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Merkel Major professor DateNov- 21'. 1977 0-7 639 1 THE RELATIONSHIP OF PLASMA INSULIN AND CORTISOL 3 TO SELECTED PLASMA CONSTITUENTS AND TO i , CARCASS COMPOSITION IN THE PIG ? n By Dennis Dale Crenwelge A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1978 ABSTRACT THE RELATIONSHIP OF PLASMA INSULIN AND CORTISOL TO SELECTED PLASMA CONSTITUENTS AND TO CARCASS COMPOSITION IN THE PIG By Dennis Dale Crenwelge Plasma from 72 pigs selected for high, low and control backfat was assayed for insulin, cortisol, free fatty acids and glucose at 56 and 115 days of age and at slaughter. These plasma data were related to carcass cut out data. Plasma insulin, cortisol and free fatty acids were higher among high backfat pigs compared to low and control back- fat lines. These differences between lines tended to be greatest at 56 days. Correlations between insulin, cortisol and carcass composition were also highest at 56 days. No significant differences in insulin, cortisol and glucose were observed between lines at the other ages. Glucose decreased significantly with age and insulin increased significantly but free fatty acids remained relatively constant. Correlations between free fatty acids and car— cass cut out data were generally significant. No signifi— cant breed or sex differences were observed for the plasma parameters of these pigs. L ACKNOWLEDGMENTS Sincere appreciation is extended to Dr. R. A. Merkel who served as supervisor for the conception, execution, and publication of the project upon which this thesis is based. The author is further indebted to his major professor for the guidance and counseling received during the course of the Master of Science program at Michigan State University and for the patience displayed during the drafting of this thesis. Gratitude is extended to Dr. H. D. Hafs who not only served as a guidance committee member but also assisted in and provided laboratory facilities for the completion of some of the serum assays. Appreciation is extended to Dr. A. M. Pearson who served as a member of the guidance committee and also pro- vided helpful suggestions for laboratory procedure. A special recognition must be forwarded to the person— nel at the USDA Research Station at Beltsville, Maryland who fed and housed the experimental animals and who also collected plasma samples and obtained carcass information. A final thank you goes to the technicians and graduate students of the Meat Laboratory and Dairy Science Depart— ment who assisted in laboratory analysis. ii TABLE OF CONTENTS LIST OF TABLES ................................... INTRODUCTION ..................................... LITERATURE REVIEW ................................ Growth and Development ....................... Carcass Composition .......................... Influence of Insulin on Body Metabolism ...... MATERIALS AND METHODS ............................ Experimental Design .......................... Transmission Values .......................... Creatine Phosphokinase ....................... Statistical Analysis ......................... RESULTS AND DISCUSSION ........................... Blood Plasma Characteristics ................. Correlations Between Blood Plasma Characteristics .......................... Carcass Quality and Cutability ............... Correlations Between Carcass Traits .......... Correlations Between Plasma Traits and Carcass Traits ........................... SUMMARY .......................................... BIBLIOGRAPHY ..................................... APPENDICES I — Reagents ................................. II — Raw Data ................................. Table 10 11 Appendix LIST OF TABLES Number, Code and Definition of Variables Used in Tables ................. Blood Plasma Values by Breed and Sex for Individual Bleedings ............. Blood Plasma Values by Line for Individual Bleedings ..................... Summary of Blood Plasma Values by Breed and Sex ............................ Summary of Blood Plasma Values by Line and Bleeding Period ................. Simple Correlation Coefficients Between Blood Plasma Values .............. Carcass Characteristics by Breed ......... Carcass Characteristics by Sex ........... Carcass Characteristics by Line .......... Simple Correlation Coefficients Between Carcass Traits ................... Simple Correlation Coefficients Between Carcass and Blood Plasma Traits .......... II—A Number, Code and Definition of II-B Raw Data Variables Used in Raw Data ............... iv Page 44 A6 A8 49 58‘ 61 63 67 71 77 INTRODUCTION The realization over the past three years that some seemingly limitless resources, energy resources in partic- ular, are indeed finite has brought on concern of over utilization of those resources. Efforts toward the con— servation of raw materials have always been in operation, although some of the more recent efforts have attained a higher priority. The production of meat animals is no exception to this effort. Perhaps some animals of past decades ap- peared to challenge the ideals of conservation. However, one must remember that animal was marketable and the excess fat, for instance, served a need in its time. Today, the producer still desires an animal which will efficiently convert his available resources into a market- able commodity. At present, that "commodity" is an ani- mal which has produced the greatest amount of lean meat and the least amount of fat and still possesses accept- able quality standards to satisfy the consumer. The prob- lem is both to define and control conditions under which this goal is achieved. The number of variables encountered in attaining that goal is phenomenal. General environmental conditions, 2 nutrition, breed, sex, and hormonal influences are but a few of the general factors, and each of these variables exhibit considerable variation. However, through persis- tence and manipulations of these variables, the scientists of today are able to define some of the ideal conditions even though not all will be answered with the tools of study available at present. This study was undertaken in an effort to better understand the influence of hormones on body metabolism and how those influences affect carcass composition. Two breeds of swine each selected for high backfat, low backfat, and unselected controls were included in the study. Endogenous circulating insulin and cortisol levels were determined in an effort to relate the effect of the two hormones to plasma glucose, plasma free fatty acids, and carcass characteristics. LITERATURE REVIEW Considerable research has been done in areas related to growth processes in animals and effectors of normal and abnormal growth. The review presented here is by no means an exhaustive expose of the field. Some of the classic studies of carcass composition are included although most references are more recent and more directly related to the aims of this study. Humoral influence on growth and- development of body tissues in meat animals is a field of more recent origin. Detailed information related specif— ically to meat animals is still somewhat limited. Conse— quently reference to other "non—meat" animals is included to aid in understanding body mechanisms. Growth and Development Growth per se is not easily defined in a manner which is acceptable to all individuals. Most researchers agree, however, that growth is more than merely an increase in size or weight. For instance, water metabolism (Weiss, 1949) can cause weight changes with no net body change. Brody (1945) described growth as the production of new biochemical units occurring during cell division, but at the same time proposed the deposition of non-protoplasmic substances such as fat be excluded from the definition. 3 L, Maynard and Loosli (1962) complied with this opinion by stating that true growth involves an increase in body structural tissues, although any increase due to fat pro- duction in reserve tissue should be considered separately. Pomeroy (1955), however, stated that although distin- guishing growth and fattening is sometimes convenient, the distinction is only arbitrary with no logical reasoning. Although this latter interjection does not define growth in any definite terms, it does exemplify the problem of determining the optimum point at which meat animals should be slaughtered. The sigmoidal growth curve can be used to describe the pattern of growth in all species of animals (Brody, 1945; McMeekan, 1959). The first portion of the curve represents development of the major organs and tissues. During this time the body undergoes many changes although gaining little in absolute weight until the last one-third of the prenatal period (Palsson, 1955). This sharp in- crease in weight continues from birth until the animal approaches maturity. In this region, bone is the first to cease growing and is rapidly followed by a cessation of muscle growth and ultimately increased fat deposition (Hammond. 1933). Bone Growth and Development. Johnson (1974) studied the growth impetus of cattle from 150 days gestation to 84 days after birth. Growth impetus is defined as high or 5 low, whenever tissue growth varied significantly from the average growth coefficient of the animal. Bone initially displayed a high growth impetus but was average just prior to birth and reached a low impetus shortly after birth. This would indicate that bone does not grow as rapidly after birth as do the other tissues of the body. Tulloh (1964) agreed with these findings and he reported that, in beef cattle, bone increased with body weight but at a decreasing rate. Callow (1949) investigated the effects of age of slaughter on the amount of bone in pigs. Animals were slaughtered over a range of 16 to 46 weeks of age. For pigs on the same diet, he found there was a decrease in the percentage of body weight represented by bone. For pigs slaughtered at three different weights, Cuthbertson and Pomeroy (1962) expressed bone development on a gram per day of age basis. The grams of bone in the carcass per day of age were 14.26, 20.36, and 14.81 for slaughter weights of 50, 68, and 92 kg, respectively. The actual percentage of bone at each of the three weights was 10.4, 9.5, and 8.3, respectively. They concluded that the rate of bone production increased to 68 kg and there- after the rate decreased indicating that other tissues were beginning to comprise a higher percentage of the car— cass. Richmond and Berg (1971a) found that bone as a percentage of pig carcasses decreased from 13.2 percent in a 92.5 kg live weight pig to 9.2 percent in a 114 kg 6 live weight pig. Hansson gt 31. (1975) found similar re- sults when studying Norwegian Landrace and Yorkshire pigs. He found that percentage bone continued to decrease in pigs up to 130 kg live weight. Muscle Growth and DeveIOpment. Muscle is the major tissue in the body from a weight standpoint (Hedrick, 1968). Since it is the tissue of most value and impor- tance, determining at what stage the highest yield and optimum efficiency can be achieved is the primary concern of the livestock industry. Hammond (1960) reported that the maximum size of an animal (and consequently volume of muscle) is fixed at birth since muscle fibers increase primarily in diameter postnatally. One would therefore expect a rapid increase in muscle volume and a decelera- tion as maturity is attained. According to the work of Johnson (1974) the growth impetus of muscle in cattle was low from 150 to 210 days gestation but from 210 to birth the growth impetus of the muscle was very high indicating very rapid growth. However, after birth muscle tissue no longer proliferates at the same rate according to Richmond and Berg (1971a) who dissected pig carcasses at differing weights and found that the percentage muscle in the car- cass decreased as weight increased. By complete physical separation of fat, lean, and bone, it was found that muscle represented 60.4% of the carcass at 23 kg live weight. Pigs slaughtered at 114 kg only had 48.7% muscle in the carcass. 