\A .I . . . . . l|.v..‘:, . . , . . Y .. \\ WW L l l ... m llBRARY Michigan State AUniversity This is to certify that the dissertation entitled INFLUENCE OF NEGATIVE ENERGY BALANCE AND BODY CONDITION ON LUTEAL FUNCTION AND ESTROUS BEHAVIOR IN DAIRY CATTLE presented by Alejandro Villa-Godoy has been accepted towards fulfillment of the requirements for Ph.D. degreein Animal Science f/W Ma' professor Date /3 / yW MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. in: I I it“; ,1 1-“ Slfl L: '----C WNWW L LIBRARY 3 1293 00 Mid‘igcm State I University This is to certify that the dissertation entitled INFLUENCE OF NEGATIVE ENERGY BALANCE AND BODY CONDITION ON LUTEAL FUNCTION AND ESTROUS BEHAVIOR IN DAIRY CATTLE presented by Alejandro Villa-Godoy has been accepted towards fulfillment of the requirements for Ph.D. degreein Animal Science f/W Majprfimfmor Date /3 / y” usabmmma'w Action/Equal Opportunity Institution 0-12771 IVIESch RETURNING MATERIALS: Place in book drop to LIBRARJES remove this checkout from w your Y‘ECOY‘d. FINES WT” be charged if book is returned after the date stamped below. mo! 0 "Liar-fl v3 1 ': .. _, l.‘ *I‘}:\ I A -« ‘3 '._‘ l J ‘ ‘ ' 3.! V 4151. .'§" Pig aria-I Q E .Lfl: -- Eur (1 23-2.? 0 4:13..-.F" QJQNV ‘ & I25K) *‘.§Q;*¢f ”11“-,"r 1‘ y ‘1 - 5:99 1 TI ABSTRACT INFLUENCE OF NEGATIVE ENERGY BALANCE AND BODY CONDITION ON LUTEAL FUNCTION AND ESTROUS BEHAVIOR IN DAIRY CATTLE By Alejandro Villa-Godoy Three studies were conducted to determine independent and associative effects of energy balance (EB) and body condition (BC) on luteal function and behavior at estrus .of dairy cattle. In experiment I, 32 Holstein cows were studied from parturition to day 100 postpartum or conception, whichever occurred first. Energy balance was estimated daily (energy intake-energy required for maintenance and lactation) and luteal function was monitored by quantifying progesterone in milk sampled every third day. Cows were fed 29 libitum but, within 100 days postpartum, 8196 of the cows experienced negative EB (NEB). Most variation in BB was explained by intake of dietary energy (r = .7). Energy balance and progesterone in milk were correlated positively. Thus, NEB may reduce luteal function in lactating dairy cows. In Experiments II and III, 20 postpubertal Holstein heifers were used in a 2 x 2 factorial experiment. Main effects were: 1) EB, positive or negative and 2) BC, moderate (MOD) or fat (FAT). Heifers were studied for 3.5 estrous cycles. Energy balance (energy intake-energy required for maintenance) was calculated daily, BC was scored (1 to 4; 4 = fat) every two weeks and luteal function was monitored _i_r_1_ XI!!! by progesterone in serum sampled daily. Corpora lutea were removed from heifers between days 10 and 12 of the last estrous cycle for in vitro studies. Alejandro Villa-Godoy In Experiment II, negative EB and FAT reduced luteal function in .912.- Adverse effects of NEB were observed as early as the second estrous cycle in MOD heifers, but were not detected until the fourth estrous cycle in FAT heifers. Adverse effects of NEB on luteal function were delayed until BC of heifers declined below MOD. Concentrations of luteinizing hormone (LH) in serum were not altered by N EB or FAT. Thus, NEB and FAT do not limit luteal function by reducing luteotropic support. Negative EB but not FAT reduced LH-induced secretion of progesterone by luteal cells in ii}!!!- In Experiment III, to monitor estrous behavior, heifers were observed for periods of 30 minutes at intervals of 3 hours. Energy balance and BC independently or combined did not reduce duration or intensity of standing and mounting behavior. I conclude that EB and BC do not reduce expression and detectability of estrus in heifers. But, FAT reduces luteal function in heifers and N EB exerts limitations on luteal function of heifers and lactating dairy cows. Apparently, effects of NEB and FAT are exerted through different mechanisms. Moreover, adverse influence of NEB on luteal function of heifers was not detected until BC declined below MOD. Esta disertacio’n fue lograda en gran parte gracias al apoyo y carifio que mi esposa, Maria Luisa Parkman de Villa, me ha brindado constantemente. Dedico esta tesis a Ella y a mi hija Lorena, quienes lograron que una dura labor se transformara en la mejor experiencia de mi Vida. ii ACKNOWLEDGMENTS For many years I was a recipient of American generosity. I thank all Americans in this department for making me feel at home. I realize that "mi casa es tu casa" has the same meaning in this great country as in my own. To my major professor Dr. Roy L. Fogwell, my deepest gratitude for his friendship and inspiring guidance. What I learned from him is invaluable and the closest word which describe what I feel is: gracias! To the members of my guidance committee: Drs. R.S. Emery, J.J. Ireland and D.R. Romsos, I express my appreciation fortheir suggestions which, without doubt, improved my research and this dissertation. I will be always thankful to Dr. H. Allen Tucker, who with his constant . example through lectures, seminars and conduction of experiments, unveiled the true meaning of "research". To Larry T. Chapin, my sincere appreciation for his infinite patience and willingness to teach me the complexities of computer language. I am indebted to Mrs. Marnie Laurion for the accurate and fast typing of this dissertation but especially for translating into English some of my "Spanglish" expressions. I thank Trudy L. Hughes for her collaboration during most phases of my research and for making possible (with her expertise) the in 3%; part of this dissertation. I express my gratitude to my friends William J. Enright, Edward P. Stanisiewski and Steven A. Zinn who made themselves available whenever I needed help. Certainly I will miss them all. iii I am especially grateful to the department chairperson, Dr. Maynard Hogberg, who made this dissertation a reality by providing economic support during the last part of my program. I am also indebted to CONACYT and INIFAP for granting scholarships during the first years of my studies. Finally, my deepest gratitude to my mother Emma Godoy de Villa, my mother-in-law Isabel A. de Parkman and all my sisters and brothers (Villa-Godoy and Parkman-Arevalo) for their continuous support, understanding and love. iv TABLE OF CONTENTS DEDICATION000000000000 OOOOOOOOOOOOOOO 0 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO ACKNOWLEDGMENTS .......... ......................... ..... . ........... LIST OF TABLES ......... 0.0000 ......... 0 ..... 0 .......... 0 OOOOOOOOOOOOOOOOOOOOOOOOOOOO LIST OF FIGURESOOO. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000 000000000000 INTRODUCTION ........................... ...... . ................. . ................ ' REVIEW OF LITERATURE ......................................................... IntrOduction0. ..... 00.00000.0...00.000.000.000...00.0000000 ..... 0.00.0.0... 000000 Energy Metabolism in Cattle........ .............. ReQUirementS Of Energy00000. 000000 0 000000000000 0.00.00.00.00 000000 Energy balance0000000 0000000 0000000000000...0000.00.00.0000000000000000 Sources of Variation of Energy Balance................ ...... Homeorhetic Control of Growth and Lactation ..... . .............. Insu1in in cattleOO00000000000000000000000 00000000000000000 0 00000 00000 Secretory Patterns of Insulin in Cattle ............... Energy Balance and Insulin. .......... Components of Energy Balance and Insulin ......... Body Condition and Insulin...... ....... ...... Growth Hormone (GH) in Cattle ........ . ....... Secretory Patterns of Growth Hormone............. Energy Balance and Growth Hormone................ Components of Energy Balance and Growth Hormone... ........ ......... Non Esterified Fatty Acids (NEFA) in Cattle ........... Energy Balance and NEFA....... .................... Components of Energy Balance, Body Condition and NEFA Luteal Function in Cattle.......... ..................... ...... Corpora Lutea of Estrous Cycles ....................... Control of Development and Maintenance of Corpora Lutea ............................. . ................ ii iii ix 10 12 12 13 14 16 16 16 17 18 19 20 20 22 22 22 Effects of Components of Energy Balance on Luteal Function. ................ . ........... . ............. Restricted Intake of Energy .......... .......... Changes in Body Weight .................................... Body Condition........... ........... . ..... .......... Yield of Milk ......................... . ......................... Insulin, GH and Non Esterified Fatty Acids on Luteal Function........... ........... .................. Insulin... ....... . .............. . ............................ . ...... NEFA .............. ........... Estrous Behavior in Cattle ............. .................... Expression of Estrus ......................................... Factors Affecting Estrous Behavior ..... ........... Hormonal Control of Estrus summary.0.00.000...0000000000000000000000000000.0000...0000.00.00.000000 EXPERIMENT I Association Between Energy Balance and Luteal Function in Lactating Dairy Cows ..................... Introduction ..... .......... ..... . .............. . ..... ....... Materials and Methods ...................................................... General.......................... ..... . ........ ..... Milk Yield Body Weight and Energy Balance (EB)........ Luteal Function and Other Reproductive Measures ...... Assimilation of Data and Statistical Analyses ............. Results and Discussion.... ................ ....... . ........ . .............. Yield of Milk, Body Weight and EB ........... . ........ . ........ Factors Associated with Variation in EB ..................... Factors Associated with Luteal Function .......... . ......... EXPERIMENT 11 Influence of Energy Balance and Body Condition on Luteal Function in Heifers......... ...... ...... Introduction... ..... ....... ....... ................. Materials and MethOdS 00000 0000000000000000000000000000.00000000000000000000 Design and General Procedures ................ Body Weight and Body Condition ................................ vi 24 24 26 26 27 28 28 30 31 31 32 33 35 38 39 40 40 40 42 43 46 46 49 51 62 63 64 64 66 Energy Balance............... ..... . ................................... Luteal Function in 1112 ...... ..... .......... Luteal Function in mm ....... . ......... . .................. Detection of Estrus............ ................. ....... LH, GH, Insulin and NEFA........... .......... ........... Assays ....... Statistical Analyses..................... ........ .......... Results .......... Energy Balance and Body Measurements........... ..... ' ..... Luteal Function _i_r_1_ m Luteal Function _i_r_1_ mtg Luteotropic Support GH, Insulin and NEFA ....... DiSCUSSion000000000000 00000000 00000000000000.00.0...000..000000.00.00.00.00.000... EXPERIMENT III Influence of Energy Balance and Body Condition on Behavior of Heifers During Estru80000000000000000.0.0.00....0.00.0.0...0.0.0.0....000. Introduction ...... Materials and Methods Progesterone in Serum Estrous Behavior.......................... ........... . ....... .. ....... Duration of Estrous Cycles.... .......... . ......................... Ambient Temperature ..... ......... ...... ...... Statistiscal Analyses ......... . ...... . ...... .......... Results and Discussion......................................... ............. GENERAL DISCUSSION“... ...... ......... SUMMARY AND CONCLUSIONS. ..................... ...... APPENDIX A Validation of a Solid Phase Radioimmunoassay Developed to Quantify Progesterone in Milk....... vii 67 67 67 68 68 69 70 72 72 75 79 84 84 90 98 99 100 102 102 102 105 105 106 118 123 125 APPENDIX B Validation of a Homologous Radioimmunoassay Developed to Quantify Bovine Insan in Serum... 134 LIST OF REFERENCES ............ ...... . ........ . .............................. 143 viii TABLE 1. TABLE 2. TABLE 3. TABLE 4. TABLE 5. TABLE 6. TABLE 7. TABLE 8. TABLE 9. LIST OF TABLES COMPOSITION OF TOTAL MIXED RATION ....... .. ...... COEFFICIENTS OF CORRELATION FOR ENERGY BALANCE WITH PARITY, BODY WEIGHT, YIELD OF MILK AND INTAKE OF ENERGY..... ........ .. ..... COEFFICIENTS OF CORRELATION OF PROGESTERONE WITH ENERGY BALANCE, INTAKE OF DRY MATTER, YIELD OF MILK, BODY WEIGHT AND PARITY............................ ....... COMPOSITION OF TOTAL MIXED RATIONS............. EFFECTS OF ENERGY BALANCE ANDBODY CONDITION ON WEIGHTS OF CORPORA LUTEA ...... EFFECTS OF ENERGY BALANCE AND BODY COMPOSITION ON SECRETORYCHARACTERISTICS OF LUTEINIZING HORMONE IN SERUM OF HEIFERS.... ....... .............. ...... .... ..... ..... ENERGY BALANCE AND BODY CONDITION ON DURATION OF ESTROUS CYCLES IN HEIFERS.... ..... EFFECTS OF ENERGY BALANCE AND BODY CONDITION ON DURATION OF INTERVALS FROM ONSET OF ESTRUS TO ONSET OF DIESTRUS IN HEIFERS............................................. EFFECTS OF ENERGY BALANCE AND BODY CONDITION ON DURATION OF DIESTRUS IN HEIFERS.... ..... . ........ .. ...... . .......... . .......... ix 41 50 53 65 76 85 114 115 116 Figure 1 . Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. LIST OF FIGURES Energy balance and yield of milk during the first 100 d postpartum Association between energy balance and concentrations of progesterone in milk........... ............ Regression of energy balance on concentrations of progesterone in milk during 3 successive estrous cyCIeS 00000 0 0000000000000000000000 0.. 000000000000 00000.0... 00000000000000000 Changes in energy balance, body weight and body condition of heifers over four estrous cycles... ..... . ...... Effects of energy balance and body condition on progesterone in serum and duration of luteal phase during the first 10 to 12 d postestrus of four estrous cycles.. ...... Effects of energy balance and body condition on luteal development ............. ........... .. ................... Effects of energy balance and body condition on secretion of progesterone by luteal cells _ir_1_ vitro......... Effects of energy balance and body condition on secretory profiles of growth hormone in serum... ........ Effects of energy balance and body condition on profiles of mean concentrations of inulsin in serum ..... Characterization of total activity, intensity, duration and accuracy of estrus in heifers.......... ........ Effects of energy balance and body condition on standing behavior of heifers during three periods Of estrus000000000 00000 0. 000000 000.0000000000.000000000000000.0000... 000000 Effects of energy balance and body condition on mounting behavior of heifers... ..... ..... . ........ Standard curves of progesterone prepared with milk, and displacement of 125I-progesterone from specific antibody against progesterone by different dilutions of a milk pool. ......... ..................... 47 55 59 73 77 80 82 86 88 103 108 111 128 Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Concentrations of progesterone determined in samples of milk by solid phase or by a previously validated, liquid phase assay.... ..... ......... .. ..... Parallelism between concentrations of progesterone in serum and milk000000000000000000000000000000 000000 0.000000000000000 Elution pattern of 125I-bovine 1nsul1n Displacement of 125I-bovine insulin from antibody for bovine insulin by different dilutions of pooled DOVine sera.00.0.0...0.00.000.00.000000000000000.0000000000000000.00.0000 Specificity of antibody against bovine insulin... ........... 130 132 137 139 141 INTRODUCTION Causes of infertility in individual dairy herds are not easily determined and once identified, they are not easily corrected. As a result, infertility is one of the leading factors which limit productivity in dairy farms. Overall, poor fertility diminishes gross income and increases cost of production resulting in a significant reduction net income for dairy farmers. Reduction of gross income is largely due to decreased yield of milk per productive life of cows in herds with low fertility. Production of milk is reduced in these herds, because non-pregnant postpartum intervals are prolonged and cows spend more days in late lactation when yield of milk and feed to milk convertion are low. Furthermore, for each month added to non-pregnant intervals, production of calves is reduced by 8.3% (Pelissier, 1972) and cows sold because of reproductive failure do not produce offspring. Ferris and Fogwell (1984) estimated that for each day that non—pregnant intervals are prolonged beyond the optimal (82 days postpartum), gross income is reduced between $2.37 and $4.63. Applying these figures to national herd averages (10 million dairy cows; 45 days beyond optimal), indicate that dairy farmers lose between 1.1 and 2.2 billion dollars a year. Infertility increases costs of milk production largely due to increased costs of veterinary services, medication, additional artificial inseminations and the need of producing more replacements (Pelissier, 1972). Despite the magnitude of economic losses that poor reproductive ‘ performance represents to the dairy industry knowledge about causes of infertility is fragmentary. Fertility in dairy cows is determined by detection of estrus, fertilization and survival of embryos. Interestingly, these three reproductive events that largely determine reproductive performance are correlated positively with concentrations of progesterone in serum of cattle (De Silva et a1., 1981; Folman et a1., 1973; Fonseca et a1., 1983; Hill et a1., 1970; Melampy et a1., 1975). Therefore, it is important to determine sources of variation of luteal function in dairy cows. Undetected estrus is a major cause of prolonged postpartum intervals to conception (Pelissier, 1972). Known sources of failure to detect estrus are: uninformed farmers, limited number of observations of cows and variation in duration and intensity of estrus within and among cows. In addition, erroneously identified estrus and poor detection of onset of estrus contribute to low conception rates after artificial insemination (Pelissier, 1978). Consequently, identification of sources of variation for duration and (or) intensity of behavior at estrus in cattle has practical and biological implications. At least 9296 of dairy cows experience negative energy balance during early lactation (Reid et a1., 1966). During negative energy balance, homeorhetic mechanisms insure metabolic support to the lactating mammary gland and sustain synthesis of milk (Bauman and Currie, 1980). Perhaps homeorhetic controls which sustain lactation limit luteal function or behavior at estrus. In herds of dairy cattle, subclinical hepatic lipidosis has been diagnosed in as many as 3596 of the lactating cows (Reid, 1980). This condition is associated with obesity of cows at parturition and has been implicated as a source of infertility (Reid and Roberts, 1983; Roberts et a1., 1979). Fatness at parturition reduces intake of feed and dietary energy by cows (Garnsworthy and Topps, 1982). Therefore, fat periparturient cows may experience more severe negative energy balance than lean periparturient cows. 3 The primary goal of this research was to determine whether energy balance and (or) body condition reduce luteal function or limit estrous behavior in dairy cattle. If so, a second goal was to gain knowledge about mechanisms mediating effects of energy balance and(or) body condition on luteal function and(or) estrous behavior. REVIEW OF LITERATURE Introduction Although lactating dairy cows are the target animal of this research, postpubertal Holstein heifers were also used to address some specific objectives. Consequently, aspects of energy balance (EB), body condition (BC), luteal function and estrous behavior pertaining to dairy heifers and lactating dairy cows will be reviewed. Occasionally, information from other species will be used to illustrate or support some concepts. Variation of EB during growth and during lactation in cattle will be described. Importance of individual components of EB will also be addressed for growing heifers and lactating cows. Emphasis will be given to components of EB related to physiology of non-pregnant cattle (i.e. voluntary intake of feed, body weight, yield of milk, BC). If components of EB related to feeds, diets or environment are discussed attention will be minor. Most dairy cows experience negative EB (NEB) during early lactation. Due to homeorhetic controls, cows sustain milk production despite NEB. Variations in peripheral concentrations of insan and GH are consistently associated with homeorhetic control of growth and lactation. Changes in concentrations of non esterified fatty acids (NEFA) are associated positively with beginning of lactation and with NEB. Thus association