THE REUNONSHW OF SOME BEGC‘HEMSCAL MD. PHYSIOLOGECAL FACTORS T0 POSTMORTEM CHANGES KN F’ORCiNE MUSCLE Thesis for the Degree of Ph, D. MiCH‘GAN STATE UNNERSHY DUANE ELMER KOCH 19 69 Inert-1’ i’ '1' \'f :x . 35m t, 46 3 Le L, uiwrsfy This is to certify that the thesis entitled THE RELATIONSHIP OF SOME BIOCHEMICAL AND PHYSIOLOGICAL FACTORS TO POSTMORTEM CHANGES IN PORCINE MUSCLE presented by Duane Elmer Koch has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science Minor prof sor Date May 27, 1969 0-169 BINDING IV ROM; & SONS’ 800K BINDERY INC. I llflfilv IIHDFIQ ABSTRACT THE RELATIONSHIP OF SOME BIOCHEMICAL AND PHYSIOLOGICAL FACTORS TO POSTMORTEM CHANGES IN PORCINE MUSCLE by Duane Elmer Koch The results of this study were obtained from.146.market-weight pigs slaughtered in four groups. The distribution of red, white and inter- mediate muscle fibers, succinic dehydrogenase (SDH) activity, myoglobin, total lipid, phospholipid and glyceride ester (neutral lipid fraction) levels were determined on longissimus (LD) muscle samples obtained from the pigs in Group I at or shortly after exsanguination. The relationship of these parameters to 45 min postmortem pH values and to 24 hr postmortem subjective quality scores was observed on normal (slow-glycolyzing) and low quality (fast-glycolyzing) LD muscles from the three breeds (Chester White, Landrace, Poland China) of pigs included in Group I. Heart weights of the pigs in Groups I, III and IV were recorded and subsequently related to rates of postmortem pH decline and/or 24 hr transmission values of the LD muscle. Muscle (LD) or rectal temperatures were obtained on the pigs in Groups II, III and IV at the time of exsanguination and at 45 min post- mnrtem and their relationship to 45 min pH, transmission values and sub- jective quality scores was observed. The effects of sample excision, at or shortly after exsanguination, upon postmortem.pH, transmission values, glycogen, glucose-B-phosphate, lactic acid, ATP and creatine phosphate (CP) levels from.the LD and rectus femoris (RF) muscles of the pigs included in Group IV were studied. In addition, glucose-l-phosphate, fructose-6- phosphate, glucose, ADP and AMP levels were compared among normal and low quality LD and RF muscles (Group IV) that had been incised at several Duane Elmer Koch postmortem.time periods. Transmdssion values and the 2 hr postmortem.pH of the LD, RF, biceps femoris (BF) and supraspinatus (SS) muscles from the pigs in Group IV also were compared. Nbrmal LD muscles had more red and fewer white muscle fibers, higher SDH activities and greater total myoglobin contents than low quality LD muscles. The size of red and intermediate muscle fibers was larger among low quality than normal LD muscles. NOrmal LD muscles tended to have higher total lipid levels and greater neutral lipid ester contents than those of the low quality muscles. Landrace pigs tended to have more myo- globin and higher SDH activities than Peland Chinas or Chester Whites, while Poland China pigs tended to have.more red and fewer white muscle fibers than the other two breeds. Heart weights of the low quality pigs in Group I tended to be lighter than those from normal pigs. However, low and nonsignificant correlations were obtained between heart weights and either 45 min pH (Groups I and IV) or transmdssion values (Groups III and IV). From observations of several pigs with pericarditis, it appeared that heart function.may be more impor- tant than heart weight as a contributory factor in the development of low quality porcine musculature. Muscle (LD) temperatures at 45.min postmortem.were found to be more highly correlated (negatively) with ultimate quality indices than.muscle (Groups III and IV) or rectal (Group II) temperatures at the time of ex- sanguination. The 33:3312 temperatures at the time of exsanguination, as well as the effects of scalding, slaughter floor temperatures and time lapse before carcass chilling appeared to influence postmortem muscle temperatures. Duane Elmer Koch Muscle (LD) incision (Group IV), at or shortly after exsanguination, stimulated contractile activity, significantly increased the rate of post- mortem glycolysis and tended to decrease ultimate muscle qualitative pro- perties. The LD muscles incised at the time of exsanguination had lower pH values, glycogen, ATP and CP levels and higher lactate contents at corresponding time periods, through 2 hr postmortem, than LD muscles not incised until 45 min after exsanguination. This effect of muscle incision was greater among normal than low quality LD muscles. Exposure of the RF muscles (Group IV) to the atmosphere during sample excision and the concomitant chilling effects at early postmortem time periods (0 to 15 min) slowed down glycolytic rates and apparently nullified the influence of contractile activity associated with muscle incision. The RF muscles incised at or shortly after exsanguination exhibited sig- nificantly higher pH values, levels of glycogen and ATP and lower lactate contents at 2 hr postmortem than RF muscles not incised prior to this time. The effects of chilling associated with early postmortem incision of the RF muscles was greater among low quality than normal muscles. Postmortem levels of all the metabolites studied appeared to be related to the glycolytic rate in both the RF and LD muscles. Low, but significant correlation coefficients were observed for trans- mission and 2 hr postmortem pH values of the RF, BF and SS muscles with those of the LD. These data indicate that the postmortem changes occurring in these muscles within a given porcine carcass tended to parallel each other. (”v—fir THE RELATIONSHIP OF SOME BIOCHEMICAL AND PHYSIOLOGICAL FACTORS T0 POSTMORTEM CHANGES IN PORCINE MUSCLE By Duane Elmer Koch A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1969 Eur J (I: FTC-2‘ 1-)")..70 ACKNOWLEDGMENT The author wishes to express his appreciation to his major professor, Dr. R. A. Merkel, for his guidance throughout this research and for assistance in the preparation of this manuscript. Appreciation is also expressed to Drs. L. R. Dugan, Jr., A. M. Pearson, G. D. Riegle and C. H. Suelter, for serving as members of the guidance committee. The author is indebted to the American Meat Institute Foundation for financial support received during his graduate study and for this research project. Special thanks are expressed to all of the Meat Laboratory, Food Science and Animal Husbandry Department personnel who provided fellow- ship, encouragement and assistance during the author's graduate career. The efforts of Mrs. Barbara Purchas, who helped with collection of much data presented in this thesis, cannot remain unmentioned. Lastly, the author is deeply indebted to his wife, Ruth Ann, and children, Tim, Brian, Dawn and Amy for a great deal of time, which was so rightfully theirs. ii INTRODUCTION . . . . . . . REVIEW OF LITERATURE . . . TABLE OF CONTENTS Red and White Muscle Fibers . . . . . . . . Postmortem Mammalian Control of Glycolysis Postmortem Changes in Muscle Changes . . . . Porcine Muscle . . . Factors Influencing the Rate of Postmortem Changes in PorCine mSCJ-e C O O O O O C O O O I O C O O O O O O O O O Pr‘edisposition O O O O O I O O O O O O O O O O O O O O The influence of changes . . . . The influence of EXPERIMENTAL FETHODS . . . Experimental Animals Slaughter . . . . . . Sampling Procedure . Group I . . Group II . . . . Group III . . . Group IV . . . . Subjective Muscle Qua Heart weights 0 o o o wadering of Frozen Muscle and Liver Samples antemortem factors on postmortem factors on lity Appraisal . . . . postmortem muscle changes mscle pH 0 O O O O O O O O O O O O O O O O O O O O O O O 0 (Muscle Moisture Determination . . . . . . . . . . . . . . . iii Page 10 16 16 18 23 25 25 25 26 26 26 26 27 28 29 29 29 30 Transmission Values . . . . . . . . . . . . . . . . . . . . Succinic Dehydrogenase Activity . . . . . . . . . . . . . . Red, White and Intermediate Fibers . . . . . . . . . . . . Myoglobin . . . . . . . . . . . . . . . . . . . . . . . . . Some Metabolites Involved in Glycolysis . . . . . . . . . . Extraction . . . . Fluorometry . . . . . . . . . . . . . . . . . . . . . G-6—P, ATP and CP . . . . . . . . . . . . . . . . . . Glucose, G-l-P and F-6-P . . . . . . . . . . . . . . . Glycogen . . . . . . . . . . . . . . . . . . . . . . . ADP and AMP . . . . . . . . . . . . . . . . . . . . . Lactate . . . . . . . . . . . . . . . . . . . . . . . Lipids O O O O O O O O O O O O O O O O O O O O O O O O O O Eitraction O O O O O O O O O O O O O O O O O O O O O O mosphOIi-p ids O O O I I O O O O O O O O O O O O O O 0 Neutral lipid ester determination . . . . . . . . . . StatisticalAnalySiS0.0000000000000000. RESULTS AND DISCUSSION 0 O O O O O I O O O O O O O O O O O O O 0 Distribution of Muscle Fiber Types, Succinic Dehydrogenase Activity, Myoglobin and Lipid Levels . . . . . . . . . . . Red, white and intermediate fiber distribution . . . . Succinic dehydrogenase activity . . . . . . . . . . . Myoglobin . . . . . . . . . . . . . . . . . . . . . . Lipids . . . . . . . . . . . . . . . . . . . . . . . . Heart weights . O O O O O C . O . O . O O O O O O O O C O 0 Muscle Temperature . . . . . . . . . . . . . . . . . . . . The Effect of 0 hr Sample Excision on Postmortem Muscle Changes 0 O O O O O O O O I O O O O I O O O O O O I I O O O Longissimus muscle . . . . . . . . . . . . . . . . . . Rectus femoris muscle . . . . . . . . . . . . . . . . Page 30 30 31 32 33 33 33 34 35 35 36 36 36 36 37 38 39 4O 4O 42 45 46 48 50 51 55 55 75 A Comparison of Postmortem Differences Between Several Porcine Muscles within the Same Carcass . . . . . . . . . . . . . . iv 89 SWY O I O O O O O O O C O O O O O O I O O C O O O C O -. O BIBLI mm mY O O O O O O O O O O O O O O O O O O O O O O O O O APENDIX O O O O O O O O O O O O Q C O O O O O O O O O O O O O Page 92 96 110 LIST OF TABLES Table Page 1 Postmortem time periods of sample excision for right and left sides of each muscle and the number of pigs included in each time period . . . . . . . . . . . . . 27 2 The mean pH values and subjective scores of the longissi- mus muscle by breed and quality group . . . . . . . . . 41 3 The distribution of red, white and intermediate fibers and succinic dehydrogenase activity in the longissimus muscle by breed and quality group . . . . . . . . . . . 44 4 Myoglobin content of the longissimus muscle by breed and quality group . . . . . . . . . . . . . . . . . . . 47 5 Serum, liver and longissimus muscle lipids by breed and quality group C O O O O O O O O O O O C O O O O O O O O 49 6 Some rectal and longissimus muscle temperatures at several postmortem time periods . . . . . . . . . . . . 54 7 The effect of 0 hr sample excision on postmortem pH decline of the longissimus muscle . . . . . . . . . . . 56 8 Effect of 0 hr sample excision on qualitative properties of the longissimus muscle . . . . . . . . . . . . . . . 58 9 The effect of 0 hr sample excision on postmortem glycogen levels of the longissimus muscle . . . . . . . 59 10 The effect of 0 hr sample excision on postmortem lactate levels of the longissimus.muscle . . . . . . . 61 11 The effect of 0 hr sample excision on postmortem glucose- 6-phosphate levels of the longissimus muscle . . . . . 62 12 The effect of 0 hr sample excision on postmortem ATP levels of the longissimus muscle . . . . . . . . . . . 63 13 The effect of 0 hr sample excision on postmortem creatine phosphate levels of the longissimus muscle . . 64 14 The effect of 0 hr sample excision on certain qualitative assessments for normal and low quality longissimus muscle 66 15 The levels of some glycolytic metabolites in normal and low quality longissimus muscle at 24 hr postmortem . . 73 vi Table Page 16 The effect of 0 hr sample excision on postmortem pH decline of the rectus femoris muscle . . . . . . . . . . 76 17 The effect of 0 hr sample excision on postmortem glycogen levels of the rectus femoris muscle . . . . . 78 18 The effect of 0 hr sample excision on postmortem lactate levels of the rectus femoris muscle . . . . . . . . . . 78 19 The effect of 0 hr sample excision on postmortem glucose- 6-phosphate levels of the rectus femoris muscle . . . . 79 20 The effect of 0 hr sample excision on postmortem ATP levels of the rectus femoris muscle . . . . . . . . . . 8O 21 The effect of 0 hr sample excision on postmortem creatine phosphate levels of the rectus femoris muscle. 80 22 The levels of some glycolytic metabolites in normal and low quality rectus femoris muscle at 24 hr postmortem . 88 23 Transmission values and 2 hr postmortem pH values of several muscles from normal and low quality carcasses . 90 24 Simple correlation coefficients for transmission values and 2 hr postmortem pH values between some muscles . . 91 vii LIST OF FIGURES Figure Page 1 Postmortem pH curves of the longissimus muscle for the pigs in Group I 0 C O O O O O O O O O O O C O O O O O O 41 2 Pestmortem pH pattern of normal and low quality longissi- mus muscle as affected by 0 hr sample excision . . . . 66 3 The effect of 0 hr sample excision on postmortem levels of glycogen, lactic acid, glucose, glucose-l-phosphate, glucose-G-phosphate and fructose-G-phosphate between normal and low quality longissimus muscle . . . . . . . 68 4 The effect of 0 hr sample excision on postmortem levels of ATP,.ADP,.AMP and creatine phosphate between normal and low quality longissimus muscle . . . . . . . . . . . . 71 5 Postmortem pH pattern of normal and low quality rectus femoris muscle as affected by 0 hr sample excision . . 82 6 The effect of 0 hr sample excision on postmortem levels of glycogen, lactic acid, glucose, glucose-l-phosphate, glucose-S-phosphate and fructose-6-phosphate between normal and low quality rectus femoris muscle . . . . . 83 7 The effect of 0 hr sample excision on postmortem levels of ATP, ADP, AMP and creatine phosphate between normal and low quality rectus femoris muscle . . . . . . . . . 85 viii Appendix O'Ijtllb LIST OF APPENDIX TABLES Heart weight, LD pH and subjective quality scores (Group I) I I I I I I I I I I I I I I I I I I I I I Page 110 Muscle fiber types and succinic dehydrogenase activities (Group I) . . . . . . . . . . . . . . . . . . . . . Myoglobin (Group I) . . . . . . . . . . . . . . . . Lipids (Group I) . . . . . . . . . . . . . . . . . . Group II . . . . . . . . . . . . . . . . . . . . . . Group III . . . . . . . . . . . . . . . . . . . . . pH and quality scores of longissimus muscle and heart weight (Group IV) . . . . . . . . . . . . . . pH values of rectus femoris, biceps femoris and supraspinatus (Group IV) . . . . . . . . . . . . . . Moisture and temperature of longissimus and rectus femoris muscles (Group IV) . . . . . . . . . . . . . Transmission values (Group IV) . . . . . . . . . . . Glycogen levels of longissimus and rectus femoris (Group Iv) I I I I I I I I I I I I I I I I I I I I I Lactic acid levels of longissimus and rectus femoris (Group Iv) I I I I I I I I I I I I I I I I I I I I I Glucose-6-phosphate levels of longissimus and rectus femoris (Group IV) . . . . . . . . . . . . . . . . . ATP levels of longissimus and rectus femoris (Group IV) Creatine phosphate levels of longissimus and rectus femoris (Group IV) . . . . . . . . . . . . . . . . . Glucose-l-phosphate, fructose-6-phosphate and glucose levels of longissimus and rectus femoris (Group Iv) I I I I I I I I I I I I I I I I I I I I I ADP and AMP levels of longissimus and rectus femoris (Group Iv) I I I I I I I I I I I I I I I I I I I I I ix 111 112 113 114 116 118 119 120 121 122 123 124 125 126 127 128 INTRODUCTION While per capita meat consumption in the United States has been in- creasing, pork consumption has remained relatively constant during the past two decades and thus it is accounting for less of the tetal. Lack of consistent product uniformity in cutability (lean to fat and bone ratio) or palatability factors has been implicated in the static pork consumption pattern. However, the contribution of pork palatability factors to product acceptability has been poorly documented to date. During the last 10 to 15 years, pale, soft and exudative (PSE) pork has received increasingly more attention. The incidence of the "lower quality" pork is quite variable and certain strains of pigs appear to be more predisposed to PSE development than others. Additionally environ- mental stressors and extremely high ambient temperatures or widely fluc- tuating daily temperatures seem to aggravate the incidence. Some, and possibly all, muscles of PSE pork carcasses are believed to be less palatable (decreased juiciness and tenderness) when cooked; to have greater shrink during processing; to exhibit more color variability in finished processed products; and to have lower emulsifying capacity due to decreased protein solubility than muscles resulting from "normal" carcasses. Research work to date has not conclusively established the factors responsible for the "normal" conversion of porcine muscle to pork, however, rapid postmortem pH drop while muscle temperature is rela- tively high.(:_35°C), results in excessive protein denaturation and this phenomenon appears to be the immediate cause of PSE muscle. -2- If PSE pork is a problem of economic importance, two approaches to its solution are apparent. The more immediate approach is that of reduc- ing (or possibly eliminating) the incidence or severity of PSE muscle through selection toward resistant breeding stock, careful management during production, proper handling prior to and at the time of slaughter and rapid postmortem chilling of the carcasses. The other approach is to elucidate the ante- and/or postmortem factor(s) responsible for the variation in ultimate qualitative properties resulting during the conver- sion of porcine muscle to pork. This study was conducted to provide preliminary information with respect to some of the variables involved in the conversion of porcine muscle to meat by: l. Observing some aerobic and anaerobic characteristics (red, white and intermediate muscle fiber types, succinic dehydrogenase activity and myoglobin components) as well as lipid classes present iii the longissimus muscle at the time of slaughter. 2. Determining what effect heart size and muscle temperature, at the time of slaughter, have on ultimate porcine muscle qualitative properties. 3. Establishing what effect sampling techniques have on postmortem glycolytic rate and ultimate muscle quality. 4. Determining if the qualitative properties of muscles from several different anatomical locations within PSE pork carcasses are similarly affected. REVIEI OF LITERATURE Bendall (1964, 1966a), Lawrie (1966a) and MCLoughlin (1969) stated that variability in meat properties resulted from differences in post- mortem changes occurring within these muscles. They further stated that these differences are a reflection of the intended function of the parti- cular muscle. Lawrie (1966a) indicated that muscles develop and differ- entiate for definite physiological purposes in response to various intrinsic and extrinsic stimuli (genetic, physiological and nutritional). Helander (1966) stated that muscle development (changes taking place in constitution, volume, and structure of skeletal muscle) is affected by age, degree of activity, hormonal state, metabolic state and pathological conditions. The transverse elements of the sarcoplasmic reticulum conduct nerve impulses via Na/K depolarization from.the sarcolemma to the triads of the longitudinal tubules (lace like network enveloping fibrils) of the sarco- plasmic reticulum (Copenhaver, 1964; Bendall, 1966a;Lawrie, 1966a). This depolarization wave releases Ca++ which in turn diffuse from the longitu- dinal tubules. The Ca++ releases ATP from its inert complex with Mg++ and activates myosin ATPase. The subsequent splitting of ATP provides the energy necessary for interaction of actin and myosin filaments result- ing in muscle contraction. Immediately the Ca++ pump in the longitudinal tubules recaptures the Ca++ and contractile ATPase activity ceases. The ATP-Mg complex, which acts as a plasticizer in the resting state, again reforms . -4— Bendall (1966e) and Lawrie (1966a) reported that ATP is the immediate energy source in contraction. Bendall (19669 stated that ATP is required to operate the Na/K pump in the sarcolemma and transverse tubules of the sarcoplasmic reticulum. Bendall (196a) and Marsh (1966) reported that ATP is also required to operate the Ca++ pump in the longitudinal tubules of the sarcoplasmic reticulum. Bendall (1963) and Henneman and Olson (1965) observed the presence of a muscle mitochondrial ATPase. White gt a_l. (1964), Bendall (1966a) and Lawrie (1966a) reported that ATP and its immediate source of replenishment, creatine phosphate, can be maintained in slowly working muscle by oxidative phosphorylation. They further indicated that in rapidly contracting muscle, the oxygen supply becomes insufficient and ATP must be synthesized anaerobically via the glycolytic process. Red and White Muscle Fibers Needham (1926) broadly classified muscles as either red or white. Lawrie (1966a) indicated that this superficial differentiation reflected both histological and biochemical differences. The "redness" or "white- ness" of a muscle has been shown to be due to the varying proportion of red and white muscle fibers (Dubowitz and Pearse, 1961; Blanchaer gt‘gl., 1963; VanWijke 93 Q” 1963; Brooke, 1966). These authors reported that red fibers stained intensely when histochemically assayed for enzymes involved in aerobic metabolism; whereas, white fibers showed positive staining reactions to histochemical assays for the enzymes associated with anaerobic metabolism. With the same histochemical techniques, Ogata and Mori (1963), Dawson and Romanul (1964), Romanul (1964) and Beatty 33 21. (1966) reported a variable number of fibers with interme- diate staining reactions. George and Berger (1966) reported that red fibers are smaller in diameter, have higher myoglobin levels and greater concentrations of mitochondria and oxidative enzymes than white fibers. On the other hand, white fibers have higher glycolytic enzyme activities, 2.2. a greater capacity for anaerobic metabolism. Red fibers display a slow and sustained contractile activity as opposed to the fast but shorter duration con- tractile response of white fibers. Red fibers usually have higher lipid and lower glycogen contents than white fibers. Dawson and Romanul (1964) and Peachey (1968) indicated