7 The findings of Cuthbertson and Pomeroy (1962) agreed with those of Richmond and Berg (1971a). Cuthbertson and Pomeroy reported the percentages muscle of 58, 68, and 92 kg carcasses were 50.3, 47.8, and 43.5, respectively. They further expressed their data on a gram of muscle per day of age basis from 1 to 50 kg, 50 to 68 kg, and 68 to 92 kilograms. Their results showed 71.95, 119.4, and 93.6 g muscle per day of age at 50 kg, 68 kg, and 92 kg, respectively. The data expressed in such a manner are misleading especially for the first age group slaughtered at 50 kg, since the grams of muscle produced by a young animal could be quite high when expressed as a percentage of total muscle but on a gram per day basis would be low. Also the data for the 50 to 68 kg group represents an 18 kg increase in weight and the 68 to 92 group represents a 22 kg increase in weight. However, even with the dis— crepancy in the.live weight units, the difference in per- centage muscle between the two weight groups does reflect the fact that muscle growth rate had decreased after 68 kg live weight. Fat Growth and Development. At birth most animals have very little body fat stores. Johnson (1974) reported a very low growth impetus for fat in bovine fetuses during the last stages of pregnancy. For the first 28 days post- natally, the impetus is low but from 28 up to 84 days the impetus is high indicating rapid fat development. Filer gt a1. (1973) found similar results in Pitman-Moore and 8 Norwegian Landrace pigs during the first 56 days after birth. At birth the body fat of the Pitman-Moore pigs expressed on a percentage basis was 1.7%. By day 28 the fat content was 23.9%. At day 56, however, a slight de- crease was noted with 21.5% fat. A similar pattern in Norwegian Landrace pigs was noted. At birth body fat was 1.2% of the carcass weight, 18.8% at 48 days, and de- creased to 18.2% by 56 days. The only explanation stated by the authors for the decrease in percentage fat occur- ring during the latter part of the study was that the pigs could gain access to the sows' feeder and consume some of the feed thereby reducing the intake of the higher energy sows' milk. Cuthbertson and Pomeroy (1962) studied fat content of 50 kg, 68 kg, and 92 kg live weight pigs and they reported percentages carcasses fat of 31.0 to 35.0 and 41.4, respectively, for these three live weight groups, clearly showing the increased fat proliferation as the ani- mal becomes heavier. Hansson gt El' (1975) indicated a similar pattern of fattening in Danish Landrace and York- shire pigs, although actual figures were lower since only primary cuts were separated and weighed as opposed to Cuthbertson and Pomeroy (1962) who used complete physical separation of all fat from the carcass. The study by Hansson gt El- (1975) showed an increase in body fat from 29.3% at 70 kg live weight to 37.4% at 130 kg live weight, 9 also reflecting a large increase of fat in the composition of the carcasses. Carcass Composition In an effort to determine the various components of a carcass, the method giving the most reliable results would be most desirable to use. The most reliable method, therefore, would be the complete separation of lean, fat, and bone. However, this method is not only time consuming but costly. In an effort to find a simple, yet reliable, method for determining carcass components, carcass meas- urements expected to most influence or reflect most on carcass composition are taken and correlated to actual carcass composition. Much work has been done with the beef carcass. Since the lean is the most valuable portion of a carcass, most prediction equations developed for car- cass composition reflect the amount of lean in the carcass. Rate of Gain. Although rate of gain is used in many beef cattle experiments, it gives no real indication of the changes which are occurring in the body (Hedrick, 1961). Scheper and Bach (1963) reported that rate of gain was dependent on initial weight of the animal as well as the weight interval during which time the rate is deter— mined. This is explained by Joubert (1954) who stated rate of gain is a physiological age-weight relationship. Faster gaining animals, when fed for the same period of 10 time as slower gaining animals, will show a positive rela- tionship between rate of gain and deposition of fat. This observation is in contrast to Hedrick gt gt. (1963) who slaughtered Hereford steers (at a constant weight and not necessarily for the same period of time) and found signifi— cant, although low, correlation coefficients between daily gain and trimmed primal (.41) and trimmed wholesale cuts (.41). The correlation between rate of gain and fat measured at the twelfth rib was -.26 which agreed with Joubert (1954), but again the value was too low to be of practical use as an indicator of carcass composition. Carcass Measures. Attempts at relating any single carcass characteristic to the percentage of muscle have generally proven unsatisfactory. The two single measures which most closely reflect the percentage muscle in cattle are loin eye size and the fat thickness over the loin eye at the twelfth rib (Hedrick, 1968). Muscles which mature more slowly offer the most re- liable estimate of muscle growth and development and con— sequently would be a more reliable indicator of total muscle volume (Hedrick, 1968). Since the longissimus muscle is one of the latest maturing muscles (Joubert, 1956), it has been extensively studied in predicting car- cass composition. Cole gt gt, (1962) found the simple correlation coefficient between longissimus muscle and total separable lean to be .59 while Hedrick gt gt. (1965) ll established a correlation of .42 between percentage total boneless retail cuts and longissimus muscle area. Inter- estingly, Goll gt gt. (1961) showed a correlation between loin eye area and weight of untrimmed primal cuts (round, loin, rib, and chuck) of .00. It should be noted that the latter authors used untrimmed cuts, therefore, the in- fluence of fat was not accounted for. Fat measures and particularly fat depth at the twelfth rib have also been extensively studied in relation to lean content in the carcass. Several studies cited by Hedrick (1968) included fat thickness over the lumbar and thoracic area were most highly correlated to carcass com— position. Allen (1966) reported the fat measurement over the twelfth rib three—fourths the distance from the medial to the lateral edge of the longissimus muscle was the fat measure most highly related to retail yield and he reported correlation coefficients of .72 and -.68, respectively, for percentage separable fat and percentage separable bone for beef carcasses ranging in carcass weight from 227 to 250 kg and from 318 to 340 kilograms. In a study involving pork carcasses, Fahey gt gt. (1977) compared several existing methods as well as new methods for estimating percentage muscle. The lean of the carcasses was physically and chemically defatted and weighed. The amount of lean tissue obtained was used to represent 100% of the muscle in the pork carcass. Of all methods studied, the most accurate predictive 12 equation utilized loin eye area and a fat measurement taken at the tenth rib, three-fourths the distance from the medial to the lateral edge of the longissimus muscle. The coefficient of determination established was 68.4 which was more accurate than all other methods including U.S.D.A. grade (27.0) and closely trimmed ham and loin (47.0). Effect of Sex on Porcine Carcass Composition. Hiner (1971) evaluated 104 pigs for percentage carcass lean and carcass fat at mean slaughter weights of 34.0, 56.7, 79.4, 102.1, and 124.7 kilograms. He reported no significant difference between barrows and gilts for either percent- age carcass lean or fat. He also found no difference be- tween barrows and gilts for percentage lean or fat in the right ham. These observations do not agree with other published data. Sex effects on carcass composition are usually noted in most animals, the primary effect being differences in fat content. When comparing barrows and gilts Richmond and Berg (1971b) found that gilts had less backfat, less total carcass fat, and a higher percentage muscle. Hansson gt gt. (1975) reported similar results between barrows and gilts and reported that boars had more lean and less fat than either barrows or gilts. He further reported that although boar carcasses had a higher per- centage bone than gilt carcasses and gilts had a higher 13 percentage bone than barrows, when a correction for fat differences was made, the bone difference disappears. This observation indicated that the sex differences in bone content was primarily due to the higher disposition of fatty tissue. Richmond and Berg (1971b) found no sex effect between barrows and gilts for carcass bone content. They reported a similar lean to bone ratio for both sexes. Bakke and Standal (1975) reported that gilts had larger loin eye areas as well as higher percentages ham and loin than did barrows. Effect of Breed on Porcine Carcass Compgsition. Studies involving crossbred pigs with half the genetic pool from the same breed, have usually not shown a breed effect. Richmond and Berg (1971b) and Richmond and Berg (1972) studied Hampshire x Yorkshire, Duroc x Yorkshire, and Yorkshire x Yorkshire crosses and found no significant differences in carcass composition although they did note that the Duroc x Yorkshire pigs were more highly predis- posed to fatten. All pigs were slaughtered at the same weight. Hansson gt gt. (1975) reported that Yorkshire car— casses contained 1.6% less lean and 1.1% more fat than Norwegian Landrace pigs. All pigs were slaughtered at a constant weight. Bereskin and Davey (1976) reported carcass data of Duroc and Yorkshire breeds of pigs selected for high and 14 low backfat within each breed. They reported significant (P<;01) breed differences for all traits consisting of average backfat, loin eye area, carcass length, percent- ages lean, fat and bone of the ham, and percentage lean cuts. These differences were for breed differences and both high and low backfat lines were represented in both breeds. Influence gt Insulin gg Body Metabolism Fat Metabolism. Jungas and Ball (1962) were among the first to study the effect of insulin on fat metabo— lism. They noted that insulin decreased release of gly- cerol in isolated epididymal fat tissue from rats. They based further studies on the fact that Gordon and Cherkes (1958) and White and Engel (1958) had shown that epineph— erine caused hydrolysis of triglycerides into free fatty acids (FFA) and glycerol. Jungas and Ball (1963) found that a combination of insulin and epinepherine through some unknown manner reduced the release of glycerol and FFA in fat tissue culture as opposed to fat tissue with only epinepherine. Jungas and Ball (1964) used fat tissue from fasted-refed rats which in the absence of added sub- strate exhibits increased glycerol release. The addition of insulin to such cultures effectively established insu- lin as anti—lipolytic. Jungas and Ball (1963) also showed that glucose interferes with normal release of FFA. Machlin (1972) explained this by stating that when glucose 15 is present, there is an ample supply of glycerol phos- phate and any released FFA can immediately be incorporated to form triglyceride. Therefore, a lipolytic substance can cause glycerol release and not necessarily a rise in FFA. Consequently when animals are used in studies, they should be fasted to decrease the glucose level in order to maximize the lipolytic effect of a hormone. Rodbell (1964) also studied the effects of hormones ' on the metabolism of fatty tissues. He noted that in- creasing the glucose concentration of the incubation media up to 6.5 umoles per ml stimulated an increase in the formation of CO and fatty acids at the expense of glu— 2 cose. The addition of insulin caused a further three-fold increase in the uptake of glucose and its utilization to form CO glyceride-glycerol and fatty acids. As little 2. as 10 uU of insulin produced a response. O'Hea and Leveille (1968) studied the response of cultured procine fat tissue as affected by insulin admin- istration and reported a relatively unchanged tissue fatty acid synthesis. In another study, O'Hea and Leveille (1970) reported that fat tissue isolated from young pigs (12 kg) showed a significant increase in fatty acid syn— thesis. However, they found that the tissue from older pigs (40 to 64 kg) did not respond with increased fatty acid synthesis. Christensen and Goel (1972) incubated fat tissue from 110 to 120 kg pigs and studied the effect of insulin on the fat metabolism. They found that the effect 16 of insulin on the lipid, fatty acid, and glyceride- glycerol content was highly dependent on the concentration of both insulin and glucose. Insulin did increase the conversion of glucose to fatty acids and glyceride— glycerol. However, at high concentrations of glucose, they found that the effect of insulin was nullified. Benjaman gt gt. (1961), however, had previously shown that age can be a factor in lipolytic activity. They worked with tissue from rats 38 days old and 647 days old and observed decreased synthesis of acetate to lipid material as well as a decreased rate of lypolysis. Mersmann gt gt. (1973) assayed glucose incorporation into 00 and tri— 2 glycerides in fat tissue from 20, 35, 60, and 112 day old pigs. They reported tissue activity of 60 day pigs to be 2.5 times higher than 35 day pigs and 4 to 5 times higher than at either 20 or 112 days. The generally accepted theory of lipolytic response to hormones is that it is mediated through the activation of adenylate cyclase at the membrane level. This stimu- lation results in an increase in 3', 5'-adenosine mono- phosphate (cyclic AMP) which in turn activates a protein kinase to activate a triglyceride lipase (Huttunen and Steinberg, 1971). Age or weight related changes could occur through alterations in any or all of the intricately linked processes or even some unrelated event such as cell alteration (Miller and Allen, 1973). 