i‘ ‘I ‘ RDQIE» rams. 1 “u; .‘ .3: ‘ at; . '5» ‘ it} “‘1‘“ 111111111111111111111111111111111111 3 1293 014110633 This is to certify that the thesis entitled Effect of Energy Balance and SomatotrOpin on Serum Insulin-like Growth Factor-I and on Height and Progesterone of Corpus Luteun in Heifers presented by Hee-Chiann Yung has been accepted towards fulfillment of the requirements for M. S. degree in Animal Science 11111. .\ jajor professor ‘1 [hue January 13, 1995 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution UBRARY Michigan State University PLACE II RETURN BOXtomnovombchockouflun yourncond. TO AVOID FINES Mum on or More data duo. DATE DUE DATE DUE DATE DUE MSU loAn mum Maud Opportunity Institution Wan-9.1 EFFECT OF ENERGY BALANCE AND SOMATOTROPIN ON SERUM INSULIN-LIKE GROWTH FACTOR-I AND ON WEIGHT AND PROGESTERONE OF CORPUS LUTEUM IN HEIFERS By Mee-Chiann Yung A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1995 ABSTRACT . EFFECT OF ENERGY BALANCE AND SOMATOTROPIN ON SERUM INSULIN-LIKE GROWTH FACTOR-l AND ON WEIGHT AND PROGESTERONE OF CORPUS LUTEUM IN HEIFERS By Mee-Chiann Yung Holstein heifers (2 13 months old) were synchronized for estrus and fed individually 68% (n=14; NEB) or 139% (n=l6; PEB) of maintenance energy requirement during four estrous cycles. On day z 12 of third estrous cycle of each heifer, prostaglandin F20: was given, and starting the next day, half the heifers were injected daily with bST (14 mg/d) until the corpus luteum was collected on day 10 after estrus. Concentration of IGF-I in serum was 29% less (P < 0.01) in control NEB heifers than in control PEB heifers. Injection of bST increased serum IGF-I in both energy balance groups, but the increase was greater (P < 0.01) in heifers in PEB (73%) than those in NEB (35%). Weight of corpus luteum in PEB control animals was greater (P < 0.01) than that of NEB controls but not affected by exogenous bST. Serum IGF-I and luteal weight were not correlated significantly. In summary, bST injection during luteal development restored serum IGF-I of NEB heifers to the concentration observed in PEB controls but it had no effect on luteal growth in NEB or PEB heifers. We conclude that low serum IGF-I concentration during luteal growth is not the reason that NEB depresses luteal growth. To My Parents & Yu-Tsai Wang iv Acknowledgements I would like to thank my major professor, Dr. VandeHaar, for his encouragement, guidance and patience. This thesis would not have been possible without his help. I would like to thank the members of my committee, Dr. Roy Emery, Dr. Ralph F ogwell, and Dr. Thomas Herdt for their advice and expertise during my program. I thank Dr. Bal Sharma for his assistance and instruction in the lab. I also thank Mr. Greg DeMarco for assistance during the study. Also, I thank fellow graduate students: Paul Dyk, Yousif Gala], Roy Radcliff, and Christa Cook, for their assistance. My sincerest gratitude is to my family and friends. I thanks my parents for their support and love throughout all these years. Also thanks to my sister Christianne Yung, brother-in-law Steve Bessen, brother Teh-Ythung, and best fiiend Mei-Hui Chen, for their encouragement. Finally, I thank Yu-Tsai Wang for his continuous encouragement and support. v TABLE OF CONTENTS LIST OF TABLES ............................. ix LIST OF FIGURES ............................. x LIST OF ABBREVIATIONS ....................... xi INTRODUCTION .............................. 1 LITERATURE REVIEW .......................... 5 Negative Energy Balance ....................... 5 The Eflects of Negative Energy Balance on Conception . . . .5 Negative Energy Balance, Insulin-like Growth Factor-1 and Ovaries .6 Possible Role of Insulin-like Growth F actor-I ......... 6 IGF-I and Follicular Biology .............. 8 Follicular IGF-I System During Negative Energy Balance ................ 11 IGF-I and Luteal Biology ............... 12 Luteal lGF—l System During Negative Energy Balance ................ 14 Possible Role of Other Hormones and Metabolites ...... 14 Somatotropin ..................... 14 Progesterone ...................... 1 5 Luteinizing Hormone ................. l7 Insulin ......................... 18 Non Esterified Fatty Acids ............... 18 The Effect of Exogenous Somatotropin on Reproductive Function . l9 Somatotropin and IGF-1 ................... l9 Somatotropin and Reproduction ............... 