of EB with secretory patterns of insulin and GH, and concentrations of NEFA in plasma of growing heifers and lactating cows will be described. In late lactation, dairy cows may ingest excessive dietary energy. Thus, at parturition cows are in positive EB (PEB) and many are fat. Peripartum fat BC reduces postpartum intake of feed and exaggerates NEB. But fat BC affects metabolism independent of EB in many species. Consequently, BC will be discussed separate from other components of EB but in association with reproduction, insulin, GH and NEFA. Two reproductive functions are the dependent variables throughout this research: 1) luteal function and 2) estrous behavior. Control of development and maintenance of corpora lutea in non-pregnant, ovulatory cattle will be addressed in this review. Discussion will focus on actions of luteinizing hormone (LH). Behavior at estrus will be described and related with sources of variation. Hormonal control of estrous behavior in dairy cattle will be reviewed. The relationships of EB or BC with luteal function or estrous behavior in cattle have not been addressed. But effects of some components of EB on these two reproductive functions have been examined. Available information addressing the association between components of EB and luteal function or expression of estrus is discussed. Finally, evidence indicating that insulin, GH or NEFA may be involved in luteal function is reviewed. Energy Metabolism in Cattle Requirements of Energy Nutritional value of feeds is expressed in several units of energy. Estimates of energy in feeds are based on productive values (i.e. meat and milk) of feeds ingested by cattle. Each unit estimates a different level of utilization of energy but all units are interrelated and have a hierarchical nature. The relationship among several measures of energy is established in the following expression: Gross energy - energy lost in feces = digestible energy (DE) DE - energy lost in urine, heat of fermentation and methane = metabolizable energy (ME) ME - energy lost as heat increment = net energy (NE) All systems currently used to determine energy values of feeds are based on principles of NE. Net energy actually indicates partial efficiencies in which ME of feeds is used for different physiological functions (Garrett and Johnson, 1983). Fasting metabolism is the major determinant of requirements of NE for maintenance (NEm). But to define NEm, energy systems adopted by the National Research Council (NRC) includes sources of variation of NEm such as: physical activity, age, gestation and lactation (Lofgreen and Garrett, 1968; Milligan and McBride, 1985; Moe et a1., 1972; NRC, 1978). Thus energy values of feeds or requirements of energy by cattle stated in review of literature or used in this research will be expressed in NE values. Partial efficiency of energy used for maintenance is higher than partial efficiency of energy used for productionin growing cattle. Therefore two values are estimated for each feed: NEm and N Eg for energy retained in tissues during growth (Garrett and Johnson, 1983). Similar. terminology is used to state requirements of energy by animals: maintenance (NEm) and gain (NEg). Because dietary energy is used with similar efficiency for maintenance and for production of milk in dairy cows, a single value of NE, net energy for lactation (NEI) is adequate to calculate rations or to determine requirements for maintenance or production of milk (Moe, 1981). In Holstein heifers within a range of 100 to 550 kg of body weight, NEg required for daily gains of .5 kg represents 37.5 to 22.496 of NE required for maintenance (NRC, 1978). But in Holstein cows, total requirements for lactation frequently exceed two to three fold the requirements of energy for maintenance (Moe, 1981; NRC, 1978). Thus, in heifers the major proportion of ingested energy is used to satisfy requirements for maintenance. In contrast, a large proportion of energy ingested by dairy cows during early postpartum is used to satisfy requirements of energy to support lactation. Enem Balance Energy balance is generally defined as input of energy minus output of energy. Thus in lactating dairy cows, EB is estimated by subtracting requirements of energy for maintenance and lactation from intake of dietary energy. In heifers, EB results from subtracting energy required for maintenance from intake of dietary energy. In these calculations growth or gain is excluded because energy is stored not consumed by body tissues of cattle. Maintenance constitutes the largest proportion of total energy required by growing cattle (Ferrel and Jenkins, 1985). Large variation in requirements of NEm exists among cattle of similar age and weight (Ferrel and Jenkins, 1985; Ledger and Sayers, 1977). Previous and current nutritional level, body weight and body composition have been suggested as sources of variation of NEm. But results are inconsistent and causes of variation of NEm among growing cattle remain unresolved (Ferrell and Jenkins, 1985). 8 At recommended rates of growth (.5 kg/d) between 62.5 and 77.696 of total dietary energy ingested by heifers is used for maintenance and thus to achieve EB (NRC, 197 8). Dietary energy exceeding requirements for maintenance of heifers is used for growth. Thus growing heifers are in PEB. Negative EB may be induced in heifers only by reducing dietary energy to levels below maintenance. In lactating dairy cows, requirements of energy for synthesis of milk exceed requirements for maintenance (Moe, 1981; NRC, 1978). Thus, to achieve EB lactating cows must ingest sufficient energy to satisfy requirements for maintenance and lactation. Reid et al. (1966) determined that coincident with peak lactation, 9296 of lactating dairy cows were in NEB. Coppock et al. (1974) observed that during the first 6 to 14 weeks postpartum 100% of lactating dairy cows experienced NEB. Thus at least 9296 of lactating. cows experience NEB at or before peak lactation. Duration and magnitude of NEB vary greatly within and among lactating cows. Average postpartum interval to nadir of EB was 4 weeks and ranged from 2 to 6 weeks (Bauman and Currie, 1980; Gravert, 1985). Average deficit of energy of lactating cows was 9 Meal of N El at nadir of EB (Bauman and Currie, 1980) but maximal deficit of energy for an individual cow was 3.2 Meal of NE] (Butler et a1., 1981). After nadir, average EB of cows increased and became positive by 6 to 16 weeks postpartum (Bauman and Currie, 1980; Coppock et a1., 1974; Gravert, 1985). Among cows experiencing energy deficit postpartum, duration of NEB in individual animals ranged from 4 to 14 weeks (Butler et a1., 1981). Sources of Variation of Energy Balance Some sources of variation for EB of lactating dairy cows have been proposed: age (parity), body weight, peripartum body condition, yield of milk and voluntary intake of feed (Blake and Custodio, 1984; Flatt, 1966). Effects of parity on EB is equivocal. Braund and Steel (1972) reported that cows in first lactation reached EB later postpartum than older cows. But in other study, parity did not affect magnitude or duration of NEB in Holstein cows (Coppock et a1., 1974). Body weight did not alter efficiency of cows to use dietary energy for production of milk (Gravert, 1985; Hooven et a1., 1968). In addition, body weight at the beginning of lactation or changes in body weight throughout lactation were not‘correlated significantly with EB of dairy cows (Flatt, 1966; Moe et al., 197 2). Consequently, variation of energy needed for maintenance irrespective of body weight, becomes a relatively small proportion of the total variatiOn of requirements for energy of lactating dairy cows. This is consistent with the fact that intake of dietary energy exceeds up to three fold the requirements of energy for maintenance in lactating dairy cows (Reid et a1., 1966). Yield of milk and EB during the first 20 d postpartum were correlated negatively in Holstein cows (Butler et a1., 1981). At least 92% of cows with high individual yields (> 30 kg/d) were in NEB at peak lactation (Reid et a1., 1966). This implies that while cows with modest yields of milk-would have no difficulty to sustain PEB, cows with. high yields of milk would be unable to maintain PEB during early lactation (Butler et a1., 1981; Reid et a1., 1966). But, Coppock et al. (1974) observed that Holstein cows with relatively low individual yields (5 20.1 kg/d) and fed ad libitum were in NEB for at least the first 6 weeks postpartum. Thus cows with low yields of milk are as likely to experience NEB as cows with high yields of milk. Cows with similar body weights and intakes of feed differ in BB due to variation in yield of milk (Bauman and Currie, 1980), thus yield of milk affects EB. But a general concept is that intake of feed is correlated positively with 10 yield of milk (Grainger et a1., 1985; Kazmer et a1., 1986) and with EB (Kazmer et a1., 1986). Therefore, increased intake of dietary energy compensates, at least partially, for greater demands of energy to support increased yields of milk. Moe et a1. (1972) observed a high positive correlation (.9) between intake of dietary energy and EB of cows. Cows of high genetic potential for milk fed ad libitum produced more milk but ingested more feed and had similar EB than equally fed cows with low potential for production (Kazmer et a1., 1986). Although daily yield of milk was negatively associated with EB (-.34), at least similar amount of variation in BB of lactating cows was explained (.45) by intake of feed (Kazmer et a1., 1986). In summary, at least 92% of dairy cows fed ad libitum are in NEB during early lactation. Despite NEB, dairy cows sustain synthesis of milk. Thus while heifers must be in PEB to grow, NEB in dairy cows is a common, spontaneous condition which does not limit synthesis of milk. Under farm conditions, dairy heifers are able to ingest sufficient feed to satisfy requirements of energy for maintenance and growth. Thus, voluntary intake does not represent a limitation to achieve PEB in heifers. But in cows, voluntary intake of feed is the principal component of EB. Homeorhetic Control of Growth and Lactation Optimal growth and lactation by cattle depend on proper nutritional management. Central to this aspect of animal husbandry is prediction of how energy in feeds is used by cattle for the frequently competing processes of growth, lactation and reproduction. The existence of biological mechanisms that prioritize metabolic support to some functions (growth, lactation, pregnancy) have been suggested (Bauman and Currie, 1980; Kennedy, 1967; Wood, 1979). Bauman and Currie (1980) 11 proposed that utilization of nutrients by various body tissues is regulated by homeostasis and homeorhesis. Homeostatic control involves maintenance of physiological equilibrium in the internal environment. Homeorhesis was defined as the coordinated changes in metabolism of body tissues to support a physiological state (i.e. lactation or growth). An example of homeorhesis is that in cattle at early stages of growth, deposition of protein exceeds deposition of fat, while in similarly fed cattle approaching mature weight, deposition of fat- exceeds deposition of proteins (Vernon, 1986). The importance of homeorhetic control is more obvious during lactation than during growth because of marked variation in EB of cows. When lactation begins, metabolism is rapidly altered to satisfy the enhanced demands for energy of the mammary gland. If cows are unable to alter energy metabolism to support synthesis of milk, yield of ‘ milk is reduced and(or) cows experience metabolic disorders (Bauman and Currie, 1980). Current understanding of factors controlling utilization of nutrients by tissues during growth and lactation is fragmentary. Control of growth and lactation is exerted by homeorhetic mechanisms which are concurrent with short-term homeostatic factors. Metabolism is controlled by interactions among endocrine factors that are homeorhetic and(or) homeostatic. This review will focus: on insulin and growth hormone (GH) which are presently considered as major homeorhetic regulators of growth and lactation. For detailed discussion on metabolic control of growth, the reader is referred to recent reviews (Brockman and Laarveld, 1986; Hart and Johnson, 1986; Trenkle, 1981; Weekes, 1986). Metabolic control .of lactation has been thoroughly discussed by Bauman and Currie (1980), Bauman et al. (1985),Collier et a1. (1984), Trenkle (1981). Discussion will focus on effects of EB and its components on secretory patterns of insulin and GH and on concentrations of N EPA in blood. 12 Insulin in Cattle A major role of insulin in concert with glucagon and catecholamines is homeostasis regarding glucose (Weekes, 1986). Insan stimulates uptake and oxidation of glucose by several tissues (Weekes, 1986). In addition to the universally recognized homeostatic effects of insulin, a chronic, homeorhetic role of insan in regulation of growth (Weekes, 1986) and lactation in farm animals has been recognized recently (Bauman et a1., 1985; Trenkle, 1981). In coordination with other anabolic and catabolic hormones, insulin controls partitioning of available nutrients during growth and lactation. Overall, insulin stimulates deposition of glycogen, triglyceride and protein in body depots (Weekes, 1986). Secretory Patterns of Insulin in Cattle In growing cattle (McAtee and Trenkle, 1971a), feeding induces a biphasic secretory pattern of insulin. Coinciding with feeding there is a rapid but transient increase of circulating insulin followed by a second rise of insulin which lasts between 2 and 6 h. Concentrations of insulin in jugular serum of lactating cows increase after feeding. But postprandial secretion of insulin is not biphasic as in heifers (McAtee and Trenkle, 1971a). Instead concentrations on insulin increased about 2 h after lactating cows ingested feed. This rise of insulin was sustained for 2 or 3 h and then declined to preprandial concentrations (Jenny and Polan, 1975). The first postprandial secretory increase of insulin in heifers, is caused by vagal reflexes (Weekes, 1986). Because in lactating cows a rapid increase of insan is not observed after feeding, vagal stimuli may be reduced in cows. The second postprandial peak of insan observed in heifers and the single rise of insulin in cows coincide with maximal postprandial absorption of digestive products. 13 What products of digestion induce postprandial release of insan by pancreas is not clear in ruminants. Due to microbial activity in rumen, dietary carbohydrate is fermented into acetate, propionate and a small percentage of butyrate. Thus, amount of glucose derived from diet that reaches and is absorbed by intestine is negligible. But large quantities of glucose are synthesized and released at all times by liver in ruminants. Glucose injected into jugular (McAtee and Trenkle, 1971a; McCann et a1., 1986) or mesenteric arteries of ruminants (Manns et a1., 1967) increased concentrations of insulin in jugular (Manns et a1., 1967; McAtee and Trenkle, 1971a; McCann et a1., 1986) and portal blood (Lomax et a1., 1979). Thus glucose may be involved in control of insulin secretion. Among products of digestion that induce release of insulin in ruminants when injected into jugular and (or) portal veins of ruminants are propionate and butyrate (Horrino et a1., 1968; Lomax et a1., 1979; Manns et a1., 1967; McAtee and Trenkle, 1971a) and perhaps free amino acids (McAtee and Trenkle, 1971a). £19331 Balance and Insulin Heifers fasted for intervals of two to eight days had lower basal concentrations of insulin in jugular blood than during fed state (McAtee and Trenkle, 1971a; McCann and Hansel, 1986). Lambs with restricted dietary energy for > 30 days that lost weight and that were presumably in NEB, had lower concentrations of insulin in serum than lambs fed adequately (Hart et a1., 1985). Steers maintained in NEB for 120 days had lower circulating insulin during the entire period than steers in PEB (Blum et a1., 1985). Consequently, deprivation of feed for relatively short periods, and NEB for long intervals decrease concentrations of insulin in growing ruminants. Concentrations of insulin in Holstein cows are lower in early lactation (< 40 (1) than in late lactation (Bines and Hart, 1981; Koprowski and Tucker, 14 1973; Smith et a1., 1976). At least 92% of dairy cows are in NEB during early lactation but all cows are in PEB by late lactation (Reid et a1., 1966; Coppock et a1., 1974). Therefore, low concentrations of insan in serum of lactating dairy cows coincide with intervals of N EB whereas high concentrations of insulin concur with PEB. Confirming this, Vasilatos and Wangness (1981) determined that basal concentrations and amplitude of pulses of insulin were lower at 30 d of lactation when cows were in NEB than at 90 d of lactation when in average cows were in PEB. Thus NEB is associated with low concentrations of insulin in serum of growing cattle and lactating dairy cows. Components of Energy Balance and Insulin Increased proportion of concentrate in diets enhanced magnitude of postprandial rise of insulin in growing sheep (Weekes, 1986). But, homeorhetic controls during growth are superimposed on homeostatic factors controlling secretion of insulin in response to changes in diet. For example, in lambs receiving constant amounts of feed per unit of metabolic weight, the second postprandial rise of insulin in serum increased with age and body weight (Weekes, 1986). Steers approaching mature size had higher concentrations of insan than younger steers (Verde and Trenkle, 1987), and insan in serum increased as heifers aged from 2 to 4 years (McCann and Reimers, 1986). But cattle approaching mature size have greater deposition of body fat than animals in early stages of growth. Thus actual source of variation for postprandial concentrations of insulin in heifers may be body composition, body weight or age. In lactating dairy cows, influence of yield of milk on serum concentrations of insulin is equivocal. Hart et a1. (1978) reported that high yielding dairy cows had lower concentrations of insulin in serum than low producing cows. However, 15 the low producing cows were crosses of dairy and beef breeds. When Holstein cows with superior yields were contrasted with Holstein cows with good yields of milk, concentrations of insulin were not different between groups (Barnes et a1., 1985) or tended to be higher in superior cows (Kensinger et a1., 1984). Dairy cows ingesting a diet with low proportion of grain did not have a postprandial increase of insulin while cows receiving a diet with a high proportion of grain had a normal rise of insulin after feeding (Jenny and Polan, 1975). Cows fed a low-grain diet had also lower basal concentrations of insulin in serum than cows fed a high-grain diet (Jenny and Polan, 1975). Thus, composition of diet may alter serum insan in cows. However, concentrations of insan were not influenced by composition of diets but were positively associated with individual intake of DM or energy by lactating dairy cows (Smith et a1., 1976). Consequently, composition of diet have some effect but amount of DM or energy ingested may exert a greater influence on secretion of insulin by dairy cows. More important than composition of diet or level of intake of DM or dietary energy on insulin is EB and(or) lactation. For example, cows in early lactation (N EB) received a diet with higher concentration of energy and ingested more DM and energy than non-lactating cows (PEB). But cows in early lactation secreted less insulin (portal vein concetrations) in response to feeding or to exogenous propionate than non-lactating cows (Lomax et al., 197 9). Summarizing, EB is associated positively with concentrations of insulin in heifers and lactating cows. Among components of EB; age and body weight are correlated positively with insulin, but body composition is confounded with these effects in heifers. In dairy cows amount of DM and(or) energy ingested exert the greatest influence on secretion of insulin. 