exposed. The Ilfoprint paper was developed in an Ilford model 1501 rapid stabilization processor using Ilford activator and stabilizer chemicals. Selected prints were fixed in Kodak fixer, washed in Orbit bath, flattened with Pakosol, washed in running water and dried on a ferrotype dryer. Sarcomere Measurement Sample Preparation. Only the postrigor aged samples (48 hr. and 216 hr.) were utilized for sarcomere measurements. Approximately 3 g of powdered muscle were weighed into 3 stainless steel homogenization cup and approximately 35 ml. of a 0.25 M sucrose solution added. The powdered mus- cle was then homogenized at high speed with a Virtis "23" macro-homogenizer for 1 minute. A drop of the homogenized sample was placed on a 75 x 25 mm 110 microscope slide and a 22 x 22 mm cover slip was placed over the sample and tapped lightly to remove entrapped air bubbles and to help prevent the formation of more than one layer of myofibrils. The slide preparation was viewed using a Zeiss WL research microscope with a 100x phase contrast oil immersion objective. A filar micrometer was used to count 25 separ- ate lO sarcomere fields for each muscle sample. The filar micrometer was calibrated with a stage micrometer having 0.1 mm and 0.01 mm divisions. Representative areas were photographed using either Kodak Panatomic-X or Plus-X 35 mm black and white film. Protein Extraction Prior to extraction, the frozen muscle samples were placed in a Waring Blendor which had been previously cooled in 3 -29 C freezer and tempered with liquid nitrogen. The samples were powdered in a -29 C freezer as described by Borchert and Briskey (1965). Chipped dry ice and frozen muscle were placed in the blendor jar and powdered by a 30 to 45 sec. burst of the blendor and then sifted with the coarse material replaced in the blendor and the procedure repeated. All material was placed into a pan and thoroughly mixed to insure that the coarse connective tissue was equally distributed. The powdered muscle samples were placed in Whirl-Pak bags and allowed to remain unsealed 3t -29 C for 12 hr. for sublimation of the dry ice. After 12 hr., the bags were sealed and stored at -29 C until used. 111 Sarcoplasmic Protein. The powdered muscle was extracted using a mod- ification of the method of Helander (1957) and Borton (1969). A 2 g sample was weighed into a 250 ml wide mouth polypropylene bottle equipped with a screw cap. Fifty ml of pre-cooled (3 C) 0.015M PO buffer pH 7.4 (Appen- 4 dix VIII) were added to the 2 g of powdered muscle. A stirring bar was added to the bottles and they were placed on a magnetic stirrer and gently stirred at 3 C for 20 minutes. The mixture was centrifuged at 3500Xg for 25 min. in a Sorvall RC2-B automatic refrigerated centrifuge. The super- natant was then passed through 6 layers of cheese cloth and collected in a 100 ml graduated cylinder. The residue was resuspended in 50 ml of 0.015MPO4 buffer, extracted, centrifuged and filtered as previously des- cribed. The 2 supernatants were combined, the volume recorded and designated sarcoplasmic protein (SP). Myofibrillar Protein. The residue from the sarcoplasmic fraction was suspended by adding either 50 ml of 1.1M KCl or 1.1M K1 in 0.1M PO4 buffer pH 7.4 (Appendix VIII). The mixture was gently stirred for 1 hr. on a magnetic stirrer and then centrifuged at 3500Xg for 25 minutes. The super- natant was collected as described previously and the residue reextracted following the same procedure as described above. The 2- supernatants were combined and designated total myofibrillar protein fraction (MP). Non-Protein Nitrogen. Fifteen ml of the sarcoplasmic protein fraction were placed in a centrifuge tube and 5 ml of a 10% TCA solution were added (Appendix VIII). The mixture was allowed to stand for 4 hr. and then cen- trifuged at 3,000Xg for 25 minutes. The supernatant was designated as the non-protein nitrogen (NPN) fraction. 112 Total Nitrogen. Total nitrogen (TN) was determined on 0.5 g of pow- dered muscle by the micro-Kjeldahl method as described below for the SP, MP and NPN fractions. Fifteen ml of the SP, MP and NPN fractions were placed in separate micro-Kjeldahl flasks and 3 glass beads, l g solid N32804, 1 ml 10% CuSO4 solution and 7 m1 of concentrated H2804 were added to each flask. The mixture was digested for approximately 45 min., cooled and 15 ml water added. Approximately 10 m1 of a 40% (w/v) solution of NaOH were added and the nitrogen distilled and trapped in a 2% (w/v) boric acid solution containing brome cresol green (Appendix TX). The boric acid solution was titrated using a standardized H2804 solution. The results were reported as total nitrogen in the muscle sample and/or in each pro- tein fraction. Stroma Nitrogen. The sum of the SP, MP and NPN nitrogen was sub- tracted from the TP nitrogen to determine the stroma nitrogen fraction. ATPase and ITPase Activity The ATPase activity and superprecipitation assays were run on only 10 muscle samples, 5 from the tender group and 5 from the control (lean) group. The 5 bulls from the tender group with the lowest shear and high- est taste panel scores and the 5 bulls from the control (lean) group with the highest shear and lowest taste panel scores were used. Preparation of Myofibrils. The method of Stromer, Goll and Roth (1967) (Appendix X) was followed with some modification. Five g of powdered 113 muscle were weighed into a centrifuge bottle as the myofibril source rather than ground muscle, otherwise the procedure of Stromer t al. (1967) was followed explicitly. C32+LActivated ATPase Activity. One ml of myofibrils containing 0.2 to 0.8 mg protein per ml was placed in a test tube to which the following were added: 1 ml 0.2M tris-acetate pH 7.0, 1 ml 0.35M KCl, 5 m1 H20 and 1 m1 0.01MCa2+ (Appendix XI)o The assay was activated by adding 1 m1 of 0.01M ATP (Sigma Chemical Company, St. Louis). The assay volume totaled 10 ml with a final concentration of 0.02M tris-acetate, 0.04M KCl (in- cludes 0.005MKC1 in myofibril preparation), 0.001M Ca2+ and 0.001M ATP. Thirty sec. after the addition of the ATP 3 1 ml aliquot was removed and added to 1 m1 0f 15% TCA solution. The remaining solution was incubated for 15 min. at 25 centigrade. Another 1 ml aliquot was removed and treated the same as the 30 sec. sample. Both the 30 sec. and 15 min. aliquots were run in duplicate. The ATPase assay was a modification of the proce- dure of G011 and Robson (1967) and Briskey and Fukazawa (1971). + Mgz-eActivated ATPase Activigy. This assay was the same as that - 2+ 2+ 2+ described above for Ca ATPase except 1 ml of 0.01M Mg rather than Ca was used as the activator. + EGTA +'Mg? -Activated ATPase Activity, The EGTA mediated ATPase 2+ activity reaction mixture was the same as described previously for Ca ~ activated ATPase except EGTA was added to the reaction mixture to a final + concentration of 0.0002M which included 0.01M Mg2 . The EGTA was added 114 in solid form to the reaction vessel to provide the proper reaction con- ditions. EDTAoActivated ATPase Activity (high ionic strength). This assay was modified by adding 2 m1 of 2.42M KCl and 1 m1 of 0.01M.EDTA and only + 4 m1 H20 in contrast to 5 m1 H20 in the Ca2 ATPase assay described above. 2+ All other procedures were the same as described previously for the Ca mediated ATPase assay. + + Mg2-and CaziwActivated ITPase Activigy. The ITPase activities were identical to the ATPase assay except ITP (0.01M, Sigma Chemical Company, . . 