20 vi Summary .............................. 22 MATERIALS AND METHODS ..................... 24 Animals and Diets .......................... 24 Energy Balance ........................... 28 Collection of Blood Samples .................... 30 Collection of Corpora Lutea .................... 3O Radioimmunoassay of Insulin-like Growth F actor-I ......... 3O Radioimmunoassay of Progesterone ................. 31 Radioimmunoassay of Somatotropin ................ 33 Enzymatic Analysis of Non Esterified Fatty Acids ......... 33 Determination of Luteal Protein .................. 33 Statistical Analysis ......................... 34 RESULTS ................................. 36 Body Weight and Energy Balance .................. 36 Concentration of Non Esterified Fatty Acids in Serum ....... 36 Concentration of Somatotropin in Serum .............. 4O Concentration of Insulin-like Growth Factor-1 in Serum ....... 4O Concentration of Progesterone in Serum .............. 43 Weights of Corpora Lutea ...................... 43 Content of Protein in Corpus Luteum ................ 47 Concentration of Progesterone in Corpus Luteum .......... 47 Content of Progesterone in Corpus Luteum ............. 47 DISCUSSIONS .............................. 5 l Weights of Corpora Lutea ...................... 51 Progesterone in Corpora Lutea and Serum .............. S4 Luteal Progesterone ..................... 54 Serum Progesterone ..................... 56 Somatotropin and Insulin-like Growth Factor-1 ........... 59 CONCLUSION .............................. 62 vii APPENDICES ............................... 63 Appendix A. The daily feeding sheet ................ 63 Appendix B. The radioimmumoassay of IGF-I ........... 64 LIST OF REFERENCES ......................... 68 viii LIST OF TABLES Table 1. Composition of diet ........................ 27 Table 2. Body weight (BW), body weight change and final body condition score of heifers in positive (PEB) or negative (NEB) energy balance for four consecutive estrous cycles ............ 38 Table 3. Least square means of senim progesterone, luteal progesterone, and luteal protein content ....................... 44 ix Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. LIST OF FIGURES The somatotropic cascade ..................... 3 Time-line of experiment .................... 26 Average weekly body weight of heifers in positive (PEB) or negative (NEB) energy balance diets .............. 37 Least squares means of the concentration of NEF A in serum at d 9 of the fourth estrous cycle ................. 39 Least squares means of the concentration of somatotropin at d 9 of the fourth estrous cycle ................. 41 Concentration of IGF-I in serum during the fourth estrous cycle ............................... 42 Least squares means of the weight of corpus luteum at d 10 of the fourth estrous cycle .................... 45 The correlation of the weight of corpus luteum with average concentration of IGF-I in serum from d 0 to d 10 of the fourth estrous cycle in control and bST-treated heifers ......... 46 Least squares means of total luteal progesterone content at d 10 of the fourth estrous cycle ..................... 48 Figure 10. The correlation of the total luteal progesterone with average concentration of IGF-I in serum from d 2 to d 10 of the fourth estrous cycle in control and bST-treated heifers ......... 50 X bST BW CL DM EB FSH IGF-I LH mRNA NEB NEFA NEm PEB PGFh ST LIST OF ABBREVIATIONS Bovine somatotropin Body weight Corpus luteum or corpora lutea Dry matter Energy balance Follicle stimulating hormone Insulin-like growth factor-I Luteinizing hormone Messenger ribonucleic acid Negative energy balance Non esterified fatty acids Net energy for maintenance Positive energy balance Prostaglandin F2, Somatotropin xi INTRODUCTION Delayed conception extends the proportion of time cows spend in late lactation and the dry period and thus decreases milk profits. A causative factor of delayed conception is negative energy balance (Houghton et al., 1990; Villa- Godoy et al., 1988). At least 92% of dairy cows experience negative energy balance during early lactation (Reid et al., 1966; Coppock et al., 1974). During negative energy balance, homeorhetic mechanisms ensure metabolic support to the mammary gland for milk synthesis. This focus on mammary support could limit nutrient supply to ovaries and thus decrease the activity of the corpus luteum. The corpus luteum produces progesterone, which is necessary for maintenance of pregnancy. Negative energy balance also decreases the concentration of progesterone in blood and milk of lactating cows (Spicer et al., 1990; Villa-Godoy et al., 1988). Decreased luteal activity is indicated by decreased luteal weight, luteal concentration of progesterone and sermn progesterone concentration in heifers (Terhune, 1992; Villa-Godoy et al., 1990). 