16 Body Condition and Insan Associations between BC and insulin in dairy cows have not been examined. But McCann and Reimers (1985a) observed that basal concentrations of insulin in jugular blood were higher in fat heifers than in heifers with moderate BC. After intravenous injection of glucose, insulin was higher in fat heifers and lambs than in lean individuals (McCann and Reimers, 1985b; McCann et a1., 1986). Weekes (1986) determined that relative to lean lambs, increased circulating insulin in fat lambs was partially due to decreased metabolic clearance rate of insulin. But relative to lean animals, release of insan from isolated islets of Langerhans and perfused pancreas from obese rodents is enhanced (Bray and York, 197 9). Thus increased secretion and reduced metabolic clearance rate of insulin result in greater concentrations of peripheral insan in obese than in lean individuals. Growth Hormone in Cattle Growth hormone exerts a variety of effects on metabolism. ,Directly or indirectly GH stimulates anabolic processes such as cell division, skeletal growth and synthesis of protein. GH also exerts catabolic actions by stimulating oxidation of lipids and by inhibiting transport of glucose into cells (Hart and Johnsson, 1986; Spencer, 1985). Growth hormone is stored in and secreted by the somatotrophs in the anterior pituitary. Secretion of GH by pituitary is controlled by multiple factors which have been summarized by Bennett and Whitehead (1983). Secretory Patterns of Growth Hormone Mean concentrations, magnitude and frequency of episodic pulses of GH in blood vary considerably among animals. Release of GH by pituitary of young ruminants seems to be inherently episodic and is not influenced by time of feeding (Breier et a1., 1986; Zinn et a1., 1986). Secretory pulses of GH are asynchronous 17 among steers but pulsatile patterns within an animal are repeatable over time (Breier et a1., 1986). Lactating dairy cows have higher concentrations of GH in serum than non-lactating cows (Hart et a1., 1978; Sartin et a1., 1985). Growth hormone in lactating dairy cows is secreted episodically. As in heifers, pulses of GH in serum are asynchronous, do not follow diurnal patterns (Phillips and Athanasiou, 1978) and are unrelated with times of feeding (Vasilatos and Wangsness, 1981) or milking (Bauman et a1., 1979; Phillips and Athanasiou, 1978). Great variation in pulsatile patterns of OH is observed among cows (Enright et a1., 1986) but as in growing cattle, episodic pulses are characteristic of individual cows on a day to day basis (Vasilatos and Wangsness, 1981). M Balance and Growth Hormone Short-term deprivation of food (12 to 15 h) increased concentrations of GH in humans (Roth et a1., 1963). However, relative to values in fed animals, concentrations of GH in serum of heifers did not vary after an interval of 60 h of fasting (McAtee and Trenkle, 1971b). Similarly, concentrations of GH did not vary in plasma collected from growing sheep at 12 h postprandial or at 96 h of fasting (Wallace and Bassett, 1970). Therefore, unlike humans and pigs, GH may not be involved in short-term metabolic control of energy in growing ruminants. In contrast, submaintenance levels of dietary energy for prolonged periods increase secretion of GH in growing ruminants. Lambs who received a diet containing about 50% below their requirements of energy for maintenance during 42 days. had higher circulating GH than lambs ingesting adequate levels of energy (Hart et a1., 1985). Increased concentrations of GH resulting from reduced intake of dietary energy by steers who lost weight was due to enhanced amplitude of pulses of GH but not to variation in baseline concentrations or frequency of pulses of GH ( Breir et a1., 1986). Influence of NEB on secretion 18 of GH has not been examined in heifers but steers maintained in NEB for 120 days had higher circulating GH than steers in PEB (Blum et a1., 1985). Dairy cows in early lactation (< 40 (I) had higher concentrations of GH in serum than cows in later (> 60 (1) stages of lactation (Koprowski and Tucker, 1973; Smith et a1., 1976; Vasilatos and Wangsness, 1981). The decline of circulating GH as lactation progresses results from reduced amplitude of pulses rather than in basal concentrations or frequency of pulses of GH (Vasilatos and Wangsness, 1981). Apparently decreased concentrations and amplitude of pulses of OH with advancing lactation is due to a reduced ability of pituitary to secrete GH. This was supported further when Kazmer et al. (1986) determined that mean concentrations and thyrotropin releasing hormone (TRH)-induced secretion of GH were lower at late than at early lactation in dairy cows. At least 92% of dairy cows were in NEB during early lactation but 100% were in PEB at late lactation (Reid et a1., 1966; Coppock et a1., 1974). Thus, circulating GH is high when cows are in NEB and it is low when cows are in PEB. Indeed, dairy cows were in NEB and had higher concentrations of GH in serum at day 30 than at day 90 of lactation when they were in PEB (Vasilatos and Wangsness, 1981). Thus, in heifers and lactating dairy cows EB is negatively associated with concentrations of GH in serum. Components of Energy Balance and Growth Hormone Prepubertal heifers had higher concentrations of GH in serum than postpubertal heifers (Sejrsen et a1., 1983; Zinn et a1., 1986). Moreover, circulating GH after exogenous administration of TRH was higher in pre than in postpubertal heifers (Sejrsen et a1., 1983). Level of dietary energy ingested by growing cattle also affects circulating GH. Sejrsen et al. (1983) determined that prepubertal PEB heifers with restricted 18a intake of dietary energy had higher GH in serum than heifers fed identical feeds but offered ad libitum. But this influence of intake of energy on circulating GH was not evident in postpubertal heifers (Sejrsen et a1., 1983). Body composition may also. alter concentrations of GH in serum. For example, concentrations of GH in jugular blood are correlated negatively with proportion of fat in carcass of cattle (Hart and Johnson, 1986). Greater deposition of fat in body depots of postpubertal heifers (Sejrsen et a1., 1983; Zinn et a1., 1986) might explain lower levels of GH in serum and reduced response of OH to TRH than in prepubertal heifers (Sejrsen et a1., 1983). However, while rate of body weight gain or deposition of fat in body tissues was associated negatively with circulating GH of prepubertal heifers, in postpubertal heifers neither intake of feed, rate of weight gain or fat deposited in tissues affected mean concentrations or secretory patterns of GH (Sejrsen et a1., 1983; Zinn et a1., 1986). It is apparent from the previous discussion that regardless of gonadal status, circulating GB or response to secretagogues of GH in cattle diminishes with age. But effectsof age on secretion of-GH are confounded with effects of body weight and body composition. An interesting point is that secretion of GH in heifers at late stages of growth are not altered by factors which affect secretion of GH in heifers during early stages of growth. It can be speculated that once a "critical" age or weight or body composition is reached by heifers maintained in PEB, variations in intake of feed above requirements for maintenance, rate of gain or deposition of fat in tissues will not alter secretion of GH. High yields of milk were correlated positively with concentrations of GH in serum (Barnes et a1., 1985; Kazmer et a1., 1986; Kensinger et a1., 1984). Independent of EB, concentrations of GH in serum were greater in high yielding than in low yielding Holstein cows (Kazmer et a1., 1986). 19 Composition of diet did not alter mean concentrations of GH in plasma from dairy cows at early lactation (Smith et a1., 1976). But intake of DM or intake of dietary energy was associated negatively with GH (Smith et a1., 1976) and with magnitude of TRH-induced pulses of GH in lactating dairy cows (Bauman et a1., 1979). The previous discussion indicates that NEB increases GH in serum of heifers and cows. But, among components of EB, only intake of dietary energy sufficiently reduced to produce NEB affects GH in postpubertal heifers. In dairy cows, components of EB which are associated with GH are yield of milk and intake of DM or dietary energy. Yield of milk is correlated positively but intake of DM or energy is correlated negatively with GH. Non Esterified Fatty Acids (NEFA) in Cattle In heifers fed once or twice daily, concentrations of NEFA in plasma were affected by time of feeding. Concentrations of NEFA were maximal before feeding and declined rapidly after feeding (Holmes and Lanbourne, 1970). In these heifers concentrations of NEFA were maintained low for 8 h postprandial and increased thereafter. Fluctuations of NEFA in plasma due to time of feeding were suppressed when heifers received diets with high proportion of grain (Holmes and Lanbourne, 197 0). In lactating dairy cows fed once daily, concentrations of NEFA in plasma were maximal shortly before feeding and were maintained low for at least 12 h after feeding (Radloff et a1., 1966). But, concentrations of NEFA in plasma of cows fed more than once per d did not follow diurnal patterns and were not affected by time of feeding or milking (Phillips and Athanasiou, 1978). In cows, concentrations of NEFA increase as parturition approaches and peak during the first 7 d postpartum (Athanasiou and Phillips, 1978a; Radloff et a1., 1966). Subsequently, NEFA decline to reach prepartum concentrations between 20 and 35 d postpartum (Radloff et a1., 1966). 20 Ene_rgy Balance and NEFA Steers in NEB had greater concentrations of NEFA in plasma than PEB steers (Blum et a1., 1985). Dairy cows in early lactation (40 to 80 (I) had higher concentrations of N EFA in plasma than cows at late (> 120 d) lactation (Bauman and Currie, 1980; Coppock et a1., 1974). Because BB in cows changes from negative to positive after peak of lactation, it is reasonable that EB could be a major source of variation on NEFA. But Vasilatos and Wangsness (1981) determined that NEB cows at 30 d postpartum had similar concentrations of NEFA than at 90 d postpartum when cows were in PEB. In contrast, dairy cows in severe NEB during the first 56 d postpartum had two-fold higher concentrations of NEFA than cows with less severe NEB (Chilliard et a1., 1984). Thus, in growing heifers and in cows within same postpartum stage, concentrations of NEFA and EB are associated negatively. Components of Energy Balance, Body Condition and NEFA Submaintenance levels of dietary energy increased concentrations of NEFA in plasma of heifers (Holmes and Lanbourne, 1970). Daily changes in concentrations of NEFA in plasma were unrelated with levels of dietary energy offered to lactating cows (Ducker et al., 1985a, b). But effects of actual intake of energy on NEFA are untested in lactating cows. In cows, deprivation of feed for 2 or 5 d (Athanasiou and Phillips, 1978b; Brumby et a1., 1975) increased concentrations of NEFA in plasma. In addition, yield of milk is correlated positively with NEFA (Radloff et a1., 1966). Concentrations of NEFA in plasma from obese pigs are the same or lower than in non-obese pigs (Bakke, 1975; Weisemberger and Allen, 1973). But obese rats have higher NEFA in plasma than lean rats (Zucker, 1972). In cattle affects of BC on NEFA are untested. 21 In summary, EB and NEFA in plasma are correlated negatively in heifers and cows. In heifers dietary energy below requirements of maintenance increases concentrations of NEFA. In cows, fasting and yield of milk enhance concentrations of NEFA in plasma. 22 Luteal Function in Cattle Corpora Lutea of Estrous Cycles After puberty but before conception, sexual behavior in bovine females follows a rhythmic pattern (estrous cycle) that peaks regularly every 21 d with estrus and ovulation. With ovulation a new corpus luteum develops. Luteal regression is prerequisite to the next ovulation and cycle. The primary hormonal product of bovine corpora lutea is progesterone (Niswender et a1., 1980). Concentrations of progesterone in blood parallel function and lifespan of corpora lutea. Progesterone blocks ovulation by exerting negative feedback on luteinizing hormone (LH). Thus, enucleation of corpora lutea during mid—diestrus reduces concentrations of progesterone and allows precocious estrus and ovulation in cattle (Snook et a1., 1969; Hobson and Hansel, 1972). In contrast, continuous administration of progesterone prevents estrus and ovulation, and extends estrous cycles in cows (Christian and Casida, 1948). Estrus and ovulation follow. withdrawal of exogenous progesterone in postpubertal cattle (Roche, 1976). Clearly, timing of events associated with estrous cycles is influenced by progesterone and thus by corpora lutea. Thus, progesterone and corpora lutea are associated temporally and functionally with behavior at estrus, fertilization of oocytes and survival of embryos. Control of Development and Maintenance of Corpora Lutea. Function of corpora lutea depends on a balance between luteotropic and luteolytic factors. During development and maintenance of corpora lutea, availability of luteotropic factors exceeds availability of luteolytic substances. Factors secreted by the anterior pituitary gland (LH) and uterus (prostaglandins E2 and 12) affect luteal function positively. Luteinizing hormone is involved in luteinization of thecal and granulosal cells (Charming, 1980). Differentiation of follicular cells into luteal cells .is 23 marked by change of the major end product of steroidogenesis from estradiol-17 to progesterone (Charming, 1980; Ireland and Roche, 1983). Presence of LH is required for maintenance of morphological and functional differentiated state of bovine luteal cells .il‘. 31352 (Gospodarowicz and Gospodarowicz, 1975). Furthermore, LH increases synthesis and release of progesterone by corpora lutea of cattle _i_n yi_\_l_9_ (Schoemberg et a1., 1967), or when incubated as luteal slices or as dissociated luteal cells 19.11139 (Armstrong and Black, 1966; Williams and Marsh, 1978). Moreover, removal of LH by hypophysectomy (Kaltenbach et a1., 1968) or neutralization of LH by passive immunization (Hoffman et a1., 1974; Snook et al., 1969) accelerated luteal regression in cycling ewes and cows. In addition, exogenous LH extends the functional lifespan of corpora lutea in sheep (Karsch et al., 1971; Donaldson and Hansel, 1965) and cattle (Wiltbank, 1961 a, b). Thus, luteotropic support to corpora lutea in sheep and cattle is provided largely by LH. In this review of literature all mention to luteotropic stimuli or support will refer to LH. Specific receptors for LH are located in plasma membrane of bovine luteal cells (Gospodarowicz, 1973). Binding of LH to these specific receptors activates the adenylate cyclase system (Condon and Black, 1976; Marsh, 1976). Activated adenylate cyclase induces synthesis of cyclic adenosine 3',5'-monophosphate (cAMP; Marsh, 1976) which ultimately stimulates synthesis (Marsh, 1976; Williams and Marsh, 1978) and release of progesterone (Gemmell et a1., 1974; Niswender et al., 1980). Relative to other stages of a bovine estrous cycle, mean concentrations of LH are low and constant throughout metestrus and diestrus (Butler et a1., 1983; Walters et a1., 1984). But during luteal development, LH is secreted as pulses of higher frequency and lower amplitude than when corpora lutea are fully developed at mid—diestrus (Rahe et a1., 1980; Walters et a1., 1984). Changes 24 in pulsatile secretion of LH may be more important for luteal development than total concentrations of LH in serum (Hansel and Convey, 1983). Once corpora lutea develop, binding of human chorionic gonadotrophin (hCG) to luteal receptors increases progressively until about d 12 postestrus when progesterone in serum, luteal weight and binding of hCG to luteal cells are maximal (Spicer et a1., 1981). During luteal development in cattle, concentrations of progesterone in serum, binding of hCG or LH to luteal cells and luteal weight are correlated positively (Rao et a1., 1979; Spicer et a1., 1981). Between (1 12 and 16 postestrus, binding of hCG to corpora lutea declined, while progesterone in serum and luteal weight were maintained (Spicer et a1., 1981). Thus, during maintenance of fully developed corpora lutea, binding of LH is not associated with luteal weight or secretion of progesterone. Effects of Compgnents of Energy Balance on Luteal Function Influence of EB on luteal function of farm animals has not been examined. But effects of some components of EB on luteal function of cattle have been tested. Discussion will focus on influence of a) restricted intake cf energy, b) body weight, c) body condition and d) yield of milk on luteal function. Restricted Intake of Energy Influence of restricted intake of dietary energy on luteal weight has been examined. Some researchers did not detect effects of undernutrition on weight (Imakawa et a1., 1983) or size of corpora lutea from heifers (Spitzer et a1., 1978). But other workers observed that reduced intake of energy diminished luteal weight in heifers (Apgar et a1., 1975; Gombe and Hansel, 1973; Hill et a1., 1970). In addition, concentrations of progesterone in corpora lutea from underfed heifers were lower than in corpora lutea from adequately fed animals (Gombe and Hansel, 1973). Relative to values in adequately fed heifers, progesterone in serum was increased, lowered or not changed with restricted dietary energy. For example, 25 during the first diestrus after start of fasting, heifers had higher concentrations of progesterone than fed heifers (McCann and Hansel, 1986). Progesterone in serum of heifers receiving dietary energy below requirements for maintenance was not reduced for three (Spitzer et a1., 1978) or four consecutive estrous cycles (Beal et al., 197 8). But in other studies, restricted dietary energy reduced progesterone in serum of heifers immediately after (Hill et a1., 1970; Imakawa et a1., 1983) or at the second estrous cycle after onset of restriction (Gombe et a1., 1973). In cows effects of restricted dietary energy are also inconclusive. Concentrations of progesterone during first postpartum estrous cycle did not differ between lactating dairy cows receiving restricted or ad libitum dietary energy (Carstairs et a1., 1980). Corah et al. (1974) could detect no effect of prolonged under-nutrition on progesterone in postpartum beef cows. But, Dunn et al. (1974) observed a progressive decline of progesterone during four consecutive estrous cycles in underfed postpartum beef cows. Slices of luteal tissues (Apgar et a1., 1983) or dispersed luteal cells (Imakawa et a1., 1983) from underfed heifers had similar basal secretion of progesterone than cells from heifers fed adequately. But, luteal cells from underfed heifers secreted less hCG-or LH-induced progesterone than luteal cells from heifers receiving adequate diets (Apgar et a1., 1975; Imakawa et a1., 1983). Conclusions from these studies are limited because concentrations of hCG in medium with luteal cells were not reported (Imakawa et a1., 1983) or LH-induced secretion of progesterone by luteal cells from heifers under restricted diet, was affected only at very high concentrations (1000 ng/ml) of LH in medium (Apgar et al., 197 5). But basal secretion of progesterone by luteal cells _ig 33229. is not affected by dietary restrictions to heifers _i_n_ lilo.- And ability of luteal cells to respond to LH may be reduced _i_n_ vitro by restricted intake of dietary energy. 