.. ’ 't‘? . u. v. i - OVFRDUE FINES ARE 25¢ PER DAY . PER ITEM Return to book drop to remove this checkout from your record. THE EFFECT OF GROWTH RATE, SEX AND AGE ON SKELETAL MUSCLE AND ADIPOSE TISSUE GROWTH AND DEVELOPMENT By Mohammad Sadegh Mostafavi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1978 ABSTRACT THE EFFECT OF GROWTH RATE, SEX AND AGE ON SKELETAL MUSCLE AND ADIPOSE TISSUE GROWTH AND DEVELOPMENT By Mohammad Sadegh Mostafavi Sixty ewes with the fastest and 60 ewes with the slowest growing lambs from past history were mated to Suf- folk and Dorset rams respectively. Three rams and three ewe lambs of each growth rate were slaughtered at each age (birth, 35, 70, 105, 140 and 175 days). The lambs were weaned at 82 days of age and then divided into four groups; fast growing rams and ewes, and slow growing rams and ewes, respectively. Muscle and fat samples were removed at slaugh- ter, weighed and powdered. Perirenal, subcutaneous and intramuscular adipose tissues were assayed for glyceride synthetase, cellularity and chemical composition. Gastroc- nemius (GT) muscle was analyzed for nucleic acid and protein fractions. Both GT and longissimus (LD) muscles were anal- yzed for fat, protein and moisture content. Rams had more subcutaneous but less perirenal fat than ewes. Except for percentage protein, age affected the Mohammad Sadegh Mostafavi chemical composition of adipose tissues. Perirenal fat of ewes had higher percentages of lipid and lower percentages of moisture than rams. Results of glyceride synthetase activity depend on the method of expressing the activities. In general, both on a protein and cell basis, the enzyme activities increased while on a per gram of fat basis activ- ities decreased with age. Compared to the slow growing group, fast growing lambs had higher enzyme activities on a protein and a cell basis in perirenal fat and on a protein or gram of adipose tissue basis in the subcutaneous depot. Compared to ewes, rams had higher enzyme activities on a gram.of adipose tissue or the cell basis in intramuscular and on a gram basis in perirenal fat. Lipid content per cell of adipose tissues increased with age. Ewe lambs had higher lipid per cell than rams. With advancing age, the number of adipocytes per gram tissue decreased while the total number per fat depot increased. Neither growth rate nor sex affected the number of adipocytes per gram or total in the adipose tissues. Adi- pocyte diameter and volume of perirenal and subcutaneous fat increased with age. Neither growth rate nor sex affected the cell diameter or volume. Rams and ewes had similar fre- quency distributions of adipocytes. Growth and development of the adipose tissues is as follows: perirenal>subcutaneous> intramuscular. At 175 days of age hyperplasia was completed in perirenal while both hyperplasia and hypertrophy were Mohammad Sadegh MOstafavi responsible for the increase in subcutaneous fat at that age. Although muscle DNA and RNA concentrations decreased, the total in the GT muscle increased with age. Neither growth rate nor sex affected nucleic acid concentrations. Compared to the slow growing group, fast growing lambs had greater average daily gains, heavier GT and LD muscles, more total DNA and RNA, more nuclei per GT but a lower protein/ DNA ratio. Rams had higher total DNA and more nuclei in the GT muscle than ewes. Both weight/nucleus and protein/DNA were not affected by sex. The percentage moisture in the GT and LD muscles decreased while the percentage fat increased with age. Com- pared to the $1 w growing group, fast growing lambs had more marbling in the GT and LD muscles, lower protein in the GT and lower concentrations of total nitrogen and nonprotein nitrogen in GT muscle. Rams had lower percentages of pro- tein and total nitrogen concentrations but higher values for each of the total nitrogen fractions and percentage fat compared to ewes. ACKNOWLEDGMENTS The author wishes to express his sincere appreciation and gratitude to his major professor Dr. R.A. Merkel, for much support and guidance throughout the course of this study and for his assistance in the preparation of this manuscript. Appreciation is expressed to Dr. H.A. Henneman for serving as a member of the Committee and also for his help in obtaining experimental lambs for this study. The author also expresses his thanks to Dr. W.G. Bergen, Dr. A.M. Pearson and Dr. D.R. Romsos for serving as members of the guidance committee. The author is grateful to Dr. W.T. Magee for help with the statistical analysis. Special thanks go to Mrs. Dora Spooner for her assistance in the laboratory experiments. The author is indebted to Mr. G. Slowins, Mr. G. Good, Mr. J. Anstead, Mr. D. Mulvaney, Mr. D. Crenwelge, Mr. G. Ebrahimi, Mr. J. Rahimzadeh and Mr. A. Omidvar for their assistance during the experimental work or preparation of the manuscript. Appreciation is expressed to Anne and Mark Hodgins and Marcia Couture for typing this manuscript. The peOple of WKAR-FM radio provided inspiring music during the long hours of this research. The author wishes to acknowledge his parents Mr. and ii Mrs. Aboldhossein Mostafavi, for their understanding and encouragement to seek advanced degrees. The author is for- ever thankful to his wife, Ety, son Babak and daughter Beta, for their sacrifice and love which makes everything more enjoyable and meaningful. iii TABLE OF CONTENTS LIST OF TABLES ..................................... LIST OF FIGURES .................................... INTRODUCTION ....................................... LITERATURE REVIEW .................................. General Aspects of Growth and Development in Meat Animals ........................................ Adipose Tissue Cellularity ....................... Methodology of Adipocyte Sizing ................ Effect of Species on Anatomical Locations .................................... Growth rate effect ............................. Sex effect ..................................... Nutrition effect ............................... Lipid Metabolism ................................. Uptake of Triglycerides ........................ Fatty Acid Synthesis ........................... Glyceride Synthesis ............................ Lipid Mobilization ............................. Postnatal Muscle Growth .......................... Changes in Muscle Mass During Growth ........... Changes in Muscle Protein and Nucleic Acid During Growth ................................ Changes in Muscle Proteins During Growth ....... MATERIALS AND METHODS .............................. Experimental Design .............................. Slaughter Procedure .............................. Tissue Collection and Preparation ................ Powdering of Frozen Muscle and Fat Samples ....... Sample Analysis .................................. Glyceride Synthetase Activity .................. Preparation of Crude Homogenate .............. Esterification ............................... Stopping the Reaction ........................ Scintillation Counting ....................... Protein Determinations ....................... Determination of Adipocyte Size and Number ..... Fixation ..................................... iv MATERIALS AND METHODS (cont.) Page Filtration and Separation .................... 54 Counting and Sizing Adipocytes ............... 55 Determination of RNA and DNA ................... 56 Protein Fractionation .......................... 58 Sarcoplasmic Protein ......................... 59 Non-Protein Nitrogen ......................... 59 Myofibrillar Protein ......................... 59 Total Nitrogen ............................... 60 Stroma Protein Nitrogen ...................... 60 Kjeldahl Method ................................ 6O Moisture Determinations ........................ 61 Ether Extractions .............................. 61 Statistical Analysis ............................. 61 RESULTS AND DISCUSSION ............................. 63 Average Daily Gain, Feed Intake and Feed Conversion ..................................... 63 Adipose Tissue Growth ............................ 64 Chemical Composition of Adipose Tissue ........... 71 Measurements of Glyceride Synthetase Activity ....................................... 76 Conditions for Optimum Glyceride Synthesis .................................. 76 Glyceride Synthetase Activity .................. 91 Lipid Content per Adipocyte ...................... 105 Cellurlarity of Adipose Tissues During Growth .... 110 Adipocyte Number ............................... 110 Adipocyte Volume and Diameter .................. 112 Adipocyte Histograms ........................... 114 Changes in Body Weight and Muscle Weight Composition During Growth ...................... 122 Body Weight .................................... 122 Muscle Weight .................................. 123 Changes in Nucleic Acids During Growth ........... 130 Nucleic Acid Concentrations .................... 130 RNA to DNA Ratio ............................... 134 Total Amounts of Nuclei During Growth .......... 136 Changes in Number of Nuclei During Growth ........ 137 Weight per Nucleus ............................... 140 Chemical Composition of CT and LD muscles ........ 142 Changes in Nitrogen Fractions During Growth ...... 146 SUMMARY ............................................ 151 APPENDICES ......................................... 155 LITERATURE CITED ................................... 213 Table 10 LIST OF TABLES Allocation of the Lambs to the Experiment ................................ Composition of Creep Ration ............... Composition of the Ration for Growing Sheep ..................................... Average Daily Gain, Feed Intake and Feed Conversion Data of the Experimental Lambs ..................................... Effects of Growth Rate, Sex and Age on Weight and Percentage of Perirenal, Subcutaneous and Intramuscular Adipose Tissues ................................... Interrelationship of Growth Rate, Age and Sex on Weight and Percentage of Perirenal, Subcutaneous and Intramuscular Adipose Tissues ........................... Effects of Growth Rate, Sex and Age on Chemical Composition of Perirenal, Subcutaneous and Intramuscular Adipose Tissues .................................. Interrelationship of Growth Rate, Age and Sex on Chemical Composition of Perirenal, Subcutaneous and Intramuscular Adipose Tissues ........................... Effects of Growth Rate, Sex and Age on Glyceride Synthetase Activity of Perirenal, Subcutaneous and Intramuscular Adipose Tissues ................................... Interrelationship of Growth Rate, Age and Sex on Glyceride Synthetase Activity of Perirenal, Subcutaneous and Intramuscular Adipose Tissues ........................... vi Page 48 48 50 64 66 67 73 74 93 94 LIST OF TABLES (cont.) Table 11 12 13 14 15 16 17 18 19 20 Effects of Growth Rate, Sex and Age on Cellularity and Adipocyte Lipid Content of Perirenal, Subcutaneous and Intra- muscular Adipose Tissues .................. Interrelationship of Growth Rate, Age and Sex on Cellularity and Adipocyte Lipid Content of Perirenal, Subcutaneous And Intramuscular Adipose Tissues ......... Effects of Growth Rate, Age and Sex on Live Weight and Weight and Percentage of Gastrocnemius (GT) and Longissimus (LD) Muscles .............................. Interrelationship of Growth Rate, Age and Sex on Live Weight and Weight and Percen- tages of Gastrocnemius (GT) and Longissi- mus (LD) Muscles .......................... Effect of Growth Rate, Age and Sex of Lambs on the Nucleic Acid and Nuclei Data of Gastrocnemius Muscle ...................... Interrelationship of Growth Rate, Age and Sex of Lambs on the Nucleic Acid and Nuclei Data of Gastrocnemius Muscle .............. Effect of Growth Rate, Age and Sex of the Lambs on the Chemical Composition of Gastrocnemius (GT) and Longissimus (LD) Muscles ................................... Interrelationship of Growth Rate, Age and Sex of Lambs in the Chemical Composition of Gastrocnemius (GT) and Longissimus (LD) Muscle .................................... Effect of Growth Rate, Age and Sex of the Lambs on the Protein Fractionation Data of Gastrocnemius Muscle ................... Interrelationship of Growth Rate, Sex and Age of Lambs on the Protein Fractionation Data of Gastrocnemius Muscles ............. vii Page 108 109 128 129 131 132 143 144 147 148 LIST OF TABLES (cont.) Appendix 1 2 3 \OCDNOU'I 10 11 12 13 14 15 Tris-sucrose buffer preparation ........... Composition of fatty acid mixture ......... Preparation of 50 mM isotonic collidine solution .................................. Calculations for determining the number and volume of fat cells in Coulter Counter ................................... Preparation of RNA standards .............. Preparation of DNA standards .............. Preparation of 1% (w/v) orcinol reagent Preparation of 4% diphenylamine reagent Preparation of acetaldehyde solution ...... Reagents used in protein fractionation .... Results of interactions between growth rate and sex on some characteristics of perirenal, subcutaneous and intramuscular adipose tissues ........................... Results of interaction between growth rate and age on some characteristics of perirenal, subcutaneous and intramuscular adipose tissues ........................... Results of interaction between age and sex on some characteristics of perirenal, subcutaneous and intramuscular adipose tissues ................................... Results of interaction between growth rate and sex on some characteristics of astroc- nemius (GT) and longissimus (LD) muscIe ... Results of interaction between growth rate and age on some characteristics of astroc— nemius (GT) and longissimus (LD) muscIes .. viii Page 155 155 156 157 158 158 159 159 160 160 161 162 164 166 168 LIST OF TABLES (cont.) Appendix Page 16 Results of interaction between age and sex on some characteristics of gastrocnemius (GT) and longissimus (LD) muscle .............................. 170 17 Number and definition of variables used in raw data and correlation coefficients .............................. 172 18 Simple correlation coefficients between variables ......................... 174 19-A Allotment of lambs by number to growth rate group, sex and age ................... 184 19-B Raw data .................................. 185 ix Figure 10 11 12 13 14 LIST OF FIGURES De novo synthesis of fatty acids from qucose and acetate ....................... Pathways and enzymes in triglyceride biosynthesis .............................. Synthesis/mobilization in ruminant adipocytes ................................ Diagrams for the control of muscle protein metabolism ........................ Growth curves of perirenal, subcutaneous and intramuscular adipose tissues ......... Percentage lipid in the three fat depots as affected by age ........................ Glyceride synthesis as a function of pH ... Glyceride synthesis as a function of ATP concentration ............................. Glyceride synthesis as a function of Co- enzyme A concentration .................... Glyceride synthesis as a function of a-glycerol 3-phosphate concentration ...... Glyceride synthesis as a function of fatty acids concentration ....................... Glyceride synthesis as a function of BSA level ..... ................................ Glyceride synthesis as a function of MgClz concentration ............................. Glyceride synthesis as a function of glutathione concentration ................. Page 17 22 25 35 65 72 77 78 80 82 83 85 87 88 LIST OF FIGURES (cont.) Figure 15 16 17 18 19 20 21 22 23 24 25 26 Glyceride synthesis as a function of time ...................................... Glyceride synthesis as a function of homogenate volume ......................... Glyceride synthetase activity of perirenal, subcutaneous and intramuscular adipose tissues expressed on a soluble protein basis ..................................... Glyceride synthetase activity of perirenal, subcutaneous and intramuscular adipose tissues expressed on an adipose tissue weight basis .............................. Glyceride synthetase activity of perirenal, subcutaneous and intramuscular adipose tissues on a per cell basis ............... Lipid content and cell volume of perirenal adipose tissue as affected by age ......... Lipid content and cell volume of subcutan- eous (SQ) and intramuscular (IM) adipose tissues as affected by age ................ Frequency distribution of perirenal adipo- cytes as affected by growth rate, age and SEX ....................................... Frequency distribution of perirenal adipo- cytes as affected by growth rate, age and sex ....................................... Frequency distribution of subcutaneous adipocytes as affected by growth rate, age and sex ............................... Frequency distribution of subcutaneous adipocytes as affected by growth rate, age and sex ............................... Frequency distribution of intramuscular adipocytes as affected by growth rate, age and sex ............................... xi Page 89 90 92 97 102 106 107 115 116 118 119 120 LIST OF FIGURES (cont.) Figures 27 28 29 30 31 Frequency distribution of intramuscular adipocytes as affected by growth rate, age and sex ............................... Growth curves of body weights for fast vs slow growing lambs as affected by age ....................................... Growth curves of body weight for rams vs ewes as affected by age ................ Growth curves of body weight and gastrocnemius (GT) and longissimus (LD) muscles ................................... Changes in concentration (mg/g fresh muscle) of RNA and DNA as affected by age .................................... xii Page 121 124 125 126 133 INTRODUCTION The efficient production of a high proportion of mus- cle relative to fat is the principal objective of meat ani— mal production. Considerable research effort is and has been expended on the contribution of nutritional and endocrine criteria to body composition and growth. Yet the question remains as to why the dietary nutrients in one animal are shunted toward muscle growth and in another, even among littermates, toward fat growth and development. Limited data (Holmes and Ashmore, 1973; Allen gt 31., 1974; Bergen gt a1., 1975) with several animal species tend to suggest that muscle cell number and probably more importantly, age of maximum myofiber hypertrophy determines the stage of rapid adipose tissue development. These studies suggest that rate and extent of muscle development has a significant effect upon the age of onset and the development of adipose tissue. Skeletal muscle and adipose tissue mass at any given stage of growth and development is determined by the extent of hyperplasia (cell number) and hypertrophy (cell size) of the respective tissue cells. These parameters are frequently estimated by DNA (hyperplasia) and protein/RNA (hypertrophy) analyses. Muscle RNA increases prior to the period of myofiber hypertrophy and it decreases during the 1 period of maximum growth rate. Since the point of maximum growth attainment cannot be ascertained, the exact sampling time for assessing muscle cell hypertrophy cannot be accu- rately determined. Likewise, DNA of muscle or adipose tis- sue cannot be selectively determined and the assay proce- dures used include the DNA from all cells present. Thus, hyperplasia and hypertrophy of muscle and adipose tissue can only be approximated by DNA and RNA analyses. Data are needed to determine the relationship of muscle tissue hyper- plasia and hypertrophy to that of adipose tissue develop- ment by actual cell counts and size measurements of each tissue. To date adipose tissue and muscle growth and pro- tein synthetase capacity have not been studied simulta- neously in the same animals. Thus, the objectives of this study were to determine skeletal muscle and adipose tissue growth and development in the same group of animals. An additional objective was to study the effects of growth rate and sex on skeletal muscle and adipose tissue growth and development from birth to 175 days of age. LITERATURE REVIEW General Aspects of Growth and Development in Meat Animals The phenomenon of growth is the central focus of the livestock and meat industry from both the standpoint of animal growth and production of all other food materials. According to Fowler (1968), growth has two general aspects. The first is measured as an increase in mass per unit times. The second involves changes in form and composition which results from differential growth of the component parts of the body. The major attempt in the study of growth of the animals is to produce carcasses that have a high quantity of muscle combined with a desirable amount of carcass fat and a minimum of bone. Significant efforts are also being made to produce animals that gain more rapidly. These attempts are paralleled by the trend toward the production of less fat. The order of tissue growth and development follows an outward trend starting with tissues comprising vital organs and physiological processes (central nervous system) fol- lowed by bone, tendon, muscle, intermuscular and subcuta— neous fat (Palsson and Verges, 1962). However, in the case of limited nutrient supply, the tissues are affected in 3 reverse order of physiological importance. The tissues of the body of growing animals which have been retarded in development by restricted environment may exhibit a remark- able compensatory growth when changed to favorable conditions. Growth and development of the central nervous system is essentially completed at birth, therefore, postnatal growth mainly involves increases in bone, muscle and fat. Each tissue in its growth and development follows a sigmoidal curve, but the maximum growth of the various tissues occurs at different ages (Palsson, 1955). A major portion of bone growth and development is com— pleted in the early stages of postnatal life (McMeekan, 1959). Due to early development of bone and later developing of muscle, the ratio of muscle to bone at birth may be as low as 2:1. The growth pattern shows that bone grows at a steady, but slow rate, while muscle grows relatively fast; therefore, the ratio of muscle to bone increases (Berg and Butterfield, 1976). Weiss gt gt. (1971) reported that bone decreased from 32 to 15 percent as body weight increased from 1 to 137 in pigs. With the exception of excessively fat animals, skeletal muscle ranges between 35 to 65 percent of carcass weight of the meat animals (Forrest gt gt., 1975). On the basis of muscle fiber number Burleigh (1976) suggested a two-phase pattern for muscle growth from embryonic to adult develop- ment. In the first phase cells destined to form muscle are actively replicating for a significant portion of the ani- mals embryonic development, and a second phase in which the amount of muscle protein per cell increases and cell repli- cation is slow or negligible. On the basis of muscle mass, Berg and Butterfield (1976) proposed a four phase pattern for muscle growth. During the first phase (prenatal phase) muscle is mainly under genetic control. In the second phase (immediate postnatally) there is a great change in muscle weight which is completed during the doubling of the birth weight of the muscle mass, but in some muscles this phase continues to a quadrupling of the muscle weight. Phase three (pre-pubertal and adolescent phase) is characterized by uniform growth of muscle in both males and females and it is the product of gene expression and muscle function. During phase four (maturing phase), relative growth of the musculature changes dramatically in the male which results in male animals becoming much more muscular when compared to females. This phase is probably triggered by androgens. Adipose tissue is the most variable carcass tissue component both in amount and in distribution. During post- natal growth and development, adipose tissue mass increases by either hyperplasia, hypertrophy, or a combination of the two. In the pig, adipocyte hyperplasia appears to be com- pleted in the subcutaneous depot before 5 to 6 months of age (Anderson and Kauffman, 1973; Hood and Allen, 1977). Mor- phological development of adipose