that the above classification is general and exceptions exist. George and Berger (1966) and Carrow st 31. (1967) reported that red muscle fibers are supplied with a greater number of capillaries than white fibers. Numerous studies have shown that muscle fiber characteristics are neurally controlled (Buller and Lewis, 1965; Henneman and Olson, 1965; Romanul and Van Der Meulen, 1967; Yellin, 1967 ; Guth, 1968; Karpati and Engel, 1968). Muscle fiber properties have been shown to be altered as a result of cross—innervating red and white muscles. Cross-innervated muscles reversed their speed of contraction and their enzymatic profiles (Close, 1965; Romanul and Van Der Meulen, 1967; Robbins _e_t_ g” 1969). Exercise has also been shown to have an effect on muscle fiber type (Carrow gt'gl., 1967; Holloszy, 1967; Edgerton 23 21., 1968; Peter gt 21., 1968). Carrow 23 31. (1967) also showed an increase of intramuscular capillary density with exercise. -6- While Briskey (1967) stated that a biochemical property of myosin is to catalyze ATP hydrolysis, Seidel and Gergely (1963), Sreter 33.31. (1966), Seidel g; 21. (1964) and Baraxy g: 21. (1965a) showed that myosin from.red muscle has a lower ATPase activity than that from.white muscle. Perry and Hartshorne (1963), Barany'gilg1. (1965b) and Trayer and Perry (1966) reported that myosin.ATPase activity increased progressively with muscle development and age. Buller £1 21. (1960) and Close (1964) re- ported that muscles of prenatal or neonatal.mammals were all physiologi- cally red. Thus Briskey (1967) indicated that the increase in myosin ATPase activity with postnatal development probably reflected the differ- entiation of red fibers to white fibers. Furthermore, Barany e: 21. (1965b) reported a direct relationship between speed of muscle contrac- tion and myosin ATPase activity. Postmortem.Mamma1ian Muscle Changes Lawrie (1966a) reported that the immediate result of blood removal at the time of slaughter is the depletion of oxygen supply and loss of all neural and hormonal control over metabolic processes. He further indicated that even though the muscle may not be actively contracting at this time, energy was required for maintenance of homeostasis. The latter author also stated that the rate and extent to which ATP becomes depleted readily affects the rate and extent of postmortem change, 1.3. rigor mortis and glycolysis. It is generally believed that all postmortem.ATP breakdown is accomp plished by sarCOplasmic ATPase activity (Bendall, 1960; Lawrie, 1966a). However, in certain instances, especially when rate of postmortem change is greatly accelerated and ATP is depleted at an abnormally high rate, 3.3. thaw rigor, cold shortening and rapid rigor mortis development as observed in some "degenerated" muscle, the myofibrillar ATPase system also become operative (Bendall, 1960; Marsh, 1966; Newbold, 1966). Since exsanguination has inhibited most or all respiration, ATP must be replenished by anaerobic processes (Lawrie, 1966a). Bendall (1960), Newbold (1966) and Lawrie (1966a) reported that ATP is preferentially synthesized by transfer of high energy phosphate from creatine phosphate to ADP. They stated that when the supply of creatine phosphate becomes limited, anaerobic glycolysis becomes operative in order to maintain ATP levels. The rate and extent of glycolysis is dependent upon glycogen availability as well as the ATP and creatine phosphate levels at the time of exsanguination. When ATP is diminished.more rapidly than resynthesized by the above reactions, myokinase catalyzes conversion of two moles of ADP to one mole each of ATP and AMP. Measurement of postmortem pH is frequently used as an indirect estimate of the extent of glycolysis. Rigor mortis onset is dependent upon ATP disappearance from the muscle (Bendall, 1960; Lawrie, 1966a; Newbold, 1966; Davies, 1967). These same authors indicated that rigor mortis development is character- ized by the loss of muscle extensibility caused by the transformation of the freely sliding actin and myosin filaments to the rigid actomyosin complex. Cassens (1966) and Davies (1967) stated that shortening may or may not occur during rigor. Bendall (1960), Lawrie (1966a) and Newbold (1966) reported that ATP complexed with Mg++ acts as a plasticizer for actin and myosin. Davies (1967) reported that ADP has a similar effect. Bendall (1960) observed that at lower pH values, lower ATP levels are needed before onset of rigor commences. Lawrie (1966a) reported that the rate and extent of postmortem changes are a reflection of muscle function which in turn are influenced by species, breed, sex, age, anatomical location of the muscle, exercise and plane of nutrition. Lawrie (1966a, 1966b) also reported that metabolic stresses, 2.3., activity, ambient temperature, humidity, atmospheric pressure, oxygen tension, feed, injury, pathological and psychological factors during or just preceding slaughter exert an influence on postmortem change. The rate at which postmortem changes occur increases with increasing temperature, especially in the range of 5° to 43°C (Bendall, 1960; Lawrie, 1966a; Newbold, 1966). Bendall (1964), Briskey (1964), Lawrie (1966a), and McLoughlin (1969) reported that the rate and extent of postmortem changes affect the use of muscle as a food. A rapid glycolytic rate and an abnormally low ultimate pH usually result in a paler muscle color than that normally encountered (Cassens, 1966; Lawrie, 1966a); whereas, an exceptionally high ultimate pH gives rise to an unusually dark color (Lawrie, 1966a). Water binding capacity is minimal at the isoelectric point which ranges from pH 5.1 to 5.5 for muscle proteins (Hamm, 1960). Bendall (1964) and Lawrie (1966a) reported that this point is at or near the normal ultimate pH of most muscles. Lawrie (1966a) found that even at high ultimate pH values there was a diminution of water binding capacity attributable to ATP disappearance and the consequential actomyosin for- .mation. Lawrie (1966a), Bendall (1964) and Briskey (1964) indicated greater than normal loss of water holding capacity due to excessively low ultimate pH values or to very rapid postmortem pH declines, especially when these conditions were achieved at or above muscle temperatures of 35°C. They attributed this loss of water holding capacity to muscle pro- tein denaturation, especially inplicating the sarcoplasmic fraction, and to loss of semipermeability of the sarcolemma. Control of Glycolysis Since rate of postmortem glycolysis probably plays a major role in ultimate meat quality, a brief discussion of glycolytic control will be presented. White g: 31. (1964) and Conn and Stumpf (1966) indicated that the rate of each enzyme-catalyzed reaction is related to the concentrap tion of active enzyme; the availability of appropriate substrates, coenzymes and cofactors; the presence of activators or inhibitors; and the tempera- ture and pH conditions. Scrutton and Utter (1968) stated that availability of substrate or regulation of catalytic activities of rate limiting enzymes, or both, are probably the major factors contributing to regula- tion of glycolytic flux. Under normal conditions, two enzymes, 1.2., phosphorylase and to a greater extent phosphofructokinase are almost always implicated as "the" rate limiting reactions of glycolysis (Lowry 21.21., 1964; Regen gt‘g1., 1964; Helmreich and Cori, 1965; Uyeda and Racker, 1965; Williamson, 1965; Wood, 1966; Scrutton and Utter, 1968). Randle (1964) indicated that glycogen degradation was controlled by phosphorylase; whereas, subsequent steps in the glycolytic pathway were controlled by the phosphofructokin- ase reaction. Wood (1966) reported that stimulation of phosphorylase activity did not necessarily increase phosphofructokinase reaction rates. Karpatkin.31‘g1. (1964) and Ozand and Narahara (1964) found that increased reaction rates of phosphorylase stimulated by epinephrine did not necess- arily activate phosphofructokinase; whereas, muscle contraction elicited by electrical stimulation increased the rate of both reactions. -10- Atkinson (1966) and Wood (1966) reported that phosphorylase b can be converted to the active phosphorylase g by phosphorylase’kinaséhin the presence of cyclic 3', 5'qAMP, Mg++,.ATP, and Ca++. Atkinson (1966) and Scrutton and Utter (1968) noted that conversion of phosphorylase b to the g form.was not absolutely necessary for muscle contraction or increased glycolytic rate. WOod (1966) reported that phosphorylase b is activated by-AMP. Atkinson (1966) confirmed this observation and added that ATP and glucose-S-phosphate inhibited the conversion of phosphorylase b'to the 2 form. Phosphofructokinase is inhibited by.ATP and citrate (Wood, 1966; and Scrutton and Utter, 1968); whereas, activation of phosphofructokinase is accomplished by.AMP,.ADP, Pi, fructose-6-phosphate and fructose-1,6- diphosphate (Mansour, 1965; Wood, 1966; and Scrutton and Utter, 1968). - Randle (1964) observed that anoxia and inhibition of oxidative phosphorylation increased glycolysis by increasing both phosphorylase and phosphofructokinase activity. He further indicated that facilitation of oxidative phosphorylation decreased glycolytic rate. In support of the above work, Lowry.et.al, (1964), Ramaiah g1 31. (1964) and Atkinson (1966) observed that an increased ATP to ADP, AMP, Pi ratio decreased glycolysis, while a decrease in this ratio increased glycolysis. Postmortem Changes in Porcine Muscle In the homeostatic state as well as immediately postmortem all por- cine.muscles are moderately dark in color, firm in texture and dry in appearance (Briskey, 1963, 1964; McLoughlin and Goldspink, 19639. Anaero- bic conditions develop rapidly following exsanguination and the rate and -11- extent of the resultant biochemical and physiological changes are largely responsible for the variation in ultimate meat characteristics (Briskey, 1963, 1964; Briskey g_t_ 31., 1966). Forrest g 31. (1963) noted that the ultimate gross morphology of porcine muscle ranged from excessively dark, firm and dry musculature to extremely pale, soft and exudative musculature. It has been shown that the pH pattern (Briskey, 1963, 1964) and/or the pH and temperature relationships in the muscle at the onset of rigor mortis are highly associated with the ultimate muscle classification (Sayre and Briskey, 1963; Briskey, 1964). Briskey (1963, 1964) described the different pH patterns which are apparent in postmortem porcine muscle. If little postmortem.glycolytic activity occurs and rigor mortis develops at high pH values or if glycoly- sis is extremely slow and rigor mortis occurs over a long time period, the muscle remains dark red in color, firm in texture and dry in appear- ance (Briskey 31‘21., 1959a, b, c, 1962; Sayre and Briskey, 1963; Kasten- schmidt g1“31., 1964). The ultimate pH of these muscles remained at or near 6.0 to 6.5. 'When postmortem glycolysis and rigor mortis occurs under normal conditions, 1.3. at a moderate rate (4 to 6 hr for completion of rigor mortis and 6 to 12 hr to achieve an ultimate pH of 5.3 to 5.6), the muscles exhibit a grayish pink to red color (normal) and are moderately firm.in structure and dry in appearance (Wismer-Pedersen and Briskey, 1961a; Briskey and Wismer-Pedersen, 1961a; Briskey'gi 21., 1962; Sayre and Briskey, 1963). Fast onset of rigor mortis and an extremely rapid rate of glycolysis with the development of low pH values (< 5.6), at temperatures above 35°C, are associated with production of pale, soft -12.. and exudative (PSE) muscles (Briskey'gi 31., 1959b; Briskey and'Wismer- Pedersen, 1961a; Briskey 31 31., 1962; Bendall and Wismer-Pedersen, 1962; Bendall 31,31., 1963; Briskey, 1963; Sayre and Briskey, 1963; Kastenschmidt 21 3.1. , 1964). ‘Wismer-Pedersen (1959) and Briskey and Wismer-Pedersen (1961a, b) observed the development of PSE musculature with normal pH patterns. Likewise, Sayre 31 31. (1964) reported dark, soft and exudative porcine muscles resulting from rapid postmortem glycolytic rates. Briskey (1963) and Sayre 31 31. (1963b) also noted a loss of normal intermuscular binding as a result of the rapid glycolytic rate. Bendall and Wismer-Pedersen (1962) and Cassens 31,31. (1963a, b) noted no histo- logical abnormalities at the time of death for muscles which ultimately become PSE. Abnormally low ultimate pH values (Lawrie et_31,, 1958) and conditions characteristic of PSE musculature, such as "muscle degeneration" (Ludvigsen, 1955», "la myopathie exudative depigmentaire du porc" (Henry 31‘31., 1955) and "white muscle disease" (Lawrie, 1960) have been reported from.some European countries. Rapid postmortem.glycolytic rates are associated with rapid decreases of ATP and creatine phosphate (Briskey and Wismer-Pedersen, 1961a; Bendall and'Wismer-Pedersen, 1962; Bendall 31 31., 1963; Briskey and Lister, 1968). Bendall 31 31. (1963) reported that rigor mortis onset occurs when ATP is at 30% of the initial level. Kastenschmidt e_t a_1_. (1966, 1968) reported that muscles exhibiting fast glycolytic rates had-lower ATP’and creatine phosphate levels at the time oftexsanguination than .muscles which underwent slow glycolysis. They indicated that the fast -13- glycolyzing muscles.may be in a highly anaerobic state at the time of exsanguination. Briskey and Lister (1968) stated that lactic acid con- tent at the time of exsanguination was directly related to the rate of postmortem lactic acid accumulation. Although low ATP levels are asso- ciated with rapid glycolysis, it is not known whether low postmortem ATP levels result from rapid hydrolysis or inefficient resynthesis (Kasten- schmidt 31.31., 1968). Briskey (1964) and Cassens (1966) reported that a review of the literature showed no consistent histological observations which were associated with postmortem.muscle properties. They stated that some of the changes observed.may have resulted from.different states of fiber contracture rather than from.varying rates of postmortem pH decline. Nevertheless, Cassens 31 31. (1963a, b) noted greater sarcoplasmic dis- ruption and decreased myofibrillar preservation as a result of rapid postmortem.changes. Sayre and Briskey (1963) and MCLoughlin (1968) reported no differ- ences in sarcoplasmic and myofibrillar protein extractabilities at the time of death, which were attributable to subsequent rates of postmortem reactions. IPostmortem.extractability of sarcoplasmic proteins was in- versely related to rate of postmortem changes, especially glycolysis (Wismer-Pedersen and Briskey, 1961a; Bendall and Wismer-Pedersen, 1962; TMCLoughlin and Goldspink, 1963a; Scopes and Lawrie, 1963; McLaughlin, 1963, 1968; Sayre and Briskey, 1963; Briskey and Sayre, 1964; Topel 31 '31., l967).~ The decreased extractability was attributed to denaturation. Decreased myofibrillar and sarc0plasmic protein extractability occurs 'when pH decline is rapid while postmortem.muscle temperature is still high -14- [> 35°C] (Sayre and Briskey, 1963; McLoughlin and Goldspink,l963a, b; Briskey and Sayre, 1964; Bendall, 1964). Bendall and Wismer-Pedersen (1962) attributed the differences in myofibrillar protein extractability resulting from.rapid postmortem pH fall to denatured sarcoplasmic protein precipitating onto the myofibrils rather than to myofibrillar protein alterations 333‘33. However, Hart (19623, Sayre and Briskey (1963) and MbLoughlin (1963) noted a decrease in myofibrillar protein solubility above that attributed to sarcoplasmic protein precipitation. In addition to the previously discussed effects of ultimate pH (Hamm, 1960) upon water holding capacity, decreased water retention is associated with rapid postmortem muscle changes as well as the degree of actomyosin formation (loss of binding sites) during rigor mortis development (Bendall and Wismer-Pedersen, 1962; Sayre 3t 3., 1964; Bodwell 91 31., 1966). Forrest 91 31. (1966) demonstrated that stimulation by electric shock shortly after slaughter resulted in stronger contractility for a longer duration among those muscles which subsequently underwent slow postmortem.changes than for muscles which underwent rapid postmortem changes. Briskey and Wismer-Pedersen (1961b) and Sayre 31 31. (1963b, c, d) showed that glycogen content 333,33 had no effect on rate of postmortem change provided sufficient glycogen was present to attain normal ultimate pH values. Sayre 31,31. (1963a) and Kjolberg 31‘31. (1963) reported that glycogen structure had little or no effect on rate of postmortem glycolysis. Bendall 31 g. (1963) and Sayre £32 31. (1963b, 0) found no consistent association between muscle buffering capacity and rate of postmortem glycolysis. -15.. Total protein, lipid and moisture contents of the muscle do not appear to be related to rate of postmortem change (Briskey 31 31., 19590; Wismer-Pedersen, 1959; Lawrie, 1960; Wismer-Pedersen and Briskey, 1961a, b; Dahl, 1962). While Sink 31 31. (1967) found no relationship between chloroformsmethanol or ether extractable lipids and rate of postmortem change, Krzywicki and Ratcliff (1967) found that phospholipid content was directly related to rate of postmortem.pH decline. Following a review of the literature, Briskey (1964) stated that rapid postmortem changes in muscle resulted in lower ultimate myoglobin contents. He emphasized that the total quantity as well as the chemical and physical state of myoglobin as affected by postmortem changes in por- cine muscle required further study. Briskey 31.31. (1959C) found no relationship between muscle potassium content and rate of postmortem changes. However, they noted higher sodium contents among muscles which underwent rapid glycolysis. Topel.31‘31. (1967) found that neither muscle nor plasma sodium and potassium levels were associated with rate of pH decline. Cassens g a_1. (1963c, d) found no differences in zinc content of muscles exhibiting fast or slow glycolysis. Sayre 31 31. (1963c) found that when muscle extracts were obtained lO.min postmortem phosphorylase was in the 3 form. These authors as well as'WismerrPedersen (1959), Kjolberg 31,31. (1963) and Aberle and Merkel (1968b) could not relate total phosphorylase activity to postmortem glycolytic rate. Sayre 31 31. (1963b, d) found no apparent association between phosphofructokinase activity and rate of postmortem glycolysis. Kastenschmidt 31 31. (1966, 1968) studied the concentrations of glycolytic -16- intermediates in porcine muscle and they implicated phosphorylase, pyruvic kinase and especially phosphofructokinase as the rate limiting steps in postmortem glycolysis. Aberle and Merkel (1968a) reported no relationship between adenylic acid deaminase activity and rate of postmortem changes. Factors Influencing the Rate of Postmortem Changes in Porcine Muscle Predisposition. Lawrie and Gatherum (1962) and Bendall 91 _a_1. (1963) indicated that muscles from.Large White pigs normally exhibit slower rates of postmortem glycolysis than those of the Danish Landrace breed. Clausen and Thomsen (1960) and Ludvigsen (1960) reported a higher incidence of PSE muscles from.Pietrain than for Landrace pigs although both breeds have a rather high incidence. Bray (1968) in summarizing the work of Judge 31,31.' (1959), Sayre e_t 31. (l963d), and Allen 31 31. (1966) noted that in this country PSE pork is more prevalent among Poland China, Hampshire and Landrace pigs than in the Chester White or Duroc breeds. The latter author pointed out that ultimate qualitative properties might be more accurately ascribed as being due to strain rather than breed differences since the PSE condition occurs among all breeds, but some breeds or strains are more predisposed than others. Lasley (1968) and Christian (1968) stated that selection of pigs for increased lean yield may have resulted in unintentional selection toward inferior muscle quality. While the work of Omtvedt (1968) concurs with the latter observation, he reported that the genetic correlation between lean yield and qualitative -17- factors was low. He also stated that although additional investigations are needed, present heritability estimates for muscle qualitative factors are sufficiently high to be useful in the selection of breeding stock. Briskey (1964) stated that adjacent porcine muscles frequently ex— hibit pronounced variation in color, gross morphology and general appear- ance. Lawrie and Pbmeroy (1963) reported that considerable variability in sodium, potassium and myoglobin contents exists between muscles. Briskey 31 31. (1960tfl indicated that muscles which exhibited relatively high ultimate pH values showed lower levels of initial glycogen, greater juice retention and higher myoglobin contents than muscles with lower pH values. Lawrie 31 31. (1963) indicated that variation in moisture content between muscles was directly related to ultimate pH values. Lawrie 31 31. (1958) and Wismer-Pedersen (1959) noted that longissimus dorsi and semimembranosus muscles were susceptible to rapid postmortem changes. Briskey and Wismer-Pedersen (1961a) also implicated the suscepti- bility of the longissimus dorsi and biceps femoris muscles. Briskey 31 31. (1960b) found that the visual appearance of the gluteus medius was a good indicator of the water binding properties of the longissimus dorsi and biceps femoris muscles. Briskey (1964) stated that the number and location of muscles exhibiting the PSE condition varies considerably within carcasses. This observation may be related to differences in relative cooling rates, myoglobin content or oxygen storage capacity of the muscles. The latter author stated that light colored, inactive, tetanic muscles are more predisposed to development of rapid postmortem changes than dark colored, active, tonic muscles. -18- Lawrie 31 31. (1958) reported a wide range in ultimate pH values between various locations within the semimembranosus muscle. Lawrie (1960) observed lower ultimate pH values and concomitant greater degrees of exudation in the lumbar region of the longissimus dorsi muscle than in the thoracic region. Briskey (1964) reported greater severity of the PSE condition in the caudal and cranial regions of the longissimus dorsi muscle than in the mud section. Beecher 31 31. (1965b) found no relationship between initial or ulti- mate glycogen and pH values with percent red fibers, myoglobin content or succinic dehydrogenase activities for muscles of varying degrees of "red- ness". However, they noted that ultimate lactic acid content tended to be higher in "white" muscles. Beecher 