17 The study of insulin secretion through the growth period was determined best through a series of studies beginning with Alexander gt gt. (1973) who determined insulin levels in the fetal and neonatal sheep. Mean plasma insulin did not show appreciable variation except between 110 to 126 days post-conception. The highest peak recorded was 72 uU insulin on day 123 which was 49 uU higher than any other level from day 68 to birth (the period of the investigation). Upon infusion of glucose before day 110, very little change in insulin levels was noted. However, at 123 days, glucose infusion caused a marked increase (up to 200 uU) in insulin over basal levels. Infusion of glucose consistently elicited ele- vated insulin levels for any time period after the 123 day initial response. Histological examination of the pan- creas in sacrificed sheep showed a marked increase in pro- liferation of secondary islets of the pancreas and appear- ance of B—cells with islets is increased. Bassett and Alexander (1971) studied changes in basal plasma insulin levels of sheep from birth to 20 days. They reported 8.4 uU/ml in the lamb at birth. The second day insulin had increased to 35.7 uU/ml and did not change significantly up to 20 days. Bassett (1974a) showed that in 1 to 3 month old lambs, insulin increased from 20 to 30 uU before feeding to 160 uU in milk fed lambs. He also noted elevations up to 40 uU after lambs consumed dry feed. In mature wethers, Bassett (1974b) 18 also showed increased insulin levels 2 to 4 hours after feeding but not as drastically as the levels in younger sheep. Siers and Trenkle (1973) established insulin values in resting pigs of 67 to 84 kg to average .60 ng/ml with ‘a range from 1.07 to .25 ng/milliliter. Machlin gt gt. (1968) established levels of 11.0 uU/ml and a decrease to 5.8 uU/ml after 3 days of fasting. When the pigs were injected with .3 uU of insulin, plasma glucose decreased from 91 mg/100 ml to 38 mg/100 ml indicating the same hypo- glycemic effect as tg ttttg studies. Growth hormone levels rose twofold within 30 minutes but returned to normal levels in 1 hour. Romsos gt gt. (1971) injected pigs with alloxan, a drug which selectively destroys pancreatic B—cells thereby inducing insulin insufficiency. In these pigs, not all endogenous insulin was cleared from the system although a 30% reduction in insulin was observed. Insulin levels were 29 and 20 uU/ml, respectively, for the control and diabetic induced pig. A third pig which was diabetic- induced received a subcutaneous infusion of insulin and had 37 uU/ml of insulin. Glucose and free fatty acid levels were three times the control levels. The infused diabetic pig did not differ significantly from the control in either glucose or free fatty acid levels. 19 Protein Metabolism. Insulin and its relation to pro- tein metabolism has not received much attention until re— cently. Jefferson gt gt. (1972) showed the ability of insulin to affect the aggregation of ribosomes. Man- chester (1967) indicated insulin had a vital role in the transport of inter-tissue amino acid transport. Millward gt gt. (1974) found that changes in muscle RNA expressed as RNAzDNA paralleled plasma insulin changes. They pro- posed that elevated insulin levels after a meal may affect RNA concentration through the interaction with ribosomes. They reasoned that since amino acids were higher during fasting than after feeding, insulin did not exert its primary effect through amino acid transport, instead it exerted a more direct influence on the ribosomes. Genetically Obese Animals — Fat Metabolism. Rats or mice are the usual model used in studying obesity through genetic selection primarily because of the short genera- tion interval. Zucker (1972) reported on mobilization of fatty tissue in genetically fat rats ("fatties"). At 2 weeks of age glycerol and FFA levels in both control and "fatties" were normal. At 4 weeks of age, glycerol levels were higher in the "fatties" although FFA levels were normal. By the sixth week of age, both FFA and glycerol levels were above normal for the "fatties" and by 15 weeks of age the "fatties" had almost twice the circulating FFA levels (29.7 umoles/1OO ml Kg 17.5 umoles/ml) and more 20 than three times the glycerol levels (21.7 umoles/100 ml Kg 5.7 umoles/lOO ml) of the control rats. Originally it was thought that increased fat mobili- zation in the obese individual was primarily due to in— creased cell numbers (Zucker, 1967). Since then it has been established that despite increased numbers of fat cells, the "fatties" are capable of accumulating more fat on a per cell basis than normal rats (Johnson gt gt., 1971; York and Bray, 1973). It was noted, however, that in mice, an increase in age was accompanied by a decrease in fat mobilization per cell in both obese and control animals (Herberg gt gt., 1970). The mechanism for the alteration of glycerol and fatty acid levels is not yet well understood. Zucker and Zucker (1961) established the genetic basis for the dif— ference as being a single recessive gene. The role of that gene, however, has only been postulated. Treble and Mayer (1973) suggested that the presence of the enzyme glycerol kinase in the fat tissue of the obese mouse allows glycerol to be converted to glycerol phosphate, the essential precursor of triglycerides. Normal mice do not have the glycerol kinase in the fatty tissues. Steele gt gt. (1974) measured tg tttgg FFA synthesis from 100 day old lean, obese, and control pigs and found fatty acid synthesis 33% higher in the obese pigs as compared to the controls and 60% lower in the lean pigs as opposed to the controls. A similar trend was shown by 21 Standal and Vold (1973). They reported data on tg ttttg FFA release from fatty tissue of fourth generation Nor— wegian Landrace pigs selected either for high or low back— fat. The control pigs released .48 umoles/lOO mg (FFA/mg tissue) while the high and low backfat pigs released .58 umoles/lOO mg and .42 umoles/100 mg, respectively. Genetically Obese Animals - Protein Metabolism. Few data are available in the area of protein metabolism of lean and obese animals. Bergen gt gt. (1975) reported one of the few studies relating to lean development in genetically obese mice. They found that DNA content be- tween the two lines was significant. They also found that muscle growth was diminished in the obese mouse post wean- ing leading them to suggest that a possible defect in muscle as well as fat synthesis may be responsible for the obesity observed in the mice. Lean and Obese Animals - Body Composition. Work done in mice (Bergen gt gt., 1975; Romsos and Leveille, 1974) and in pigs (Weiss gt gt., 1971; Standal and Vold, 1973; Steele gt gt., 1974; and Althen, 1975) selected for or against fat, has shown the definite influence such selec- tion can make on body composition. Weiss gt gt. (1971) reported significant muscle differences in lean and fat lines of pigs. Separable muscle was 52.1% and 48.9% for the lean and fat lines, respectively. The total separable 22 fat increased from 20.2% in the lean line to 21.9% in the fat line. Standal and Vold (1973) reported compositional dif- ferences in fat, control, and lean lines of Norwegian Landrace pigs selected for four generations. The authors found the fat component of the carcasses increased from 21.5% in the lean strain to 25.3% in the control and to 30% in the fat strain. Body muscle for the three lines was 52.0, 48.1, and 46.0%, respectively. The loin eye area, however, did not differ significantly among lines nor did color scores for quality differ significantly. Duroc pigs were selected for 14 generations for high backfat, low backfat, or control and Yorkshire pigs were selected for 14 generations for the same fatness traits (Hetzer and Miller, 1973). Both breeds showed an in- crease in the proportion of lean and decrease in carcass fat for the pigs selected for low backfat Kg pigs selected for high backfat. High backfat pigs were significantly lower in percentage lean cuts and percentage lean in the ham, higher in percentage fat cuts and percentage fat in the ham, and had smaller loin eye areas than either control or low backfat pigs. Low backfat and control pigs did not significantly differ in percentage lean or fat cuts or in loin eye size. The only significant difference between the lean and control lines was the percentage lean, fat and bone in the ham. Low backfat pigs had significantly 23 higher percentages of separable lean, less separable fat in the ham, and more bone (except for bone in pigs of Yorkshire breeding) than control pigs. Endogenous Corticoid Levels. Dvorak (1972) cultured prenatal adrenal glands and found ACTH greatly enhanced the production of corticosteroid. This observation led him to conclude that the fetal pig was capable of corti— costeroid production. Ash and Heap (1975) confirmed this hypothesis by measuring corticosteroid levels in the sow and fetus. The maternal level of corticosteroid was 60 ng/ml of blood while the fetal levels for male and female were 210 and 200 ng/ml, respectively. Blood samples from the umbilical vein only contained 40 ng/milliliter. The authors, therefore, concluded that the fetus is capable of producing its own corticosteroids and that the placen- tal wall is apparently impermeable to corticosteroids. At birth the corticosteroid levels increase drasti- cally as shown in sheep by Bassett and Alexander (1971) and in swine by Dvorak (1972). In sheep, the levels of corticosteroid were as high as 81 ng/ml immediately after birth but by day 20, levels had decreased to 13 ng/ml which was similar to adult sheep levels (Bassett and Alexander, 1971). Dvorak (1972) reported values of 100 ng/ml for the pig fetus 10 days prepartum and that level rose to 20 ng/ml one day before birth and peaked at 42 ng/ml 12 hours after birth. Ten days postpartum the level 24 dropped to 18 ng/ml and at 46 to 60 days corticosteroid levels were 8.3 ng/ml which were not significantly higher than adult levels. No differences in circulating levels were noted between the intact male and female at any time either before or after birth. Fluctuation in Corticosteroid Levels. A variety of factors influence corticosteroid levels in animals. Bassett (1974b) reported daily fluctuations in sheep with no apparent pattern and no evidence of circadian rhythm. Purchas (1973) demonstrated the effect of fasting and feeding. In sheep fasted 18 to 20 hours, a three-fold increase in cortisol levels was noted. Levels dropped to normal 2 to 4 hours after feeding. However, in sheep frequently fasted for 18 to 20 hours, no significant change was noted indicating there was a conditioning ef- fect. Ray gt gt. (1972) studied the response handling steers had on corticoid levels. Blood was collected via cannula as well as venipuncture of the jugular and tail veins. The steers were collected once in the pen, moved to a squeeze chute and collected a second time with a third collection made after 15 minutes in the squeeze chute. Corticoid levels rose but did not differ signifi— cantly between the first and second collections. However, after 15 minutes in the chute the level rose from the previous 18 ng/ml to as high as 60 ng/milliliter. None 25 of the animals indicated signs of apprehension during any part of the collections. McNatty gt gt. (1972) reported the influence of a new environment on the cortisol level in sheep. Levels rose from normal values of 8 to 10 ng/ml to as high as 33 ng/ milliliter. Only after 28 days did levels return to normal. The same effect was reported by McNatty and Young (I973) when ewes were exposed to a change in light pat- tern. Twelve hours of diffuse (indoor) light and 12 hours of darkness initially elicited a rise in cortisol levels. On subsequent days, however, a gradual decrease to nor- mal cortisol levels was noted indicating a conditioning response. Topel gt El- (1973) reported daily fluctuations of hydrocortisone levels in pigs. Levels were highest in the morning and lowest in the afternoon. Similar find- ings were reported by Whipp gt gt. (1970). Morning levels of hydrocortisone in swine plasma were significantly higher (24 ng/ml) than afternoon and midnight levels of 8 and 6 ng/ml, respectively. Glucocorticoid Influence on Body Metabolism. Glucoé corticoids play an important role in regulation of blood glucose (Sasaki gt gt., 1974). They are required as activators of other hormones which control gluconeogene- sis, glycogneolysis, and lypolysis. Part of the function of glucocorticoids is to maintain the normal sensitivity 26 of the metabolic pathways to cyclic AMP. Corticoids in general may exert their effects primarily through the preservation of the intracellular ionic concentration (Exton gt gt., 1972) Monder and Coufalik (1972) reconfirmed that cortisol, one of the primary and most abundant of the glucocorti— coids (Bassett and Alexander, 1971; McNatty gt gt., 1972; Ash and Heap, 1975), stimulated the synthesis of glycogen in fetal rat liver cultures. An increased incorporation of labeled carbon from alanine into glycogen was noted. Also, the quantity of labeled carbon detected in glucose was diminished indicating a reduced ability to produce glucose in the presence of cortisol. Reilly and Black (1973) reported similar results using 14C—alanine in adre- nalectomized sheep. The authors reported increased in— 14 corporation of C into glucose, reduced CO production 2 and decreased oxygen consumption. Apparently the influ- ence of glucocorticoids then is more indirect than most other hormones. Mayer EI.