tissue in fetal lamb, 6 calves, and pigs are generally similar, but with a different time sequence. It has been found that the depot sequence of adipocyte development in red meat animals from early to late is perirenal, subcutaneous, intermuscular and intramuscular (Lee and Kauffman, 1974a). Adipose Tissue Cellurlarity Methodology of Adipocyte Sizing Techniques for estimating adipocyte size and number ha 5 provided valuable information about the fattening pro- ce ss and the study of lipid metabolism. Quantitation of DNA c<3tntent of adipose tissue has been used to estimate adipose Ce 11 number. The limitation of this method is that it over- ES timates the number of adipose cells because of the large 1"‘l-ltmber of stromal cells which are difficult to separate from ad ipocytes and will contribute to tissue DNA level (Stern and Greenwood, 1974). Although treatment of adipose tissue ‘71 th collagenase has been reported to improve the DNA esti— mation of adipose cells (Smith gt 11;. , 1972; Ashwell gt g1. , 19 76) , it has been concluded that collagenase preparation will rupture adipose cells, especially large ones (Ashwell e\t g_l_. , 1976). The microscopic technique of measuring fat Ce3L1 diameter from conventional thick or thin frozen sections (Ashwell _e_t_ _at. , 1975) and stained sections (Ashwell gt gt. , 1976), has been criticized (Sjostrom gt _a_l_. , 1971) because the fixation procedure may cause shrinkage and some mathema— tical assumptions have to be applied to account for varia- bility in shape. In addition, there would be error due to the fact that not all cells have been cut through their equa- tor. Finally, only a small proportion of cells may be count- ed which might not be the representative of the population of fat cells in the respective depot. The osmium fixation method of Hirsch and Gallian (1968) is more objective than other methods. However, it requires an expensive apparatus and the cost of osmium tetroxide is re Elatively high. In addition, this method fails to measure Small cells, generally those less than 25 pm in diameter. MO difications to this method have been suggested to improve the isolation of fat cells in osmium (Etherton gt fl~ , 1977). may reported that treatment of osmium fixed adipocytes with 8 N Urea and mild heat (50C) solubilized the connective tis- Sue and resulted in a debris-free suspension of fixed adipo- c3r‘tes. In addition, this modification greatly reduced the time for removing the adipocytes from the connective tissue m~a-1:.rix and appeared to have no effect in the structural integrity or size of the fixed cells. Ef:Eect of Species and Anatomical Locations Several investigations have confirmed that adipose tissue mass increases in cell number (hyperplasia) and Q9 11 enlargement (hypertrophy) or a combination of both (Efirsch and Han, 1969; Hubbard and Matthew 1971; Anderson 21nd Kauffman, 1973; Hood and Allen, 1977). Different fat cieposits of the animal body have different fat cell sizes. flangebak gt gt., (1974) observed that the pattern of adi- p>ocyte volume in lamb depots were: perirenal > subcutaneous > :iJitermuscular. Hood and Allen (1973) showed that the mean cijLameter of bovine adipocytes from the intramuscular depot :iss smaller than those in subcutaneous, intermuscular and p>£acrirena1 depots. The observations of Moody and Cassens C ]_‘968) with bovine intramuscular fat and those of Lee and ICiaL1uffman (1971) with porcine intramuscular fat also showed that the fat cell size of this depot is smaller than sub- <=1;1:taneous fat. Lee and Kauffman (1974a) concluded that the I>flcfwesence of small adipocytes in intramuscular fat indicates tllfluat this depot is later developing than other adipose tis- Sue depots. Moody and Cassens (1968) reported that the increase in marbling score was associated with both hyper- t53=fiophy and hyperplasia of intramuscular adipocytes. These a"~:I.‘t:hors suggested that once a muscle begins to increase fat c(D‘l'ltent, both size and number of fat cells increase. They El:IL..so reported that the largest average adipose cell diameter iJrl. the intermuscular depot was associated with the largest adipose cell mass within a particular muscle. Anderson and I('aaiffman (1973) reported that the changes in total carcass El(lipose tissue in l to 2 month old pigs were due primarily ‘:<> increase in the number of adipose cells. Between 2 and 5 Inonths, changes in intramuscular fat depot mass were due to 23 combination of hypertrophy and hyperplasia. After 5 months, tihere was no increase in adipose cell number, and adipose tzissue mass increased solely by hypertrophy. Similar results veere reported by Hood and Allen (1977). Hirsch and Han (T1969) concluded that the plateau of adipose cell number in zréits was reached at 15 weeks of age. More recently Green- vvc>¢od and Hirsch (1974) reported that the majority of adi- po cyte hyperplasia is completed by the fifth postnatal week 11:1 rats. In a comparison between rats, guinea pigs and fleastmsters, Di Girolamo and Mendlinger (1971) found that rats and hamsters had a considerable capacity to enlarge the fat Ce :11 size with increasing age between 6 weeks and 1 year, Vflezile guinea pigs showed limited capacity in this respect. 1hr), the same interval, guinea pigs had a marked increase in the number of fat cells in the epididymal fat pads, while the rat and hamster had a limited increase. These results S'L1.ggest species differences in that guinea pigs increased i~t2l£s epididymal adipose tissue mass mainly by an increase in the number of fat cells with little change in cell size, “'11 file the rat, hamster and pig (Hood and Allen, 1977) do so Ina~i.nly by an enlargement of individual adipose cells. G]? thh Rate Effect Comparing obese and non-obese humans, Hirsch and 1tl-detectable in adipose tissues from lambs fed at main- t:e‘lnance and slaughtered at the end of the growth period. I{CDVJeveL when the lambs fed at maintenance were subsequently giVen a finishing diet _ag libitum, the increase in carcass adipose tissue was paralleled by an increase in total lipo- p“Totein lipase activity in all adipose tissues. Results of el't‘periments (Haugebak gt gt. , 1974; Merkel gt _a_1_. , unpub- lished data) have indicated that lipoprotein lipase activity varies among anatomical fat depots with dietary manipulation. Lipoprotein lipase activity as expressed on a cell number 01‘ soluble protein basis was greater in subcutaneous adipose 15 tissue of lambs than in perirenal and intramuscular depots (Haugebak e_t_ gt. , 1974) . In an experiment with lambs (Merkel gt gt. , unpub- lished data) , lipopretein lipase activity in subcutaneous fat did not change as lambs grew from 8 to 32 weeks. Activ- ity of the enzyme in perirenal fat was similar in 8- and 16- week lambs, but decreased markedly in 32-week lambs. In a study with pigs (Lee and Kauffman, 1974a) lipoprotein lipase activity increased in subcutaneous fat from birth to 4 weeks 0 f age, but only slightly thereafter up to 16 weeks. It then declined in subcutaneous fat, while it remained unchanged in muscle tissue (Lee and Kauffman, 1974a). Quantitatively, lipoprotein lipase activity in adipose tis- sue of lambs was greater than in muscle (Parr, 1973; Lee and Rauffman, 1974a). Lipoprotein lipase activity in rat ep ididymal fat decreased with increasing body weight ( Chlouverakis, 1962; Nestel gt gt., 1969). Results of lipo- protein lipase activities in different species of animals Show that the activity correlated with the fat deposition of the animals. (Nestel gt fin 1969; Lee and Kauffman, 1974a, 19 74b). Fa t ty Acid Synthesis Adipose tissue is the major site for g novo fatty ac id synthesis in ruminants (Payne and Masters, 1971; Ingle e \t a\1., 1972a, 1972b; Martin e_t g, 1973). The pattern of 16 fatty acids synthesized is similar to the fatty acid composi— tion of the tissue (Pothoven gg gl., 1974). The pathways of fatty acids syntheses in ruminant adipose tissue are differ- ent from nonruminants. The classic experiments of Hanson and Ballard (Hanson and Ballard, 1967, 1968; Ballard gg gl., 1972) first showed that adipose tissue from mature cows and sheep utilized acetate but not glucose as a carbon source for g 9239 synthesis of fatty acids. The pathways of dg mg fatty acid synthesis from glucose and acetate is shown in figure 1 (Bauman, 1976). These pathways are also confirmed by other workers who have studied adipose tissues from ovine, bovine and caprine species (Hood gt_:_ fl~ , 1972; Ingle g; g1. , 1972b; Baldwin g g1. , 1973; Young and Baldwin, 1973). Fatty acid 8 ynthesis can be described in a two step reaction by the malonyl CoA pathway (Kumar g5 g_1_. , 1972). In the first step, ma lonyl CoA is formed from acetyl CoA plus HOD; by the enzyme acetyl coA carboxylase. In the second step, one mo lecule of so ca11ed "primer" acetyl CoA condenses with 8 even molecules of malonyl-CoA to form palmitic acid. The s econd step is catalyzed by a multienzyme complex (fatty ac id synthetase) which is composed of six enzyme subunits (Mayes, 1977). The overall reaction for this synthesis which yields palmitic acid from acetyl CoA is: “Q etyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+———-r palmitic acid + 7 002 + 3 CoA + 14 NADP++ 6 H20 17 Glu o e-6-P’ ‘\ NADPL— —— —NADP+4""'\ 11 Pentose \ Phosphate 9 cycle , I Fru tose-6-P I NADPH— -- --NADPH I e---” l I Tridse-P— — — — fia-Glycerol-P NADi \ Influx ‘g I Malonyl CoA Primer I \ ,’ I ADH” ‘ ’ 8 NADPH N. P+ NAD+ PyruvatefiA—aglpialate I 6 Acetyl Call 5 NADH l Oxalacetate 7 Pyruvatg\:k I 4 cetyl CoA 1 c tyl CoA A 3 Ox alace ate itratee— -—+Citrate Acetate 12 Tricarboxylic + // acid cycle Isoc tratebu—qlsoci rate NADP 13 ( a-Keto NADPH gluta7te 4-— -) a-ketoglutarate \__—— / Mitochondrion Cytosol Figure 1. 2g novo synthesis of fatty acids from glucose and acetate (Bauman,1976). (1) Pyruvate car- boxylase. (2) Pyruvate dehydrogenase. (3) Citrate synthetase. (4) Citrate cleavage enzyme. (5) NAD- malate-dehydrogenase. (6) Malic enzyme. (7) Acetyl- CoA synthetase. (8) Acetyl-CoA carboxylase. (9) Fatty acid synthetase. (10) Hexokinase. (11) Glucose phos- phate isomerase. (12) Aconitase. (13) NADP-isocitrate dehydrogenase. BHBA - 8-hydroxybutyrate. The negligible activities in ruminant adipose tissues are denoted by X. 18 Some of the enzymes which are necessary for lipo- genesis to occur from glucose as substrate are absent in ruminants. The enzymes which are absent in ruminant liver (Ballard and Oliver, 1964) and ruminant adipose tissues (Hanson and Ballard, 1967; Ingle gg g1. , 1972b) are malic enzyme and citrate cleavage enzyme. The activities of these two key enzymes have been shown to be 50-fold higher in rat éiclipose tissue than in ruminants (Hanson and Ballard, 1968). The two enzymes show dramatic changes in activity during development of young ruminants. Ballard g_t_ £11. (1969) found a considerable quantity of these two enzymes in fetal calf 1 iver which does possess the capability for lipogenesis from g1 ucose. The activities of these enzymes diminish as the runnen develops (Hardwick, 1966). Bauman gg §_]___. (1970) also reported very low activities of malic and citrate cleavage enzyme in mammary tissue of ruminants which is indicative of ve ry little incorporation of glucose into fatty acids in this tissue. The inability of ruminants to incorporate glu- cos e as a substrate for $3 132.12 fatty acid synthesis seems me t abolically adapted to conserve glucose for metabolic functions such as energy production in nervous tissue and e3"~"371.’:hrocytes, lactose synthesis in mammary glands, and pro- d‘Llction of NADPH and a-glycerol phosphated for triglyceride SnYthesis in lipogenic tissues (Allen gig g1. , 1976). The source of reducing equivalents for _d_e m fatty ac - 1d synthesis in ruminant adipose tissue also differs from l9 non-ruminants. In ruminant adipose tissue, NADPH is gener- ated in the hexose monophosphate shunt pathway (via glucose- 6-phosphate dehydrogenase and 6-phosphog1uconate dehydro- genase). The activity of NADP-isocitrate dehydrogenase has been reported to be extremely high in ruminant adipose tissue relative to its activity in non-ruminants (Bauman, 1976). The advantage of the isocitrate cycle in ruminants is that acetate can be utilized to generate NADPH. It has been estimated that at least 25 percent of the NADPH necessary for lipogenesis in ruminants is supported by the isocitrate cycle, with the remainder generated from the pentose phosphate cycle (Young and Baldwin, 1973). Adipose tissue appears to be the major organ for gg novo synthesis of fatty acids in ruminants (Ingle g g1. , 1 972b) and nonlactating pigs (O'Hea and Leveille, 1969) , While the liver is more important in birds (Leveille g gl. 1. 9 68; O'Hea and Leveille, 1968). In rats both organs con- tribute significantly for g n_o_\Lg synthesis of fatty acids (Leveille, 1967; Chakrabarty and Leveille, 1968). The intracellular sites of fatty acid synthesis are cytoplasmic, mitochondrial and microsomal components. Al though ctyoplasmic and mitochondrial enzyme systems are S imilar, they have different end products which are palmitic and stearic acid for “cytoplasmic and mitochondrial systems re S‘pectively (Masoro, 1968). Fatty acid synthesis has been shown to be influenced 20 by breed (Chakrabarty and Romans, 1972; Hood and Allen, 1975L age (Ingle gg g1., 1972b; Pothoven gg gl., 1975), diet (Allee g5 g1., 1972; Ingle gg gl., 1973; Pothoven and Beitz, 1973) and anatomical site of the depot (Anderson gg gl., 1972; Hood and Allen, 1975). In contrast to non-ruminants, the fatty acid composition of adipose tissue in ruminants is rust markedly affected by the fatty acid composition of the criet. This is because rumen microorganisms are able to fujydrogenate the unsaturated fatty acids. Therefore, the ];r3:eformed fatty acids which are taken up by adipose tissue are predominantly saturated (Dawson and Kemp, 1970). G cheride Synthes is There are two known pathways in the mammalian system ;iE?<:>r triglyceride synthesis. The 2-monog1yceride pathway <:'<:2].ark and Hubscher, 1961) and glycerol 3-phosphate pathway (Weiss g5 91., 1960). Biosynthesis of triglycerides via the 13:31.:9'cerol phosphate pathway appears to be the major route in adipose tissue (Shapiro, 1965; Vaughan and Steinberg, 1965), jLSIfiL liver (Weiss and Kennedy, 1956; Marinetti, 1970) and in the mammary gland (Howard and Lowenstein, 1965). In intes- 1t1::i-‘-1|l".lal mucosa both pathways are functional, but, studies have Show that the monoglyceride pathway is more important than 1E;j]LCS'cerol phosphate pathway for re-synthesis (Johnston and JES:I=.<>VHI, 1962; Senior and Isselbacher, 1962; Senior, 1964). The pathways and enzymes for triglyceride biosynthesis 21 are shown in figure 2. Triglyceride formation from fatty acids is dependent on ATP, CoA and Mg2+. Glycerol cannot replace a-glycerol phosphate in the glycerol phosphate path- way, however, it has been shown that there is the possibil- ity of some ester formation when millimolar amounts of glyc- erol are added (Margolis and Vaughan, 1962). Even in the absence of a-glycerol phosphates, a small amount of fatty acids has been shown to be incorporated into triglyceride (Steinberg gt_ g1. , 1961). This has been suggested to be due to endogenous a-glycerol phosphate which might not have been removed completely upon dialysis or also may be due to e strification of diglycerides, preformed or generated by 1. ipolysis during incubation (Vaughan and Steinberg, 1965) . Therefore, formation of glycerol phosphate is an obiligatory S tZep in the synthesis of triglycerides. This may be formed by phosphorylation of glycerol (derived from hydrolytic b reakdown of lipids) with ATP in a reaction controlled by glycerol kinase (ATP-glycerol phosphotransferase) , and also by reduction of dihydroxy -acetone phosphate which is gen- erated by the glycolytic sequence of reations, with the NAD- 1 inked dehydrogenase as the enzyme. It has been reported that glycerol kinase has limited distribution in animal tis- sues, but it is found in liver (Bublitz and Kennedy, 1954), 1'<:—L<1‘ney (Wieland and Suyter, 1957) and intestinal mucosa ( Clark and Hubscher, 1962) mainly in the cell sap. It is e s sentially absent from adipose tissue (Margolis and Vaughan, 22 2-monog1yceride Glycerol 3-phosphate pathway pathway 2g nova Plasma F.A. ’/////F.A. \\\\\\L.Fatty acids pool ATP ( ) AMP 2-monog1yceride 1 a-glyerol-B-phoshpate CoA Pi (2) \/ Lysophosphatidic acid ‘ CoA (2) Fatty-acy (5) 00A CoA pool Phosphatidic acid (3) 1,2-dig1yceride V l - 2—diglyceride CoA CoA (4.) (4) Triglyceride EE§1ure 2. Pathways and ensymes in triglyceride biosynthesis Fatty acid CoA ligase, (2) acyl-CoA-L-glycerol 3 phos- 45LWte o-acyl transferase, (3) L-a-phosphatidate phosphohy- <:>‘1yase, (4) acyl-CoA-1,2-diglyceride o-acyl transferase, acyl-CoA-Z-monoglyceride o-acyl transferase. AQUAW WW 23 1962), therefore, glycerol phosphate must be formed from the dihydroxyacetone phosphate via glycolysis of glucose. It has been reported that the level of a-glycerol phosphate may be a key control for triglyceride synthesis in adipose tissue (Leboeuf, 1965). This suggests that triglyceride synthesis in adipose tissue is closely associated with carbohydrate metabolism including gluconeogenesis. This latter is particularly important in fat metabolism of rumi- Iiants. Results reported by Packter (1973) indicated that 'tflne rate of triglyceride synthesis is increased following ifeaeding, which is accompanied by increased levels of blood glucose and insulin. Although the subcellular site of triglyceride synthesis in adipose tissue in not known, the subcellular site of glyceride biosynthesis in mammary gland, liver and intestinal mucosa of several species has been identified. Studies on marrunary gland of cow (Gross and Kinsella, 1973), goat and sow (Bickerstaffe and Annison, 1971), rat (Tanioka gig g1. , JLEE’ 77'13) and guinea pig (Kuhn, 1967) indicate that the main sub- <::‘E=13L-Ilular site of triglyceride synthesis in the above mentioned SZEDQcies is the microsomal fraction. The microsomal fraction has been found to be responsible for glyceride synthesis in ‘thisaL‘:l-, guinea pig and rat livers (Daae, 1973) and sheep, chicken EIIT“<5l 'pig intestinal mucosa (Bickerstaffe and Annison, 1969). 24 Lipid Mobilization Lipid synthesis and mobilization in ruminant adipocytes are not independent, but their control must be coordinated. These two functions tend to be reciprocal process (figure 3). Synthesis and mobilization of fat depot triglycerides are in a dynamic state. After feeding, a hyper-insulin state ensues especially in non ruminants. Insulin increases :fatty acid synthesis and ultimately triglyceride synthesis. IIhnsulin is believed to involve at least two possible mecha- Iujism of action in the synthesis of triglyceride. First it :jllncreases glucose permeability which stimulates glycolysis and hexose monophosphate shunt. The former yields acetyl (22¢:1A.and glycerol phosphate and the latter produces NADPH. (::<:>risequent1y, the tricarboxylic acid cycle produces more ATP. 49L:1_31_ of these processes result in higher rates of fatty acid ‘EllTlAc3.trig1yceride synthesis. The second mechanism of action 0 f insulin is believed to be at the level of gene expression (:354[£51rinetti, 1970). Injection of insulin has been shown to :jL3‘3l<::rease the activity of certain enzymes such as acetyl CoA (zzfaldtrboxylase and citrate cleavage enzyme, both of which are ijJ‘IIJE30rtant for fatty acid synthesis (Tepperman and Tepperman, l 9 65; Olson, 1966). Insulin also leads to increased glucose- ES"“Ibhosphate dehydrogenase and 6-phosphogluconate dehydro- g e‘lhase activity. The rate of influx and efflux of non-estrified fatty 25 Adipocyte Triglyceride (3 (4) Glycerol-P 1ycerol i I Fattyacyl CoA:: Fatty acids ‘1 (1) Glucose Acetate Fatty acids A Q n Lipoproteih x Triglycerides 17 v Glucose Acetate Fatty acids Albumin Glycerol & NEFA Glycerol Pla 81113 I ’11 H ‘Qg novo fatty acid synthesis Uptake of plasma fatty acids Fatty acid estrification Fatty acid mobilzation NEFA = non estrified fatty acid §L1re 3. Synthesis/mobilization in ruminant adipocytes (Buuman, 1976) I) I) I) AAAA AWNH 26 acids in adipose tissue is under hormonal control, particu- larly insulin and epinephrine (Newsholme and Start, 1973). Insulin increases influx of NEFA while epinephrine increases efflux of NEFA from adipocytes. The effects of epinephrine are always opposed to those of insulin. The primary effect of epinephrine is to increase the hydrolysis of triglycerides in adipose tissue by a mechanism in which the hormone (epinephrine) is believed to stimulate the adenyl cyclase ssystem by direct interaction at or near the cell membrane (liobinson gg g1., 1967). This interaction results in pro- cit1ction of more cyclic AMP which in turn stimulates the gauc:tivation of hormone sensitive lipase. Feedback control of triglyceride synthesis has been described by Newsholme and Start (1973). They indicated that fatty acids or fatty acyl-CoA esters have a negative fe edback effect on acetyl CoA carboxylase. The rate of ci txate formation and its effect on acetyl CoA carboxylase 3i-53 éanother control point affecting the formation of acetyl ‘(3‘2’495— carboxylase which is believed to be a rate limiting e“:3-257me in fatty acid synthesis. However, _ig y_i_t_rg studies Show that the level of citrate needed to convert acetyl CoA czhéal‘3="t:oxylase from a monomer to a more active trimer is much tjl:i;'JE§]b1er than the physiological level of citrate in the cell (vagelos, 1964). The control of fatty acid synthesis in adipose tissue I? 1=‘<:>1Dn dietary carbohydrate or lipid has a two-fold effect. 27 Ingestion of these nutrients influences the hormonal state of the animal together with a favorable substrate concentra- tion and causes enzyme activities to increase and hence the rate of fatty acid synthesis. In short, the activity of the esterifying enzymes in starvation and refeeding parallels the activities of other enzyme systems involved in lipo- genesis and lipid mobilization. Enzymes of the hexose mono- phosphate shunt pathway (Hollifield and Parson, 1965) , fatty acid desaturating enzymes (Benjamin and Gellhorn, 1966) and lipoprotein lipase activity (Hollenberg, 1959) are all decreased in adipose tissues of fasted rats. These activ- it :ies have been shown to be restored to levels close to, or above normal with refeeding (Hollenberg, 1959; Hollifield and Parson, 1965; Benjamin and Gellhorn, 1966). Release of fatty acids from triglycerides is catalyzed by triglyceride (hormone sensitive), diglyceride and mono- leCeride lipases. The rate limiting step in lipolysis is the hormone sensitive lipase reaction. This enzyme has been Suggested to be a cytoplasmic enzyme in lipid rich matrix Ce l 15 (Khoo gg g1. , 1972). The regulation of this enzyme Ciepends on the intracellular level of cyclic AMP (Patton, 19 7 0; Robinson e_t_ g1. , 1971). In this mechanism, cyclic AMP St imulates protein kinase which in turn activates hormone sen eitive lipase by converting from the non-phosphorylated inactive form to a phosphorylated active state (Robinson e t 631. , 1971). Lipolysis in adipose tissues removed from rim Lb“ M ‘ I- hula ”a lg. \~§ 28 lambs differing in propensities to fatten has been measured by Sidhu at El' (1973). They reported that lipolysis in— creased with age and fatness in lambs. In contrast to non- ruminants, relatively few studies have been conducted with ruminants. However, it is apparent that ruminant adipose tissue is much less sensitive to lipolytic hormones than non-ruminants (Prigge and Grande, 1971). Postnatal Muscle Growth (Zldanges in Muscle Mass During Growth Regardless of size, muscle tissue constitutes approx- :ingIately 25 percent of human and rat body weight at birth (E lliott and Cheek, 1968). This percentage changes to 45 percent in the adult mammal (Young, 1970). Therefore, there Zi~53 .a substantial increase in the proportion of muscle during 130 S tembryonic period. Postnatal growth of mammalian muscle fibers is almost ent irely due to hypertrophy of pre-existing muscle fibers 'Eiltjl<5l not by hyperplasia (Stromer 33 al., 1974). However, 8 Qme postnatal increase in muscle fiber number has been reported (Goldspink, 1962; Chiakulas and Pauly, 1965; Bridge {aarjl‘il Allbrook, 1970), which appears to depend on the state of "1tl1urity of animal at birth, which can be considered as an £2=H= 1: ension of the embryonic differentiation of the tissue. 