2+ 2+ . St. Louis) was substituted for ATP and the Mg and Ca activators were added in the same proportions as previously described. Phosphate Determination. Inorganic phosphate was determined by a modification of the Fiske and Subbarow (1925) procedure as described by Leloir and Cardini (1957)(Appendix XII). The values were reported as micrograms phosphate (ug) per milligram (mg) of protein per minute. Myofibril Protein Determination. The biuret reaction was used to determine the protein for standardization (Gornall, Bardawill and David, 1949)(Appendix XIII). 115 Superprecipitation E3tural Actomyosin Preparation. Myosin B (natural actomyosin) was extracted by following a modification of the procedure of Arakawa _£ _1. (19703). Five g of the powdered muscle were suspended in 30 m1 of W-E solution (Appendix XIV). This suspension was stirred magnetically at 2 C for 16 to 24 hours. Ten m1 of the extracted solution were removed after 16 to 24 hr. and centrifuged at 15,000Xg for 20 minutes. The supernatant containing myosin B and SP was diluted with distilled water to 0.15M KCl and centrifuged at 15,000Xg for 20 minutes. The precipitate (myosin B) was dissolved in 1.0M KCl and then diluted with distilled water to a final concentration of 0.5M KCl. The precipitation and dissolution cycle was repeated twice and the final precipitate was adjusted to 0.5M KCl and dissolved by gentle magnetic stirring overnight and then clarified by centrifugation at 15,000 Xg for 20 minutes. Superprecipitation Assay. All readings were obtained from a Beckman DU spectrophotometer equipped with a Gilford attachment and a Sargent SR recorder. All solutions except myosin B and ATP were kept at 27 C prior to the assay determinations. Myosin B was stored at 3 C and ATP was stored at -20 C prior to their use. The superprecipitation assay followed the procedure outlined by Ara- kawa t 1. (19703, c) and Briskey and Fukazawa (1971). Myosin B concen- tration was determined by a modification of the biuret procedure of Gornall t 1. (1949). 116 2+ . . . Mg, + EGTA superprec1p1tation. The total reaction volume of each assay was 3 ml in a Beckman cuvette. The final concentration of the reagents, added in order, were as follows: myosin B, 0.4 mg/ml; tris-acetate, .OlM pH 7.0; KCl, 0.1M; MgClz, 0.001M; EGTA, 0.001M; and ATP, 0.001M. The solution was mixed after the addition of each reagent except for ATP which was added to the cuvette after it was placed in the spectrophoto- meter. Prior to the addition of ATP, 3 reading was made and then the ATP was carefully added to the center of the cuvette and the reaction plotted on the recorder. 2+ . . . . Low Ca Superprecgpitation. The procedure for this assay was identical to the previous assay except that CaCl2 was added to a final concentration of 0.00005M and EGTA was deleted from the assay mixture. Statistical Analyses Data were analyzed on the CDC 6500 Computer at Michigan State Univer- sity. Product moment correlations and a one way analysis of variance was first performed on the data and then the data were treated as a split plot design with treatments as main effects and time as the subeffect. Duncan's New Multiple Range Test (Steel and Torrie, 1960) was applied when analysis of variance data were significant in order to detect the significantly different means. The more important correlation coefficients are listed in Appendix XVI through XIX. RESULTS AND DISCUSSION Electron Microscopy 1 Hr. Postmortem. The samples obtained approximately 1 hr. postmortem were designated as controls to which the postrigor aged samples, 48 hr. and 216 hr., respectively, could be compared. The control samples (1 hr.) will be compared to literature observations whenever possible since these samples were not obtained immediately postexsanguination. Mitochondria. Figure 1 is a group of myofibrils (X17,500) with an intermyofibrillar row of mitochondria. Several of the mitochondria, as indicated by the single arrows, are swollen and considerable cristal damage can be seen. Other mitochondria, as indicated by double arrows, appear to be approximately normal in size and the cristae are in a more normal and organized condition. Figure 2 shows a higher magnification (X37,800) electron micrograph with several subsarcolemmal mitochondria which are oriented in various planes. Several mitochondrial areas, as indicated by arrows, are disrupted and the cristae have been degraded. The intact cristae tend to be some- what denser and in a slightly different configuration than the cristae shown in figure 1. Figure 3 represents another myofiber area showing mitochondria sec- tioned in longitudinal and transverse planes. Some swelling can be seen in many of the mitochondria and areas of disrupted cristae, indicated by arrows, can be seen. 117 118 Figure 1. 1 hr. postmortem bovine longissimus muscle fiber. M ' inter- myofibrillar row of mitochondria. Swollen mitochondria shown by single arrows; double arrows show less swollen mitochondria. (X 17,500) Figure 2. 1 hr. pastmortem bovine longissimus muscle subsarcolemmal mito- chondria. Arrows indicate areas of cristal degradation. (X 37 ,800) 119 Little attention has been given to the morphology of postmortem bovine muscle mitochondria. Considerable research has been reported on other species relative to the determination of fiber type by mitochondrial number and cristae density. Gauthier (1970) reported the division of rat diaphragm muscle fibers into three classifications, partially by the quantity and location of mitochondria. Extrapolating her observation for rat muscle to include bovine muscle is difficult, however, mitochondrial disruption is obvious in the 1 hr. sample when compared to the mitochondria in fibers reported for rat muscle. Dutson 33 31. (1974) investigated postmortem changes in normal and low quality porcine longissimus muscle mitochondrial ultrastructure. They found no obvious changes in mitochondrial continuity in either normal or low quality animals at 15 min. postmortem. The swelling and disruption of the mitochondrial membranes in the 1 hr. samples suggest that an altera- tion in the cell environment has occurred between exsanguination and sampling. It is difficult to ascertain whether the changes in the mito- chondria are the result of sample excision, fixative osmolarity, or post- mortem changes inherent to the animals tissue. Z—line. In an effort to more effectively discuss changes in Z—line morphology, the fibers in this study have been arbitrarily classified as type I and type II based on Z—line density and width and number and loca- tion of mitochondria. This classification is based entirely on subjective evaluation of the ultrastructure of the Z-line and mitochondrial population. Dubowitz (1970) classified fibers as type I and type II by using reciprocal 120 Figure 3. 1 hr. postmortem bovine longissimus muscle fiber. Arrows indi- cate areas of mitochondria cristae degradation (X 17,500) Figure 4. 