§l- (1974) and Mayer gt gt. (1975) had shown that glucocorticoids have specific binding sites in the cytosol of muscle tissue. Based on that fact, Mayer and Rosen (1975) investigated the interaction of catabolic cortisol with anabolic androgens. Labeled cortisol was injected into rats or to cultured rat muscle media. Ad- ministration of androgens, both tg ytttg and tg tttg, re— duced the percentage bound, labeled cortisol in the muscle 27 cytosol. The authors concluded that androgens were cap— able of competitively replacing glucocorticoids on cytosol binding sites of muscle tissue thereby reducing the cata— bolic effect of the glucocorticoids. Glucocorticoid Influence on Body Composition. As discussed previously large fluctuations in glucocorticoid levels for little or no apparent reason pose particular difficulty in determining the role of glucocorticoids on body composition. It is assumed that all animals are stressed but probably not to the same degree during plasma or serum collection. Consequently, large differences in glucocorticoid levels are necessary in order to show sig- nificant differences. The majority of the work which has been done with corticoid levels in pigs has been the role of corticoids in the PSE and PS8 conditions. Biochemical changes occur- ring with respect to the PSE condition was reviewed by Symbesma (1972) while Topel (1972) reviewed some of the physiology associated with the quality changes of PSS pork. Weiss gt gt. (1971) were among the first to relate corti— coid levels to growth and composition characteristics of the pig. As pigs increased in weight from 1 to 137 kg, plasma corticoid levels decreased from 45.90 ug/100ml at 1 kg to 12.25 ug/lOOml at 9 kilograms. Subsequent levels at increased weights fluctuated from between 14.09 ug/ 100ml at 18 kg and 25.78 ug/lOOml at 114 kg live weight. 28 Similarly Dvorak (1972) reported decreasing corticoid levels from birth to 70 days of age. After 70 days plasma corticoid levels plateaued and approached normal adult levels. Data analyzed for lean or fat strain pigs in the study of Weiss gt gt. (1971) showed cortisol levels to be higher (P<.01) in lean pigs (26.80 pg/100ml) than in the fat strain (15.56 ng/lOOml). Longtssimus muscle color was also significantly lighter in the lean than in the fat strain. Results of Jedlicka gt gt. (1976) agree with some of Weiss gt El- (1971) although the former induced stress and measured corticoid response, whereas the latter evalu- ated endogenous corticoid levels. Jedlicka gt gt. (1976) divided pigs into a two by three factorial design for the following variables: high and low backfat, large and small longissimus muscle area, and light and dark meat color. The pigs were subjected to 40 C temperature and 100% relative humidity for 40 minutes. The major differences in corticoid levels appeared in plasma samples taken at 40, 70, and 90 minutes post—treatment. Corticoid levels were higher in the animals with 1) less backfat, 2) larger longissimus muscle, and 3) with lighter color of lean. The difference in corticoid levels was attributed to the abil— ity of the leaner animals to produce greater quantities of steroids and consequently lower quality meat. MATERIALS AND METHODS Experimental Design A total of 72 pigs representing three genetic lines and two breeds and two sexes were included. Twenty—four pigs were represented in each of the three lines; one line selected for high backfat, one line selected for low back- fat, and a third unselected line served as a control group. Thirty-six pigs were of Duroc breeding and 36 pigs were of Yorkshire breeding. Barrows and gilts were equally repre- sented in all three lines and in both breeds. The Duroc breed represented the 19th generation of selection and the Yorkshire breed represented the 17th generation of selec- tion. No adjustment of data was made for the differences in generation interval. All the pigs were bred, reared, and slaughtered at the U.S.D.A. Research Station in Belts- ville, Maryland. All blood samples and carcass data were collected by U.S.D.A. personnel at the Station. Plasma Collection. The pigs were bled at three dif- ferent time periods. The first bleeding was at 56 days (weaning), the second bleeding at 115 days (approximately the midpoint between weaning and slaughter), and a third bleeding at 98 to 102 kg (the day before slaughter). Collection of blood (for all analyses except creatine 29 3O phosphokinase, CPK) was accomplished by snaring the pig and collecting 10 ml of blood by vena cava puncture into Vacu-Tainer vials (Bectron-Dickerson, Rahway, New Jersey). The vials contained 14 mg NaEDTA and 5 drops of 4% NaF (4g/1OO ml). Prior to blood collection, the tube was evap— orated to dryness. Blood was collected at 1:00 pm on each bleeding day. The blood was centrifuged at 3000 x g and the plasma decanted, frozen, and shipped to Michigan State University. Bldod samples to determine CPK were collected independently but at the same time periods as described above. One ml of blood was collected with no additives in the tube. _After clotting the blood was centrifuged at 3000 x g and the serum assayed immediately to determine CPK activity. Slaughter Procedure. All slaughtering occurred on Thursday mornings of the week the pig attained 100 kilo- grams. Slaughter procedures were in accordance with con- ventional methods. Carcass measures were in accordance with procedures as outlined by the American Meat Science Association (Hiner, 1952) except for backfat which was de- termined in five locations taken at the first rib, seventh rib, first lumbar, mid lumbar, and last lumbar. The con- ventional three point backfat measure was also calculated from the average of the first rib, first lumbar vertebra and last lumbar vertebra measurements. The carcasses were chilled 96 hours before breaking. The lean cuts (hams, loins, Boston butts and picnic shoulders) were removed and 31 trimmed according to A.M.S.A. procedures and careful ac— count kept of the weights (to the nearest .05 kg) and of all the trimmings. The fat cuts (leaf fat, plate, total trimmed backfat (including skin), and other fat trim) were also cut in accordance with A.M.S.A. standards and weighed to the nearest .05 kilogram. Physical separation of muscle, fat, skin, and bone of the right ham was also done on the day of cutting and the weight of each recorded to .005 kilogram. A 10 g sample of the longissimus muscle was removed the day of cutting and prepared immediately for transmis— sion value determination as described later. Backfat at the 10th rib was calculated from the re- gression of backfat (Fahey gt gt., 1977) to 3 point back— fat measures. Percent muscle was then calculated in ac- cordance with the procedure of the National Pork Producers Council (1976). Insulin Radioimmunoassay. Plasma insulin concentra- tion was determined by radioimmunoassay according to the procedure described by Meiburg (1973). The procedure was as follows: 1. Disposable culture tubes (12 x 75 mm, Scientific Products, McGaw, Illinois) were used for all samples and standards. Tubes 1 and 2 (non-specific binding tubes) re- ceived 500 pl buffer B1 (Appendix I. A. 2). All standard tubes received 400 pl buffer B1 and 100 ul of porcine 32 insulin standards (Appendix I. A. 3; Eli Lilly) ranging from 1.43 to 239.0 pU. All sample tubes received 250 or 350 pl and 250 or 150 pl buffer B1 (for a total of 500 pl in each tube). 2. On day zero (first day), 200 pl of guinea pig anti-porcine insulin serum (GPAPI; Appendix I. A. 7; Miles Laboratories) was added to all sample and standard tubes. Tubes 1 and 2 received 200 pl of 1:400 normal guinea pig serum (NGPS, Appendix I. A. 6). Tubes were gently vor- texed and incubated at 4 C for 24 hours. 3. On day one (24 hours later) 100 pl 125I-insulin (Appendix I. A. 8; Amersham Searle, specific activity of 50 pCi/pg) were pipetted into each tube; the tubes were gently vortexed and incubated at 4 C for 24 hours. 4. On day two (36 hours later), 200 pl sheep anti- guinea pig gamma globulin (SAGPGG, Appendix I. A. 9) were pipetted into all tubes except tubes 3 and 4, gently vor- texed, and incubated at 4 C for 96 hours. 5. On day five (sixth day), 3.0 m1 of PBS (Appendix I. A. 4) were added to all tubes (except 3 and 4) and cen- trifuged at 2500 x g for 30 minutes in a Sorvall RC3 re- frigerated centrifuge. The tubes were then decanted and left inverted on absorbent paper for 30 minutes. Any excess liquid was wiped from the upper half of the tube and then all tubes were counted for 4000 counts for 4 min- utes in a Nuclear-Chicago Model 4320 autogamma scintillation spectrometer. 33‘ 6. Standard curve values were entered into a CDC 6500 computer which was programmed to calculate a regres- sion equation with linear, quadric, and cubic components. Regression equation values were entered into an Olivetti computer (Programme 101, Olivetti Underwood, New York) which automatically calculated insulin values and cor- rected for dilution of samples. Free Fatty Acid Assay. Plasma free fatty acids (FFA) were determined by the method of Ho (1970) as modified by Bieber and cited by McGuffey (1975). Plasma samples were extracted by pipetting 200 pl into 12 x 75 disposable culture tubes and adding 1.0 ml Dole extraction mixture (Dole, 1956; Appendix 1.0. 1). The tubes were then vortexed and placed in an ice bath. After 10 minutes, 200 pl distilled water and 200 pl hep- tane were added, the tubes vortexed and again placed on ice. After 10 minutes, a 200 pl aliquot of the heptane (upper) layer was removed and placed in a 1.5 ml polypro- pylene centrifuge tube with a snap cap (Brinkman Instru- ments Inc., Westbury, New York). Then .8 ml chloroform plus .1 ml 63Ni reagent (1.01 mCi/lOO mg 63Ni; Amersham Searle Corporation, Arlington Heights, Illinois, Appendix I. C. 2) were added to the tubes, each was vigorously vor— texed for 45 seconds and then centrifuged at 500 x'g for 10 minutes. The 63Ni (upper) layer was aspirated off and a .5 ml aliquot of the lower (chloroform) layer was transferred to a 22 x 50 mm scintillation vial (Packard 34 Instrument Company, Downers Grove, Illinois). The chloro— form was allowed to evaporate in a fume hood before 10 ml of scintillation fluid (Appendix I. C. 3) were added. The samples were then counted 10 minutes in a Nuclear-Chicago liquid scintillation counter. A standard curve was determined by pipetting suffici- ent standard solution (Appendix I. C. 4) for a working curve between 0 and 100 nmoles. Ethanol was evaporated and palmitic acid reconstituted with 200 pl distilled water. Standards were then subjected to the same proce— dure as the unknown plasma samples. A regression curve of palmitic acid (y) and counts (x) was entered into a Texas Instruments SR-51 and unknown samples calculated from the regression curve. Plasma Glucose. Plasma glucose was determined by the GOD-PERID method as described in the Boehringer Mannheim Corporation bulletin BMC 7453 (1974). The enzyme was re- constituted in distilled water (Appendix I. D. 2). Five ml of the enzyme solution were added to 20 pl blood plasma in 16 x 100 mm disposable culture tubes. The samples were vortexed and the color allowed to develop for at least 25 minutes. Absorbance was read at 600 nm on a Beckman Model 24 Spectrophotometer. A standard curve was determined by pipetting 20 pl of each standard glucose solution (Appendix I. D. 1). A blank containing 20 pl water was used as the reference. A 35 standard curve was plotted and extinction coefficients determined to calculate sample values. Plasma Cortisol. This method is a modification from that described by Smith gt gt. (1972). To account for procedural losses of cortisol, 10 pl of tracer cortisol (Appendix I. B. 3) were added to six 16 x 100 mm disposable tubes (Scientific Products) and also to two scintillation vials. The methanol was evap- orated to dryness. Six unknown plasma samples were ran— domly selected and 100 pl plasma were added to each of the six 12 x 75 mm tubes. Two ml iso—octane (nanOgrade) were added and vortexed for one minute. The contents of the tubes were frozen for approximately 1 hour, decanted and the iso-octane discarded, being careful not to thaw the serum. The serum was allowed to thaw after decanting, 2 ml methylene chloride added and then vortexed for one min- ute. The methylene chloride was evaporated to dryness and 5 ml scintillation fluid (3a, 70B, Research Products Inter- national Corporation, Elk Grove Village, Illinois) were added to each of the six vials and also to the two vials with tracer cortisol. Extraction efficiency was calculated from these tubes and samples were adjusted for losses. Three sets of standards were included per assay. Stan- dard cortisol (Sigma Chemical Company) was diluted to 10 ng/ml in methanol. A standard curve was determined from .05, .10, .20, .25, .50, .75, 1.0d, 1.50, 2.00, and 2.50 ng cortisol into 12 x 65 mm culture tubes. 