31 31. (1965b) reported that rigor mortis developed earlier in the light portions of the biceps femoris and semitendinosus muscles than in the dark portions. Beecher 31 31. (1965a) further indicated that the light portion of the semitendinosus muscle exhibited faster glycolytic rates following exsanguination than the dark portion. Beecher 31 31. (1968) found that myoglobin content, percentage of red fibers and succinic dehydrogenase activity in the dark portion of the semitendinosus was approximately twice the corresponding levels in the white portion of the muscle. They also noted less moisture and sodium, but more lipid in the white portion, while calcium, potassium and magnesium contents were similar for the two sections of the muscle. The influence of antemortem factors on postmortem changes Thyroid (Ludvigsen, 1953) and adrenal (Ludvigsen, 1957) insufficiencies have been implicated in the susceptibility to rapid postmortem changes. -19- Ludvigsen (1960) postulated that in muscular pigs the increased muscle development was probably due to increased pituitary growth hormone pro- duction which would suppress thyrotropic and adrenocorticotropic activi- ties. Wismer-Pedersen (1968) concurred with this postulation and further stated that the more muscular pigs are more predisposed to PSE develop- ment. Ludvigsen (1953) reported that he increased the incidence of PSE pigs by feeding methylthiouracil 10 to 14 days prior to slaughter. Topel and Merkel (1966, 1967) followed the same experimental design and were unable to substantiate these findings. Hedrick 31.31. (1963) and Ramsey 31'31. (1964) reported an improve- ment of porcine muscle color and increased ultimate pH by adrenaline injection 24 hr prior to slaughter; however, Aberle and Merkel (1968b) could not change ultimate qualitative characteristics by injecting epine- phrine 10 min before exsanguination. Topel 31 31. (1967) reported that decreased levels of plasma l7- hydroxycorticosteroids were associated with more rapid muscle postmortem pH declines. However, induced suppression of plasma l7-hydroxycorticos- teroid levels showed no relationship to the rate of postmortem changes (Topel and Merkel, 1967; Aberle and Merkel, 1968b). Judge 21 31. (1966, 1968a, b) noted that thyroid and adrenocortical insufficiencies were related to development of PSE muscle. Cassens 31 39? (1965) reported that adrenal glands displaying large lipid masses, ‘which they interpreted as indicative of degenerative changes, were directly associated with rate of postmortem.changes. Howe 31 31. (1969) -20- produced similar adrenocortical alterations by subjecting stress susceptible pigs to variable combinations of temperature and humidity environments. Forrest e_t_ _a1. (1963) noted that the incidence of PSE paralleled the daily environmental temperature fluctuations and the incidence was highest when fluctuations were greateSt. Sayre 31 31. (1961) decreased initial glycogen and ultimate lactic acid levels by subjecting pigs to an ice water bath prior to slaughter. Heat treatment (45°C) for 30 to 60 min prior to slaughter greatly accelerated postmortem glycolytic rates (Kastenschmidt g1 31., 1964, 1965). Sayre 21 31. (1963b) produced a similar response to heat treatment in Hampshire and Poland China pigs, but not among Chester Whites, even though the internal muscle temperature of all breeds was above 41°C. Kastenschmidt 31'31. (1964, 1965) greatly reduced the rate of postmortem pH decline by subjecting pigs to heat treatment followed immediately by cold treatment. Improvement in ulti- mate muscle properties was noted by this combination of preslaughter treatments even when internal muscle temperatures were not decreased (Kastenschmidt 31,31., 1965). Ultimate muscle quality was adversely affected when growing pigs were subjected to alternating temperatures (Thomas 31,31., 1966; Addis g1 31., 1967a, b; Howe g 31., 1968; Judge, 1968). This effect was most noticeable at moderate (40%) as opposed to low (17 to 30%) or high (85%) relative humidities. High relative humidity in combination with warm environmental temperatures had no detrimental effect on ultimate muscle quality. The latter authors indicated that it was possible to acclimate pigs to the heat stress. They ascribed this response to increased aerobic metabolic capacity. -21- Forrest 31 g. (1965) reported that greatly elevated heart and re- spiration rates, immediately prior to slaughter were associated with rapid rates of postmortem pH decline. They further noted that both heart and respiration rates tended to increase as muscle temperature increased. Forrest (1965) and Forrest e_t 31. (1968) concluded that circulatory and respiratory difficulties leading to increased blood PCOZ and decreased sz and pH could be major contributors to production of PSE muscle, particularly when animals are subjected to warm temperatures immediately preslaughter. These authors observed that with the strains of pigs studied, the Chester Whites were able to maintain homeostatic conditions under heat stress to a greater extent than the Poland Chinas and thus the Chester Whites were more resistant to development of rapid postmortem muscle changes. Engelhardt (1966) reported that domestic pigs have a smaller heart weight per unit of body weight when compared to wild pigs or other domestic animals. He indicated that this gave an unfavorable relationship between cardiac capability and body need. Briskey and Lister (1968) reported that the development of anoxia associated with the death struggle and exsanguination process accounts for most of the muscle lactic acid content at slaughter. Meyer 31 31. (1962) reported that hogs with high glucose tolerance levels tended to have increased muscle glycogen levels and faster rates of postmortem glycolysis. Wismer-Pedersen (1968) concluded that the occurrence of PSE musculature does not appear to be related with any known nutritional deficiency. -22- Briskey'31 31. (1959a, b, 1960a) reported that exhaustive exercise immediately preslaughter decreased initial glycogen levels and increased ultimate pH values. Lewis 91 31. (1959, 1961) improved ultimate muscle color as a result of periodic preslaughter electric shock. Sayre 31'31. (1963c) decreased initial glycogen levels and rate of postmortem.changes by fasting for 70 hr prior to slaughter. Judge 31 31. (1967) increased rate and extent of postmortem glycolysis by physically restraining hogs prior to slaughter. Sayre 31 31. (1963c) increased the rate of postmortem glycolysis by short term exercise and excitement immediately prior to slaughter. Wismer-Pedersen (1959) stated that fright and shock rather than mere exercise were responsible for rapid postmortem.pH fall. Wismer- Pedersen (1968) stated that exercising pigs (long term) during the feeding period might improve ultimate muscle characteristics but only if the ex- ercise was relatively extensive. Briskey (1963) and Lawrie (1966a) indicated that there appears to be little or no effect of stunning procedure upon rate or extent of post- mortem.changes as long as the medulla oblongata in the brain was not destroyed. Wismer-Pedersen and Rieman (1960) reported an increase in the incidence of low pH immediately after exsanguination when the medulla oblongata was cut. McLoughlin (1964) reported that stunning with a cap- tive bolt pistol before exsanguination resulted in more rapid postmortem pH declines than when no stunning was performed. He also indicated that electrical stunning caused a more rapid pH decline in the muscle post- mortem than the carbon dioxide immobilization method. Both methods pro- duced more rapid pH falls postmortem than when stunning was not performed before exsanguination. -23- Briskeyu41963)‘ indicated that a slow rate of bleeding or retention of blood increased the rate of postmortem glycolysis. Wismer-Pedersen and Rieman (1960) reported increased rate of pH decline as the time be- tween exsanguination and evisceration increased. Sayre g 31. (1966) reported that muscles Of 5 to 10 kg pigs contained more glycogen and less myoglobin and total nitrogen than similar muscles from.250 to 300 kg pigs. They noted that muscles from.more.mature pigs tended to have faster postmortem.g1ycolytic rates, with increased color loss and decreased protein solubility than muscles from younger pigs. The influence of postmortem factors on muscle changes. Briskey (1963) reported that removal of the skin from.pork carcasses as opposed to conventional slaughter procedures Giehairing) facilitated faster chilling rates, which subsequently resulted in slower postmortem pH drops and darker colored musculature. Subjection of excised muscles or intact carcass sides to elevated temperatures (37°C) immediately post- mortem, accelerated rate of pH drop (Wismer-Pedersen and Briskey, 1961a; Bendall and Wismer-Pedersen, 1962; Briskey, 1964; Beecher 31.31., 1965a; Bodwell 31 31., 1966). On the other hand, partial freezing of loinscn‘ hams in liquid nitrogen immediately postmortem was effective in improving ultimate meat qualitative properties (Borchert and Briskey, 1964). Additionally,Borchert and Briskey (1965) reported higher sarcoplasmic and myofibrillar protein extractabilities and improved emulsifying pro- perties when the muscles were rapidly chilled or partially frozen (liquid nitrogen). Lewis 31‘31. (1967) observed a decrease in muscle quality due to conventional freezing (-30°C) immediately after slaughter. -24- Direct electrical stimulation of excised muscles greatly accelerates postmortem glycolytic rate (Hallund and Bendall, 1965; Bendall, 1966b; Forrest and Briskey, 1967; McLoughlin, 1969). Forrest and Briskey (1967) reported accelerated postmortem changes after electrically stimulating the spinal cord of the split carcass immediately postevisceration. MCLoughlin (1969) noted that contraction of muscle postmortem.markedly increased glycolytic rate. EXPERIMENTAL METHODS Experimental Animals Pigs, ranging in weight from 148 to 281 lb and originating from different management regimes, were slaughtered in four separate groups. Group I included 18 Poland China, 16 Landrace and 6 Chester White pigs, which were obtained from three Michigan hog producers. Group II included 40 pigs which were slaughtered for the 1968 Michigan Spring Barrow Show. These pigs represent 34 different Michigan hog producers. Group III included 44 pigs of various breeding, originating from either the ‘Michigan Swine Testing Station or the Michigan State University swine herd. Group IV included 22 Ybrkshire pigs which were obtained from the University swine herd. Slaughter The pigs in Groups II and III were slaughtered at the University Meat Laboratory in accordance with conventional procedures, 133., they were electrically stunned, bled, scalded, dehaired, eviscerated and split into halves prior to washing and placing in a 3 to 4°C cooler. Animals in Groups I and IV were shackled and bled without stunning. Be- cause of the muscle sampling procedure, these carcasses were not scalded but skinned, eviscerated and left unsplit, then washed and placed in 3 to 4°C coolers. -25- -26- Sampling Procedure Group I. Samples of the right longissimus (LD) muscle were excised from the 4th to 5th lumbar region at the time of exsanguination (0 hr). Subsequent LD samples were excised cranially from the initial sample site at 15.min, 45 min and 3 hr postmortem. A 24 hr postmortem sample was excised from the left LD in the 10th rib region. Histochemical samples were excised from the approximate geometric center of the right LD at 30 min postmortem. All muscle samples were frozen in liquid nitrogen imme- diately after excision. Blood samples were collected at the time of exsanguination, allowed to clot, centrifuged and the resultant serum frozen and stored at -20°C. Within 5.min postexsanguination, a liver sample was excised and frozen in liquid nitrogen. Group II. Rectal temperatures were recorded for the pigs in this group at the time of exsanguination. At 45 min postmortem, the right LD temperature of each pig was recorded and a sample was then excised from the right LD (10th rib region) for surface pH determination. Group III. Muscle temperature was obtained at the time of exsan- guination by inserting a thermometer directly into the right LD. LD temperature was also obtained on 20 of the carcasses in this group immediately after dehairing. At 45 min postmortem, LD temperature was recorded for all 44 Carcasses in this group and a sample (right side) 'from the 11th rib region of the LD muscle was then excised for surface pH measurement. Transmission values (measure of water extractable pro- teins) were determined on 24 hr muscle samples excised from the 9th rib region of the LD (right side). Group IV. Samples from both the right and left LD and rectus femoris (RF) muscles were excised and frozen immediately in liquid nitro- gen at the postmortem time periods shown in Table l. The 0 hr LD samples were obtained at the time of exsanguination from the 2nd to 3rd lumbar region. All subsequent LD samples were excised by moving progressively cranial to the 0 hr sample. Samples were removed from the left LD directly opposite those of the right LD for corresponding postmortem time periods. The 0 hr RF samples were also obtained at the time of exsanguination. The anterior third of the RF was used for 0 hr or 15 ,min samples, the medial third for 45 min or 2 hr samples and the posterior third for the 24 hr samples. Table 1. Postmortem time periods of sample excision for right and left sides of each muscle and the number of pigs included in each time period. —4- -— .1 _ Lo ngisa@L(£QT 1 -- "“ ' Right ‘ ‘ ‘“ " ' Left .Bsstmortem. . ., ‘ . , . , . I .. time 0hr 15min45min2'hr 24hr 15min45min2hr 24hr No. of pigs 12 12 ‘ 12 12 12 12 12 12 No. of pigs 5 5 5 5 5 5 5 5 5 No. of pigs 5 5 5 5 5 5 5 __1 _A"Rectus femoris (RF) No. of' pigs 10 10 10 lo 10 No. of pigs 5 5 5 5 5 No. of pigs _ 6__ .. 6 6 ., 6 6 -28- LD temperatures (right side) were recorded at the time of exsan- guination and at 45 min postmortem (both sides). The RF temperatures from both sides of six pigs were recorded at 2 hr. Samples removed from.the right LD and RF at the time of exsanguination and from.both right and left sides at 24 hr postmortem were used for.moisture deter- .minations. Samples of the biceps femoris (BF) and supraspinatus (SS) muscles from.the right side were excised shortly after exsanguination (4 to 5 min) and at l, 2 and 24 hr postmortem for pH determinations. Twenty- four hour (postmortem) samples from.the LD and RF muscles from both right and left sides and right side BF and SS muscles were used to obtain transmission values. SubjectiveMuscle Quality Appraisal At 24 hr postmortem, LD muscles from all carcasses were subjectively evaluated using a system similar to that described by Forrest 31 31. (1963). A score of 0-5 was recorded for each of the following three factors: structure and firmness, marbling, and color. Highest values were assigned to the normal or ideal for each of these characteristics, i.e., dry and firm, moderate or higher degrees of marbling, and grayish pink color. Scores of 0 were assigned to very soft exudative muscles which were devoid of visible marbling and very pale in color. -29- Heart Weights The hearts from.3l pigs in Group I and all pigs (66) in Groups III and IV were removed, trimmed of excess fat and weighed to the nearest 0.1 g. Powdering of Frozen Muscle and Liver Samples The frozen muscle and liver samples (stored at -20°C following liquid nitrogen freezing) were powdered in a -20°C room as described by Borchert 33,31. (1965). Shattered pieces of the frozen samples were placed in a Waring blendor jar with chipped dry ice, pulverized for approximately 30_sec and then sifted. The coarse material which re- mained on the sieve was discarded. The powdered samples were placed in sterile polyethylene bags and 12 hr were allowed for 002 sublimation before sealing the containers or using the samples for subsequent analyses. The powdered samples were stored at -20°C until used for analysis. Muscle pH Approximately 5 g of frozen, powdered muscle were suspended in 25 ml 0.005 M sodium iodoacetate. The pH estimates were obtained from these suspensions with a Corning Model 12 expanded scale pH meter.' For pH determination of fresh tissue, approximately 5 g of muscle were blended in 25 ml 0.005 M sodium iodoacetate in a small Waring blendor jar and pH of the suspension was recorded. -30- Muscle Mbisture Determination Approximately 2 g of finely ground muscle were dried in a 100°C oven for 18 hr. After cooling in a desiccator for approximately 1 hr, weight loss was recorded as moisture (AOAC, 1965). Transmission Values The transmission value procedure described by Hart (19623 was used as an objective measure of muscle quality for the carcasses in Groups III and IV. Ten g of finely ground muscle sample (24 hr postmortem) were weighed in a centrifuge tube and cold distilled water added to bring to a total volume of 40 ml. The mixture was thoroughly stirred and held at 2 to 4°C for 20 hr after which time the mixture was restirred, centri- fuged, and the supernatant filtered through Whatman No. 1 filter paper. One ml of clear filtrate was mixed in a test tube with 5 ml of pre- chilled (20°C) pH 4.6 buffer (9.35 parts of 0.2 M NaZHP04 and 10.65 parts of 0.1 M citric acid). This mixture was incubated in a 20°C water bath for 30 min and after thorough mixing, percent transmission was read on a Bausch and Lomb Spectronic 20 calorimeter at 600 mu against a blank (1 ml muscle filtrate and 5 ml distilled water). Succinic Dehydrogenase Activity The 0 hr LD samples from.the carcasses in Group I were analyzed for succinic dehydrogenase (SDH) activity (Bonner, 1955). Approximately 4 g of powdered, frozen sample were extracted for 30 min with 15 ml 0.02 M phosphate buffer (pH 7.2). The samples were then centrifuged at 1500 x G -31- for 20 min and the supernatant (3°C) was adjusted to pH 5.7 with l N acetic acid. The resultant mixture was centrifuged at 1500 x G for 15 min and the precipitate suspended in 3 ml of 0.1 M phosphate buffer (pH 7.2). A 0.3 ml aliquot of buffered suspension was added to a tube contain- ing 1.9 ml of 0.15 M phosphate buffer (pH 7.2), 0.3 ml of 0.1 M ch, 0.3 ml of 0.01 MIK3 Fe (CN)6 and 0.2 ml of 0.2 M sodium succinate. Re- duction of K3 Fe (CN)6 was read spectrophotometrically at 420 mu with a Beckman DU Spectrophotometer. Absorbance was obtained initially and after 30 min of incubation at 35°C against a blank [identical to the sample tubes except distilled H20 replaced K3 Fe (CN)6]. Red, White, and Intermediate Fibers The fresh frozen LD samples for histochemical analysis of fiber type were sectioned (10 u) on a Slee freeze microtome (-20°C). The sections were placed on coverslips and stained for SDH activity (Nachlas 31 31., 1957). The sections were incubated in a 0.05 M phosphate buffer (pH 7.6) at 37°C. This buffer also contained 0.05 mM of sodium succinate and 0.5 mg of Nitro BT [nitro-2,2:5,5'-tetraphenyl-3,3'-(3,3'-dimethoxy- 4,4'-biphenylene) ditetrazolium chloride] per ml. After incubation (l to 2 hr) the sections were washed in physiological saline (8.5 g NaCl, 0.2 g CaClz and 0.1 g KCl in l 1. of distilled water).and nixed in 10% formol saline for 10 min, rinsed in 15% ethanol for 5 min and mounted in glycerine jelly. Prints (10 1/2' x 13 3/4") were made from the slides which were photographed from.a magnification of 80X. Pictures were obtained from -32- three different areas of each muscle sample (LD) for the determination of red, white and intermediate fiber types. Relative numbers as well as relative areas of fiber types were determined. Area was measured with a compensating polar planimeter. Relative fiber size for each type was calculated from the number and total area of the respective fiber type. Myoglobin Total myoglobin concentration as well as estimates of the relative amounts of reduced myoglobin (Mb), metmyoglobin (MMb) and oxymyoglobin (02 Mb) were determined on the 0 hr LD samples from Group I carcasses according to the absorbancy ratio method of Broumand g; 31, (1958). Five g of frozen, powdered muscle were extracted in a stoppered Erlenmeyer flask with 20 ml cold distilled water by vigorous shaking for 1 1/2 mun and the.mixture was then filtered through Whatman No. 1 filter paper. The first 2 to 3 ml of filtrate were discarded and absorbance of the remaining filtrate was spectrophometrically determined at 473, 507, 573 and 597 mu against a water blank. After obtaining the above readings, one drop of 0.5% KCN and one drop of 2% K3 Fe (CN)6 was added to the blank and each sample tube. Absorbance at 542 mu was recorded as a mea- sure of total myoglobin compounds converted to cyanometmyoglobin (CMMb). The absorbancy ratio 507/573 mu was used to estimate the % MMb while the ratio 473/597 mu estimated the % Mb. These percentages were read directly from standardized curves presented with the original method. Percent 02 Mb was determined by difference [% 02 Mb = 100 - (% MMb + % Mb)]. The millimolar extinction coefficient of CMMb is 11.3 at 542 mu. -33- Some Metabolites Involved in Glycolysis Some of the metabolites involved in the glycolytic pathway were ex- tracted from the frozen, powdered LD and RF samples obtained from the carcasses of Group IV and enzymatically determined by fluorometry in accordance with the procedures of Lowry'31 31. (1964) and Maitra and Estabrook (1964). The metabolites assayed included glycogen, glucose, glucose- 1 -phosphate (G-l-P), glucose—6-phosphate (G-6-P), fructose-6- phosphate (F-6-P), lactic acid, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and creatine phosphate (OP). Extraction Frozen,powdered muscle samples were placed in test tubes containing 3 M 110104 (prechilled to 3%) and after sample weight was obtained addi- tional 3 M H0104 was added to give a HClO4:muscle ratio of 2.86:1 (V/W). Following centrifugation (1500 x G for 15 min), 3.14 ml of 2 M KHCO3/g of initial muscle sample weight were added to the decanted supernatants. After 002 evolution, 4.0 m1 of 2 M Tris base/g of the initial muscle same ple weight were added to bring the pH to 7.5 to 8.0. The supernatants containing 1 mg of muscle equivalent/0.01 ml were decanted from the KC104 precipitates and then stored at 3°C until analyses were completed (within 2‘days). Fluorometry Analyses were conducted with 1 ml of solution which included reagents, enzyme(s) and neutralized sample extracts in 7 x 