1 hr. pOstmortem bovine longissimus muscle type 1 fiber. A = A‘band; M = M-linc; I = I-band; Z : Z-linv; S = sarcomere (X 17,500) 121 histochemical stains. However, the type I and type II fibers in the pre- sent study are similar to the type I and type II fibers of Dubowitz (1970) only if the ultrastructural observations and histochemical stains identify the same fiber characteristics. Figure 4 shows a group of myofibrils having thick and dense Z-lines characteristic of type I fibers. The Z-lines have a dense fibrillar con- figuration that is readily evident 3nd a sharp division between the I-band and Z-line is apparent. The Z-lines are continuous across the myofibril which imparts an unaltered appearance to their entire structure. Figure 5 is another representative type I fiber which is similar in most respects to the fiber in figure 4. The Z-lines are very distinct, wide and dense, which is characteristic of type I fibers and a number of intermyofibrillar mitochondria (indicated by arrows) are also very appar- ent. Figure 6 shows several myofibrils which are representative of the type II fibers. The Z-lines in these myofibrils appear to be moderately wide and dense, but are clearly more diffuse and distinctly less fibrillar than type I Z-lines. The demarcation between the I—band and Z-band is not as obvious as that observed for type I fibers. Figures 7 and 8 shows 2 additional examples of type II fibers differ- ing only slightly from that seen in figure 6. The myofibrils in figure 7 have Z—lines that are more diffuse and in certain areas,as indicated by arrows, only limited structure can be seen. The Z-lines of the myofiber in figure 8 are thinner, but less diffuse and slightly more distinct than those in figure 7. 122 Figure 5. 1 hr. postmortem bovine longissimus muscle type I fiber. Arrows indicate several intermyofibrillar mitochondria (X 17,500) Figure 6. 1 hr. postmortem bovine longissimus muscle type II fiber. A = A-band; M - M-line; I = I-band; z = Z-line; S = sarcomere (X 17,500) 123 Figure 7. Type 11 fiber in 1 hr. bovine longissimus muscle. A ' A-band; M ' M-line; I ' I-band; Z ' Z-line; S ‘ sarcomere. Arrows indi- cate areas of loss of z-line structure. Figure 8. Type II fiber in 1 hr. bovine longissimus muscle. A = A-band; M ' M-line; I ' I-band; z = Z-line; S = sarcomere (X 17,500) 124 Electron micrographs of early postmortem muscle fibers in the liter- ature show distinct Z-lines which frequently illustrate a prominent fibrillar configuration. A similar condition can be seen in type I fiber samples at 1 hr. postmortem in the present study, however, the type II fibers appear to be altered slightly in fibrillarconfiguration. Gauthier (1970) reported that 3 types of fibers were present in rat diaphragm muscle. The Z-lines in all 3 fiber types of rat muscle are distinct and fibrillar and do not tend to be diffuse as was seen in several of the type II fibers in the present study. Dutson 3£_31. (1974) reported ultrastructure data that showed no change in Z—line ultrastructure of red and white fibers of por— cine 1ongissimus muscle at 15 min. postmortem in either normal or low qual- ity carcasses. Goll (1968) and Henderson 3£_31. (1970) reported no dis— cernible changes in at death samples of bovine, porcine and rabbit muscle. The samples in the present study appear to have fibers that are divided into 12 relatively distinct populations having divergent Z-line morphology and stability at 1 hr. postmortem. Contractile State. The contractile condition of the fibers at 1 hr. postmortem varied from little or no contraction to maximal contraction. Figure 9 represents a group of myofibrils with a clear and distinct I-band, however, the H-zone is indistinct suggesting that some contraction has occurred in this fiber. The fiber in figure 10 has a very small I-band and the close proximity of the A-band to the Z-line indicates that maximal contraction has occurred. Figure 11 is a fiber area that appears to be relaxed or slightly stretched as indicated by a wide I-band and a distinct H-zone. 125 Figure 9. 1 hr. postmortem bovine longissimus muscle type 11 fiber illus- trating partially relaxed muscle sarcomeres. A ' A-band, M‘ H-line; I - I-band; z ' z-line; H ' H-zone; s ' sarcomere (X 17,500) Figure 10. 1 hr. postmortem bovine longissimus muscle type 1 fiber illus- trating contracted muscle sarcomeres. A = A-band; M = M-linc: I = I-band; Z = Z-linv; H = H'ZOhc; S = sarcom-rc (X 17,500) 126 Figure 11. Type 11 fiber, 1 hr. postmortem, illustrating relaxed c0ndi- tion. A ' A-band, M ' M-line; I ' I-band; Z ‘ Z-line; H 3 H-zone; S ' sarcomere (x 17,500) 127 The contractile condition of the 1 hr. samples indicates that many of the fibers were not adequately restrained before fixation. Since no mechanical device was used to restrain the samples, the appearance of contracted fibers was expected and not easily prevented. The contracted condition did not seem to alter the ultrastuctural morphology of the fibers. MrLine. The Mrline was readily apparent in 311 preparations as a dark line bisecting the center of the A—band as shown in figure 4. No distinct differences in M-line morphology were found between fiber classifications, treatments or contractile condition of the fibers. Glycogen. Generally, at death samples have an abundant supply of 15 to 30 nm diameter glycogen granules (Lehninger, 1970) in the intermyofibrillar cytoplasm and in the cytoplasm in other locations within the muscle fiber. These granules were conspicuously absent in the 1 hr. postmortem samples. The dearth of glycogen in the 1 hr. samples can be interpreted in several different ways. Since all animals were bulls, the possibility exists that initial glycogen reserves were low, and by the time the samples were taken, glycolysis had proceeded far enough to deplete all or most of the reserves. Sample removal may have contributed to the glycogen loss since contraction was rather violent during excision. The literature of at death muscle samples reported by Goll (1968), Henderson 3£_31, (1970) and Dutson 3£_31, (1974) have a variable but obvious quantity of glycogen in the cytoplasm. No firm conclusion can be drawn concerning the lack of glycogen in the 1 hr. samples from the data obtained in this study. 128 Treatment Effects. The 1 hr. postmortem samples tend to show no dis- cernible effects due to treatment. The group of animals selected for tenderness appeared to show an equal amount of mitochondrial disruption, Z-line alterations and glycogen depletion as that found in the group not selected for tenderness. Likewise, contractile state did not appear to differ between tenderness groups. 48 Hr. Samples Mitochondria. Considerable mitochondrial morphological variation was present in the postrigor (48 hr.) samples, however, there was more of a tendency for all of these organelles to be disrupted as compared to the 1 hr. samples. Figure 12 represents a myofiber area showing several mito- chondria in various states of disintegration. The intermyofibrillar mito— chondria, as indicated by M2, appeared to retain more of the characteristic morphological features than the mitochondria encircling the I-band-Z-line area, which are indicated by M1. The mitochondria, indicated by M, in figure 13, are more disrupted than those in figure 12. The outer membrane in several mitochondria appears to be broken and the cristae in most of the mitochondria are severely disrupted. Cassens 3£_31, (1963) reported mitochondrial disruption in normal porcine loggissimus at 24 hr. postmortem. Dutson 3£_31, (1974) reported that some mitochondrial disruption or loss of cristal density could be found in all 24 hr. samples, but was particu- larly obvious in the white fibers (type II). Although not investigated thoroughly, it appeared that the mitochondria in type I fibers are less labile than mitochondria in type II fibers in the present study. 