2 A possible mediator of negative energy balance on reproduction is insulin-like growth factor-I (IGF-I), a peptide that mediates many actions of somatotropin (Baxter, 1986). Somatotropin and IGF-I are regulated by nutritional status (Ronge et al., 1988), and the concentration of IGF-I is positively correlated with the concentration of progesterone in serum of cows (Spicer et al., 1990). Furthermore, adding IGF-I to granulosa cells in vitro enhances some of the effects of follicle stimulating hormone and luteinizing hormone (Adashi et al., 1991; Hammond et al., 1991; Giudice, 1992). The major source of serum IGF-I is the liver, but IGF-I is also produced by many other tissues including granulosa cells and luteal cells (Hammond et al., 1985; Einspanier et al., 1990; VandeHaar et al., 1995). Synthesis of IGF-I by ovarian cells raises the possibility that IGF-I may act through an autocrine and/or paracrine as well as an endocrine role. However, in this thesis, only the endocrine role will be examined (Figure 1). In vitro, IGF-I enhances progesterone secretion in murine (Adashi et al., 1985), bovine (Schams et al., 1988) and porcine (V eldhuis et al., 1985 ) granulosa cells. IGF-I also stimulates progesterone secretion of bovine luteal cells collected within 1-5 day post- mtrusand incubated for 72 hours (McArdle and Holtorf, 1989). In addition, in vitro perfusion of IGF-I to bovine luteal cells increases progesterone secretion Pituitary Gland V . Somatotropin Negative Nutritional Status IGF-I’ l 5' v Ovarian F olliclc or Corpus Luteum — Affect May affcc I V IGF-I Figure 1. The somatotropic cascade 4 (Sauerwein et al., 1991). In lactating cows, exogenous bovine somatotropin (bST) increases the concentration of IGF-I and progesterone in blood (Gallo and Block, 1989; Schemm et al., 1990). The objective of this thesis was to test the hypothesis that serum lGF-I mediates the effect of energy balance on ovarian function and to determine the effect of exogenous bST during the luteal phase on luteal weight and luteal progesterone content. I postulated that bST would increase the concentration of IGF-I in serum of heifers in negative energy balance to the normal concentration of heifers in positive energy balance and thus provide enough IGF-I to the ovary to support normal luteal growth. If so, then perhaps I could alleviate the impaired luteal development in cattle in negative energy balance by injection of bST just before estrus to restore concentrations of IGF-I in serum to normal. To avoid the effect of energy balance confounding with postpartum interval, lactation, sensitivity of the anterior pituitary gland to luteinizing hormone releasing hormone, or reproductive diseases in dairy cows, pubertal heifers were used to determine these effects of bST and negative energy balance on luteal development. LITERATURE REVIEW Negative Energy Balance Energy balance (BB) is. defined as the difference between the energy intake of an animal and its energy expenditure. Energy lost from the body includes the loss of heat, urine, feces, and hair growth. When the energy intake exceeds the losses, an animal is in positive energy balance (PEB). On the other hand, when the energy intake is lower than the animal's energy expenditure, an animal is in negative energy balance (NEB). The Eflects of Negative Energy Balance on Conception Most dairy cattle experience NEB in early lactation because of the insufficient intake of energy for production of milk (Coppock, 1985). Cows which are in severe NEB take a longer time to return to normal cycling in the early postpartum period (Staples et al., 1990). Butler et al. (1981) reported that ovulation and initiation of the first normal luteal phase occurred approximately 10 days after EB began returning toward a positive value. This result of Butler 5 6 et a1. (1981) agrees with the observation by Lucy et a1. (1991) that higher EB is associated with more ovulation in dairy cows in early lactation. Moreover, restricted dietary intake in postpartum cows