29 The increase in the length of muscle fiber is primarily asso- ciated with an increase in the number of sarcomeres along myofibrils (Goldspink, 1968), as well as a small increase in the length of the individual sarcomeres (Aronson, 1961; Shafiq, 1963). However, the increase in the length of indi- vidual sarcomeres is more important in invertebrates than vertebrates (Aronson, 1961). The changes in sarcomere length may vary in different species and strains of animals accord- ing to their rate of growth. There are different schools of thoughts concerning the addition of sarcomeres to the myofibrils. Some authors (Buska and Edwards, 1957) have suggested that the myofibrils grow interstitially; in other words, new sarcomeres are added to the myofibrils at some point along their length. They have based their theory on the fact that the sarcomeres Of adjacent myofibrils are often out of register because of S 3— ight differences in sarcomere length. In this case, there wi 11 be some of the myofibrils with additional sarcomeres for a given length of muscle which is taken as evidence that the S arcomere has been inserted. However, in order to insert a new sarcomere in this way, it is necessary for the myofiber n91: only to divide transversally, but, also it should involve 111° difications of sarcoplasmic reticulum and transverse tub- ular system (Goldspink, 1972). Other workers (Holtzer gt a1. , :L 9 S7; MacKay e_t_ a1. , 1969) have suggested that the lengthen- i 11g of myofibrils occurs by serially adding sarcomeres to 30 the myofibrils. The mechanism by which the new sarcomeres are added has been discussed by Goldspink (1972). He suggested that the ends of fibers are the regions of longitudinal growth and that the new sarcomeres are most probably added serially to the ends of the pre-existing myofibrils. This hypothesis fits with fact that the terminal sarcomeres of myofibrils are shorter than those in the middle. Presumably the terminal sarcomeres are the most recently formed ones which have not had time to increase in length. The latter hypothesis is confirmed with the experiments (Williams and Goldspink, 1971) in which tritiated adenosine was injected into growing mice. Autoradiography and scintillation counting from these expe- riments showed that most of the label was incorporated into th e end regions of the muscle fibers, suggesting that these regions are more active in the synthesis of actin and ribo- SOmal RNA. The mechanism of sarcomere assembly is not well under- stood. Legato (1970) suggested that in cardiac muscle, Z- di S‘ks are the centers for the assembly of the new sarcomeres. Th1 s assembly is accomplished by hypertrophy of Z-disk mate- }: it’s-.13 which occupy the areas where the sarcomere will ulti- mat ely develop and then by gradual replacement of Z-substance, th ick and thin filament form the new sarcomere (Ezekwe and 1» La): tin, 1975). The increase in girth of muscle fiber is almost 31 entirely by the increase in the number and size of myofibrils (Goldspink, 1972). Studies by Goldspink (1970) showed that the 11111311361? of myofibrils in mouse biceps brachii muscle may increase up to lS-fold during postnatal growth. Goldspink (1972) suggested that when the myofibril reaches a certain thickness, it splits longitudinally by the force originated from stress placed on the Z-disk by the oblique pull of the actin filaments during contraction. This tension is suffi- cient to tear a small hole in the center of the disk which then spreads longitudinally and causes splitting of the entire myofibril. Large animals tend to have larger muscle fibers than those Of small animals (Luff and Goldspink, 1967; Byrne _et al, 19 73; Hanrahan gt; a1. , 1973; Ezekwe and Martin, 1975); but the. difference in muscle size between the large line and small line is mainly due to difference in the total num- b er of fibers in the muscle and not to difference in the f :‘Lber Size (Luff and Goldspink, 1970). Although there is 1'16 difference in the total number of fibers between the same anatomical muscles of males and females in mice, the mean f :iber diameter in the male is greater than in females (Rowe arid Goldspink, 1969). Adrian gt a. (1969) reported that from a physiological 8 tandPOint, it is not feasible to have development of fiber beyond a certain diameter, because the distance from the Q enter 0:5 the fiber would be too great to allow for oxygen 32 diffus :ion and also impulse transmission down the T-system, to the center of the myofibril. This confirms the fact that somehow during the evolution of the larger animals it has been necessary for the fiber to increase in number rather than size. It has been shown in rodents (Rowe and Goldspink, 1969) that muscle fibers grow in a discontinuous way rather than in a gradual and continuous manner. Very soon after birth all of the fibers are approximately the same size. As the animal grows postnatally some of the muscles such as the biceps brachii will undergo extensive hypertrophy as compared to other muscles such as soleus and extensor digitorum lon- 811.: Which will essentially retain their original size throughout the life of the animal. The population of small and large fibers can be changecl by exercise or changing the level of nutrition of the animal (Goldspink, 1970). The stimulation for hyper- trophy of muscle fibers is believed to be due to the inten- 8 it)’ Of the work load to which the fiber is exposed. This 1 S apparant, because as the animal grows there will be a QonSiderable increase in body weight in the animal, there- Sore, the work load on skeletal muscle will be increased. I~3:ypert1:‘0phy of striated muscle fiber due to exercise has I) een accepted for many years (Morpurgo, 1895). Morpurgo C 1895) attributed muscle fiber hypertrophy to an increase in S arcoplasm rather than myofibrils. Later, cytological 33 studies showed that hypertrophy was mainly associated with increases in myofibrillar portion of the muscle fiber (Richter and Kellner, 1963; Goldspink, 1964, 1970). However, under certain conditions of exercise some hypertrophy of the fibers has been found to be partly or wholly due to increase in mitochondrial and sarcoplasmic proteins (Gordon e_t a1. , 1967) . The effect of exercise on hypertrophy of muscle fibers has been studied at the molecular level by Goldberg (1968, 1969) and Hamosh _e_t_ a1. (1967). Goldberg (1968, 1969) has reported that during work induced hypertrophy, the incorpor- ation of leucine - 14C into both sarcoplasmic and myofibril- lar prOteins is enhanced and also the rate of degradation of these proteins is reduced. Hamosh _e_t_:_ a1. (1967) reported that during hypertrophy Of muscle fibers, there is an increase in RNA concentration and also there is a greater ability for the cell-free system t O SWithesize proteins. They found that L-phenylalanine was incorPOrated into microsomal protein at a faster rate by the microsomal fractions prepared from hypertrophied muscle, b 0th in the presence or absence of artificial RNA (Poly U.). rhey also reported an increased RNA content in the micro- & omal fractions. H(>1'mones may exert a direct effect on muscle growth or L11dirfict via regulation of food intake in the animal. Sev- Q 131 hormones affect protein metabolism and the growth of 34 skeletal muscle. Insulin and growth hormone are considered to have the greatest effect on protein synthesis of mammal- ian skeletal muscle. Insulin stimulates amino acid uptake in muscle via its interaction with the cell membrane (Figure 4). Individual amino acids are taken into the intracellular compartment in proportion to the amino acid composition of the muscle protein, rather than in proportion to the amino acid in the extracellular compartment (Turner and Munday, 1976). Insulin also stimulates the translation process inde- pendent: 1y of amino acid intake, by an action possibly medi- ated by inhibition of adenyl cyclase and stimulation of guan- osyl cyclase (Cuatrecasas, 1974). Although _i_n_ v_it_:_1;9_ experiments using hypophysectemized animals have lead to the conclusion that growth hormone stim- ulates the transport of both amino acids and glucose, as well as stimulating the incorporation of amino acids into proteins, 1 t should not be concluded that growth hormone mimics the actions of insulin. It has been shown that administration 0 f growth hormone to whole animals results in both protein anabolism in muscle and lipolytic effects in adipose tissue ( Reeds gt _a_l__. , 1971). The anti-insulin action of growth hor- hone 0n adipose tissue in the absence of an increase in insulin is a protective mechanism for body protein during EaStj-ng , exercise and stress in that they prevent the exces- & :‘Lve 1~18e of amino acids or substrate for the generation of g :lucose and metabolic energy. Nevertheless, when insulin Aggregat i on Binding and Initiation E longation W W Cortisol ’ F :igure 4 , 'Zl‘urner and \ \ ‘Stimulation; —— - -OInhibition; 22 35 [ NUCLEUS ] Degradation Products 4 Thyroid ,I’Hormones‘x‘ Steroid Hormones\\‘ mRNA Ribosomes<——b Sub-units / f —— —- Growth Hormones a’ insulin 4! P01 somes Growth Hormone / insulin / Bound Complex Chain Elongation \W\\\\N~.LnRNA Cortisol_.._.__._o Insulin Somatomgdins ii 9 . \ka” Protein _4—5Amino acid? . I3Amino acid -..: - AR W» I /’ Cortisol I Insulin Diagram for the control of muscle protein metabolism Munday, 1976). Cell membrane 36 and growth hormone secretion occur simultaneously, such as in the case of after feeding, the action of growth hormone on muscle is truly anabolic, which leads to the conclusion that there may be an important interaction between insulin and growth hormone in stimulation of muscle protein synthesis (Reed _e_t_ a_l_. , 1971). Just as growth hormone needs insulin for its protein anabolic effect, insulin also depends on growth hormone for its protein systhetic action, which indi- cates that insulin and growth hormone are mutually dependent for increasing protein systhesis. In addition, it seems that they have a synergestic effect on protein synthetic action when the concentration of both hormones increases (Turner and Munday, 1976). Aridrogens have direct or at least an indirect effect 01:1 muSCIe development (Goldspink and Rowe, 1968; Grigsby El; é}: . 1976). The degree of responsiveness of different IImuscles to androgens varies considerably. Kochakian _e_t_:_ a_l_. C 1961) working with guinea pigs reported that temporal and 111881361? muscles are very sensitive to castration and subse- Qment replacement therapy. However, the response of muscle 1: '0 androgens is more uniform (Kochakian, 1966) , with the Q 2(ception of lavator ani muscles (Venable, 1966, a,b). film-”gens increase the rate of protein synthesis in most IIIIJSCIES . Results of experiments (Novak, 1957; Kochakian, l 955) indicate that following administration of adrogens, th ' e lnc30rporation of labelled amino acid into muscle proteins 37 is increased in both intact or castrated animals. Florini and Breuer (1966) reported that ribosomes obtained from cas- trated animals are less active in protein synthesis than those of intact animals. They also showed that the combin- ation of testosterone and growth hormone can modify the pro- tein synthesis ability of ribosomes. They concluded that the main factor for increasing protein synthesis by these hormones is the increase in messenger RNA production. The influence of early nutrition on growth and develop- ment of muscle fibers has received considerable attention in recent years. Muscle is one of later developing tissues and may be affected by nutritional deprivation imposed during hyperplasia. In an experiment with pigs, Robinson (1969) reported that undernutrition during pregnancy does not affect muscle cell number, while stress during pregnancy and lactation caused the termination of muscle cellular hyper- plasia to be earlier than those of the control. It is well known that the reduction in food intake by animal or human causes a considerable reduction in muscle mass (Allison e_t_ El; : 1962). The decrease in muscle mass is shown to be associated with a decrease in muscle mean fiber diameter (Joubert, 1956; Montgomery, 1962; Goldspink, 1964, 1965). ck’ldspink (1964) and Rowe (1968) reported that the starva- tion effect on those mouse muscles that are normally com- DOSEd Of large and small fibers, causes a reduction in the number of large phase fibers in the muscle so that the fiber 38 size distribution tend to become unimodal again. Goldspink (1965) has also reported that the increase in the fiber size is due to reduction in the number of myofibrils in the fiber and that this accounts for the decrease in the contractile strength which is normally associated with starvation or atrophy. The mechanism of reduction of myofibrils from mus- cle fiber during starvation is not clear, however, Bird _e_t a_l. (1968) reported that levels of cathepsins was increased five days after reduction of food intake. On the other hand, DeDuve e_t _a_l_. (1962) postulated that lysosomal enzymes were functioning in the normal economy of cell catabolism or renewal - Therefore it seems that there should be a mechanism under which the release of these enzymes could be increased in case of fasting (Bird _e_t 511., 1968) or retarded in case of refeeding. Changes in Muscle Protein and Nucleic Acids During Growth True cellular growth is estimated by measuring weight, protein’ DNA and RNA content of tissues and organs (Mirsky and Ris, 1949). The increase in mass of protein during hypertrophy of muscle cells may rise from changes in the :rates of either protein synthesis or degradation or changes in both. The contribution that changes in the degradation rate made during hypertrophy of skeletal muscle has not been C31early established. Turner and Garlick (1974) and 39 Mi11ward gt g. (1975) calculated that protein degradation rate doubled during the period of rapid muscle growth in rats- In contrast Goldberg (1969) reported a decrease in degradation rate which contributed towards the increased protein mass in rat Soleus muscle during hypertrophy, because more radioactivity was retained in the proteins of hypertrophying muscle than of the controls eight days after pulse labeling with 3H-leucine. The amount of DNA per diploid nucleus is generally considered to be consistent in tissues (Mirsky and Ris, 1949; Vendrely, 1955) and since there is no evidence of poly- ploidy during skeletal muscle growth (Enesco and Puddy, 1964). the increase in DNA reflects an increase in number of nuclei - Results of experiments show that the total content of DNA is increased during growth (Enesco and Puddy, 1964; Gordon e_t_ gt., 1966; Buchanan and Pritchard, 1970; Johns and Bergen, 1976; Harris gt gt., 1977; Laurent and Sparrow, 3977) . Harbison gt gt. (1976) reported that total DNA increased approximately 2.0 (obese pigs) - 2.7 (muscular Figs) fold between 23 and 118 kg of live weight. These :tzesults agree with the data for rats (Enesco and Puddy, 19643 Enesco and LeBlond, 1962; Harris gt g_l_., 1977), mice 3' 3 3 'Iotal 18 18 18 18 = 72 lambs total TABLE 2. COMPOSITION OF THE CREEP RATION. Ihngredient _ .Percentage of total, Alfalfa Meal 25 (horn 28 Soybean Meal (49% protein) 29.5 Crimped Oats 10 Molasses 6 High Zn-Trace-Mineral Salt 1 'Bone‘Meal .5 10.0.0 h aThe vitamin and mineral supplement furnished the following quantities per kg of feed: Vitamin A, 2200 I.U.; Vitamin D , 660 I.U.; Vitamin E, 11 I.U. In addition approximately 4.4 g ASP (250) was added per kg feed. 49 and group feed intake was recorded at 140 and 175 days of age. The composition of ration for growing sheep is shown in table 3. Slaughter Procedure The neonatal lambs were slaughtered within 10 or 12 hr of birth. The rest of the lambs were fasted approxi- mately 15 hr prior to slaughter. In order to avoid the effect of electrical stimulation, the animals were not immo- bilized prior to exsanguination. Bleeding was accomplished by severing the carotid artery and jugular vein. Following exsanguination the pelt was removed as rapidly as possible. Tissue Collection and Preparation The desired samples were rapidly removed from the ani- mals and weighed. These samples included the following: ggstrocnemius (left leg) and longissimus muscles, perirenal, subcutaneous and intramuscular fats. The entire left longis- giggg muscle was removed and then freed of adhering surface fat before it was weighed. For fat, protein and moisture determinations, one third of the muscle from the lumbar region was saved (except for neonatal lambs the whole longis- giggg_muscle was saved in order to obtain sufficient samples). Subcutaneous fat was removed from.the dorsal thoracic and 50 TABLE 3. COMPOSITION OF THE RATION FOR GROWING SHEEPa Ingredients Percentage of total. Dehydrated alfalfa (17% protein) 30.0 Corn, grain 32.5 Oats, grain 19.5 Soybean Meal (50% protein) 12.5 Molasses 5.0 High Zn-Trace-Mineral Salt .5 100.0 aThe vitamin and mineral supplement furnished the following quantity per kg of feed: Vitamin A, 5500 I.U.; Vitamin D3, 687 I.U.; Vitamin K, 11 I.U. 51 lumbar regions of the carcass except at birth where the absence of external fat made it impossible to obtain subcu- taneous fat samples. Intramuscular fat was obtained only from the lambs at 140 and 175 days of age. This fat was physically separated from the entire right longissimus mus- cle. The whole sample or subsamples were placed in poly- ethylene bags, frozen in a mixture of dry ice and 2—methy1- butane, and stored at -85C for subsequent analyses. Powdering of Frozen Muscle and Fat Samples The frozen muscle and fat samples (except for the intramuscular fat) were powdered in a '250 room as described by Borchert and Briskey (1965). Chipped dry ice and shat- tered pieces of frozen muscle or fat were pulverized in a Waring Blendor jar for apporximately 30 to 60 sec. After sifting the samples, the coarse material which remained on the sieve was again placed in the blendor and the process repeated. After the second pulverization and sifting, the coarse material was discarded. The powdered samples were placed in polyethylene bags and were not sealed until 12 hr after filling to allow carbon dioxide sublimation. After sealing, the samples were stored at -85C for later analyses. 52 Sample Analyses Glyceride Synthetase Assay A modification of the assay method of Bennink (1973) was used to determine glyceride synthethase. Preparation of Crude Homogenate Preparation of the crude homogenate was carried out at 2 to 3C. Depending on the adipose tissue, approximately 1 to 2 g were sliced with a razor blade and weighed while frozen. The sliced tissue was homogenized in three volumes of Tris-sucrose buffer (Appendix 1) in a Brinkmann Polytron (Model PCU-2-110, and saw-tooth model PT-lO-ST) for 45 sec at setting 5 (50% of full speed). The sample was further homogenized by three strokes in a Thomas teflon glass homogenizer. In order to separate cell debris and nuclei from crude homogenate, the hemogenate was filtered through glass wool (prewashed with Tris-sucrose buffer, pH 6.6). Esterification In a preliminary study, the time, pH, and concentration of cofactors necessary for a maximum rate of glyceride synthesis were determined by varying the con- centration of each cofactor while the other cofactors were held constant. The labeled precursor used in this assay was L-glycerol-14C(U)3-phosphate with a specific activity of approximately 20,000 dpm/mole. The cofactor concentrations for the 3 fat depots as determined in the preliminary 53 studies were 1.75 mM.ATP;3.3 mM MgC12;.ltmiCoA; 20 mg/reac- tion tube BSA; 3.3 mM glycerol 3-phosphate; 100 mM potassium phosphate buffer (pH 6.6) and .67 mM mixture of fatty acids. In addition 15 mM glutathione (GSH) was used for subcutaneous and perirenal but, 7.5 mM GSH was used for intramuscular fat. The fatty acid mixture (Appendix 2) was solubilized and neu- tralized with KOH (1 m1 of 2 M KOH per 100 ml of free fatty acid mixture) and sonicated for 1 min with a Bronson Sonifier (Model 350; duty cycle setting 2). The fatty acid mixture was immediately pipetted into 25 ml Erlenmeyer flasks which contained all cofactors. This mixture was sonicated as described above and .5 ml of the crude homogenate (enzyme) was added and the flasks were stoppered. The enzyme assay was conducted in a total volume of 3'ml at 37C: for 45 min with gentle shaking in a Eberbach Shaker Bath (Eberbach Corporation, Ann Arbor, Michigan). Duplicate flasks with- out ATP and CoA were run as blanks along with the samples. Stopping the Reaction After the 45 min incubation period, the reaction was stopped by adding 8 ml of a solvent mixture consisting of isopropanol and heptane (1:1 v/v), and then shaken vigorously. Five ml of .03 M NaOH were added to the flasks and shaken to wash the solvent and the upper layer (heptane) allowed to separate from the lower aqueous layer. Scintillation Counting, A 2 ml aliquot of the heptane layer was transferred to scintillation vials containing 10 ml of scintillation cocktail (.5% PPO in toluene). The 54 scintillation vials were counted in a Packard Tri-Carb Liquid Scintillation Spectrometer (Model 3310, Packard Instrument Company, Downers Grove, Illinois). Counting ef- ficiencies were calculated by channels-ratio-method. Protein Determinations Protein concentrations of the crude homogenates were determined by the Lowry method (Lowry gg gl., 1951). Bovine serum albumin was used as standard for these determinations. Determination of Adipocyte Size and Number Fixation Fresh samples of perirenal, subcutaneous and intramuscular fat were fixed in 5 ml of 3% osmium tetroxide (Appendix 3) in scintillation counting vials which contained 3 ml of 50 mM collidine buffer (Appendix 3). Fixation was allowed to proceed for 72 hr under a hood. Filtration and Sgparation After fixation, adipocytes were filtered through two different pore sizes of Nitex nylon screens (Tetko Inc., Elmsford, N.Y.) with the smaller pore size (15 um) being placed on the bottom and the larger pore size (either 150 or 250 um) on top. The 150 um.pore size top screen was used for perirenal and subcutaneous adipocytes from the birth, 35 and 70 day age groups and also for the intramuscular fat cells from the 140 day age group. The top pore size screen for the remainder of the samples was 250 uflL The pore size screens were chosen based on preliminary studies using microscopic and Coulter Counter observations. 55 The osmium fixed adipose tissue was transferred to the upper filtration screen and the fat cells were washed free of connective tissue with a stream of distilled water and gentle prodding with a blunt glass stirring rod. The released cells 'were collected on the lower 15 um screen while the very small particles and cell fragments passed through. The fixed cells which remained on the bottom screen were trans- ferred to a tared 250 m1 beaker and weight of the suspension ‘was brought to 240 g by adding .9% sodium chloride. The suspended cells were then ready for sizing and counting on the Coulter Counter. Counting and Sizing Adipocytes The procedure of Hirsch and Gallian (1968) was followed and counting was done on a Model B Coulter Counter. Calibration of the Coulter Counter ‘was made with corn (large particle size) and pecan (small particle size) pollen. Appropriate settings for the Coulter Counter were determined by prior trials. Two aperture tubes 'were used. The aperture diameters used were 250 um and 400 um.for samples that filtered through 150 pm and 250 um top screens, respectively. In a series of calculations (Appendix 4) the number and the volume of the cells per gram of adipose tissue for different selected size ranges were calculated. 