129 Figure 12. Type I fiber, 48 hr. postmortem. M 1 ' mitochondria which encircle the myofibril; M 2 ' intermyofibrillar mitochondria. (X 17,500) Figure 13. Two type II fibers, 48 hr. postmortem. M = disrupted lnlvr' myofibrillar mitochondria (X 7,840) 130 Z-Line. Z-line line ultrastructure in some postrigor (48 hr.) fibers has undergone considerable modification when compared to 1 hr. samples. Again, however, 2 relatively distinct fiber groups are apparent as dis- tinguished by Z-line morphology and mitochondrial density. Type I fibers, having wide and dense Z-lines, as indicated by figures 14, 15 and 16, appear essentially unaltered and are similar in ultrastructure to pre- rigor type I fibers. The only discernible alteration in type I fibers appeared to be a less distinguishable fibrillar nature of the Z—line. Type II fibers, although somewhat more variable in appearance than type I fibers, are easily distinguishable from type I fibers by the appearance of breaks in the Z-line evident at 48 hr. postmortem. Figure 17 is a group of myofibrils having Z—lines approaching the thickness and density of type I fibers, however, as indicated by arrows, a number of breaks can be seen in the Z—line. Figure 18 is a higher magnification of a Z-line from the same fiber shown in figure 17. The arrows indicate 2 areas of loss of continuity in Z-line structure. Figure 19 is an electron micro- graph of a portion of 2 fibers exhibiting differing amounts of Z-line breakage. Fiber A is similar to that in figure 17 in that only minor Z- line breakage had occurred, (shown by arrows), whereas fiber B had more evidence of Z-line breakage, as indicated by arrows. Figure 20 shows another group of myofibrils representing the type 11 fiber group. The major difference between this fiber and previous examples is the plethora of Z-line disintegrations. Several areas, as indicated by arrows, are almost devoid of distinguishable Z-line material. Figure 131 Figure 14. Type 1 fiber from 48 hr. postmortem bovine longissims mscle. A ' A-band; I ‘ I-band; H ’ H-zone; u " H—line; z ' z-line; S " sarcomere (x 17,500) l“Sure 15. Type I fiber from 48 hr. postmortem bovine MM mscle. A ’ A-bana; I = I-band; H = H-zone; H = M-line: Z = Z-line; S = sarcomere (x 17,500) 132 .7- A‘; a v ' Q _ ,2 . at .35 s \ .lb ‘ 5-5' 9 . .. ‘- ‘ . 3‘" ,.- . ~ J’ 3 r ‘ , \ - \ h, , V\\ _ ‘.. .. Figure 16. Type I fiber from 48 hr. postmortem bovine longissimus muscle. A ‘ A-band; I = I-band; M = M-line; z ‘ z-line; S ‘ sarcomere (17,500) FiSure 17. Type II fiber, 48 hr. postmortem, showing Z-line degradation. Arrows indicate breaks in Z—line structure. A = A-band: I = I-band; M = M-line; Z = A-line; S = sarcomere (x 17,500) 133 Figure 18. Higher magnification of Z-line area from figure 17. Arrows indicate breaks in the z-line (X 37,800) Figut‘e 19. Two type II fibers, A and B. Arrows indicate breaks in [LU Z-line. A = Z-band; I = I-lmnd; M = M-l ine; 7. = JZ-lim-; S = sarcomere (x 9330) 134 21 shows a fiber exhibiting a similar condition to that seen in figure 20, however, the remaining Z-line material appears to be slightly less dense and considerably more diffuse. Figures 22 and 23 differ only slightly from previous examples of type II fibers, except for the proportion of Z-line disintegration. The double arrows in both figures indicate areas of longitudinal splitting. In figure 23, the single arrows indicate Z-lines that are almost totally devoid of typical Z-line material. Even without obvious Z-line material, several sites can be seen in this figure where filamentous material appears to be continuous through the Z—line area. I-Z Breakage. The ultrastructural change common to both types of fibers is the disruption of the connection between the thin filaments and the Z-line. The type I fibers exhibit distinct I—Z breakage since in many cases the Z-line can be found intact on one or the other half of the I- bands. In figure 24, the arrows indicate an area of an I—Z junctional breakage which is unilateral and the Z-line material remains attached to I-band filaments only in one-half I-band. The I-band-Z-line junction has almost completely separated with little I-filament remaining attached to the Z-line. Figure 25 exhibits a similar but more massive case of I-Z breakage, however, as indicated by the arrows, some connection still exists between the Z-line and thin filaments. It appears that the thin filaments have been stretched rather than totally severed from the Z-line. Figure 26 is a low magnification electron micrograph of a portion of 2 .fibers. The arrows indicate several areas of apparent I-Z junctional 135 Figure 20. 48 hr. postmortem type II fiber. Arrows indicate areas of Z- line degradation. A = A-band; I = I-band; M = M-line; Z = Z- line; S = sarcomere (X 17,500) Figure 21. 48 hr. pastmortem type II fiber. A = A-band; I = I-band; M = M-line; Z = Z-line; S = sarcomere (X 17,500) 136 Figure 22. Type II fiber in 48 hr. postmortem bovine lo_ngissimus muscle‘. Double arrows indicate areas of longitudinal splitting (x 17,500) Figure 23. Type 11 fiber in 48 hr. postmortem bovine longissimus muscle. Single arrows show Z-lines that are practically devoid of typical dense Z-line material, double arrows indicate areas of longi- tudinal splitting. Z = Z-line (X 17,500) 137 Figure 24. Type I fiber in 48 hr. postmortem bovine longissimus muscle. Arrows indicate a break at the junction of the I-band and 2- line. A * A-band; I = I-band (X 17,500) Figure 25. 48 hr. postmortem bovine longissimus muscle type I fiber. Arrows indicate filamentous material connecting Z-line and I-band. A = A-band; 1 = I-band (x 17,500) 138 breakage, which are not easily discerned at this level of magnification. Figure 27 is a higher magnification of these areas which clearly shows that the breakage has occurred at the I-Z junction. The Z-lines are typical of type I fibers as indicated by their width, density and unaltered ultra- structural appearance. Generally, as in previous examples, the Z—line remains on one side of the original I-Z-I band connection and in most ex- amples the breakage is not total as illustrated by a residual connection between I-band and Z-lines. This observation is clearly depicted in figure 27 (arrows), however, this is a general rule applicable to the majority of postrigor type I fibers and occasionally a I—Z breakage can be seen in which the I filaments and Z-line material have completely separated from each other. Figure 28 shows a type I fiber exhibiting an atypical Z-line breakage or splitting. The single arrow indicates the Z-line that appears to be undergoing symmetrical Z-line division rather than an I-Z junction break- age. This is an uncommon condition and it is difficult to ascertain whether it results from postmortem storage or is an abberation inherent to the in 3132 fiber. The double arrows indicate an area of breakage at the 1-2 junction, however, the Z material adheres to both halves of the I—band filaments rather than one as shown in figure 27. All previous examples represented type I fibers exhibiting I-Z junc- tional splitting, however, type II fibers exhibit a similar condition. Figure 29 shows a fiber with considerable Z-line breakage and I-Z junctional sPlitting. In several areas, indicated by single arrows, breaks have 139 Figure 26. Low magnification electron micrograph of a portion of 2 fibers in 48 hr. postmortem bovine lgggissimus muscle. Arrows indicate areas of I-z junction breakage (x 1,120) Figure 27. Higher magnification of figure 26 showing 1-2 junction breakage. Arrows indicate filamentous connection between the I-band and Z-line. I ‘ I-band; Z = Z-line (X 7,840) 140 in 48 hr. poa‘ on shows sy-atrie . ‘ ‘ ”Arms of breakage at {line (H.500) r '3 ;_ ‘. » Figure 29. Type 11 fiber in 48 hr. postmortem bovine longissimus muscle. Single arrows indicate breaks that occur at the junction of the I-band and x-linw, dnuhlv JFYOWS show Z-linc material on both portions wt l‘hJHJI l ; l-hund; d ‘ [-linu (X 17,500) 141 occurred at the junction of the thin filaments and Z-line. The double arrows indicate an area of breakage that has allowed Z-material to partially remain on both sides of