increases the interval from calving to first ovulation (Butler and Smith, 1989), first estrus (Whitemore et al., 1974) and conception (Haresign, 1979) when compared to ad libitum intake. Also in another study, postpartum cows that were fed a higher energy diet required fewer inseminations per conception and conceived 19 days earlier than those fed a lower energy diet (F ohnan et al., 1973). Thus, NEB could delay conception in lactating cows. Negative Energy Balance, Insulin-like Growth Factor-I and Ovaries Possible Role of Insulin-like Growth F actor-I Mature IGF-I is a single-chain polypeptide of 70 amino acids that is structurally similar to proinsulin and contains B and A domains which are homologous to those of insulin. Liver is the main source of circulating IGF-I, and IGF-I from liver was originally proposed to mediate the somatogenic actions of somatotropin (ST) (Salmon and Daughaday, 1957). However, IGF—I protein and IGF-I mRNA are also found in many other tissues (D'Ercole et al., 1984; 7 Lund et al., 1986). The local production of IGF-I is not only regulated by ST but also by other modulators such as follicle stimulating hormone (F SH) and luteinizing hormone (LH) in ovaries (Giudice, 1992) or is independent of ST (Murphy and Friesen, 1988). Multiple production sites and various regulators of IGF-I raise the possibility that in addition to endocrine actions, IGF-I may have autocrine and/or paracrine roles in tissues. Actions of IGF-I tentative influence both positively and negatively by a family of binding proteins that are found in the circulation and in extracellular fluids (Baxter et al., 1989). Binding proteins modulate the action of IGF-I through prolonged half-life of IGF-I and variation of binding of hormone to its receptor (Giudice. 1992). Thus, concentrations of IGF-binding proteins can modulate the action of IGF-I. In lactating dairy cows, the level of IGF-I in serum is positively correlated with EB (Ronge et al., 1988). Steers (Breier et al., 1986), heifers (V andeHaar et al., 1995), and cows (Shanna et al., 1994; Rutter et al., 1989) that are in NEB have lower concentrations of IGF-I in serum than those animals in PEB. As in cattle, concentrations IGF-I in plasma are decreased in underfed humans (Clemmons et al., 1981), rats (Macs et al., 1983), dogs (Eigenmann et al., 1985) and sheep (Pell et al., 1993). Decreased concentrations of IGF-I in blood during 8 underfeeding may signal to reduce anabolic reactions and to focus on survival rather than production. IGF-I and Follicular Biology Granulosa and thecal cells are the steroidogenic cells of a dominant ovulatory follicle. After ovulation and during luteinization these follicular cells become the large and small steroidogenic cells, respectively, of the corpus luteum (CL; Median et al., 1990). During early diestrus (d 4 to d 6), granulosa cells develop into large luteal cells and thecal cells develop into small luteal cells. In mid-diestrus (d 10 to d 12), a portion of the small luteal cells develop into large luteal cells. Since granulosa and thecal cells are the parental cells for luteal cells, the variation in number or function of the parental cells could contribute to the variation in number or steroidogenic ability of luteal cells. Removal of granulosa cells from preovulatory follicles in heifers (Milvae et al., 1991) and monkeys (Kreitrnann et al., 1981; Marut et al., 1983) reduces serum progesterone concentration in the succeeding luteal phase. Furthermore, precocious induction of ovulation decreases the concentration of progesterone in serum in the succeeding luteal phase of ewes compared to control group (Murdoch et al., 1983). Presumably, decreased development of preovulatory 9 follicles would decrease the number of follicular cells and would decrease subsequent luteal development. Bovine (Spicer et al., 1993) and swine (Hsu et al., 1987; Hammond et al., 1985 ) ovarian granulosa cells synthesize immunoreactive lGF-I and specific binding proteins for IGF-I. In addition, there is a positive correlation (r = 0.69) between IGF-I in serum and follicular fluid (Echtemkamp et al., 1990), and concentrations of IGF-I are significantly lower in follicular fluid than in serum of cattle (Echtemkamp et al., 1990), pigs (Hammond et al., 1988) and humans (Owen et al., 1991). Thus, IGF-I from serum may also be a source of IGF-I in follicular fluid. Concentrations of IGF-I in blood are unrelated