36 Samples were run in duplicate using 100 pl plasma pipetted into 16 x 100 mm disposable culture tubes. Two ml iso—octane (nanograde) were added and the tubes were vortexed for one minute. The tubes were frozen for approx— imately 1 hour, decanted and the iso—octane was discarded being careful not to allow the plasma to thaw while de- canting. The plasma was allowed to thaw, 2 ml methylene chloride added, vortexed for 1 minute and frozen for ap- proximately one hour. The methylene chloride was decanted into 12 x 75 mm culture tubes and evaporated to dryness. The procedure was continued with both the standard and the sample tubes at this point. Five-tenths ml labeled cortisol (Appendix I. B. 1) were added, vortexed and in— cubated at least 12 hours at 4 C. After incubation, the tubes were placed in an ice bath and as quickly as possi- ble .5 ml dextran coated charcoal were added (Appendix I. B. 4), vortexed and centrifuged 15 minutes at 2900 rpm in a Sorvall RC3. A .5 ml aliquot of supernatant was removed, 5 ml scintillation fluid added and the vials were counted for 10 minutes for total counts. Calculation of samples was done on a CDC 3600 computer with a standard program to correct for extraction loss. The computer was programmed to calculate a regression curve of the standards using cubic components. Sample values were automatically printed. 37 Transmission Values The transmission value method described by Hart (1962) was used in this investigation as an objective determina- tion of porcine muscle quality. Since low quality procine muscle has lower protein solubility than normal muscle, the protein extract of the muscles should reflect a dif- ference in light transmission through the extracted sample. Low quality muscle results in higher transmission values because of less extracted muscle protein in solution. Conversely, high quality muscle has a lower transmission value due to more extracted muscle proteins in solution. Reagents for the test included .2 M dibasic sodium phosphate and .1 M citric acid. The reagents were combined in the ratio of 9.35 ml sodium phosphate and 10.65 ml citric acid solution resulting in a pH 4.6 buffer. Ten g of finely ground longissimus muscle were placed in a cen- trifuge tube. Cold distilled water was added until the total volume was 40 ml and the mixture was then thoroughly stirred. The mixture was held at 2 to 4 C for 20 hours after which it was stirred again and then centrifuged at 2000 x g for 20 minutes. The supernatant was filtered through S & S No. 588 filter paper. One ml of the clear filtrate was mixed with 5 ml of pH 4.6 buffer solution at 20 C in a colorimeter tube. This mixture was held at exactly 20 C for 30 minutes. After this incubation, the solution was mixed again by inversion of the tube and read at 600 nm in a Bausch and Lomb Spectronic 20 colorimeter 38 against a blank containing 1 ml of muscle extract and 5 ml distilled water. Percentage transmission was recorded as the transmission value. Creatine Phosphokinase The analysis of creatine phosphokinase (CPK) was in— cluded in this study as an objective measure of stress susceptability of pigs since studies have shown elevated CPK activity values to be associated with stressed pigs (Reddy gt gt., 1971; Addis gt gt., 1974; Beermann gt gt., 1975)- The procedure used was according to Sigma Technical Bulletin No. 520 (1967). For reagents see Appendix I. D. Standard Curve Determination. A creatine standard solution (.40 pmoles per ml) was prepared by reconstitut— ing a creatine standard vial, Stock No. 520-60 with 100 ml'water. Either .O, .2, .4, .6, .8, or 1.0 ml of the stock solution was added to each tube and then water was added so the initial volume of each tube was 1.0 milliliter. One ml of a-naphthol solution (Reagent E1), 1.0 ml of diacetyl (Reagent G1) and 7.0 ml water were then added to each tube and the contents of each tube were mixed im— mediately after adding the reagents. They were allowed to stand at room temperature. After 15 minutes had elapsed, the standard tubes were read at 520 nm using the .0 tube I as the reference tube. All tubes must be read within 10 39 minutes of the end of the 15 minutes incubation time. The results were plotted on graph paper using optical density values (y - axis) tg corresponding Sigma value (x - axis; .0 = 9, .2 = 32, .4 = 64, .6 = 96, .8 = 128, 1.0 = 160). Test Procedure for Samples. Two 15 ml centrifuge tubes were labeled "TEST" and "BLANK," respectively. Five- tenths ml phosphocreatine solution (Reagent A1) were added to each tube. Then .1 ml of a l in 10 dilution of serum to water (1 part serum and 9 parts water) was added to the TEST tube only. One-tenth ml water was added to the BLANK tube only. Both tubes were placed in a 37 C water bath for a few minutes to allow the solutions to warm up. To each tube .2 ml ADP-glutathione solution (Reagent Cl) was added which starts the reaction. The tubes were thoroughly mixed by swirling and the exact time noted. At the end of 30 minutes incubation time, .2 ml P-HMB solution (Reagent D) were added to each tube (stops reaction) and thoroughly mixed. One ml a-naphthol solu- tion (Reagent E1), 1.0 ml diacetyl (Reagent G1) and 7.0 ml water were added to each tube and the tubes were swirled after the addition of each reagent. They were placed in a 37 C water bath for 15 to 20 minutes to develop the color. The tubes were centrifuged for approximately 5 minutes at 2000 x g. The TEST sample was read at 520 nm using the BLANK as the reference. The readings were made within 10 40 minutes after removing from the centrifuge and the CPK activity was determined from the established standard curve . Statistical Analysis The design of the experiment was a factorial arrange- ment with two sexes, two breeds, and three lines. These main effects and interactions were tested statistically using an analysis of variance procedure. For cases where line effects were significant, the student range test was used to evaluate differences between specific lines (Duncan, 1955). The data were analyzed separately for three different bleeding times (56 days, 115 days, and at slaughter). The data were then analyzed with the first three factors as the main effect and bleeding time as a subeffect in a split—plot analysis. In addition, linear correlation coefficients were calculated between pairs of dependent variables. All statistical analyses were preformed on a Control Data Center 6500 comuter at the Michigan State University Computer Center. RESULTS AND DISCUSSION Blood Plasma Characteristics The values for the plasma traits by breed, sex, and line for all three bleeding periods (56 day, 115 day, and immediately prior to slaughter) are shown in tables 2 and 3. At 56 days of age circulating insulin levels were signifi— cantly (P<.O5) higher for barrows compared to gilts. The high backfat line also had significantly higher (P<.05) levels of insulin than either the low or control backfat group. In lean and genetically obese mice, circulating in— sulin levels in obese lines may be as much as 100 times higher than circulating insulin levels in the lean strain (Abraham gt gt., 1971). Although the pigs in this study do not show such a drastic elevated level, circulating in- sulin levels of the high backfat line are significantly elevated. The influence of sex or breed on insulin levels in cattle was determined to be nonsignificant (Irvin and Trenkle, 1970). Tables 4 and 5 summarize the data across the three bleeding periods (56 days, 115 days, and slaughter). In- sulin levels for Durocs were slightly higher at 108.7 uU/ ml although the difference was not significant (Table 4). Breed effect in cattle as determined by Irvin and Trenkle (1971) were also found to be nonsignificant. Sex differ- ences of 108.0 pU/ml for gilts and 103.2 pU/ml for barrows 41 42 TABLE 1. NUMBER, CODE AND DEFINITION OF VARIABLES USED IN TABLES Number Code Definition 1 FIG NO Individual pig identification number 2 BREED 1 = Duroc; 2 = Yorkshire 3 LINE 1 = High Backfat; 2 = Low Backfat; 3 = Control 4 SEX 2 = Gilt; 3 = Barrow 5 INS WEAN Plgsmg insulin concentration at 56 days U ml 6 INS MPG Plagma insulin concentration at 115 days (pU/ml) 7 INS SLAU Plasma insulin concentration at slaughter (pU/ml) 8 COR WEAN Plasma cortisol concentration at 56 days (Hg/ml) 9 COR MPG Plasma cortisol concentration at 115 days (ng/ml) 10 COR SLAU Plasma cortisol concentration at slaughter (ng/ml) 11 FFA WEAN Plasma free fatty acid level at 56 days (nmoles/ml) 12 FFA MPG Plasma free fatty acid level at 115 days (nmoles/ml) 13 FFA SLAU Plasma free fatty acid level at slaughter (nmoles/ml) 14 GLU WEAN Plasma glucose level at 56 days (mg/ 100ml 15 GLU MPG Plasma glucose level at 115 days (mg/ 100ml 16 GLU SLAU Plgsma glucose level at slaughter (mg/ 00ml 17 CPK WEAN Plasma CPK at 56 days (Sigma units) 18 CPK MPG Plasma CPK at 115 days (Sigma units) 19 CPK SLAU Plasma CPK at slaughter (Sigma units) 20 TNS VAL Muscle transmission value (Recorded as OD; report as %T) 21 BF10 Backfat at 10th rib; transformed from average 3pt backfat (cm) 22 NOINGRP Number in group; identifies pigs of same breed, line, and sex as 1, 2, 3, 4, 5, and 6 for computer coding purposes 25 ADG Average daily gain (kg) 30 LEAF FAT Leaf fat (kg) 35 TOT BFWT Total backfat weight (kg) 36 HAM LEAN Weight of physically separated lean of right ham (kg) (continued) 43 TABLE 1 (Continued) Number Code Definition 37 LEAN CUTS Total weight of lean cuts (kg) 38 FAT CUTS Total weight of fat cuts (kg) 39 HAM FAT Weight of physically separated fat from right ham (kg) 40 HAM BONE Weight of bone from right ham (kg) 41 HAM SKIN Weight of skin from right ham (kg) 42 CARC LEN Carcass length (cm) 43 BF 3PT Average backfat measure taken at 3 points (cm) 44 BF 5PT Average backfat measure taken at 5 points (cm) _ 2 46 LEA Loin eye area (cm ) 47 COL FIRM Color-firmness score for quality 48 MARB Marbling score for quality 49 AVE QUAL Average of color-firmness and marbling scores 62 F CUT Total fat cut weight (kg) 63 PCT HAM Right and left hams expressed as percent of chilled carcass weight 64 PCT LOIN Right and left loins expressed as per— cent of chilled carcass weight 65 PCT HL Percent ham and loin expressed as per- cent of chilled carcass 66 PCT LC Lean cuts expressed as percent of chilled carcass weight 67 PCT LEAF Leaf fat expressed as percent of chilled carcass weight' 68 PCT BF Total trimmed backfat expressed as per- cent of chilled carcass weight 69 PCT FTCU Fat cuts expressed as percent of chilled carcass weight 70 RT HAM Weight of right ham (kg) 71 PCT LRH Physically separated lean from right ham expressed as percent of right ham weight 72 PCT FRH Physically separated fat from right ham expressed as percent of right ham weight 73 PCT BRH Bone from right ham expressed as percent of right ham weight 74 PCT SKRH Skin from right ham expressed as percent of right ham weight 75 PCT MUSC Estimation of the percent muscle with 10% intramuscular fat on a chilled carcass basis 44 Acmscflp:ovv as. oo.v em.mmm m.ooH H.em em.eoH we.mm agoofi\ms .msme mHH .mmoozau we. oo.v mm.aom e.sfia e.mafi es.emfi mm.HHH Asoofi\me .msme em .emoesao me. em. He.mmm m.mm m.em e.mm H.mm HE\meHoec .empgmsmam .mq mUZHQmmgm QHQZH mom xmm Qz< Qmmmm Mm mMDQ¢> mg macaw Rom no woman mean one QHQPHS cam mcfla ass Co mQMoE p.m so. we. ma.mess m.mm m.mm m.mm m.mm megs: «swam .eeeemzmam .xmo me. ma. mm.emmea e.moa s.mfia m.smH o.mm megs: memfim .msme mfia .mmo we. as. 00.0mms s.ms o.mm m.mm m.ms mean: memam .msme em .mmo mm. He. mo.eem H.sm m.Hm m.ew s.mm agoofi\we .neesmsmam .emoosao xmm Umohm paflw aohhmm mmfinmxnow oopsm ohmsvm smos moCMOHLHCme nohhm Rom voohm QHpmHsopnmno mamwam Mo Hm>mq AUoSQHPCOoV N mqmHQZH mom mZHQ Mm mmDQ<> <2m<fim Dooqm .m mgmmq nonnm OHPmHhopowhmzo mammam msflq Aeesefleeoov m mqmoq Nmm QZ< Qmmmm Mm mmDQ¢> <2moq m QOHmmm wzHommqm aze mqu wm mmpae> «ameqm mooqm mo wm GHQMHHN> mooo manwahm> mmDH<> .Amo.vmv pamoAMHQwfln one mm.mn mo mooam> oo.H Ho. so.