the divided I-band as compared to the type I fiber I-Z breakage which predominately leaves Z material attached to only one of the halves of the I—bands. Figure 30 shows a low magnification electron micrograph of 2 fiber portions. In fiber A, arrows indicate several areas of I—Z breakages in contiguous myofibrils. The I—Z breakage in this fiber tends to follow a definite pattern, whereas previous examples appeared to indicate a more random distribution of breaks in that only rarely did 2 I-Z junction breaks occur in contiguous sarcomeres. Figure 31 shows a higher magnifi— cation of the area represented in the square in figure 30. The higher magnification electron micrograph shows that the breakage occurs at the junction of the thin filament and the Z—line. The double arrows indicate Z-line breakage both at an I-Z junction and at another Z-line where I-Z splitting has not occurred. The single arrows indicate portions of Z- material that has remained on both halves of the I—band. M-Line. The M—line in most preparations seemed to be unaltered, how- ever, occasionally, as seen in certain myofibrils of figures 22 and 23, the M—line is not easily discerned. Generally, the M-line can be seen, in an apparent unaltered state, in all samples. Contractile State. The postrigor fibers appeared to have undergone little perceptible alteration in sarcomere length when compared to prerigor that! 30. Figure 31. 142 Low magnification electron micrograph of a portion of 2 fibers, A and B. The arrows indicate myofibril breakage (x 1,400) Higher magnification electron micrograph of the area within the Square in figure 30. Single arrows show Zrlinv material remaining on both hJIVvs ol the I-hnnd, lhv double arrows in- dicalr Z—Hm- degradation. 1 : l‘lmml; .'. * flinc (X 9,5203 143 fibers. Fibers with distinct and wide I-bands (figure 14 and 15) were frequently encountered, however, many fibers (figures 21, 22 and 23) with narrow, almost indistinguishable I—bands, could be found in most samples. Glycogen. No granules having the dimensions or morphology commensur- ate with that reported for glycogen could be found in any of the postrigor samples. These results indicate an apparent dichotomy in Z-line condition of postrigor samples. Davey and Gilbert (1969) reported that the Z—line condition in bovine sternomandibularis muscle was dependent somewhat on the animal from which the myofibrils were isolated. Some preparations contained Z-lines that were unaffected by storage, whereas others appar- ently suffered total Z—line loss. No direct comparison can be made between this study and that reported by Davey and Gilbert (1969) since they used a homogenization procedure for myofibril isolation. This procedure might contribute to the leaching of partially degraded Z-lines (Goll, 1968) and as such either the Z—lines are completely removed or are unaffected. Dutson g£_§l, (1974) reported that Z-lines of red fibers (type I) from both normal and low quality porcine longissimus muscles were virtually unaltered by 24 hr. of postmortem storage. However, white fibers from the same ani- mals underwent substantial Z-line alterations after 24 hr. of storage. Henderson at al. (1970) found no changes in bovine semitendinosus myofibrils after 24 hr. at 2 or 16 C, however, considerable disruption could be found in electron micrographs after 24 hr. when the samples were stored at 25 or 37 centigrade. Hay et_al, (1973a) found little evidence of morphological 144 alterations in chicken leg muscle during postmortem storage, however, considerable alteration in Z-line structure was found in chicken breast muscle as early as 48 hr. postmortem. Fukazawa _£‘_l. (1969) found that Z-lines in myofibrils prepared from chicken pectoral muscle were either degraded, completely removed, or were broken at the junction of the Z- line and I-filaments. The degradation of the Z-line, as considered previously, and I-Z breakage seem to be independent events. The occurrence of the latter phenomenon appears to be a function of the linkage between the Z-line and I-filament and not related to Z-line degradation. Fukazawa _£‘_l. (1969) considered that a breakdown of structures at the junction of the I-band and Z-line was a prerequisite for myofibril fragmentation during homogeni- zation. Davey and Dickson (1970) observed that aged bovine muscle broke at the I-Z junction when tension was placed on muscle strips. These authors found no indication of breakage in unstretched samples prior to application of tension to the muscle strips. In unaged samples the strips lengthened predominately by withdrawal of thin filaments from the A-band, although considerable tension was necessary to force this stretching. As reported by Hay _£Hal. (1973a) and Dutson _£.il- (1974) the condi- tion of the Z-line in postmortem fibers is dependent on the fiber type. Hay gt al. (1973a) reported that the chicken leg muscle red fibers main- tained Z-line continuity even after several days of postmortem aging. The type I fibers in the present study reacted similar to chicken leg muscle fibers (Hay t l., 1973a) and porcine longissimus muscle red fibers 145 (Dutson 35 21,, 1974). On the other hand, the type II fibers.lose Z-line structure similar to that of chicken breast muscle (Hay 35 a1., 1973a) and porcine longissimus muscle white fibers (Dutson 35 31., 1974). Implica- tions of this phenomenon with postmortem aging and tenderness will be considered later. Little evidence could be found that would suggest any consistent M— line alterations occurring as the result of 48 hr. postmortem storage. An occasional fiber, as mentioned earlier, could be found that contained altered M-lines, however, these appeared to be the exception and not the general situation. The glycogen content and contractile state were apparently unchanged after 48 hr. of storage. The glycogen reserves were considered earlier and the contractile state will be considered later in association with sarcomere length changes and tenderness. 216 Hr. Samples Mitochondria. The morphology of mitochondria after 216 hr. of post- mortem storage varies from fiber to fiber. Figure 32 shows portions of 2 fibers with the arrows indicating 2 mitochondria in 1 fiber. The mito- chondrion indicated by the single arrow is relatively dense and the cris— tae have been altered very little. The mitochondrion indicated by the double arrow has an area devoid of cristae indicative of severe degrada- tion. Figure 33 shows portions of another 2 fibers with the arrows indicating several intermyofibrillar mitochondria. These mitochondria appear swollen 146 Figure 32. Mitochondria in 216 hr. postmortem bovine lgggissimus muscle. M ' mitochondria, single arrow indicates a mitochondrion with recognizable cristae, double arrows show a portion of a de- graded mitochondrion (X 17,500) {v Figure 33. Two fiber portions of 216 hr. postmortem bovine longissimus muscle. Arrows indicate disrupted intermyofibrillar mitochon- dria (X 9,530) 147 and the cristae are either completely missing or degraded sufficiently to make organelle identification difficult. Figure 34 shows several myo- fibrils with a few mitochondria that are intermediate to those in the previous 2 examples. The organelle is easily identified, but the cristae show considerable disruption. Few data can be found in the literature concerning postmortem mito- chondrial morphology. The variable condition of the mitochondria suggests a fiber type-degradation relationship, however, this was not investigated and can not be substantiated without further investigation. Z-Line. The condition of the Z-lines in the aged samples (216 hr.) resembles that seen for postrigor samples (48 hr.) in many respects. The type I fibers in the aged samples are almost indistinguishable from the type I fibers at 48 hr. postmortem. Figure 35 shows a group of myofibrils representing a type I fiber. The Z-lines are virtually unaltered and still maintain the thickness and density observed for the same fiber types in 1 hr. and 48 hr. samples. Figure 36 is a higher magnification of several myofibrils having very thick and dense Z-lines that have maintained much of the fibrillar structure seen in the 2 previous time periods. Figure 37 is another representative sample of type I fibers. Although the Z-lines in this fiber are not as dense as those seen in figure 36, they are almost completely unaltered from that at 1 hr. postmortem. The fibrillar nature of the Z-lines is discernible, however, this fiber has a tendency to be more diffuse than type I fibers in prerigor and early postrigor (48 hr.) samples. 