to the stage of the estrous cycle (Huang et al., 1992); however, concentrations of IGF-I in follicular fluid increase with follicular size (Spicer et al., 1988; Hammond et al., 1988). Hammond et al. (1985) observed that the concentration of IGF-I in follicular fluid is greater in preovulatory follicles than in immature follicles. In contrast, Rutter and Manns (1991) observed no relationship between concentrations of IGF-I in follicular fluid and size of follicles in postpartum beef cows. However, most studies show that the size of follicles is positively correlated with the 10 concentration of IGF-I in follicular fluid but the cause-effect relationship between this two is not clear. For IGF-I to act on a tissue, receptors for IGF-I must be present. Type I IGF receptors are found in rat and ovine granulosa cells (Monget et al., 1989; Adashi et al., 1988). Type I IGF receptors bind IGF-I, IGF-H and insulin, but the affinity for IGF-I is 50 to 100 times greater than the affinity for IGF-II or insulin (Rechler and Nissley, 1985). The number of IGF-I receptors per cell on granulosa cells of ovine antral follicles is not influenced by the size of follicles (Monget et al., 1989). In vitro, IGF-I increases ovine granulosa cell multiplication both in small and large follicles (Monniaux and Pisselet, 1992). IGF-I also stimulates the secretion of progesterone of bovine granulosa cells (Schams et al., 1988). Cholesterol is the precursor of progesterone and its uptake also is positively affected by IGF-I. IGF-I increases F SH-stimulated aromatase activity and binding of the LH receptor to LH of rat granulosa cells (Adashi et al., 1985). Additionally, IGF-I also increases basal and FSH-induced secretion of progesterone of ovine granulosa cells, (Monniaux and Pisselet, 1992). Furthermore, both P SH and LH enhance IGF-I binding to its receptor in rat granulosa cells (Adashi et al., 1986). Thus, IGF-I, through changes in receptor 11 binding, may modulate some of the effects of FSH and LH on ovaries. IGF-l augments the absolute rates of biosynthesis of progestin in swine granulosa cells by enhancing four mechanisms: 1) de novo synthesis of cholesterol in granulosa cells, 2) cellular uptake of low and high density lipoprotein-bome sterol substrate, 3) activity of cholesterol side-chain cleavage, and 4) turnover of cellular cholesteryl esters (Veldhuis et al., 1989). Concentrations of IGF-I in dairy cattle vary with age and stage of lactation (Abribat et al., 1990). In heifers, IGF-I in serum increased at puberty (Jones et al., 1991), but heifers that have delayed puberty because of malnutrition have lower concentrations of IGF-I (Granger et al., 1989). In addition, cattle selected for high twining frequency have 47% greater concentrations of IGF-I in peripheral blood than those of normal cattle (Echtemkamp et al., 1990). These studies suggest that IGF-I is associated with reproductive function in cattle. Follicular I GF -I System During Negative E nergv Balance Severe restriction of dietary energy intake in postpartum cows decreased follicular fluid volume in medium-sized (7-9.9 mm in diameter) and large-sized (> 10 mm) follicles (Rutter and Manns, 1991). Restricted dietary energy also negatively influences the development of dominant follicles. Low dietary intake 12 decreases the diameter and persistency of dominant follicles during the estrous cycle of beef heifers (Murphy et al., 1991; Burns et al., 1994). Savio et al. (1993) suggested that the decreased persistency of the dominant follicle is caused by low concentrations of progesterone in plasma and is partly responsible for the impaired fertility of heifers during NEB. Short-term fasting significantly decreases concentrations of IGF-I in plasma but does not affect concentrations of IGF-I in follicular fluid of heifers (Spicer et al., 1992). Severe NEB for 35 days, which normally would decrease serum IGF-I, does not affect concentrations of IGF-I and progesterone in follicular fluid of beef cows (Rutter and Manns, 1991). Granulosa cells of rats (Oliver et al., 1988), cattle (Spicer et al., 1993) and swine produce IGF-I (Hsu et al., 1987; Hammond et al., 1985 ). If local production of IGF-I is a major source of intraovarian IGF-I, these data suggest that the regulation of IGF-I synthesis is different in ovary and liver, so that the concentrations of IGF-I in serum and follicular fluid do not decrease in parallel during