- mm. mm. mo. mo.- no.- on seam mmo oo.a , on. ma. mm. mo.- so.- am. ma om: mmo oo.a mo. Ho.- Ho. HH.- oo. ma zmmz mmo oo.a mm. oo. ma. mo. on omqm oqo oo.a em. oa. mm. ma om: poo oo.a mo. on. an z mannanm> coco manmflnm> AUQSQHPQOUV @ mgmge 60 (r = .28) but was still rather low. However, by slaughter time the correlation between the two factors was .51. Carcass Quality and Cutability Table 7 summarizes the carcass parameters by breed. Average daily gain (ADG) for Durocs (.67 kg/day) was sig— nificantly (P<.O5) greater than for Yorkshires (.58 kg/day). Durocs, however, were significantly (P<.O5) fatter than Yorkshires as indicated by more backfat (5.36 cm 13 4.81 cm for the three point measurement), more leaf fat (3.8% .y§_3.4%), a higher % fat cuts (24.5% 1£.22.1%) as well as a higher % right ham fat trim (34.8% y§_32.7%). Yorkshires had significantly more lean than Durocs (Table 7). Yorkshires had higher percentages (P<.05) ham and loin (36.8% y§_35.0%), lean cuts (54.4% yg 53.0%), lean 0f the right ham (57.1% 13 54.1%) and separable muscle (44.3% yg 41.2%). Yorkshires also had significantly (P<.O5) larger loin eye areas (LEA) with 27.94 cm2 compared to 24.87 cm2 for Durocs. Jensen gt gt. (1967) compared Duroc and Yorkshire carcasses and reported similar results for backfat thickness, percentage lean cuts, color, firm- ness, and marbling. No differences in LEA was found, al— though Yorkshires (24.26 cmz) were slightly larger than Durocs (23.81 cm2). Table 8 summarizes the ADG and carcass parameters for sex. Average daily gain was higher (P<.O5) for barrows (.65 kg/day) than for gilts (.61 kg/day). In general, 61 nooSQMPCOOV oo.v OH.m 9H.mm mH.:m R .Em: gamma we swoq oo.v H©.N 93.:m mo.mm & .mezo swag oo.v os.H Qw.wm wo.mm R .cfloa out 8mm oo.v am. po.sa so.oa a .saoa oo.v om. ps.oa ma.oa s .emm oo.v OH.: 8H.NN Dm.dm R .mpSo Pam oo.v mm. ss.m em.m s .osm when oo.v mm.m Qm.:m mm.fim So .zpmsmfi mmmonmo oo.v mH. p:m.sm www.mm NEo .monm who Smog oo.v mm. mmo.s p:m.m So ..mtos m we .m>m .pmwmomm oo.v 3N. tam.: ppm.m So ..mmos m Mo .w>m .pt%&omm oo.v so. sss.m poo.o so .ean meoa as esmmosm oo.v :00. 8mm. ppm. mm .cfimm madam mwmnm>< .0MMOLWMMMam .ttaWthm.s .tasmg.oa sores OHPmHt.t.tt.ao mm....s omohm Qmmmm Mm mOHBmHmmBo commmflSWcmme opoom hPflHMSU owMHo>< whoom msflanums ohoom mmoQShH% osm Hoaoo R .oaomss manmnmmom R .86: games no damn R .Em: Psmflh mo mcom R .ese emmaa to ram mo. mo. . o.mm m.mm oo.v om. sm.m pa.m oo.v on. no.m po.m as. as. a.m m.m oo.v om.m am.ee sm.ae oo.v mo. so.m pm.m oo.v om. am.o eo.o oo.v mm.m ss.mm pm.am ohflnmxpow condo ooQtomchmHm onmsom some . %o Ho>oq Mohhm coohm oprHMopomnmso mmwonwo Assessesoov s mamme 63 wooscflpsoov oo.v ofi.m pm.mm mo.:m R .88: anfln Mo somq oo.v Hw.m Qw.:m mw.Nm R .mPSO Gama oo.v os.a po.om so.sm R .saoa one som oo.v Hm. ea.ma so.oa R .saoq oo.v os. nm.oa so.oa R .emm oo.v 0H.: mN.NN Qm.:m R .mPSO pmm oo.v mm. m:.m Qw.m R .wwM mood mH. wR.N :.mm w.mm so .mpmsoa mmtohto oo.v mH. Qm©.mm mwa.mm N80 .mohm who Smog mo. mm. mm.: mo.m so ..mtms m mo .w>m .pmmmomm ma. 3N. Ho.m mH.m So ..mMoE m Mo .m>m .mexomm on. no. ma.o om.o so .eaa neon as esmmosm Ho. :00. mam. Qmw. mm .camw %Hflmo ommmm>< oomwomwwwmflm osmSWMMMMms paflw xmwohnwm oflpmflnopomnmso mmmonwo xmm Wm moHBmHmmeo soamnasmssnm No. om. mm.m nm.m spoon zpflamso owmso>< Ho. 0:. mm.m QR.N spoon wcflannmz Rd. at. H.m N.m onoom mmos8hfl% ocm poaoo oo.v mm.m Qm.md mm.fid R .oaomzs camshmmmm mm. mo. m.m H.N R .88: psmmh Mo cflmm Ho. om. mm.w pm.m R .88: inHA mo osom oo.v mo.m sm.mm . em.mm R .ssm games no emm mosseflwflsmflm ohmsom cmms PHHw sonpmm . mo Ho>oq 808nm ompmflnmpomnmno mmtopmo Rom moossHPsooo m mqmee 65 barrows were fatter as shown by the average of three meas- urements of backfat thickness (5.15 cm yg 5.01 cm) although the difference was not significant. Fatness indicators were significantly (P<;O5) greater for barrows than for gilts (leaf fat, 3.8% lg 3.4%; fat cuts, 24.3% y§_22.2%; and fat of the right ham, 35.2% 15 32.2%). Muscle differences showed gilts to be more muscular than barrows (Table 8). Gilts had larger (P<;O5) LEA 2 lg 25.16 cmz), higher percentages than barrows (28.6 cm ham and loin (36.9%.yg 34.9%), higher percentages lean cuts (54.8%_y§,52.6%), higher percentages lean of right ham (57.3% yg 54.0%), and higher percentages separable muscle (43.9% yg 41.6%). Bone of the right ham was higher (P<.O5) for barrows (8.6%) than for gilts (8.3%). Bere- skin and Davey (1976) reported no difference in percent- age bone of the ham although Hetzer and Miller (1973) re- ported a highly significant sex effect with barrows haVing higher percentage bone within the Yorkshire breed. Richmond and Berg (1972) also reported that barrows had a higher percentage femur than gilts although the difference was not significant. Quality scores generally favored the fatter barrow carcasses. Color and firmness scores tended to be higher for barrows (3.2) than gilts (3.1) although this difference was not significant. Marbling score was higher (P<.05) for barrows (2.7) than for gilts (2.3). Average quality score was higher (P<.O5) for barrows (2.9) than for gilts (2.7). 66 Table 9 summarizes the ADG and carcass characteristics by backfat line. Average daily gain was significantly (P<.O5) lower for the high backfat line (.58 kg/day), in— termediate for the low backfat line (.62 kg/day), and highest for the control line (.68 kg/day). Bereskin gt gt. (1975) reported high backfat pigs gained slightly faster than low backfat lines although the difference (.70 kg/day 1g_.69 kg/day) was not statistically significant. Backfat thickness for the three genetic lines (Table 9) was significantly (P<.O5) different for the high (7.61 cm), low (3.03 cm), and controls (4.60 cm). Significant differences existed among the three lines for percentages leaf fat, fat cuts, and fat of the right ham, with the high line being fattest, the low line trimmest, and the control line being intermediate. Correspondingly, measures of leanness including percentages ham, loin, ham and loin, lean cuts, lean of right ham, and separable muscle were all significantly (P<.05) different among the three lines. The high backfat line had the lowest percentage lean, the con- trol line was intermediate, and the low line was highest. Loin eye area followed the same pattern as that for percent- ages of lean among the lines. Standal and Vold (1973) re— ported similar differences in three lines of Norwegian Land- race pigs selected in a similar manner for as little as 4 generations. Bereskin and Davey (1976) found similar dif- ferences in Durocs and Yorkshires selected for backfat thickness. Acoscfipcoov 67 oo.v oa.m em.mm os.so so.oe so: games so soon oo.v Ho.m em.am oa.mo ma.sa R .meso smog oo.v oR.H oo.om om.as sa.om R .saoa one 86m oo.v am. as.oa om.oa so.ma R .caoq oo.v or. om.oa oo.mm sa.oa R .esm oo.v oa.: po.mm so.ma oo.sm R .neoo pom oo.v mm. nm.m so.m oo.s R .988 Room oo.v wu.m QN.MR ow.mm mm.m© 80 .nmeoH mmmohmo oo.v ma. pmm.mm 000.:m mmm.ma N80 .8688 who Smog oo.v mm. pw:.: www.m om:.n 8o ..m868 m Re .m>m .pmmxomm oo.v am. 900.: ono.m oH©.R 8o ..mto8 m %o .m>m .Pmmmomm oo.v so. ems.m mos.m oma.oa so .pan room as eommosm oo.v soo. omo. omo. sow. mm .sasm maaso omsao>< .smwownwwwa. .t..Wthm.s aortaos eon swag oapmatstomtss. m...t.s osaq MZHQ Mm mOHBmHmmeo soammflsmsmne mo. om. ©.N m.m o.m onoom zpflamso 0mt~0>¢ oo.v 0:. wa.m mm.m :H.m choom mcflapuwz oo.v He. :N.m :m.m mm.m onoom mm088hflm osm noaoo oo.v mm.m 93.3: ow.mm 0N.mm _ R .0H0m58 manmnmmom oo.v mo. :m.m :m.m mm.a R .80: p:wflh Mo sflxm oo.v om. as.m oo.o so.R R .sms armas mo osom oo.v mm.m ea.mm so.mm om.aa R .esm emman no pom ooQtomhmcme meandm 8008 Homecoo 309 :mfim wo H0>0q nonmm OHP mflfimu-ONLSNSO mmNOM-NU osaa Aoossaesoov o mmmge 69 Cutability differences between backfat lines show sig- nificant differences among all three lines (Table 9). The high line had the greatest amount of fat trim followed by the control and then the low line. Percentage lean of the carcass was directly related to selection against fat with all three lines being significantly different. The low line yielded the highest percentages of lean, the control was intermediate, and the high line had the lowest percentages lean. Each of the individual parameters was significantly different among the three lines. Loin eye area was also significantly (P<.O5) different for the three lines with the high backfat group having the smallest LEA (19.29 cm2), the control intermediate (26.52 cm2), and the low line the largest (34.90 cmz). Selection for or against backfat tended to select for higher quality than was observed in the control line. The high backfat line was lower in color and firmness score (2.8) than either the low (3.5) or the control (3.2). Mar- bling score was higher (P<.O5) for the high backfat line (3.1) than for the low (2.2) or control (2.1). Average quality score approached significance (P<.O6) among the lines with the high backfat line having had the highest score (3.0) followed by the low line (2.8) with the control line having had the lowest score (2.6). Similarly, transmission values were not significantly different, although the high line had the highest value (38.2) followed by the low line (30.5) with the control line (28.3) the lowest value. 7O Correlations Between Carcass Traits Correlation coefficients between ADG and carcass para- meters are listed in Table 10. Average daily gain was negatively and nonsignificantly correlated with the fat— ness indicators of backfat, % leaf fat, % fat cuts, and % fat of the right ham. Loin eye area, % ham, % loin, % ham and loin, % lean of the right ham, and % muscle were all positively but nonsignificantly correlated with ADG. Only % bone in the right ham (r = .41) and % skin of the right ham (r = .34) were significantly correlated with ADG. Mor— tensen and Madsen (1976) similarly reported ADG positively and nonsignificantly correlated to lean content of pig car- casses. Hedrick gt gt. (1963) also found positive correla- tions (r = .41, P<.01) between trimmed primal cuts and ADG in cattle. In contrast, Bereskin and Davey (1976) reported negative correlations between ADG and several measures of lean including LEA (r = -.31, P<.01), % lean of ham (r = -.25, P<.01) and % lean cuts (r = -.17, P>.O5). Positive correlations were reported between ADG and % fat of the ham (r = .23, P<.01) and backfat (r = .05, P>.O5). Such appar- ent contradiction could be the result of the feeding regime since Bereskin and Davey (1976) slaughtered after a pre- determined time period, whereas Mortensen and Madsen (1976) and Hedrick gt gt. (1963) slaughtered at a constant weight. Hedrick (1968) hypothesized animals fed to a constant weight will have positive correlations between ADG and yield of lean cuts. 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Fat thickness was more highly correlated with P>.O5) and positively correlated at 393 days (r plasma insulin at 118 days (r .35) and at 198 days (r = .40) than at 393 days (r = .05) although none of the corre- lations was significant at the .05 level. Grigsby (1973) reported a positive correlation (r = .37, P<.01) between average daily gain and serum insulin in cattle while 80 measures of carcass lean tended to be positively correlated, whereas carcass fat indices were generally negatively re- lated to serum insulin. Plasma cortisol followed a pattern similar to plasma insulin. At 56 days all parameters except ADG, average quality, and transmission value were significantly corre— lated to corticoid levels. At 115 days and at slaughter the correlations were not significant but trends paralleled the 56 day values. Fat parameters positively correlated (P<.01) with 56 day cortisol values were backfat (r = .34), % leaf fat (r = .30), % fat cuts (r = .34), and % fat of the right ham. Muscle parameters correlated (P<.01) to 56 day cortisol levels were loin eye area (r = -.36), % ham and loin (r = —.36), % lean cuts (r = -.37), % lean of right ham (r = -.36), and % muscle (r = —.36). Color and firm- ness (r = .34, P<.01) and marbling (r = .24, P<.O5) at 56 days were significantly correlated to cortisol levels whereas average quality and transmission value were not signifi- cantly correlated. At 115 days only marbling (r = .25, P<.O5) was significantly correlated to plasma cortisol while no significant correlations were noted with plasma cortisol at slaughter time. Purchas gt gt. (1970) noted a decreased width of the glomerulosa region of the adrenal gland in heifers fed MGA and a consequent reduction in plasma cortisol. Reduced circulating cortisol levels were associated with increased growth rate although Hafs gt gt. (1971) suggest the MGA may 81 have increased growth rate by a means other than solely by reduction of cortisol. Weiss gt gt. (1971) reported significantly higher plasma 17-hydroxycorticosteroid levels in a lean strain of pigs as opposed to a fat strain. Correlations between total body protein and the corticoid levels were positive but nonsignificantly higher for the lean strain. Correlation of plasma corticoid with total chemical fat was negative for the lean strain (r = -.47) and positive for the fat strain (r = .20) although neither value was significant. Plasma free fatty acids were positively correlated to carcass fat parameters, negatively correlated to muscle and lean indicators, while no pattern was established for quality parameters. Correlations progressively increased from 56 days (all P<.O5) to 115 days to slaughter where all values were highly significant (P<.01) except for % bone of the right ham and transmission value which were significant at the .05 level. At slaughter FFA were corre- lated with backfat (r = .33), % leaf fat (r = .36), % fat cuts (r = .36) and % fat of the right ham (r = .36). Cor— relations to muscle and lean indicators at slaughter were highly significant (P<.01) for LEA (r = -.30), % ham and loin (r = -.33), % lean cuts (r = -.36), and % muscle (r = -.34). At slaughter the only quality parameter sig— nificantly correlated to FFA was transmission value (r = —.23, P<.O5). 