56 Determination of RNA and DNA A modification of the method Munro and Fleck (1969) was used to determine RNA and DNA in gastrocnemius muscle samples. Approximately .2 g of powdered muscle were weighed in dupli- cates in Corex test tubes and 2 m1 of cold deionized water 'were added. The tubes were stoppered and vortexed. After adding 5 ml of cold 2.5% perchloric acid (PCA) (w/v) the tubes were stoppered, vortexed and placed in an ice bath for at least 10 min and then centrifuged at 34,800 x g_for 15 min. The supernatant was discarded. The pellet was broken up with an applicator stick and 5 m1 of cold 1% PCA were added. The tubes were stopped, vortexed and centrifuged at 34,800 x g for 15 min and the supernatant was discarded. The pellet was broken up and 4 ml .3N potassium hydroxide were added and the tubes stoppered, vortexed and sealed with tape to prevent popping. The tubes were incubated at 37 C in a ‘water bath for 2 hr. At the end of the incubation time, the tubes were vortexed and placed on ice for 5 min. Five ml of cold 5% PCA were added and the tubes stoppered, vortexed and placed on ice for 15 min. The tubes were centrifuged at 34,800 x g for 10 min. The supernatant was decanted into 25 ml graduated tubes and saved. The pellet was broken up and washed twice with 5 ml of 5% PCA each time followed by stoppering, vortexing and centrifuging at 34,800 x g for 10 Inin. The supernatant from each of these 2 centrifugations 57 ‘was added to the 25 ml graduated tubes and the total volume 'was brought to 20 ml with 5% PCA and then mixed. This frac- tion contained RNA. The pellets were saved for DNA extrac- tion. For DNA extraction, the pellet remaining from the RNA isolation was broken up and 5 ml of cold 10% PCA were added. and the tubes were vortexed and marbles were placed on the top of the tubes to act as condensers. The suspension was digested in a water bath at 70 C for 25 min. At the end of digestion, the tubes were placed on ice for 5 min, then cen- trifuged at 34,800 x g for 10 min. The supernatant was decanted into 15 ml graduated tubes and saved. The pellets were broken up and washed with 4.75 ml of 10% PCA, stoppered, vortexed and centrifuged at 34,800 x g for 10 min and the supernatant was added to the 15 ml graduated tubes and the total volume was brought to 10 ml with 10% PCA and mixed. This fraction contained the DNA. For determination of RNA concentration, orcinol was utilized in a calorimetric procedure. Two ml of the RNA fraction were pipetted into 16 mm pyrex test tubes in dupli— cates, as well as a reagent blank using 2 ml of 5% (w/v) PCA instead of sample, and a set of duplicate test tubes containing RNA standards (Appendix 5) of 12.5, 25.0, 37.5 and 50 mg RNA/ml was used“ To all of the above tubes 2 ml of 1% (w/v) fresh orcinol reagent (Appendix 7) which were made up just prior to use, were added. 58 Marbles were placed on the top of the tubes to act as condensers and the tubes were placed in a boiling water bath for 30 min. After boiling, the tubes were cooled in running cold water for 5 min and allowed to reach room temperature and then read immediately at 680 nm on a Beckman Model 24 Spectrophotometer. For determination of DNA concentration, diphenylamine and acetaldehyde were utilized in a colorimetric procedure. Two m1 of the DNA fraction were pipetted into 16 mm pyrex test tubes in duplicates. In addition, a reagent blank using 2 ml of 10% PCA instead of sample and a set of dupli- cate test tubes containing DNA standards (Appendix 6) of 12.5, 25.0, 37.5 and 50 mg DNA/ml were used. To all of the above tubes 2 ml of 4% (w/v) diphenylamine in glacial acetic acid (Appendix 8) and .1 ml of acetaldehyde solution (Appendix 9) were added and vortexed. Marbles were placed on top of the tubes to act as condenser and the tubes were incubated over- night at 30 C in a water bath. After incubation, the tubes were cooled to room temperature and read at 595 nm on a Beck- man Model 24 Spectrophotometer. Protein Fractionation The protein fractionation procedure was a modificaiton of the method of Helander (1957). All fractionation proce— dures were carried out at 2 to 3 C with cold extraction solutions. 59 Sarcoplasmic Protein Five g of powdered frozen muscle were weighed in 250 ml polyethylene wide mouth centrifuge bottles equipped with screw caps. Fifty ml .015 M potassium phosphate buffer (Appendix 10) were added to the bottles and extracted on a magnetic stirrer for 3 hr. After centrifugation at 1400 x g for 20 min, they were filtered through eight layers of cheese cloth into 100 ml graduated cylinders. The residue was re-suspended in 50 ml of potassium phosphate buffer and extracted on a magnetic stirrer for 3 hr. After extraction, they were centrifuged at 1400 x g for 20 min and filtered as described above. The volume of the combined supernatant was recorded. Duplicate 15 ml samples were used to determine the amount of sarcoplasmic protein nitrogen present in the sample by the Kjeldahl method. The residues were saved for myofibrillar protein nitrogen determination. Non-Protein Nitrggen Fifteen ml duplicate aliquots of sarcoplasmic protein supernatants were pipetted into 50 ml polyethylene centrifuge tubes to which 5 ml of 10% (w/v) tri- chloroacetic acid (TCA) were added. The solution was allowed to stand for 2 to 4 hr, then centrifuged at 12,100 x g for 20 min. The supernatant was carefully decanted into Kjeldahl flasks for non-protein nitrogen determination by the micro-Kjeldahl method. Myofibrillar Protein The residue from the sarco- plasmic protein extraction was suspended in 50 ml 1.1 M potassium iodide (KI) phosphate buffer (Appendix 10) and 60 extracted on a magnetic stirrer for 3 hr. After extraction, the bottles were centrifuged at 1400 x g for 20 min and fil- tered through eight layers of cheesecloth into 100 ml grad- uated cylinders. The residue was resuspended in 50 m1 of 1.1 M KI phosphate buffer, extracted for 3 hr, centrifuged at 1400 x g for 20 min and filtered as described above, and the combined volume of the supernatants was recorded. Dupli- cate 15 ml samples of the suspension were used to determine the amount of myofibrillar protein nitrogen in the sample by the micro-Kjeldahl method. Total Nitrqgen Total nitrogen was determined on approx- imately .5 g of powdered muscle by the micro-Kjeldahl method. Stroma Protein Nitrogen Stromal protein nitrogen was calculated by subtracting the sum of sarcoplasmic, myofibril- lar and non-protein nitrogen from the total nitrogen. Total nitrogen was expressed as milligrams per gram of fresh mus- cle tissue. All protein fraction nitrogen values were expressed as a percentage of total nitrogen. Kjeldahl Method The American Instrument Company (1961) Micro-Kjeldahl method was used for nitrogen determinations. 61 Moisture Determinations Moisture and ether extract determinations were per- formed on powdered gastrocnemius and longissimus muscle sam- ples, powdered perirenal and subcutaneous fat and on dis— sected intramuscular fat from the longissimus muscle. Approx- imately 1 to 5 g samples were weighed into previously dried aluminum dishes and dried in a JIKMZ oven for 24 hr. Weight loss was recorded after cooling the samples in a desiccator and the moisture was calculated as percentage of fresh tissue (A.O.A.C., 1970). The dried samples were saved for the ether extract determinations. Ether Extraction The fat content was determined by extraction of the dried samples with anhydrous ether for 4 hr in a Goldfisch fat apparatuses outlined by A.0.A.C. (1970) and the data were expressed as percentage of fresh tissue. The fat con- tent of adipose tissues was also expressed as grams per cell. Statistical Analysis A factorial experiment was designed with two growth rates, two sexes and six ages. There were also three repli- cates per experimental unit. The main effects and their interactions were analyzed by the analysis of variance 62 method (Steel and Torrie, 1960). When significant differences were observed between more than two means, Duncan's Multiple Range test (Duncan, 1955) was performed to determine which means were signifi- cantly different. In addition, linear correlation coeffi- cients were calculated between pairs of dependant variables (Steel and Torrie, 1960). The statistical analysis was per- formed at the Michigan State University Computer Center. RESULTS AND DISCUSSION Average Daily Gain, Feed Intake and Feed Conversion Average daily gain, daily feed intake and feed conver- sion for the lambs are presented in table 4. Since the lambs were group fed, individual feed intake could not be obtained. Therefore, average daily gain is the only feed lot character- istic that could be analyzed statistically. Since the Suffolk-sired lambs grew faster than Dorset—sired lambs in this study, they were catagorized as fast and slow growth rate groups, respectively. This reference to growth rate groups will be followed thoughout the remainder of results and discussions. Between 105 and 140 days, average daily gain of the fast growing rams and ewes and slow growing rams were similar and all were significantly (p<.05) higher than the slow growing ewes. However, between 140 and 175 days only the fast growing rams gained more rapidly (p<.05) than the other groups. As would be expected, in both age periods (105 to 140 days and 140 to 175 days) feed intake tended to be related to body weight gain. Average feed intake of the lambs between 140 and 175 days was higher than between 105 and 140 days (1835 2g 1442 g/day/lamb, respectively). Feed 63 64 to gain ratio during both periods were similar for fast and slow growing lambs (table 4). Although rams and ewes had similar feed conversion between 105 and 140 days, ewes were superior to rams in feed conversion (5.52 2g 4.81 for rams and ewes, respectively) between 140 and 175 days of age. These data indicate that the fast growing lambs had higher feed to gain ratios than the slow growing rams. TABLE 4. AVERAGE DAILY GAIN, FEED INTAKE AND FEED CONVER- SION DATA OF THE EXPERIMENTAL LAMBSa Growth Rate Growth Rate Fast Growigg Slow Growing Fast Growing Slow Growing Sex Sex Sex Sex Ram Ewe Ram Ewe Ram Ewe Ram Ewe 105 to 140 dgys 140 to 175 days Number of lambs 7 9 7 9 4 6 4 6 Average daily gain, g 262b 242b 263b 200C 411b 346C 350C 302C Feed intake, g/day/lamb 1592 1392 1519 1265 2324 1725 1888 1405 Feed/gain 6.08 5.79 5.77 6.32 5.65 4.98 5.39 4.65 aMeans of the average daily gains for each period having the same superscripts are not significant (p>.05). Adipose Tissue Growth Adipose tissue accretion and its percentage of live body weight are presented in tables 5 and 6 and figure 5. The weights and the percentages of both subcutaneous and 65 280 x10 195 130 - 135 210 235 WEIGHT [G] 70 31 7o“ 105 . HGE (DRYS) 4 140 155 Figure 5. Growth curves of perirenal, subcutaneous and intramuscular adipose tissues. 665 .oocuuauucwum mo Ho>oa new huuafinanouml.um .Amo. Aavucmuuwacwam haamuaumaumum uo: mun muoauomuoosm mama msu acqumon sou mama can so uuouuo aqua sumo segue: nauozwuovu .oaama NH «0 owuuo>o as» can snooze .mmsmouu amonava umaaumaauuucq new space «a can oaoucauauaao no Annoyance new onsua on no smote goo.v uwmo. sumo. vmmo. vao. vmmo. choc. an. mac. owe. o_. cmo. one. unasoonawuucn doo.v oo~.n cum.m cem.~ oc<.~ one. 1 mm. ~m.~ nm.~ co. m~.~ mn.~ nsoocausoasm doo.v uo~._ vow. ode. nun. can. one. goo.v vcm. ode. Nm. em. as. Hucouuuom uuwaucouuua usmnuu umoaav< doo.v u~m.m~ oqo.n~ vaa.m ooa.m vuom.~ cum. mm. ma.w n~.¢ Ne. n—.o e~.w unaauoaaauucu _oo.v venom unama cone ooou uswu I no. e_w nuO— an. mna doe uaoocouaunam soo.v moon CAN” eel" memes some emu so. om~ em” on. ooN e_~ Hacoeeeee "Auvuzwuoa o:unuu.umoa«v< soo.v ca~.e< mm~.em moo.m~ wq~.o~ beo.m~ umm.m _oo.v ,bmo.- owe.e~ goo.v WWm.NN onc.e~ Amxvuzmaoa o>wa .um msu o<~ nod es mm o .um 03m Bum .um casouullm:waoum uuaoauuzmawz Beam umum pamwmvwmm< snow couch auscuo mmammufi mmomHa< mw ago who woman: cue. nee. «No. aNO. mac. moo. mac. ~mo. -o. «no. Nwo. moo. 03m moo. nmo. ewe. umo. ego. woo. «no. Nqo. o—o. mmo. mmo. coo. sum uwflsumsawuucn mo.n oc.n am.~ o~.~ a_.~ I e~.¢ oe.~ oo.~ em.~ w~.~ 1 63m eo.n oc.e oe._ em._ on. 1 m~.n om.m so.“ ma.“ do.~ I aux maoocnuaunsm ao.~ cm. on. on.” on. uc. um.~ No. cc. am. On. Nm. 03m mm. ~c.~ en. cm. on. me. co. mm. on. on. cm. «a. an: Huaouauom "uwwuaouuon mamnuu omoauvc so.wN on.- am.m do.m aN.~ «N. ma.o_ oo.- nm.o o~.o ow.~ cm. can so.m~ o~.- mo.m o~.o o~.~ mm. on.w~ Nu.w~ cm.o ne.m co.n hm. Bum uaaauaaaauucH nemm owed ch can find I News mac“ oNn new om_ 1 can «com cued wwm Nam mm 1 o~m~ nnn~ mmn omu me“ 1 sum msoocouaoasm sec omw o- do“ ac ma soc sum me~ «0— cm _N was he: won hog ee~ ea ma Non awn on no co m— an: Accouauom "Amvuzwwoa manna» uuoaa1< ne.~e ww.m~ n~.n~ om.- c~.- ~m.m cm.c¢ n~.om w~.o~ mo.o~ m~.m~ -.o 03m oe.m< o~.mn N¢.~N _w.o~ co.~_ ee.< e~.mm em.me oo.~n Nu.e~ ~w.<~ mm.m aux Amxvunwaoa o>wa mn— ecu no“ em mm o mug can mod 05 mm 0 now ouaoaousmsuoz mmhovvww< 1~mwwovom< ,lwdasouo 36am wcaaouo ummh mh<¢ :H3OKU ammammwh mmomHD< ¢subcutaneous>intramuscular>perirenal>pelvic fat. In their study, the 140 to 152 g of fat dissected from fetal calves was quite evenly distributed between intermuscular and perirenal fat. The most notable observation in their study was the complete lack of subcutaneous fat which agrees with the findings of the present study. There was a high correlation (p<.01) between live 69 weight and perirenal fat weight (r=.82) as well as with sub- cutaneous fat weight (r=.89). Several investigators includ- ing Barton and Kirton (1958) and Kirton and Barton (1962) have also observed a high positive correlation between weight of lambs and amount of carcass fat. The pattern of perirenal and subcutaneous adipose tis- sues weight and percentage increases in relationship to the live weight were similar (figure 5). However, between 70 and 105 days (when body weight increased from 20.2 to 28.1 kg), there was a decrease in perirenal fat weight from 148 to 117 g, while subcutaneous fat increased from 296 to 430 g. Nevertheless, neither of these changes was significant (p>.05). These nonsignificant changes in perirenal and subcutaneous fat are probably due to the effect of the stresses associ- ated with weaning. The data (table 5) suggests that perirenal fat is more sensitive to these stresses than subcutaneous fat. Growth rate of the lambs did not significantly (p>.05) affect weight of perirenal and subcutaneous fat. However, the percentage of subcutaneous fat was higher in slow growing than fast growing lambs (P=.06). Makarechian gg g1. (1978) found that when breed of sire influenced growth rate, car- cass composition of the progeny was not necessarily affected. They observed that Dorset-sired lambs grew slower, had less bone and more fat than Suffolk-sired lambs. These data agree ‘with the results of the present study. However, their 70 results were based on carcass weight rather than live weight, as in the present experiment. They also reported a high dressing percentage for Dorset-sired lambs, which would be expected because of the greater amount of fat. Lambuth ggigl. (1970) reported that fast gaining lambs had no significant increase in total retail yield or edible portion, but had lower percentages of total fat trim.and higher percentages of total bone than slow growing lambs. Sex affected the perirenal and subcutaneous adipose tissue weights differently (table 5). Rams had more (P=.05) subcutaneous, but less (P=.09) perirenal fat than ewes. On a percentage basis, perirenal fat of ewes was significantly (P<.01) higher than rams. This is in agreement with the results reported by others (Shelton and Carpenter, 1972; Kemp gg gl., 1976). In general, female cattle and sheep fat- ten at lighter live weights than castrated males, whereas castrated males fatten at lighter weights than intact males (Bradley gg gl., 1963; Prescott and Lamming, 1964; Wilson gg gl., 1969). Berg and Butterfield (1976) reported that fat had the greatest effect on carcass composition between sexes. Weight and percentage of perirenal fat were affected by the interaction between age and sex (P<.05, Appendix 13). No other interactions were significant (P>.05). 71 Chemical Composition of Adipose Tissue Proximate analysis of the fat depots are shown in tables 7 and 8. Except for percentage protein of intramus- cular fat, age had a significant (P<.01) effect on the per- centage lipid, protein and moisture of the three fat depots (table 7). During the 175 days of the experiment, percentage lipid in the perirenal and subcutaneous adipose tissues increased 1.7 and 22 fold, respectively (figure 6). The rea- son for this large difference in lipid deposition during postnatal growth is due to the difference in the fat content of the two adipose tissues already present at birth. Peri- renal adipose tissue had accumulated 34% lipid prenatally, while subcutaneous adipose tissue had only 3.5% at birth. The correlation between percentage lipid and adipose tissue weight was higher in perirenal (r=.82, P<.01) than that in subcutaneous (r=.53, P<.01) or in intramuscular (r=.38, P>.05). Body weight was also significantly (P<.01) correlated with percentage lipid in perirenal (r=.72) and subcutaneous (r=.76) fat but not intramuscular (r=.38, P>.05) fat. The pattern of increase in percentage lipid of perirenal and sub- cutaneous fat was highly correlated (r=.96, P<.01). Both of these fat depots were affected by the stresses associated with weaning (figure 6). The decrease in fat deposition immediately following weaning may be due to caloric reduc- tion that resulted from reduced feed intake during the first 72 120 <>D)( rdCD'O ZCI'TI HUT/'0 SOCUrfl :DC‘JU Z—HTI C2132 (0233 J PERCENTHGE 60 OMI— CO ‘b' 3% Figure 6. 55 tbs 120 RGE (DHYS) Percentage lipid in the three fat depots as affected by age. 75 .wocmowwacwam mo Hw>mH How hufiafinmnoumuum .Go. A evecmudeawam kHHmowumfiumum uo: mum muofluomumaom mamm mnu wcaummn Sou meow osu co uommmm Came :omw :HnufiB mammewmto .mpfima NH mo amaze .oommflu mmoawom “manomDEmuucw pom mnama NH paw moomcmuoonom uo Hmamuaumo How mnSmH on «0 cmem 73 Hosav o~.mm om.q¢ I I I I ma. w.om ~.~q oe. 0.5m q.oq umaaomaamuucH doom. mm.mH mum.wfi oo.o~ mom.HN em.mm 00.0w mm. m.~m w.mm om. H.¢~ m.~m msomcmuoonom aoko mm.n mN.~H w~.o~ mo.m~ u~.ma o~.mm aoo.v N.wH N.HN mm. w.mH m.mH Hmcwuwumm "muoumaoa mwwucmoumm Hm. mq.w~ mo.ofi I I I I om. qo.wH o~.mH om. H¢.oH mm.w~ umaoomoamuuaH doom. moq.m mum.m mumm.q ooqm.q oom.m owm.- mm. m©.o mm.© we. wo.o cm.c mnomGMuoonsm seedy mac.fi m~m.~ ooc.m wow.~ omq.m omH.HH «a. oq.m Nu.m Hm. Nc.m cm.m Hmcmufiumm "samuoum mwmuawouwm No. o~.om o~.wq I I I I mm. w.mm q.fim cm. o.qm N.mm umaoowoamuucH Hoko mm.ow w~.mm om.mo mm~.cm wo.mm um.m mo. o.~o N.oo mm. o.mm o.~c maooomusonom goody wN.Hm www.mw woo.~w www.mw oc.mn oo.qm doo.v o~.wn om.qm Nm. 0.05 n.0n Hmcmuaumm "UHQHH mwmucmouwm .pm mug oqfi moH 0m mm o .um 63m 5mm .um wcwzouw wcfi3ouw mucoEouommmz nAmxmovmw< mxmm 30am ummm moumu nuBOHQ mmammHH MmomHQ< mm msu mum mammZm 0.00 0.00 I I I I 0.00 0.00 I I I I m3m 0.00 0.00 I I I I 0.50 5.00 I I I I Ema amasomoEmuucH 0.HH 0.00 0.00 0.00 0.00 0.55 5.0a 0.00 0.50 0.0a 0.00 0.00 ~30 0.00 5.00 5.00 0.00 0.00 0.00 5.50 0.00 0.00 0.00 0.00 0.05 3mm msoocmusonom 0.0 0.00 0.00 0.0a 0.00 0.00 0.0 0.00 0.00 0.00 0.00 5.00 030 0.0 00.00 0.00 0.00 0.00 0.00 0.0 00.00 0.00 5.00 0.50 0.50 5mm Hmomuwumm “wuoumwoe wwmuomoumm 00.5H 00.50 I I I I 50.00 05.50 I I I I ~30 00.00 05.00 I I I I 00.00 00.00 I I I I 5mm amazomosmuucH 55.0 00.0 00.0 00.0 50.5 00.0H 00.0 00.0 00.0 05.0 05.0 00.00 mzm 00.0 00.0 00.0 50.0 00.0 00.00 05.0 00.0 05.0 00.0 00.0 00.00 Ema moomcmuoonom 00.0 05.0 00.0 00.0 00.0 50.00 00.0 00.0 50.0 00.0 00.0 00.00 mam 00.0 c5.H 00.0 00.0 00.0 ~0.HH 00.0 05.0 50.0 00.0 50.0 00.00 800 Hmcmuauom "GHMuoua owmucmoumm 0.00 0.00 I I I I 0.00 5.00 I I I I m3m 0.00 0.00 I I I I 0.00 0.00 I I I I 8mm umaoomSEmuuaH 0.00 0.05 0.05 5.05 0.00 0.0 5.05 0.05 0.00 0.05 0.00 0.0 030 5.00 0.55 0.00 0.05 0.00 0.0 0.05 0.05 0.00 0.05 0.00 5.0 8mm moomamusonom H.00 0.50 5.00 0.00 5.55 0.00 0.00 0.00 0.00 0.00 0.00 0.00 oBM 0.00 0.00 0.00 5.00 0.05 0.00 0.00 0.50 0.05 5.00 0.00 0.00 800 Hmcmuaumm "0aofia mwmuamopmm 050 000 000 05 00 0 05a 000 00a 05 00 0 xmm unmEmuommmz Amhmwvmwa Amwwvvmw< wmwzouu scam wcwaouo ummm my.05) by sex for either subcutan- eous or intramuscular adipose tissues (table 7). Also, per- centage protein in perirenal fat was not affected by sex (P>.05). Chemical composition of fast and slow growing lambs were similar (P>.05). There was a significant (P<.05) interaction between age and growth rate on the percentage lipid and moisture of the subcutaneous adipose tissue (Appen- dix 12). No other significant interaction was observed (P>.05). 76 Measurements of Glyceride Synthetase Activity Conditions for Optimum Glyceride Synthesis A study to determine the assay conditions for optimum glyceride synthesis was conducted on sheep perirenal, sub- cutaneous and intramuscular adipose tissue. The conditions studied included pH of the assay medium, concentrations of ATP, CoA, glycerol 3-phosphate, MgClz, glutathione, fatty acid mixture, length of the assay in minutes and levels of BSA and the adipose tissue homogenates. The results of these experiments are presented and discussed below. The optimum pH for glyceride synthesis of the three fat depots was similar (figure 7). Maximum rates of glyceride synthesis occured at pH 6.6. This value is similar to the results reported by Bennink (1973) for rabbit mammary gland. An optimum pH of 7.0 has been reported for adipose tissue glyceride synthesis in the bovine (Benson, 1969) and rats (Steinberg gg gl., 1961; Daniel and Rubinstein, 1968). How- ever, optimum pH values above 7.0 have been reported in mam- mary gland tissue of several different species (Askew gg a1., 1971; Bickerstaffe and Annison, 1971; Bennink, 1973; Gross and Kinsella, 1973). Triglyceride formation was highly dependent upon the presence of ATP (figure 8). This finding agrees with the data reported for bovine adipose tissue homogenates (Benson, 100 G O a o ‘ b 0 Percentage 0' Optimum n: o 02 77 8.4 8.8 8.8 7.6 pf. Figure 7. Glyceride synthesis as a function of pH. 78 100 Ir -‘ \ E 80 a E E 080 '3' o 040 .——. Peri. D o s A Subcut. 2 :20 .——_- IH'YO. 0 mM ATP Figure 8. Glyceride synthesis as a function of ATP concentrations. 79 1969) and those for rats (Steinberg gg gl., 1961; Angel and Roncari, 1967). Glyceride synthesis without added ATP was approximately 5% of that for the optimum concentration (1.75 mM). Concentrations of ATP greater than 2.5 mM inhibited esterification. The results confirm other work performed on bovine (Benson, 1969) and rat (Angel and Roncari, 1967) adi- pose tissue. This inhibition of higher concentrations of ATP can be partially reversed by increasing Mg2+ concentra- tion (McBride and Korn, 1963). The concentration of ATP needed per milliliter of homogenate for triglyceride forma- tion in sheep (present study) and bovine adipose tissue (Ben- son, 1969) seems to be considerably lower than that of mam- mary tissue (Askew gg gl., 1971). This observation may be due to the high requirement of ATP for triglyceride forma- tion in mammary glands compared to that in adipose tissue because of the greater quantity of fat synthesized per unit of time. There is an absolute requirement for CoA in the tri- glyceride formation (figure 9). The essentiality of CoA (and ATP) confirms the fact that triglyceride synthesis in sheep adipose tissue occurs via the a-glycerol phosphate pathway since the initial reaction of this pathway requires ATP and CoA for its activity. The assay system is very sen- sitive to the added ATP and CoA (figures 8 and 9). Similar results have been found in bovine adipose tissues (Benson, 1969), rats (Steinberg gg gl., 1961) and also in mammary 100 I. O a O O O Porcofag. of Optimum M O 80 s—0 a 0—0 Peri. ‘——-A Subcuf. l———. Intro. 0 .2 .4 6 .8 mM CoA Figure 9. Glyceride synthesis as a function of Coenzyme A concentration. 