40 mm fused quartz -34- fluorometer tubes. The Aminco-Bowman Spectrophotofluorometer was employed to read the changes in concentration (fluorescence) of either the reduced nicotinamide dinucleotide phOSphate (NADPH) or reduced nicotinamide dinu- cleotide (NADH). An excitation wavelength of 376 mu and an emission wavelength of 464 mu was used for all analyses. Standards and enzyme blanks were run with each analysis. All reactions were conducted at room temperature (25 to 28°C). Reaction media, containing 0.01% bovine serum albumin, were prepared and stored at -20°C until needed. Where NADH was required, it was added shortly before each series of analyses. All enzymes (Sigma Chemical Co.) were diluted for use in these analyses with 0.02 M Tris buffer (pH 7.5) containing 0.02% bovine serum albumin to provide the required amounts in 0.01 ml. G-6-P, ATP and CP These compounds were measured on the same sample aliquot (0.02 ml of neutralized H0104 extract). Reactions were carried out in 0.1 M Tris buffer (pH 7.5). This buffer solution contained l.mM of glucose, 0.3 mM of NADP and 5.mM of MgC12 per liter. Before adding 2 ug of yeast G-6-P dehydrogenase to each tube, the initial fluorescence reading was obtained. After the completion of NADPH increase (5 to lO.min), fluorescence was again recorded. Yeast hexokinase (2.5 ug) was then added and following an additional increase in NADPH (approximately 20.min) another reading was recorded. Ten ug of muscle phosphocreatine kinase and 0.03 uM of ADP (prepared withinlhrof use) were added to each tube. Upon completion of this reaction (20 to 30 min), a final fluorescence reading for this series of reactions was recorded. -35- Glucose, G-l-P and F-6-P Aliquots of the previously described muscle extract (0.02 ml) were added to 0.1 M Tris buffer (pH 7.5). This buffer solution contained 0.3 mM of ATP and NADP and 5 mM of MgClp/l. Initial fluorescence was obtained after completion of the reaction following the addition of 2 ug yeast G-6-P dehydrogenase. Additional fluorescence was recorded upon completion of the reaction initiated by the addition of 10 ug of muscle phosphoglu- comutase to each tube (1 to 2 hr). Still another 1 to 2 hr elapsed following the addition of 10 ug of yeast phosphohexose isomerase to each tube before reading again. The last fluorescent reading for this series of compounds was obtained following the completion of the reaction catalyzed by the addition of 2.5 ug yeast hexokinase. Glycogen Approximately 25 m1 of 1 N HCl.wen3added to the insoluble residue which remained after H0104 extraction as previously described. This mixture was placed in a 100°C oven for 3 to 4 hr to convert muscle glyco- gen to glucose and after heating, additional 1 N HCl was added to obtain a HC1:muscle ratio of 20:1 (V/W). Aliquots (0.005 to 0.01 ml) of clear HCl hydrolysate (obtained by centrifugation) were added to the reaction media as previously described for glucose. Initial fluorescence was obtained 5 to lO.min after 2 ug of yeast G—6-P dehydrogenase had been added. Approximately 20 min after the addition of 2.5 ug of yeast hexo- kinase to the reaction media a final reading of NADPH fluorescence was recorded. -36- ADP and AMP Reactions for ADP and AMP were conducted in a 50 mM potassium.phos- phate buffer (pH 7.0). This buffer also contained 3 uM NADH, 0.02 mM ATP, 0.02 mM phosphoenolpyruvate and 2 mMIMgClZ/l. A 0.02 ml aliquot of neutralized H0104 extract was used for these analyses. Fluorescence was obtained 10 min after the addition of heart lactic dehydrogenase (8 ug/ tube). Additional fluorescence readings were recorded- after the come pletion (10 min) of NADH decrease following the introduction of 1 ug of muscle pyruvate kinase. The final decrease in NADH was recorded about 10 min after the addition of 1 ug of muscle adenylic acid kinase to each tube. Lactate Lactic acid concentrations were fluorometrically determined according to an enzymatic procedure described by Hohorst (1963). Aliquots of neu- tralized H0104 extracts (0.005 to 0.01 ml) were added to a hydrazine- glycine buffer (7.2 g glycine, 5.2 g hydrazine sulphate, 0.2 g EDTA-NaHz .2 H20 and sufficient 2 N NaOH to attain a pH of 9.5 in a total volume of 200 ml). This buffer contained 0.3 mM of MAD/l. Readings were recorded upon completion of the fluorescence increase (approximately 2 hr) after the addition of 10 ug heart lactic dehydrogenase. Lipids Extraction Lipids were extracted by modification of the methods described by Ostrander and Dugan (1962) and Masaro 31 31. (1964) from the 0 hr LD, liver -37- and serum samples of the pigs in Group I. Sixty ml of chloroformrmethanol (lslfW/V) was added to either 20 ml of serum or 10 g of muscle or liver and stirred for 10 min. Twenty ml of 5% zinc acetate were added to the extraction mixture and stirred for an additional 1 min. The mdxture was then filtered with the aid of suction through a Buchner funnel fitted with Whatman No. 1 filter paper. The precipitate was extracted an addi- tional two times and filtered as described above. The combined filtrates were transferred to a separatory funnel and the chloroform layer removed. The water-methanol layer was washed twice with 75 ml chloroform. The chloroform fractions were combined and evaporated to dryness with a rotating flask evaporator. Traces of the solvent were removed by heating the residue in a 100°C oven for 5 min. Upon cooling in a dessicator, weight of the lipid material was obtained. This lipid material was re- dissolved in 10 ml chloroform and stored at -20°C for further analysis. Phospholipids Phospholipids of the LD samples were separated from neutral lipids by modification of the methods described by Bates (1958), Reiser 31.31. (1960) and Choudhury and Arnold (1960). Lipid material was dissolved in 50 ml of chloroform and mixed with 10 g of silicic acid [heated (110°C) for 2 hr]. The mixture was stirred for 10 min and filtered through a sintered glass funnel (medium porosity) by suction. Four-50 ml portions of chloroform.were used to elute the neutral lipids. Phospholipids were then eluted with four-50 ml portions of methanol. Both fractions were evaporated to dryness, restored to a 10 ml volume with chloroform, and stored at -20°C for further analysis. -33- Phospholipid phosphorus was determined as described by Beveridge and Johnson (1949). One ml of concentrated H2804 was added to dry phospho- lipid fractions in a microdigestion flask. The samples were placed on a burner (micro-K5eldahl unit) and allowed to digest for six hours. When digestion was partially complete, 1 to 2 drops of 30% H202 were added and the flasks were shaken between the addition of each drop. Upon comp pletion of the digestion, the samples were cooled and 9 ml of distilled water added. The samples were transferred to a 50 ml volumetric flask with 15 ml of distilled water. Twenty ml of freshly prepared molybdate- hydrazine sulfate reagent [25 ml of 2.5% (I/V) sodium molybdate dihydrate in 3 N H2804 and 10 ml 0.15% (II/V) hydrazine sulfate in 3 N 112304 brought to a total volume of 100 ml with distilled water] were added with a rapid delivery pipet. The samples were adjusted to volume with distilled water, .mixed and heated in a boiling water bath for 10 min. Within 24 hr after cooling, color development was measured with a Spectronic 20 calorimeter at 830 mu. Neutral lipid ester determination Ester concentration was measured on the neutral lipid fractions (Lands, 1958). Equal volumes of 4% NaOH and 4% hydroxylamine hydrochlor- ide (H/V) in 95% ethanol were mixed and the resulting NaCl was removed by filtration. Two ml of the clear filtrate were added to a dry lipid- sample in a screw cap culture tube. The tubes were shaken, heated (65°C) for 5 min and then cooled. Five ml of ferric perchlorate solution [4 ml of stock ferric perchlorate (Goddu_ gt al., 1955) and 2.5 ml of 70% HC104 diluted to 100 ml with cold absolute ethanol] were added as the tubes -39- cooled. Stock ferric perchlorate was prepared by dissolving five g of ferric perchlorate in 10 ml of 70% HC104 and 10 ml of H20. This.mixture was diluted to 100 ml with anhydrous 2-butyl alcohol while cooling under a water tap. Twenty min after the addition of the ferric perchlorate solution, color development was measured by recording absorbance with a Spectronic 20 colorimeter at 530 mu. Triolein was used as a standard. Statistical Analysis The statistical procedures followed were those discussed by Steel and Torrie (1960). Duncan's new multiple range test was applied when analysis of variance data were significant in order to detect the signi- ficantly different means. Student's i values were obtained when only two means were compared. Where appropriate, simple correlation coeffi- cients were obtained. RESULTS AND DISCUSSION Distribution of Muscle Fiber Types, Succinic Dehydrogenase Activity, Myoglobin and Lipid Levels The pigs in Group I were categorized by breed according to the rate of postmortem.pH decline in the LD muscle. Five pigs from each of three breeds (Poland China, Landrace and Chester White), which had 45 min post- mortem.pH values greater than 6.0 were called "normal". Five pigs frmm each of the Poland China and Landrace breeds were called "low quality" since they had 45 min pH values less than 6.0 and possessed ultimate LD muscle properties tending toward the pale, soft and exudative (PSE) condi- tion described by Briskey (1964). The source of Chester White pigs used for this study were known to have a very low incidence of PSE and thus no attempt was.made to obtain a low quality Chester White group. The means of 45 min postmortem.pH values and 24 hr subjective quality scores of the LD are presented in Table 2. As expected from the report of Briskey (1964), a significant (P'< .01) difference for both 45.min pH values and subjective quality scores was noted between quality groups. These data are presented to justify the categorization of the pigs used in this study. A significant (P < .01) correlation coefficient of 0.63 was obtained between 45 min pH values and subjective quality scores of all the pigs in Group I (40). The rate of postmortem pH fall was more rapid among the low quality pigs than for the normals (Figure 1). Comparable (pH fall) curves were observed within quality groups for the breeds included in this study. -40- pH 6.0 5.5‘ 5.0 - 41.. Table 2. The mean pH values and subjective scores of the longissimus muscle by breed and quality group.l Breed Chester Poland China Landrace White Trait Nonmal Low quality Normal Low quality Nermal 45 min pH 6.193 5.4910 6.233 5.67b 6.26" Subjective quality score2 10.03 5.613 11.0a 6.8b 13.0a 1Means with the same superscripts are not significantly different (P:> .01). Score based on a 15 point scale with 5 possible points for each of the three muscle properties: marbling, color and structure (firmness and exudation). Poland China Chester White Landrace normal low quality "' 52593 PC-N :3 -fi - ---A'.-. C — *- ----- -‘— 11 l l_J I | | 15 45 min ' 3 hr ' mln Postmortem time Figure l. Postmortem.pH curves of the longissimus muscle for the pigs in Group I. -42.. While the initial (0 hr) pH values of the low quality pigs were slightly lower than normals, the rate and/or extent of decline was markedly differ- ent at 45.min postmortem. The rate of pH decline between quality groups became progressively narrower with postmortem time after 45.min and ulti- mate pH values (24 hr) were essentially the same. These data concur with the reports of Briskey and Wismer-Pedersen (1961a), Sayre and Briskey (1963) and Briskey (1964) and further substantiate the quality grouping used in this study. Red, white and intermediate muscle fiber distribution. White muscle fibers have a much greater anaerobic metabolic capacity and lower myoglobin and lipid levels than red fibers (George and Berger, 1966) and Beecher £2 al. (1965a, 1968) reported that differences in red and white fiber contents between the light and dark portions of the semi- tendinosus muscle were related to postmortem glycolytic rate. 'Thus it seems logical that differences in white or red fiber content between LD muscles could possibly be related to differences in ultimate quality characteristics. However, Briskey and Lister (1968) reported that a higher aerobic capacity (larger red fibers, higher levels of cytochrome oxidase and succinic dehydrogenase) existed for Poland China pigs than for Chester Whites and that the higher aerobic potential was related to development of PSE musculature. Thus this phase of the present study was undertaken to compare indices of aerobic metabolic capacity between normal and low quality LD muscle within and among several breeds of pigs. In this study muscle (LD) fiber types were categorized as red (posi- tive), white (negative) or intermediate on the basis of their staining -43- reaction for SDH activity. Intermediates were designated as those fibers which were not distinctly positive or negative but were intermediate in their staining reaction. The three fiber types were determined as a per- centage of the total for both number and area. The relative fiber type size could then be calculated from these data. When expressed as a percentage of total number, the normal Poland China pigs had more red fibers (P < .05) than either the low quality Poland China or Landrace pigs (Table 3). The low quality Landrace pigs had more white fibers than the normal groups and fewer intermediates than the other groups (p.< .05). On a percentage of total area basis (Table 3) both Poland China quality groups had signifiCantly (P < .05) larger red fiber areas than all other groups. Both Poland China quality groups also had smaller white fiber areas than the normal Chester Whites or low quality Landrace pigs (P < .05). The low quality Poland Chinas had significantly (P < .05) larger intermediate fiber areas than the low quality Landrace pigs. On a percentage of total area basis, Poland Chinas had more red and less white fibers than either Chester White or Landrace pigs; however, no significant (PI> .05) differences exist between quality groups. On a percentage of total number basis, differences between breeds are apparent. Additionally, muscle from normal pigs contained more red and intermediate and less white fibers than that from low quality pigs (P < .01). From these data it is apparent that differences in relative areas or numbers of muscle fiber types vary considerably between breeds as well as quality groups. - 44.. Table 3. The distribution of red, white and intermediate fibers and succinic dehydrogenase activity in the longissimus muscle by breed and quality groups. Breed Chester Poland China Landrace White Measurement and _ fiber type Normal Low quality Normal Low quality Normal Percent of total number Red 27.52a 22.67b 24.03a:b 20.34b 23.79a.b White 63.13b 68.51a.b 65.86b 74.11a 67.22b Intermediate 9.34a 8.83a 10.10a 5.54b 8.99a Percent of total area Red 16.59a 16.79a 14.56b 14.09b 13.78b White 76.51b 74.95b 78.22a.b 81.37a 80.39a Intermediate 6.90a,b 8.25a 7.223:b 4.53b 5.83%b Relative size2 Red 10.023:b 7.91b 10.97a 8.883’b 10.31a White 5.00 5.36 5.58 5.52 4.97 Intermediate 8.25a:b 6.50b 9.52a 7.87a’b 9.36a Total fibers 6.05 5.87 6.63 6.07 5.95 Succinic dehydro- genase activity3 21.68b 18.49b 33.55a 17.91b 24.61351D 1Means with the same superscripts do not differ significantly (PI> .05). 2Number of fibers per square inch of picture. 3Millimicromoles succinate oxidized/min/g of muscle (0 hr). These data explain why Briskey and Lister (1968) reported more red fibers among "stress-susceptible" pigs or those which ultimately developed PSE.musculature. These authors used Chester Whites as "normal" or "stress -resistant" pigs, while Poland Chinas were used as the "stress-susceptible" pigs. The data in the present study supports_the breed differences, but not the differences between quality groups. -45.. Data for the relative size of each muscle fiber type are also pre- sented in Table 3. Higher values correspond to relatively smaller fiber sizes. The relative size of the red and intermediate fiber types differ between breed-quality groups, whereas the white fiber size appears markedly similar. The "t" test analysis shows that normal muscles had smaller red (P'< .01) and intermediate (P < .05) fibers than the low quality muscles. There do not appear to be any breed differences in relative size of the three fiber types. A significant (P < .05) correlation coefficient (r = 0.48) was ob- served between percentage of red fiber numbers and 45.min pH; whereas, the percentage of red fiber area was negatively, but nonsignificantly (P > .05) correlated (r = _.24) with 45 min pH. Succinic dehydrogenase activity. The mean SDH activities between breed and quality groups are shown in Table 3. The Landrace pigs had significantly (P < .05) higher SDH activities than the normal Poland Chinas or the low quality pigs. Normal muscle possessed higher SDH activity than that from low quality muscle (P‘< .05). These data for SDH activities support that of the relative numbers and areas of fiber types previously discussed. LD muscles with higher percentages and/or smaller areas of red fibers tended to have higher SDH activity. Since the red and intermediate fibers from low quality muscle are larger than those from normal muscle, they are perhaps not as "red" or aerobic in nature as indicated by SDH activity. The correlation between SDH activity and 45 min pH for all the pigs in Group I (40), although low (r = 0.33) was significant (P‘< .05). The -45- correlation coefficient for SDH activity and subjective quality scores (r = 0.42) was highly significant (P < .01). Myoglobin. Table 4 presents the mean levels of total Mb as well as relative amounts and absolute values of MMb, reduced Mb and Osz. The normal Landrace pigs had significantly (P < .05) more total Mb than the other quality groups, while the low quality Poland Chinas had significantly (P < .05) less than all other groups. Breed differences were apparent since normal Landrace pigs had.more total Mb than normal Chester Whites or both Peland China quality groups (P < .05). Low quality Landrace pigs had more total Mb than low quality Pbland Chinas. Significant (P < .05) differences between quality groups were also apparent. Nermal Landrace pigs had more total Mb than either the low quality Landrace or Poland Chinas and normal Chester White and Poland China pigs had more total Mb than low quality Poland Chinas. There were no significant (PI> .05) differences in percents MMb, reduced Mb or OgMb between any of the breeds or quality groups. Differ- ences in levels of these Mb fractions reflect total Mb differences. Even though there appeared to be no consistent pattern among the percentages of these Mb fractions there was a trend toward the normal pigs having higher MMb and 02Mb levels than the low quality groups. Total Mb from 0 hr LD muscles (40 pigs) was significantly (P < .01) correlated with 45 min pH (r = 0.41). From the data in Tables 3 and 4 it can be seen that higher proportions of red muscle fiber types were associated with increased SDH activities -47.. Table 4. Myoglobin content of the longissimus.muscle by breed and quality group.1’2 Breed Chester Poland China Landrace White Normal Low quality Normal Low quality Normal Percent of total myoglobin Metmyoglobin 33.3 33.7 39.3 36.1 34.8 Reduced myoglobin 16.5 19.3 25.1 20.9 19.0 Oxymyoglobin 50.2 46.9 35.6 43.0 46.2 Millimicromoles /g of muscle . b c a b b Total myoglobln 80.2b 55.4 114.8a 89.4b 79.4b c Metmyoglobin 26. 3 ’C 19. 2° 45.8 32.4 28.0 ' Reduced b b a b b myoglobin 12.8a 11.2b 29.2a b 19.3a b 15.5a b Oxymyoglobin 41.1 25.0 40.1 ’ 37.7 ’ 36.0 ’ IMeans with the same superscripts do not differ significantly (PJ> .05): 20 hr samples. and higher total Mb levels in normal muscle than in low quality muscle at or shortly after the time of exsanguination. While all of these observa- tions were not significantly correlated with each other, individually they were significantly related to 45 min postmortem pH values. Thus it appears from these indices that a greater aerobic metabolic potential existed in normal muscle than in low quality muscle at the time of exsanguination. This may at least partially explain the postmortem differences observed between normal and low quality LD muscle. Also, this observation.may explain why the 0 hr pH of the low quality muscles was lower (P < .01) -48- than that for normal muscles (Figure 1). Additionally, Kastenschmidt gt 2;. (1966, 1968) indicated that "fast-glycolyzing" muscles may already be in a highly anaerobic state at the time of exsanguination. However, it remains to be established to what degree these differences are inher- ent or attributable to management practices during growth and development. Table 5 summarizes the serum, liver and LD lipid analysis of the pigs by breed and ultimate quality group. The lower quality Landrace pigs had higher serum lipid levels than the normal Landrace or both Poland China quality groups (P > .05). Liver lipid content did not vary signifi- Cantly (P1>.05)between any of the breed or quality groups. While muscle lipid levels did not vary significantly (Pl> .05) be- tween groups, there was a trend toward normal muscle among the Poland China and Landrace breeds to have higher concentrations of lipids than the low quality groups. Percent of muscle lipid was significantly (P < .05) correlated with 45 min pH (r = 0.39). There were no significant (PJ> .05) differences in phospholipid content when expressed on either a total muscle basis or muscle lipid basis (Table 5). Phospholipid content on a total muscle was negativeLy,but nonsignificantly (P > .05) correlated with 45 min pH (r = -.23). These data do not support the highly significant relationship between muscle phospholipid content and 45 min pH observed by Krzywicki and Ratcliff (1967). However, their phospholipid values representuionly the myofibrillar and reticular fractions of muscle, whereas the values reported in this study were determined on the whole muscle. -49.. Table 5. Serum, liver and longissimus muscle lipids by breed and quality group. Breed Chester Poland China Landrace A White Normal Low quality Normal Low quality Nermal Lipid sourcel"2 Serum 0.305b 0.3l4b 0.308b 0.374a 0.330%b Liver 5.38 5.36 5.29 5.58 5.63 Longissimus 4.20 3.20 4.77 4.18 3.98 Lipid component Phospholipids (9- eq P/g LD lipid) 164 242 171 164 146 (u eq Pyg LD) 6.16 7.13 7.69 6.56 5.94 Glyceride esterszi3 (u eq /8 LB b c lipid) 763 : 702C 87la:b 8208,b.c 915a (u eq/g LD) 33.5a,b 22.0b 40.7a 34.4%b 36.6a I%~of lipid material on a fresh weight basis. 