26 Reduced concentrations of LH in blood as cause of adverse effects of restricted diets on luteal function has been tested. In ovariectomized heifers, some authors observed that underfeeding increased LH (Imakawa et a1., 1987). But other workers did not detect effects of dietary restrictions on LH in serum (Beal et a1., 1978). In cycling heifers, effects of restricted dietary energy on serum LH vary with stage of estrous cycles. Relative to adequately fed heifers, peri-ovulatory LH was high in underfed and fasted heifers (Gombe and Hansel, 1973; McCann and Hansel, 1986). Similarly, gonadotropin-releasing hormone (GnRH)-stimulated secretion of LH was higher in heifers with deficit of dietary energy than in properly fed heifers at estrus (Bale et a1., 1978; Rasby et a1., 1986). Dietary restriction did not affect secretion of LH during diestrus (Hill et a1., 1970; Spitzer et a1., 1978). Changes in Body Weight The relationship between changes in body weight and luteal function is not clear. Losses in body weight were associated with decreased concentrations of progesterone in serum of heifers (Beal et a1., 1978; Hill et a1., 1970; Imakawa et a1., 1983, 1986) and postpartum beef cows (Donaldson et a1., 1970). But in other studies, declining body weight of heifers (Spitzer et a1., 1978) or cows (Corah et a1., 1974) was not accompanied by changes in progesterone. Relative to values in animals gaining weight, concentrations of LH were reported to be reduced (Imakawa et a1., 1986), increased (Beal et a1., 1978) or unchanged (Hill et a1., 1970; Spitzer et a1., 1978) in serum from heifers losing weight. Body condition In women (Frisch, 1984; Hartz et a1., 1979) and rodents (Bray and York, 1979) obesity has been associated with infertility. In dairy cows it was suggested that obesity at parturition is related with postpartum reproductive problems 27 (Reid and Roberts, 1983; Roberts et al., 197 9). Although the link between obesity and infertility exists, the underlaying mediating effects of obesity and specific reproductive functions affected in obese individuals have not been determined. McCann and Reimers (1986) and Spicer et a1. (1984) observed that concentrations of progesterone at mid—diestrus in fat heifers was similar than in moderately conditioned heifers. In rodents, obesity has been associated with low concentrations of LH in blood (Bray and York, 1979). But in heifers, fatness did not alter mean concentrations or GnRH-induced secretion of LH (Rasby et a1., 1986; Spicer et a1., 1984). Existing information is not conclusive regarding association between fatness and luteal function in cattle. But luteal function is associated positively with fertility and fatness is associatedwith infertility. Does limited luteal function mediate adverse effects of fat BC on fertility? If so, fat BC would be expected to affect luteal function. Yield of Milk The relationship between yield of milk and reproductive functions in dairy cattle is equivocal. Some researchers reported a negative influence of high yield of milk on fertility (Berger et a1., 1981; Laben et a1., 1982; Morrow 1969; Spalding et a1., 1975). But other workers did not find any association between reproductive performance and level of milk production of dairy cows (Carstairs et a1., 1980; Dachir et a1., 1984; Fonseca et a1., 1983). Yield of milk and progesterone in serum during first postpartum diestrus were not associated in dairy cows (Carstairs et a1., 1980). But first postpartum corpora lutea are functionally subnormal in cattle and subjected to many negative influences (Rutter et a1., 1985). Therefore results obtained by Carstairs et a1. (1980) are not conclusive. 28 InsulinJ Growth Hormone and Non Esterified Fatty Acids on Luteal Function During NEBIconcentrations of insan decrease but GH and NEFA increase in jugular blood of cattle. If NEB reduces luteal function in cattle, do changes in concentrations of insulin, GH and(or) NEFA mediate effects of NEB on luteal function? Evidence supporting these assumptions will be discussed. M11 Uptake and utilization of glucose are necessary for viability and function of bovine luteal cells _i_r_1_v_i_g'_o_ (Armstrong and Black, 1966, 1968; Flint and Denton, 1969; Savard et a1., 1963). Uptake and utilization of glucose by luteal cells are mediated by insan in cattle (Armstrong and Black, 1966; 1968) and rats (Flint and Denton, 1969). Increased uptake of glucose by luteal cells stimulates incorporation of 140 from [1-14C1'acetate into sterols and steroids (Flint and Denton, 1969) and enhances supply of reducing equivalents; both essential for -d_e_ £019 synthesis of. cholesterol and production of progesterone by luteal cells of ruminants (Armstrong and Black, 1966, 1968; Savard et a1., 1963). Specific receptors for insulin have been identified in luteal cells of rats (Ladenheim et a1., 1984) and in luteinized granulosal cells of gilts (Veldhuis et a1., 1984). These observations provide evidence for direct actions of insulin in luteal cells. Effects of insan on luteal function are not restricted to control of glucose metabolism. Insulin may affect steroidogenesis. Steroidogenic actions of insulin include increased supply of extraovarian cholesterol. -Cholesterol is the main precursor for progesterone and is delivered to bovine corpora lutea by low density lipoproteins (LDL; Savion et a1., 1982). Binding sites for LDL were identified in corpora lutea of pigs (Veldhuis et a1., 1986) and rats (Christie et a1., 1979; Rajendran et a1., 1983). Insan increases binding of LDL to adrenal glands of mammals (Brown et a1., 1981) and porcine granulosal cells (Veldhuis et a1., 29 1986). Thus insulin may increase binding of LDL to corpus luteum, increase supply of cholesterol and support, if not stimulate, synthesis of progesterone by luteal cells. Insulin also increases binding of hCG in porcine granulosal cells (May and Schomberg, 1981). Overall, responsiveness of luteal cells to luteotropic stimuli should be increased by insulin. Accordingly, _i_n_ y_i_t_r_o_ insulin induced luteinization of porcine granulosal cells (May and Schoemberg, 1981), increased proliferation of bovine granulosal cells (Savion et a1., 1981) and enhanced secretion of progesterone by porcine and bovine granulosal cells (Charming et a1., 1976; Savion et a1., 1981). Moreover, insulin is required for maximal steroidogenic actions of gonadotropins on cultured granulosal cells from pigs (May and Schoemberger, 1981). _I_n_ 1119, exogenous insan blocked adverse effects of restricted dietary energy on FSH-induced ovulation in cattle (Harrison and Randel, 1986) and on spontaneous ovulation in gilts (Jones et a1., 1983). Basal and LH-induced secretion of progesterone were depressed in streptozotocin induced diabetic rats (Tesone et a1., 1983). Injections of xyalazine, an inhibitor of insulin secretion, reduced insulin and progesterone in serum of heifers (McCann, 1984). Therefore, insulin exerts positive effects on ovarian and luteal function. In cattle experiencing N EB, concentrations of insulin are reduced. Thus, NEB could affect luteal function adversely because of reduced concentrations of insan in serum. Growth Hormone Growth hormone antagonizes actions of insulin on uptake of glucose by adipose tissue of sheep (Vernon, 1978). But in liver of rats, GH and insulin synergize to induce synthesis of somatomedin-C (Maes et a1., 1986). Furthermore, GH stimulates formation of receptors for LH and synthesis of progesterone by granulosal cells of rats (Jia et a1., 1986). These roles of GH are perhaps mediated by increasing ovarian somatomedin-C (Davoren and Hsueh, 1986) which 30 mimics actions of GH in granulosal cells of rats (Adashi et a1., 1985). Study of actions of GH on luteal functions has been neglected. Thus there is no information indicating the direction of GH effects, if any, on luteal function. Based on actions of OH on other tissues, increased GH during NEB in cattle might affect luteal function in at least three ways: a) should GH act on corpus luteum, as it does on adipose tissue, then GH might oppose actions of insulin and reduce luteal function; b) if GH acts on corpus luteum as in granulosal cells, greater concentrations of GH during NEB might affect luteal function positively; and c) if as occurs with hepatic synthesis of somatomedin, actions of GH on corpora lutea require presence of insulin at concentrations observed in non—obese, adequately fed individuals; incresed GH during NEB would not alter luteal function due to concommitant reductions of insulin. 212.12 ' High concentrations of NEFA in plasma were associated with poor fertility in dairy cows (Ducker et al., 1985b). But cause-effect relationship between increased NEFA and infertility has not been tested. Addition of NEFA to human serum albumin devoid of lipids, reduced binding of progesterone by albumin (Ramsey and Westphal, 1978). Decreased binding affinity of albumin for progesterone was proportional to the amount of NEFA. Thus increased NEFA during NEB may reduce binding of albumin to progesterone and increase free progesterone in blood. Consequently, metabolic clearance rate of progesterone may be increased and concentrations of progesterone in serum reduced. 31 Estrous Behavior in Cattle In most mammalian females, sexual receptivity is confined to estrus, the stage of an estrous cycle immediately preceding ovulation. At or near estrus, females will seek out a male and engage in behaviors which enhance the probability of copulation. These behaviors have been defined as proceptivity when estrous behavior of laboratory species is described (Clemens and Gladue, 1979). Proceptivity corresponds to a group of behavioral signs classified as "initiating behavior" in cattle (Esslemont et a1., 1980; Glencross et a1., 1981). If a sexually active male is present, the receptive female stands so the male can achieve vaginal penetration (penile intromission; Clemens and Gladue, 1979). The group of behaviors of a sexually receptive cow has been classified as "receiving behavior" (Esslemont et a1., 1980; Glencross et al., 1981). In cattle, a female in estrus stimulates other females who mimic the sexual behavior of males. Thus, in groups of bovine females, individuals in estrus have the opportunity to display the receptive as well as initiating behaviors in absence of males. The practical importance of detecting estrus in groups of bovine females is to appropriately schedule artificial insemination relative to ovulation. Estrous behavior of dairy heifers and cows in absence of males will be discussed in this review. Expression of Estrus Esslemont et a1. (1980) classified the most frequently observed components of estrous behavior as: a) agressive behavior which is expressed mainly as butting and chasing; b) investigatory behavior that includes sniffing, rubbing, licking and chin-resting; c) disordered mounting, consisting in mounting cows not in estrus or disoriented mounting (i.e. head to head mounting), and d) oriented mounting with standing. Clearly each component of behavior includes an initiating and a receiving individual. 32 With the possible exception of standing, and in presence of a male, penile intromission, all behavioral signs of estrus may be displayed at all stages of an estrous cycle by bovine females. But all signs of estrus are displayed most frequently in proestrus and estrus (Esslemont et al., 1980; Glencross et al., 1981). Standing and mounting are the behavioral components most definitive of estrus. For example, in all heifers studied by Glencross et a1. (1981), onset of standing behavior coincided with estrus (peak concentrations of estradiol-17B) in serum. Based on concentrations of progesterone and estradiol, other authors determined that few standing events occur at non-estrus stages in heifers (Esslemont et al., 1980). Therefore, near 100% of standing events occur .at estrus in heifers. Between 70 (Helmer and Britt, 1985) and~79% (Hurnick et al., 197 5) of all mounting events were exhibited at estrus in cattle. Duration of standing estrus in individual heifers ranged from 3 to 21 h (GlencrOss et al., 1981) and averaged 10.2 h (Esslemont et al., 1980). For lactating dairy cows, duration of standing behaviorranged from 7.5 to 10.1 h (Hurnick et al., 1975). Estrous behavior is a continuum throughout an estrous cycle, but frequency and intensity of estrous activity, especially standing and mounting, peak immediately before and during estrus. Factors Affecting Estrous Behavior Many environmental factors affect expression of estrus in dairy cattle. Time of day when cattle are observed alters efficiency on detection of estrus (De Silva et al., 1981; Helmer and Britt, 1985; Hurnick et al., 1975) as does duration (Hurnick et al., 1975) and frequency of observations (Donaldson, 1968). High (> 25°C) ambient temperatures reduce expression of estrus (Bond and McDowell, 1972; Ganwor et al., 1965) and percentage of cows detected in estrus is altered by type of housing (De Silva et a1., 1981; King et al., 1976). As numbers 33 of animals simultaneously in estrus increased up to 4 or 5, mounts per period of observation increase proportionally (Esslemont et al., 1980; Helmer and Britt, 1985; Hurnik et al., 1975). Effects of EB, intake of dietary energy, body weight or BC on estrous behavior of cattle have not been examined. But, influence of other components of EB on expression of estrus were tested. Carstairs et al. (1980) did not observe any association between levels of dietary energy offered to dairy cows and detection of estrus. Some workers found a negative correlation between yield of milk and detection of estrus (Morrow et al., 1966) but others observed that estrous behavior was not influenced by yield of milk in dairy cows (Carstairs et al., 1980; De Silva et al., 1981; Fonseca et al., 1983). Experiments addressing the association between estrous behavior and levels of dietary energy offered to cows (Carstairs et al., 1980) or yield of milk (Morrow et al., 1966; De Silva et a1., 1981; Fonseca et al., 1983), do not allow definitive conclusions because estrus was considered a discrete rather than a continuous variable. To draw valid conclusions from those data, many more animals must be observed. In addition, duration of estrus averages approximately 10 h in heifers and lactating. cows (Esslemont et al., 1980; Hurnick et al., 1975). However in previous reports, cattle were only observed for periods of 1 h at intervals of 12 h. Thus in existing research, number of cattle and number of periods of observation were insufficient to characterize adequately variation of estrous behavior under different conditions. Hormonal Control of Estrus Removal of gonads reduced or suppressed sexual activity in mammals. With the exception of primates, mammalian females become sexually unresponsive to males after removal of ovaries (Clemens and Gladue, 197 9). In cattle, removal of ovaries eliminates estrus (Katz et al., 1980). Manifestation of estrous behavior 34 in ovariectomized cattle is restored by exogenous estradiol-17B (Katz et al., 1980; Melampy et al., 1957), estrone (Wiltbank et al., '1961) or testosterone (Katz et al., 1980; Kiser et al, 1977). Estradiol is several fold more potent than testosterone or estrone in eliciting estrous behavior in ovariectomized cattle (Katz et al., 1980; Wiltbank et al., 1961). Thus among the ovarian steroids capable of restoring sexual activity in ovariectomized cattle, estradiol-178 is the most potent and thus may play the most important role in regulating estrous behavior of gonadally intact cattle. Concentrations of estradiol in serum increase during estrus (Chenault et al., 1975; Glencross et al., 1981). Passive immunization of ewes against estradiol inhibited estrus (Scaramuzzi et al., 1975). Large doses of progesterone antagonize stimulatory effects of exogenous estradiol on estrous behavior of ovariectomized cows (Melampy et al., 1957). But positive effects of exogenous estradiol on estrous behavior are enhanced when given simultaneously with progesterone in low doses (Melampy et al., 1957). In cycling cows, concentrations of progesterone at estrus ranged between 0.4 and 1.0 ng/ml of serum (De Silva et al., 1981). Within this range, circulating progesterone at estrus was correlated positively with mounting activity in dairy cows (De Silva et al., 1981). Thus, concentrations of progesterone in peripheral blood of female cattle at estrus may enhance estrous behavior. Repeated administration of estradiol suppressed estrous behavior in ovariectomized cows. But pretreatment with progesterone abolished refractoriness caused by repeated injections of estradiol (Carrick and Shelton, 1969). In addition, pretreatment with progesterone enhanced display of estrous behavior induced by single injection of estradiol in ovariectomized heifers (Melampy et al., 1957). Thus, variation in concentrations of progesterone during luteal phases preceding estrus may also influence estrous behavior in cycling female cattle. 35 Estradiol is the major hormone determining estrous behavior but progesterone modulates effects of estradiol on expression of estrus. Summary At least 92% of dairy cows fed ad libitum experience N EB in early lactation. Large variation in magnitude and duration of NEB is observed among cows. Yield of milk and intake of feed have been implicated as factors with major impact on EB in dairy cows. But the fact that NEB is equally frequent among cows with high and low yields of milk, indicates that the major determinant of EB is intake of feed. Homeorhetic mechanisms support synthesis of milk during NEB. Perhaps homeorhetic mechanisms that support lactation alter reproductive functions in dairy cows. Luteal function is correlated positively with expression of estrus, conception and embryo survival. Associations between individual components of EB (i.e. reduced dietary energy, changes in body weight or yield of milk) and luteal function in cattle are not consistent. Perhaps these inconsistencies are due to unexplained or uncontrolled variation in other components of EB. As a single variable, EB should integrate multiple components of variables such as diet, feed intake, body weight and yield of milk. Thus EB is integrative. A high proportion of dairy cows experience NEB during early lactation and normal luteal function is important to fertility in cattle. The first objective of this dissertation was to determine the relationship between EB and luteal function in lactating dairy cows. Fat periparturient dairy cows ingest less DM and dietary energy during early lactation than lean cows. Thus fatness at parturition may accentuate postpartum NEB of cows and further limit luteal function. Fat BC may affect luteal function in cattle by enhancing NEB or by mechanism unrelated with EB. Thus the second objective of this research was to determine the independent and associative effects of EB and BC on luteal function in dairy heifers. 36 Luteinizing hormone is the main luteotropic factor in cattle. Effects of reduced intake of dietary energy on LH are equivocal. But it is possible that reduced luteotropic support to corpora lutea might mediate effects of EB or BC on luteal function. Thus, a third objective of the present dissertation was to determine independent or associative effects of EB and BC on LH in dairy heifers. Alternatively, responsiveness of luteal cells to luteotropic stimuli might be reduced directly or indirectly by energy deficit. The fourth objective of this research was to determine effects of EB, BC and (or) their interactions on basal and (or) LH-induced secretion of progesterone from luteal cells 3113312.- From reviews cited, it is apparent that in cattle insulin and GH are involved in homeorhetic control of growth and lactation. Additional literature indicates that secretion of insan and GH vary with changes in EB and BC. Concentration of NEFA changes in response to various stimuli associated with lactation or EB. Thus, the fifth objective was to determine effects of EB, BC and (or) their interactions on profiles of insulin and GH and in concentrations of NEFA in blood of dairy heifers. Insulin exerts an important role in luteal function of cattle and other species. Whether or not GH exerts any effects on corpora lutea has not been addressed. But known effects of GH on ovarian follicles or on non-reproductive tissues provide rationale that GH might be involved in luteal function. Increased N EFA have been associated with infertility in cattle. Because luteal function and fertility are positively associated, NEFA may alter luteal function. Thus changes of insulin, GH and(or) NEFA associated with homeorhetic control of lactation might mediate effects of NEFA on luteal function. The sixth objective of the present dissertation was to determine associations between luteal function and changes of circulating insulin, GH or NEFA. 37 Knowledge of hormonal control of estrous behavior in cattle is fragmentary. However, behavior of bovine females at estrus or other stages of an estrous cycle has been assessed and described thoroughly. Many environmental factors which alter estrous behavior of cattle have been identified. But whether metabolic status of cattle affects expression of estrus has not been tested adequately. Therefore, the seventh objective of the present dissertation was to determine the independent and associative effects of EB and BC on estrous behavior in dairy heifers. EXPERIMENT I: Association Between Energy Balance and Luteal Function in Lactating Dairy Cows 38 EXPERIMENT I: Association Between Energy Balance and Luteal Function in Lactating Dairy Cows INTRODUCTION Yield of milk and levels of dietary energy have been implicated as causes of infertility in lactating dairy cows. But effects of yield of milk on fertility of cows are equivocal. Some researchers observed that reproductive efficiency declines as yield of milk increases (Berger et al., 1981; Morrow, 1969; Spalding et a1., 1975), whereas other workers did not confirm this association (Dachir et al., 1984; Fonseca et al., 1983; Hansen et al., 1983). Similarly, there are inconsistent effects of low dietary energy on reproductive performance. Ducker et al. (1985a) and King (1968) observed that diets with low energy were detrimental to fertility of dairy cows. In contrast, other workers did not find a relationship between diet and reproductive performance (Carstairs et al., 1980; Ducker et al., 1985b; Gardner, 1969). Energy balance is the net result of associations among diet, intake and use of nutrients by cows and yield of milk. Thus energy balance as a single measure should integrate homeorhetic changes that occur during lactation. At least 92% of dairy cows experience negative energy balance during early lactation (Coppock et al., 1978; Reid et al., 1966), but magnitude and duration of negative energy balance are variable among cows (Butler et al., 1981). In dairy cattle, luteal function is associated with three events that determine fertility: 1) success of detecting estrus (Melampy et al., 1957), 2) rate of conception (Folman et al., 1973; Fonseca et a1., 1983) and 3) embryonic 39 40 survival (Hill et al., 1970). Therefore, it is important to identify factors that affect luteal function in dairy cows. The main objective of this study was to determine the relationship between energy balance and luteal function in lactating dairy cows. MATERIALS AND METHODS General. Eight primiparous and 24 multiparous lactating Holstein cows were studied from parturition to 100 day ((1) postpartum or conception, whichever occurred first. Cows calved normally and remained healthy throughout the study. Cows were housed in stanchion stalls and received water and feed ad libitum. Cows were fed a total mixed ration (TMR; 50% roughage: 50% grain on DM basis) at .0200 and 1400 h (table 1) formulated to satisfy all requirements of nutrients for maintenance and lactation. Intake of feed by individual cows was recorded daily. Throughout the study ingredients of TMR were sampled and DM of ingredients was determined weekly. Amount of ingredients in TMR were adjusted according to variation in DM to maintain a 50:50 ratio of roughage to grain. On alternate weeks during the experiment, TMR was sampled for chemical analysis (table 1). Milk Yield, Body Weight and Energy Balance. Cows were milked twice daily (0400 and 1500 h) and yields of milk were recorded. Concentrations of fat in milk were determined monthly by Michigan Dairy Herd Improvement Association (East Lansing) using an infrared analyzer (Multipec, Wheldrake, England). For each cow, it was assumed that samples of milk within :2 15 d from sampling (1 had the same concentrations of fat than the analyzed sample of milk. Milk was adjusted to 4% fat (4% FCM) by formula derived from Tyrrell and Reid (1965): 4% FCM = yield of milk [136 + 5 (actual % of fat)/340]. 