NEB. IGF-I and Luteal Biology After ovulation, the CL is formed. The number of steroidogenic cells in a bovine CL increases until 8 d after estrus (Lei et al., 1991). Bovine CL express 13 the mRNA and protein for IGF-I and the abundance of mRNA for IGF-I in luteal tissue is increased from d 1-5 until (1 12-17 of the luteal phase (Einspanier et al., 1990). IGF-I binding sites are in CL and the specificity, affinity and capacity of IGF-I binding sites are constant throughout the estrous cycle (Sauerwein et al., 1991), in contrasts to granulosa cells (Adashi et al., 1986). In addition, the number of IGF-I receptors in CL does not correlate with serum or luteal progesterone concentrations in humans (Obasiolu et al., 1992). Incubating a primary culture of bovine luteal cells collected 4 - 10 d after estrus with IGF-I for 4 h does not affect the secretion of progesterone (Schams et al., 1988). However, adding insulin or IGF-I for more than 24 h to bovine luteal cells collected (1 1-5 after estrus does increase the secretion of progesterone (McArdle and Holtorf, 1989). Also, adding IGF-I to luteal tissue in vitro increases the secretion of progesterone (Sauerwein et al., 1991), and IGF-1 increases DNA synthesis by luteal cells (Chakravorty et al., 1993). Furthermore, the stimulative effect of IGF-I on luteal cells is dose dependent (McArdle and Holtorf, 1989). Thus, IGF-I has a positive effect on luteal development. 14 Luteal I CF -I System During Negative Energy Balance During NEB, the abundance of IGF-1 mRNA is decreased in liver. But the abundance of IGF-I mRNA in CL is not changed by NEB (VandeHaar et al., 1995). The regulation of IGF-I synthesis is on the mRNA level (Pell et al., 1993). This supports our previous suggestion that the regulation of IGF-I synthesis is different in ovary and liver. It is also suggested that autocrine and/or paracrine IGF-I does not mediate effect of NEB on CL. Possible Role of Other Hormones and Metabolites Somatotropin Somatotropin (ST) is stored in and secreted by the somatou'ophs in the anterior pituitary gland. The protein and mRNA of ST receptors are found on bovine granulosa cells and luteal cells (Lucy et al., 1992). In vitro, ST increases the synthesis of protein in granulosa cells and increases the number of granulosa cells in the presence of insulin (Langhout et al., 1991). Moreover, ST stimulates progesterone biosynthesis in cultured human luteal cells (Lanzone et al., 1992). Dietary restriction increases pituitary ST mRNA and concentrations of ST in senrm of cows (Kirby et al., 1993). Reducing feed intake in steers and heifers 15 to the point that they lost body weight significantly increases mean concentrations of ST in plasma (Breier et al., 1986; Villa-Godoy, 1987; VandeHaar et al., 1995). This increase of mean ST is due to the increased amplitude and duration of pulses of ST but not the baseline concentrations or the frequency of pulses of ST(Breier et al., 1986; Villa-Godoy, 1987). Progesterone Small and large cells are defined within 3 CL according to their morphological and biochemical difference. Small bovine luteal cells range from 10 to 25 pm in diameter and large luteal cells range from 25 to 50 pm in diameter, which is 13 times bigger than small luteal cell in cell volume (Koos and Hansel, 1981). Without the stimulation of LH, large luteal cells secrete 20- fold more progesterone on a per cell basis than small luteal cells in cattle and sheep (Koos and Hansel, 1981; Fitz et al., 1982). Large luteal cells are responsible for approximately 78% of the progesterone secreted into serum (Niswender et al., 1985; O‘Shea et al., 1987). Progesterone is necessary for three events that influence fertility in dairy cattle: estrus detection (Melarnpy et al., 1957), conception (Folrnan et al., 1973; Fonseca et al., 1983), and embryonic survival (Hill et al., 1970). Furthermore, 16 concentrations of progesterone in the estrous cycle before insemination are also associated positively with conception rate (Carstairs et al., 1980; F onseca et al., 1983). Thus, low concentrations of progesterone in serum may limit fertility. Studies show that the diameter of ovulatory follicles and CL are decreased by dietary energy restriction (Burns et al., 1994; VandeHaar et al., 1995). Moreover, the fact that NEB decreased the ratio of large to small cells in the CL (Terhune, 1992) indicate that there are fewer large cells for progesterone