148 Figure 34. 216 hr. postmortem bovine longissimus muscle fiber. The arrows indicate several mitochondria in various states of degradation (X 17 ,500) Figure 35. 216 hr. postmortem bovine Ioneissimus muscle type I fiber showing essential 1y no change (X 17.3“”) Figure 36. Higher magnification of a portion of a 216 hr. postmortem bovine longissimus muscle type I fiber showing essentially no change. (X 28,000) Figure 37. Type I 216 hr. postmortem bovine _l_eneivssimus muscle fiber showing essentially no change (X 17,500) 150 Figures 38 and 39 show representative myofibrils of type II fibers in the 216 hr. samples. The myofibrils in figure 38 have relatively broad and dense Z-lines, however, as indicated by the arrows, several breaks have occurred in the Z-line. Figure 39 shows a group of myofibrils that are similar to those in figure 38. The large arrows indicate breaks in the Z-line which are associated with what appears to be longitudinal splitting (small arrows) of a portion of the myofibril especially in the I-band. Figures 40 and 41 show 2 additional representative electron micrographs of type II fibers, however, these myofibrils show considerably more Z-line degradation (large arrows) than the previous micrographs of type II fibers. The small arrows indicate areas of longitudinal splitting which appear to be primarily associated with areas of Z-line breakage. Figure 42 shows a portion of 2 fibers, A and B, that represent the variation in Z-line breakage found in aged (216 hr.) type II fibers. Fiber A has relatively broad and dense Z-lines with only minor breakage, however, fiber B has narrower and less dense Z-lines and numerous areas of Z-line degradation. I-Z Breakagg. The breakage occurring at the junction of the thin filament with the Z-line follows a similar pattern to that seen in the 48 hr. samples. The principal difference between 48 hr. and 216 hr. samples is the greater abundance of I-Z splitting which was found in most fibers of the 216 hr. samples. Figure 43 shows a type I fiber exhibiting several areas of I-Z breakage as indicated by the arrows. The small arrows indi- cate areas that are breaking on both sides of the Z-line. This type of 151 Figure 38. 216 hr. postmortem bovine longissimus muscle type 11 fiber. Arrows indicate breaks in the Z-line. Z ' Z-line-(X 17,500) Figure 39. Type 11 fiber in 216 hr. postmortem bovine longissimus muscle. Large arrows show breaks in the Z-line, small arrows indicate longitudinal splitting. Z 2 /.-line (X 17,50”) 152 Figure 40. 216 hr. postmortem bovine longissimus muscle type 11 fiber. Large arrows indicate Z-line degradation, small arrows show areas of longitudinal splitting. Z ' z-line (X 17,500) A! W.“ ~ '. was - I . Figure 41. Type 11 fiber in 216 hr. pestmortem bovine longissimus muscle. Large arrows Show Z-line degradation, small arrows indicate longitudinal splitting. Z = z-line (X 17.500) fibers. A and I, in 216 hr. poetmortem bovine ' much (1: 11,480) Type I 216 hr. postmortem bovine loin'issimns flflhu 1e 11%. r. Large arrows indicate l‘7. breakage Ut'eui'rine, en '1 \ldi' .1 ' Z-llnt“, small .U‘rt‘W‘i itHlit’le I-If l\1’- .Il‘_.l'.'i' .‘tilirr‘t‘x "' l~ " sides of Z‘llne. [ 7 l-lvmll; ,"_ _'ll"|‘ l): i531” 154 breakage generally is not seen in type I fibers, however, this particular fiber represents an exceptional case and was not observed to occur in most type I fibers. Figures 44 and 45 show additional representatives of type I fibers, however, in these myofibrils the breakage has progressed to the point that they have been completely severed at the I-Z junction. The single arrows in figure 44 indicate 2 areas that appeared to have pulled away from the Z-lines but have not been completely severed. The double arrows in the same figure indicate areas that have broken in the I band region rather than at the I-Z junction. The single arrows in figure 45 indicate several areas that have broken predominately at the level of the I-2 junction. Figures 46 and 47 are type II fibers that have broken at the I-Z junction. The single arrows in figure 46 indicate areas of breakage at the I-Z junction and which have retained the majority of the Z-line on the visible myofibril fragment. The double arrows indicate areas of Z- line breakage and longitudinal splitting. Figure 47 is another type II fiber having an area of I-Z breakage, but it has not progressed to the point of total I-Z severance as shown for the myofibrils in figure 46. The arrows indicate areas of Z-line breakage within Z-lines which are ad- jacent to the I-Z junction breakage. Figure 48 shows a portion of a fiber similar to that in figure 46. The single arrows indicate areas of Z-line breakage and the double arrows indicate an area of myofibrils that are obliquely sectioned. 155 Figure 44. 216 hr. postmortem bovine M mole type 1 fiber. single arrows indicate incomplete I-z break-3e, double arrows indicate apparent breakage in the I-band area. I ' I-bend; Z ' Z-line (X 17,500) Figure 45. 216 hr. postmortem bovine longissimus muscle type I fiber. The single arrows indicate areas that have broken at the lchl of the I-2 junction. 1 I I-hand; Z = Z-line (X 7,340.) 156 Figure 46. 216 hr. postmortem bovine longissimus muscle type II fiber. The single arrows indicate I-Z breakage and the double arrows show z-line degradation and longitudinal splitting. I ‘ I-band; Z ' Z-line (X 9,520) I"igure 47. Type II fiber in 216 hr. postmortem hovim- longissimus muscle. The single arrows indicate nrvns of Z-lim- breakaw- .uliact-nt (0 tlw [-7, junction hn-zlkagv. I = l-h.nnl; f. l Lfi-Iinv (X 9.33”) 157 Fiber Breakage. Several of the 48 hr. postmortem samples had numerous areas of I-Z breakage, however, most of the breakages involved only random Z-lines and only rarely were areas found that suggested incipient trans- verse fiber division. Figure 49 shows a low magnification electron micro- graph of portions of three fibers. As indicated by the arrows, fiber A has several areas of partial transverse breaks. One of the areas of fiber breakage (double arrows) shows greater myofibril separation near the trans- verse center of the fiber and the break decreases in width as it extends in one direction towards the sarcolemma. Figure 50 shows another fiber exhibiting oblique breakage that extends across the entire fiber. The arrows indicate a small area of complete I-Z breakage that has occurred near the sarcolemma. Figures 51 and 52 show low magnification electron micrographs of several fibers that have complete transverse fiber breakage. In figure 51, fiber C has not only broken completely but it has also been pulled apart leaving a large gap between the 2 broken halves of the fiber. Figure 52 shows a different fiber, which broke similarly to that in figure 51 and likewise has a gap between the two broken fiber fragments. Figure 53 shows a higher magnification of a fiber similar to the 2 previous fibers. The double arrows indicate areas that have obviously broken at the level of the Z-line, whereas the single arrows indicate the adjacent portions of these myofibrils which apparently had moved sufficiently from the longitu- dinal orientation during breakage and consequently they were sectioned obliquely. Figure 48. 