81 glands of cows (Askew gg gl., 1971; Bennink, 1973) and rab- bits (Bennink, 1973). In the absence of a-glycerol phosphate there is no tri- glyceride formation (figure 10). This could be expected, becasue a-glycerol phosphate is probably the main fatty acid acceptor in adipose tissue. However, Steinberg gg g1. (1961) showed that even in the absence of a-glycerol phosphate there is a low level of incorporation. This may be due to the pre- sence of endogenous a-glycerol phosphate in the adipose tissue homogenate. Steinberg (1962) reported that the requirement for a-glycerol phosphate in adipose tissue could be replaced by ADP + NADH but not by glycerol or monoolein. These results demonstrate biosynthesis of triglycerides in adipose tissue goes through the glycerol phosphate pathway and that the monoglyceride pathway is not important. This inability of adipose tissue to utilize glycerol can be explained by the fact that glycerol kinase is essentially absent in adi- pose tissue (Margolis and Vaughan, 1962). When the mixture of fatty acids as described in mate— rials and methods was added to the assay medium, there was a sharp increase in a-glycerol phosphate incorporated into triglycerides (figure 11). In the absence of fatty acids, there was no incorporation of a-glycerol phosphate into tri- glycerides in the intramuscular fat, but in perirenal and subcutaneous fat there was approximately 20% incorporation (figure 11). This observation may be explained by the fact 82 100 ‘fi *1 O E 8 a E o- I “80 O - l o . 0 I u 40 o———-o Peri. D I .8 L 4 Subcul. U L o 20 I-_-I Intro. 0 I! 0 0 2 4 6 8 1o mMGlycotol 3—Phosphate Figure 10. Glyceride synthesis as a function of d-glycerol 3-phosphate concentration. Percentage of Optimum 83 100 80 \ I \ 60 \ 40 9—‘0 Pen 20 ' L0 4 Subcut I-—-—. Intro 0 0'0 A 3 1.2 1.6 2,0 m M Fat ty A c id Figure 11. Glyceride synthesis as a function of fatty acids concentration. fa ex th tr P0 an SUI St] hax Ste 84 that perirenal and subcutaneous fat depots are early deve- 1oping adipose tissues compared to intramuscular fat, and they may have more endogeneous fatty acids than intramuscular fat. In addition, intramuscular fat is more sensitive to small increments of fatty acid concentrations (figure 11). Due to the presence of endogeneous fatty acids, Bennink (1973) concluded that acyltransferase activities must be measured with labeled glycerol 3-phosphate rather than labeled exogeneous fatty acids if true acylating capacities are to be determined. The use of labeled a-glycerol 3-phos- phate has also been emphasized by Davidson and Stanacev (1972). Despite different sensitivities of intramuscular fat versus perirenal and subcutaneous fat to the addition of exogeneous fatty acid concentrations, the optimum level for the three fat depots was the same (.67 mM). Higher concen- trations of fatty acids inhibited a-glycerol phosphate incor- poration into triglycerides. The response of the enzyme system to the addition of BSA was different in the 3 adipose tissues (figure 12). However, they had the same concentrations for optimum condi- tions of the enzyme assay. High levels (>20 mg) of BSA had an inhibitory effect on glyceride synthesis of adipose tis- sue homogenates. This might be due to enhancement of sub- strate emulsification. Similar results of BSA inhibition have been reported in other experiments (Daniel and Rubin- stein, 1968; Benson, 1969). 100 E 3 .§ 3° 3 o '3' 60 O a D g 40 O U 0 °' 20 o 85 0 IO Figure 12. of BSA level. 20 3O 40 mg BSA/nae t ion Glyceride synthesis as a function 86 The amount of esterification was increased when MgClz ‘was added to the assay medium (figure 13). However, even in the absence of MgC12 there was approximately 50%or-glyce- rol phosphate incorporation, which suggests that endogenous Mg2+ was present. There was a rather broad plateau after the optimal concentration (3.3 mM) of Mg2+. Endogenous Mg2+ has been reported to be higher in liver (Benson, 1969) than in adipose tissue. The concentration of glutathione (GSH) needed for optimum enzyme assay conditions in the intramuscular fat was different than perirenal and subcutaneous depots (figure 14). High concentrations of GSH inhibited triglyceride synthesis as shown in figure 14. The function of GSH in the process of esterification is to increase activity of the enzyme system by protecting susceptible thiol groups of CoA from oxidation and it also protects lipids from autooxidation. Glutathione can be reversibly oxidized by the loss of two hydrogens, which results in formation of a disulfide bond. The latter functions as a hydrogen donor in oxidation-reduction reactions. The amount of a-glycerol phosphate esterifed increased linearly from 0 to 45 min of incubation time followed by a plateau from 45 to 60 min (figure 15). The relationship between enzyme source (adipose tissue homogenate) and glyceride synthesis was linear between 0 and 1.0 ml of perirenal and subcutaneous homogenate, but between 0 and .6 ml for the intramuscular homogenate (figure 16). 87 l_. I g 4-0 E ‘a C) '5 o ‘, .40 3 5 s subcuI. c o 3 l—i Intro. 8’. 20 h- 0 4 8 I2 16 MM MQC '2 Figure 13. leceride synthesis as a function of MgCl2 concentration. 100 Percentage of Optimum G O 60 20 88 / A o I I C . " Pbrl e. A Subcut '——""-. lfl'ffl. 0.0 7. 5 1 5.0 2 2.5 3 0.0 m M Glutathione Figure 14. Glyceride synthesis as a function of glutathione concentration. 100 80 Percentage of Optimum O O .5 O N 0 Figure 15. time. 15 89 30 45 60 Iimo(min) Glyceride synthesis as a functicn of 90 .wEoHo> mumCmmoEon wo coauocom m mm mwmwnucem A .52288581 2 ‘9 up? £1 0 q b .3230 mkuoo>au .0H ouowfim é wnwggdo go command .eoa 91 In the present experiment .5 ml of crude adipose tissue homo- genate was used per reaction vial for all depots. The optimum assay conditions observed in these experi- ments were presented in the materials and methods section. Glyceride Synthetase Activity Changes in glyceride synthesis activity are shown in tables 9 and 10. The pattern of enzyme activity changes with age, depends on the method of expressing the activities. When expressed per milligram protein, enzyme activities increased (P <.05) from birth to 35 days in both perirenal and subcutaneous adipose tissues (table 9, figure 17). The increase in enzyme activity was much greater in subcutaneous fat as compared to perirenal fat (.77 fold 3g 113 fold increase). The enzyme activity in perirenal adipose tissue plateaued at age 35 days and remained essentially unchanged thereafter. However, there was a significant (P <.05) increase in the activity between 70 and 105 days for subcu- taneous adipose tissue which was then followed by a plateau through 175 days of age. The effect of age on the enzyme activity of intramuscular adipose tissue was nonsignificant (P >.05). Even at the last period of the experiment (175 days) the enzyme activity of intramuscular adipose tissue was negligible. This fact necessitates the lenghtening of experimental periods if the pattern of enzyme activity in this adipose tissue is of interest. 92 MIN/HG PROTEIN 1 2:40 2.80 3.20 <>Dfi( HCO'U 2.00 1.60 1.20 l RRTE UTILIZED/ 0.80 L NMULES SUBST l 0.40 .00 “b 3% 70 TIES 140 HGE (URYS) Figure 17. 'Glyceride synthetase activity of perirenal, subcutaneous and intramuscular adipose tissues expressed on a soluble protein basis. 175 93 .000000003000 00 00>00 now huanwaaaoumlum .000.A nvucuuuuunwan 0000000000000 00: 000 nua0uonu0050 0500 0:0 0:00000 so» 0800 0:0 :0 000000 name £000 =«s»«3,0c0020000 .mnlua 00 00 0000090 0:» 000 060020 .0030000 0000000 uaaauoaauuuca ~00 00:00 00 0:0 000060000030 no 006000000 000 wanna 00 00 00020 00. 00. 00. I I I I 000. 000. 000. 00. 00. 00. 0000000900060 000.v 00.00 05.00 00.00 00.0 00.0 I 50. 0.00 0.00 00. 5.00 0.00 000000090050 000.v 00.00 00.00 00.00 00.00 00.00 00.50 00. 0.00 0.00 00. 0.00 0.00 00:0uwu00 uuuuhuoam0< 500 00. 00. 50. I I I I 00. 000. 000. 00. 00. 50. M00auosaouucm 000.v 00.00 00.00 00.00 00.5 00.0 000. 00. 0.0 0.0 00. 0.0 0.00 0900:0090030 000.v 05.00 000.00 00.00 00.00 00.50 05.05 00. 00.00 05.00 00. 0.00 0.00 000000000 ”030000 0000004 0 00. 000. 000. I I I I 00. 000. 000. 00. 000. 000. u00=003fl00000 000.v 050.0 050.0 000.0 000.0 000.0 000. 00. 00.0 50.0 000. 000.0 000.0 090000030090 000.v 000.0 000.0 005.0 005.0 000.0 000.0 00. 00.0 00.0 000. 050.0 005.0 00:000000 "cu0uoum 08 "~00 .c0a 000 0000000: 000000050 000032 .um 050 000 000 05 00 0 .um 039 600 .um usuaouw 0:030u0 nuaoauuauauz 3600 0000 000030000 exam 033 5380 mmbmmHH mmOmH9< m<990092<¢HzH 92¢ msowzHHQ< 90.05). During all periods of the experiment, enzyme activities were higher for perirenal than for subcutaneous fat (irrespective of how the activities were expressed). This difference was especially marked at birth because the enzyme activity (per milligram protein) for perirenal adipose tis sue was approximately 57% of its adults value while that for subcutaneous adipose tissue was .67. of adult value. In other words, the postnatal increase in enzyme activity per mi IL ligram protein was greater for the subcutaneous depot than for perirenal adipose tissue. The accumulation of fat Was also much greater for subcutaneous depot than for peri- ran all fat during the 175 days of the experiment. In both perirenal and subcutaneous fat, but not intra- mus cular adipose tissue, fast growing lambs had higher CP< - 01) enzyme activities (per milligram protein) compared to the slow growing group (table 9). Merkel gt _a_l. (unpublished data) concluded that subcu- ta‘tleous adipose tissue from Southdown lambs (higher propen- sity to fatten) synthesized more glycerides than Suffolk (lower propensity to fatten) sired lambs. Effect of sex on 96 enzyme activity per milligram protein was not significant (P > .05). However, Merkel gt al. (unpublished data) observed higher glyceride enzyme activities (per milligram protein) for ewes than for wethers or rams. Results of interaction between growth rate and sex are presented in Appendix 11. Only in perirenal adipose tissue was the interaction between growth rate and sex significant (P < .05). In this adipose tissue, the fast and slow growing ewe lambs had the highest and lowest average values for glyc- eride synthesizing activities (per milligram protein) (2.79 vs 2.20 respectively). There was a significant interaction (P < .01) between growth rate and age in both the perirenal and subcutaneous adipose tissues (Appendix 12). In both of the se adipose tissues the fast growing lambs had higher enzyme activities at most ages compared to slow growing lambs. Gl)VCeride synthesizing activity per milligram protein in Perirenal adipose tissue was also affected by the interaction betineen sex and age of the lambs (Appendix 13). When the enzyme data are expressed per gram of adipose tis Sue, a different pattern was observed (table 9 and 10, fightre 18). The enzyme activity (per gram tissue) decreased at amatically (647.) in perirenal adipose tissue between birth and 35 days (table 9). This marked decrease in enzyme activ- ity per gram of adipose tissue (figure 18) was also accom- Famed by the period of greatest accumulation of lipid (figure 6) from birth to 35 days of age. The decrease in 00 0.5. . 0.0 Qw 0.? 0m. ON Du UDWWHF UWOLHDQ 0\ZHZ\DUNHJHF3 MFQKFWQDW WUJOZZ 97 80 <39)( tht ZIHn —mmm 2wm~ Imzm ZEN“ cnxz our: 29 39 LIEED/HINég RDIqufi TISSgF NHOLES SUBSTRRTE UTI 10 1 OF"— [—C DU) 70 “b 55 Figure 18. perirenal, subcutaneous and intramuscular adipose tissues expressed on adipose tissue weight basis. _’l 65' {be 140 HGE (DRYSJ Glyceride synthetase activity of 375 98 enzyme activity continued from 35 to 70 days followed by an increase between 70 and 105 days and then gradually decreased again and to a low value at 175 days. The reason for these changes may be explained as follows: the number of adipocytes of perirenal fat per gram of adipose tissue of new born lambs was 2.4 fold larger than those at 35 days (table 11). On the other hand, enyzme activity per gram of perirenal adipose tis- sue of new born lambs was 2.67 fold larger than at 35 days. Also there is a high correlation coefficient (r=.89, P<;Ol) between the number of adipocytes and the enzyme activity per gram of perirenal adipose tissue. Since the number of adi- pocytes per unit weight of adipose tissue decreased between birth and 35 days of age because of the increase in their size, expression of the enzyme activity per unit weight decreased at 35 days when fewer fat cells were present per unit weight of tissue. However, when the data were expres- sed on adipocyte number basis, higher enzyme activities were observed for larger adipocytes than small cells (table 9). This general trend of decreased enzyme activity per gram of adipose tissue was seen over the 175 days of the present experiment. However, between 70 and 105 days, the enzyme activity increased (P<.05). This observation can be explained by the fact that the effects of weaning and the consequent restricted caloric intake suggests that fat mobilization has occured as shown by the decrease in weight of this tissue (table 5). Thus, the percentage of small adipocytes (<25 uuo a be mi Ce Co 99 should have increased at age 105 days and this trend was actually observed (figures 22 through 25). However, the number of adipocytes per gram should also have increased because of the higher percentages of small cells. But the data in table 11 show a decrease (P<.05) in adipocytes per gram had occurred between 70 and 105 days of age. This observation may possibly be explained as follows: as men- tioned earlier, the effects of weaning stress (82 days of age) resulted in mobilization of lipid and consequently the size of adipocytes was reduced. Reduction in size of the adi— pocytes may have resulted in some of the very small fat cells (<25um) having passed through the filter screens and thus they were not counted. Consequently, the proportion of large cells had increased between 70 and 105 days which resulted in an artificially greater diameter or volume of the adipocytes. Therefore the number of cells per gram of tissue probably was underestimated in both the subcutaneous and perirenal depots at 105 days of age (table 11). The underestimated fat cells per gram of tissue also resulted in a decrease in numbers of total cells in each adipose tissue depot (total number of fat cells is the product of weight of the adipose tissue depot and number of fat cells per gram) between 70 and 105 days (table 11). Another possibility which ‘might have contributed to the underestimation of total fat cells is that at age 105 days the aperture size used in the Coulter Counter was changed from 250 um to 400 um” This 100 change in aperture size was made because from trial and error, past experiences and other works (Hirsch and Gallian, 1968) it was believed that at 105 days the diameter of some adi- pocytes was so large that they created problem with passage through 250nm orifice. However, this decision was made with- out considering the effects that weaning might have on the results. The 400 um orifice probably underestimated the number of fat cells because clumps of small cells which are not uncommon and are difficult to estimate, would not be individually counted. This difficulty also affects the size data (table 11). In contrast to perirenal fat, the enzyme activity per gram of subcutaneous adipose tissue was very low in newborn lambs. This observation was not unexpected, because at birth no dissectable subcutaneous adipose tissue was present (table 5). The connective tissue layer in which the subcu- taneous fat would develop later was physically dissected from the carcass of lambs at birth for use for the enzyme studies. This connective tissue layer was also fixed with osmium tetroxide and the cells separated as described in the mate- rials and methods for the adipose tissues. Microscopic examination of the tissue showed that very few adipocytes ‘were present and all were very small. Insufficient numbers of adipocytes precluded any Coulter Counter data on this tissue at birth. However, in this fat depot there was a marked increase (82 fold) in the glyceride enzyme activity 101 per gram of adipose tissue between birth and 35 days (table 9). This 82 fold increase in enzyme activity was accompanied by over a hundred fold increase in weight (table 5) and a 17 fold increase in the percentage lipid in the subcutaneous fat depot (table 7) during this dame 35 day period. Growth rate of the lambs did not have an effect on enzyme activity per gram of perirenal and intramuscular fat (p>.05). However, enzyme activity was higher (P=.O6) in the subcutaneous adipose tissue of fast growing lambs as compared to the slow groups (table 9). In both perirenal and intra- muscular adipose tissues the values of glyceride synthesis activity per gram of tissue were higher for rams than for ewes (P<.05). However, the sex effect on the enzyme activity of subcutaneous adipose tissue was not significant (P>.05). The enzyme activity per gram of both perirenal and subcutaneous fat was affected by the interaction between growth rate and age (P<.01, Appendix 12). In addition, enzyme activities of perirenal adipose tissue were affected (P<.05) by the inter- action of sex and age (Appendix 13). No other interactions were significant (P>.05). When enzyme activities were expressed on a per cell basis, in both the perirenal and subcutaneous depots the enzyme activities increased with age (P<.01, tables 9 and 10 and figure 19). However, no significant change in enzyme activity occured for the intramuscular fat between 140 and 175 days (table 9). The enzyme activity expressed on the 102 80 PERIRENRL SUBCUTHNEOUS INTRHHUSCULRR <>D)( 70 l 60 l 7 N/lO CELLS LIZED/HI 4p 51 RHTE UTI 30 l NHOLES SUBST 2o v I “b 35 7b-. lbs 140 1 5 HGE (DHYSJ Figure 19. Glyceride synthetase activity of peri- renal, subcutaneous and'intramuscular adipose tissues on a per cell basis. 103 basis of 107 cells in perirenal adipose tissue did not change significantly (P>.05) from birth to 70 days or in the sub- cutaneous fat from 35 to 70 days (table 9). Between 70 to 105 days, enzyme activities per 107 cells in perirenal and subcutaneous depots increased (P<.05) 2.2 and 4.2 fold, respectively. Between 105 and 140 days the enzyme activity in both adipose tissue depots increased but only that for the subcutaneous fat was significantly different (P<.05). The enzyme activity for both perirenal and subcutaneous fat expressed on a cell basis showed further increases between 140 and 175 days (P305) between fast growing lambs and the slow growing group. Similar results were observed by Merkel £2,3l- (unpublished data) in subcutaneous adipose tissue in lambs. Effect of sex on the enzyme activity on a per cell basis of perirenal and subcutaneous adipose tissue was also not significant (P>.05). However, rams had higher enzyme activities per cell in the intramuscular adipose tissue 104 compared to ewes. In contrast, Merkel £5 al. (unpublished data) found higher glyceride enzyme activities on a per cell basis in subcutaneous fat of ewes compared to rams. Enzyme activity per cell in both subcutaneous and intramuscular adi- pose tissues was affected by the interaction between growth rate and age (P<.05, Appendix 12). The enzyme activities on a per cell of perirenal adipose tissue were also affected (P<.05) by the interaction of sex and age, (Appendix 13). None of the other interactions was significant (P>.05). When comparing the three basis of expressing the glyc- eride synthetase activity, the data per unit of protein and per gram of perirenal and subcutaneous adipose tissues are quite constant after 70 days of age, but when the data are expressed on a cell basis, enzyme activities increased with fat accretion and adipocyte hypertrophy between 70 and 175 days of age. Thus these data indicate that adipocytes of perirenal and subcutaneous depots maintained the capacity for the glyceride synthesis throughout the experimental period in the present study. Additionally, glyceride synthesis activities on a cell basis of the large cells at 175 days of age (figure 19) were greater than at all other ages and the activity parallelled the increase in lipid accumulation (figure 21). 105 Lipid Content Per Adipocyte Changes in lipid content per adipocyte of the three adipose tissues with age are shown in tables 11 and 12 (also figures 20 and 21). As would be expected, in all adipose tissues, lipid content per cell increased with age. The lipid content per cell was significantly (P<.01) correlated with the percentage lipid in the perirenal (r=.55) and subcu- taneous (r=.48) depots but not for intramuscular (r=.27, P>.05) fat. The greatest increase in lipid content per cell for the perirenal fat (16 fold) occurred during the first 35 days. Undoubtedly, the subcutaneous fat increased similarly between birth and 35 days even though no observations of the birth adipocytes could be made. Fat content per cell increased significantly (P<.05) between all periods for the three adipose tissues, except no difference (P>.05) occurred between 35 and 70 days for subcutaneous fat. Growth rate did not significantly (P>.05) affect the fat content per cell of the three adipose tissues. However, the fat content per cell of perirenal adipose tissue of ewe lambs was higher (P<.05) than that of rams. This observation is consistent with the larger volume (P=.04) and diameter (P=.07) of the fat cells in ewes as compared to rams (table 11). Lipid content per cell was highly correlated (r=.96, P<¢Ol) with the volume of the perirenal fat cells (figure 10). 106 40° 2L4 350‘ . 2 0.3 3004 ~ .2 0.2 {:3 ‘3 48.4 1 S L 1 lipid Content/Coll(n9)(v———v) ‘3' .7: a Cell Volumdpmaxlo‘HH) 100 ' . .17. 8 50‘ 17-2 0 1, f T 16.6 0 35 70 105 MO I 5 A90 (do vs) Figure 20. Lipid content and cell volume of perirenal adipose tissue as affected by age. 107 280 , 46.8 (H) so- lipid Comm ('—-') lM-lipid Content (H) SQ-Coll Volume 245 r (. .) IM— Cell Volume "'3 2104 "36.8 A 175‘ " d I- g 31.8 ‘3 =. ,,'>'< g 5. § '40" “26.8 E 5 3 .12 S a. _- 3105“ «#213 $3 70“ ' 1 P163 35 ‘ "' -I r-".8 O‘L V l l ‘1 6'8 0 35 70 105 I40 I75 A90(days) Figure 21. Linid content and cell volume of subcutaneous (SQ) and intramuscular (IM) adipose tissues as affected by age. l()8 acquauucwwm maauuuunuuwum yo: man munwuuwuoasm mama onu wcaummn aou mama man so uoouwo cums sumo cusuua scam: .mwammfiu amonwvm umaaumSEMHucfi uOu .oocmouugcmwm uo Hm>oH ham auwaunonouml.um .3 .A3 cmuovo .mnaua Nd uo owoum>w «nu mun unmozn mnEmH Nu can maomcmusopam uo annoyance how mason on wo award doo.v v_.e_ no.5 I I I I «o. vw.w u¢.N~ "N. n._~ m.o umaaumaamuucm Hoo.v um.mm~ om.n-. cs.so~ uo.ms um.mn I on. «.mNS ~.an~ as. m.o- o.mm~ maomcuusunam ~oo.v cm.nmn w~.oe~ um.mo~ ow.~c v0.0m um.m no. vc.¢m~ o~.eo~ cm. n.c- q.o- Hmcmufiuom "chvmuxuoafivuuoa ucwucou van“; fioo.v v~.¢~ ow.o I I I I mm. <.o— n.o~ ow. m.o~ e.o~ um~=omafiwuucn doo.v um.mm om.on vo.m~ ad.N um.m I oo. ~.oN e.cN mm. w.o~ m.oN maoucmuaoaam _oo.v co.mm oa.o~ um.- as.“ om.o ca.~ so. so.o_ u~.m~ NN. m.m_ s.m_ Hacuuauua "Aqodxmenv 0E:Ho> mahoonwn< moo.v v~.¢o o~.om I I I I do. 0.0m c.5m mm. n.5n ¢.om umaaomaaauucm ~o0.v un.oo o~.nm vo.on u~.~m oe.~m I co. a.oc ~.¢e um. o.oo m.mo unoccuusonam zoo.v un.wm uo.me om.an ve.~m c~.om uo.~m so. m.~s m.wm NN. ~.om ~.ze annoyance "AEnv pmuwam«p ouzoonwv< o“. c.wo~ n.e- I I I I mm. N.qm~ a.oo~ cg. ~.N- q.-~ amasomaswuucu doo.v on.omo v~.w~e oo.oo~ vc._mq o~.mm~ I cc. e.mo< m.qqe no. n.9mc m.m~e maoocmuaonsm doo.v vo.o~_ va.~n_ ue.mo_ vo.¢o~ o¢.¢- o~.q~ ms. ”.mefi w.wmd ms. m.mc— ~.wm~ Hmcwuuumm "Ahofixvmsmmwu omoauvm voumuwawm floo.v vwm.u oo~.e I I I I c_. n.m ~.m co. mm.m ~m.m uwaaomaamuucu ~oo.v vo.~ cc.m cm.c o~.o~ o~.a~ I Nu. o.o o.od mm. m.c_ m.o maomcwusoaam _co.v um.n w~.m um.a us.sz em.hz on.ss as. ~.s~ o.o_ a~. a.m_ o.s_ Hmcoufiuma "A odxvusmmau omonuv< w “woo monsooaav< mo honesz .um mud 08— mod oh mm o .um 83m Emm .um.wdwaouwlw:«3oum. mucwEousmwoz 3o~m umwm namvaMow< mxmm wmumx Luaouo mmommmh mmomuo< m<4=umnzn osu was ncwozw 109 oo.m ma.m I I I I o_.~d o<.n I I I I 03m c~.- no.o~ I I I I oo.n_ n~.a I I I I In: unasooaluuucu c.no~ 5.55— a.~c— ~.mc m.c~ I N.qa~ n.cm_ <.un o.wm o.mm I 03E q.om~ c.oo~ w.mo_ n.5n ¢.nn I «.mnw m.On~ n.0o— ~.wn n._e I an: oaoocauaunam c.ao< ~.on_ a.-_ ~.mo a.oe m.o n.~ne a.oo_ ~.oo c.oo n.oc m.o 03m o.c- o.oo~ o.mw m.wn «.mn o.n o.oo~ ¢.oc~ o.n~— ¢.mm n.8n m.o an: accouauom ”AmcvmuAuonuva you ucoucou vunwa e.e~ o.c~ I I I I ~.¢~ ~.s I I I I 03m o.m ~.h I I I I e.n~ o.o I I I I an: uoflsuoaaouucn «.mn n.c~ °.- M.“ o.~ I ~.an n.~n ~.w~ c.~ m.w I 03u n.mn q.on o.n~ _.n o.“ I ~.~c c.o~ m.- a.n o.o I as: waoocnusoaaw n.ce _.u~ o.o_ ~.o m.o a.~ e._m m.o~ n._~ ¢.m ~.m n.~ 8:» o.n~ m.o~ ~.~e a.s n.m o.~ n.o~ m.n~ n.- m.c n.o n.~ 11¢ uncouuuom "A¢o~xna: Vuazao> ousoonan< a.¢o _.mc I I I I o.no e.cn I I I I 83m n.mo ~.