production in NEB animals. Villa—Godoy (1987) found that in lactating dairy cows, the concentration of progesterone in milk is reduced proportional to the magnitude of the energy deficiency. Furthermore, Spicer et al. (1990) reported that the concentration of progesterone in serum during early postparttun period was positively associated with EB. However, other studies have observed different results. Restricted dietary intake in cattle also has been reported to increase (Donaldson et al., 1970; McCann and Hansel, 1986) or have no effect (Lucy et al., 1993; Spitzer et al., 1978) on concentrations of progesterone in blood. Thus, the effect of restricted dietary intake on the concentration of progesterone in serum of cows has no consistent results. During ovemutrition, cattle either have greater (F ohnan 17 et al., 1973) or no change (Spicer et al., 1984) in concentrations of progesterone in blood. What causes these different results is not clear. Luteinizing Hormone Luteinizing hormone is the primary luteotmpin in most domestic animals (Charming et al., 1974). The fiequency of pulsatile LH secretion is important for the final phase of maturation of ovarian follicles (Randel, 1990) and thus for induction of estrus and ovulation. In addition, LH is required for maintenance of CL (Hoffman et al., 1974) and stimulates the secretion of progesterone fi‘om CL. Thus, the concentrations and pulses of LH are associated with the development of follicles and the production of progesterone in CL. . McCann and Hansel (1986) found that during the luteal phase, concentrations of LH are lower in fasted animals than in fed cattle. In support of this report, Irnakawa et a1. (1986) reported that dietary energy restriction suppresses mean concentrations of LH and amplitude of LH pulses in heifers. Furthermore, even small differences in energy intake influence the timing of the prepubertal surge of LH (Hall et al., 1994). In contrast, Gombe and Hansel (1973) found that progressive increases in mean LH occur when heifers are fed low energy diet for three successive estrous cycles. However, Terhune (1992) 18 found that NEB does not affect basal concentrations of LH. As with progesterone, the effect of restricted dietary intake on LH is not clear but the variance in methods of studies may be the cause. Insulin In woman, diabetes is associated with low fertility and hyperinsulinemia is associated with polycystic ovaries (Poretsky and Kalin, 1987). Concentrations of glucose and insulin in plasma of fasting heifers are lower than in fed heifers (McCann and Hansel, 1986). Additionally, low energy intake can decrease concentrations of insulin in plasma even when no change in glucose is observed (McGuire et al., 1991). In general, concentrations of insulin are positively associated with energy balance (Lucy et al., 1991; VandeHaar et al., 1995). Non Estertfied Fatty Acids High concentrations of non esterified fatty acids (NEFA) in plasma are associated with poor fertility in cows (Ducker et al., 1985). Heifers in NEB have higher concentrations of NEF A in plasma than those in PEB (V andeHaar et al., 1995). Exogenous NEFA decreases the binding of progesterone by albumin (Ramsey and Westphal, 1978). Thus, increased concentrations of NEF A during l9 NEB may reduce the binding of progesterone to albumin and increase the concentration of free progesterone. As a result, the metabolic clearance rate of progesterone may be increased during NEB, which, in turn, would decrease the concentration of total progesterone in blood of NEB animals. The Effect of Exogenous Somatotropin on Reproductive Function Somatotropin and I GF -1 Administration of bST to dairy cattle increases the concentrations of somatotropin and IGF-1 in serum (Davis et al., 1984; Gong et al., 1991) and increases the mobilization of NEFA from adipose tissue (Peel et al., 1981). ST acts partly through specifically binding to receptors in liver, and thus increasing the synthesis of IGF-I and the concentration of circulating IGF-I. Plasma insulin level is also increased by exogenous bST in heifers (Gong et al., 1993) and in lactating cows (VanderKooi, 1993). The specific binding of ST in liver is greater in steers fed a high energy diet because of the appearance of a high-affinity binding site for ST compared to those steers which were fed a lower energy diet (Breier et al., 1988). At restricted energy intake, bST does not increase liver