2Means with the same superscripts do not differ significantly (P1> .05). Determined on the neutral lipid fraction only. Glyceride ester contents expressed on a total muscle and muscle lipid basis are presented in Table 5. On a muscle lipid basis, Chester White pigs had higher glyceride ester contents than both Poland China quality groups (P < .05). Normal Landrace muscles had greater glyceride ester contents (muscle lipid basis) than the low quality Poland Chinas (P < .05). Low quality Poland China samples contained less glyceride esters than_ those for either normal Chester White or Landrace pigs on a whole muscle basis (P‘< .05). On a whole muscle basis glyceride ester levels were significantly (P‘< .01) correlated with 45 min pH values (r = 0.55). -50- These data indicate that if muscle lipid content is a factor in determining the rate or extent of postmortem changes this effect would result from.the neutral lipid fraction. Fritz 33 El; (1958), Issekutz 23 El. (1964), Spitzer and Gold (1964) and Masaro (1967) reported that lipid oxidation.may provide an important energy source for muscle activity. While the phospholipids of muscle are structural-functional elements, they are not.mobilized for energy purposes; whereas triglycerides serve as the ultimate lipid energy reservoir (Masaro, 1967). Beatty 21 El. (1963) stated that red muscle fibers, commensurate with the greater aerobic metabolic capacity, would more readily oxidize fat for energy than white fibers. Thus the trend toward increased glyceride ester and total lipid contents in normal muscle may be associated with or are a result of the greater aerobic potential among these muscles. Heart Weights A deficiency of cardiovascular capacity (anatomical and physiologi- cal) has been implicated in the etiology of PSE musculature (Forrest gt 31., 1965, 1968; Merkel, 1968). Additionally, Engelhardt (1966) reported that wild pigs have heavier heart weights, and thus more favorable rela- tionships between cardiac capability and body need, than domestic pigs. The above reports prompted a collection of heart weights in this study. In order to minimize live weight differences, all heart weights were converted linearly to that of a 200 lb live weight pig. Mean heart weights for the normal and low quality Poland China pigs were 274.9 g and 270.7 g, respectively; 282.2 g and 236.4 g for the normal and low quality Landrace, respectively, and 286.1 g for the normal Chester Whites in Group I of -51.. this study. Since only three heart weights were obtained for the normal Poland Chinas, this group was not included in the statistical analysis. Analysis of the above data indicate that low quality Landrace pigs had significantly (P‘< .05) lighter heart weights than normal Landrace or Chester Whites. However, heart weights were nonsignificantly (P1> .05) correlated with either 45 min pH values of LD muscles from Groups I and IV or transmission values from Groups III and IV (r = 0.07 and -.07, respectively). These results indicate that heart weight pg; 22 was not associated with the rate of postmortem.pH decline or ultimate muscle quality. Two pigs in Group III, which had moderate to severe cases of pericarditis (detected upon postmortem examination) resulted in carcasses with severe PSE musculature. The heart weights of these two pigs (248 g and 235 g, respectively) when adjusted to a 200 lb live weight basis were comparable to the mean (253 g) of the low quality Landrace and Poland Chinas in Group I. These lower weights are contrasted to the 281 g mean heart weight of the normal pigs in Group I. However, it is likely that the impaired heart function rather than heart weight peg s3 could have contributed to the low quality muscle of these carcasses. Muscle Temperature The adverse effects of high temperature-low pH relationships on ultimate muscle quality have been well documented (Briskey and Wismer- Pedersen, 1961a; Bendall and Wismer-Pedersen, 1962; Briskey, 1964). The inCidence of low quality muscle development was reported to be markedly -52- increased when body temperature at the time of exsanguination is near or exceeds 42°C (Merkel, 1968). Hoernicke (1966) observed insufficient temperature regulation among some pigs. Thus, a phase of the present study involved the relationship of muscle temperature at the time of exsanguination and during early postmortem periods to some muscle quality parameters. Temperature of the LD muscle 45 min postmortem was significantly (P < .01), but negatively correlated with (Groups II, III and IV) sub- jective quality scores (r = -.32) and with (Groups II and III) 45 min pH (r = -.52). Muscle temperature 45.min postmortem was also significantly (P < .01) correlated with (Groups III and IV) transmission values (r = 0.56). The higher LD temperatures 45.min postmortem either played a role in development of low quality muscle or they possibly resulted from the exothermic reactions associated with increased glycolysis. Temperatures at the time of exsanguination showed lower relationships to rate of pH decline and ultimate quality properties than those at 45 min postmortem. Rectal temperature (Group II) was nonsignificantly (I’> .05) correlated with 45 min pH (r = -.29). LD temperature at exsan- guination (Group III) was nonsignificantly (Pl> .05) correlated with 45 min pH (r = -.29). A low, but significant (P‘< .05) correlation (r = 0.27) was observed between LD temperatures at the time of exsanguination and transmission values (Groups III and IV). Thus it appears from this study that muscle temperature at the time of exsanguination was not as important as that at 45 min postmortem in influencing ultimate muscle qualitative properties. -53- LD temperatures at 45 min postmortem (Table 6) were significantly (P'< .01) higher than 0 hr rectal temperatures (Group II) or 0 hr LD temperatures (Group III). The greater temperature differential between 0 hr and 45 min postmortem in Group II possibly resulted from the fact that 0 hr temperatures were obtained rectally rather than directly from the LD muscle. However, pigs in Group II were slaughtered as a group (similar environmental temperature conditions); whereas, the pigs in Group III were slaughtered (in lots ranging from 2 to 10) over a three month period. The relatively "hot and humid" conditions associated with extensive use of hot water and steam on the slaughter floor while the large number of pigs in Group II were slaughtered may be at least par- tially responsible for the elevated 45.min temperatures observed in this group. The prolonged exposure (1 to 1 1/2 hr) of the pigs in Group II to "hot and humid" slaughter conditions before chilling is unusual since the time required for dehairing, evisceration and cleaning before chilling varies to some extent with the number of pigs and normally far fewer are slaughtered at any one time. The scalding operation which involved soaking the entire pig in hot water (60°C) for 5 to 10 min prior to dehairing could possibly have elevated the 45 min muscle temperature. From limited observations (20 pigs) in Group III, the temperature after scalding (15 min postmortem), while not significant (P > .05), tended to be higher than the initial temperature. The pigs in Group IV were not scalded or the carcasses skinned and eviscerated until approximately 2 hr postmortem, and the 45 min temperatures were essentially the same as the initial temperatures -54.. (Table 6). Thus subjection to scalding and/or "hot and humid' slaughter floor conditions may have contributed to increased muscle temperatures at 45 min postmortem. Table 6. Some rectal and longissimus muscle temperatures at several postmortem time periods. Timegperiods No. of_pigs 0 hr 15 min 45 min 402 39.3ID —- 40.6a 443 39.9b —- 40.4aL 203 40.3 40.7 40.5 194 39.6 -- 39.6 lM'eanswith superscripts are significantly different (P < .01). 2Group II pigs, 0 hr temperature was rectal rather than LD. 3Group III pigs, LD temperatures. roup IV pigs, LD temperatures. Two Yorkshires, which were intended to be slaughtered among the pigs in Group III, died prior to slaughter while either in transit to or shortly after arrival at the Meat Laboratory. One of these pigs had an LD temperature of 44.7°C approximately 1 hr postmortem with a pH value of 5.45; the other had a LD temperature of 44.0°C and a pH of 5.70 within 30.min postmortem. Two additional Yorkshire pigs had LD temperatures of 41.9°C and 42.2°C, respectively, at the time of exsanguination, while 45 min temperature was 41.7°C for both pigs. The LD pH at 45 min postmortem was 5.65 and 5.70 for these pigs, respectively. These latter two pigs were the same ones previously indicated to have had pericarditis and which ultimately possessed severe PSE musculature. Cardiovascular -55... impairment may very likely have contributed to the elevated temperature in the latter two pigs. While observations in this study indicate that temperature 45.min postmortem appeared to exert a significant influence on ultimate porcine muscle properties, the factor(s) which contributed to elevated muscle temperatures (45.min postmortem) have not been positively identified. The Effect of 0 hr Sample Excision on Postmortem Muscle Changes Observations.made while excising muscle samples from the pigs in Group I indicated that many of the postmortem changes which occurred were affected by the sampling technique. The 3 hr LD sample (excised from the same muscle which was incised for the earlier postmortem samples) frequently exhibited a trend toward PSE development, while the 24 hr sample from the opposite LD (not previously incised) appeared more "normal'. Thus the pigs in Group IV were slaughtered to determine what, if any, effect(s) sampling procedure had on glycolytic rate and ultimate muscle qualitative characteristics. ‘nggissimus muscle. Table 7 summarizes the postmortem pH patterns and illustrates the experimental design. A combination of three different initial sampling times were used to compare the right (LD-R) and left (LD-L) longissimus muscles as follows: 0 hr LD-R with 45 min LD-L (line 1, Table 7), 0 hr LD-R with 15 min LBeL (line 2', Table 7) and‘15 min'LDl-stith 45 min LD-L (line .30. v n: Escher-semen cote- see so coats-tease meow one see: memo: . ”w H _ omm.m p00.0 oom.w In ohm.m now.m omv.o ohm.o nu m mmH.m c-cHw.m n-owm.m om¢.o me.m omm.m o-ovo.o o-QNH.m o-n-aom.w m emu m nmo 0 new w an pom m 0mm m nae o an w an m «a no em as m as- me as- 3 no em .3 m 5.3- 9. cs- 3 .m- o owe-W mo .02 oofihom mafia Boyhoaomom coauom,osfip sawhoawmom ill m38fimmwwcoa who; mssflmmfimcoa «swam H.3Homsa mosfimmfimooa on» me oofiHooo mm.fiowuoSvmom so defimfioxo oflgsdm a: 0 mo woommo one .b canoe -57- 3, Table 7). Hereafter these three initial sampling times from the LD—R and LD-L will be referred to as 0-45, 0-15 and 15-45, respectively. It is readily apparent from the data in Table 7 that 0 hr sample excision had a significant effect on the rate of postmortem pH decline. In the 0-45 group, the LD-L 45 min pH was significantly (P < .05) greater than LD-R 0 hr, 15 min or 45 min pH. The LD-L pH at 2 hr postmortem.was comparable to the LDhR 0 hr pH. A significant (P < .05) difference be- tween the two sides at 2 hr was readily apparent. In the 0—15 group, LD-L 45 min pH was significantly (P'< .05) greater than LD-R 45.min pH. When comparing pH values among the three combinations of initial sampling times, it is apparent that LD muscles not incised at 0 hr maintained a high pH for at least 2 hr postmortem. No marked differences in 24 hr pH values were observed for any LD muscle indicating that only rate and not extent of postmortem glycolysis was affected. Table 8 presents the means for transmission values and subjective quality scores as affected by 0 hr sampling. While none of the differ- ences were significant (P1> .05), there was a trend toward lower ultimate quality as a result of 0 hr sampling in the 0-45 group. One of the carcasses in the 0-45 sampling group had 2 hr pH values of 5.31 and 6.31, transmission values of 95.0 and 18.5 and subjective quality scores of 5 and 10 for the LD-R and LD-L, respectively. Two other carcasses from the same sampling group had transmission values of 85.2 and 38.8, and 56.0 and 19.2 for LDuR and LD-L, respectively. Thus it is apparent that ulti- mate muscle pr0perties of some carcasses were.markedly affected by 0 hr sampling of the LD muscle. -58- Table 8. Effect of 0 hr sample excision on qualitative properties of the longissimus muscle. iTransmission Subjective LD-R, LD—L initial values1 scores sampling_time LD-R LD-L LD-R LD-L 0-45 49.7 30.9 8.4 9.9 0-15 21.4 24.3 11.2 11.0 15-45 22.7 20.7 10.4 1106 1Lower values correspond to more normal musculature. 2Higher scores correspond to more normal musculature. To support the pH data shown in Table 7, glycogen, lactate, G-6-P, ATP and CP levels for the corresponding sampling groups are presented in Tabl$9 through 13. Glycogen levels of the LD—L at 45 min postmortem were significantly (P < .05) greater than those at 0 hr, 15.min or 45 min for the LD-R.muscle (Table 9). Glycogen content of the LD—L at 2 hr was significantly (P‘< .05) greater than that of the LD-L muscle at 2 hr postmortem. There were no significant differences within the 0-15 and 15-45 sampling groups between the LD-R and LD-L muscles at the same postmortem time periods. The LD-L muscles tended to maintain higher glycogen levels than the LD-R in the 0-15 group, especially at the early postmortem time periods. From a comparison of the glycogen levels of the LD-L and LDbR.muscle samples in the 15-45 group at all postmortem time periods with levels in the other two sampling groups, it is readily apparent that glycogen content remained higher when no 0 hr sampling was performed. There appeared to be no marked differences in glycogen content among any of the muscle samples at 24 hr, thus indicating that only the rate and not the extent of glycolysis was affected by sampling schedule. .Amo. A-mv havcmoHMflcmflm sommfio we: co mumfisomuomsm made on» :9“: meme: N .oHomna mo M\duooao>flsvo omoosaw mo moHQSOpofla mo oommoamxo mosao>a . 9 o o o o o o o .w 8% N no em oo um oN N pm wN ma mm oN mm m o>.v o-nN.oN om.wv ob.Hm cm.N om.mN mm.o¢ n-ov.mm om.Ne m on m no mm mN mm pH N on ma no hm ob ow pH mm NH no em as N ses.me see.mfi an em on m see.ms see.mH as o swam] mo .oz weapon mafia soppoawmom mzaammflwcoa whoa weapon asap sophoswmom asamnowwsoa amuse . emu mo mHo>oH comoozfiw Eowhoepmoq no scam«QXo mammom .oaomaa mnaammfiwcoa on 0 mo woommo one .m oases -50- The lactate values are presented in Table 10. Lactate levels in the LD-L 45 min muscle samples of the 0-45 group were significantly (P‘< .05) lower than in either 0 hr, 15 min or 45 min LD-R samples. A significant (P'< .05) difference between sides was observed at 2 hr. There were no significant (Pl> .05) differences in lactate content between the LD-R and LD-L muscles within the same postmortem time periods in the 0-15 or 15-45 sampling groups. Lactate levels of the 15-45 group were consis- tently lower than the levels of both the 0-45 and 0-15 groups through at least 2 hr postmortem. No marked differences in lactate levels were evident between 24 hr means. These data support the observations for pH and glycogen levels. Table 11 summarizes the effects of 0 hr sample excision on G-6-P levels. It can readily be seen that the levels were relatively high at 0 hr, then reached a low between 15 min and 2 hr and increased-again at 24 hr. This pattern agrees with that reported by Kastenschmidt gt El. (1968). However, these authors reported slightly higher values than those observed in this study. As observed with the other glycolytic metabolites, the 24 hr G-6-P values for all muscle samples were similar. In the 0-45 group, the LDbL muscles 45 min and 2 hr postmortem levels were significantly (P < .05) lower than LD-R 45 min and 2 hr levels, re- spectively. When comparing sides (LD-R vs LD-L) of all the sampling groups, it again is apparent that 0 hr sample excision resulted in elevated G-6-P levels at least through 2 hr postmortem. -61.. .Go. A .3 senescenesmfl note- eon co neat-canoes..- oson ofi fie. ammo: N .oeomos M\ooaoaonoea_mo commongxo mosao>H om.om no.mv oe.mN ow.mw no.em om.Hm oH.NN m oN.mw o-n-om.mo oo.mm om.Nm mo.mw n-om.mm o-o-no.mm o-o-nm.m¢ o-ow.o¢ m oe.mm o-oH.mm om.om om.Hm nN.¢e ow.mm o-om.m¢ om.N¢ NH .2 em .2- N are me ce- 3 .3 sm 9- m see me one 3 a- o numfi noenom,oaep aopuoauoom weapon ofiee.fioehoaemom mo 02 mason-ween to.- 3333- N-H.oeom:a mnafimoemnoa one mo mHo>oH oeopooa sovnoaumom no deemeoxo oeoaom an 0 mo woommo one .OH oenoe .Amo. A-mv eHHQoQNMNoMHo nowheo we: op memenomthSm oadm one nee? mono: N .oeomza w\woeoaonoea.mo oommongxo mosao>a _ m.- ome e coo..- N one o omN e a? m oNo 0 56cm H m oNo.» p.omo.s omH.N cem.N c.3mc.c o.oNN.s ccc.N c-o.oms.s o.o.omm.m m era.» N-NNH.N mmN.H o-ome.c o-omo.m e.omc.N c.5me.m o.osm.m NH no «N on N see_me can we on sN on N sas.ms ces_ma or o anew no .cz noenom osnv aownoaumom powwow mane Eoenoaumom assenoemmofi enoa essenaemsoa semen one we mHo>oH oeonmoonanouomoouem sowhoaeoom no noemeoxo oegaom an 0 mo woommo one N-H.oHoo:a osaemmewnoe .HH oenoe -53- Table 12 shows the effects of 0 hr sample excision on ATP levels of the longissimus muscle. In the 0-45 sampling group, the LD-L 45 min ATP levels were definitely higher than those of the 0 hr, 15 min or 45 min LD-R muscle samples (P < .05). Levels of ATP in the 2 hr LD-L muscles were significantly (P < .05) higher than those in 2 hr LDbR. While no significant differences existed within the same postmortem time period in the 0-15 sampling group, there was an obvious trend for ATP levels in ID-L to remain higher than those at corresponding postmortem times in the LD-R muscles. When comparing sides (LD-R vs LD-L) of all three sampling groups, it is readily apparent that 0 hr sampling was responsible for lower ATP levels. This effect upon ATP level was probably attributable to the vigorous muscle contraction attendant with LD sample excision at or shortly after the time of exsanguination. The lower ATP levels very likely resulted from.the activation of myofibrillar ATPase required to Table 12. The effect of 0 hr sample excision on postmortem ATP levels of the longissimus muscle. 1 2 Right longissimus Left longissimus ‘ Postmortem time period Postmortem time period 5353:: 0 hr 15 min 45.min 2 hr 15 min 45 min 2 hr 12 2.10b 2.00b 1.58b 0.43C 3.72a 1.52b 5 2.16a b 2. 03a b 1.65""b 0.92b 3.26a 3.46a 1.16b 5 3.62a 3.28a 0.92b 3.48a 1.63b IValues expressed as micromoles/g muscle. Means with the same superscripts do not differ significantly (P1> .05). -64- operate the contractile mechanism. The lower ATP levels, in all proba- bility, could be responsible for the increased glycolytic rate observed as a result of 0 hr sample excision. The effect might be expected from the reported controlling influence that ATP levels have on glycolysis, especially on the phosphofructokinase reaction (Wood, 1966; Scrutton and Utter, 1968). Electrical stimulation of the contractile mechanism also has been reported to activate the phosphorylase and phosphofructokinase enzymes (Karpatkin gt_31., 1964; Ozand and Narahara, 1964). Table 13 summarizes the CP levels observed at various postmortem time periods as a result of 0 hr sample excision. In the 0-45 sampling group, LD-L 45 min CP levels were significantly (P'< .05) higher than those at 0 hr, 15 min or 45 min in the LD-R.muscles. In the 0-15 group, LD-L 15.min CP levels were considerably higher (Pi> .05) than those at 0 hr or 15 min in the LD—R muscles. The CP levels at 2 hr postmortem were quite similar in all sampling groups from both right and left LD muscles. Table 13. The effect of 0 hr sample excision on postmgrtem creatine phosphate levels of the longissimus muscle. ’2 Right longissimus Left longissimus Postmortem time period Postmortem.time period figiggf 0 hr 15.min 45 min 2 hr 15 min 45.min 2 hr 12 0.49b 0.36b 0.24b 0.19b 1.85a 0.30b 5 0.30b 0.10b 0.11b 0.13b 1.81a 0.59b 0.20b 5 2.20a 0.922"b 0.04b 1.562"b 0.09b gValues expressed as.micromoles7g muscle. Means with the same superscripts do not differ significantly (Pi> .05). -65- When comparing all three sampling groups, it is apparent that the 0 hr sample excision drastically reduced CP levels. Thus higher CP levels among the muscles not incised at 0 hr provided for a ready source of ATP. This observation could possibly help account for the slower rate of glycolysis observed among those pigs not sampled at 0 hr. The CP levels observed in this study were lower than those reported by Kastenschmidt 31,31. (1968), but no explanation was apparent except for possible breed differences. To further examine the effects of 0 hr sample excision on rate and extent of postmortem glycolysis of the LD muscle, three pigs from the two sampling groups, 0-45 and 0-15, were designated as "normal" and three pigs from.these same sampling groups were designated as "low quality" based upon rate of postmortem.pH declines and 24 hr transmission values and subjective quality scores. Nermal LD muscles had significantly (P'< .05) slower pH declines, lower transmission values and higher subjective quality scores than the low quality LD muscles (Table 14, Figure 2). In addition to the glycolytic metabolites previously discussed levels of G-l-P, F-6-P, glucose, ADP and.AMP were also determined on samples from both (right and left) LD muscles of these pigs. These data are presented graphically (Figures 3 and 4) to illustrate the results previously dis- cussed and to show the differences in response to the effects of 0 hr sample excision between normal and low quality LD muscles. Figure 2 shows that there was a difference in the postmortem pH pattern of LD muscle between the normal and low quality pigs. The 2 hr pH values of normal LD muscles were significantly (P‘< .01) higher than those of the low quality pigs (Table 14). At 45 min postmortem, only the pH -66- N = normal LQ = low quality 6.50__ N—LD-L LD-R = right longissimus LD-L = left longissimus 6.25 \. 6.00 \\ \ ‘\\ 5.75 \ ‘~ \. \\ \ \ 5.50 \ \\ \ \ .. “.\ ‘\, 5. 