216 hr. postmortem bovine longissims mscle type II fiber. The single arrows indicate z-line breakage and the double arrows show an area of obliquely sectioned myofibrils. Z ' Z-line (X 17,500) Figure 49. Portions of 3 fibers, A, B and C, from 216 hr. postmortem bovine longissimus muscle. The arrows indicate areas of par- tial fihcr breakage and the double arrows indicate an area extending across l/Z of tho filn-r (X 780) Figure 51. Low magnification electron micrograph of .l portion m h Ill‘x'l‘h A, B, C» D, F. and F, of 31') hr. P'HQIRIUI'IUIH huvim- L1"? muscle. Fiber (1 has: hruln-u lx‘.|H‘.‘.'«-1‘~;vl\ .unl n-lrntHi Figure 52. Figure 53. ssimus muscle. The arrows indicate the 2 broken halves of the fiber (X 1,400) Portion of a broken fiber from 216 hr. postmortem bovine 1233 - J Higher magnification electron micrograph of a trunsvursely divided 216 hr. postmortem bovine longissimus muscle fiber. —_—¥—.—— _ ' The Single arrows show obliquely sectioned portions ut mvotx- brils and the double arrows indicate Z : Z-line (X 5,400) l-/. hreakmze. l 5’ l-lmnd, 161 M-Line. The M-line did not appear nearly as discrete and prominent as in earlier postmortem time periods, however, it was present in all fibers at 216 hours. Contractile State. The actual sarcomere lengths of all the samples D will be presented later, however, the ultrastructural observations suggest that little sarcomere lengthening has occurred after 216 hr. of post- mortem aging. Glycogen. As was the case at 1 hr. and 48 hr. postmortem, no glyco- gen granules could be found in any sample at 216 hours. The absence of glycogen is consistent with other published data in postrigor muscle. The absence of these particles in the 1 hr. samples has been discussed previously. The structure of the Z-line changed very little between 48 and 216 hr. postmortem. The type I fiber Z-lines became slightly more diffuse, however, the alteration was very subtle. The type II fibers were similar in most respects to the 48 hr. samples, however, the Z-line degradation tended to be more constant from fiber to fiber although some fiber varia- tion still existed. As mentioned previously, Davey and Gilbert (1969) found 2 fiber populations, based on the presence or absence of Z-lines, in myofibrils of muscle aged for 20 days. The predominate myofibril popu- lation was devoid of Z-lines, while these authors found that other samples contained Z-lines. Fukazawa and Yasui (1967) reported that homogenized chicken muscle myofibrils lost all evidence of Z-line material after 24 162 hr. of postmortem storage. In contrast to these observations, no sample in the present study could be found that was devoid of Z-lines, although many had lost as much as one-half of the Z-line material. This apparent observation can be partially explained by different methods of sample preparation since Davey and Gilbert (1969) and Fukazawa and Yasui (1967) used myofibril preparations that possibly allowed leaching of material from the labile Z-lines. Henderson _£._l. (1970) reported a total loss of Z-lines from rabbit muscle after storage at 37 C for 24 hr., however, lower storage temperatures prevented the complete loss of Z-lines. The latter authors observed that bovine muscle Z-lines were not removed from myofibrils by homogenization until 168 hr. postmortem. These results are confusing, however, an apparent fiber type-species relationship seems to exist in postmortem muscle Z-line lability. The amount of I-Z breakage became much more prominent in muscle at 216 hr. than was observed at 48 hr. postmortem. At 216 hr., there appeared to be a tendency for considerable numbers of I-Z breakages to be present in those muscles where they were observed at all since some fibers showed no I-Z breakages. This contrasts somewhat to the 48 hr. samples where only isolated and seemingly random I-Z breakages occurred. The most obvious difference among the three time periods is the appearance of total trans- verse fiber splitting as the result of massive breaks at the I-Z junction in the 216 hr. samples. Hanson _£‘_l. (1942), Paul _£._l' (1944) and Rams- bottom and Strandine (1949) reported the presence of fiber breaks in post- mortem muscle. Paul t 1. (1944) found these breaks occurred as early as 24 hr. in bovine muscle and increased with postmortem storage time. 163 Ultrastructure and Tenderness. Most of the ultrastructural observa- tions on muscle have been concerned with postmortem structural alterations and not the direct association between ultrastructure and tenderness. The association between these alterations and tenderness has primarily been by inference and not by objective or direct measurement. Goll (1968) suggested that since Z-line degradation and postmortem tenderization followed a similar time sequence that a cause and effect relationship could exist. Davey and Gilbert (1967b) found that bovine sternomandibu- l§£i§_muscle aged for 3 days at 15 C suffered a complete loss of Z-lines. These authors suggested that the loss of normally refractory Z-lines was closely associated with the effects of aging. Weidemann _£‘_l. (1967) found that the degree of disruption of myofilaments was a good indication of tenderness. Davey and Gilbert (1969) suggested a possible relationship between loss of myofibrillar lateral linkages and tenderness. Davey and Dickson (1970) substantiated this observation but concluded that other changes probably were more important to overall tenderness variation. Hay _£._l' (1973a) reported that chicken breast myofibrils underwent sub- stantial alterations, particularly at the Z-line, whereas leg myofibrils underwent little perceptible morphological change up to 168 hr. postmortem. Since the leg muscle, adductor longus, is considered to be a predominately red muscle and the breast muscle, pectoralis superficialis, predominately white the authors attributed the postmortem muscle changes to a possible difference in lability between red and white fibers. The red and white fibers presented by Hay t al. (1973a) corresponds closely to the type I 164 and type II fibers, respectively, in the present study both morphologically and the type of postmortem alterations. If in fact postmortem aging and ultrastructural alterations have a cause and effect relationship, then the observation that Z-line degradation is a primary facet of tenderization must be evaluated in a different perspective. This is not to suggest that Z-line degradation does not impart fragility to muscle fibers, but the presence of a population of fibers that apparently do not undergo perceptible Z-line alteration suggests that another, at least equally important, alter- ation exists which could account for tenderization in these fibers. The relationship between Z-line degradation and tenderness in the present study is difficult to assess. The fibers classified as type II were extremely variable in susceptibility to Z-line degradation although all fibers within this group were affected to some degree. The amount of degradation seen for any one sample was dependent on the presence of de- gradation in the particular section of the fiber that was sampled and particularly dependent on the fiber types present in each sample. No fibers were seen in the 48 hr. and 216 hr. samples that were classified as type II fibers that were free of Z-line degradation. If Z-line degradation imparts fragility to the fiber system, then the type I fibers apparently do not become fragile or tenderize by the same mechanism as that observed for the type II fibers. In addition to the degradation and disappearance of the Z-line in 24 hr. postmortem chicken pectoral muscle, Fukazawa t l. (1969) found that the junction of the I-Z bands was weakened and broke during homogenization. 