41 TABLE 1. COMPOSITION OF TOTAL MIXED RATION. Item Concentrationa Ingredient , % Alfalfa hay 12.5 Corn silage 37.5 High moisture ear corn 30.0 Protein supplement (44%) 17.0 Dicalcium phosphate and vitamin premix 3.0 Chemical analysesb» 0 Dry matter, % 52.9 Energy, Mcal/ 1.6 Crude protein, % 17.8 Acid detergent fiber, % 21.0 8Dry matter basis. Values are means from 10 samples of total mixed ration taken in alternate weeks during the study. bMethods for chemical analyses in Pritchard and Staubus (1978). cConcentrations of minerals in total mixed ration were Ca (.6%), P (.4%), K (1%), Mg (.2%), S (.2%), Na (.15%), Mn (32 ppm), Fe (217 ppm), Cu (10 ppm) and Zn (45 ppm). dAs NEl (estimated from total mixed ration). 42 From d 4 to 7 postpartum until the end of the study, body weight of cows was measured (6 to 7 h after 0200 h feeding) on two successive d per week. Weekly changes in body weight were extrapolated to estimate daily body weight (weekly change/7 (1). Energy balance (EB) of cows was estimated daily by subtracting net energy required for maintenance and lactation from intake of NEl. Requirements of NE] for maintenance were based on daily body weight and calculated as suggested by NRC (1978): NE] (Meal) = 80 (Kcal NE/kg-75). Due to requirements for growth, cows in first and second lactation were fed 20 and 10%, respectively, above requirements of NE for maintenance indicated for mature cows (NRC, 1978). To determine N El for lactation, daily yield of 4% FCM (kg) was multiplied by .74 Mcal (NRC, 1978). Daily intake of NEl (DM basis) was calculated by multiplying N El (Meal) Per kg of TMR (table 1) by kg of TMR ingested by cows. Luteal Function and Other Reproductive Measures. Concentrations of progesterone in fat-free milk parallel concentrations of progesterone in serum of cows (Appendix A; Pope et al., 1976). Therefore, luteal function can be monitored by progesterone determined in milk or serum. To quantify progesterone in milk, samples of milk were obtained every third d from d 5, 6 or 7 postpartum until the end of the experiment. Collection and processing of samples of milk and radioimmunoassay techniques used to quantify progesterone are described in Appendix A. To detect estrus, cows were observed for three daily periods of 30 min (0600, 1800 and 2400 h). Estrus was when a cow stood to be mounted (3 2 sec) during intervals of low concentrations of progesterone in milk (< 1 ng/ml). Day of ovulation was recorded as 3 d before concentrations of progesterone in milk exceeded two standard deviations above basal progesterone. Basal progesterone (.175 :1: .01 ng/ml) was the mean of progesterone in milk sampled I— , 'I_|nu‘— 43 during the first week postpartum, when all cows were anovulatory. Estrous cycle was defined as the interval between two consecutive periods of estrus. When estrous behavior did not accompany ovulation, d 0 of an estrous cycle was recorded as one d before ovulation. At weekly intervals from d 5, 6 or 7 postpartum to first artificial insemination, reproductive organs of cows were examined rectally to determine presence of corpora lutea I in ovaries and to determine uterine involution. Presence of corpora lutea indicated resumption of ovarian functions postpartum. To determine uterine involution, diameter and length of uterine horns were estimated. Uterine involution was when previously gravid uterine horn had diameter and length similar to opposite uterine horn and neither horn declined further. Assimilation of Data and Statistical Analysis. 'To characterize changes in EB of cows throughout the study, E3 was expressed as Mcal of NEl/d and could be positive (PEB) or negative (NEB). To explain variation of EB, daily EB was the dependent variable in multiple linear regression using backward elimination procedures (Gill, 197 8). Independent variables were daily yield of 4% FCM, daily body weight, daily intake of DM and parity (1, 2 or Z 3 parturitions). Concentrations of progesterone in milk were plotted over time within an estrous cycle per cow and area under the curve was calculated. Area included values that were 2 standard deviations above basal progesterone and that were sustained for _>_ 3 consecutive samples of milk. Maximal concentration of progesterone within an estrous cycle (peak) and duration of luteal phase (interval of progesterone exceeding basal progesterone by 2 standard deviations) were calculated for first to third postpartum estrous cycles. Together, peak progesterone and duration of luteal phase accounted for 84% of the variation 44 in area under the curve of progesterone (r2 = .84; P < .001). Area, peak and duration were used as measures of luteal function. To determine sources of variation in luteal function, area, peak or duration were used as dependent variables in multiple linear regression analyses, including backward elimination procedures (Gill, 1978). Independent variables were mean EB per estrous cycle, mean intake of DM per estrous cycle, mean yield of 4% FCM during the first 100 d postpartum, mean body weight per estrous cycle and parity (1, 2 or _>_ 3 parturitions). Concentrations of progesterone during first postpartum estrous cycle are lower than in subsequent cycles in cattle (Edgerton and Hafs, 197 3). Thus by regression analysis, I examined the association between progesterone (area, peak or duration) as dependent variable and consecutive estrous cycles (first, second or third) as an independent variable. To determine sources of variation on duration of postpartum anovulation I used interval from parturition to first ovulation postpartum as dependent variable and used same analysis and independent variables described in the previous paragraph. The preceding analysis indicated confounded effects of EB, duration of postpartum anovulation and number of consecutive estrous cycles postpartum on progesterone (see Results and Discussion). To reduce these confounding effects, concentrations of progesterone were blocked within first, second and third estrous cycle and mean EB was limited to the interval when all cows were anovulatory (d 1 to 9 postpartum). Additional advantage of this reorganization of data was that we were able to address whether or not EB and progesterone were associated within and among estrous cycles per cow. Thus with cow as experimental unit, we examined the regression of progesterone (area, peak or duration) during first, second or third estrous cycle on mean EB during anovulation (3 0, -0.1 to -3.0, -3.1 to -6.0 and < -6 Mcal of NEl). Unless stated, 45 all reference to EB will be mean during anovulation and will be discussed as mean EB. For these data, specific comparisons were between means of progesterone for control (_>_ 0.0 Mcal) and means of progesterone for other levels of mean EB during anovulation (-0.1 to -3.0, -3.1 to -6.0 or _<_ -6 Meal). Contrasts were made by Dunnett's test (Gill, 1978). To determine association between EB and luteal function over time, we examined changes of progesterone within level of mean EB across estrous cycles as follows. Slopes of regressions of progesterone (area, peak or duration) within level of EB were contrasted with zero slope (Gill, 1978). Significant increases of progesterone were indicated by positive slopes while negative slopes indicated decreases of progesterone over time. In addition, slope for regression lines of progesterone within level of NEB (-0.1- to -3.0; -3.1 to -6.0 or < -6 Meal) were contrasted with corresponding slopes of cows in PEB during anovulation (_>_ 0.0 Mcal). Lack of parallelism between slopes indicated difference in rate of change of progesterone over time. To further examine the association between EB during anovulation and luteal function of cows, duration and magnitude of EB were determined for cows grouped by level of EB (>0.0, -0.1 to 3.0, -3.1 to -6.0 or < -6 Mcal) during postpartum anovulation. For this we defined onset of NEB as the first 3 consecutive d postpartum when cows were in NEB. End of NEB was when cows were in PEB for at least 8 d of two consecutive weeks. Duration of NEB was the interval between the first d of onset and the first d of end of NEB. Magnitude of NEB was indicated by nadir of EB within the interval of NEB. To determine the relationship between these aspects of EB and luteal function, area of progesterone within first, second or third estrous cycles was the dependent variable in a regression analysis by backward elimination, in which the independent variables were day of nadir, nadir and duration of NEB. 46 Using chi square (Gill, 1978), proportions of ovulations associated with estrous behavior were contrasted among levels of mean EB, mean intake of DM per estrous cycle, mean body weight per estrous cycle, mean yield of 4% FCM during first 100 d postpartum or parity (1, 2 or _>_ 3 parturitions). RESULTS AND DISCUSSION Yield of Milk) Body Weight and EB. Mean yield of 4% FCM during the experiment was 32.4 :t .9 kg/d (range: 20.8 to 40.7 kg). Peak yield of milk (40.2 i: 3.4 kg) occurred at 44.8 t 3.5 d postpartum. Initial body weight of cows averaged 601 :t 30 kg. Four cows maintained or gained weight throughout the study. All other cows lost 29.4 d: 3.9 kg (range: 8.6 to 106 kg) from first week to 43.4 t 4.2 d postpartum when nadir for body weight occurred. From nadir, body weight increased and by the end of the study all cows averaged 591 i .31 kg. Mean intake of DM increased from 19.1 i .9 kg in first week to 27 :1: 2.2 kg by d 100 postpartum. When EB was averaged for all cows (figure 1), mean EB was positive during the first 2 d postpartum, became negative on d 3 and reached nadir by d 10 postpartum. Starting on d 11 postpartum, mean EB increased from nadir and was zero or positive by 80 d postpartum. Approximately 81% (n = 26) of the cows were in NEB for at least 4 consecutive d postpartum. But at the end of the study 69% (n = 22) of the cows were in net energy loss. Thus by d 100 postpartum, 69% of cows did not recover energy lost at earlier stages of lactation. Overall, daily EB was highly variable within and especially among cows. For all cows, the observed range of daily EB was -35 to 31 Meal of NE. Among the 26 cows experiencing NEB, nadir of EB (-16.3 t 1.2; range: -4 to -35 Meal) and duration of N EB (69.6 3: 5.9, range 4 to 98 d) were similar to values reported previously (Butler et al., 1981; Kazmer et al., 1986). In the present study, cows 47 Figure 1. Energy balance and yield of milk during the first 100 d postpartum. Daily mean of energy balance (Meal of net energy for lactation) was calculated for all cows (top panel) or for cows grouped according to mean energy balance during postpartum anovulation (1 to 9 d). In parenthesis are numbers of cows. Yield of 4% FCM is the mean during first 100 d postpartum. Pooled standard error of energy balance for all cows was 1.3. Pooled standard error for yield of milk was .9. ENERGY BALANCE (Mcol) 48 20 All Cows (n=32) YIELD A “W 32-4 -|O-* 20 20 Mco|(n=13) IO 0 32.3 -IO 20: -O.l to-3 Mcol (n=6) IO:\ ‘ o 3|.8 -IO-‘IIIIIIITIIFTIIIIIIII 20] -3.l to -6 Mcol (n=5) IO:\ O_W34.8 -2|glllrillr11ijlerlTrl <-6 Mcol (n=8) .1 O ' WW 3|.3 4qu ‘l rlllllilrrilllllfl O ' 20 4O 60 80 ‘ IOO DAY POSTPARTUM 49 experienced NEB even when feed offered was not limiting. Thus the large variation in EB among cows was spontaneous. Consequently I was able to examine the relationship between luteal function and different levels of EB without dietary manipulation. This is consistent with examining effects of homeorhetic control of lactation on luteal function. To further examine EB throughout the experiment, cows were grouped according to mean EB during postpartum anovulation. In cows which averaged _>_ 0 Meal of NE during anovulation, magnitude of energy deficit (nadir: -8.7 i: 1.6 Meal) was less (P < .01) and interval of NEB was shorter (38.6 (1) than in cows with <0.1 Meal during anovulation (figure 1). Magnitude and duration of NEB did not differ among groups of cows that were in energy deficit during anovulation. But, average EB throughout the study was lower (P < .05) in groups of cows with _<_ -3.1 Meal than in cows with marginal energy deficit (-0.1 to -3 Meal) during anovulation (figure 1). For all cows with _<_ -0.1 Meal during anovulation, EB at nadir averaged -l9.1 :1: 1.3 Meal and mean duration of NEB was 78 t 6 d. But day of nadir was earlier (P < .01) in cows with < -6 Meal during anovulation (d 12 i 4.4 postpartum) than in cows with -3.1 to -6 Meal (d 30.2 i: 8.5) or with -0.1 to -3 Meal during anovulation (d 40 t 10.5). Thus, level of energy deficit during anovulation seems to be determined primarily by when nadir occurs postpartum and, to a lesser extent by actual of nadir. Factors Associated with Variation in EB. Parity and body weight were not significantly associated with EB (table 2). But variation in BB of cows was explained largely (table 2) by intake of energy (P < .01) and, to a lesser extent by yield of milk (P < .05). Throughout the study, only 19% (n = 6) of the cows were in PEB. Yield of 4% FCM (32.8 :t 1.9 kg) of cows always in PEB was not different than mean yield of 4% FCM for the 26 cows in NEB (32.3 t 1.01 kg). Interestingly, the cow with highest yield (40.7 kg 4% FCM/d) ingested more 50 TABLE 2. COEFFICIENTS OF CORRELATION FOR ENERGY BALANCE WITH PARITY, BODY WEIGHT, YIELD OF MILK AND INTAKE OF ENERGY.a Body Yield of Intake of Parity Weight Milk Energy Energy balance .10 -.13 -.25b .73c aDaily energy balance was regressed on estimated daily body weight, daily yield of milk (4% FCM), daily intake of dietary energy and parity (1, 2 or _>_ 3 parturitions). bSignificant correlation (P < .05). cSignificant correlation (P < .01). 51 DM (30.9 kg/d) than any other cow and sustained PEB during the study. These observations on individuals and the fact that yield of milk did not differ among cows with distinctly different EB (figure 1), illustrate that some cows with large yields of milk ingest sufficient energy to satisfy requirements for high yields of milk and to sustain PEB. Conversely, some cows with average or low yields of milk do not ingest enough energy to maintain PEB even with modest yields of milk. In general our observations are consistent with results reported by Kazmer et a1. (1986) and indicate that variation in BB is largely due to voluntary intake of DM by cows. Yield of milk, although associated negatively, has a minor effect on variation in EB of cows. Thus, yield of milk would not be highly predictive of EB or metabolic status of lactating cows. Factors Associated with Luteal Function. All cows ovulated at least twice during the study. The interval from parturition to first ovulation averaged 23.9 :1: 2.2 d (range 10 to 52 d). Behavioral signs of estrus were detected in association with 64.9% of ovulations. A lower proportion (P < .01) of behavioral estrus was associated with first postpartum ovulation (34.4%) than with second (71.9%) or third (83.3%) postpartum ovulations. Duration of postpartum anovulation and detection of estrus associated with ovulation were not correlated significantly with parity, body weight, yield of 4% FCM, intake of DM or EB. My observation that all cows ovulated and resumed estrous cycles by 23.9 :1: 2.2 d postpartum is consistent with previous reports (Butler et al., 1981; Carstairs et al., 1980; Rosemberg et al., 1977). Thus, postpartum anovulation is not a source of extended intervals non pregnant in most dairy cows. Success of detecting estrus associated with ovulation in the present study was low as in previous works (Butler et al., 1981; Carstairs et al., 1980) and confirms the importance of understanding sources of variation for expression of estrus. 52 First, second and third estrous cycles began at 23 i 2, 42 :l: 2 and 63 i: 3 d postpartum, respectively. Duration did not differ among postpartum estrous cycles and averaged 21.3 t 8 d. Mean duration of luteal phases was 12.8 t .6 d and did not vary among postpartum estrous cycles. But concentrations of progesterone (area and peak) increased (P < .05) from first to third postpartum estrous cycle. This agrees with previous reports that, concentrations of progesterone in serum during luteal phases after first ovulations are lower than during subsequent luteal phases of postpartum dairy cows (Edgerton and Hafs, 1973). Yield of 4% FCM, mean body weight per cycle and parity were not correlated significantly with area and peak of progesterone or with duration of luteal phase (table 3). In addition changes in body weight from parturition to nadir of body weight were not associated with progesterone in milk (data not shown). Yield of milk affects EB and changes in body weight are altered by EB. Thus these variables are associated with EB and might be expected to affect reproductive events. But in the present study, there was no evidence that duration of postpartum anovulation, detection of estrus or luteal function * were influenced by yield of milk or body weight. Thus, as independent factors, yield of milk or body weight may not influence reproductive performance in lactating dairy cows. Alternatively, yield of milk and (or) body weight may affect reproductive performance by mechanisms related to reproductive variables not examined in this study. Since duration of luteal phases did not vary among cycles, it is not surprising that duration of luteal phase was not associated with EB or intake of DM. But variation in area and peak of progesterone were correlated positively (P < .05) with mean EB and intake of DM per estrous cycle (table 3). However, intake of DM was the most important component of EB (table 2). Thus, coefficients 53 TABLE 3. COEFFICIENTS OF CORRELATION OF PROGESTERONE WITH ENERGY BALANCE, INTAKE 0F DRY MATTER, YIELD OF MILK, BODY WEIGHT AND PARITY. Independent Progesteroneb variablesa Area Peak Duration Energy balance ' .28c .25c .07 Intake of DM .230 .13 .04 Yield of milk -.01 -.04 -.01 Body weight .07 .09 .05 Parity -.02 -.01 .03 aEnergy balance, intake of DM and body weight were averaged per estrous cycle. Yield of milk is average of 4% FCM during the first 100 d postpartum. Parity was 1, 2 or Z 3 parturitions. bVariables describing progesterone were means per estrous cycle of area under the curve of progesterone (progesterone > basal concentrations), maximal concentration of progesterone (peak) and duration of luteal phase. cSignificant correlation with independent variable in same line (P < .05). 