25. -'-~- ‘:::‘~ \ 5.00 ! *J* L. : } 9 0 hr 1 45 min 2 hr 24 hr min Postmortem time Figure 2. Postmortem pH pattern of normal and low quality longissimus muscle as affected by 0 hr sample excision. Table 14. The effect of 0 hr sample excision on certain qualitative assessments for normal and low quality longissimus muscle. _‘iNormal quality Low quality Quality Right Left Right Left assessment longissimus longissimus longissimus longissimus Transmission b b a a valuesli4 12.7 11.3 69.9 66.8 Subjective a a b b quality scoresz’3 11.3 12.3 6.0 6.3 pH, 45 min3 6.10b 6.56a 5.84C 6.16b pH, 2 hr4 5.85b 6.29a 5.29C 5.37c iLower values correspond to more normal musculature.. 2Higher values correspond to more normal musculature. 3Means with the same superscripts are not significantly different (P:> .05). 4Means with the same superscripts are not significantly different (P1> .01). -67- LD-L pH values were significantly (P < .05) different between normal and low quality pigs. The pH values of the LD-L muscles 45.min postmortem were significantly (P < .05) greater than those of the LD-R muscles within quality groups. At 2 hr postmortem only the LD-L pH values of the normal muscles were significantly (P‘< .01) higher than those of the LD-R. These data indicate that 0 hr sample excision had a greater effect on normal muscles than on the low quality LD muscles. Postmortem changes in muscle glycogen and lactate quantities (figure 3) corresponded to the magnitude of the pH declines, 3.3. glycogen dimin- ution essentially paralleled the pH drop, while lactate accumulated in inverse proportion to these levels. Glycogen values were consistently lower and lactate levels uniformily higher in the low quality.musc1es than those of the normal pigs at least until 2 hr postmortem. The 0 hr sample excision had a.markedly greater effect on both of these metabolites in normal muscles than in those of lower quality. At 2 hr postmortem, glycogen and lactate levels were similar between the low quality LD-R and LD-L samples; whereas normal LD-R had considerably less glycogen and more lactate than those of the LD-L muscles. The glycogen and lactate curves (Figure 3) support the pH curves (Figure 2) in that low quality muscle exhibited a faster rate of postmortem glycolysis than normal muscle, while 0 hr sample excision more readily affected postmortem glycolysis in normal LD than LD muscle in the low quality carcasses. The postmortem hexosemonophosphate (G-l-P, G-6-P and F-6-P) curves are presented in Figure 3. No explanation is apparent as to why the F-6-P curves did not parallel those of G-6-P as observed by Kastenschmidt 33 21. 60 45 3O 15 100 75 25 Micromoles/g muscle -68- N-LD-L ' NeLD-R )— DQ-LD-R Glycogen - (glucose equivalents) ll 11 cAJ -LD-R NQLD-L Lactate Glucose l, l Figure Ti. 1r 15 45.min min l. OIhr 15 (min Postmortem time N-LDbR G-l-P I I 3. The effect of 0 hr sample excision on postmortem levels of glycogen, lactic acid, glucose, glucose-l-phosphate, glucose -6-phosphate and fructose-6-phosphate between normal low quality (LQ) longissimus muscle LD-L = left longissimus). N) and (LD-R = right longissimus; 45 min 2 hr -69- (1968). The latter authors reported considerably lower levels of F-6-P in porcine LD muscle than those obtained in this study. The only differ- ences observed for the F-6-P curves in the present study was that the low quality group LD-L 45.min postmortem muscles contained higher levels (F-6-P) than the other muscle samples at 45.min postmortem. Except for the 0 hr samples, G-6-P levels were consistently higher among low quality muscles than normal LD muscle, especially at 45 min and 2 hr postmortem. In general, the G-6-P levels were relatively high at 0 hr and minimal between 15.min and 2 hr postmortem; however, in all cases the 2 hr samples had higher G-6-P levels than those at 45 min postmortem. The G-6-P levels of the low quality LD muscles were essentially the same or slightly higher at 2 hr than at 0 hr; whereas, the G—6-P levels of the normal LD muscles were considerably lower 2 hr postmortem than at 0 hr. The LD-R muscles, which were incised at 0 hr, exhibited higher G-6—P levels at 45 min and 2 hr postmortem than the LD-L muscles. This effect of 0 hr sample excision on G-6-P levels was particularly obvious among the normal muscles. The 0 hr G-l-P levels (Figure 3) were similar between normal and low quality LD muscles. The G-l-P levels of low quality LD-R.muscles increased .markedly until 15.min postmortem, remained relatively constant between 15 min and 45 min and then gradually decreased until the 2 hr postmortem sampling period. The G-l-P levels of the normal LD-R muscles increased gradually from 0 hr until 45.min and then increased more rapidly until 2 hr postmortem. The LD-L muscles of both quality groups gradually increased -70- in G-l-P level between 45 min and 2 hr; however, the low quality muscles had consistently higher values at both postmortem time periods. At 2 hr the normal LDbR muscles had higher G-l-P levels than the low quality LD-R muscle samples. There were no differences in 0 hr glucose levels between the normal and low quality LD-R muscles (Figure 3). Glucose levels and the post- mortem patterns agree favorably with the work reported by Kastenschmidt 23,21. (1968). Glucose levels increased.more rapidly with time postmortem among the low quality muscles than in normal muscle and they were higher in the low quality muscles than in the normal LD muscles at all of the postmortem time periods studied. The LD-R samples (0 hr incision) had more glucose than LD-L muscles at corresponding postmortem time periods. This difference was most obvious among normal muscles than for the low quality muscles. Accumulation of glucose postmortem can result from a- amylase activity upon glycogen as reported by Lawrie (1966a). Levels of ATP (Figure 4) were consistently higher among normal LD muscles especially the LD-L muscles than the low quality muscle samples. Except for the normal LD-anuscles, ATP levels gradually decreased from 0 hr until 2 hr postmortem. The normal LD-anuscles exhibited a considerably higher ATP level at 45 min than all other groups. Even though a marked (lecrease in.ATP levels occurred in these normal LD-L muscles between 45 min and 2 hr postmortem, the (ATP) levels at 2 hr were comparable to those of the normal LD-R.muscles at 0 hr. The LD-L muscles contained consistently more.ATP than the LD-R.muscle, within quality groups, especially among normal muscle samples. -71- 6 - 5 m: LQ‘LD'R V’A ‘ N-LD-R fl 4 )- 3 n - -L 2 .— .3 8 l - ADP 3 on I l J a (D '5' g 205‘- 1. n— 8 CP «4 =1 2.0”- .8 b N-LD-R 1.5 - .6 N-LD-L -LD-R ‘ 1.0 - .4 — LQ—LD-L 005 b .2 _ N—LD-R AMP fl I . 1 J 0 hr 45 min 27hr 0'5; i5 45 min ‘2 hr _ .min .min Postmortem time Figure 4. The effect of 0 hr sample excision on postmortem levels of ATP,.ADP,.AMP and creatine phosphate between normal (N) and low quality (LQ) longissimus muscle (LD-R - right longissimus, LD-L = left longissimus). -72- The levels of CP (Figure 4) were unexplainably low among all of the muscles included in this phase of the experiment, and in fact, CP levels were not detectable in any of the low quality muscle samples. The normal LD-R.muscles had very low levels of CP at 0 hr, but no detectable quanti- ties after 15 min postmortem. The normal LD-L samples had the highest CP levels at 45 min postmortem, and though its levels decreased, CP was still detectable at 2 hr. The ADP levels (Figure 4) were unexplainably higher than those re- ported by Kastenschmidt gt_gl. (1968). The normal LD-L muscles had appreciably less ADP than all the other muscle samples. While ADP levels remained relatively constant among the normal LD-R and low quality LD—R and LD-L muscle samples through 2 hr postmortem, the ADP levels of the normal LD-L muscles increased between 45 min and 2 hr. The AMP levels (Figure 4) were similar among all the LD-L muscle samples studied, except that normal LD-R muscles had an obviously greater AMP content than low quality LD-R.muqcles at 15 min postmortem. ‘While the LDbL muscles had higher.AMP levels than the LD—R.muscles at 45 min and 2 hr, the AMP levels gradually decreased in the muscles of all quality groups during this postmortem time interval. Table 15 summarizes the levels (24 hr postmortem) of all the glycolytic metabolites studied, except those of ATP and CP, since the latter two compounds were detected in very minute amounts and then only in a few of the samples. Levels of the glycolytic metabolites present at 24 hr post- mortem.should provide an indication of the extent of glycolysis. Although no marked differences existed between quality groups or between sides -73- Table 15. The levels of some glycolytic metabolites in normal and low quality longissimus muscle at 24 hr postmortem.1 Qualitygroup ____rgg Nermal Low quality Right Left Right iLeft Metabolite longissimus longissimus longissimus longissimus Glycogen2 3.2 5.6 1.9 2.1 Lactate 93.1 90.9 98.5 93.9 G-l-P 0.12 0.19 0.29 0.25 G-6-P 6.01 6.89 5.75 5.92 F—6-P 2.36 2.53 2.37 2.30 Glucose 5.80 4.68 6.31 6.30 ADP 5.54 4.10 5.86 5.72 AMP 0.28 0.26 0.20 0.24 1Levels expressed as.micromoles7g muscle. 2Glycogen levels expressed as micromoles glucose equivalents/g muscle. (LDbR vs LD-L) for these metabolites, several small differences were ob- served. The low quality LD muscles had higher levels of lactate, G-l-P, glucose and ADP’and lower levels of glycogen, G-6-P and AMP at 24 hr postmortem than normal muscles. Within quality groups, the LD-R.muscles had consistently higher levels of lactate and ADP and lower levels of glycogen and G-6-P than the LD-L muscle samples. The normal LD-R muscles also exhibited higher levels of glucose than the normal LD-L samples. From the data for glycogen and lactate levels, it appeared that the low quality muscles underwent (slightly) more extensive glycolysis than normal muscles, and that the LD-R.musc1e samples exhibited (slightly) more extensive glycolysis than LD-L samples. -74- In summarizing the effects of 0 hr sample excision on the LD muscles, it was obvious that resection of LD muscle at the time of exsanguination definitely resulted in a more rapid rate of postmortem glycolysis and that the ultimate muscle properties were altered. Nermal muscles or those exhibiting a relatively slow rate of postmortem.g1ycolysis were more markedly affected by 0 hr sample excision than the low quality muscles or those which showed a relatively fast rate of postmortem glycolysis. Excising the 0 hr muscle sample resulted in stimulation of contractile activity within the entire incised LD muscle; however the excised sample BEE 23 exhibited especially marked contractile activity before it was frozen in liquid nitrogen. ATP and CP are utilized by the contractile mechanism (Bendall, 1966; Lawrie, 1966a) and "slow-glycolyzing" muscle was reported to contain more ATP and CP than "fast-glycolyzing" muscle at the time of exsanguination (Briskey and Lister, 1968; Kastenschmidt 31 21., 1968). Thus the stimulation of contractile activity by the 0 hr sample excision could have conceivably reduced the ATP and CP levels to those normally observed in "fast-glycolyzing" muscle at the time of ex- sanguination. From an examination of Figures 2-4, it appears that the rate of glycolysis was inversely proportional to ATP levels. The considerably higher ATP levels, present in normal LD muscles not sampled at 0 hr, were most likely responsible for the reduced rates of glycolysis. The increased G-6-P levels after 45 min postmortem would be expected if inhibition of the phosphofructokinase enzyme occurred (Wilson gt 31., 1967). Phospho- fructokinase activity is reportedly pH sensitive and to be inhibited by -7 5- pH values of 6.0 or lower (Mansour, 1965). In the present study these low pH values were attained at or shortly after 45 min postmortem in all the LD muscles except for the normal LD-L samples. Rectus femoris muscle. The RF muscle is considered a "red" muscle as opposed to the LD which is classified as a "white" muscle. As such, the RF is implicated (Briskey'gt,§l., 1960b; Beecher 23.31., 1965b) as being more resistant to the development of low quality musculature than the ID. Thus, the RF muscle was included in this study to compare its response to 0 hr sample excision with that of the LD. The experimntal design for sample excision from the RF muscle was similar to that previously described for the LD. A combination of three different initial sampling times were used to compare the right (RF-R) and left (RF-L) rectus femoris muscles as follows: 0 hr RF-R with 45 min.RFLL (line 1, Table 16), 0 hr RF-R with 2 hr RF-L (line 2, Table 16) and 15 min RF-R with 2 hr RF-L (line 3, Table 16). Hereafter these three initial sampling times from the RF-R and RF-L will be referred to as 0-45, 0-2 and 15—2. The effects of 0 hr sample excision on postmortem pH decline of the rectus femoris muscle are presented in Table 16. The 0-45 group showed no significant (P1> .05) differences in 0 hr or 45 min pH values between the two sides (RF-R vs RF-L). The 24 hr pH values for this sampling group were lower (P'< .01) than the earlier postmortem.va1ues, but were nearly identical between the two sides. In both the 0-2 and 15-2 groups the 2 hr pH values for the RF-L were significantly lower than 2 hr -75- Table 16. The effect of 0 hr sample excision on postmortem pH decline of the rectus femoris muscle. Right rectus femoris Left rectus femoris Postmortem time period Postmortem time period gig? 0 hr 15 min 45 min 2 hr 24 hr 45 min 2 hr 24 hr 10 6.44a 6.39a - 5.44b 6.41a 5.43b 5 6.55a 6.32a 5.48c 5.84b 5.39C 6 6.62a 6.32b 5.56d 5.90c 5.47d lMeans with the same superscripts do not differ slgnlflcantly (P1> .01). pH for the RF-R muscles. While the 2 hr pH value of the 0-2 sampling group was not significantly (Pi> .05) different from the 0 hr pH among the RF-R.muscles, the 2 hr pH values of the 15-2 group were significantly (P < .01) lower than the 15 min pH of the RF-R muscle samples. While the 24 hr pH values of the latter two sampling groups (0-2 and 15-2) were significantly (Pl> .01) lower than the earlier postmortem pH values in these groups, the 24 hr pH values of the RF-R samples tended to be slightly higher than those of the RF-L samples. These data indicate that the RF muscles were affected opposite the LD muscles, 2.3. 0 hr sample excision appeared to inhibit rather than stimulate postmortem glycolysis. These differences in results between the two muscles were not expected since the RF.muscles appeared to contract just as violently or even more so than those of the LD following 0 hr sample excision. Subsequent to the detection of the 2 hr pH differences between the RF muscles in the 0-2 sampling group, muscle (RF) temperatures were ob- tained at 2 hr postmortem in the 15-2 sampling group. The temperatures -77- at 2 hr postmortem were 34.2% and 38.4°C for RF-R and RF-L samples, respectively. This difference was statistically significant (P < .01). The removal of skin and subcutaneous fat to facilitate excision of the RF-R muscle samples apparently allowed the remaining portion of the in- cised RF-R muscles to dissipate heat, while the RF-L which were not exposed until 2 hr postmortem tended to maintain ip’xizg temperatures. Despite the differences in postmortem pH declines no significant (I’> .05) effect on RF transmission values were noted. The RF-R and RF-L transmission values for the 0-45, 0-2 and 15-2 sampling groups were 15.6 and 18.4, 11.3 and 9.9, and 11.2 and 12.4, respectively. The RF values of glycogen (Table 17) and lactate (Table 18) appeared to substantiate the pH patterns. In the 0-45 sampling group neither glycogen nor lactate levels were significantly (Pi> .05) different among 0 hr RF-in or 45 min RF-R and RF-L muscle samples. Significantly (P < .05) less glycogen and more lactate was found at 2 hr postmortem in the RF-L muscles as opposed to the RF-anuscle samples in both the 0-2 and 15-2 sampling groups. Significantly (P < .05) more lactate accumulated at 2 hr than at 15.min postmortem in the RF-R muscles of the 15-2 sampling group. Comparable values for the 0-2 sampling group between 0 hr and 2 hr were nonsignificant (I’> .05). No.marked differences in 24 hr glycogen or lactate levels were apparent between sides (RF-R vs RF-L) in any of the sampling groups. Thus, it appears that the rate rather than the extent of postmortem glycolysis was affected by 0 hr sample excision. _73_ Table 17. The effect of 0 hr sample excision on postmortem glycogen levels of the rectus femorls muscle.1: Right rectus femoris Left rectus femoris Postmortem time period Postmortem time period fgiggf 0 hr 15 min 45.min 2 hr 24 hr 45.min 2 hr 24 hr 10 30.5a 33.7a 6.6b 35.8a 5.1b 5 36.4a 30.8a 4.9C 15.2b 3.8C 6 39.2a 32.9a 5.1C 17.8b 3.5C iLevels are expressed as.micromoles glucose equivalentsig muscle. 3Means with the same superscripts do not differ significantly (PI> .05). Table 18. The effect of 0 hr sample excision on postmortem lactate levels of the rectus femoris muscle. ’ Right rectus femoris Left rectus femoris Postmortem time period Postmortem time period NEiggf 0 hr 15 min 45.min 2 hr 24 hr 45 min 2 hr 24 hr 10 27.2b 34.2b 73.2a 32.2b 76.8a 5 19.8b 34.6b 68.8a 69.3a 77.6a 6' 18.5C 34.6b 71.2a 67.2a 74.2a lLevels are expressed as micromoles/g muscle. 2Means with the same superscripts do not differ significantly (PJ> .05). Table 19 summarizes the G-6-P levels in the RF-R and RF-L muscle samples. The G-6-P levels appeared to be relatively high initially, dropped to low levels between 15 min and 2 hr postmortem and then reached higher levels at 24 hr than those found initially. No significant (I’> .05) differences at 45 min (0-45 sampling group) or 2 hr (0-2 and 15-2 sampling -79- groups) for G-6-P levels were noted between RF-R or RF-L samples. However, at 2 hr postmortem in both the 0-2 and 15-2 sampling groups the RF-L muscles had more than twice as much G-6-P as the RF-R muscle samples. While the differences in G-6-P levels at 24 hr postmortem were only sig- nificant (P < .05) in the 15-2 sampling group, the levels tended to be higher among RF-R than RF-L muscle samples in the other two sampling groups. Table 19. The effect of 0 hr sample excision on postmortem.glucose-G- phosphate levels of the rectus femoris muscle. ’ Right rectus femoris Left rectus femoris Postmortem timegperiod Postmortem time period fziggf 0 hr 15 min 45.min 2 hr 24 hr 45 min 2 hr 24 hr 10 4.07b 0.91C 7.02a 0.91C 6.04a’b 5 3.12b 1.12b 7.02a 2.94b 5.70a 6 1.740 1.12C 8.24a 2.66c 5.78b ILevels are expressed as micromoles/g muscle. 2Means with the same superscript do not differ significantly (P1> .05). The ATP and CP levels of the RF muscles are presented in Tables 20 and 21, respectively. No significant (I’> .05) differences in ATP or CP levels at either 0 hr or 45 min postmortem.were evident between RF-R and RF-anuscle samples for the 0-45 sampling group. These reSults as well as the other glycolytic.metabolite data for the RF muscles indicate that stimulation of the contractile machinery by sample excision did not cause a reduction of ATP levels and thus did not enhance the glycolytic rate- -80- like that observed for the LD muscle. If myofibrillar ATPase was stimulated to any extent, then a very efficient maintenance of ATP levels occurred or the chilling effect associated with the RF muscle excision may have offset any stimulatory effects of the 0 hr muscle incision. Table 20. The effect of 0 hr sample excision on postmortem ATP levels of the rectus femoris muscle. ’ Right rectus femoris Left rectus femoris Postmortem timegperiod Postmortem time period fgiggf 0 hr 15 min 45.min 2 hr 24 hr 45 min 2 hr 24 hr 10 2.73“ 2.50“ 0.12“ 2.64“ 0.08b 5 3.72“ 2.51“ 0.20“ 0.72“ 0.13“ 6 3.60“ 2.49b 0.19“’d 0.76“ 0.10d ‘k ELevels are expressed as.micromoleé7g muscle. Means with the same superscript do not differ significantly (PJ> .05). Table 21. The effect of 0 hr sample excision on postmortem creatine phosphate levels of the rectus femoris muscle}:2 Right rectus femoris Left rectus femoris Postmortem time period Postmortem timegperiod jgiggf 0 hr 15 min 45 min 2 hr 24 hr 45 min 2 hr 24 hr 10 0.80“ 0.78“ 0.07“ 0.45“'“ 0.06“ 5 2.32“ 0.39“ 0.09“ 0.19“ 0.11“ 6 2.13“ 0.18“ 0.14“ 0.14b 0.09“ Ji.evels are expressed as micromoles/g muscle. 