~n I I I I n.mo w.om I I I I and unnaunalauucn o.oa e.an c.cs n.~m ~.~m I e.oo c.ca m.o~ o.~n ~..05). Cellularity of the Adipose Tissues During Growth Adipocyte Number Number of adipocytes was expressed either on a per gram of adipose tissue basis or as the total number for each adipose tissue depot (tables 11 and 12). As would be ex- pected, the number of fat cells decreased with age when ex- pressed on per gram of tissue (table 11). This is due to the increasing size of the cells with age, consequently fewer numbers of adipocytes were present per unit weight with advancing age. This observation is verified by the signif— icant (p<.01) negative correlation coefficients between adi- pocyte number (per gram.basis) and volume of the fat cells in perirenal (r--.69), subcutaneous (r=-.79) and intramuscular (r=-.58) adipose tissues. Neither growth rate nor sex of the lambs significantly (p>.05) affected the concentration of 111 adipocytes per gram of adipose tissues for any of the three depots. The interaction between growth rate and sex resulted in a significant (P<.Ol) effect on the number of adipocytes per gram of intramuscular adipose tissue (Appendix 11). No other interactions were significant (P>.05). 0n the basis of total number of adipocytes per depot, the total number of adipocytes in perirenal and subcutaneous fat generally tended to increase with age (table 11). Although the total number of adipocytes in the perirenal adipose tissue increased from.birth to 35 days, the increase was not significant (P>.05). The largest increase (83%) in this fat depot occurred between 35 and 70 days (P<.05) which was followed by a 50% decrease (P<.05) between 70 to 105 days. This latter decrease apparently is due to the events discussed earlier for the data at 105 days of age. The total number of fat cells in the subcutaneous adi- pose tissue depot increased (150%, P<.Ol) between 35 and 70 days, but decreased between 70 and 105 days. The latter observation probably is attributed to the explanation pre- sented earlier. The number of adipocytes in the subcutaneous connective tissue removed from the lambs at birth was very low and the few adipocytes present were extremely small in diameter as observed by microscopy. Thus, a considerable increase in subcutaneous adipocytes had to have occurred between birth and 35 days of age to account for the 195.1 x 107 cells present at 35 days of age in this depot. In 112 contrast to perirenal fat, the number of fat cells in sub- cutaneous adipose tissue increased (P<.05) from 140 to 175 days. These data suggest that the perirenal fat hyperplasia has plateaued while subcutaneous adipose tissue still has an increase in cell number occurring between 140 and 175 days. This observation also indicates the earlier maturity of perirenal fat compared to that for subcutaneous fat. The total number of adipocytes in intramuscular adipose tissue decreased (although not significantly) from 140 to 175 days. This latter decrease could be due to the change in aperture tube from 250 um to 400 um for intramuscular adi- pose tissue at age 175 days. Neither growth rate nor sex significantly affected the total number of adipocytes in any of the three fat depots. The interaction between growth rate and sex (Appendix 11) and between growth rate and age (Appendix 12) affected the total number of adipocytes in intramuscular adipose tissue (P<.05). No other interactions were significant (P>.05). Adipocyte Volume and Diameter The mean diameter and volume of adipocytes in the three adipose tissue depots were significantly (P<.01) affected by age (table 11 and 12). The diameter of the perirenal and subcutaneous adipocytes increased significantly with age, except between 35 and 70 days, until 175 days (P<.05). 113 Between 35 and 70 days the diameter of perirenal and subcu- taneous adipose tissue cells were similar, but at 105 and 140 days subcutaneous adipose tissue had larger adipocytes than perirenal fat. However, at 175 days fat cell size of the two depots was similar. Intramuscular adipose tissue fat cell diameter at 175 days was 30% less than that from subcutaneous and perirenal fat. These data indicate that intramuscular adipocytes hypertrophy occurs at later ages than for subcu— taneous and perirenal fat. During the last two periods of the experiment, when fat cell diameter or volume of the three fat depots were increasing significantly, a significant (P<.05) increase in adipocyte number also occurred in subcu- taneous fat but not in the perirenal or intramuscular adipose tissues. The results of fat cell number and diameter suggest that when the lambs weighed between 36 and 46 kg (140 and 175 days), hyperplasia apparently had been completed in perirenal adipose tissue and the increase in the fat depot during this time was primarily due to hypertrophy. However, when the lambs were younger, both hyperplasia and hypertrophy were responsible for the increase in weight of perirenal adipose tissue (table 11). On the other hand, both hyper- plasia and hypertrophy continued to contribute to the increase in subcutaneous adipose tissue at 175 days. Hypertrophy contributed significantly to the increase in intramuscular fat, whereas hyperplasia data were beset with the events associated with change in aperture as discussed previously. 114 Perirenal adipose tissue of ewe lambs had larger dia- meter (P=.07) fat cells and volumes (P<.05) than rams. These data are consistent with the total mass of the perirenal adipose tissue since ewe lambs had significantly (P<.05) more fat in this depot. Diameter and volume of subcutaneous and intramuscular adipose tissue were not affected by sex (P>.05). Growth rate of the lambs did not affect the dia- meter or volume of the fat cells in any of the adipose tis- sues (P>.05). Fat cell diameter and volume of the perirenal adipose tissue was affected by the interaction between age and sex (Appendix 13). None of the other interactions was significant (P>.05). Adipocyte Histograms Figures 22 through 27 depict the frequency distributions of adipose cells isolated from the three adipose tissues. Each bar of the histogram represents the contribution in per- centage of total adipocyte number made by the cells within a specified diameter range (abscissa). The histogram patterns for ram and ewe lambs were similar for each of the three adi— pose tissues. As shown in figures 22 and 23, approximately 95% of the adipose cells of the perirenal depot at birth had diameters of less than 401nm. With age up to 70 days, the percentage of small cells decreased while larger cells increased. This change caused a shift in bar height to the right at each age up to 70 days. At 105 days, the AId i Piocty'ssIs Numbsr of To! cl Psrssnloos of tho 115 liAIS'l GFR{DVVI HUG “M EWE .0 ,. .0 P .0 F- -:_'.m .0 ,_ .'.'N ‘0 ... " ‘0 .. X=3z4 so P so - so .- so - Io I- Io - o E l ‘ o I . so '- so P so - 1’ ”V so »- ssosv X- _ 40 '- 50p ‘0 h- i-Sa: so } so - so - so - I ~-l ~~I I o , ‘ - o , 7 . so ~ so I- so P- :0 DAY so — 700” X: 7 -= so F 5Q 40 b X “C2 80 '- 39 .— 30 r- | I so — l I l Io Io - l s: I. ' 7 J5; ° 7 ' so F' so P Ion on .o _ Ios on .0 _ ‘izsm “ _ i=5” 30 ’- 39 .— so - l I so I- to ‘- l l to r- J o , , ' 1 o J l J l . so F so so I- go on .0 NO on so - x=77°6 ‘0 2:72.? so - so so b so 1o — l l I so so so .0 E 17. 0" ‘0; '73 DAY ‘0 I— x=.‘.7 ‘6 b I=9‘7 so - so - IO P- 20 v- “1- so - o M o o so Ioo '00 s so '00 Iso Adi Pos yts Dismstsr' (pmstsr) figure 22. frequency distribution of perirenal adipocytes affected by growth rate, age and sex. AdiPOCny. of Numbsr Psrcsntogs of tho Total 116 5 low GROWING RAM ...,“ i=37-0 35 DAV X=47.3 70 DA V has '05 DAV i=5‘2 ML. "0 DAY i: 73.1 [111']? |75°AV i=79fi ll.l|'JJ_I+ so no no A d i P o s y t s Frequency distribution of perirenal adipocytes as affected by growth rate, age and sex. Figure 23 . .II'N i=317 35 DAY i=5” 7° DAY 1:516 l‘o DAV i=6” F '75 DAV i=93.‘ (,umstsr) 117 distribution pattern had changed because the percentage of small cells had increased compared to that at 70 days of age. This observation is consistent with the explanation discus- sed earlier for the perirenal fat data from the lambs at 105 days. An interesting observation is that the percentage of small cells (less than 30 um) that had increased at 105 days essentially maintained this level (between 30 and 40Irm) throughout the remainder of the experiment. However, dis- regarding the very small cells, after 105 days, the remainder of the cells increased in diameter and the distribution grad- ually shifted to the right with age. The distribution of the cells at the last two ages (140 and 175 days) had a bimodal shape with the first mode being represented by the small cells (less than 30 um) and the second mode by the larger cells. The adipocyte distribution observed for perirenal was similar to that observed for subcutaneous adipose tissue (figures 24 and 25). In intramuscular fat both small and large diameter fat cells were present. Compared to the data at 140 days, at 175 days the percentage of small cells had increased and the histogram bars had also shifted to the right (figures 26 and 27). Thus it appears that at 175 days both hyperplasia and hypertrophy probably contributed to the development of intra- muscular adipose tissue. Adipocytes of Numbsr Psrssntago of Total FAST RAM 11 8 GROWING so so so 35 0" so I 3SDAY Y:so.s ‘= so so — x “’2 so so - so so ~ 10 I 10 .. o J ‘ o I so so ~ 50 70 on ,0 _ 70 on izsm i252: so ‘0 ‘ so so — so so — 10 I I 10 e 1 I 1 o , I o . - so so 50 I05 on so __ w: on X:69.0 i=70.3 co so - so so - so so b 10 l J 10 - J I LI I . - I I I I I— so so r so HODAY so _ I_so DAY i=7._9 X=B4.‘ so so - so so - so so - 10 10 - s I L I I I I I l - I s I I I I I L so so - so '75 9" so — I75 DAY i=9” 33:90.6 so so - so so - so so - 1o 10 h I s I I I I I I I s [I I I II o . - fi— 0 . I # so 100 no 0 so 100 150 Figure 24. Adiposyts Diomstsr llamas-rt Frequency distribution of subcutaneous adipocytes as affected by growth rate, age and sex. Psrsontogs of tho Total Number of Adipocytes 119 SLOW G ROWING - RAM ,0 . EWE so ,0 _ 85 osv 5° _ 35 on i=50,6 7:51»? so— so - so- so - so - so - I .. 7 7 I o "I so r so _ 70 DAY so _ 70 DAY Fzssj 7:51-5 so - so - 3O — 3° .— so — so — 10 ~ 10 »— l I o I o 50 " .0 . 5° _ l05 DAY so L 125 DAY 7268-2 X:74.l so — so — so - so ~ so - so - I l I o I I I l I . so 5 80 ' 5° . no on so _ no on i=9o.o 7:794 so - so - so - so ~ so - 20 ~ Io - Io I o I I I I I I I o. ILJI I I I 9° so ' so _ I75 on .0 _ I75 on F:ay.¢ 7:90-0 so - 4° *- so - so - so - so - L ‘° ” l I l J '° | l I I I o .‘ I I . I o 'I I I ' 0 so 100 I50 0 so too 180 Adi posyts Diomstsr ( pmotsr) _ Figure 25. Frequency distribution of subcutaneous adipocytes as affected by growth rate, age and sex. 120 .Xom new own .ouou nusouw an wouoowmo om mou%ooawmo uoasoosaouuaw mo 60335.5»: xenon—wok .oN ouswfi El» ..o.oEo.n o.>ooa_1< On O n «sous on. so. a...w__wf o 5.00" x >(D mu— 0.— low I on r. 0.00".“ >(o o: _u>>u O Op I .2. Io. 1 on go. 05 02.3080 our 60p 0 k . . .. m. _ 1 0.00"“ >‘O mk— J I _ _ 0.0nnm. >(O 0'- (‘4;— has. Op on 00 0' on 00 Oh Or 0' Oh so sflotussssd |0301 Oq; Isqmnu ssusodgpy go .xom was owm .oumu Susouw hp nousommm mm mouhooawow 121 umHSUmsEmuucw mo cowuznauumav unconvoym .NN whammy «so .oElv ..osoEomn oa>oon_1< on. cop on 0 amp cap on o .d s 3 Op s u ON ..Do .5 Au» s o oéoflm as «....on I on u. :3 ...: o... :3 mt . Ave 00 I. o .1. o N o 6. Op .I “N .u« n .I o a w I now O». “W .1 0.. .v. m. wooflm. pémflm V. :3 o! 3 :3 3. on w as so u 3 at: < I OZ.BO¢O 30..“ u 122 Changes in Body Weight and Muscle Weight and Composition During Growth Body Weight Results of body weight gain as affected by growth rate, age and sex are presented in tables 13 and 14. At all ages fast growing rams and ewes (except for new born rams) were heavier than those of the slow growing group (table 14). As can be seen in figure 29, the fast growing group was heavier at all ages from 35 days onward than the slow growing rams. In addition, the overall effect of growth rate resulted in significantly (P<.01) heavier lambs in the fast growing group compared to the slow growing group (26.4 y_s_ 22.8kg, table 13). This difference in body weight was due to differences in daily gain (260 yg 230 g/day) rather than the differences in birth weights (4.2 yg 3.9 kg). The greatest percentage increase in body weight occurred between birth and 35 days. The growth rate of rams was significantly (P<.01) greater than ewes (table 13). Average daily gain of rams and ewes was 260 and 224 g, respectively, for the entire experimental period. The growth rate of fast growing lambs and rams was greater than slow growing lambs and ewes. The highest (230%) and the lowest (27%) percentage increase was seen during the first (0 to 35 days) and the last (140 to 175 days) age peri- ods, respectively. There was a significant (P<.05) 123 interaction between sex and age (Appendix 16) on body weight. No other significant interaction was seen (P>.05). The growth curves presented in figure 28 to 30 indicate that the usual sigmoidal shaped curve was not observed in this experiment mainly because the experimental period was not long enough. This can be seen from the data (table 13) which show that even at the end of the experiment the lambs still had significant (P<.05) body weight-and gastrocnemius (GT) and longissimus (LD) muscle weight increases. Muscle Weight The increase in GT and LD weights was very closely correlated with body weight (r = .98, r = .97 for GT and LD respectively, P<.01). This in agreement with the results reported by Hammond and Appleton (1932) in sheep and Orme 25 a1. (1960) in cattle. Butterfield (1962) showed that age, weight and breed of the animal had no effect on the correlation between muscle weight and body weight. The regression equa- tion for body weight (X) and GT weight of the 72 lambs in the present experiment is as follows: GT weight (g) = 3.40 x body weight (kg) + 8.05. By applying the equation one can estimate the GT weight from live body weight of the lambs. As was true for live body weight, GT and LD weights were significantly (P<.01) affected by growth rate, sex and age (table 13). During the early stages of growth when the lambs were tripling their birth weights, the greatest 124 80 KG) 5.0 40 I LIVE HEIGHT ( 30 I 20 I ch 3'5 7'0 1'05 1140 HGE (DRYSJ Figure 28. Growth curves of body weights for fast y_s_ slow growing lambs as affected by age. 75 125 51.50 45.50 39.50 39.50 KG) I HEIGHT ( 27.50 E .50 LIV 2} 15.50 9.50 D)( nun zzn nu: at 120 175 cp.50 7b 105 RGE (DHYSI Figure 29. Growth curves of body weight for rams gs ewes as affected by age. LIVE WEIGHT (KG) 126 S on) X L IVE WEIGHT 0 GT A L0 g-lr- J'56O 8... ~L—480 si- .-.... §-_ --320 g I 9. Ill 3 8”. --240 _"j u on a E 2‘:- 160 moir- "'80 T— I o c:lJ a? 140 1‘75 70 105 HGE (DHYSI Figure 30. Growth curves of body weight and as- tocnemius (GT) and Longissimus (LD) muscles. 127 increase in rate of GT and LD weights also occurred. These data are similar to those reported by Butterfield (1976) and the emphasize that this is the period of maximum growth. There was no significant (P>u05) interactions between the main affects, that is, growth rate, sex and age on muscle growth. Another expression of muscle growth is the calculation of percentage of muscle relative to total body weight at various stages of growth (tables 13 and 14). On the latter basis, neither GT nor LD weights were affected (P>.05) by either growth rate or sex. However, age had a significant effect (P<.Ol) on the percentage GT and LD weights (table 13). The percentage GT of body weight increased sharply from birth to 35 days. After 70 days the percentage CT of body weight plateaued, and the final percentage was significantly (P<.05) lower than initially. The percentage LD of body weight increased between birth and 70 days followed by a nonsignificant (P>.05) decrease between 70 and 105 days. The highest rate of increase in percentage LD was observed between 105 and 140 days, therafter the percentage plateaued (table 13). These data indicate that CT and LD were relatively constant proportions of live body weight after 70 and 105 days for GT and LD muscles, respectively. 8 2 maamufiumwumuw uo: mum mumwuomumasm 08mm ozu wcfiumon Bow mamm mxu co .Amo. Aavucmowmwcwfim uoowmm cams comm :fiSuH3 mcmoznwmovo .mnEMH Nfi mo mwmuo>m mnu mum snooze .mnEmH om mo owmuo>m may mum mcmozm Hoo.v owm.~ wn¢.~ v-.~ wtqm.H woqm.~ oNH.H mm. om.~ om.H mm. am.H mN.H wN GA A v Hoo.v :N.mmo wo.mmm m~.¢mm mm.mm~ nq.mmH om.qq Ho. vmfim om.qmm coo. w<.o~m om.nmm uswfioa GA fico.v 0mm. onm. vomm. wow. owe. mom. mm. oq. mm. no. es. oq. N Ho 3 Hoo.v :~.mm~ wo.emH mq.mofi oq.mw mm.mo oo.m~ ~oo.v vw.nw o~.ooH moo.v um.om UN.~o~ uswfim3 90 wav doo.v nm.oq www.cm .Mmo.wm oqm.om nqo.mfi uma.m goo.v .wmo.mm omo.oN Hoo.v www.mm um«.o~ uswfims o>fiA .um mmH oqfi mom ow mm o .um 03m 8mm .um wGHBOHw wcfisouw mucmfiousmwoz nAmzmcvmw< mxom 30Hm ummm mmumm nuBOHU mmqumbz ADAV mDZHmmHUZOA Qz< AHuv mDHZMZUOMHmHA zo wa Dz< m0< .mHm on» mum mammZM ms.~ as.z sN.H z~.H ~N.~ mH.H ms.z om.z ~m.~ os.z o~.a as. 83m mm.z ms.s w~.z sm.a mz.a w~.3 HN.~ mm.a wz.s mm.z z~.a mo.z amm mwmucmooma as was mos mam HEN ass mm «so smm «am New «on as mam one mmm Ham mAN Hmz mm Nms was mam msm mes ms awe vauawsms as mm. am. am. cs. as. am. on. em. mm. mm. «m. mm. mam mm. am. am. ms. ms. ms. mm. am. an. as. sq. mm. 5mm mwmocmooma yo ass mos om as 00 Ms cos wNz nan me me mu 83m ass ass sea mm an as was own man son as ma awe Amounwsms so ms.zs ww.w~ as.mN am.es «N.~z Nm.m m.qs Ne.sm wn.o~ mo.mz mz.m3 Hz.q mam so.ms oz.wm NQ.NN sm.sz «$.33 ss.s s~.mm sm.ms mm Ne.¢~ zm.sz ww.m 8mm wavunwsms 8>Ha was oqs mos oh mm 0 was ecu was en mm o xmm uamamosmmmz Amhmtvow< Ammmmvmw< wCH3OHO BOHm wCH3OHO ummm meHA ZO Nmm Dz< mo< .mH.05). Muscle RNA and DNA concentrations decreased (P<.05) by 62% and 50%, respectively, between birth and 35 days (table 15, figure 31). Thereafter, concentration of RNA plateaued, while DNA showed a further, but small (P<.05) decrease in nucleic acid concentrations. These decrease in muscle nucleic acid concentrations during postnatal growth agree with results reported previously (Enesco and Puddy, 1964; Robinson and Bradford, 1969; Powell and Aberle, 1975; Aberle and Doolittle, 1976; Johns and Bergen, 1976; Harbison gg 51., 1976). The high concentration of DNA in the baby lambs might be attributed, at least in part, to the presence of more muscle fibers per unit weight of muscle. In addition, skeletal muscle tissue of new born lambs is similar to that of embryonic muscle in which the nuclei and nucleoli consti- tute a high proportion of the muscle compared to that of later ages. During the period between birth and 35 days, the lambs showed the maximum muscle growth rate, and since 131 .oocooauwcwuo uo Ho>oH you auwawnmnoums.um .Ano. Aavucoouuacwwo haaouaumwuoum uo: oum muaauomuoasm oEom osu wcauoon sou oaom ozu :o uoowuo case sumo cuzufio ocoozuovu .mnaoH mg no omouo>o osu one ocoozn .opEoH on «o owouo>o ox» ouo ocoozo Ammuo_xv doo.v «n.om u¢.oo «c.0q oc.mn no.mn om.w~ om. o.os N.¢m c_. n.0q c.mm msoao=:\u:w«o3 Asosxvsssusc ~oo.v wm~.~m umm.o~ oom.m~ ovo~.- voa.- on~.m coo. vuq.o~ u~m.m~ doo.v tun.m~ umo.o~ mo uonE=c annoy no. vooo.~ o—o.n omm.m vmm.~ noe.~ oo~.m o“. mm.N on.~ oh. ow.~ hm.~ m ocu oum mcmozo «.mm m.nq ¢.¢q m.oq m.am m.n~ o.mq n.0c c.mm ~.~m o.wm o.- o3m Awlofixv ~.cm m.om o.m¢ n.¢q N.wN 5.5H n.m< m.~q m.mq w.om H.mm m.n~ 6mm mooaooc\unwflo3 m~.~N mm.~N om.o~ om.m~ No.m~ on.“ mm mc.nm mm.- mm.m~ m~.m~ oq.n osm Amofixvfioaosa mm.o~ NH.wN mo.o~ mo.- nfi.m~ Ha.o~ mn.Ho Hm.mm ww.cm Nm.wm mw.wfi m~.o Eom «0 “opens Hmuoh mm.~ NN.m m~.c ~5.N co.~ mw.~ ~o.~ mw.m -.m o.~ No.~ o~.m osm mw.N Ho.m ww.~ oo.~ om.N mo.N qm.~ mo.~ m~.m mm.~ mm.~ mm.m 8mm .05), however, age had a significant (P<.05) effect on this ratio. The ratio decreased from birth to 35 days (P<.05) then remained unchanged until 70 days at which time it increased again (P<.05) and essentially regained and maintained the initial value thereafter. The percentage increase of total DNA in the GT between birth and 35 days was approximately twice that of RNA, thus the ratio decreased. The highest ratio of RNA/DNA was observed at 105 days of age. This may be attributed to the following explanation. The lambs were weaned at 82 days of age which obviously reduced feed intake for the next few days. RNA concentration has been shown to decrease rapidly 135 following dietary restriction; whereas, DNA is much less sensitive to dietary intake (Howarth and Baldwin, 1971). Restriction of dietary intake to normal ad libitum levels has been reported to result and a marked increase in RNA synthesis (Howarth and Baldwin, 1971). Undoubtedly the RNA concentration at 105 days of age in the present study resulted from the compensatory increase in RNA synthesis to above nor- mal levels when the lambs returned to normal feed intake levels during the week or days just prior to the 105 day sampling period. Other data also indicated that RNA/DNA ratio increased when stressed (Logan ggpgl., 1952; Gluck 35 31., 1964). Goldspink (1964) reported that stress due to borderline protein intake caused a reduction in DNA and an increase in protein/DNA or RNA/DNA ratios. The present results were in agreement with the above observations (table 15). The results of several experiments shows that total muscle growth is more closely related to total DNA than to rate of protein synthesis (Cheek 33 51., 1971; Buhlinger g; 31., 1978). The present experiment also confirms the above statement (Appendix 18) regarding the relationship of DNA to protein accretion. High ratios of protein/DNA and RNA/DNA both provide an indication of high rates of protein synthesis. 136 Total Amounts of Nucleic Acids In addition to concentrations of DNA and RNA, the total amount of each in the GT was calculated. When compared to the slow growing group, fast growing lambs had more total RNA and DNA in their muscle (P>.05). Rams had more total DNA in their GT muscles than ewes (P>.01). These observations are a reflection of the increased muscle weight among the fast growing lambs and rams. Although the total muscle RNA of rams was higher than ewes, the difference was not statis- tically significant (P>.05). Both total RNA and DNA contents of the GT were affected by age (P.05) increase up to 175 days of age. The increase in DNA was more than twice (109%) that of RNA during the first period (birth to 35 days). Between 35 and 70 days DNA showed a further significant (P<.05) increase (23.4%) but only a slight nonsignificant (P>.05) increase (approximately 6%) between 70 and 105 days of age. Between 105 and 140 days, DNA increased (27.6%) significantly (P<.05) followed by a nonsignificant (p>.05) increase (8.7%) between 140 and 175 days of age. These nucleic acid data essentially parallel the increase in muscle weight with growth. The only signif- icant (P<.05) interaction observed was between growth rate and age on the total DNA content of the GT (Appendix 15). 137 There was a high correlation (P<.Ol) between muscle weight and total DNA (r = .89) or total RNA (r = .81). Changes in Number of Nuclei During Growth In tissues that are composed of mononucleated cells, total DNA content is a direct indication of the total number of cells because their diploid nucleus contains a constant amount of DNA (Mirsky and Ris, 1949; Thomson 25 gl., 1953; Vendrely, 1955). However, skeletal muscle is multinucleated and tfluu; the relationship between the quantity of DNA and the number of muscle fibers is more complex. Cheek 33 El. (1971) introduced the "DNA Unit Concept" which considers the cytoplasm—to-nucleus ratio as a cell unit within the muscle cell. The number of nuclei in a given mass of muscle tissue can be calculated from the total DNA content. Thus, in multinucleated muscle cells the number of nuclei provides an indication of muscle growth potential. In order to study this relationship in the present experiment, the total num- ber of nuclei was calculated by dividing the total DNA con- tent of the GT muscle by 6.2, since 6.2 is the amount of DNA in picograms in a single diploid nucleus (Enesco and Puddy, 1964). This index is used to estimate the cellularity (num- ber of nuclei derived from total DNA) in the skeletal muscle tissue (Trenkle 2E gl., 1978). The results of the total number of nuclei in the GT muscle are presented in table 15. 