54 of determination were not strengthened by including intake of DM or NE with EB as an independent variable in statistical models (data not shown). Associations of intake of DM with EB and luteal function observed in this experiment may explain inconsistent results about effects of dietary energy on reproduction (Carstairs et al., 1981; Ducker et al., 1985a, 1985b; Gardner, 1969; King, 1968). Generally, researchers assume that cows fed ad libitum are in PEB or in less N EB than cows which receive restricted amounts of feed. But, based on evidence from this study, intake of feed or appetite are not related linearly with feed offered to cows above 100% of requirements of NE for maintenance and lactation. Therefore, actual EB and reproductive performance of cows may be similar although feed offered to cows varies. Energy balance per estrous cycle and progesterone in milk were correlated positively (table 3). But successive postpartum estrous cycles were correlated positively with EB (r = .5; P < .05) and with progeSterone in milk (r = .3; P < .05). Furthermore, duration of postpartum anovulation determines when estrous cycles occur and EB varies eolinearly with postpartum interval. Therefore, effects of EB and successive postpartum estrous cycles on luteal function of cows are confounded. To reduce these confounding effects, cows were segregated among 4 categories of mean EB during postpartum anovulation (1 to 9 d). Among these four groups of cows we contrasted progesterone blocked within postpartum estrous cycle. During the first estrous cycle postpartum, variation of progesterone in milk (area) was not correlated significantly with mean EB (figure 2). But within second or third estrous cycle, variation of progesterone (area) was correlated positively (P < .08) with changes in EB during anovulation (figure 2). In fact, during second and third cycles, cows with most severe NEB (< -6 Meal) during anovulation had lower (P < .05) progesterone in milk (area) than PEB cows (figure 2). In addition, cows with EB between -3.1 and -6 Meal during 55 Figure 2. Association between energy balance and concentrations of progesterone in milk. Progesterone was measured in free-fat milk collected every third d. Energy balance (Meal of net energy) was averaged during interval of postpartum anovulation (1 to 9 d). Association examined was between levels of EB during anovulation and progesterone within first, second or third postpartum estrous cycle. In parenthesis are numbers of cows within estrous cycle per level of energy balance. Values are mean 1: SE. Energy balance and progesterone were correlated positively within second and third cycle (r = .3; P = .07). I"This value differs (P < .05) from value for _>_ 0 within estrous cycle. 56 1 CYCLE I 100- " (5) 60- ‘ T (a) 20- _ [TI 1 a c» v II I I III LIJ A 21; ‘1 CYCLEZ 8 5'00" (13) 11.1 ‘53 - T (6) 55‘? 60‘ I (TI) (5) CD :IJ ‘ 33¢: 20- [I] 0' d I I I l m “ CYCLES IOO“ T (5) * ‘ T "‘ (7) (3) , 60- T. "D 20- l I I I 20 -o.| -3.| <-6.0 to to -3.0 -6.0 ENERGY BALANCE 57 anovulation had less progesterone (P < .05) during the third estrous cycle than PEB cows. Duration of luteal phases and EB during anovulation were not correlated significantly, but associations between peak progesterone and mean EB (data not shown) were similar than for area and EB. Similarly, in underfed heifers length of diestrus was normal but progesterone in serum was lower than in adequately fed heifers (Hill et al., 1970; Imikawa et al., 1983). One interpretation of these data is that second and third postpartum corpora lutea in cows which were in severe NEB during anovulation, had normal lifespan but secreted less progesterone. Effects of NEB on magnitude of peak progesterone could be due to a) reduced luteal development, b) decreased secretory activity per luteal cell or c) a combination of these effects. Why was EB during anovulation not related with luteal function during first estrous cycle postpartum? Why was apparent influence of NEB delayed for 40 to 70 d when second and third postpartum corpora lutea developed? Concentrations of progesterone in serum (Edgerton and Hafs, 1973) and in milk (this study) during the first postpartum diestrus were lower than during subsequent estrous cycles. It is possible that the first postpartum corpus luteum is inherently limited independent of NEB. Thus adverse effects of NEB observed on second and third postpartum corpora lutea are inconsequential or undetectable during first postpartum diestrus. In contrast, modulating factors present during first diestrus (Rutter et al., 1985) are absent or reduced by 40 to 70 d postpartum, when adverse effects of NEB on luteal function in second and third postpartum diestrus are exerted or detectable. Alternatively, NEB may not have immediate influence on luteal function of cows. This is possible because in heifers with restricted dietary energy and presumably in N EB, progesterone did not decline until second and third diestrus after dietary restriction (Gombe and Hansel, 1973x 58 In cows that were in PEB, marginal NEB (-.1 to -3 Mcal) or most severe NEB (<-6 Mcal), contrasts between zero slope and slopes of regression of progesterone within level of EB during anovulation over time postpartum (figure 3), indicated that progesterone increased (P < .05) from first to third estrous cycle. But in cows with EB of -3.1 to -6 Meal during anovulation, progesterone tended (P = .09) to decline overtime (figure 2). When contrasted with PEB cows, slopes of progesterone in cows with marginal NEB (-.1 to -3 Mcal) were parallel. But slopes of cows in severe NEB (-3.1 to -6 or <-6 Mcal) during anovulation did not parallel (P < .05) slopes of cows in PEB or in marginal NEB. Thus in cows with severe NEB (_<_ -3.1 Mcal), progesterone did not increase with sequential postpartum estrous cycles as in cows with marginal (-.1 to -3 Meal) and PEB during anovulation, or increased at a lower rate. Based on results from cows in PEB and moderate NEB, luteal function is affected positively by progressive postpartum estrous cycles or advancing postpartum interval. But severe NEB appears to modulate adversely the positive effect of progressive postpartum estrous cycles on luteal function of dairy cows. Variation in concentrations of progesterone in milk (area) was not associated with changes in nadir or duration of NEB in cows. But progesterone during second estrous cycle was correlated positively (r = .51; P < .01) with postpartum interval when nadir of EB occurred. Moreover, changes in progesterone during second and third estrous cycles were associated positively (re = .69; P < .01) with interactions between postpartum interval to nadir and EB at nadir. An interpretation of these results is that luteal function in second and third postpartum diestrus will be most limited in cows with early postpartum nadir of EB and severe NEB at nadir. Therefore early occurrence of nadir relative toparturition and severity of NEB at nadir (are components of EB during anovulation that potentially limit luteal function in dairy cows. 59 Figure 3. Regression of energy balance on concentrations of progesterone in milk during 3 successive estrous cycles postpartum. Energy balance was averaged during postpartum anovulation (1 to 9 d postpartum) and cows were grouped .332." (.;n= 13), -.1 to-3 (O;n= 6), -3.1 to-6(A;n=5)or<-6 Mcal(A;n = 8). Relative to zero slope, slopes of regression lines for 3 0 and -.1 to -3 were positive (P < .01), slape for < -6 was positive (P < .05) and slope for -3.1 to -6 Meal tended (P = .09) to be negative. Slopes of regression lines of _>_ 0 or -.1 to -3 differed (P < .05) from slopes of -3.1 to -6 (P < .01) or from < —6. 60 |40q . Mod/d I20: IOO- PROGESTERONE Area (ng - d- mli') G) <3 I N 0‘") POST PARTUM ESTROUS CYCLE 61 In this experiment, diet was formulated to satisfy all requirements for energy and was offered _a_q libitum. But NEB occurred in 81% of the cows and was spontaneous rather than due to dietary restriction. Although yield of milk was associated negatively with EB, most variation of EB was determined by intake of energy. There was no evidence that parity, body weight or yield of milk affect reproduction because these variables were not correlated significantly with duration of postpartum anovulation, detection of estrus or luteal function. Mean EB during postpartum anovulation was not associated with duration of luteal phases. But EB during anovulation was correlated positively with l) luteal function within second and third postpartum estrous cycles and 2) changes of luteal function among successive postpartum estrous cycles. My primary conclusion is that severe NEB (_<_ —3.1 Mcal) during postpartum anovulation may reduce level of luteal function during second and third postpartum diestrus. Nadir of EB occurred earlier postpartum in cows with severe NEB (< -6 Meal) than in cows with less severe NEB during anovulation. Moreover, d of nadir but not EB at nadir was correlated positively with luteal function within second estrous cycle. Interactions between d of nadir and E8 of nadir were associated positively with luteal function within second and third cycle. Therefore, timing and magnitude of NEB appear to be important determinants of the extent that NEB limits luteal function in cows. Severe NEB was associated with reduced luteal function during second and third estrous cycles, when most dairy cows are artificially inseminated for first time postpartum. Luteal function is associated with reproductive events that determine fertility in cattle (Folman et al., 1973; Hill et al., 1970; Melampy, 1957; Rosemberg et al., 1977). Consequently I suggest that NEB is a potential source of infertility in cattle. EXPERIMENT 11: Influence of Energy Balance and Body Condition on Luteal Function in Heifers ' 62 EXPERIMENT 11: Influence of Energy Balance and Body Condition on Luteal Function in Heifers Introduction Negative energy balance (NEB) is associated with reduced luteal function in lactating dairy cows (Experiment I). This is particularly significant since 3 80% of cows are in NEB during early lactation (Coppock et al., 1974; Reid et al., 1966; Experiment I) and reduced concentrations of progesterone in serum are associated with low rates of conception (Folman et al., 1973), embryonic death (Hill et al., 1970) and failure to detect estrus (Melampy et a1., 1957). Thus NEB may be a source of infertility in lactating cows. Intake of dietary energy is an important source of variation of energy balance in lactating dairy cows fed ad libitum (Experiment I). Cows that are overconditioned at parturition consume less feed and energy during early lactation than moderately conditioned herdmates (Garnsworthy and Topps, 1982). Consequently overconditioning may enhance adverse effects of N EB on luteal function. In dairy cows, influences of NEB, time postpartum and sequential estrous cycles on luteal function are confounded (Experiment I). Therefore, I used postpubertal Holstein heifers to determine the independent and associative effects of body condition and energy balance on luteal function. In addition, I examined the relationship between body condition and energy balance with concentrations of insulin, growth hormone (GH) and non-esterified fatty acids (NEFA) in blood serum. 63 64 Materials and Methods Design and General Procedures. Twenty postpubertal Holstein heifers were grouped by body weight and assigned to treatments within a 2 x 2 factorial experiment. Main effects were: 1) body condition: moderate or fat, and 2) energy balance: positive or negative. Heifers were housed in a free stall barn that was divided into two pens. Each pen was equipped with 16 gang-lock stanchions at the feed bunk. In addition, feed bunks were partitioned into 16 individual mangers. Thus, heifers could be fed individually. Throughout the study heifers were fed total mixed rations (TMR; table 4) at 0500 and 1700 h and had free access to water and salt supplemented with trace minerals. The study consisted of three phases: conditioning, adaptation and experimental. The conditioning phase lasted about 6 mo during which heifers were fed to produce two groups of animals with distinct body conditions (BC), moderate or fat. To achieve this goal, 12 heifers with. an average body weight of 275.8 i: 5.6 kg received diet A (table 4) in amounts that supported daily gains of .78 kg and maintained moderate BC (MOD). In addition, 8 heifers with average body weight of 279.1 1 7.6 kg received diet B (table 4) ad libitum to support daily gains of .9 kg and to produce fat BC (FAT). The conditioning phase ended when BC of the two groups was different (P < .05). To determine energy balance (EB), heifers were fed individually. Thus during an interval of 20 d, heifers were adapted to gang-lock stanchions and trained to consume their daily dietary allowance within two intervals of 90 min (0500 to 0630 and 1700 to 1830 h). Variation in intake of dry matter (DM) per unit of body weight or in levels of starch in diets alters patterns of rumen fermentation in cattle (Robinson et al., 1986). Because changes in rumen fermentation may affect ovarian functions in cattle (McCartor et al., 1979), during the period of adaptation MOD and FAT heifers received the same diet TABLE 4. COMPOSITION OF TOTAL MIXED RATIONS. 65 Dieta __ Item B C Ingredients, 96 Alfalfa haylage 24. 4.4 22.4 Corn silage 53. 16.9 , 48.9 High moisture ear corn 16. 73.1 12.0 Protein supplement (4496) 5. 5.1 16.2 Vitamin premix .5 .5 Chemical analysisb9C Dry matter, 96 40. 50.9 46.4 Energy, Mcal/kgd 1. 1.6 1.5 Crude protein, 96 13. 13.3 17.7 Acid detergent fiber, 96 21. 14.3 15.5 aDry matter basis. bMethods for chemical analysis in Pritchard and Staubus (1978). cConcentration of minerals in total mixed rations were: Ca (.7196), P (.3996), K (1.1%), Mg (.26%), S (.2196), Na (.1396) Mn (57 ppm), Fe (228 ppm), Cu (8 ppm) and Zn (48 ppm). dAs NEm (estimated from total mixed rations). 66 (A, table 4) and received similar amounts of DM per unit of body weight. Therefore, at the end of the adaptation phase, all'heifers received the same diet for at least 20 d but BC of heifers was still different. The experimental phase lasted 3.5 estrous cycles and ended when corpora lutea of heifers were removed surgically (lutectomy) 10 to 12 d postestrus of the fourth estrous cycle. From the first d and throughout the experimental phase, 6 MOD and 5 FAT heifers individually received diet A (table 4) to sustain daily gains of .78 kg and positive EB (PEB). The remaining MOD (n = 6) or FAT (n = 3) heifers individually received diet C (table 4) in amounts calculated to cause NEB without deficiences in protein, vitamins or minerals. Thus at the beginning of the experimental phase four treatment combinations were established: MOD-PEB (n = 6), FAT-PEB (n = 6), MOD-NEB (n = 5) and FAT-NEB (n = 3). Twice daily throughout adaptation and experimental phases, heifers were restrained to receive TMR individually and to record daily intake of feed. During the entire study, weekly samples of ingredients in TMR were collected and dried in oven at 100°C for 12 h to determine DM. Based on changes in DM of ingredients, TMR were adjusted to maintain a constant ratio of ingredients. During the entire experiment, TMR.was sampled weekly. For each TMR, aliquots from individual samples were obtained and pooled so at the end of the study there was a pooled sample from each TMR. Duplicates from each pool of TMR were analyzed chemically (table 4). Body Weight and Body Condition. Throughout the study, body weight and BC of heifers were used to monitor diets. Heifers were weighed (1500 h) on two consecutive d per week. Weekly changes in body weight were extrapolated to estimate daily changes in body weight per (1 of the experiment. Methods devised by Mulvany (1981) were modified and used to determine BC of heifers. 67 Body condition of heifers was scored every other week in a scale of 1 to 4 (4 = fat), including half points. For scoring BC of heifers, thickness of subcutaneous adipose tissue was estimated by palpating loin and tailhead. Loin consisted of spinal and transverse processes of lumbar vertebrae. Tailhead was a triangular area defined by anterior coccygeal vertebrae and both Tuber ischia (pin bones). Average scores for loin and tailhead indicated BC of heifers. Scores used for statistical analyses of BC were mean of 4 observers. Energy Balance. During the experimental phase, EB was estimated daily for each heifer. Energy balance was calculated by subtracting energy required for maintenance from intake of energy. Net energy required for maintenance (NEm) were based on daily body weight and calculated by: N Em (Mcal) = (.077) body weight-75 (National Research Council, 1984). Daily intake of energy was calculated by multiplying NEm in feeds (table 4) by intake of DM. Luteal Function in vivo. To determine the influence of EB, BC and(or) their interactions on progesterone, jugular blood was sampled daily (1500 h) for experimental phase (3.5 estrous cycles). Progesterone was quantified in serum by radioimmunoassay. Luteal Function in vitro. To determine effects of EB, BC and (or) their interactions on basal and LH-induced secretion of progesterone from luteal cells _in _v_igg, corpora lutea were collected via supravaginal incision between (1 10 and 12 postestrus of the last estrous cycle. Immediately after collection, corpora lutea were rinsed with medium 199 (Gibco Laboratories, Grand Island, NY), pH 7.35 with Hank's salts, NaCHOg (.36 g/liter), HEPES (4.7 g/l), bovine serum albumin fraction V (1 mg/ml), penicillin G (sodium salt, .7 g/l), streptomycin sulfate (.1 g/l) and neomycin sulfate (.05 g/l). Rinsed corpora lutea were placed in tubes with fresh medium and transported on ice to our laboratory. Subsequently, adherent interstitial tissue was removed and weights 68 of corpora lutea were determined. Corpora lutea were bisected and sliced (Stadie-Riggs microtome). For dissociation of cells, slices of luteal tissue were placed in Hank's medium devoid of Ca” and Mg++ but containing collagenase (Worthington type IV, 125 U/ml, .05 to .396), deoxyribonuclease (.00596) and bovine serum albumin (.0596; Sigma Chemical Co., St. Louis, MO). Luteal slices were incubated at 37°C in an atmosphere of 95% 02 and 596 C02 for 4 to 6 h with frequent agitation by aspiration to disperse cells. Viability of luteal cells (> 9096 in all corpora lutea) was determined by Trypan blue exclusion (Patterson, 1979) and cells were counted by hemocytometer. Dissociated luteal cells from each corpus luteum were suspended in medium 199 and incubated at 37°C in an atmosphere of air for 2 h. Incubation medium contained 0,- .1, 1, 10 or 100 ng/ml of bovine luteinizing hormone (NIH-LH-B8) and each dose was replicated 5 times per corpus luteum. After incubation, cells and medium were frozen together in bath of dry ice-methanol and stored at -60°C until assayed for progesterone. Detection of Estrus. To monitor intervals between estrus, heifers were observed daily for signs of estrus for at least two periods of 30 min. A heifer was considered in estrus when she stood to be mounted for at least 2 sec and concurrently had < 1 ng of progesterone per ml of serum. In addition, ovaries and uteri of heifers were examined rectally at least twice before experimental phase to confirm postpubertal status. Only postpubertal heifers were included in the experiment. LH, GH, Insan and NEFA. Energy balance, BC and(or) their interactions may alter luteotropic support to corpora lutea during diestrus. To determine effects of EB and (or) BC on LH, polyvinyl cannulae were installed in a jugular vein of heifers and on d 9, 10 or 11 postestrus of fourth estrous cycle, approximately 24 h before lutectomy. Samples of blood were collected every 69 15 min during an interval of 12 h (0430 to 1630 h) for quantification of LH. In addition, GH and insulin were measured in serum from these same samples. Feed was presented to heifers immediately after collecting blood at 0500 h. Jugular blood was taken before (0430 and 0500 h) and every h (0600 through 1600 h) after feeding, placed in heparinized tubes and resulting plasma was assayed for NEFA. Am. Concentrations of progesterone in serum were quantified by a solid-phase radioimmunoassay described in Appendix A. From 21 assays, concentrations of progesterone in serum from heifers in estrus was .15 :t: .002 ng/ml. The intraassay coefficient of variation (CV) was 9.696 and the interassay CV was 12.7%. In serum from pregnant cows’ concentrations of progesterone were 6.7 :t .05 ng/ml (intraassay CV = 4.296; interassay