82 Average daily gain and % bone of the right ham were the only significant correlations to glucose at weaning. Only % bone of the right ham was significantly correlated to plasma glucose at 115 days. Glucose was not signifi- cantly correlated to any parameter at slaughter. CPK at 56 days was negatively correlated (P<.O5) to average quality score (r = —.24). At 115 days CPK was negatively correlated (P<.O5) to backfat (r = -.25) and % leaf (r = -.27), and positively correlated (P<.O5) to lean of right ham (r = .23) and % muscle (r = .25). At slaughter only marbling (r = -.27) was significantly (P<.O5) to CPK levels. SUMMARY A total of 72 pigs from three lines which had been se- lected for high backfat, low backfat, and a control line were included in this study. Duroc and Yorkshire breeds and barrows and gilts were equally represented. Blood was obtained at 56 days, 115 days of age, and immediately prior to slaughter at approximately 100 kilograms. Plasma samples were stored for later analysis of insulin, cortisol, free fatty acids, glucose, and creatine phosphokinase activity. Carcasses were fabricated into wholesale cuts and all trim and wholesale cut weights were recorded. The right ham was separated into lean, fat, skin, and bone components. Insulin levels were higher for Durocs than for York- shires at slaughter. High backfat lines had higher plasma insulin at 56 days than either low or control lines. Corti- sol was higher in high backfat lines than low backfat lines at 56 days and barrows were higher than gilts. No differ- ences were detected at 115 days or at slaughter. Free fatty acids (FFA) were not different at any age period for breed and sex but were higher (P<.O5) at 115 days and at slaughter for the high backfat line than either the low or control line. Glucose levels were higher at both 56 and 115 days for the Durocs but showed no sex or line differences. 83 84 Creatine phosphokinase (CPK) was lower for the control line at slaughter only. Overall plasma values were evaluated across all three bleeding periods for breed, sex and line, in addition to evaluation within each bleeding period. Durocs showed lower levels of glucose and CPK than Yorkshires. Cortisol and FFA were both higher in the high line, lower in the low line while the control was not different from the low or high line. Insulin levels were lower at 56 days than either 115 days or at slaughter. FFA was lower at 115 days than at 56 days or slaughter. Glucose levels were lowest at slaughter, intermediate at 115 days and highest at 56 days. CPK was higher at 115 days than either 56 days or slaughter. Correlations between plasma parameters were mostly nonsignificant. Those factors which were significant showed low correlations and no trends were apparent. Dur- ing the growth period there was little change of one plasma characteristic dependent on or influenced by a change in another plasma characteristic. Carcass data was analyzed for fatness, lean yields, and quality score. Durocs were significantly fatter and lower yielding than Durocs. For most characteristics, except backfat, barrows had more fat trim and less lean yield than gilts. The low backfat line yielded the least fat trim and the highest yield of lean, the control was intermediate, and the high backfat line was highest in fat yield and low- est in lean yield. Marbling was higher in Durocs than 85 Yorkshires and also higher for barrows than gilts. The high backfat line had higher color scores than either the low or control backfat lines. No significant color or transmission value differences occurred between breed, sex, or line. Correlation coefficients between carcass parameters indicated backfat was highly positively correlated to all fat trim parameters and highly negatively related to lean parameters. Similarly, fat yield parameters were negatively correlated to lean indices and positively related to each other. Marbling, color and firmness scores were negatively correlated with lean indices and positively correlated with backfat and other fat indices. Transmission value was positively correlated (r = .23) with backfat, color and firmness (r = .58). Relationships between carcass and blood plasma traits were also determined. Insulin and cortisol at 56 days were highly significant (P<.01) and negatively correlated to carcass yield (r = -.19 for % muscle). At 115 days and slaughter, correlations were low and nonsignificant. Insu- lin was not significantly correlated to cutability or qual- ity factors at any age and cortisol at 56 and 115 days was correlated (P<.O5) only to marbling score. FFA were gen- erally significantly (P<.O5) correlated to carcass yield at 56 days of age and by slaughter the correlations were highly significant (P<.01). Marbling and average quality corre- lated to plasma FFA at 115 days. 86 Glucose showed no correlation to cutout data or car- cass quality scores. A correlation of r = —.32 (P<.01) was, however, noted between ADG and glucose at weaning. CPK values were correlated to backfat (r = -.25, P<.O5) and % muscle (r = .25, P<.O5) at 115 days. BIBLIOGRAPHY BIBLIOGRAPHY Aberle, E. D., R. A. Merkel, J. C. Forrest, and C. W. Alliston. 1974. Physiological responses of stress susceptible and stress resistant pigs to heat stress. J. Anim. Sci. 38:954. Abraham, R. E., E. Dade, J. Elliot, and D. A. Hems. 1971. Hormonal control of intermediary metabolism in obese hyperglycemic mice. II. 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Fatty, a new muta- tion in the rat. J. Hered. 52:275. APPENDICES APPENDIX I REAGENTS A. Reagents for Insulin Radioimmunassay 1. Buffer A1 NaHgPOh'ZHZO ............................. 6.2 g Merthiolate (Thimersal) .................. 0.25 g Bovine Serum Albumin (Albumin, Bovine, 35% Sterile Solution, Sigma Chemical Co., St. Louis, Missouri) ............. 14.6 ml Distilled H20 ............................ 950 ml Adjust pH to 7.5 with 5 N NaOH Bring final volume to 1 liter Store at 4 0 (up to 3 months) Buffer B1 NaCl ..................................... 9.0 g Dissolve with 1 liter Buffer A1 Store at 4 0 (up to 3 months) Insulin Standards Rinse a small screw cap vial with Buffer B1 and dry Weigh 200-500 pg of insulin on a Cahn Electro— balance and transfer to the screw cap vial Add saline solution (0.85%, pH 5.0) for a 1 mg/ml solution Add sufficient Buffer B1 for a 500 mg/ml solution Transfer appropriate amount of 500 mg/ml solution to 100 ml volumetric flasks containing Buffer B1 to attain the working standard concentrations of: .06, .08, .1, .2, .3, .4, .6, .8, 1.0, 1.5, 2.0, 2.5, 4.0, 5.0, and 10.0 ng/100 pl Multiply each standard concentration times 23.9 pU/mg and express serum values in pU/ml Dispense each standard in 2 ml quantities (enough for one assay) Freeze and store at -20 C 97 4. 98 0.01M Phosphate Buffered Saline (PBS), pH 7.0 NaCl ..................................... 143 g Merthiolate (Thimersal) .................. 1.75 g NaH2P04 (69.005 g diluted to 1 liter in distilled water) ...................... 120 ml NaZHOPu (70.98 g diluted to 1 liter in distilled water) ...................... 240 ml Dilute to 17.5 liters With distilled water Store at 4 C Adjust pH to 7.0 before using 0.05M Disodium Ethylenediamine Tetraacetate (0.05 M EDTA-PBS) Disodium EDTA ............................ 18.612 g Add 0.01 M PBS ........................... 950 ml Adjust pH to 7.0 with 5 N NaOH Bring final volume to 1 liter Store at 4 0 Normal Guinea Pig Serum (NGPS) Dilute serum 1:400 in 0.05 M EDTA—PBS Freeze and store at —20 C Guinea Pig Anti-Porcine Insulin Serum (GPAPI) Dilute serum 1:400 in 0.05 EDTA-PBS Dispense in small quantities, store at -20 C On day of use, dilute further in 1:400 NGPS for a final dilution of 1:100,000 125I-Insulin Dilute 125I-Insulin to activity of 15,000 cpm using Buffer A1 as diluent Sheep Anti—Guinea Pig Gamma Globulin (SAGPGG) Dilute serum 1+25 on day of use (one part SAGPGG serum plus 25 parts 0.05 M EDTA-PBS) Reagents for Corticoid Determination 1. 1,2,6,7-3H Cortisol Set up three disposable glass syringes (2.5 cc) as columns Place a 2.1 cm disc of filter paper in the bottom (out from Whatman G-F/A glass fiber papers) 99 Wash the sides of the column and the filter paper with 90:10 benzene-methanol (Nanograde or glass distilled) Pour a column to a height of 3.2 cm using Sepha- dex LH-20 (Pharmia Fine Chemicals, Uppsala, Sweden) equilibrated at least 12 hours with 90:10 benzene-methanol Place another disc of filter paper on top of the column and wet with solvent Allow the solvent to drain off Apply 50 pl 1,2,6,7—3H-Hydrocortisone (New England Nuclear, Boston, Massachusetts) to each of the three columns Apply 0.5 ml solvent to each column and collect the fraction in a 12x75 mm disposable culture tube (Scientific Products) until the column stops dripping Repeat the above steps for a total of 12 fractions per column Take a 10 pl aliquot from each fraction collected and count for 1 minute to determine the fraction with most radioactivity (usually fractions 8, 9,10) using 5.0 ml preblended scintillation fluid (3a 70B, Research Products International Corporation, Elk Grove Village, Illinois) Combine the contents of the three most radioactive tubes from each column into a single tube and dry off the solvent Add 1.0 ml 0.1% Knox Gelatin-PBS (1.0 g Knox into 1 liter PBS) at pH 7.0 Vortex at least 1 minute Take three 10 pl aliquots, add 5.0 ml scintillation fluid and count for 0.1 minute All three aliquots should have similar counts, if not, vortex again and repeat the above step Dilute the cortisol in 2.0% dog serum (see below) for an activity of 12,000 cpm/ml (stable for up to one month at 4 C) 2.0% Dog Serum Dog serum (Grand Island Biological Co.) was diluted to 5.0% (1:20) with distilled water Vortex for 3 hours with 45 g Florisil (80 mesh; Matheson Coleman and Bell) per 400 ml diluted dog serum Centrifuge at 2500 x g for 15 minutes Add distilled water to give a final dog serum con- centration of 2.0% Use immediately or store frozen at -20 C 100 Tracer Cortisol Follow procedure as outlined in Appendix I. B. 1. with the exception of the last step Instead of diluting in 2.0% dog serum, dilute the 1,2,6,7-3H-Cortisol to 2,000 cpm/10 pl using methanol Store at 4 C Dextran Coated Charcoal Wash neutral norit with distilled water, then with methanol and oven dry Washed neutral norit (Fischer Scientific Co.) .................................. 0.50 g Dextran T 70 (Pharmia Find Chemicals, Uppsala, Sweden) ...................... 0.100 g Glass distilled water .................... 100 ml Mix and store at 4 C for at least 12 hours before use C. Reagents for Nonesterified Fatty Acids 1. Dole Extraction Mixture Isopropanol .............................. 400 ml Heptane .................................. 100 ml H2804 (1.0 N) ............................ 40 ml 63N1 Solution NiC12'6H O ............................... 0.4050 g Add 1.0 M triethanotamine to make 100 ml Add 63N1 to give 10 cpm/100 pl reagent Scintillation Fluid Napthalene ............................... 160 g PPO (2,5 diphenyloxazole) ................ 10 g Dimethyl POPOP (1,4-Bis-2-(r-methyl-5— phenyloxazolyl)-benzene) .............. 0.1 g Zylene ................................... 770 ml P-dioxane (1,4 Dioxane) .................. 