138 Fast growing lambs had more nuclei per GT (P:901) than the slow growing group (24.65 x 109 yg 19.77 x 109). There was a significant interaction (P<.05) between age and growth rate for the total number of nuclei per GT (Appendix 15). Although the number of nuclei in the GT at birth was lower in fast growing lambs than the slow growing group, the high- er rate of muscle growth in the fast growing lambs resulted in greater nuclei numbers during the remainder of experi- mental periods. The maximum increase in the number of nuc— _lei occurred between birth and 35 days of age which was 163% Hand 89% for fast and slow growing lambs, respectively. As would be expected, rams had more nuclei in their muscles than ewes (P<.01) because they had heavier muscles. The number of nuclei increased (P<.Ol) with increasing age, how- ever, between 140 to 175 days the increase was nonsignifi- cant (P>.05). High correlation coefficients were observed between number of nuclei and muscle weight (r = .89) and with body weight (r = .86). Some of the increase in muscle DNA may originate from the increase in nuclei associated with connective tissue and other cell types present in muscle tissue (Jablecki 23 $1., 1973). However, Enesco and Puddy (1964) reported that a major proportion of the postnatal DNA increase was due to increases in nuclei within the muscle fibers. They also found that the proportion of DNA in muscle and connective tissue cells does not change during growth of skeletal 139 muscle. The results of the present experiment showed the percentage of connective tissue evaluated as stroma protein nitrogen in the GT was low (less than 1%). The actual per- centage of stroma protein nitrogen which was highest at birth (.6%) decreased to approximately .5% at 175 days of age. Since connective tissue contains relatively few cells, therefore few nuclei, it can be concluded that the contribu- tion of nuclei from connective tissue in total muscle DNA was negligible. With increasing age the cellularity (number of nuclei derived from total DNA) of the GT muscle increased (P<.01). The total number of nuclei increased threefold during muscle growth from birth to 175 days, however, the greatest increase occurred between birth and 35 days (120%). After 35 days the total number of nuclei per muscle continued to increase (P<;05) up to 105 days and remained unchanged thereafter (P>.05, table 15). Buhlinger 25 El. (1978) concluded that the difference in number of muscle nuclei rather than differ- ences in protein synthesis probably accounted for the differ— ences in protein deposition of obese and lean pigs. In the present experiment it was found that measurements such as protein/DNA, muscle DNA or RNA concentrations and the ratio of RNA/DNA are not as highly correlated to muscle weight as total number of nuclei in the GT (Appendix 18). This obser- vation is in agreement with the data reported by Ashmore and Robinson (1969) , Ezekwe and Martin. (1975:), Powell and Aberle (1975) and Buhlinger 3E 31. (1978). 140 These conclusions support the DNA unit concept proposed by Cheek E; El. (1971) that the total amount of muscle gained during growth is associated with the total DNA present. Muscle fibers do not undergo mitosis (Stromer gt 31., 1974) during postnatal growth, therefore the increase in total DNA during growth cannot originate from mitotic division of mus- cle nuclei. Recent data indicate that satellite cells are capable of postnatal mitosis and that one or both of their daughter cells resulting from this mitosis may be incorporated into the multinucleated muscle cell (Reger and Craig, 1968; Moss and LeBlond, 1970, 1971; Schultz, 1974). Thus, the DNA Unit Concept of Cheek g3 El- (1971) amplifies the importance of satellite cells for postnatal muscle growth. Weight Per Nucleus Since the ratio of cell protein/water is quite constant (Cheek g; 31., 1971), it can be deduced that protein/DNA ratio is an index of hypertrophy of muscle cells. Another parameter which also is an index for cell size is weight of the cellular constituents. This is an index of the amount of material associated with each nucleus, and therefore, it is influenced by size of the cell and the quantity of its intracellular materials. To calculate the weight per nucleus, weight of the muscle was divided by the total number of nuclei in the GT. The protein/DNA and weight/nucleus data 141 are presented in tables 15 and 16. Weight per nucleus and protein/DNA ratios increased (P<.05) after birth until 70 days and remained unchanged thereafter (P>305). There was a high correlation (Pu05), the significant difference (P<.05) in protein/DNA ratio suggests that there was more hypertrophy in slow growing lambs than the fast growing group. The effect of sex on both the protein/DNA and weight/nucleus ratio was nonsignif- icant (P>.05). The weight/nucleus ratio was affected by the interaction between growth rate and age (Appendix 15). Other interactions were nonsignificant (P>.05). In summary, it is concluded that the greater GT muscle mass of fast growing lambs and rams was due to more cellularity (DNA content) rather than size of the muscle fiber. The larger fibers of these lambs were associated with high concentrations of myo- fibrillar proteins and to a lesser extent to the sarcoplasmic proteins (table 19). 142 Chemical Composition of CT and LD Muscles The percentage fat, protein and moisture of the GT and LD muscles are presented in tables 17 and 18. During muscle growth the percentage mositure decreased (P<.Ol) (from 79.4 to 72.9 in CT and from 79.1 to 73.6 in LD), while the per- centage fat increased (P<.01) from .96 to 5.07 in GT and from .62 to 3.66 in the LD. The percentage fat in the GT did not change significantly (P>.05) between 35 and 140 days, but then increased significantly (P<.05) between 140 and 175 days. In contrast to the GT muscle, the percentage fat in the LD showed a further increase (P<.05) between 35 to 70 days of age and then was followed by a decrease (P<.05) between 70 to 105 days. The decrease in fat content of LD muscle between 70 and 105 days may be attributed to weaning stresses suggesting that marbling in the LD muscle is more sensitive to these stresses than the GT. However, between 105 and 140 days the percentage fat in the LD returned to the value at 70 days and showed a further increase (P<.05) between 140 and 175 days. The decrease in moisture content is a well established phenomenon (Callow, 1947; Dickerson and Widdonson, 1960; Reid g_t_ a_l. , 1968; Hafez andDyer, 1969) and can be explained by concomitant increase in fat and protein content as the muscle grows and develops. The negative correlations between mois- ture and fat in the GT or LD muscles are presented in 143 .oocmofiMHome mo Ho>oH pom hufiawnonoumu.um .Amo. onucmoHMficmfim >Hamofiumfiumum uos oum muofiuomuoonm oEmm ofiu wowuoon 30H oEmw osu co uoommo came nowo cwnufia mcmozwmouo .mnEMH Na mo owmuo>m onu ouo mamozn .mnaoH om mo owouo>m onu oum mooozo 94 :H oHSumHoa Hoo.v m©.mm w~.mn on.ou oq.on um.mn o~.mm so. u~.cn oo.on mo. m.on 0.0m owouaoouom DA ca awououm Hoo.v mm.H~ wm.- oo.oN oo.o~ um.oH uo.o~ om. H.oN w.mH mm. o.o~ ¢.aH ommucoouom GA CH now Hoo.v moo.m oom.w umo.~ om~.~ voo.H owe. om. oo.N mo.m mo. u-.~ ooa.~ owmucoouom Hm :H ououmfioa Hoo.v mm.~n oo.qm ufi.om u~.om u~.cm oe.mn mo. m.mu m.mn mm. 5.05 w.mn omoucoouom Ho ca owououm Hoo.v oq.o~ oH.o~ ov¢.ad hm.oH uH.mH on.ofi mo. um.m~ o~.m~ moo. um.mH oo.m~ owmuooouom 90 :a mom doo.v omo.m .wmm.m wcm.m b~m.m bum.m uoa. No. ww~.m owo.m mo. bm~.m oeo.m owouaoouom .um ohm cod mom an mm 0 .pm ozm 8mm .Hm wofisouw wofi3ouww muaoaousmooz xom 30am umom Amhoovow< n moumm :uBOHU m mmuomsz Ange mszmmHozou oz< Aeov msezmzoomemo on» one mcoozo 0.~5 0.05 H.05 0.05 5.55 0.05 0.m5 5.05 0.05 0.05 H.55 5.05 mam 5.m5 N.m5 5.05 m.05 0.55 0.05 0.05 0.05 ~.55 o.05 0.05 ~.ow 5mm ea c“ ounuowoe omouaoouom N.~N N.H~ 0.o~ m.o~ <.0H 0.0H 5.- m.- 0.0N m.o~ o.o~ n.0fi osm H.HN m.HN 0.0m 0.o~ w.m~ 0.0H H.HN H.~N N.o~ 0.o~ ~.w~ c.0H Box 94 0H :Hououo owmucoouom 00.0 mo.m mm.~ 0m.~ 0m.~ m0. Ho.m o~.~ H0.H 5m.H m5.H m0. oBm 5m.q 5N.~ m0.H HN.N mq.H O0. m0.~ w5.N 00.~ m0.~ O5.H #0. Box nu ma umm owmucoouom m.~5 0.05 0.05 0.05 0.05 o.m5 o.M5 0.05 0.05 0.05 H.05 N.m5 o3m m.M5 0.M5 m.m5 m.05 0.05 0.05 o.m5 0.05 0.05 5.05 «.05 m.ow Ema BU SH ousumaoa owmucoonom q.- 0.o~ 0.05 «.mfi o.mH 0.0H 0.0g m.o~ m.o~ 0.05 0.mH m.0~ osm o 5.oN o.o~ ~.o~ s.o~ ~.aH ~.5H o.o~ q.au o.m~ o.m~ m.wa N.0H sum so as cemuooa mmmucouumm 0.0 5.m o.m H.m w.m o.H H.m m.m N.m 5.m m.m m. ozm 0.0 m.q w.m 0.5 0.m w. 0.0 0.0 5.0 «.0 m.m H.H Box 90 :« uow owMucoouom 05H 00H 00H 05 mm o 05H 00H 000 O5 mm o xom mucosousmmoz Ammovvow¢ Amhouvow< wcfi3ouu 3oam wcfi3ouo ummm MH.05). Although the percentage protein in the GT of fast growing lambs was lower (P<.01) than the slow growing group the differences in the LD was nonsignificant (P>.05). Percen- tage moisture of the GT and LD muscles was not affected by growth rate (P>.05). Rams had higher (P<.05) percentages of moisture in the LD compared to ewes. The effect of sex on the percentage moisture of the GT was nonsignificant (P>.05). The maximum change in moisture and fat was observed during the first and the last 35 days of the experiment. However, the change during the first 35 days was greater than that of the last period. The percentage protein in both GT and LD muscles increased sharply after birth until 35 days, but then the increase was more gradual thereafter. There was a signifi- cant (P<;05) interaction between growth rate and sex on the percentage protein in both GT and LD muscles. No other significant (P>.05) interactions were observed. 146 Changes in Nitrogen Fractions During Growth The nitrogen fraction data are expressed as: milligrams nitrogen per gram GT, milligrams nitrogen per GT and each fraction expressed as a percentage of total nitrogen (tables 19 and 20). Age had a significant effect (P<.01) on all nitrogen fractions. As the GT growth progressed, the concen- tration of total, myofibrillar and sarcoplasmic nitrogen increased (table 19). The increase in concentration of myo— fibrillar nitrogen was essentially parallel to total nitrogen with a high correlation coefficient of .91 (P<.01). The con- centration values are similar to those reported by Helander (1957) on gastrocnemius muscle of cattle. However, the per- centage increase in sarcoplasmic nitrogen (46%) from birth to 175 days was greater than for myofibrillar nitrogen (31%). This is in agreement with data of Lawrie (1961) for the beef longissimus muscle. Stroma nitrogen per gram GT was the most variable when compared to the other nitrogen fractions (table 19). This might be explained by the fact that the stroma nitrogen is determined by difference which would reflect the combination of experimental errors in other fractions. How- ever, the general trend of changes in stroma nitrogen con- centration during GT growth were similar to those reported by Helander (1957) for this same muscle in cattle. Stroma nitrogen was negatively correlated (P<.05) with myofibrillar 147 .o0couquucwuo uo ao>oH 000 huuuuaopoums.um .A00.A 0000000006000 adaoouuouuouo go: ouo oue«uuouonao oaoo osu 0000000 300 0:80 050 do uooumo can! nooo 60:00: ocooznwmo00 .onaoa 5g 00 owouo>o 000 sec scoot0 .00305 00 mo omouoso 000 one ocooxo cowouuac owaooamoouom ~00.v 0000.5 0005.5 0005.5 000.5 000.5 050.5 00. 05.5 ~5.5 mm. 05.5 05.5 \00000000 uoHH«HA«NO5z so. 2:2 o~.2 392 85.2 392 no.2 ca. 5.2 ....2 Z. .92 ....2 comes? 539:.-.an 000. 005.5— o5.m~ 005.0u o000.0~ 000.05 05.~5 M0. 5.0— 0.0— 00. 5.0g 0.0g 0000000: oamuum N co ouuac ~00.v 00.0— o0~.0_ 000.50 00.0~ 00.0~ 05.0~ 0m. 0.0~ 0.0~ 50. 0.0~ n.0~ oaaooaooouom n cowouuuu :5.v ooodn 0.0.2 35...; coon 05.5.0 013 2. 5...; too om. 93 0.3 035230821... 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The ratio of myofibrillar to sarcoplasmic protein nitro- gen varied during growth. The highest ratio was observed in the GT muscle of the new born lambs. Neither growth rate nor sex had an effect on these ratios (P>.05). Concentration of total and nonprotein nitrogen was higher in slow growing lambs than the fast growing group (P<.05). The other nitrogen fractions were not affected by growth rate (P>.05). Only total nitrogen (milligrams per gram) was significantly affected by sex (P<.05) which showed that rams had lower total nitrogen than ewes. On the basis of the total milligrams of nitrogen in the GT muscle (table 19 and 20), all nitrogen fractions increased significantly (P<.Ol) during growth. Both sarcoplasmic and myofibrillar nitrogen fractions had a higher rate of increase in GT weight. This is in agreement with the results reported by Gorden 25 31. (1966) in rats. During the 175 days of the experiment the GT weight increased 9.2 times, while total sarcoplasmic and myofibrillar nitrogen increased 13.6 and 12.2 times, respectively. On the other hand, the increase in stroma (9.4 fold) and nonprotein nitrogen (9.7 fold) was similar to that of the GT increase of 9.2 fold. High cor- relation coefficients were obtained between the individual nitrogen fractions and they ranged from .92 to .99 (Appendix 150 18). These data suggest that estimating the total amount of one nitrogen fraction may be a useful tool for predicting another nitrogen constituent. In addition, there was high correlation coefficients between GT and individual nitrogen fractions (r values ranged from .94 to .99). Fast grwoing lambs had higher values for the nitrogen fraction compared to the slow growing group (P<.05). Rams had higher values for nitrogen fractions than ewes (P<.05). The only signifi- cant interaction was observed between sex and age (Appendix 16) which affected (P<.05) total myofibrillar and nonprotein nitrogen. When expressed on the basis of percentage of total nitrogen, the percentage of both myofibrillar and sarco- plasmic fractions increased significantly between birth and 35 days and maintained a relatively constant percentage thereafter (table 19). The percentage stroma and nonprotein nitrogen tended to decrease during GT growth, however, the changes were not consistent. Neither growth rate nor sex significantly (P>.05) affected the percentages of the nitro- gen fractions. No significant (P>.05) interactions were observed for these data. SUMMARY This study was designed to determine the effects of growth rate, sex and age on muscle growth and fattening of lambs from birth to 175 days of age. Sixty ewes with the fastest and 60 ewes with the slowest growing lambs were mated to Suffolk and Dorset rams respectively. Three ram and three ewe lambs of each growth rate were slaughtered at each age (birth, 35, 70, 105, 140 and 175 days). The lambs were weaned at 82 days of age and then divided into fast growing rams and ewes, and slow growing rams and ewes. Each group was penned and fed separately until slaughter time. The new born lambs were slaughtered within 10 to 12 hr after birth, while the lambs of the other age groups were fasted approximately 15 hr prior to slaughter. All muscle and fat were rapidly removed, weighed, frozen, powdered and stored at -85 C for subsequent analyses. Perirenal, subcutaneous and intramuscular adipose tissues were assayed for glyceride synthetase, cellularity (size and number of fat cells) and fat, protein and moisture. The GT muscle was analyzed for nucleic acid and protein fractions. Both GT and LD muscles were analyzed for fat, protein and moisture contents. 151 152 Fast growing lambs and rams had higher average daily gains than the slow growing group and ewes, respectively. Ewe lambs were superior to rams in feed conversion between 140 and 175 days of age. Body weight, muscle and adipose tissue weights increased with increasing age. Although fast growing lambs deposited more total protein than the slow growing group,the increase in total adipose tissue of the fast and slow growing lambs was similar. However, higher percentages of subcutaneous fat were detected in the slow growing group compared to fast growing lambs. Rams had more subcutaneous but less perirenal fat than ewes. Rams also had greater muscle weights than ewes. When expressed per milligram protein or cell basis, glyceride synthetase activity increased with increasing age, but on a gram of adipose tissue basis the activity decreased. The effect of growth rate and sex differed between depots and among the expression of the enzyme activities. The data for fat cell number and diameter suggest that when the lambs weighed between 36 to 46 kg (140 to 175days) hyperplasia apparently had been completed in perirenal adipose tissue and the increase in this depot during this period was primarily due to hypertrophy. On the other hand, hyperplasia and hypertrophy contributed to the increase in subcutaneous adipose tissue at age 175 days. 153 However, cellularity in both perirenal and subcutaneous adipose tissues was affected by the stresses associated with weaning of the lambs. Hypertrophy contributed to the increase in intracellular fat. Results of different adipose tissue measurements indicated that the growth sequence is perirenal> subcutaneous > intramuscular. Muscle RNA and DNA concentrations decreased while the total RNA and DNA increased in the GT muscle with increasing age. Although, growth rate and sex had no effect on the concentrations of nucleic acids in the GT muscle, fast growing lambs and rams had more total RNA and DNA and also more total nuclei in the GT muscle than slow growing lambs and rams, respectively. Although weight/nucleus and protein/DNA increased with age, these ratios were not affected by sex of the lambs. On the other hand protein/DNA (hypertrophy) of the slow growing lambs was higher than the fast growing group. As GT growth progressed, the concentration of total myofibrillar and sarcoplasmic nitrogen and the total of each fraction in the GT increased. The highest value of myofibrillar/sarcoplasmic ratio was observed in the GT muscle of the newborn lambs. Concentration of total and non-protein nitrogen was higher in slow growing lambs than the fast growing group. Rams had lower nitrogen concen- trations but higher values for total nitrogen fraction than ewes. 154 From the results of the present study it is concluded that although Suffolk-sired lambs grew faster and had heavier GT and LD muscles than the Dorset-sired lambs, weight of the adipose tissue depots of the two groups of the lambs were similar. However, because the slow growing lambs were lighter in weight and had similar quantities of adipose tis- sue, compared to fast growing lambs, the fat depots expres- sed as a percentage of body weight were greater for the slow growing group. Even so, the unexpected results for the adi- pose tissue mass in the present study suggests that the two groups did not differ greatly in their genetical pro- pensity toward fatness. APPENDICES 155 APPENDIX 1 TRIS-SUCROSE BUFFER PREPARATION, pH 7.2 Ingredient g/liter 30 mM tris 3.63 .3 M sucrose 102.69 1 mM glutathione (GSH) .3073 1 mM EDTA .3722 Dissolve and dilute to 1 liter with distilled water. Adjust pH to 7.2 and store at 2 to 3 C. APPENDIX 2 COMPOSITION OF FATTY ACID MIXTUREa Fatty acid Molecular description % g/100 ml Myristic 14 4.38 .0216 Palmitic 16 25.58 .1402 Stearic 18 17.74 .2372 Oleic 18:1 44.57 .0971 Linoleic 18:2 5.86 .0296 Linolenic 18:3 1.84 .0081 aThese data are for medium weight lambs and has been calculated from the tables reported by Tichenor ggy§1., (1970) 156 APPENDIX 3 PREPARATION OPSO mM ISOTONIC COLLIDINE SOLUTION, pH 7.4 Ingredient ml (a) .2 M Collidine (2,4,6-trimethyl pyridine) 37.5 (.609 g per 100 ml distilled water) (b) .3 M NaCl 39.4 (1.753 g per 100 ml distilled water) (c) .l M HCl 25.0 (.833 ml 12 N HCl per 100 ml distilled water) (d) Distilled water 48.1 Adjust to pH 7.4 To obtain a 3% osmium tetroxide solution, dissolve l g of 0304 in 33.3 ml of the above collidine buffer. 157 APPENDIX 4 CALCULATIONS FOR DETERMINING THE NUMBER.AND VOLUME OF FAT CELLS IN COULTER COUNTER (a) (b) (e) (d) (e) (f) (g) (h) (1) Calculate mean radius (r) of the cells in each range by changing I and A in the following equation: v 4/3nr3 = T.A.T. = I.A.T. volume of the cell (03) radius of the cell aperture current setting amplifier setting lower threshold at 50% count Subtotal no. of the cells per range = F x height of the peak F, the average no. of cells per line = no. of cells in a particular window (this window usually has an average peak)% distance of the peak from the base line. Sum all the subtotals for part (b) % of cells per range = (b) x 100 (C) Total cells per range = Total no. of cells in total volume x (d) Total no. of fat cells = no. of cell in 2 ml suspension x total volume of suspension/2 Weight in mg of total cells per range = {2(a)}3. (e). .4719 x 10-9 % recovery = Sum of (f) x 100 Sample weight in mg Adjusted total no. of cells per range = (e) (85 Volume of cells per range = (h). {(a)}3. (4/3) . n 158 APPENDIX 5 PREPARATION 01' RNA STANDARDS (a) Dissolve 12.5 mg RNA in 250 ml 5% (w/v) PCA This solution contains 50 mg RNA per ml. (b) Add 12.5 ml 5% (w/v) PCA to 37.5 ml of (a). This solution contains 37.5 mg RNA per ml. (c) Add 25 ml 5% (w/v) PCA to 25 ml of (a). This solution contains 25 mg RNA per ml. (d) Add 37.5 ml 5% (w/v) PCA to 12.5 ml of (a). This solution contains 12.5 mg RNA per ml. (e) Store all the above solutions at 2 to 3 C. APPENDIX 6 PREPARATION OF DNA STANDARDS (a) Dissolve 12.5 mg DNA in 250 ml of 10% (w/v) PCA. This solution contains 50 mg DNA/m1. (b) Add 12.5 ml 10% (w/v) PCA to 37.5 ml of (a). This solution contains 37.5 mg DNA per m1. (c) Add 25 m1 of 10% (w/v) PCA to 25 ml of (a). This solution contains 25 mg DNA per ml. (d) Add 37.5 ml of 10% (w/v) PCA to 12.5 ml of (a). (e) This solution contains 12.5 mg DNA per ml. Store all the above solutions at 2 to 3 C. 159 APPENDIX 7 PREPARATION OF 1% (W/v) ORCINOL REAGENT (a) (b) (C) Make 10% (w/v) of FeCl3 in concentrated HCl. Make .05% FeCl3 solution by taking S‘ml of (a). and diluting to 1 liter with concentrated HCl in volumetric flask. This will be stock solution. Make 1% orcinol solution by adding 100 ml of (b) to 1 gm orcinol in a volumetric flask and stirring vigorously with a magnetic bar for about 20 min. This solution must be made fresh just prior to use. APPENDIX 8 PREPARATION OF4% (w/v) DIPHENYLAMINE REAGENT Make 4% (w/v) diphenylamine solution by adding 100 ml glacial acetic acid to 4 g of diphenylamine and store at 2 to 3 C. 160 APPENDIX 9 PREPARATION 017' ACETALDEHYDE SOLUTION (a) Add .4 m1 of acetaldehyde to a 250 ml volumetric flask. (b) Bring to 250 ml and store at 2 to 3 C. APPENDIX 10 REAGENTS USED IN PROTEIN FRACTIONATION .015 M Potassium phosphate buffer pH 7.5 KZHPO4 2.16 g KHZPO4 .326 g Dissolve and dilute to 1 liter with distilled water. Adjust pH to 7.5 and store at 2 to 3 C. 1.1 M KI, .lM phosphate buffer pH 7.5 KZHPO4 14.631 g KHZPOA 2.178 g KI 182.6 g Dissolve and dilute to 1 liter with distilled water. 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I I I I uuaaunsauuucm 00. n.ne c.a5 c.05 5.c 5.c I n.en c.an c.c“ c.c 5.c I unoccuusucsm 00. 