2Means with the same superscript do not differ significantly (PI> .05). -31- The ATP levels at 2 hr postmortem were significantly (P < .05) higher among the RF-R muscles than in the RF-L muscle samples in the 0-2 and 15-2 sampling groups. The ATP levels in the RF-R samples were significantly (P < .05) lower at 2 hr postmortem than at 0 hr or 15 min for both groups. The CP levels paralleled ATP concentrations. In contrast to the LD muscle previously discussed, low, but detectable levels of ATP and CP were found in most 24 hr RF muscle samples. A comparison of the glycolytic metabolites discussed above, together with levels of G-l-P, F-6-P, glucose, ADP and AMP was made for the RF muscle between normal and low quality groups similar to that presented for the LD muscle. In both the normal and low quality groups the same three carcasses included in the discussion of the LD muscles plus one additional carcass were used in each group. The 0 hr values represented the same three carcasses used for the LD muscle; 45.min values included two of the three carcasses; 2 hr values included the remaining carcass of these three plus the additional carcass (indicated above); and the 24 hr values included all four carcasses in each group. This approach was followed because 45.min and 2 hr RF samples were not obtained from the same carcasses with the sampling procedure used for this phase of the study. Thus, it should be kept in mind during the subsequent discussion that direct comparisons of glycolytic metabolites in the RF muscles be- tween postmortem time periods are limited by this sampling procedure. However, direct comparisons between muscles (RF-R vs RF-L) and quality (normal vs low quality) groups can be made for individual postmortem time periods. -82- N normal LQ low quality RF-R = right rectus femoris 6.5 \\ RF-L = left rectus \ femoris \ \ '\ \\ \ a. \ \ \ \s \ \ \\\ \ x. \ \ \\\ \ \\\\ \ 5.5 \ -__,_______\ -a I I 1 I 1 2'41 15 45 2'hr I f hr hr min min Postmortem time Figure 5. Postmortem pH pattern of normal and low quality rectus femoris muscle as affected by 0 hr sample excision. The pH patterns for normal and low quality RF-R and RF-L muscles are presented in Figure 5. There appeared to be a more rapid pH decline in the low quality RF muscles than among normal muscles. This observation was especially obvious in the RF-L muscle samples. Thus it appears that the temperature effects (cooling) caused by 0 hr sample excision influenced the rate of postmortem pH decline more among low quality RF muscles than in normal muscles. Glycogen and lactate levels (Figure 6) substantiated the results ob- tained for the pH patterns. The normal muscle samples retained more glycogen and accumulated less lactate than the low quality samples through the 2 hr postmortem time period. These observations were more obvious for RF-L than for RF-R muscles. Normal RF muscles contained more glycogen and less lactate than the low quality muscle samples at both 45 min and 2 hr postmortem. Differences in glycogen and lactate levels between sides 20 10 80 1.6_ G-l-P LQ-RF—L LQ-RF-L FGlycogen (glucose equivalents) l J - Lactate Micromoles/g muscle Figure Glucose ' !: J I .J r .mln EFhr 0 hr 45 mln 2 hr Postmortem time 6. The effect of 0 hr sample excision on postmortem levels of glycogen, lactic acid, glucose, glucose-l-phosphate, glucose- 6-phosphate and fructose-6-phosphate between normal (N) and low quality (LQ) rectus femoris muscle (RF-R = right rectus femoris; RF-L = left rectus femoris). -84- (RF-R vs RF-L) at 2 hr postmortem were greater in low quality than in normal RF muscles. The G—l-P levels (Figure 6) decreased from 0 hr to 45 min postmortem and then increased markedly from 45.min to 2 hr among all muscle samples. Normal samples had lower G-l-P levels than low quality muscles at each postmortem time period. The G-6-P levels (Figure 6) decreased from 0 hr to 45 min postmortem in all muscles and remained relatively constant between 45 min and 2 hr in the RF-R muscles. The RF-L muscles showed marked increases in G-6-P levels from 45 min to 2 hr postmortem. At 2 hr postmortem, the levels of G-6-P were higher in the RF-L muscle samples than in RF-R muscles and these higher levels were greater among low quality muscles than the normals. While the normal RF-L muscles exhibited an increase in F-6-P levels (Figure 6) from 45 min to 2 hr postmortem, the F-6-P levels of all the other samples gradually decreased from 0 hr to 2 hr. Additionally, the low quality.muscles exhibited slightly higher F-6-P levels than the normal RF-R samples. Glucose levels (Figure 6) remained relatively constant in the RF-R muscles from 0 hr to 2 hr, but they increased between 45 min and 2 hr in the RF-L muscles. This increase was slightly greater among the low quality muscles than in the normal RF muscle samples. Glucose levels of low quality muscles were consistently higher than those of normal RF muscles. The ATP levels (Figure 7) of the normal RF samples were consistently higher than those of the low quality muscle samples. While normal RF-R muscles showed little change in ATP levels from 0 hr to 2 hr postmortem, the ATP levels of the low quality RF-R samples decreased gradually during -85- 5.0 _- 1.0.- ATP .h. o O Micromoles/g muscle 9’ c: 2.0 1.0 !_~ I I I 0 hr 45.min 2 hr 0 hr 45 min 2 hr Postmortem time Figure 7. The effect of 0 hr sample excision on postmortem.levels of ATP, ADP, AMP and creatine phosphate between normal (N) and low quality (LQ) rectus femoris muscle (RF-R = right rectus femoris; RF-L = left rectus femoris). this same postmortem time period. The RF-L muscles showed a definite decrease in ATP levels from 45 min to 2 hr postmortem. The ATP levels of low quality RF-L muscles were almost completely diminished at 2 hr postmortem. -86- The CP levels (Figure 7) of the normal RF-R samples remained higher than those of the other muscle samples and they decreased steadily from 0 hr to 2 hr postmortem. The CP levels of the low quality RF-R muscles gradually decreased from 0 hr to 45.min and remained relatively constant until 2 hr postmortem. The CP levels of the normal and low quality RF-L samples were low and remained esdentially the same between 45.min and 2 hr postmortem. The ADP levels (Figure 7) increased from 0 hr to 45 min and then decreased from 45.min to 2 hr postmortem in all samples. Low quality RF-R muscles consistently had higher ADP levels than the normal RF-R samples. ‘The low quality RF-L muscles had higher ADP levels at 45.min postmortem and lower levels at 2 hr than all Other muscle samples; whereas the normal RF-L samples had lower levels at 45.min and higher levels at 2 hr than all other muscle samples. The AMP levels (Figure 7) increased from 0 hr to 45 min postmortem and then decreased in all muscle samples from 45.min to 2 hr postmortem. Low quality RF-R muscle samples consistently had higher AMP levels than the normal RF-R.musc1es. The low quality RF-L muscles had higher AMP values at 45 min and slightly lower levels at 2 hr postmortem than the normal RF-L muscle samples. While the postmortem curves of AMP and ADP were similar, levels of both of these nucleotides showed a more marked change between 45.min and 2 hr postmortem in low quality than in normal RFBL muscles. From Figure 5 it can be seen that normal muscle samples had slightly higher 24 hr pH values than low quality RF muscles and that the RF-L 24 -87- hr pH values tended to be lower than corresponding RF-R values. Table 22 summarizes the 24 hr levels of the metabolites studied. Normal muscles had slightly more residual glycogen, especially among the RF-L samples, and less lactate than the low quality muscle samples. Additionally, the RF-L muscles accumulated more lactate than the RF-R.muscles especially among the low quality samples. The 24 hr G-l-P and G-6-P levels were higher among the normal than the low quality muscle samples. The RF-R muscles tended to have higher levels of G-l-P and G-6-P than the RF-L muscles. Glucose levels were higher among low quality muscle samples than in normal muscles and the RF-L muscles contained slightly more glucose than the RF-R samples. The ATP and CP levels were slightly higher in normal than in low quality muscle samples at 24 hr postmortem; whereas, low quality samples had higher levels of ADP and AMP than normal muscles. From the data presented in Tables 16-22 and Figures 5-7, it is apparent that pH decline and glycogen degradation paralleled the post- mortem.ATP diminution. Although lactate levels were inversely related to the above metabolites, the accumulation of lactate was also propor- tional to pH fall and the reduction of glycogen and ATP. The ATP levels apparently played a role in the control of glycolysis among the RF muscles similar to that previously discussed for the LD muscles. The higher ADP and AMP levels in the low quality muscles, at least until 45.min post- mortem, in all probability enhanced the glycolytic rate among these muscles. With the introduction of the temperature variable in the RF muscles, it was not possible to determine if sample excision, at or shortly after -88- Table 22. The levels of some glycolytic metabolites in normal and low quality rectus femoris muscle at 24 hr postmortem.1 Qualityggroup ____ Normal __:_Low qualityg Right Left Right Left rectus rectus rectus rectus Metabolite femoris femoris femoris femoris Glycogen2 3.3 3.4 3.1 2. 2 Lactate 68.8 69.7 71.1 75.0 G-l-P 1.22 1.09 0.94 0.80 G-6-P 5.29 4.40 4.82 3.83 F-6-P 2.30 1.95 2.48 2.00 Glucose 3.18 3.74 4.36 4.60 ATP 0.17 0.09 0.07 0.03 CP 0.19 0.22 0.12 0.10 ADP 0.66 0.47 1.60 1.54 AMP 0.12 0.16 0.16 0.22 1Levels expressed as.micromoles/g.muscle. 2Glycogen levels expressed as.micromoles glucose equivalents/g muscle. exsanguination, affected the RF muscle similarly to that previously described for the LD muscle. Levels of glycolytic metabolites and the pH values at 45 min tend to indicate that the RF-R exhibited slightly faster rates of glycolysis than the RF-L at this postmortem.time period. However, much of the difference was, in all probability, nullified because of the lower muscle temperatures already existing in the RF-R than in the RF-L muscles at 45.min postmortem. -89- A Comparison of Postmortem.Differences between Several Porcine Muscles within the Same Carcass Briskey (1964) suggested that some muscles are more resistant to the development of PSE conditions than others. He attributed this muscle difference to variation in ”cooling rates or oxygen-retaining capacities between muscles. From observations of the carcasses in Groups I, II and III of this study, it appeared that most, if not all, of the muscles of a low quality carcass were affected when rapid glycolysis occurred in the LD.muscle. Thus, in addition to the LD and RF, two other muscles, 2.3., the biceps femoris (BF) and supraspinatus (SS) were sampled (0, l, 2 and 24 hr postmortem) from the carcasses in Group IV to study some qualitative properties. The six most "normal" and the six "lowest quality" carcasses of the pigs in Group IV were compared. The basis for categorization of these 12 carcasses into the two quality groups included the combination of transmission values, rate of postmortem.pH declines and subjective quality scores of the LD muscle only. Transmission values and 2 hr pH values of each muscle were compared between normal and low quality carcasses (Table 23). It was decided to use 2 hr pH values rather than the pH at some other postmortem time period, because the transmdssion values of both LD muscles of the pigs in Group IV were more highly correlated with 2 hr pH than with 45 min pH (r = -.69 and r = -.49, respectively). To justify this categorization of the car- casses into normal and low quality groups, normal LD muscles had signifi- cantly (P‘< .01) lower transmission values and higher 2 hr pH values than low quality LD muscles. There was a definite trend for all of the muscles -90- Table 23. Transmission values and 2 hr postmortem pH values of several muscles from normal and low quality carcasses. Transmission value. pH (2 hrpostmortem) Low Low Muscle Nermal quality Normal quality Right longissimus1 13.6 66.2 5.86 5.42 Left longissimus1 12.9 56.5 6.15 5.56 Right rectus femoris 10.4 15.4 Left rectus femoris2 9.7 20.7 Biceps femoris3 8.1 14.7 6.45 6.04 Supraspinatus 13.6 20.3 6.31 6.22 1Means were significantly different (P < .01) for both transmission and 2 hr pH values. 2Means were significantly different (P < .05) for transmission value only. 3Means were significantly different (P'< .05) and (P < .01) for trans- mdssion values and 2 hr pH values, respectively. studied (BF, SS, RF-R and RF-L) to have lower transmission values and higher 2 hr pH values among normal carcasses than those of low quality carcasses. Normal BF muscles had significantly (P < .05) lower trans- mission values and higher (P‘< .01) 2 hr pH values than the low quality BF muscles. The normal RF-L muscles had significantly (P < .05) lower transmission values than the low quality RF-L muscles. Because of the sampling procedure used, there was an insufficient number of 2 hr pH values to allow for an evaluation of the RF muscle. However, from the postmortem pH patterns shown in Figure 5, it is apparent that the low quality RF muscles had more rapid pH declines than normal RF muscles. Simple correlation coefficients for transmission and 2 hr pH values of the LD-L muscles with those of the LD-R, RF-L, RF-R, BF and SS muscles - 91- from all of the carcasses (22) of Group IV were calculated (Table 24). Although low, correlations of 2 hr LDaL pH with 2 hr pH values of the LD—R, BF and SS were significant. Correlations between transmission values of the LD-L and those of the LD—R, RF-R and BF were also signifi- cant (P < .01), but low. Thus, it appears that none of the muscles studied (BF, RF and SS) was. entirely resistant to rapid rates of post- mortem glycolysis or development of low quality muscle characteristics when the LD of a given carcass was affected. The BF and SS muscle inci- sion and exposure (cooling) shortly after exsanguination most likely affected these muscles similarly to the sampling effects found in the LD and.RF muscles. Table 24. Simple correlation coefficients for transmission values and 2 hr postmortem pH values between some muscles.1’2 Transmission values 2 hr postmortem.pH values Left Left Muscle longissimus Muscle longissimus Right longissimus 0.64 Right longissimus 0.65 Left rectus femoris 0.35 Right rectus femoris 0.67 Biceps femoris 0.67 Biceps femoris 0.58 Supraspinatus 0.40 Supraspinatus 0.47 ICorrelation coefficients > 0.423 are significant—(P < .05). 2Correlation coefficients > 0.537 are significant (P < .01). SUMMARY The results of this study were obtained from l46.market-weight pigs slaughtered in four different groups. The distribution of red, white and intermediate muscle fibers, succinic dehydrogenase (SDH) activity, myo- glObin, total lipid, phospholipid and glyceride ester (neutral lipid fraction) levels were determined on longissimus (LD) muscle samples ob- tained from the pigs in Group I at or shortly after exsanguination. The relationship of these parameters to rates of pdeecline and subjective quality scores of the ultimate muscle properties was observed on normal and low quality LD muscles from the three breeds of pigs included in Group I. Heart weights of the pigs in Group I, III and IV were recorded a n :d'r their relationship to rate of postmortem pH decline and/or 24 hr transmission values observed. Muscle or rectal temperatures were obtained on the pigs in Groups II, III and IV at the time of exsanguination and at 45 min postmortem and their relationships to 45 min pH, transmission values and subjective quality scores was also observed. The effects of sample excision, at or shortly after exsanguination, upon pH and transmission values and glycogen, glucose-6-phosphate, lactate, ATP and creatine phos- phate (CP) levels from the LD and rectus femoris (RF) muscles were studied for the pigs included in Group IV. In addition to the above observations for Group IV pigs, glucose-l-phosphate, fructose-6-phosphate, glucose, ADP and AMP levels were compared among normal and low quality LD and RF muscle samples excised at several postmortem time periods. Transmission values and 2 hr postmortem pH values of the LD, RF, biceps femoris (BF) and supraspinatus (SS) muscles from the pigs in Group IV were compared. -92- -93- Nermal LD muscles had more red and fewer white muscle fibers, higher SDH activities and greater total myoglobin contents than low quality LD muscles. The fiber size of red and intermediate muscle fiber types was larger in low quality than in normal LD muscle. Nermal LD muscles tended to have higher total lipid levels and greater glyceride ester contents of the neutral lipid fraction than those of the low quality LD muscles. Landrace pigs tended to have more myoglobin and higher SDH activities than Poland Chinas or Chester Whites, while Poland China pigs tended to have more red fibers than the other two breeds. Heart weights of the low quality pigs in Group I tended to be lighter than those of the normal pigs. However, no significant correlations were obtained between heart weights and either 45.min pH (Groups I and IV) or transmission values (Groups III and IV). Observations from several pigs which had pericarditis indicated that heart function.may be more important than heart weight in contributing toward development of low quality porcine musculature. Muscle (LD) temperature at 45.min postmortem was found to be more highly related (negatively) to ultimate muscle quality indices than muscle (Groups III and IV) or rectal (Group II) temperature at the time of exsan- guination. While no identification of the factor(s) contributing to the differences in LD temperatures was.made in this study, in all likelihood, the ip_vivo temperatures at the time of exsanguination, as well as scalding, slaughter floor temperatures, and time lapse before carcass chilling con- tributed to postmortem muscle temperatures. Muscle (LD) incision of the pigs in Group IV, at or shortly after the time of exsanguination, stimulated contractile activity, significantly -94- increased the rate of postmortem.g1ycolysis and tended to decrease ulti- mate muscle qualitative characteristics. The LD muscles incised at the time of exsanguination had lower pH values, glycogen, ATP and CP levels and higher lactate contents at corresponding time periods through 2 hr postmortem, than LD muscles not incised until 45.min after exsanguination. This effect of muscle incision was greater among the normal than low quality LD muscles. Excision of RF muscle samples involved removal of skin and subcu- taneous tissues thus exposing the RF muscles to the atmosphere. The chilling effect resulting from exposure of the RF muscles tended to slow down glycolytic rates and apparently even nullified the effects of con- tractile activity resulting from muscle incision at an early postmortem time (0 to 15 min). The RF muscles not incised until 2 hr postmortem exhibited significantly lower pH values and levels of glycogen and ATP and higher lactate contents than RF muscles incised at or shortly after the time of exsanguination. The effect of chilling associated with early postmortem incision of the RF muscles was greater among the low quality than normal muscle samples. Postmortem levels of all the metabolites studied appeared to be related to the glycolytic rate in both the RF and LD muscles. Transmission and 2 hr postmortem.pH values of the RF, BF and SS muscles showed low, but significant correlations with those of the LD muscles. 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APPENDIX -110- 1b) LD pH and subjective quality scores Group Ia Appendix A. ost-mcrtem pHV Heart weight, .ID Quilit Heart Slaughter wt A 24; 3 hr 'hr C F 4 V. min 15 min O'hr th, (lbs) raise: 3O 53891610865236056 12112232332222322 «connotes-00.0.. 5555555555555555 noose-ounce.- 5555555555555 5 243621 02364882842 312312122 32332222223 coco-o... 55555555555555 (g)' 195 1 3 1 0 o. 2 9 2 7 9 m, 1 7 a. l 1 2 or 6 7 1 3 l a 5 a. 1 1 1 or A. 7 1 0 1 II ) .5 mm In! ( m3 5 .ma m : .fl ) 3 tN ....l S( W e nu 0. a1 r em e n o w en 1 D." n as S Na na ¢.+.fl.t OX x e ’8 etOt S 4 0n n dol ,.1 r 9 6.03.0 ve e S... A... mama .n in d.1.o.1 e 3 13 S labig team *a .0 22, 24, 25, 30. -110- Appendix A. Heart weight, LD pH and subjective quality scores Group Ia’b). SlaugHtEr" Heart .ID posthOrtem EH‘ w 45 ‘7 wt t 15 24, Qudlit Afiihai ’(1bé) (g) o’hr min min '3 hr ‘hr' H'”d"F l-PC-B 195 — 6.44 6.18 6.06 5.55 5.58 3 5 5 2-PC-G 180 — 6.18 5.92 5.80 5.64 5.56 1 3 3 3—PC-G 190 - 6.44 6.54 6.55 5.92 5.26 3 4 4 4-PC-G* 194 — 6.35 6.28 6.14 5.24 5.22 1 2 2 5-PC-G 172 - 6.02 5.52 5.72 5.26 5.28 1 2 2 6-PC-G 180 — 6.10 6.12 6.06 5.29 5.25 2 3 3 7-PC-G 148 — 6.15 5.94 5.67 5.48 5.30 1 4 4 8-PC-G 161 — 6.28 6.18 6.10 5.55 5.56 2 4 5 9-PC-G 168 - 6.19 5.88 5.63 5.54 5.37 1 3 3 lO-PC-G 163 249.7 6.26 6.14 6.05 5.40 5.28 1 3 3 ll—PC-G 163 535.7 6.36 6.11 6.05 5.31 5.27 2 4 4 12-PC-G 210 291.3 6.06 5.76 5.65 5.28 5.23 1 2 2 13-PC-G 209 ' 249.6 6.21 6.15 6.22 5.37 5.25 3 4 4 14-PC-G 191 237.3 6.14 5.80 5.61 5.34 5.30 1 2 1 lS-PC-G 204 255.1 6.19 5.58 5.42 5.40 5.33 1 2 2 16-PC-G 165 222.1 6.20 5.68 5.27 5.34 5.24 2 3 3 17-PC-G 198 311.2 6.02 5.66 5.33 5.26 5.20 1 2 2 18-PC-G 205 327.8 6.35 5.90 5.50 5.40 5.29 2 5 4 19-PC-G 201 272.9 6.33 6.14 5.80 5.30 5.20 1 2 3 20-LR-G 216 248.1 6.30 5.98 5.48 5.14 5.19 1 3 2 21-LR-G 211 269.4 6.48 6.43 6.26 5.20 5.23 2 4 3 22-LR-G 220 248.2 6.22 6.05 5.70 5.32 5.27 1 3 3 23-LR-G 226 282.7 6.40 6.33 6.08 5.14 5.15 1 3 2 24LLR—G 226 258.9 6.17 6.12 5.97 5.23 5.23 3 5 3 25-LR-G 240 276.3 6.20 5.84 5.20 5.16 5.18 1 2 2 26CLR-B 208 258.2 6.34 6.30 6.16 5.22 5.19 1 3 2 27-LR-B ‘ 230 312.7 6.36 6.31 6.26 5.21 5.21 3 4 3 28-LR-G 183 251.8 6.22 6.18 6.18 5.27 5.26 3 4 3 29-LR-G 196 264.5 6.36 6.28 6.18 5.30 5.31 3 5 3 30-LR-B 196 262.1 6.34 6.27 5.98 5.22 5.20 1 2 2 31—LR-B 191 299.5 6.36 6.29 6.27 5.33 5.38 3 5 4 32-LR—G 190 285.0 6.30 6.32 6.20 5.36 5.36 4 5 4 33—LR-B 192 231.6 6.28 6.24 6.02 5.24 5.25 2 4 3 '34-LR-G 201 258.5 6.36 6.26 6.12 5.28 5.22 2 4 3 35-LR—G 192 278.9 6.32 6.26 6.10 5.28 5.23 1 4 3 36—CW—G 204 305.5 6.14 6.09 6.07 5.22 5.26 3 5 4 37-CW-B 225 263.4 6.34 6.36 6.45 5.28 5.30 3 5 5 38-CH—B 213 265.1 6.14 6.22 6.16 5.24 5.25 3 2 2 39-cw-G 198 304.6 6.31 6.30 6.26 5.32 5.26 4 5 5 40-CW-G 192 322.6 6.28 6.32 6.38 5.58 5.36 4 4 5 4l-CW—G 210 265.6 6.31 6.24 6.13 5.22 5.26 4 5 4 ERilled by overdose of Na pent. anesthesia. aHogs included in text as "normal" (N): 3, 8, 10, 11, 13, 21, 27, 29, 31, 32, 36, 37, 39, 40, 41. ‘ 1 bHogs included in text as "lower quaiityu (10): 14, 15, 16, 17, 19, 20, 22, 24, 25, 30. .JIIII:::1_______________________________________________________________________________ g“."I‘otal 6.8 5.3 I. 9.7 7.3 Relat ive si zeU w , 9.1 4.2 Area % -lll- Muscle fiber types and succinic dehydrogenase activities (Group I). ! Muscle fiber types 9.1 15.6 78.0 6.4 12.0 5.6 2 60.1 10.7 16.8 75.4 7.8 Number % 27.3 63.6 R 29 15min" activityg' mm Appendix B. 6A71140250 n o o o o 500055676565 2521868801 87666755m5 6648843HI-68 0....- 4554575454 0307233908 soot-onco- 99767797m5 cunénonb nv4.0unvouow I 0 I D O I I O _D 8V7.:unonb mwAenD.b 3232773923 DUO-IO... 7.2a ouacouRvanvO 7.": 7.0.nlno R87.pu 2612398178 %&&&%m%553 1111121111 2646769440 0 o o o 0 8986662496 5605757649 I O no.b 7.7.7.7.:un..o 2869694021 .0000. 868900 346 7288371752080042079784915009 .0. 5993134204514525 8524 586 G$$$4$£4$$4 C mmammmnflm 45678901} 111111222 6.8 9.3 9.8 12.6 80.3 7.1 10.8 5.9 20.2 70.0 4. 3. 4 2 3 O 7 9 7 9 1 44 887 _.uo_ on. _.75.407 66 67.6 52 246 —.uo_ co. __2 .512 2 222 02321591 amu moles succinate oxidized/min/g frozen;'powdered muscle at 37°C. Fibers/sq. in. of picture analyzed. ‘Killed by overdose of Na pent. anesthesia. b -112- Myoglebin (Grbup I). Appendix C. OgMb . T Amount 7‘ ”'Kmo'unt“a 4 unt‘ 49978622791337471039074689869206177971606 conceal-9w. .00. 0.0. o o 222 372211533 W32334323343342 flue-.02 00.20w85858758553202208000.580072505500528 . I. I 0"... I .0. O. C... 0.... 8345293091.241827798956931o 89596220929198 4435524455656435454343355 22333344545632 09447008806247063128465955855831000881756 .0. 00.000.00.00... 0 37878996605 30 048324045 9224254636685 86 55222530088075856222250025825755855032538 0...... I 0.0.0.... 0 O 0.. 064461121683 6303849 44469 853450 202128 02410674284878098346663414286618425036456 C 0.. .Ifi‘OIIAHIOOOOO .910318888 440092 3529 enamel 90 9608 5058827255827030955852028522 83 o to... one... o 0.0 l 0 Que—.0 9182 4 91 365 2433410v06 o 00%. o 0 on!” 8 8 8 33 32 2m34 416252946713324324030.03.847.89.17.465928.8om790w DIOOOIOOOOOOOOIOOOO O O...