CV = 6.796). Tubes containing luteal cells and medium were thawed and frozen repeatedly to disrupt membranes of cells. Media and disrupted luteal cells were extracted twice (benzene:hexane, 1:2) and quantified for progesterone (Louis et al., 1973). From 25 radioimmunoassays, concentrations of progesterone in a pool of media plus luteal cells from heifers in late diestrus was 13.7 :I: .14 ng/ml (intrassay CV = 7.4596; interassay CV = 9.9996). Extraction efficiency among assays was 95.3 t .596. Concentrations of LH and GH in serum were determined by radioimmunoassays described by Convey et a1. (1976) and Purchas et a1. (1970), respectively. From a single assay, concentrations of LH in serum from cows in estrus were 10.5 i .3 ng/ml (CV = 8.596). Concentrations of GH in serum from lactating cows was 5.4 i .1 ng/ml (CV = 6.2%). Methods to quantify insulin are described in appendix. Concentrations of insulin in serum from 5 h fasted beef heifers was 66.7 t 1.9 uU/ml (CV = 6.896) but was 177.5 1 5.9 uU/ml (CV = 4.596) in serum from recently fed beef heifers. Concentrations of NEFA in 70 plasma were determined by colorimetric techniques (Brunk and Swanson, 1981). From 13 assays, concentrations of NEFA in plasma from fed heifers were 135.8 :t 4.3 qu/l (intraassay CV = 296; interassay CV = 16.496). Statistical Analyses. Data on EB, BC and body weight change were averaged per estrous cycle and analyzed by split-plot analysis of variance with estrous cycles as subplots (Gill and Hafs, 1971). In addition slopes of regression lines of EB, BC or body weight change across estrous cycles were determined. Positive slopes indicated that EB, BC or body weight increased. Negative slopes indicated that EB, BC or body weight decreased. Lack of parallelism of slopes between treatment combinations (Gill, 1978) was interpreted as a difference in rates of change of EB, BC or body weight. Results from this analysis will indicate whether or not the treatment combinations needed to address our objective were established. To analyze concentrations of progesterone in serum, area under the curve of progesterone was calculated for first 10 to 12 d of 4 estrous cycles within heifer. Area included values that were 2 standard deviations above baseline (.125 + (2) .089 = 0.3 ng/ml). Baseline (.125 ng/ml) was the mean concentrations of progesterone. in samples of blood taken during estrus fl: 1 d. These data were arranged in a 2-factor design with 4 estrous cycles as a subplot and examined by a split-plot analysis of variance (Gill, 1978). Area under the curve is determined largely by maximal concentration of progesterone (peak) and duration of diestrus (interval that progesterone exceeds baseline by 2 standard deviations). Therefore, for all four periods of diestrus during experimental phase, peak concentrations of progesterone and duration of luteal phases during first 10 to 12 d postestrus were calculated and were analyzed in a 2-factor design with 4 estrous cycles as a subplot. To evaluate luteal development I used rate of increase (slope) of progesterone in serum during first 10 d postestrus of each cycle. Origin of 71 these slopes (onset of diestrus) was day when concentrations of progesterone exceeded baseline by 2 standard deviations. Slopes for progesterone within estrous cycle were contrasted among treatment combinations according to methods described by Gill (1978). Parallelism between slopes of two treatment combinations was interpreted as similar rates and (or) extent of luteal development. In contrast, lack of parallelism between slopes of 2 treatment combinations indicated different rates and (or) extent of luteal development. Concentrations of progesterone in medium were analyzed by split-plot analysis of variance with doses of LH as subplots. Results of this analysis will determine existence of independent or associative effects of EB and BC on luteal function _i_r_1_y_iy_9_ and(or) _ig _vi_t59_. Luteotropic support may be altered by EB and(or) BC. Insulin, GH and (or) NEFA likely are affected by EB, BC and(or) their interaction. Therefore, concentrations of NEFA in plasma and concentrations of LH, GH and insulin in serum were analyzed by analysis of variance for repeated sampling over time (Gill and Hafs, 1971). Furthermore, baseline concentrations, frequency and amplitude of pulses of LH, GH and insulin were calculated and then analyzed by analysis of variance for 2-factor experiments with fixed effects (Gill, 1978). Pulse of LH (Hughes et al., 1987) was an increase in concentrations of LH that exceeds a value of LH in previous 30 min by twice the within assay standard deviation (2 (0.55) = 1.1 ng/ml). Amplitude of pulses was the difference between nadir and subsequent maximal value reached during a pulse of LH. Frequency of pulses was the number of pulses per heifer occurring during an interval of 12 h. Basal LH was mean concentration of LH in all samples not included in pulses. Amplitude, frequency and duration of pulses of GH and insulin and basal GH were identified by the Pulsar program which uses alogarithms for pattern recognition (Merriam and Watcher, 1982). Basal insulin was set at mean concentrations in preprandial samples. 72 Energy balance and(or) BC may affect luteal function and metabolic factors (GH, insulin, NEFA). Are luteal function and metabolic factors associated? To determine associations (between luteal function and GH, insulin or NEFA in heifers, regression analyses were used in which mean concentrations of progesterone in serum at d of lutectomy was the dependent variable. Independent ‘ variables were mean concentration of GH, insulin and N EPA in blood of heifers. For area and peak of progesterone and duration of luteal phases within treatment combination, specific contrasts among estrous cycles were made by Bonferroni t statistics (Gill, 1978). These contrasts will identify specific interactions of EB with BC that alter progesterone over time in 1132. But for area, peak and duration of luteal phases within cycle, Dunnett's test (Gill, 1978) was used to contrast controls (MOD-PEB heifers) versus other treatment combinations. Results from these comparisons will be to. know specific interactions of EB with BC that alter progesterone within estrous cycle 111mg. To determine whether specific interactions of EB with BC affected luteal function ill m, basal and LH-induced progesterone in controls (MOD-PEB) were contrasted with other treatment combinations by Dunnett's test. Effects of specific interactions between EB and BC on luteal weight, LH, GH, insulin and NEFA were determined by contrasting treatment combinations with Bonferroni t test. For statistical models with split-plot structure, error terms and critical values for Dunnett's and Bonferroni t tests were modified as recommended by Gill (1986). Results Energy Balance and Body Measurements. As expected, EB was positive throughout the experimental phase (figure 4) in heifers that received diet A (table 4). In contrast, heifers offered diet C (table 4) were in PEB during the 73 Figure 4. Changes in energy balance, body weight and body condition of heifers over four estrous cycles. Heifers had moderate (O) or fat (0) body condition and were in positive (--) or negative (- - -) energy balance. Body condition at the beginning (I) of the experimental phase is included in the bottom panel. For each variable, treatment combination means within estrous cycle (or I) with different superscripts differ (a,b b9°P < .05 or 3’CP < .01). Pooled standard errors are: .6 for energy balance, .1 for body weight and .06 for body condition. ENERGY BALANCE (Meal/d), BODY CONDITION BODY WEIGHT (kg/d) 74 M o—W o I 50 \\ \\\ ESTROUS CYCLE 75 first experimental estrous cycle, approached EB during the second cycle and were in NEB during the third and fourth estrous cycles (figure 4). Parallelism between slopes indicates that rates of decline in EB were not different between MOD-NEB and FAT-NEB heifers. However, duration of NEB (46 :t .6 d) and net accumulated loss of energy (-98.1 at 12.6 Mcal) for FAT-NEB heifers were greater (P < .01) than for MOD-NEB heifers (38 :I: 2.8 d; -57.3 3: 4.1 Mcal). In agreement with changes in BB, MOD-PEB and FAT-PEB heifers gained weight during the experimental phase (figure 4). In contrast, MOD-NEB and FAT-NEB heifers gained weight during the first two estrous cycles, but lost weight during the third and fourth estrous cycles (figure 4). As indicated previously, BC at the beginning of the experimental phase (figure 4) differed (P < .05) between FAT (3 at .06; range 2.8 to 3.5) and MOD heifers (2.5 i .03; range 2.3 to 2.6). Heifers in PEB maintained initial BC with no significant change throughout the experimental phase (figure 4). In contrast, - BC of MOD-NEB and FAT-NEB heifers declined (P < .05) and was less (P < .05) than BC of corresponding control heifers (MOD-PEB and FAT-PEB, respectively) by the second experimental estrous cycle. Luteal Function in vivo. Discussion of effects of BC on luteal function or on any other dependent variable will refer to BC of heifers at onset of experimental phase. Otherwise, it will be indicated. Luteal weight was not influenced by EB or BC independently. But corpora lutea of FAT-PEB and MOD- NEB heifers weighed less (P < .05) than corpora lutea of MOD-PEB heifers (table 5). As single effects, EB or BC did not alter luteal function among estrous cycles (figure 5). Within first, second and third estrous cycle, EB or initial BC did not influence luteal function. But during the fourth estrous cycle, NEB heifers had less (P < .05) area and peak progesterone than PEB heifers and FAT heifers had less (P < .05) area and duration of luteal phase than MOD heifers 76 TABLE 5. EFFECTS OF ENERGY BALANCE AND BODY CONDITION ON WEIGHTS OF BOVINE CORPORA LUTEA.a Treatment combination n Luteal weightb', 8’ MOD-PEB 6 6.2 t 0.70 FAT-PEB 6 4.6 :l: 0.6d MOD-NEB 5 4.5 a: 0.6d FAT-NEB 3 5.1 :t 1.009d aCorpora lutea were collected on d 10, 11 or 12 postestrus of the fourth estrous cycle. bData are expressed as mean t standard error. c:dMeans with different superscript differ (P < .05). 77 Figure 5. Effects of energy balance and body condition on progesterone in serum and duration of luteal phase during the first 10 to 12 d postestrus of four estrous cycles. Heifers had moderate (O) or fat (0) body condition and were in positive (—) or negative (- - -) energy balance. Data are mean 1 standard error. For area, peak or duration means within an estrous cycle with different superscript differ (89b P < .10; 8’6 P < .05; and P < .01). 78 c 228 326w FIIITIIIITFIIIT .ZO_._._OZOU >oom #4.... 00.2 L _ L. ...... .H n £26 26th I’rITTITIIUIUIII‘ #4.". 00.2 L N 226 mace—mm Iriltlrrlrriilr‘ _ «.26 323m (m Mouvano BSVHd 1‘73an UuVOU)XV3d 3N0831$3908d (.-Iw ~p-DUI vaav 79 (figure 5). Therefore, during fourth cycle N EB and FAT independently exerted adverse effects on luteal function and there were no interactions. In contrast, during the first three cycles EB and BC did not exert independent effects on luteal function but several interactions were observed: relative to MOD-PEB heifers, MOD-NEB heifers had less (P < .05) area and peak progesterone from second throughout fourth estrous cycle (figure 5). But FAT-NEB heifers had less (P < .05) area, peak and duration of luteal phase than MOD-PEB heifers only during the fourth estrous cycle (figure 5). Except for third cycle, FAT-PEB had less (P < .05) area and peak progesterone during the study than MOD-PEB heifers. In addition, luteal phases of FAT-PEB heifers were shorter during first (P < .10) and fourth (P < .05) cycles than for MOD-PEB heifers (figure 5). Rate of increase of progesterone during the first 10 d postestrus (figure 6) did not differ among estrous cycles in MOD-PEB heifers. During the first two estrous cycles, MOD-NEB heifers had similar rates of increase of progesterone than MOD-PEB heifers. But during third and fourth estrous cycles progesterone of MOD-NEB heifers increased slower: (P < .05) than in MOD-PEB heifers (figure 6). Relative to values for MOD-PEB heifers, progesterone increased slower (P < .05) during all estrous cycles in FAT-PEB heifers (figure 6). Progesterone in FAT-NEB heifers increased at similar rates than in MOD-PEB heifers during the first three estrous cycles. But for the fourth estrous cycle, rate of increase of progesterone of FAT-N EB heifers was slower (P < .005) than in MOD-PEB heifers (figure 6). Luteal Function in vitro. Energy balance or initial BC of heifers did not influence basal secretion of progesterone by luteal cells _ig 3193 (figure 7). But the interaction between MOD and NEB affected adversely the ability of luteal cells to secrete basal progesterone _i_n y_i_t_1;<_>_. Evidence of this was that luteal cells from MOD-NEB heifers secreted less (P < .01) basal progesterone 80 Figure 6. Effects of energy balance and body condition on luteal development. Luteal development was measured by rate of increase of progesterone in serum during the first 10 d of four estrous cycles (for clarity only values for d 4 and 10 postestrus were plotted). Heifers had moderate (O) or fat (0) body condition and were in positive (-) or negative (- - -) energy balance. Coefficients of determination (r2) for regression equations of estrous cycles 1, 2, 3 and .4, respectively, were: .9, .8, .7 and .8 for MOD-PEB heifers; .4, .6, .7 and .2 for MOD—NEB heifers; .4, .5, .6 and .4 for FAT-PEB heifers; .6, .6, .6 and .1 for FAT-NEB heifers. Within estrous cycle, slopes with different superscript differ @fip_._.<4mm mi: N. n . QMZIhd... mum I #4... n _ o bk s .N r3 2 m h n n .0 bL - — p p n p p _ - \ um r0 r0. r . . . . . b . b . b . \ Elm 1w L. ro— muauoos. .2 (wmas )0 ("J/5U) ENOWBOH Hlmoas 88 Figure 9. Effects of energy balance and body condition on profiles of mean concentrations of insan in serum. Heifers were in MOD-PEB, FAT-PEB, MOD-NEB or FAT-NEB. Jugular serum was taken on d 9, 10 or 11 after the fourth estrus, during a 12 h period at intervals of 15 min. Pooled standard error was 2.9 uU/ml serum. INSULIN (uU/ml of serum) 89 70- - -I -( 60- . 504 MOD-PEB -I FAT-PEB 4o« .. so» - d 4 207! 7! I f I fir I—F F1 I I I I I I I I I I I I I I* 40‘ MOD-NEB FAT-NEB 30a 207, I I f I r I I I I I I If T I f I I r I I I T I I— o l 3 5 7 9 II o I 3 5 7 9 n TIME RELATIVE TO FEEDING (h) 90 interactions between EB and BC were observed for any measurements of insulin in serum. But during the first 3 h postprandial, amplitude (42.9 1: 4.2 uU/ml) and duration (137.5 1 39.8 min) of pulses of insulin in FAT-PEB heifers tended (P = .08) to be greater than in MOD-PEB heifers (amplitude = 30.6 t 6.4 uU/ml; duration = 76.3 :I: 20.1 min). As single effect, EB but not BC altered amplitude and duration of pulses of insulin during the first 3 h postprandial. During this interval, amplitude (36.7 3: 6.4 uU/ml) and duration (116.7 :t 3.1 min) of pulses of insulin in PEB heifers were greater (P < .01) than for NEB heifers (amplitude = 15.9 3: 4.2 uU/m; duration = 72.3 i 15 min). Body condition did not influence NEFA in plasma and there were no interactions of EB and BC. But heifers in NEB had higher (P < .01) mean concentrations of NEFA (404 i 120 qu/l) in plasma than PEB heifers (84 :I: 36.4 qu/l) Insulin and GH were not correlated significantly with concentrations of progesterone in serum at lutectomy. In contrast, concentrations of N EPA in plasma were correlated negatively (r = .52; P < .01) with concentrations of progesterone in serum at lutectomy. Discussion Heifers in PEB maintained BC and sustained daily gains with minimal variation throughout the experimental phase._ Independent of initial BC, PEB heifers received the same diet and similar amounts of feed per unit of body weight. Therefore, I was able to determine the independent influence of BC, MOD or FAT, on luteal function without bias by diet or EB. Within each category of initial BC, I produced heifers in sustained PEB during the entire study or in NEB for the last two estrous cycles. Thus it was possible to determine independent effects of EB and associative effects of EB with BC on luteal function. 91 In the present study, diets were formulated based on requirements for growth and maintenance from NRC (1984). Accordingly, diets given to NEB heifers were deficient in energy from the beginning of the experimental phase. But, during the first estrous cycle, heifers committed to NEB gained weight but when amounts of feed offered were reduced further, heifers lost weight. Retrospective chemical analysis of feeds indicated that TMR used contained more NEm than averages estimated from NRC (1984). Thus, calculated restrictions of dietary energy may not cause NEB in all cases due to variation of nutrients in' feeds and(or) in requirements for maintenance of heifers. These observations may explain variable effects of restricted intake of dietary energy on luteal function in previous studies in which NEB of heifers was presumed (Apgar et al., 1975; Beal et al., 1978; Gombe and Hansel, 1973; Hill et al., 1970; Spitzer et al., 1978). During NEB, losses of body weight paralleled decreases in EB of heifers (r = .72; P < .01). Therefore, if heifers are losing weight, dietary energy is insufficient to satisfy requirements for maintenance. Thus, a discrete marker for positive or negative EB is gain or loss of weight. Declines of BC paralleled decreases of EB of heifers (r = .54; P < .05) during NEB. Because BC is a measurement of subcutaneous fat, decreased BC during NEB indicates that concomitant losses of body weight are due, at least in part, to decreased fat. In fact, as cattle lose weight, up to 7096 of tissue lost is fat (Reid and Robb, 1971). During the fourth estrous cycle, NEB heifers had less progesterone in serum than PEB heifers and FAT heifers had less progesterone than MOD heifers. Therefore, NEB and FAT have independent adverse effects on luteal function. Because rate of increase of progesterone was reduced in MOD and FAT heifers, it is likely that NEB and FAT as independent effects inhibit development of corpora lutea. Moreover, luteal phases were shortened in FAT heifers. Reduced 92 duration of luteal phases during fourth estrous cycle was due to delayed onset of diestrus indicating that ovulation could be delayed in FAT heifers. Our results are consistent with earlier reports that spontaneous NEB reduces progesterone in serum of lactating dairy cows without altering duration of luteal phases (Experiment 1). Whether induced in heifers or spontaneous in lactating cows, NEB exerts adverse effects on luteal function. Reduced luteal weight at mid-diestrus from MOD—NEB and FAT-PEB heifers is consistent with adverse effects of NEB or FAT on luteal development. Perhaps reduced mass of luteal tissue diminishes total amount of progesterone secreted from corpora lutea and in part explains reduced concentrations of progesterone in serum of NEB and FAT heifers. From previous reports, effects of restricting dietary energy on luteal function were equivocal in heifers. Some researchers observed negative association between restricted dietary energy and progesterone in serum (Gombe and Hansel, 1973; Hill et al., 1970; Imakawa et al., 1983) but other workers did not (Beal et al., 1978; Spitzer et al., 1978). I have shown that restricting dietary energy does not guarantee NEB in heifers. Perhaps in studies where luteal function was normal during restricted dietary energy, heifers were not in NEB. But, in studies where luteal function was reduced, dietary energy was restricted sufficiently that heifers were in NEB. Thus, luteal function will probably be normal unless diet is restricted sufficiently to cause NEB in heifers. Duration of NEB and net energy deficit in FAT-NEB heifers were greater than in MOD-NEB heifers. During fourth cycle, it is interesting that FAT-NEB heifers had lowest numeric increase, smallest area and peak of progesterone and the shortest luteal phase of all treatment combinations. Consequently, secretion of progesterone is associated negatively with severity and(or) duration of energy deficit in heifers. 