165 Davey and Dickson (1970) and G011 SE El: @970) reported that the weaken- ing of the junction of the I-filaments with the Z-line could account, at least partially, for the loss of tensile strength and rigor resolution. Davey and Dickson (1970) studied the breakdown at the I-Z junction and suggested that the weakness at this point was primarily responsible for the effects of meat aging. Generally, the morphological expression of I-Z weakness is breakage and separation at the junction of these myofibrillar components. However, these same authors found that the inherent weakness imparted by postmortem aging, at the I-2 junction could be present without ultrastructural expression. If, in fact, the observations by Davey and Dickson (1970) are true, then the interpretation of the ultrastructural data in the present study becomes very tenuous and difficult to relate to tenderness. The ultra- structure of 1 hr. and 48 hr. bovine longissimus muscle was variable and no association between morphological changes and tenderness groups could be found. However, at 216 hr. a subtle association was found between the amount of fiber breakage (maximal I-Z splitting) and tenderness. This association showed that the most tender (shear) sample contained more fiber breakage than the toughest sample, Early work by several groups 1., 1942; Paul t 1., 1944; and Ramsbottom and Strandine, (Hanson‘gg 1949) reported a relationship between fiber breakage and shear values. Hanson _£._l' (1942) reported that chicken breast muscle had more and an earlier occurrence of fiber breakage than thigh muscles and generally longer storage periods resulted in more breakage. This latter group also 166 found that breast muscle became tender sooner than thigh muscle when scored by a taste panel. The most tender muscle in the study, the pector- alis secundis, was found to microscopically exhibit postmortem changes earlier than the other muscles studied. Moller _£Mél- (1973) reported that myofibril fragmentation was positively associated with tenderness. This myofibril fragmentation was induced by homogenization and is diffi- cult to compare with unhomogenized muscle samples. The amount of fiber breakage and shear value in the present study did not appear to be related when the differences in shear value were less than that observed for the most tender and toughest sample. However, the observation by Davey and Dickson (1970) that the weakness at the I-Z junction did not necessarily express itself ultrastructurally leads one to question the classification of samples as tough if fiber breaks or I-z breaks are not apparent. Thus, it appears that a muscle sample was tender if a large percentage of the fibers broke during storage. However, the lack of fiber breakage, in the present study, appears to be ineffective as a criteria for classi- fying a sample as tender or tough. A combination of biochemical and ultra- structural data may be necessary to effectively evaluate shear and taste panel differences between samples, particularly if these differences are small. A combination of several biochemical parameters and ultrastructural data and their interrelationship with tenderness will be considered later. 167 Sarcomere Length Sarcomere lengths were measured only on the 48 and 216 hr. longissi- mus muscle samples. The sarcomere length means of the tender and control groups at 48 and 216 hr. postmortem time periods are presented in table 1. Table l. MEAN SARCOMERE LENGTH OF THE LONGISSIMUS MUSCLE OF EACH LINE AND AGING PERIODa Postmortem time Tenderness line Tender Control Overall 48 hr. 2.12 i 0.02 2.13 i 0.03 2.14 i 0.02 216 hr. 2.10 i 0.02 2.09 i 0.03 2.10 i 0.03 aMean sarcomere length is expressed in um : S.E. Sarcomere length of the 48 and 216 hr. samples were similar, however, sarcomere lengths of the 216 hr. samples were slightly although nonsigni- ficantly (P > .05) shorter than those at 48 hours. The treatment means (control and tender groups) are essentially the same within each aging time period with the 48 hr. samples having slightly longer sarcomeres than the 216 hr. samples. These observations are not in agreement with those of Gothard t l. (1966) who found considerable lengthening of bovine longissimus and semimembranosus sarcomeres during postmortem aging. Parrish t l. (1973) reported no change in bovine longissimus muscle sarcomeres after 7 days aging, however, bovine semitendinosus muscle sarcomeres in- creased in length during each postmortem aging interval examined. The lack of a difference between the sarcomere lengths of the control and tender 168 groups was surprising in light of the effect of sarcomere length on ten- derness (Locker, 1960). However, Marsh (1972) reported that shortening of 20 percent or less had little effect on tenderness. If the approximate resting sarcomere length of bovine muscle is considered to be 2.4 pm (Bendall, 1971), then the sarcomeres in the present study have contracted less than the 20 percent reported to be necessary to have an effect on shear values. Some correlation coefficients between sarcomere length and muscle properties are presented in table 2. Sarcomere length of the 48 hr. sam- ples was significantly (P < .01) and negatively correlated (r = -.78) with the amount of KCL extracted myofibrillar N and also with the KCL extracted myofibrillar N to sarcoplasmic N ratio (MF-N:SP-N) (r = -.71, P < .05). The 216 hr.: KCL and KI extracted myofibrillar N were significantly negatively correlated (r = -.62 and r = -.66, respectively) with sarcomere length at 48 hr. but nonsignificantly correlated with sarcomere length at 216 hr. postmortem. Cook (1967) reported that postrigor (48 hr.) bovine sternoman- dibularis myofibrillar protein extraction increased with an increase in sarcomere length. In the present study, sarcomere length of 48 hr. aged samples was significantly correlated (r = 0.74, P < .05) with 1 hr. post- mortem temperature. The sarcomere lengths at 216 hr. were significantly correlated (r = -.64, P < .05) with shear values, but not with taste panel scores (r = 0.22). The shear value correlation concurs with previous observations for the re- lationship with sarcomere length (Locker, 1960), however, the taste panel and sarcomere length correlation disagrees with that reported by Herring t al. (1965a). 169 Ho. V max mo. V ma coaumCHomcmmxoumoa .u: H um msaflmmwwcoq mo chaumuanon oHumu z oHEmmHQOoumm Ou z Hmaawunwmoze wmuomquo 40M .n: mom .uz cam um z wouomuuxw HM n mev .un wq um z wouomuuxo HM u NHMo .u: mam ad 2 wouomuuxm ADM u mquxn .u: wq um z cmuomuuxm Aux n NAUMQ qq.o NN.o xqo.- mN.- mo.- mH.- HN.- s¢.- A.u£ came :uowH ouoEooumm «ea.o Ho.- mm.- «as.. «00.- so.- «mo.- .lma.- A.u; qu suwooa ouoEooumm mmze Hmcmm pmmnm mz-mm eme ume nmqom «Nous mummH uoHNumum "Zumz unocumz .000< UO0000 8000080000 000C00030H mQOHMMm UZHU¢ 92¢ mzHA Wm mMHHH>HHU< mmwuu< mmmcumccmH vofiuom EmuuoEumom mQOHMmm 02H0< oz< mzHA Mm mmHHH>HHO< mm