770 ml Absolute ethanol ......................... 460 ml Standard Solution Dissolve 0.0256 g palmitic acid in absolute ethanol to a final volume of 100 ml (10-3 M) Take 10 ml of 10-3 M solution and bring to a f'nal volume 0 100 ml with absolute ethanol (10' M) Use the 10‘ M solution as the standard stock solu- tion D. 101 Glucose Reagents 1. Standard Solutions Dissolve appropriate amount of glucose in 100 ml distilled water to give the following mg% solutions: 50mg% ............................. 50 100mg% ............................. 100 200mg% ............................. 200 300mg% ............................. 300 500mg% ............................. 500 GOD-PERID Enzyme Dissolve one bottle Cat. No. 15756 in distilled water and bring final volume to 1000 ml Store up to 6 weeks at 4 C mg mg mg mg mg NUMB ER, APPENDIX II-A CODE AND DEFINITION OF VARIABLES USED IN RAW DATA Number Code \0 CD \7 O\ \J‘k-P WNH N NHHH HHH l—‘ H r—A H H O\OCI)\) GUI-(3' \0 N H O N N PIG BREE LINE SEX INS INS INS COR COR COR FFA FFA FFA GLU GLU GLU CPK CPK' CPK TNS BF10 NOIN NO D WEAN MPG SLAU WEAN MPG SLAU WEAN MPG SLAU WEAN MPG SLAU WEAN MPG SLAU VAL GRP Definition Individual pig identification number 1 = Duroc; 2 = Yorkshire 1 = High Backfat; 2 = Low Backfat; 3 = Control 2 = Gilt; 3 = Barrow Plasma insulin concentration at 56 days (ms/ml) Plasma insulin concentration at 115 days (ms/ml) Plasma insulin concentration at slaughter (ns/ml) Plasma cortisol concentration at 56 days (ns/ml) Plasma cortisol concentration at 115 days (ms/ml) Plasma cortisol concentration at slaughter (ms/ml) ‘ Plasma fatty free acid level at 56 days (nmoles/ml) Plasma free fatty acid level at 115 days (nmoles/ml) Plasma free fatty acid level at slaughter (nmoles/ml) Plasma glucose level at 56 days (mg/100ml) Plasma glucose level at 115 days (mg/100ml) Plasma glucose level at slaughter (mg/100ml) Plasma CPK at 56 days (Sigma units) Plasma CPK at 115 days (Sigma units) Plasma CPK at slaughter (Sigma units) Muscle transmission value (Recorded as 0D; report as %T) ' Backfat at 10th rib; transformed from average 3pt backfat (cm) Number in group; identifies pigs of same breed, line, and sex as 1, 2, 3, 4, 5, and 6 for computer coding purposes (continued) 102 103 APPENDIX II—A (Continued) Number Code Definition 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 ON TEST OFF TEST ADG SLAU WT HCARCWT CCARCWT HD WT LEAF FAT RT HAM LFT HAM BELLY WT LOIN WT TOT BFWT HAM LEAN LEANCUTS FAT CUTS HAM FAT -HAM BONE HAM SKIN CARC LEN BF 3PT BF 5PT BF10 RIB LEA COL FIRM MARB AVE QUAL Weight of pig at start of feeding period (lb at 56 days of age) Off test weight of pigs (lb) Average daily gain while on test (lb/day) Slaughter weight of pigs (1b) Hot carcass weight (lb) Chilled carcass weight (lb) Head weight (lb) Leaf fat (lb) Weight of right ham, untrimmed (lb) Weight of left ham, untrimmed (lb) Belly weight (lb) Loin weight (lb) Total backfat weight (lb) Weight of lean as separated from the right ham (lb) Weight of lean cuts: ham, loin, Boston butt, and picnic shoulder (lb) Weight of fat cuts: leaf fat plate, total backfat trim, and other fat trim (lb) Weight of fat trimmed from right ham (lb) Weight of bone in right ham (lb) Weight of skin from right ham (lb) Carcass length (cm) Average backfat of measures over first rib, last rib, and last lumbar verte- bra (in) Average backfat of measure over first rib, seventh rib, last rib, mid lumbar, and last lumbar (cm) Extrapolated backfat over the longissimus muscle at the tenth rib in) Longissimus muscle area (in ) Color and firmness score (Wisconsin system) Marbling score (Wisconsin system) Average quality score; mean value of 47 and 48 104 moo. om.ss os.om oo.om o.oo m.mo o.so o.oo o.os o.oo os.om oo.sm oo.o so.o oo.o moo. sm.oo os.mm so.oo m.so o.oo o.omm o.omm o.os o.os mo.mm oo.om oo.o oo.mm om.m ooo. os.os oo.oo oo.oom m.ms o.ss o.oo s.oo s.so o.oo mo.mm oo.o sm.o mo.m mo.m mom. oo.oo oo.omm oo.mo o.ss o.smm o.oom m.mo o.mom o.oom oo.om mo.om os.om oo.o oo.o ooo. oo.oo sm.oom oo.s o.so o.so m.mom o.os s.oom m.omm om.sm mm.o om.om mo.mm oo.o osm. oo.ms mo.so oo.oom s.mo o.oo s.oom o.ss o.so o.oom os.om om.mm so.sm oo.s os.m oms. ss.mo om.oo ms.om o.oo s.os s.oom o.oo o.os m.oo ms.om sm.om oo.oo sm.m om.om ooo. oo.som os.om mo.om s.os o.oo o.omm o.mo s.os o.omm mo.sm ms.o ss.om oo.o oo.o ooo. oo.om sm.os so.omm o.os s.oo o.smm o.os o.so o.os mo.om ss.mm oo.o os.o so.s oso. oo.oo mm.mo so.mm s.mo o.omm o.smm s.oom o.oo o.ms mm.om om.sm om.mm oo.m so.om ooo. oo.os oo.mo mo.om o.ss o.os s.som m.os s.om s.oo mo.om om.mm oo.mm mo.s oo.o mss. oo.oo os.oom oo.oom o.ss o.oo o.oom o.oo m.oo o.mo oo.s mm.mm om.sm om.m oo.s omo. oo.so mo.mo oo.mom o.so o.osm o.osm o.so o.oo s.omm oo.om sm.mm so.om sm.o om.s omo. ms.omm oo.omo os.mo o.oom o.omm o.omm o.os o.oo o.os No.mm mo.mm oo.sm oo.o so.m moo. mo.oom mo.so os.so o.oo o.so o.omm o.oo o.oo o.oo om.mm oo.mm oo.om sm.o oo.o oos. oo.os oo.ss so.so s.mo o.som s.mmm o.os o.oo o.oo oo.om os.om ss.sm oo.o oo.o omo. os.ss mm.ooo so.mmm o.oo o.smm m.oom o.oo o.oo o.oo om.om oo.mm oo.o oo.o oo.o moo. sm.osm ms.oo os.mm o.oo o.omm m.mmm o.oo m.ms m.mo os.oo oo.mm om.o om.o os.s moo. oo.som mo.so oo.som s.mm m.mom s.omm m.oo o.oo o.oom ms.o sm.om oo.sm oo.o oo.s oos. oo.omo om.mo mo.mom o.omm s.mmm o.mmm o.ss o.os o.os so.om om.sm os.om oo.o os.o oos. oo.om oo.mmm os.sm o.so s.mom o.smm o.oo s.os o.oo oo.sm sm.om om.mo mm.m .om.m om om om sm - 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NN.m mo.o s.No N.os os.s o.om o.NN o.NN o.om o.sm o.o o.om o.som o.msm ooN os.m o.oo oN. os. oo.o s.so o.oo oo.o o.oo N.om o.oN s.mm o.mm o.o o.om o.ssm o.Nom NmN oo.m o.oo oN. so. os.o o.oo m.oo oo.s o.oN o.om o.oN o.om s.Nm o.s o.Nm o.osm o.som omN oN.m s.os oo. oo.m so.s o.ss o.oo mo.o s.o s.sN s.om s.mm s.om o.o o.om o.oom o.sse smN oo.m Nsmsos oo oo s-oloolNNIsDoo No mooMoN oN sMoNnN-sw- Honsmz 0H:0HH0> oN N m oH N H sN o m sN N o oo o o mN N N oN o N sN N m oN o m so N N oN N m sN o m oN N o oN o o oo N o os o o oo N o so o o so N m oo o m so o N ownmww m m m N N N N m m m m m m m m m N N m m m mm oosN smsN mosN ONsN omsN oosN NosN oooN oooN sNoN omoN NmoN soNN soNN ssNN msNN oNNN oNNN omNN HHNN NsHN H A0058quoov m.- HH NHQEanHe. 110 s.ss oo. oo.m mm.s o.ss o.mo mo.o o.o o.sN m.om m.om o.om o.o o.om o.oom o.ssm NmN oo.m NNN o.ss oo. mo.m om.s Nsss s.so oo.o o.o s.oN m.om N.om N.om s.s o.sm o.som o.oom ooN oo.m omN o.Ns so. mo.m ss.o s.so o.oo ms.o o.sm o.oN o.om s.om m.om o.o o.sm o.osm o.osm omN.so.m HNN s.os oN. no” oo.o s.os m.os sm.s o.oN m.oN s.NN N.om o.sm o.s o.Nm o.osm o.oom omN No.m smN o.os oN. oo.m oo.o o.os o.os os.o o.sN s.oN m.oN o.sm m.sm o.s o.mm o.ssm o.mmm mmN sN.m HNN s.ss oo. oo.m ss.o o.oo o.oom oo.Nm o.o N.No o.om s.mm m.om o.o o.om o.Nsm o.osm omN Nm.m omN o.os oo. ss.m oo.s o.os o.oo oo.mm o.s o.mo o.oN o.sm o.sm o.s o.sm o.Nsm o.osm smN sN.m oNN s.os oo. Ns.m No.s N.mo o.oo oo.o o.om syoN s.oN o.om s.om o.s o.om o.oom o.msm ooN os.m omN s.os No. Ns.m oN.o s.os N.No mN.o o.sm N.sN s.oN o.om N.om o.s o.sm o.msm o.osm omN os.m oNN s.os os. so.m so.o m.so o.so Nm.mm o.o o.oo m.sm o.sm o.sm s.s o.om o.oom o.osm smN so.m oNN s.ms oN. mo. sm.o o.os o.os No.s o.mN o.sN s.NN o.sm o.om o.o o.om o.ssm o.osm omN om.m omN s.os sN. oo.m so.o s.os m.os om.s o.sN N.oN o.sN N.sm o.sm o.o o.Nm o.ssm o.mom omN om.m omN o.os oo. ms.m os.o o.mo o.oo os.o s.om o.oN m.oN s.om o.om m.s o.om o.osm o.Nom omN oo.m oNN N.ss oo. oo.m oo.m N.mo o.oo No.o. o.om o.oN o.mN o.om m.om o.s o.sm o.osm o.osm NmN sN.m oNN N.ss oo. oo.m No.o o.oo o.som oo.Nm N.o N.oo o.om o.om s.mm o.N o.om o.Nsm o.omm smN so.m oNN o.os oo. mo.m mm.s s.so o.Nom os.mm o.s o.oo o.om o.mm o.sm o.o o.sm o.osm o.mom omN om.m sNN o.os oo. oo.m oo.o o.so N.som oo.Nm o.o o.oo o.om s.om o.os s.N o.om o.osm o.oom omN oN.m oNN o.Ns oN. oo.m oo.s m.os o.oo ss.om m.mm s.oo N.oN.s.om N.om o.o o.om o.msm o.osm mmN so.m smN o.os oo. os.m oo.o o.oo s.so oo.om o.o N.oN N.sm N.sm s.om o.o o.om o.som o.ssm omN oo.m oNN N.os oo. mo.m sN.o o.No o.mo oo.om N.o o.oN o.om s.om o.om o.o o.om o.som o.som ooN oo.m omN o.so oN. oo. oo.o N.oo N.oo mo.o o.oN o.om o.sN o.Nm o.Nm o.o o.Nm o.ssm o.Nom NHN so.m oNN Nsmsosoo oo so oo oosooo No mooooN N N 8 Hopssz 0H:0Hmd> sN o mm o no on mN N mm so so mo oN oo oN oN mo so oo oN oo oo oo N MMNMNNMMNNMNMNMN H H H N N N N N N H N N N N N N N N H H H NmoN mmoN oooN ome MNMN oooN nomN mmmN HmmN msmN ommN mmmN oNnN anN oonN NooN NosN NosN oNoN mosN NosN m A0058Hp800v ml HH Nannmmme. 111 s.ms sm. oo. No.o s.os o.ss mm.s s.sN o.oN m.oN o.sm o.sm o.s o.mH o.osm o.mom omN sm.m oNN sN N m N ossN s.ms mN. oo. oN.o s.os s.os oo.o N.oN s.sN o.NN o.sm s.sm o.s o.om o.mmm o.oom omN oo.m sNN oN o m N NssN o.ms oN. oo. Ns.o m.os N.os No.o o.sN o.NN o.sN s.sm o.om o.s o.mm o.osm o.oom omN om.m sNN No N m N omsN o.os mm. oo. os.o m.os o.os oo.o s.oN N.oN o.sN o.om N.om o.o o.Nm o.osm o.ssm omN os.m NNN so o m N mmsN s.ss No. oo.mos.s o.os o.oo oo.o o.o o.oN m.om s.om o.om N.s o.sm o.som o.ssm mmN so.m omN oo o N m sosN o.os oo. oo.s so.o N.mo o.so .oo.s o.sm m.oN o.om s.sm o.sm o.o o.om o.oom o.msm ooN mo.m smN so o o m oooN N.os ss. oo.m Ns.s o.os s.oo oo.o o.om o.oN o.sm o.om o.om o.s o.om o.som o.oom NmN Ns.m omN so N o m oooN o.os oo. os.H oo.o m.so o.oo os.s o.sm s.oN o.om o.om o.om o.o o.om o.oom o.ssm NmN oo.s omN oo o o H osoN o.os oo. so.m oo.o m.oo o.No NN.om s.s s.oN m.sm o.om m.om s.o o.om o.oom o.oom ooN oo.m omN No N N m omoN N: A“ 0: on mm sm on mm mm mm mm Hm -o|mu 0N N N N N 3N MNdMN H H0983: 0H90HH0> Aeoofimsoov m- mm mmmemmme 112 o.N N o oo.s oo.m oN.o ss.o N No.o N N m osmN o.o N s ms.o so. oN.N oN.N m ss.m N N m somN o.o N s so.s os. ss.N so.N m mo.N o N m NomN o.s o o oo.N so.o NN.s oo.s m No.om N m m mmmN o.s o o os.N sm.s No.s so.s m oo.om o o m NmmN o.N N N os.o oo.N NN.o sN.o N oo.o N o m oooN o.N o N oo.s oN.N oo.o os.s N oo.o o o m mooN o.N N o so.o sm.s oN.s so.s N oo.om N m N osoN o.o N s os.o oo.o oo.o oo.o N Ho.s o m N msoN o.o o o mo.o so.o ss.o so.o m sN.o N m N oooN o.N N o oN.o oo.o oN.s os.s m om.om o m N oooN o.N m o oo.o oo. Nm.N so.N N so.m N N N oooN o.N N o so.o om.m oo.N sm.o N No.o o N N oooN o.N N o oo.o oo. oo.N so.N m so.m N N N osoN o.o N s so.s oN.m oo.N oN.o m Nm.o o N N NsoN o.N N N mo.s mo.m oo.o oo.o N oo.s N o N NooN o.o N s om.o No.m om.s sN.s N so.s o o N mooN o.N N o oo.o NN.N No.s os.s H so.o N o m oNoN o.o o s No.s sm.o No.o sm.o m oo.o o o H NNoN o.N N o oo.o oo.m sm.s sN.s m so.s N o N smoN o.N N o Nm.o oN.N oo.s os.s m oo.o o o N omoN Nflmmmm os os ss os NNN-oNNN H0983: 0H90HHO> Hoossmmsoov m- Hm xmmzmmme 113 o.o o.N o.o o.N o.N o.N o.o o.s o.s o.N o.N o.s o.o o.o o.N o.o o.N o.o o.o o.o o.o NI...- «3 .s lMJQNHMNMMWNdeNNHN-S'Nm es _sler co co :r :r 0\ ex ex s» «N 0\ ex am a: e: aw c\ cw cw 0a 0\ ms.N os.s om.m ss.o mo.N mo.o os.o os.o sN.N oo.s No.0 os.N ss.o mo.m Ns.o oo.o No.o om.H so.s oN.s oo.o oo.H oN.N so.N oo.o sm.m No.N om.o so.N so.o so.s sm.s oo.N No.s oo.o oN.o oo.o oN.m sm.o oo.o oo.N oo.s oo.s oo.o om.N sm.s so.s oo.s oN.o ss.N os.o so.o oN.s oN.N so.s ss.s oN.s sN.N oo.s oo.s NN.s oN.N os.s oo.s om.s oo.N No.s oo.s NH.o No.N ss.s so.s ms.N sm.s oo.s so.s oo.m os.s oo.s so.o oo.s mN.m sm.o oN.o ososssos.NlN- NNNMMMM33MMMd’d'MM-fl'fimmm H09ssz 0H90HH0> mo.HH N oo.:H Nw.HH 00.: 00.0 No.N oo.N no.0 md.HH sN.o nw.HH Nm.oH HN.0 Hs.o os.o Hm.o wo.o oo.o oo.oH HN m oosN sosN mosN oNsN mHsN oosN NosN oooN oooN sNoN oHoN NmoN soNN soNN ssNN msNN oNNN oNNN omNN HHNN NsmN m A00smH9000v mIHH xHszmm< 114 o.o o s oo.s oo.m NN.o oo.o o oN.o o N NmoN o.s s s os.s ssm No.o oo.o s ss.o oN mmoN o.N N o No.s os.N os.s om.o o mo.o N o oooN o.o o o oo.o oo.o oo.o os.o s mo.o N m oooN o.N o m os.o so.o Nm.s so.s s oo.om o m oooN o.N m s mN.o oo.m os.N oo.o o os.N N N oooN o.o N s ss.o HN.m oo.N oN.o o so.o o N oooN o.o N s os.o No.m No.s om.s o oo.s N o oooN o.o N s oo.o No.m om.s sN.s o so.s o o mooN o.o o o oN.o oN.H so.o oo.o o sN.o N N osoN o.N N N so.o oo.o oo.o oN.s o oo.o N m oooN o.o o o No.o Ns.o No.o oo.s o os.o o m oooN o.N N N so.o so.N oo.s oo.s o oN.o o o oNoN o.N N o os.s Ns.m so.o so.o o oo.s N o smoN o.o N s so.o mo. No.N oo.N o oo.N N N oooN o.N m o sm.o Nm.m oo.N so.o o oo.N .o N NooN o.N N o os.o oo. ss.N so.N s om.N N N oosN o.N N o oo.o ms.m os.o oo.o s so.o o N NosN o.o o o No.o oN.m Nm.o sN.o o om.o o N oosN o.o N s os.o oo. so.N so.N s om.N N N mosN o.o o o os.N so.s ss.s so.s o oo.mm o m NosN N.os-N18 0s es os NMnlm-leormll H0952 mdpmflfidmw soosemmeoov mm.HH Hmmzmmme 115 O m mmV-INNNNNN MMJMfiMNNM oo.o os.o oo.o om.s o No.o oo.o No.s sN.s o oo.o ms.s oo.s oo.o o mo.N mo.s om.s os.s o oo.s os.m so.o so.o o oo.s oo.N os.s oo.s o oo.s os.m oo.o oo.s o oo.o om.N oo.s oo.s o so.o oo.m oo.N oo.o o H09sdz 0H90HH0> A0058H9:00v mIHH XHszmm< mmlfillilillll“111willrllllilllllllllllillm 3 1293 03046 9880