93 In NEB heifers, loss of body weight paralleled declines in EB. Large losses of body weight should indicate great deficits of energy and should be associated with more severely limited luteal function. However, I detected adverse effects of NEB on luteal function (area, peak and rate of increase of progesterone) as early as the second estrous cycle in MOD-NEB heifers, but not until the fourth estrous cycle in FAT-NEB heifers. Therefore, temporal associations between NEB and reduced luteal function depends apparently on BC of heifers when NEB starts. Interactions between levels of EB and FAT indicate other potential sources of variation for progesterone in serum of heifers. For example, compared to MOD-PEB heifers, FAT-PEB heifers had slower increases and lower peaks of progesterone during three estrous cycles, supporting the concept that FAT reduces luteal function independently. But in FAT-NEB heifers, adverse effects of FAT on luteal function during first to third estrous cycles did not exist or were masked by NEB. Perhaps the key to understand interactions between FAT and N EB is variation of BC throughout the study. Body condition and subnormal luteal function of FAT-PEB heifers were sustained during the experimental phase. But reduced intake of dietary energy caused BC of FAT-NEB heifers to decline to MOD. Because BC and consequently amount of fat in depots were reduced, adverse effects of excess fat on luteal function were removed. But NEB continued, so by fourth estrous cycle BC of FAT-NEB heifers decreased below MOD, with a concomitant reduction of all variables used to examine luteal function in 3129: Thus effects of NEB on luteal function of FAT-NEB heifers were delayed until BC. declined below MOD. An interpretation of these data is that effects of EB on luteal function are exerted indirectly by altering BC of cattle. 94 There were no independent or associative effects of EB or BC on mean concentrationsand pulsatile patterns of LH. Thus, adverse effects of NEB and FAT on luteal function are not due to limited luteotropic support. These results agree with previous reports that LH in serum of heifers was not reduced by restricted dietary energy (Hill et al., 1970; Spitzer et al., 197 8) or FAT (Spicer et al., 1984). But our results were determined at mid—diestrus and do not examine the possibility that periestrus secretion of LH is altered by NEB and(or) FAT as with restricted intakes of energy (Gombe and Hansel, 1973) or fasting in heifers (McCann and Hansel, 1986). As single effects EB or BC of heifers did not influence basal secretion of progesterone by luteal cells _ifl 11113. Body condition of heifers did not alter LH-induced secretion of progesterone from luteal cells _in $132. But NEB in heifers reduced secretion of progesterone in response to LH _i11_ 11132. How might N EB reduce LH-induced secretion of progesterone _ig £113? Corpora lutea are composed by small and large luteal cells (Alila and Hansel, 1984). Large luteal cells secrete most basal progesterone. Numbers of receptors for LH and LH—induced secretion of progesterone _ip_ FEES. are greater in small than in large luteal cells (Fitz et al., 1982). Because NEB did not alter basal secretion of progesterone from luteal cells _ig _v_i_t_r_o_, it is not likely that N EB affects steroidogenic activity of large luteal cells. But potential mechanisms by which NEB reduced LH-induced secretion of progesterone _in 11119 are: a) reduced numbers of small luteal cells, b) reduced numbers and(or) affinity of receptors for LH on small and possibly large luteal cells, c) altered postreceptor event(s) involved in LH-induced steroidogenesis or d) a combination of some or all the above mechanisms. Basal progesterone secreted _ifl 11112 by luteal cells from MOD-NEB heifers was less than in other treatment combinations. Thus, BC of heifers at the 95 beginning of dietary restrictions modulates effects of NEB on basal secretion of progesterone from luteal cells _12 11112. Consequently, NEB limits LH-induced secretion of progesterone and NEB interacts with MOD to reduce basal steroidogenic activity _in v_it_1_'_o. In the present study, NEB decreased progesterone in serum, reduced luteal weight and development, and reduced LH-induced secretion of progesterone _in 1132. Similarly, FAT decreased progesterone in serum reduced luteal weight and slowed luteal development but did not alter basal or LH-induced secretion of progesterone _in 111132. Thus, NEB and FAT affected luteal function _ifl lilo but only N EB affected luteal function 13m. Because NEB but not FAT reduced luteal function _12 1133, adverse effects of NEB and FAT on luteal function are apparently exerted through different mechanisms. In FAT heifers, low concentrations of progesterone in serum were associated with reduced luteal development with no apparent reduction in secretory activity of luteal cells. But in NEB heifers, low concentrations of progesterone in serum were associated with reduced luteal development and with reduced secretory activity of luteal cells. Pulsatile secretory activity of GH in serum from PEB heifers was unrelated with time of feeding and followed similar patterns than those reported previously (Sejrsen et al., 1981; Zinn et al., 1986). In the present study, FAT did not alter mean concentrations or pulsatile secretory patterns of GH in heifers. These results support previous observations that body composition does not influence GH in post-pubertal heifers (Sejrsen et a1., 1983; Zinn et al., 1986). But NEB increased concentrations of GH in heifers as in steers (Blum et al., 1985). Secretion of insulin in meal-fed cattle increased shortly after feeding (McAtee and Trenkle, 1971). In the present study, insulin increased to maximal concentrations within 3 h postprandial in PEB heifers. In contrast, relative 96 to basal levels, postprandial insulin did not change in NEB heifers. Thus NEB blocks postprandial increase of insulin in heifers. Response of insulin to feeding is modulated by energy status of cattle and by amount of energy in diet (Brockman and Laarveld, 1986; Lomax et al., 1979). McCann and Reimers (1986) observed that glucose-induced secretion and basal concentrations of insulin were greater in fat than in moderately conditioned heifers. In the present study, postprandrial concentrations of insulin tended (P = .08) to be greater in FAT than in MOD heifers. Heifers studied. by McCann and Reimers (1986) were older (2.5 to 4 yr of age) than heifers in our experiment (< 2 yr of age). In'obese individuals hyperinsulinemia progresses with advancing age and increasing obesity (McCann and Reimers, 1986). Thus FAT heifers used in the present study could be in early stages of hyperinsulinemia. Concentrations of NEFA in plasma of NEB heifers were greater than in PEB heifers. This is in agreement with earlier research in steers (Blum et al., 1985). Body condition did not alter concentrations of N EPA in plasma of heifers and there were no interactions of BC with EB. To my knowledge this is .the first report of effects of BC on N EPA in plasma of cattle. Consistent with adjustments expected during energy deficit, NEB heifers had increased concentrations of GH and N EFA but reduced insulin in blood. It was of interest to examine, by correlation, the possibility that GH, insan and (or) NEFA mediate adverse effect of NEB on luteal function. Lack of significant correlations between progesterone in serum and insulin or GH could be interpreted that changes in GH and insulin are not involved in reduced luteal function during NEB. But insulin exerts positive effects on luteal function _111 3332 (McCann, 1984) and 19.1312 (Savion et al., 1982), and GH antagonizes actions of insan in some tissues (Rizza et al., 1982). Thus, it would seem fruitful to test directly the hypotheses that decreased secretion of insulin or antagonism of insan by CH mediate adverse effects of NEB on luteal function. 97 Increased concentrations of NEFA in plasma of NEB heifers correlated negatively with progesterone in serum. This agrees with data indicating a negative association between NEFA in plasma and reproductive performance in dairy cows (Ducker et al., 1985). Negative associations between NEFA and progesterone may be causative or coincidental. Overall I determined that NEB and FAT independently reduced luteal function of heifers 111 3332- Because LH in serum was not reduced by EB or BC, adverse effects of NEB and FAT on luteal function are not due to reduced luteotropic support. Negative EB and FAT slowed rate of increase of progesterone in serum and this was interpreted as reduced luteal development. Adverse effects of N EB were first detected during second cycle in MOD heifers but not until fourth cycle in FAT heifers. Basal secretion of progesterone _in _v_i_t_rg was not influenced by EB or BC. But basal progesterone _ig mg was reduced in MOD-NEB heifers. Thus initial BC of heifers determined time from initial dietary restriction when adverse effects of NEB on luteal function occurred _i11_ 11313 and determined effects of NEB on basaL. secretion of progesterone _in 1111-2. FAT had no effect but N EB reduced LH-induced secretion of progesterone _i_n m. In FAT heifers, low progesterone in serum was associated only with reduced luteal development with no apparent effect on activity of luteal cells. But in NEB heifers, low progesterone in serum was associated with reduced luteal development and reduced secretory activity of luteal cells. Because NEB but not FAT reduced luteal function 111 11152, effects of NEB and FAT on luteal function are apparently exerted through different mechanisms. My primary conclusion is that N EB and FAT exert independent limitations to luteal function. Apparently, adverse effects of NEB and FAT on luteal function are exerted through different mechanisms. In addition, BC of heifers determines temporal manifestation of adverse effects of NEB on luteal function. EXPERIMENT III: Influence of Energy Balance and Body Condition on Behavior of Heifers During Estrus 98 EXPERIMENT III: Influence of Energy Balance and Body Condition on Behavior of Heifers During Estrus Introduction Estrus is detected in association with 50 to 6496 of ovulations when dairy cows are observed two (King et al., 1976; Williamson et al., 197 2) or three times per d (Experiment 1). Under constant surveillance, estrus is associated with 94 to 10096 of second and third postpartum ovulations (Hurnik et al., 1975). Because farmers commonly observe cows twice daily, a high proportion of estrus is undetected in dairy herds. Together, undetected and inaccurately detected estrus lower fertility (Macmillan and Watson, 1971; Pelissier, 1978), prolong postpartum intervals to conception and represent a major source of economic loss to dairy farmers (Barr, 1974; Esslemont, 1974; Williamson et al., 1972). Many methods to improve detection of estrus have been developed (Foote, 1975; Kiddy, 1977; Holman et al., 1987). But failure to detect estrus still limits reproductive performance in dairy cows. Some workers proposed that inefficiency of farmers is the leading cause of undetected estrus (Pelissier, 1976; Zemjanis et al., 1969). But a large range in duration of estrus among dairy cattle, 3 to 21 h (Glencross et al., 1981; Hurnick et al., 1975), indicates that there are biological sources of variation for expression and detection of estrus. At least 8096 of dairy cows are in negative energy balance during early lactation (Reid et a1., 1966; Experiment I). Homeorhetic mechanisms insure metabolic support for lactation during negative energy balance (Bauman and Currie, 1980). My hypothesis is that homeorhetic events of postpartum dairy cows may limit expression and detection of estrus. 99 100 Cows that are overconditioned at parturition ingest less feed and energy during early lactation than moderately conditioned cows (Garnsworthy et al., 1982). Intake of dietary energy is a major source of variation of energy balance in postpartum dairy cows fed ad libitum (Experiment 1). Thus overconditioning may enhance negative energy balance and indirectly alter expression of estrus in lactating cows. In lactating dairy cows, display of behavioral estrus in association with ovulation varies among postpartum estrous cycles (Experiment I). Effects of successive estrous cycles may interfere with detection of other sources of variation of expression of estrus. In heifers these interfering factors do not exist. Thus, this experiment was designed to determine independent and associative effects of energy balance and body condition on estrous behavior of heifers. Collaterally, I examined the influence of energy balance, body condition and their interactions on duration of estrous cycles. Materials and Methods Twenty nulliparous, Holstein heifers were grouped by body weight and assigned to treatments within a 2 x 2 factorial experiment. Main effects were: 1) body condition (BC): moderate or fat, and 2) energy balance (EB): positive or negative. Materials, and general procedures regarding housing, nutrition and measuremetns of EB, BC and body weight were as described previously 1 (Experiment II). Briefly, heifers were housed in a free stall barn equipped for individual feeding. Before the study heifers were fed so BC (1 to 4; 4 = fat) remained moderate (MOD = 11) or became fat (FAT; 11 = 9). After approximately 6 mo of these regimens, BC of MOD and FAT heifers differed (P < .05). Subsequently, during 20 d of adaptation, FAT and MOD heifers were trained to use gang-lock stanchions and to consume their daily dietary allowance within 101 2 intervals of 90 min (0500 to 0630 and 1700 to 1830 h). This allowed me to determine intake of DM and EB daily. But during 21 h per d, heifers were not confined and were free to exhibit all signs of estrus. Pubertal status of heifers was determined by two daily observations for estrus (0700 to 0730 and 1900 to 1930 h), and by rectal examination of reproductive organs at least twice during the adaptation period. Thus at the end of the adaptation period, heifers with two distinct BC (MOD or FAT) and that were postpubertal were available. The experiment lasted 3.5 estrous cycles and ended between d 10 and 12 after the last estrus. Throughout the experiment, 6 MOD heifers and 6 FAT heifers were fed (Experiment H; table 4) to gain weight and to be in positive EB (PEB). The remaining MOD (n = 5) and FAT (11 = 3) heifers received a- diet (Experiment II; table 4) to satisfy requirements for protein, vitamins and minerals but was limited in energy to produce and sustain negative EB (NEB). Thus, from the’ beginning of the experiment four treatment combinations were established: MOD-PEB (n = 6), FAT-PEB (n = 6), MOD-NEB (n = 5) and FAT-NEB (n = 3). To estimate EB, feed intake was measured daily and body weight weekly throughout the experiment. Daily body weight was calculated by extrapolation of weekly changes in body weight (weekly change/7). Throughout the experiment, EB was estimated daily for each heifer. Energy balance was calculated by subtracting requirements of energy for maintenance from intake of energy. Requirements of net energy for maintenance (NEm) were based on daily body weight and calculated as follows: NEm (Mcal) = (.077) body weight-75 (NRC, 1984). Daily intake of energy resulted from multiplying NEm in feeds by intake of DM. Body condition was scored in alternate weeks using a scale of 1 to 4 (4 = fat) according to methods developed by Mulvany (1981) and modified to determine BC in heifers (Experiment II). 102 westerone in Serum. Jugular blood was sampled daily by venipuncture. Progesterone was quantified in serum from all these samples by radioimmunoassay (Appendix A). Concentrations of progesterone in blood were used to define diestrus (see Duration of Estrous Cycles) and to define estrus (see Estrous Behavior). Estrous Behavior. Heifers were housed in two separate but adjacent pens so heifers in the two pens were observed simultaneously. One pen contained MOD-PEB and FAT-PEB heifers and the other pen held MOD-NEB and FAT-NEB heifers. For 3 consecutive estrous cycles (3 periods of estrus), heifers were observed daily for periods of 30 min at intervals of 3 h (beginning at 0100 h). Events recorded during periods of observation included standing to be mounted (_>_ 2 sec), mounting and mounted but not standing. A heifer was considered to be in estrus when standing behavior coincided with concentrations of progesterone < 1 ng/ml of serum. Duration and frequency of mounting is correlated positively with number of animals up to 5 in estrus simultaneously (Helmer and Britt, 1985; Hurnick et al., 1975). Number of animals in estrus simultaneously could bias analysis of effects of EB and(or) BC on estrous behavior. Therefore, during each period of observation, number of heifers standing and(or) mounting during each period of observation was recorded and used as a covariate in statistical analyses of behavioral data related with estrous behavior. Duration of Estrous Cycles. I examined the influence of EB, BC and their interactions on duration of complete estrous cycles and selected stages of estrous cycles. An estrous cycle was the interval between onset of two consecutive periods of estrus. Diestrus was the interval within an estrous cycle when progesterone in serum exceeded basal concentrations of progesterone by two standard deviations (.125 + .09 (2) = .3 ng/ml). Basal progesterone was the mean 103 Figure 10. Characterization of total activity, intensity, duration and accuracy of estrus in heifers. Onset of estrus was when first standing event was observed during basal progresterone. Duration was the interval from first to last (END) standing or mounting event during basal progesterone per estrus. Area under frequency distribution of standing or mounting was used to measure total activity. - Intensity (PEAK) was maximal number of events (standing or mounting) within area. Accuracy was the percentage of standing or mounting events within 12 h from onset of estrus. All behavioral data were collected when progesterone in serum was basal (< 1 ng/ml). 104 :E\O:.A. EDOI :E\o:.V. mzommpmmooma m... . o mzomuemmooma O n O S Y L V\\\\\\ .. w. ON a 53.384 1.. N M \ I . O R I ozm Emzo o u. u 1 C om .. I w m .. low M N 00... G I ma xh.>_._.U< 45.0% 2\m.co>ov xdma 2: zo_._.U._._>_._.o< 43.0% 2\mEo>3 xU 0.10) slope of standard curves (figure 17) between .02 and .4 ng of insulin/tube (b = -11.0; r2 = .99). Coefficient of variation (CV) of insan within dilution of serum from fasted heifers was 6.296 whereas interdilution CV was 6.596. For insan in serum of fed heifers, within dilution CV was 4.596 and interdilution CV was 13.396. From five assays, intraassay CV was 7.696 and interassay CV was 13.796. Specificity of antibody against bovine insan was determined by testing the degree of cross-reactivity with 6 hormones (figure 18). Bovine GH (purified, 15825-AJP-152, Upjohn, Kalamazoo, MI), bovine LH (NHI-LH-B8), bovine FSH (BP3, USDA), bovine TSH (NIH-TSH-BS), bovine prolactin (NIH-PRL-B4) or bovine-porcine glucagon (Sigma Chemical 136 Co., St. Louis, MO), in amounts up to 50 ng/tube did not cause significant displacement of 125I-insulin ((196 cross-reaction). Recoveries from serum supplemented with .1, .5 and 1.0 ng of insulin/tube (4 replicates each) averaged 97.6 1 2.1. Samples of jugular blood were collected from heifers every 15 min during an interval of 12 h surrounding a meal. From each sample (n = 1000), duplicates of 200 111 of serum (diluted in 0.5 M phosphate buffer, .596 bovine serum albumin, .996 NaCl, pH 7.3) were quantified in a single radioimmunoassay. Average concentration of insulin for all samples was .93 1 .02 ng/ml (25.1 uU/ml) and range was .26 to 5.01 ng/ml (6.99 to 134.8 uU/ml). 137 Figure 16. Elution pattern of 125I-bovine insulin. Bovine insulin and 125I-iodide were mixed and eluted through a 10 (1/10) ml column of bio-gel P-60. Before elution, iodination reaction was initiated by chloramine—T and stopped 15 sec later by adding sodium metabisulfate. Volume of elution fractions was 1 ml. Radioactivity was determined during .10 of a min in a 20 III aliquot from each elution fraction. Fractions 7 and 8 contained 125I-bovine insulin whereas fractions 10 and 11 contained free 1251. 137 Figure 16. Elution pattern of 125I-bovine insulin. Bovine insulin and 125I-iodide were mixed and eluted through a 10 (1/10) ml column of bio-gel P-60. Before elution, iodination reaction was initiated by chloramine-T and stopped 15 sec later by adding sodium metabisulfate. Volume of elution fractions was 1 ml. Radioactivity was determined during .10 of a min in a 20 111 aliquot from each elution fraction. Fractions 7 and 8 contained 125I-bovine insulin whereas fractions 10 and 11 contained free 1251. 138 Radioactivity (counts/.10 min 1 1000) 400 350 300 250 200 150 100 50 F- P Elution Fraction 139 Figure 17. Displacement of 125I-bovine insulin from antibody for bovine insulin by different dilutions of pooled bovine sera. Slope for eight standard curves of insulin (0) paralleled (r2 = 9) slope for regression line of insan in dilutions of pooled serum from fasted (D; 42 replicates) or fed heifers (A; 36 replicates). 140 .3259: 2382. O.N O._ m. m... N. 9. _. DO. ONO. _ . _ _ _ _ _. — — _ o o 08 ow. 8.8 om oc ow /o/ Dd .3223 :3me I o/ ./_u .8 4, ID I o 4/ /D / IO? /