5.55 0.5c 0.55 c.c— 5.55 c.a_ 5.cc c.cc 0.5g c.c— 5.a~ m.c_ aucuuauom "ouumuomucu 50— "uon 0u565I you von«55u: aunuumcao 05062 .um N50 dad nod. 05 Ifim 0 IHNA 005 nod 05 IIwn 0 oucoaouaouu: nmhovvuw< Ammocvow¢ ~30 Ham 5a.couc .50 xHazmmm< 166 APPENDIX 14. RESULTS OF INTERACTION BETWEEN GROWTH RATE AND SEX ON SOME CHARACTERISTICS OF GASTROCNEMIUS (GT) AND LONGISSIMUS MUSCLESa GROWTH RATE Fast Growing Slow Growing Gastrocnemius (GT) Sex Sex muscle data Ram Ewe Ram Ewe Pr. Live weight (kg) 28.67 24.20 24.56 21.07 .56 GT weight (g) 108.5 94.0 91.9 80.4 .69 GT Percentage .39 .40 .39 .40 .47 Percentage fat 4.01 3.27 3.35 3.10 .26 Percentage protein 18.54 19.53 19.61 19.46 .001 Percentage moisture 75.93 75.77 75.80 75.55 .87 Concentration of nitrogen(mg/g) 29.8 30.8 30.9 31.1 .10 Concentration of myofibrillar 14.9 15.4 15.5 15.5 .13 nitrogen (mg/g) Concentration of sarcoplasmic 5.57 5.77 5.76 5.82 .33 nitrogen (mg/g) Concentration of stroma 5.58 5.62 5.58 5.73 .79 nitrogen (mg/g) Concentration of non-protein 3.84 3.97 4.03 4.01 .09 nitrogen (mg/g) Total nitrogen (mg) 3360 2960 2930 2600 .87 Total myofibrillar nitrogen(mg) 1700 1500 1480 1310 .89 Total sarc0plasmic nitrogen(mg) 630 570 560 500 .95 Total stroma nitrogen (mg)"7 600 510 510 460 .50 Total non-protein nitrogen (mg) 430 380 380 330 .87 %Tmyofibrillar nitrogen 49.9 49.7 49.9 49.7 .96 %_§arcoplasmic nitrogen 18.4 18.5 18.4 18.7 .74 % stroma nitrogen 18.7 18.9 18.8 18.5 .79 %finon-protein nitrogen 13.0 12.8 12.9 13 .39 Myofibrillar nitrogen/ 2.70 2.70 2.71 2.70 .90 sarc0plasmic nitrogen Concentration of RNA (mg/8) 5.10 4.95 4.83 5.14 .51 Concentration of DNA (mg/g) 1.89 1.77 1.75 1.78 .30 Total RNA (mg) 453 363 353 343 .48 Total DNA (mg) 168 139 131 114 .40 Protein/DNA 113.7 128.7 131.8 129.1 .07 RNA/DNA n 2.76 2.77 2.76 2.92 .72 Total number of nuclei (x107) 26.85 22.45 21.08 18.47 .46 Weight/nucleus (x10-8g) 37.4 39.8 40.9 40.2 .29 167 APPENDIX 14. (con't) GROWTH RATE Fast Growingr Slow Growing Longissimus (LD) Sex Sex muscle data Ram Ewe Ram Ewe Pr. LD weight (g) 376 339 334 287 .74 LD percentage 1.27 1.32 1.34 1.29 .21 Percentage fat 2.01 1.78 1.09 2.33 .10 Percentage protein 19.5 20.2 20.0 19.9 .02 Percentage moisture 76.8 76.4 76.4 76.1 .80 aMeans are average of 18 lambs. 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Prov 0950592009905 99 mUHHmHmmHU05 505 5055ucucouml.um .00305 c 00 00050>0 0:0 050 000020 50. 5.55 5.05 0.05 5.05 5.55 5.55 0.05 5.55 5.05 5.05 5.55 0.55 00000500 0550000005 50. 5.55 5.55 0.05 0.05 0.55 5.05 0.55 5.55 5.05 5.05 5.55 5.05 0500005 0500000005 500. 50.0 00.5 55.5 55.5 50.5 50. 55.5 00.5 00.5 5.5 55.5 50. 000 0500000005 55. 00.5 50.5 55.5 05.5 55.5 05.5 55.5 55.5 55.5 55.5 55.5 00.5 0555000005I05 55. 050 500 005 555 555 55 000 000 505 505 505 05 505 00550: 55 0000 050008 5050 00550050005 55. 5.55 5.55 5.55 55 55 5.55 5.55 5.55 5.55 5.55 5.05 55 500-0555 0005000500550: 50. 55.05 00.55 55.55 55.55 55.55 55.5 55.55 50.55 55.05 50.55 55.55 00.5 500500 505000 00 000000 50005 55. 05.5 55.5 55.5 00.5 55.5 55.5 55.5 5.5 55.5 55.5 50.5 55.5 I <255<25 500. 5.055 5.005 5.505 5.505 5.005 5.50 0.055 0.005 0.555 0.005 5.555 5.55 <2050500005 50. 505 555 555 505 505 55 555 505 055 505 555 50 5500 <20 50005 50. 550 550 550 055 055 055 555 055 550 505 555 555 5500 <25 50005 00. 05.5 55.5 50.5 50.5 05.5 05.5 55.5 50.5 05.5 05.5 50.5 05.5 555005 <25 00 0050000000000 55. 50.5 05.5 55.0 55.5 50.0 55.05 50.5 55.0 05.5 05.0 50.0 50.05 555000 oH new huwawnunouml.um .onflaH o «o owuu0>u usu mun accord nN. ~.m~ _.m~ ¢.o~ $.95 «.55 ~.w~ o.e~ c.m~ o.5“ ~.o~ ~.mn m.a~ «usumfios ammucouuom oo. e.- m.- ~.o~ o.o~ hwm_ 5.o" ~._~ ~.- c.cN n.o~ o.o~ n.o~ aflououm‘ummucmuuum on. m~.n an.~ ~5.~ ea._ co." no. mn.n mm.~ «o.~ ~q.~ on._ do. uau ammucmuuam ~N. Be." ma.“ on.~ "a." ¢~.~ oc.~ on._ we._ n~.~ on.~ ¢~.~ o~.d omqucouuma n4 m~. one see c¢n man ~n_ an «#0 com mon aom ~o~ on Amv usmfiua a; uuuv uaumau Andmxw:e«nnqmcoa o~. a.cq m.oe m¢ ~.nm “.mm m.o~ a.om o.me ~.e¢ ~.ne _.mn o.5. AmmumHMM mammmmw\mmwwaz dc. N~.on «.mN eo.o~ hm._n e.- n.~ n~.qn um.nn m~.m~ ~.- ~m.m~ nu.” Aoo~xu «odes: uo uoaasm Hauoa no. mm.~ o.~ no.n o~.~ mn.~ mm.n mo.~. so.~ "c.n ¢m.~ mq.~ mm.~ mm4m zmgmm mBZmHUHhmmOD ZOHBjmmmOU MHmZHw 0H NHszanH< NH. NN. NO.- ON.- OO. ON. OO. HO. NN. ON. OO. NN. NO.- OO. NN. ON HN. NN. NN.- NN.- NN. HO. NN. NO. NN. NN. NN. NN. OO.- OH. - NN. ON NN. NO.. NN. NO. ON.- NN.- NN. NN.- ON.- OO. ON. NN.- HH.- OH.- NN.- NN NN. HN. NN.- ON.- NN. NN. NN. HN. NO. ON. NN. NN. NN. NH. NN. NN NN. NN. NN.- NN.- NO. NO. NN. OO. H NO. NN. NO. ON. NO. NH. NN. NN NN. NO. NO.- NO.- NN. ON. ON. ON. ON. NO. NN. NN. OO. NN. NN. NN NN. N9 NH.- ON.- HN. NN. NN. NN. NH. NO. HN. HN. NH. - OH.- HN. ON NN. NN. NN. ON.- ON. ON. N9 NN. OO. NN. N9 NO. NO. NO. NN. NN NO. NO.- HH. NH.- NH. NO. HO. NO. O9 - OH. NH. OO. NH. OH.- NO. NN HN. NN. ON.- NN.- O9 NN. NN. NN. ON. N9 ON. NN. HN. NH. ON. HN NN. NN. ON.- ON.- NN. NN. ON. NN. NN. NO. N9 NN. H9 - NO. NN. ON NN. NN. NN.- NN.- ON. ON. NN. NN. NN. NO. O9 NN. NO.- NO. NN. ON NN. NN. OO.- NO.- ON. NN. NO. ON. NO. NN. ON. NN. NO. HN. NN. NN OO. NO. NN.- NN.- NN. ON. ON. ON. NO. NH.: NN. NN. NH. NH. NN. NN NN. NN.- NN. NN. HN.- HN.- NN. ON.- NO.- NN. H9 - NN.- H9 - OO. - NN.- NN NN. NN.- NN. NN. NN.- NN.- NN. NN.- NO. - N9 NN. HN.- NO.- ON. - HN.- NN a. ON. ON. NN.- ON.- HN. NN. HN. NN. NN HO. H9 NN. OH. NO. NO. ON 7, NN. OH. NH.: NH. NN. NO. NH. ON. ON. HO. NN. OO. OO. NN.- NO. NN :. OO. HO. NN.- NN.- NN. NN. NN. N9 ON. NN. NN. NN. N9 - OH. NN. NN NN. NN. NH. - OH.- NN. HN. NN. NN. NH. NO. NN. ON. NO.- NH. ON. HN HN. NN. ON - NN.- NN. NN. NN. ON. NO. HN. NN. NN. NO.- ON. NN. ON ON. NN. NH.- NNO- NN. NN. ON. NN. HH. ON. NN. NN. NO. NH. ON. NO ON. NN. NN.- NH.: NO. HN. N9 NN. NO. OO. NO. ON. ON. NH. NN. NO ON. NN.- NN. NN. HO.- ON.- ON. OO.- NN. HN. - HO.- HO. ON. - ON.- OO. NO OO. NN. NO. NH.- NO. NO. NN. NH. NO. ON.- NO. ON. OO.- OO.- HH. NO NO. NN. NH.- NN.- HN. ON. ON. NN. NH.- ON. HN. ON. NH. NH. NN. NO ON. NN.- NN. OO. NN.- ON.- NN. HN.- NN.- NN.- NN.- ON. NO.- NH.- ON. OO OO. NO.- ON. NN. NN.- NN.- NN. NN.- HN.- NN.- NN.- NN. NO.- OO.- NN. NO NN. NN. NN.- NN.- NN. NN. HN. ON. ON. NN. NN. NN. NO. NH. NN. NO NN. NN. - HN. NO. HN.- NN.- NN. NN.- NN.- OO.- ON.- NN. NH.: OH.- ON. HO NO. NO. - NN. ON. ON.- NN.- ON. NN.- NO.- ON.- ON.- ON. OH.- HH.- OO. OO NN. NN. NN.- NN.- HN. NN. NN. NN. NN. NO. HN. NN. NH. OH. NN. ON NN. NN. NN.- ON.- OO.H NO. NN. NO. NO. NN. OO. H ON. NO. OH. ON. NN HO. NO. HH. NH. OO.- HO.- OO. NO.- NH.: NO. OO. - NN. OH. ON. NO. NN NN. ON. ON.- NN.- ON. NO. OO. NO. NN. NO.- ON. NN. NH. HH.- OO. NN NH OH NH NH HH OH O N N N N O N N H .02 OHNNHNO> N.uaooo NH NHOzNNN< 176 NN HO.- HN.- NN.- ON. NN. HN. NN. NN. NN. NN. NN. HO. NO. NN. ON ON. NN. OO.- OO.- ON. HN. NN. NN. ON. NN. ON. NN. HO. NH. NN. NN NO. NN.- OH.- NH.- HO. NO. HN.- HH. NO. NH.- HO. NO.- NO. OO. NO. NN HN.- NO.- NO. OO.- NN.- OO.- OO.- NO.- OH.- NO.- NN.- NN.- OO.- ON.- NN.- NN NH. NN.- NH.- NH.- HN. HN. NH.- NN. NO.- NO. HN. OH. NH. NO.- NH. NN NN. OO.- NH.- OO.- OO. NO. NO.- ON. ON. NH. OO. ON. HO. NN. ON. ON HN. NN. NN.- ON.- NN. ON. NN. NN. ON. NO. NN. NN. NO. HO. NN. NN NN.- NN.- NN. ON. ON.- NN.- HN.- HO.- ON. ON. ON.- NH.- NO. NH. ON.- NN NH.- NN.- NN. ON. NN.- NN.- ON.- NN.- ON. HN. NN.- ON.- OO. ON.- ON.- HN NH OH NH NH HH OH O N N N N O N N H .02 mHnmNum> N.ucooO NH xHOzmmm< OO. NO. - N9 NN. NO. ON. HO. NN.- NO. NN. NN. NN. ON. - NN. - NN. ON NN. NH. - ON. HN. OO. NN. OH. NN. - NO. NO. NN. HN. NN. - ON. - OO. OO ON.- NN. ON.- NN. ON. ON. HH.- HN. - OO. ON. - OH. NO. ON. - NN.- NO. NO NH.- NN.- NO.- OO. - N9 - NN.- ON. NH. NH. HN. NH.- ON. - HO. ON. ON.- NO NO.- OO. OO. NH. - NN. - NO.- NO. OO. ON. - OO.- OH. NO. NN.- NO.- OH. NO OH. NN. NO.- NN. OO. ON. O9 - NH.- NN.- NN. - NH. ON. ON.- NN.- NO. NO HN. - N9 - OO.- ON.- ON.- ON.- OO. H9 NN.- NN. - ON.- NN.- ON. NO. ON.. OO NN. - O9 - OO.- NN.- NN.- NN.- HH. H9 NN. - NN. - NN.- NN.- HN. OO. NN.- NO ON. NO. OO. ON. NN. NN. OH. - HO.- NN. NN. ON. HN. NN.- NO.- NN. NO ON.- OO.- NO.- NN.- NN.- ON.- HH. NN. HN.- HN.- NN.- HN. - NN. NO. NN.- HO NN.- OH.- NO.- HO.- ON.- NN.- OH. NN. ON.- HN.- NN.- NN.- ON. OO. NH.- OO ON. OO. NO. NN. NN. ON. HH.- NN.- NN. NN. NN. NN. NN.- OO.- NO. ON NN. H9 NO. NN. NN. HN. NO.- NN.- NN. OO. ON. NN. NN. - NN.- OO. NN NO.- H9 NH.- NO. OO.- N9 - NO. NH. NO.- HO. OH.- NO. NN. - NH. NN. NN NN. NH. NO. OH. NN. ON. NH. NN.- NO. ON. NN. NN. OO.- ON.- NN.- NN NN. OO. NN. NN. NN. NN. NO.- NN. - NN. NO. HN. ON. NH.- NN.- OO. NN NH.- OO. OH.- OO.- HN.- NN.- OH. NN. HN. - NN.- NN.- ON.- NN.- NN. NN.- ON "u OO.- OH.- NN.- NH. NN.- HN.- NH. ON. NN.- NN.- NN.- NN.- NH.- NN. OO. NN 1; NH.- NO.- NH. NO. NN. NN. NH.- ON. OO. OO.- NN. NH.- NN. NN.- NO.- NN HN.- NH.- NO. ON.- NN.- NH. NO.- NN. NO. OO.- OO. NH.- HN. HN. ON.- HN OO.H OH. NO. ON. NN. NN. OH. NO.- NN. NN. NO. ON. NN.- NN.- ON. ON OO.H OO. NO.- NO. OO. - NO.- OH.- NO.- O9 - NO.- NO. HH. OO.- NN. ON OO.H NN. ON. ON. OO.- NO.- NN. ON. OO. NN. ON.- NO.- NN.- NN OO.H NO. N5 NH. NO.- NN. NO.- NN. HN. NN. - NN.- NN. NN OO.H ON. NH.- HN.- NN. ON. NN. NN. NH. - NN.- HN. NN OO.H NO.- HN.- NN. NN. ON. NN. NN. - HN.- ON. NN OO.H OO.- NO.- OH.- NH. - NO. ON.. OO. NH.- ON OO.H NN.- NO.- ON. - NN.- ON. ON. NN.- NN OO.H NN. N5 ON. NO.- ON.- HO.- NN OO.H ON. NO. ON.- NN.- NN.- HN OO.H NN. HN.- NN.- OH. ON OO.H NN.- NN.- OO. OH OO.H NN. NN.- NH OO.H NN.- NH OO.H NH ON ON NN NN NN NN ON NN NN HN ON OH NH NH NH .02 mHanum> NuucooO NH anzmmm< NN. HO. NN9 ON5 NN5 NO” NO.- NN.- ON. NN. NN. NN. NN.- NN.- NN. ON NN. OOW NN. NN. NN. NO. OO.- ON.- ON. NH. NN. NN. NN.- NN.- NN. NN OO.- NH.- NH. NO. NO. NH.- NH.- OO. NH. OH. NO. OO.- NN.- OO. OH.- NN ON.- NO. NO.- NN.- ON.- ON.- NH.- OH. NH.- NO.- NN.- NN.- ON. OH. HN.- NN HH.- NN.- HH. OO. HN. HH.- ON.- NH. ON. NO. NN. NN.- ON.- NO.- NH.- NN NO. OO.- NN.- ON. OO. NN.- NH.- NO.- HN. NN. NN. NH.- NN.- NH.- NH. ON NN5 NO. NN. HN. HN. NN. OO. NN.- NN. NO. HN. HO. NN.- ON.- HN.- NN ON.- OO. ON.- NN.- HN.- NN.- NH. NO. HN.- OO.- NN.- NO.- NH. NN. ON. NN NH.- NO.- OO.- ON.- NN.- OO.- NN. ON. NN.- ON.- NN.- NN.- OO.- ON. ON.- HN ON. OO. ON. NN. ON. NO. NO.- OO.- NO. NN. ON. NO. OH. ON.- NN. ON ON. NO.- NN. HN. ON. NN. OH. HO.- NN. OO. NN. NO. NN.- ON.- HO.- ON mm NN.- OH.- NO.- OO.- NN.- NN.- HH. NN. ON.- NN.- ON.- NN.- NH. NN. ON.- NN 1. NN. OH. NO. NN. ON. ON. NO.- NN.- NN. ON. ON. NN. NH.- NN.- NN. NN NN. HO. NN. NN. ON. NN. HO.- NN.- NN. NN. ON. NN. NN.- ON.- NN. NN NN. OO. OO. NN. ON. NN. HO.- ON.- NO. NN. NN. HN. ON.- NN.- NN. NN NO. NH.- ON. HN. NO. ON. NH. NO.- NN. NO. HN. NN. NN.- NN.- NN. ON NN. NO. ON. NN. NN. ON. HO. NN.- NN. NO. NN. ON. NN.- NN.- NN. NN NO.- OO. HN. OO. NH.- HN. OH.- ON. OH. NH.- HO. OH.- NO. OH.- ON.- NN OO. HH. OO. NN. ON. NN. OO. NO.- NN. NN. NO. NN. NN.- NN.- NN. HN NO. NO.- OO. NN. NN. ON. NH.- ON.- HN. ON. OO.H HN. HN.- NN.- OH. ON NN. NO.- NN. NN. NN. NN. NO. ON.- NN. NN. NN. HN. ON.- NN.- NO.- ON ON. OO. NO. NN. ON. NN. NO.- HN.- OO. NO. ON. ON. ON.- NN.- ON. NN NN. OH. HN. OO. ON. NN. NO.- NN.- NN. HN. NN. ON. NO.- ON.- HN. NN NN.- NO.- ON.- ON.- NN.- NN.- NO. NN. HN.- NO.- NN.- NN.- NO. HN. NN.- NN HN.- NO.- NN.- ON.- NN.- NN.- HO.- ON. HN.- NN.- NN.- ON.- ON. HN. NN.- NN NN. HH. NN. NN. ON. NN. OH.- HN.- ON. NN. NN. NN. NN.- NN.- NO.- ON OO. HH. ON.- OO. NN. NN. HH. NO.- NH.- ON.- HO.- HN. NN.- NN.- ON. NN HN. NO.- NN. NN. HN. OO. NO. NN.- OO. ON. NN. NN. NO.- NN.- NO. NN NO. OO.- NN. NN. ON. NO. NH. HO.- NN. HH. ON. NN. NN.- HN.- NN. HN ON ON NN NN NN NN ON NN NN HN ON OH NH NH NH .02 OHNOHNO> N.ucouu NH xHOzmmm< OO. NN. - NO.. NN. NN. - OO. - NN. NN. NO. ON. ON. OH.- o9 - NO. NH.- NN NO. NN - NO.- NN OO. - NO. - NO. ON. NH. - NN. NO. NO. HN. NH. NH.- ON NN. ON.- NN.- NN. ON.- ON.- ON. OO. OO.- NN. ON. NH.- ON.- HO. NN.- NN HO. NO. NH.- HH.- OH. - OH.- NH. NH. NH. - NO. NH.- ON. OH.- N9 ON. NN HN. NN.- NN.- NN. HN. - NN.. HN. NN. NO. - NN. ON. OH.- NN.- NH. - NO.- HN NH. ON.- NN.- ON. NN.- NN. - NN. ON. OH.- NN. HN. NN.- NN.- NN. OO. ON NO. ON.- ON.- NN. ON.- NN. - ON. ON. NO.- NO. HN. NH.- NH.- NN. OH. ON ON. ON.- NO.- NN. NN.- NO.- NN. NN. NO.- ON. HN. ON.- OH.- NO. NN.- NN NH. NN.- ON.- NN. ON.- ON.- HN. NN. OO. NN. ON. NN. - OO.- NO. NO. NN NN.- ONO NN. NN.- NN. HN. NN.- HN.- NO. NO.- ON. - NN. NN. HN. NN. NN NNNI NN. NN. NN.- NN. NN. NN.- NN. NO. NO.- NN.- NN. ON. NO.- NO. NN NON NN.- NN.- NN. NN.- NN.- NN. HN. OH.- NN. NN. NN. - NN.- NN.- HO.- ON NN. NN.- NHN ON. NH.- HO. NH. ON. NN. NH.- NN. NO. - HH. HN. NN.- NN NN.- ON.- NN.- ON. ON.- NN.- ON. NN. NO.- NO. ON. NN. - NO.- OO.- NO.- NN OH.- NN.. HON- NN. NO.- NN.- NO. ON. OO. NH. NO. N9 NO. NN.- NN.- HN HN. ON.- HN.- NN. HO.- NN.- HO. NN. OH. ON. ON. NO. - NO. NO. OH.- ON OO. NN.- NO.. NN. ON.- ON.- NN. NN. OO. NN. HO. HH. ON. NN. ON.- OO m” ON. ON.. NN.: HN. NN.- NH.- OO. HO. NN. NN.- NO. ON. - OO. HO.- NN.- NO 1. ON.- NO. NO. NO.- NO. NH. NN - ON.- OO. - NN. O9 - NN. HN. NH.- NO. NO NO. ON.[ NH.- NN. NH. OO. ON.- NH. NH.- NN. NN. ON.- NO. NH.- NH.- NO OO.H NN.; HN.- NN. NN.- NO.- NN. NN. NN. NN.- OO. ON.- NN.- NO. OH.- NO OO.H NO. OO.H- N9 HO. NO.- ON.- NH. HN.- NN. - NN. ON. HN.- NN. OO OO.H NO.- NO. NO. OO.- NN.- ON. HN.- ON. - NN. NN. OO. NO.- NO OO.H N9 - NO.- NO. NN. NH.- NN. NN. NN.- ON.- NN. ON.- NO OO.H NO. OO. H HN.- NO. NN.- NN.- NN. ON. OH.- OO. HO OO.H N9 - ON.- OH. NN.- ON.- NN. ON. NN.- HO. OO OO. H ON. NO.- ON. NN. NN.- NN.- NH. HO.- ON OO.H NO.- NO. NN. ON.- ON.- NO. NN.- NN OO.H NN.- HN.- NH. H9 - OO.- ON.- NN OO.H ON. NO.- NN. - OH.- NN. NN OO.H NN.- NN.- OH.- NN.- NN OO.H OH. ON. NO. ON OO.H NN. - OH.- NN O9 H NO. NN OO.H HN NO OO NO NO HO OO ON NN NN NN NN ON NN NN HN .oz OHNOHHN> NuucooN NH xHOzmmm< 180 Nm. Oc.n 00.: mo. Oo.u on u Oo. we. mo.n NN. no. NN. ON.: NN. mN. mm mm. on.: mO.u Om. um.u mO.u mm. mm. 00. mN. mm. mH. mH.u mm. Hm. mm 0H.: mN. NH.: NN. 0H. NN. NH. no. 0H.: oo.- mo. mo. NN. Hm.u mo. NN No.u co. No.L Oo. NH. Oo. HH. mm. NN.: NH.: mo. NH. mo.u 0O. Hm. on mo.u ON. mH.u 0N. co. mN. No. mN. NH.: mH.u NH. mH. OH. ON.n mo. mm 0H.: mo.u 0N.: No. No. NN. mo. OO. HH.: ON.u No. no. oN. NH.: mO.u ON mN.u mN.a 0N.: an. mm.u NN.: NN. on. mN. on. NN. mo. Om.u No.1 ON. mm NN. Om. Om. Om. NN. Hmw mN. mm. mm. Nm.u mO. mo. wc. mH. OH.: NN mH.u mm. mN. Hm. mm. Om. Om. NN. Nm.u NN. ON. HN. mN. mH.n mH. HR 00. Om.n mm.u Nm. OO.: mO.u mO. HO. mO.u Hm. mm. ON. 0H.: NN.: NN.: 0N Om.s om.u NN.: NN. mN.n Hm.u 0N. om. mO.n BO. Nm. mN. mc.n OO.: mN. mo mm.1 Nm. Ha. No. mm. mm. om. ON. mH. mm.u mm. mm. mm. 00.: NC.: mo mH. 0N.: ON.: on. 0N.: mN.u on. we. oo.u OO. om. Hm. mO.u NH. mN.u no wN. HN.: Oo.u on. mN.u 00.: mm. om. OO.: 0O. 00. OH. mN.u Nm. mo.u we mO OO mO NO HO 0O mm mm mm mm mm Om mm Nm Hm .oz OHNOHHO> NOucooN NH xHazmmm< NO.- NN.- NN. NN.- OO.- OO. NN. ON.- ON. ON. ON. NN. ON. NO. HN. ON m“ N9 OO. HN. NN. HN. NN. ON. ON. - NN. NN. NN. NN. NN. OO. ON. NN 1. N9 ON. HH. OH. - N9 OO. NH. NO. O9 OH. OH. - NH. H9 OO. OO.- NN NO. NO.- NN.- HN. - OO.- HN. NO.- NO.- NO.- NO.- HO.- NN.- NN.- NN. NN.- NN OO.H NN. NO. NO.- OO. NH.- NH. NO.- NO. NN. NH. - NN. HN. NH. NH.- NN OO.H NO.- NO. OH.- HO.- NO. NH. HH. OH. NO. - NN. ON. HN.- OO. ON OO.H ON.- OH.- OO. NN. HN.- NN. NN. ON. ON. ON. NH. ON. NN OO.H NO.- NN.- NO.- ON. HN.- OO. NN. - ON.- HO. - NO.- NN.- NN OO.H NO.- NN. ON. HN.- NN.- ON. - HO. HN. - NO. NN.- HN OO.H OO. ON.- ON. HO. ON. ON. NO. NH.- NN. ON OO.H NN.- NH. NO. ON. NO. NO. NH. NN. ON OO.H NN.- NN.- OO.- HN.- ON.- OH.- ON.- NN OO.H HN. HN. ON. ON. NH.- NN. NN OO.H ON. NN. NO. NO. NN. NN OO.H ON. ON. NO. NN. NN OO.H NN. NO. NO. ON OO.H ON. ON. NN OO.H OO.- NN OO.H HN NN ON NN NN HN ON ON NN NN NN NN ON NN NN HN .Oz OHNOHNN> A.uaoov mH anzmmm< NN. OO. NN. NN. NN.1 NN.1 ON. NN. NN. NO. ON. OO. ON. ON.1 NO.1 ON NN. NN. NN. NN. NN. 1 NN. 1 NN. NN. NO. NN. NN. NN. NN. NN.- NO.1 NN NO. NO. 1 NH. 1 NH. 1 OO. 1 OH. NH. NN. 1 NO. ON. NH. 1 HO. NH. 1 OH. HN.- NN NN.- HN.- NO. 1 HH.- NN. NN. NH.1 ON. 1 OH.1 HN.- OO. 1 NN.- NN. 1 HO.1 HH. NN NN. OH. NH. 1 NN.- HH. 1 NO. NO. HN.- NO. NN. NH.1 HH. NH.1 NO. ON.1 NN NN. NH. NO. ON.1 HN. 1 OH. NO. NH. NN. NN. NO. NN. NH. NH. ON.1 ON HN. HN. ON. HN. HN.1 NN.- NN. NO. 1 NN. NN. NO. NN. NH.- HH. NO. NN NN.1 NN.1 HN.- OH.1 ON. HO. NN.- ON. NN.- ON.1 ON.1 NN.- ON. HN.- HO.1 NN HN.- HH.- ON.1 NO.1 HN. HN. NN.- NN.1 NN.- NH.1 ON.1 HH.- NN.- ON. ON. HN ON. ON. NO. NN. ON.1 HN.- ON. NH. NN. NH. NN. OO. NN. NO.1 NO. ON NN. NN. ON. NH. HN.- OO.- NN. HN.- NN. ON. ON. ON. OO.- NN. NN. ON ON.1 ON.1 HN.- NN.- ON. NN. ON.1 NN.1 NN.- OO.- NN.- NN. 1 NN.- ON. OH.1 NN OO. OH. NN. ON. NN.- NN. 1 NN. NN. HO. NH. ON. NN. NN. HN.1 OH.1 NN ON. NN. NN. ON. ON.1 NN. 1 NN. NN. ON. NN. ON. NN. NN. OO.- OO. NN NN. NN. NO. NN. NN.- NN.- NN. NH. OO. NN. NN. ON. NN. NN.- HN. NN HN. ON. ON. NN. NN.- ON.1 NN. OO. NN. ON. NN. NN. NN. NH.1 ON. ON m" NN. HN. NN. ON. ON.1 ON.1 HN. NN. NN. ON. NN. NN. NN. NN.- HN. NN 1. HO. HN. OO. HO.1 NH. NH.1 NO.1 NO.1 HO.1 NH.1 NO.1 NO.1 NN.- OO.- OO.- NN NO. ON. HN. ON. NN.- ON.1 ON. NO. NN. NO. NO. NN. NN. NN.- NH.1 HN OO.H NN. NN. NN.NN.1 NN.1 NN. HO.1 NN. ON. NN. NN. NH. NH.1 OH. ON OO.H NN. HN. ON.1 NN.- NN. NO. ON. ON. NN. NN. NO. NO.1 NH. ON OO.H ON. ON.1 NN.- ON. ON. NO. NN. NN. NN. NN. NN. 1 NN. NN OO.H ON.1 NN.- HN. NN. ON. NN. OO. NO. NN. NN. 1 HN.- NN OO.H ON. ON.1 ON. ON. 1 NN.- ON.1 ON.1 NN.- NN. NO. NN OO.H NN.- NN.- NN. 1 NN.- NN.- ON.1 NN.- NN. NH.1 NN OO.H OH.1 NN. NO. NO. NN. NO.1 HO. 1 HN. ON OO.H NO. NH. HO. ON. NN. NN. 1 NO. NN OO.H ON. NO. OO. NO. NH. NH.1 NN OO.H HN. NN. NN. NO. 1 NO. HN OO.H NN. NN. OO. 1 HN. ON OO.H NN. NN.- NO.1 OO OO.H ON.1 OH. NO OO.H NO.1 NO OO.H NO ON ON NN NN NN NN ON NN NN HN ON OO NO NO NO .02 OHNOHHO> N.uaoov NH anzmmm< 183 OO.H mm. mH.1 NN.1 mm OO.H mo.1 OO.: mm OO.H No. NN OO.H on mm mm NN on .02 wHHMNHm> H.ucoov mH xHszmm< 184 .mHnmu m>onm msu CH cBonm Hmvuo mnu CH umnEda nEmH 50mm mom vmumHH mum mmHnmu unmsvmmndm SH wumc HH NNH OOH NOH ON NN O HNNNNO ONO mo< 92¢ me .mDOMU MH NNH OOH NOH ON NN O ANNOOO ONO ¢H NNH OOH NOH ON NN O HNNONO ONO NNH OOH NOH ON NN O NNNONN ONO O.HON 3.2 9383 N-OH NHOZNNHNO OO.H OH.m OO.m ON.m NO.N OO.m NO.N O0.0 OO.m O0.0 O0.0N Nm.NH ON.H ON.N NO.N OO.N ON.O NO.m ON.N OO.N NN.N NH.m Om.NH NN.OH OO.N OO.m NH.m NO.N OO.N OO.N ON.N ON.H ON.O NO.m Om.NH NH.NH NH wcH30HO 3oHN NN.H ON.N NN.N NH.m OO.m NN.m ON.H NO.m NH.m OO.m Nm.NH O0.0H OO.N NN.m OO.m OO.N OO.m ON.O ON.N ON.m ON.N NO.m O0.0H NN.NH Om.N OO.m ON.N OO.N Nm.N NO.N NN.N NO.N Om.N ON.N N0.0H Om.NH NH NCHBOHU ummm NN.ON OO.Nm ON.OH OO.NN N0.0H N0.0H mH.NH m0.0H ON.HH HN.N NN.H ON.N OH.mm Nm.mm Om.NN N0.0N m0.0H ON.HN OO.NH ON.NH Om.OH NN.HH ON.N Hm.O NN.Hm N0.0N HN.ON NN.Hm m0.0N NN.NN Om.HH NO.NH mN.N O0.0 NN.N HO.N HH me30HO BOHN 8 mw NN.mm Om.HO O0.0m OO.NN mN.OH OO.NN NN.OH OH.NH N0.0 NO.N mO.N OO.m HN.NN NN.Nm Nm.ON O0.0m O0.0N NN.NH OO.NH ON.OH OO.NH NO.NH NN.N NO.N NO.Hm Nm.Om NN.mN ON.mm NN.NN NN.NN ON.mH NN.OH ON.NH OO.NH NO.N NO.H HH NCN30HO ummm mN.HO mN.HO NN.NN OH.Nm ON.NN NN.NN ON.ON OH.NH ON.NH N0.0 NN.N N0.0 N0.00 ON.HN O0.0N N0.0m ON.OH HN.NN mm.OH ON.OH O0.0H O0.0H ON.m OO.N OH.Nm O0.00 ON.Om OO.mO O0.0N N0.0N N0.0H NN.HN m0.0H ON.HH NN.m NN.m OH NCHBOHO 30HN NO.NO NH.NN OH.Nm NO.Nm NN.HN N0.0m ON.OH NN.ON Om.HH ON.OH O0.0 NH.O NN.OO HN.HN N0.0m N0.00 Nm.Nm NN.NN ON.OH N0.0N O0.0H ON.NH OO.m NN.O OH O0.00 NN.HN OO.Nm mO.NO ON.Nm O0.0m OH.NH O0.0H O0.0H ON.ON OO.m NN.m m3m 8mm m3m 8mm m3m 8mm msm 8mm 03m 8mm m3m 5mm .05 wcH3ouo umwm mHamHum> NNH OOH NOH ON NN O HNNNNO ONO OHOO 3 NNH OOH NOH ON NN O NNNONO ONO ON NN O NONNNN ONO NNH OOH NOH ON NN O HONNON ONO O AmNmOO ONO mna qu mOH o“ mm Amkmcv mw< o Amchv mm< sum nna oqa mOH o“ mm o AmNmnv mw< mNH qu mOH on mm o Amhmuv mw< <93 32 H.283 MTE finzmmfi 197 H.Nm m.mN m.wN n.0N m.NN 3.mo m.NN H.HN m.m3 O.mm 3.H m.3 3.mm H.mm o.om m.3N N.mw N.Nm o.3N 3.HN m.mo 3.oo H.N H.m m.Nm 3.mm m.mN m.Hm m.Hm m.mm m.Nm H.mN N.Hm o.mm 3.0 3.3 mm wCHzopU 30Hm H.mN 3.Hm m.mN N.3N m.om H mm m.NN N.HN N.H3 N.Nm m. N.m m. NN o.mN m.om m.Hm H.ow m.Nm H.NN N.NN N.Hm m.mw m.H H.m . 3. NN m.NN O.NN m.om N.mm m.mN N.NN N.ON .mN m.wN N.N m.~ mm mcHzohU Pmmm O.mNH N.mmH N.Nm «.33H 3.NN 0. 3m w.om o.om m.om N.m3 w.m n.0H m.mmH m.moH 3.omH m.HNH m.wN m. ooH N. No m.3m m.mN o.om m.mH H.mm m.NmH m.omH m.ONH m.omH m.3a a. mmH N.Nm H.Nm m.N3 o.on m.oH N.mH mm MCHZOMU 30Hm N.HNH 3.00m o.o3H o.mmH m. mm m.mHH m.MN m.HoH O.mm N.N3 O.mH 3.NH m.m3H N.NNH O.NNH .mNH m. HmH m.NN m.Hm O.mHH O.NN H.mo H.NH m.oH .3mH w.3mH m.mHH .ONH m. mNH N.HmH w.mo o.om N.mm 3.3m N.mH 3.oH mm wstomo pmmm mO.m mH.N ow.3 N3.3 mH.m Hm.m m3.m Nm.m m.3 mm.3 NO.N 3o.m mN.m NO.m mm.3 NH.3 3m.m Nm.m mH.m No.3 m.3 0.3 Nm.m Hm.o m3.m Nm.m mN.m Hm.3 No.0 NH.N NN.m mN.m «.3 m.o mN.m mO.m dezouu BOHm NM 3N.3 mN.m mN.m mN.m mm.m HH.m Nm.m NH.m 33. 3 mo. 3 NN. m No.0 OH.m mm.3 m3.3 om.3 3H.o mN.m om.o Nm.m NN. 3 Nm. 3 Hm. w 3m.3 mm.3 ~3.m 3N.m HH.m mH.N mm.w OO.N No.3 0N. m mm. m 0H. m ~3.m Nm mam ewm mzm 8mm mam 8mm mzm 8mm mzm 8mm mzm 8mm .oc mama 3% 28> mNH 03H moH ON 0 Amhmuv mw< 30) :32 Va}. ...-H2 .034 <£> MUN-2T HHH NW5 HHH NOO NNN \O\OO\ HHH O\O\("\ HHM 10mm N.3N \O\O\O HHH CDNO HNN 00(1) HHN NO\C'\ NNN Inmm O\CDN ®\O\O O O I M0?! H H 43 w1ng Slow Gro “if“ I HHH H\OO NHN H'TCDH v-‘lv-IN BBQ) HHH “Bin NNN OH-d' NNN (\(DW HHH O\(x)V" o HHN IAN-3’ NNN NNN (WNW 11.0 10.7 11.5 12.1 10.5 12.2 w1ng Fast Gro 44 199 Slow Growing NNN (\\O\O O\-':)'O\ PHOU'N H 70 10.3 2. 16.0 11.9 12.4 12.2 16.3 17.5 11.h 13. 9. 9. 12. 16.9 20.2 6. 18.4 “:01 (IDHH :ann NNO NO\('\ WWW an W1ng Fast Gro who comm 5:?“ HN\O \Od’lfi 45 mng Slow Gro (xv-mm :rmm WNW) 0:2'0\ 33B \OUWVN O\:}’N 0CD: -:)'\O-:7’ with \O-fi'\0 :rmm 45 N.mm 3.Nw N.N3 N.m3 3.Nm N.mw H.m3 N.Nm N.Nm N.mm N.Hm 3.Nm N3 @3395 30Hm N.mm N.mm N.o3 N.m3 N.ON m.mm 0.3m m.Nm m.3m N.Nm 3.wm o.mm m3 mangohw Pmmm m.NN 0.3m m.m3 N.om o.mH o.om m.Hm N.Hm o N.NN N53 N33 N.m3 N3 0 2 mgmzoho BOHm H.H3 w.om m.om N.N3 m.o3 H.om N.m3 m.Nm m.NH N.Nm N.Hm N.m3 N3 wsfisohu pmwm O.NH m.NH N.oH N.3H m.NH N.N N.NH m.mH 3.NH 3.0N N.NH N.mH o3 MNHHBONHU 30Hm O.NH 3.NH m.NH 3.NH N.NH o.oH 3.NH N.mH N.NH N.mH O.mH N.3H 03 25 83mm 95 sum 25 5mm mzm Nam 25 Edm 03m 5mm .oc msHSEHo pmmm memHHm> mNH 03H moH 0N mm o AmNmuv NNN 0NH 03H 00H 0N mm o Ammmuv NNN 393m 33m A.0coov N-NH xHazmmm< 204 NNN 3NN NOH NON NOH OOH NON NN N0 HN 3H NN NON ONN H0N 0o3 00 0N NN NNH H0 N0 NH 0H N00 NNN H3N N00 03H 0NH 3ON 0NN N0 NN 3H 0H O0 mcHsopw BOHm 030 ONm HNN NNN 00 HHH 0NH 0N 30 NN 0H NH 3ON N00 0NN ONN. NOH N 00H 0N NHH 00 NN 0N 0N0 00m ONN HN3 0NN N H O0H 0N 3HH ONH 0N NH O0 mezouo Pmmm 3H0 030 NNN NHm OHN 0ON H0N OmN N3H H3H ON N3 OON NON HH3 O00 mmN NNN N0H mON N0H 30H NN NN 0N0 N00 o33 NNO 0NN 0o3 NON NON NNH 03H 33 03 N0 wmfisohu SOHm 3ON NNN 3N0 0N0 0NN N3m NNN m3m 30H NNH mm N0 300 0N3 NH3 0NN H03 0NN OmN NN3 ONH 0NH 03 03 330 NN0 NN3 NNO 033 N03 30N 00N N0H NON N3 Hm N0 mmmzohc Pmmm N.0H N.0H H.NH O.NH N.0H N.0H N.0H 0.0H N.3H N.3H 0.NH N.NH N.0H N.0H N.NH 0.NH N.0H O.0H 0.0H 0.0H O.0H N.3H 3.NH H.NH 3.NH N.0H 3.0H 3.0H 0.0H H.0H N.0H N.0H N.NH N.NH O.HH H.3H 00 mmHSouc 30Hm 0.0H N.0H O.NH 0.0H 3.0H 0.0H 3.0H N.0H 0.3H N.3H H.NH N.NH 0.0H 0.NH H.NH 3.0H N.0H 0.0H 3.3H N.0H N.0H H.3H N.NH O.NH O.NH 3.0H N.0H 0.0H H.0H N.NH N.3H N.3H N.NH N.NH N.NH N.OH 00 wsm 8mm mzm 8mm mam 8mm ozm 5mm msm 6mm 03m 8mm .0: wCHzoho pmwm mHanhm> 0NH O3H NOH ON 0N O HONNOO ONO OOOm 3ON H.anwv N-NH mezNNNO 205 NOH ONH OHH NON NmH OHH NO NOH NO 00 mm 00 0NH NNH mOH OOH OHH O0H 0NH NHH 0NH 00H Nm mm O0H 00H HOH 00H mmH O0H HOH 0O 00 NOH 00 O0 O0 wcHBOHU 3on mHN OHm OON mNH NO 0NH NmH 0NH MHH N0 00 00 «OH OmN O0H mmN 0NH NOH NMN O0H HOH OHH N0 00 NHN ONN 00H NOm ONH OON NHH HON NOH NmH Nm N0 O0 mmHBOHO ummm 00m OmO 0mm 0mm OON 0N0 NmN HON OON mMH NO 00H Omm NOO 00m O0m OOm 00¢ NON 0mm NOm Nmm ON OON O00 Hmm OO0 000 0H0 NOO 0ON OON OOH NNN HON ONH O0 wCH30Ho BOHm N00 mmN mmq N00 ONN H00 «Hm O00 mmN 0NH NmH 00H NHm 0mm ONm NON NON 00m OON 0N0 m0N 00m OON 00H mq0 N00 ON0 0mm HON ONm 0NN 00¢ 0NN NmN O0H NN O0 wcH3ouu ummm NmN OON NOH OON mmH OOH OON mN O0 Hm 0H mN OON ONN HON OOO Om 0N ON mmH H0 N0 NH 0H N00 NNm HON N00 00H 0NH «ON 0NN N0 mm 0H 0H 00 meono 30Hm 000 OOm HNm ONN 0m HHH 0NH 0O 00 Nm 0H NH «ON Nmm 0mN OOm NmH N0 00H mm NHH 00 NN 0N 0N0 00m Omm HNq 0NN ONH O0H 0N OHH OmH 0N NH 00 m3m 80m mam Ema mzm 8mm msm 80m m3m Emm m3m Emm .oa wstouw ummm mHanHm> 0NH O3H mOH an O HONNOO 004 0NH O3H 0OH ON OO O ANNOOO mw< 0NH O3H mOH ON ON O HONOOO ONO 0NH O3H OOH HONNOO ONO ON.OO 32 I H.083 HH-OH xHOszOO 209 ON HN OH HH 0H OH OH HN NH OH ON OH OH 0H 0H NH OH OH ON HN 0H ON OH ON NH OH OH OH ON NN 0H NH OH NN ON 0H NN wdH3onu 30HO OH HN 0H HH OH ON ON OH 0H OH OH ON OH NH 0H NH OH OH ON O OH NN NN OH O HN NH 0H HN ON ON NN ON HN ON OO NN OdH3ouu ummm HH NH OH OH OH OH OH OH OH OH NH OH NH NH OH NH OH OH OH NH OH NH OH OH NH OH NH OH OH NH OH OH OH OH 0H 0H HN wcHzouo 3oHO OH NH OH 0H OH OH HH OH OH OH OH 0H NH OH OH OH OH OH HH OH OH NH OH 0H OH NH OH OH OH OH OH HH NH NH OH HH HN wdHBOHO ummm OH OH OH ON OH OH OH OH HN OH NH OH ON ON OH NN OH OH NH NH HN ON OH 0H ON ON OH OH OH OH OH OH HN 0H NH NH ON me3090 SOHO HN OH OH OH NH OH OH ON ON NH OH NH OH ON OH OH OH NH ON HN OH ON 0H OH NN OH OH OH OH NH OH ON 0H ON OH 0H ON m3m 8mm m3m 5mm mBM Sam 03m 8mm 03m 8mm m3m 8mm .oc OmHBOHO ummh MHQOHHM> NNH O3H 0OH ON ON O NONOOO ONO ONH O3H OOH ON mm O OONOOO ONO O NONOOO mw< mNH O3H moH cu mm o Am>mwv mw<