- 3%52308619455144MQ726364653940369783W7535 7556876565H673 96MM9 MB 67MBHMNM8777 5779 82850578053 *Killed by overdose of Na pent. anesthesia. any. moles/g froz'en', 'powdered muscle. -113- Appendix D. Lipids (Group I), ,. , Phospholipid _ 5i§5eride esters Lipid (% fregg wt) (0 hr LD) (0 hr LD _ ueqYZ—ueqP/ ue ue _Animal Serum Livgg, LD (045;) 4g LD g lipid g LD g lipid l-PC-B - - - _ - - - Z-PC—G _ - - - - - - — 3-PC-G 0.31 4.92 5.04 5.40 107.1 42.9 851.2 4.100;» - - - - - — - 5-PC-G - — - - _ - - 6-PC-G - - - - - 7-PC-G - - - - _ - - 8-PC-G 0.27 5.16 3.96 7.92 201.0 31.5 795.4 9-PC-G - — — - - — - lO-PC-G 0.32 5.41 2.62 5.48 211.4 15.6 595.4 ll—PC—G 0.29 5.70 3.39 7.71 227.3 22.1 651.9 12-PC-G - - - - - - _ 13-PC-G 0.34 5.69 6.01 4.30 71.5 55.4 921.8 14-PC-G 0.35 5.79 2.22 4.16 162.0 19.1 860.4 lS-PC-G 0.36 4.87 5.25 7.32 139.4 31.6 601.9 16-PC-G 0.26 6.01 2.14 8.18 383.2 13.5 630.8 17-PC-G 0.30 4.53 2.61 8.48 325.8 17.0 651.3 18-PC-G - - - - _ - _ 19-PC-G 0.31 5.62 3.78 7.49 198.1 29.0 767.2 20-LR-G 0.34 4.97 3.26 6.25 192.8 23.3 714.7 21-LR-G 0.29 5.15 3.83 6.28 164.0 37.5 979.1 22—LR-G 0.33 6.38 5.52 7.14 143.6 42.7 773.6 23-LR-G - - - - - _ - 24-LR-G 0. 40 4. 93 4. 30 4. 52 105 . 4 40. 5 941. 9 25-LR—G 0.38 5.97 3.98 7.68 192.9 33.2 834.2 26-LR—B _ - - - _ — — 27-LR-B 0.26 5.20 6.92 7.18 103.8 52.0 751.4 28-LR-G - - - - - _ - 29-LR-G 0.32 4.81 4.20 8.34 198.8 35.6 847.6 30-LR-B 0.43 5.63 3.85 7.20 186.6 32.1 833.8 31-LRPB 0.34 4.81 3.65 8.80 241.4 34.4 942.5 32-LR—G 0.33 6.48 5.27 7.85 149.1 44.0 834.9 33-LR-B - _ _ - — — — 34-LR-G _ - - - — — _ 35-LR-G - - - — - - — 36-CW-G 0.31 5.61 3.56 4. 8 137.3 31.5 884.8 37-cw—B 0.37 5.89 4.11 8.40 204.5 33.1 805.4 38-CW—B - - — - — — — 39-cw—G 0.26 5.66 5.25 5.28 100.6 53.1 1011.42 40-cw.G 0.34 5.67 3.75 3.25 88.0 32.2 858.7 41-cw—G 0.36 5.34 3.25 7.88 202.3 32.9 1012.3 ¥Ki11ed by overdose of N3 pent. anesthesia. -ll4- oo.o m.oOH 3.m0H H N H m3.m m.HN mm.H m.mN omH =-0N Hm.o m.NOH m.HOH 3 m H N3.3 o.mN NN.H w.mN 3mH mxumH ON.o N.mOH m.NOH 3 N N me.m 3.HN ON.H 3.3N NNH muwH Hm.o c.3OH m.NOH 3 m m 3N.N N.HN NN.H m.om HmH =-3H mm.o N.mOH w.HOH m 3 3 mm.m m.NN 3H.H 0.0m 33H =-oH 3H.o w.wOH w.3OH m m m aw.m H.0N NN.H w.mN me mq-mH No.0 w.mOH N.NOH 3 3 N HN.m 3.HN b3.H N.wN 33H mx-3H N3.o c.3OH H.N¢H m m m Ho.3 m.HN mm.H o.Hm mmH =-NH mo.m 3.mOH o.NOH N N N mm.m m.HN bH.H o.mN mmH n-NH mm.o m.3OH o.3OH m m N 33.3 o.mH mm.H m.om me mx-HH 0H.o m.m0H H.NOH N N N eo.m N.NN NN.H N.wN NNH mx-0H oe.m N.3OH o.m0H 3 N H >N.N o.NH mm.H m.wN H3H 04-3 NN.3 m.oOH o.3OH m m 3 03.3 w.wH No.H w.mN mmH mm-w NH.o m.N0H N.NOH m N m pm.m w.HN NN.H m.wN oNH no-3 3m.m w.mOH c.3OH m N H NN.3 N.NN NN.H m.Hm 33H =-o Ho.o m.oOH 3.NOH 3 3 3 No.3 o.ON mo.H m.Hm 3mH >-m om.o N.mOH 3.N0H m m N NN.3 H.HN NH.H H.Nm 33H NN.3 3N.m o.3OH N.HOH 3 3 3 NN.3 m.NN oo.H N.¢N NNH =-m om.m o.mOH 3.NOH 3 3 m mm.m m.HN NN.H o.Hm me =-N mm.m H.3OH m.NcH m 3 3 03.3 e.NN 3N.H N.NN mNH »-H AcHa,m3v AnHe_m3v Ad: ov a o z AN.=HV a 28: A.=HV .pHv ApHv :opmmm ma NH n4 propm N«.Hp=o 38pm pmpprHsp apmaog as “how whflwdhwgwe Ohm QMOA HflMxoflm mmdohdo .HH 95.5 .m fiwcomn: -115... mH o o.mOH m m m Ho w ¢.NN O.mN mfiH BUIO¢ HN.m m.v0H m ¢ m vb.¢ onN N.Hm NoH Elam Ob.m o.oOH fl m H mm.¢ m.mH 0.0m ObH omlwm ON.o w.¢OH m m m w¢.¢ H.HN m.mN me lem $0.0 H.mOH m ¢ ¢ mm.m H.0N womN mmH Mlem ww.m ¢.mOH m m H N¢.¢ ©.wH woom bmH omlmm Nmow m.wOH fi m N Hwofi b.0N NoaN wwH Elfin Nm.m w.¢OH m m H Nm.¢ moON oumN mMH mlmm m¢.m H.¢OH m m m mmofi m60N moon NmH Bole ow.m o.mOH ¢ w m ma.¢ w.ON m.om owH VIHm flo.@ mum0H m V m ¢¢.m boHN mon me mlom O¢uo m.VOH m m m Haod b.0N bN H m.mN mMH MNImN Em.m m.m0H m m N mm.m o.NN ON.H m.mN NfiH FINN mHom «.mOH w v w bb.v N.HN mm.H m.wN me mNIbN Nmom m.mOH m m N omov N.NN bH.H m.om wflH mle b¢.w o.¢OH m fi m mm.m H.wH mm.H w.®N O¢H QIMN No.w m.dOH m w m omcv ooHN ON.H m.wN mmH mlvN wN.o VovOH ¢ ¢ m m.bH mvoH m.mN wwH BDImN NH.o wmeH m fi v N.NN NN.H mch NfiH mNINN mm.m m.¢OH m m H bomN ONoH OcNm me EIHN 3.3 30.3.3 #3 a; $30.80 A 60:59:03 HH 9.5.5 .H NHEGQE .mfifiufimfihom 63.83:: aofivoamfi fiovuofimoma. N.NO OO.O OO.H 3 O O O.OO ON.O O.OOH O.3OH N.3OH O.OOO OHN O-N-ON O.O3 ON.3 OH.H 3 3 O O.3O ON.O O.OOH O.OOH O.3OH H.OON OON O-=-OH O.OO OO.3 O3.H O O O O.NO OO.O O.OOH N.3OH 3.OOH O.3ON OHN N.>-OH O.OO NN.3 NH.H O 3 3 O.OO O3.O O.OOH O.3OH O.3OH O.OOO NNN N-=-OH O.O3 OH.O OH.H O O O O.NO OO.O O.3OH O.OOH N.NOH O.OOO ONN O-=-OH O.O3 HO.3 OO.H N N N O.OO OO.O N.OOH 3.3OH O.NOH O.O3N OON O-N-OH O.OO OO.3 NO.H O 3 O O.Ne OH.O 3.3OH O.3OH O.OOH O.HON NON N-N.3H 3 3.OO O3.3 O3.H O O N O.NO OO.O O.3OH O.3OH O.OOH O.OHN OON O-N-OH n O.OO OO.O ON.H N N H O.OO OO.O 3.OOH 3.OOH O.3OH O.OON OON O-ON-NH . O.O3 3O.3 O3.H O O 3 O.N3 O3.O O.3OH O.3OH O.3OH O.HON NON O->-HH O.OO O3.O OO.H 3 3 N O.OO OO.O O.3OH H.OOH 3.OOH O.OON OON METOH O.ON OO.O OO.H H H H O.OOH ON.O H.5OH N.NOH O.OOH O.OON OOH *m-N-O H.OO 3O.3 OO.H H H H O.OO OO.O O.NOH O.OOH O.NOH O.OON OON 3N->-O N.N3 NH.3 OH.H H N N O.OO OO.O N.OOH O.OOH N.OOH O.OON OOH O.N-O N.N3 3H.O OO.O O O O O.N3 O3.O O.NOH O.OOH O.OOH O.OON NOH O-=-O O.OO 3O.3 OO.H N 3 N O.OO OO.O O.OOH O.OOH O.OOH O.OHN OOH O->-O O.O3 OO.3 OO.H 3 O O O.O3 OH.O O.3OH 3.OOH O.3OH O.HNN OOH O->-3 N.O3 NN.3 ON.H N 3 O N.ON OO.O O.3OH O.3OH O.3OH O.OON OON N.N-O O.O3 33.3 OO.H O 3 O -- ON.O H.3OH N.3OH O.3OH O.O3N HOH m.n-N O.N3 OO.O NO.H N 3 N -- OO.O O.OOH O.OOH O.OOH H.OON NOH O-=-H a OHOH AN.3HV H.3Hv N O m p=H3> pHa O3 3H5 O3 OHNOO p: O HOV «3 HOHV HuaH=3 65 Ed: 60h“ mm0fl¥0fl£uv PHHdR—G GOMmmfiSmflth Saw 3 .WO #3 #thm #3 6N3 pHoH H3mxodm Hmov paupadmmmmp OH downmsuHO .HHH anode .m xH333OO3 -ll7- a 5m x MN H w m m m.mb mN w w.vOH N.NOH m.mmN wNN wimlwv m.bm N b¢.H m m N m.wm ON.@ H.mOH m.NOH mobvN wNN m|=|m¢ ¢.wm x ON.H v m m m.Hm mo.m m.¢OH N.NOH ©.wHN OHN 0I>IN¢ 5.0% N NN.H N N N O.Nm Ow.m NomOH O.wOH w.owN ©NN ullev w.wm X m¢.H v ¢ ¢ m.mm mH.o O.VOH H.m0H F.5fiN mNN u|>|o¢ N.Nw N mN.H m m ¢ m.mw mo.@ m.vOH o.¢OH w.HmN VNN wlwlmm m.wm N bM-H v fi m m.©w mb.m ®.VOH O.NOH b.bNN NON mlwlmm bowm N mm.H N N N o.mm Ow.m b.mOH w.m0H m.¢wN wMN mlhlbm NoHfi x bO.H m m m o.bH mm.m womOH b.¢OH m.me NmH ¢|>Iom m.O¢ Nm.m MH.H v ¢ ¢ m.mN Om.® m.mOH N.¢OH m.wa NmH Qlkolmm n.0v om.¢ hm.H N ¢ H o.mm OH.o 0.30H o.m0H w.w¢N me oimlwm m.mv mN.¢ OO.H 3 v m m.No ov.w NomOH N.OOH m.mwN NwH Olmlmm N.mm HH.m mm.H v m m m.©¢ Om.m w.¢OH $.m0H ¢.ONN me mIWINm ®.om om.m hm.H m m m 0.0% om.m v.wOH O.NOH o.m¢N MHN mIMIHm N.©¢ b©.¢ mw.o w w N O.Nb Ob.m m.¢OH w.mOH m.mbN NwH wnwlom 0.0v 0H.v Om.H v m N m.m¢ oo.w N.NOH v.mOH N.NmN QON wIWImN H.N¢ Nm.v mm.o w m m w.VN mm.w v.mOH H.¢OH ©.bwm mmH Olmle w.mm mm.m ON.H m fi N N.Nm om.o V.VOH H.NOH H.mmN me mlmle H.0fl hm.m wN.H m w m m.~N mm.o w.mOH O.VOH m.omN va mlwloN w.mm OH.m mm.H m m m w.mH om.m w.vOH 0.60H N.NHm O¢N mIWImN v.bm mm.m bfi.H m m m w.om Ow.m N.NOH O.mOH w.mwN ovN mIWIVN m.H¢ mo.v OH.H m w v N.m© oo.m N.NOH N.NOH ©.mwN fiHN mlwlmN H.0w wo.v wH.H m m v N.Om om.® Nov0H m.mOH m.HwN mmH wIBUINN 0.0w bN.¢ mN.H fl m m N.mm mm.w woNOH N.HOH m.OoN mHN mlleN N OHOH Na: 7:: N O z SHE fie O3 .fia O3 .3 O 1 3 t. 3: H9533 cad ad: Nona mmocx0H53 mewmmm :OHmmflamcape mm 94 vane: 93 who :Hoq wmmxoam may ohswaho How 94 powsmsaam .AwoSQchoov HHH macaw .h xfiuconm< pH and quality scores of longissimus muscle and heart weight (Group IV). Appendix G. scores LD-L M C :3 3 ‘1‘ Q A 45 min 2 hr 24 hr M C F pH 2 hr 24 hr 15 min 45 min LD-R ostmortem time 1n wt (g) 0 hr 15 m Heart Slaughter wt (lbs) Animal 5 4 X 6.42 6.14 5.18 5.80 5.19 5.92 5.25 5.85 6.19 5.91 6. 243.9 193 4 6.13 5.85 5.13 6.10 6.25 6. 188 213.9 4-B 3 X 5.52 5.12 6.00 5.11 6.42 211 6-G 6.12 266.4 4 6.13 5.30 6.32 5.26 6.51 6.59 5.94 6.14 6.10 6.13 261 198 10-B* ll-G 4 6.19 255.6 -118— V‘LO 03% 5.87 5.13 6.35 l 6.17 6.16 5.82 5.12 6.23 6.16 2 6.20 2 7 196 254.1 12—G LO V‘ 6.64 5.96 5.12 13-G 4 240 240 l4—G lS-G 6.31 6.00 5.25 6.13 5.92 6.31 6.45 6.53 5.90 6.15 5.96 6.48 6.56 6.59 6.10 6.55 6.66 6.18 251.8 5.33 5.18 5.67 5.18 6.16 6.32 6.69 6.36 5.25 5.20 5.66 5.20 16-G 3 180 l7-G 6.34 5.31 5.82 5.25 216 18-B 5.90 5.32 5.31 5.30 243.1 198 279.2 4 4 3 5.50 5.30 272.3 00 O0 NN mm OH NN 4 4 X 6.49 6.02 5.27 2 6.13 5.26 6.59 237.0 174 22-G *Postmortem inspection indicated slight pericarditis. X Samples not obtained. -119- .Uofidwno «o: monadm N .mfifiuadofinom #333 68803623 qofloommnfl fiowhofiwomz. N66 66 N 2.6 3m6 .36 N 36 >36 No6 N 666 m36 N 3.6 N wumm m36 om6 N $6 36 3N6 N m36 >36 36.6 N 36 NH6 N 36 N muHm 366 m36 N 366 86 56 N 636 mm6 $6 N m36 86 N 66.6 N muom 066 56 N 366 86 066 N 86 366 3H6 N 66 036 N 666 N uan 666 mm6 N 666 636 >36 N 3w6 666 HN6 N mw6 m36 N >56 N muwH $6 mo6 N 36 om6 86 N 366 ~36 066 N «66 mm6 N 36 N an: 366 86 056 666 mm6 $6 H66 «36 3m6 36 N 036 .36 N N 366 uan 066 606 86 $6 $6 666 2.6 $6 336 N06 N 66 H36 N N $6 66H 666 86 336 36 >36 om6 366 N36 N36 86 N 36 3H6 N N wm6 wu3H No6 mm6 066 666 336 mo6 mp6 mw6 mm6 86 N . m36 66 N N 56 wuma 86 3N6 mm6 336 636 $6 $6 $6 wm6 mm6 . N. 666 m36 Mn N 86 oINH N66 wm6 mm6 «66 $6 $6 H36 066 w36 N 36 636 N mo6 N 636 at: 2.6 :6 N36 636 36 006 mm6 N.N6 $6 N 36 mb6 N w36 N 636 $73 666 N36 36 m36 $6 36 H66 636 «36 N 666 066 N m36 N wm6 aim 366 36 $6 $6 86 366 3b6 N66 036 N 666 mm6 N m36 N 666 $6 86 86 .66 M66 636 m36 066 w36 536 N m36 36 N 336 N mm6 awn. wm6 mm6 066 066 mm6 3N6 m36 m36 om6 N 3m6 mm6 N 636 N 636 $6 $6 3m6 $6 66 336 mH6 666 636 mm6 N HN6 636 N om6 N wm6 cum m36 wm6 mm6 666 wm6 wm6 $6 5.6 H36 N 066 N.N6 N . 366 N 636 m3 56 E6 006 $6 3m6 366 86 >36 36 wm6 mm6 om6 $6 $6 6H6 mm6 Mum 5.6 636 $6 86 mm6 036 $6 066 $6 NH6 $6 36 om6 $6 $6 a36 arm N mm6 636 366 N 66 $6 56 N 306 N N 56 636 66 mm6 “VIN .3 3m an m OH: H .E o .E 3m .5 N .E H a: o .3 3m .3 m 2d: a: 3m .3 N ad: 5m: a: o 35% m3 m3 3 we: Eowhoswwom 95.6. Eotoapmom 2:3 Sotoawmom we: Smtofimo Nina Ammv mswwgmmmhasw as. 3.583% 33on 7 Alma , , E5 3.553 mswoom mm .CHH 9.6th Oswauflmmdhwa 9.8 3.588 mmoofin .wfiaoaom 9308 me $335 an .: 53:233. -120— .uoqfiupno «on moamsdm N .mfiwficudowuom #nwfifim cwdeMucfi :oawowmmad amwuoawmom* 88H ~88 8.2: ~43 8.3 8.8 8.8 8.8 8.8 8.8 8.8 88 88H 32 8.82 38 8.8 8.2. 8.8 8.2. 8.8 8.2. 8.3. 88 o.~S o.~8 888 8.8 8.8 8.2. 8.2. 8.8 8.8 8.8 8.8 88 o.~8 H88 22: o.~S 18 8.2. 8.3. 8.2. 8.2. 8.2. 3.2. 88 ~.~S H88 ~.~S ~.~S 88 8.2. 8.2. 8.8 8.8 8.8 8.2. 88 883 8.88 3.8 848 8.8 8.8 8.8 8.2. 8.8 8.3. 8.8. 88 8.83 «:2: of: x a 8.8 8.8 8.2. 8.2. 8.8 8.8 88 2:: ~52 8.08 x x -.2. 8.2. 8.8 8.8 ~12. 8.8 88 8.2: 8.2: 888 x x 8.2. 8.2. 8.8 8.8 8.8 8.8 83 0.2: 0.2: ~88 x x 8.2. 8.2. 8.8 8.8 8.8 8.8 88 v.83 3.3 8.88 x x 8.2. 8.2. 8.8 8.8 ~22. 8.2. 88 3.8 8.88 8.88 x x 8.8 8.8 -.8 8.2. 8.8 8.8 8: 848 8.88 8.82 x x 8.8 8.8 8.8 8.8 8.8 8.2. 8-8 ~42 ~88 ~48 x x 8.8 8.2. 8.2. 8.8 8.8 8.8 88 ~82 8.2: 8.88 x x 8.2. 8.2. 8.2. 8.8 8.2. 8.2. 88 H.8H 8.2: ~82 x x 8.8 8.8 3.8 8.8 8.8 8.8 82. 8.on 0.88 38 u x 8.2. 8.2. 8.2. 8.2. 8.8 8.8 88 ~.~S 3.8 2.88 x x 8.8 8.8 8.8 8.8 3.2. 8.8 88 .88 8.2: 8.2: x x 8.8 8.2. 8.2. 8.8 8.8 8.8 mi. x 8.88 3.3 x x 8.8 8.2. ~88 8.8 8.2. 8.8 88 x 88 889” x x 8.8 8.8 8.8 8.8 8.2. 8.8 o-~ x “.42 3.2 x x 8.8 8.8 8.8 8.8 8.2. 8.2. 8H Es 8 fig 8 .E o .78 «-8 .E 8 .E 8 .2 o E 8 .3 8 E o 85% .73 ~75 8.; EL 938.588 .83 .2 ~ .A>H nachuv moHomna mflposmm unpack can musfimmfiwcoa Mo ouswuhmmsmw can mhswmfio: .H Nfiucwmm< SS LD-L RF-R RF-L Transmission values (Group IV), LD—R Appendix J. Animal -121- (DI-ONO“) wmomowomwmowwmmo oooooooooooao-uo-ooooo cacoHNwHfl'ooommwac-lmmfi‘ LO Q HNNMNHNNNHHHHHHHHHHH i mwOONOONDOEOmmwmwwmeN 'H .uooooococooooooooouooP b VHV‘WOO‘DNQO’QQ ”WWI-[fin PH HNHHHHHN HHH g 0 ‘2 ”Noel-DONG NmewowlflONNOI-D O uoncooooou-oo-ooooooooa. HQNHmwfi‘mmmwmu-(wao: me H ”NNHHHHH H H HHHH :5 DD 0H I; ONOQNO Coo mwlDLOCDOONol-Dom oouooooouo-oo-ooooooou'd b‘omNgmfi'Nme‘c’NNmNI-{OHHNH 0.) N HHHHHH H HHHHHHH {a O 'H E OWOLOIDQN O ONLOLONNQNIDNO °H ooooooo-ouooosocoooooo wmfl'cocoood'mmwcnmmbm 05:”me G mH‘DmNHHHle-‘H LOH Hm S .p i ONIDCDONIDOQNIDOOLOwwNQI-Oomo V) 00. 0.00000IOOOIOOUOOU: NQDV‘CDMIDHCDNNCNOSHQV‘HHMLOWW “4 H F Lowfi‘l-OwaH N LOHNHOQV‘H E 0) +5 ‘4 a wommouceagocaowwomummo +' 1 '(‘5' II I II I I I Iél I l I I I I I m HN V‘LD‘DFWOIOHNM mwhmeHN O Transmission values (Group IV). Appendix J. BF SS RF-L LD-L Animal 12.0 1-G 54.0 21.0 14.0 22.8 28.5 53.0 31.5 85.2 38.8 19.0 6-G 7—G 24.2 14.5 14.2 .0 24.8 41.5 8-G 9-G lo-B-x- 16.5 17.5 19.2 16.0 23. 32.2 —121— 80.5 2. 9.0 12 15—G 12.0 11.2 17-G 10.2 11.0 11.8 10.2 11.2 11.0 13.5 19-G 11.5 18.5 20-B 15.2 12.0 18 0 11.5 33.2 48.8 13.2 18.0 13.5 18.0 22-G ted slight pericarditis. ica d inspection in *Postmortem -122- Q) stooNI-Isrmcnbmboo E ‘4 0.0.0.0...- ‘H .C NHHLOL‘WOV‘MV‘NN +4 H E (D .p S-I $4 Mm O) o u: '0. I E HIDN‘I‘NNNNxNxNL‘ +4 N NH H V) 0 v4 wabmHObI—Im I C: Iota-0.... a -H >u>c><‘r4x B UDquaoaoao><¢aaaaoa CDHWQDGNNHCOWCD V‘E 0.0.00.0... N xHNwmmcooommNm g H H -H E V503 N 4" Co I o HO<><><><><><><><8 5 N CON 4.! I-I OC‘OOOQDLDCDIDUJQDCDCO 335 6défi£o"'é'x ,4 ‘Q a fi‘aaoauaoaflagzgzggoqsi E 0 COO“) Q LDC: on o 5 Q‘r4'H x>m)a3><><><><><><><><>< o I E fibOJFi s. 1% (5 v E VommebNNr—Iow COCOOOOOIOI. In V‘Hfi‘lfiml‘mmgfi‘fiofil O a K)®53§8In§Bf3L0coIn g .4 . I N m SLO: o In H'H ><><><><><><><><><><>< a) a) ‘P (DUDHNQLOCDQ‘CDCDNO r-I km: 0. u o I o o. o o o o Ofl'l'i NLOCO‘DngQDNIDQOIS g E E, V‘C‘ONV‘ fi‘mLONCOr-I b0 0) O O NLDV‘NalDl‘NODQDLOQD a £5 ssdadssssgaq E; a: E fi'aacu <‘<$~¢ oauacn aaco‘¢ S E «ammooobtomsrbmco 0 0.0.0000...- :4 fi‘fiDflDr1fi‘b-FIO>OJWJ<‘m x O mmmd‘d‘srmwmmmv 'H 'U H C d omnwuowomow m 5 I I I I I I I I I I I I Q. ‘ HNCOV‘LDQOb-meI—IN £4 5:: I-II-II-I HOGDV‘ com-3'0: 33.8 2. 6. 5.5 X X X X 5.9 29.9 8.7 2.4 44.8 37.5 34.8 38.2 45.4 33.1 3.8 45.0 45.8 28.8 38.8 33.5 37.1 14-G 16-G 17-G 18-B 40.4 19-G 5.5 23.7 44.0 6.6 1.8 40.1 39.0 32.9 X X 20-B 21-B 2.5 17.8 54.7 33.6 1.8 X 47.8 X 34.2 4.9 X 57.1 59.2 37.2 5.1 22-G ,_powdered muscle. frozen lu moles glucose equivalent/E X Samples were not obtained. of longissimus and rectus femoris (Group IV). 1 Glycogen levels Appendix K. -122- a) VCDNHVCOCDNLDFQDHOW LDQ‘l—ILDCDCD E E o o I .0 to o o o n u no. a o n o o a I: NHHBb-WCOV‘MV‘NNCOMVNV‘NV'LOHN 5 4.) 8 E m H @wwHLOLDOV'E‘COW . Q U I I C. I . O I . I . E vaNNNxxNNmeNmemcfimwb 4;, N NI-I I-ICO H HNNN H O )—7 wbmme—Iobfim I C 0.0.000... a -:-I Nr—Icomsrcommasrr-IxxNNNNNNNNN E co oqto oaoacn dIaaaocu v WHWQQNNHQWwWL‘V‘OL‘QmNQO} . O ' .. . O . C I I O I O ' NE Namgmmgoommmcrgmco'oim'moicooonisr a) E -H E V503 NVQOL‘OMCDGQDN 4" on o c o a. o o o o 0.. 3 N CON VII—IN mmmmmm 4.) $4 OC‘OC’JCDLOCDLDCDQDQDCD 335 ddéddd"°é°xxxxxxxxxxx ‘3 S VIoaquaoaoag33:ggoa53 0 Q1 (OOH) WEfi‘Ol—Iw 235 £56Nxxxxxxxxxxxx§ééé°§ ha 8 anoma m-¢r103u>b-c>c>r1r1«>rIoau>b-a3c>«> m ,C: outta-ooo-oooocooocooor-i ‘P [VWV‘HMMV‘OSQDNORwaWI-IMFML‘M o 8 N V‘N UDNC'J‘I‘V‘NV‘I-I LOP-IN mfi‘fi‘fl‘I—Im E S 44 cowmmwbmmmmlxme—Ioamooomfi'I-Ip mLOG cola-alooooooooooooooo‘c OV'H I—Imooob-Scoooomcumtxcnmgmmvmbd‘ OJ E to V‘OOQOID UDu5 S I—‘I 'U I Nooooo 3 BLOC p 0.0. O H-H NNNNNNNNNNNGJHHLDV‘NNNNN” Q a LDb-‘I‘V‘V‘ : ‘3‘ fife Nsl‘cnmbovmcocoHcooomoooofi‘NomooOI—I (D N oo'ooo-o-oooooooooo-cooN VHHmI—IHOHHMI—IOCDOWNHNHHHLD 8 O.) 'I-I 5 _é NCOb-l-DV‘IDCD‘DNL‘WHNIDHL‘V‘NwQDwN - oooooaoaooooooooo-oooqm +3 vmmHLDMHcoOLomoI—IbmoomooI—Icovg \o E N (OI-i mHNNNI-IN NQ‘ 03 NNW I-I 2'8 0) a): +’ (DDHNODLOQDV‘QDmNOHW‘Q'HWODmeN Fin-i gm: oo-ooooooooooo-oooo-oodd Ofi'r'i Ntnmtogmcommmcvgwmmgovmmwm >

o>53co'¢ $3636303u>u3OJ<0 fi‘UDtnco <3N V€C>><>< to O mmmsrsrsrmwmmmv'mmmm V‘ m3 3H 3 wommaoo omwoouwoamwmmw 0% IIIIIIIIIIIIIJ‘IIIIIIIIE "4 PIN vmcotsmeI-Imm IDCOL‘WC’OHN (D é: HHHHHI—IHHHHNNN 3N d rectus femoris (Group IV). d levels1 of longissimus an icmi Lmt Appendix L. RF-L ostmortem time 4 2 hr hr 2 45 min LD-L ostmortem time RF-R ostmortem time. LDhR ostmortem time 45 Animal 2 hr hr min ggg; 24 hr 0 hr min min 24 hr min min 2 hr 0 hr min J (0 2X 8X 8.0 15.9 42.1 90.0 11.9 X 21.0 32.1 48.1 103.8 59.0 l-G 24.2 29.0 38.6 z-G 38.0 38.2 48.7 B -123- ¢)C>O>C>b-C>«DUDFIFIC ><0;u>o>a>u>¢>a§b-¢>474 pr 0) New w g§§xxxxxxxxm 41b «>01»ch CO 3,0)P1b-QDD-UDODrI05 #893 O NI-IN w b-<‘¢>F10)m)< I gunIDI4I4N N Mbbb O‘N ‘6NNNNNN 5% CD NNOwML‘NN ssfimémfié 03 co &5NNNNMN 44 ”ONOLOceNr-I N m o m 050 96.0 Hg” ‘0“) o 0 Nu) 05 H 78.4 35. 71.6 X 35.8 59.9 73.3 5-G 47.8 44.6 51.0 (DIDLO NQDLOU) coo. wm'd'u') MQV‘CD 00>") SH? Q Q N54“ 0000) o o NNr—I 0000 o 0 MN COCO p to 91:00 MN NM 90.8 31.9 84.6 20.3 76.6 24.0 38.1 36.5 <‘ 51.3 101.0 10.5 20.5 66.3 80.6 X 33.0 75.2 X 62.8 38.7 41.7 [s 77. 13—G 37.9 39.8 47.4 55 .3 .6 NNNNNN (ONCDECOIO 05mm“ bbmbwg OfllI-IL‘UDO') 8mg NSC; xxxxxx 61.5 78.8 1 . 47.6 111.2 49.6 1105.6' X 29.2 77.0 12.0 X X .9 28 X. 20.3 25.0 X 22-G 1 u moles/g frozen, powdered muscle. X Samples were not obtained. d rectus femoris (Group IV). issimus an Glucose 6-phosphate levels1 of long Appendix M. fF-L ostmortem time RF—R ostmortem time LD—L ostmortem time LD-R ostmortem time 2 hr 24 hr 2 hr 24 hr min min 2 hr hr min min 2 hr 24 hr 0 hr min min 0 hr min 9.20 5.08 2.01 1.56 1.65 4.80 2.24 0.81 0.14 0.41 4.71 4.67 4.12 1.69 2.97 7.04 3.68 0.57 1.81 2. 36 X 2.56 0.14 0.95 3.50 0.68 3.40 X X X X X l-G 9.09 5.42 4.12 6.38 9.63 2-G 4.18 2.07 1.08 2.51 4.36 3-B 3.37 3.41 3.05 4.40 3.95 4-B 3.84 1.61 1.25 2.59 7.78 5-G 3.93 2.14 1.52 5.54 6.66 0.54 0.90 0.54 3.55 4.58 0.18 0.63 3.50 10.74 X 7.66 0.44 8.74 2.11 7 7.27 4.74 1.25 4.16 10.68 X 7-G 7.04 4.40 2.98 5.55 6.23 8-G 5.63 3.11 1.83 6.13 9.40 9—G 6.90 4.56 2.74 4.36 4.87 lO-B 4.89 5.96 2.67 4.21 7.69 6.38 0.30 8.20 0.40 5.22 2.98 0.71 3.47 2.74 0.61 1.67 7.98 0.18 X X X 13—G 5.20 3.73 2.05 2.80 8.80 0.84 0.84 0.48 10.65 1.41 7.36 4.15 15-G 6.65 4.13 2.04 2.67 7.79 3.34 1.99 4.62 16-G 6.67 6.15 3.57 6.55 6.62 4.47 3.31 6.48 5.12 5.96 3.90 1.43 1. 5.73 9.82 4.67 2.08 0.70 14.57 1.71 0.58 X 6.21 3.42 9.75 6.80 5.52 6.50 5.25 0.51 4.57 0.38 0.33 0.31 0.51 0.41 0.82 X X 2.56 0.72 5.63 8.22 17-G 5.26 9.06 1.29 0.76 3.40 7.44 18—B X X 2. 50 19-G 20-B 8.12 5.75 3.19 6.99 7.14 5.40 2.50 7.38 X 0.40 0.45 X 7.58 0.91 0.28 1.60 8.12 X 0.45 2.64 X 22—G l u moles7g frozen, powdered muscle. X Samples were not obtained. .o4navomwmc «on who: m4 o>mAII II 05.0 N II 44.0 N 00.0 00.0 N 00.0 00.4 N 00.0 05.4 N 04.0 04.0 N II 50.0 N 00.0 00.0 N 44.0 II N _m 00.0 00.0 N H II 00.0 N _ 00.0 N 00.4 00.0 N 40.0 II N 04.0 II N 00.0 00.0 N 40.0 II N 00.4 II N 00.4 II N 00.0 50.0 00.0 05.4 00.0 04.4 00.4 0 40 04 g AIhM .oon4avao won who: mm408u0 N .o4omufi cwuocsom .cwnoum M\wo4os 1 4 00.0 00.0 N 00.0 N II 00.0 50.0 N II 04.4 00.0 44.0 N 0I00 II 04.4 N 04.0 N II II 04.0 N II II 00.0 40.0 N mI40 00.0 04.0 N 00.0 N II 05.0 04.0 N II 40.0 00.0 00.4 50.0 mI00 04.0 00.0 N 00.0 N II 00.0 00.0 N II 40.4 05.4 00.0 N 0I04 00.0 00.0 N 04.4 N II 00.0 00.0 N II 40.0 50.0 00.0 N mI04 II 44.0 N 00.4 N II 00.4 40.4 N II 00.0 00.0 00.0 N 0I54 II 04.4 N N 04.0 50.0 44.0 40.4 04.0 II 00.0 04.4 00.4 00.0 0I04 40.0 00.0 N N 04.4 II 00.0 00.0 00.0 II 44.4 00.4 00.4 04.0 0I04 00.0 04.4 N N 04.0 II 00.0 04.4 40.4 II II II 05.0 00.0 0I44 40.0 00.0 N N 00.4 II 00.0 04.0 00.4 II 55.4 00.0 00.0 40.0 0I04 00.0 00.0 N N 00.0 II 40.4 05.0 00.4 II 04.4 00.0 00.0 40.0 0I04 00.0 N 04.4 N 00.0 II II 00.0 N II 40.0 00.0 00.4 00.0 0I44 00.0 N 44.0 N 00.4 II 00.0 44.0 N II 04.4 05.0 04.0 05.4 mI04 II N 00.0 N 00.0 40.0 54.4 05.4 N II 00.0 00.4 00.4 55.4 0I0 II N 00.0 N 00.0 II 44.4 00.0 N II II 04.4 00.4 00.0 0I0 00.0 N 00.0 N 00.0 II 00.4 00.0 N II II 00.4 40.0 54.0 0I5 II N 04.0 N 04.0 II 00.4 50.0 N II 05.0 00.0 55.0 00.0 0I0 II N 05.4 N 05.4 II 00.0 04.0 N II 50.0 00.0 55.0 44.0 0I0 II N 00.0 N 40.0 II 00.0 00.4 N II 50.4 44.0 00.0 00.0 mI4 00.0 40.0 00.0 04.4 00.4 II II 05.0 N II II 40.0 40.4 44.4 mI0 II 50.0 04.4 00.4 40.0 II 00.4 40.4 N II 00.0 40.0 05.0 00.0 0I0 N 00.4 04.0 04.0 00.0 II 05.4 00.0 N II 45.0 00.0 00.4 00.4 0I4 an Imm 0 =46 :45 p: 0 p: u: 0 :48 c424 a: u: 0 :45 :4E u: 0 40 04 04 40 04 04 40 04 04 4da4n< @544 Eowhofipmo NINN mB4v Emwhoswmo AIQA @844 Emwnoawmo NIQA .A54 msohwv w4koaom waoop can mua4mm4mqo4 mo 4m4o>m4 094 .2 N4uzwma< .w4navow4mc «on who: m4o>oqII .Uoc4w4no we: mum: mo40§d0 N 640mg cwhmusoa 5394.4 m\mw4oe 1 4 II II N II II N 00.0 N II II 04.0 N II II 00.0 00.0 N 0I00 II 50.0 N II 04.0 N 00.4 N II II 54.0 N II II 04.0 00.4 N mI40 II II N 40.0 II N II N II 04.0 00.0 N II II II 00.0 00.0 mI00 44.0 04.0 N II II N 00.0 N II 44.0 05.0 N II II 04.0 05.4 N 0I04 50.0 04.0 N 50.0 00.0 N 00.0 N II 00.0 00.0 N II 44.0 00.4 50.0 N mI04 54.0 II N II II N 00.0 N II 00.0 40.0 N II 00.0 II 00.0 N 0I54 II 00.0 N II 04.0 N N 40.0 II 00.0 II 54.0 II 00.0 II II II 0I04 II II N II II N N II II 00.0 40.0 04.0 II 40.0 00.0 50.0 40.0 0I04 44.0 00.0 N II II N N II II 00.0 40.0 00.0 II II 04.0 04.0 00.0 0I44 & 04.0 00.0 N 00.0 00.0 N N 45.5 II 40.0 00.4 05.0 II II II II 00.0 0I04 H II II N 54.0 00.0 N N 00.4 II 04.0 00.0 44.0 II 00.0 04.0 II 00.0 0I04 . 04.0 N II 04.0 N II N 00.4 II II II N II II II II II oI44 II N 00.0 II N 00.0 N 44.0 II 00.0 00.0 N 00.0 40.0 44.0 04.0 04.0 mI04 04.0 N 00.0 04.0 N 44.0 N 40.0 II II 00.0 N II II II II II 0I0 II N 45.0 II N II N 00.4 II 00.0 00.4 N II II 50.0 40.0 40.0 0I0 II N 04.0 II N 00.0 N II 00.0 40.0 00.0 N 00.0 II 50.0 50.0 40.0 0I5 II N 04.0 II N 00.0 N 00.0 II 00.0 05.0 N II 00.0 44.0 50.0 05.0 0I0 II N 00.0 II N 40.0 N 00.0 II 00.0 00.4 N II 00.0 40.0 00.0 00.0 0I0 II N 05.0 II N 04.4 N 00.4 II 00.0 40.0 N II 50.0 04.0 40.0 00.0 mI4 00.0 40.0 40.0 50.0 44.0 40.0 54.0 04.0 II II II N II II II II II mI0 II 40.0 00.0 II 40.0 00.4 40.0 00.4 II II 00.0 N II II II 04.0 00.0 0I0 N II N N II 00.0 00.0 50.0 II 00.4 00.4 N II 04.4 00.0 00.4 45.4 0I4 an A: 0 :42, h: u: 0 G48 G45 an 0 A: as 0 :45 :48 4: Mm‘0 :48 :48 mm 0 40 04 04 40 04 04 40 04 04 4ua4n< mE44 Sowuofiwmo NImm $844 Emwhofipwo AIDA wE44 Bewpoaumo NIQA .A>H machov m4hoEwm wswomh and msa4mw4mao4 mo 4w4m>m4 wwmnmwosm mq44mwh0 .0 x4wzomm< ,1. 13‘ V) 'H LI woofi‘fiommfi' lDOCO O 0%.: OLDV‘ (0310103 VCOH 8 EN .9000... on. d) 'H OHOONOHO OHN ’H +4 (0 AS E 000 MHV‘H ODCD 5 Id) NV” «30005 HO +3 lg...) .oNN one. 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N a c>c>c>c>0<0 ua4I4 I o m 53 .2 rIrI4drIo>aa I4<>< N>< MN NR NN MN MN NN Glucose 0.71 1.56 4.19 1.05 1.36 0.60 0.68 3.41 0.34 1.18 3.13 2.76 4.36 6.26 1.55 2.62 2.21 3.72 5.32 1.78 3.88 4.90 X 2—G 1.66 1.85 1.98 3.02 4.93 3-B 2.30 2.41 2.77 4.37 5.61 9 3 4 1.82 2.85 X X X X 2.82 3.88 6.07 2.27 1.65 2.32 4.32 2.23 X X X 3.40 5.07 5.27 7.09 31 11 .38 .95 1.89 2.07 2.14 2.81 7.39 0.55 1.54 0.88 5.53 1.05 3 2 27 86 3 2 X 1.82 3.74 0.76 3.24 (56!!) 11 3.. HEDGE cc. 4N0 R58 to. 440 2.50 4.33 5 6 X X X KN NN NN NN MN 16-G 1.80 2.58 2.95 4.73 6.24 1.76 2.49 4.91 6.58 1.81 X X 18-B 21-B i u moles/g frozen, powdered muscle. X Samples were either not analyzed or were not obtained. ——Levels were not detectable. -128- SOLOQNNNV‘ 040000000 (DV‘E 546100340! 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