weight, but it does at high 20 energy intake (Pell et al., 1993). Breier et al. (1988) and Ronge and Blum (1989) demonstrated that undernutrition decreased concentration of IGF-I in blood and abolished the response of IGF-I to exogenous bST. However, other studies have shown that bST increases IGF-I in serum of early lactation cows in NEB (Gallo and Block, 1991; Rouge and Blum, 1989; Vicini et al., 1991). In addition, exogenous somatotropin increases IGF-I in serum of lambs in NEB (Bass et al., 1991) and of humans in NEB (Snyder et al., 1990). Therefore, the effect of bST on IGF-I in serum is diminished but not absent during NEB. Somatotropin and Reproduction Studies on the effects of bST on reproductive function are not conclusive. Administration of bST, in a sustained-release vehicle, to seven-month old heifers does not alter the age at puberty (McShane et al., 1989) or the pattern of LH release (Hall et al., 1994). In contrast, administration of bST exerts some negative effects on reproductive function in cows. Days open and the number of cystic ovaries tends to increase as the dose of bST increased in dairy cows (Hansen et al., 1994). BST-treated cows, compared to those not treated, need twice as many inseminations for every conception (McGuffey et al., 1991). 2] BST increases the frequency of LH pulses but decreases the baseline and average concentrations of LH during the first follicular phase after treatment started (Schemm et al., 1990). Long-term administration of bST also increases the concentration of LH in serum in response to gonadotropin releasing hormone at 14 d postpartum in cattle (Gallo and Block, 1991). Gallo and Block (1989) and Schemm et al. (1990) found that bST- treatrnent increases plasma progesterone concentrations in lactating cows. Furthermore, progesterone in plasma was increased during the first two estrous cycles post-treatment and during pregnancy by long-temr bST treatment (Gallo and Block, 1991). However, short-term administration of bST from ~14 d before ovulation until the end of next cycle did not affect the concentration of progesterone in serum in the following estrous cycle (Gong et al., 1993). Long-term treatment with bST does not affect lifespan of the CL, length of the follicular phase, length of the estrous cycle or the diameter of the largest follicle in cows (Schemm et al., 1990). Thus, the effects of bST treatment are not the result of changes in the time required for follicular and luteal development but of changes in the activity of the follicle or CL. Treatment with bST doubles the population of small follicles (2-5 mm) (Gong et al., 1991) in the ovary of heifers. This increase in small follicles is 22 most likely the result of increased recruitment of small follicles (< 5 mm) because bST does not alter the turnover of follicular waves nor the inhibitory action of the dominant follicle on its subordinate follicles in heifers (Gong et al., 1993). Furthermore, this increase in small follicles occurred after the concentration of ST in serum had returned to control values but the concentration of IGF-I in serum was still high, suggesting that the effects of bST on follicles are through circulating IGF-I. Studies in cattle (Davoren et al., 1986), gilts (Spicer et al., 1992), and humans (Owen et al., 1991) suggested that ST increases intraovarian concentration of IGF-I. Concentrations of IGF-I in follicular fluid of the dominant follicle are decreased in cows immunized against growth hormone- releasing factor with low concentrations of ST and IGF-1 in serum (Kirby et al., 1993). However, porcine somatotropin did not affect estradiol or progesterone concentrations in follicular fluid of pubertal gilts (Bryan et al., 1989). Summary Negative energy balance in cattle reduces the concentration of progesterone in serum, which in turn may decrease fertility by impairing the detection of estrus, the ability to conceive, and (or) the survival of embryo. 23 Factors that may be responsible for this decrease in serum progesterone include: 1) changes in the concentration of luteotropic hormones in serum and the number of receptors for these hormones on luteal cells, 2) changes in the cell population of the CL, 3) decreases in growth of the CL, and 4) decreases in activity of luteal cells. NEB also decreases the concentration of IGF-I in serum, and IGF-1 promotes both growth and differentiation of granulosa cells and secretion of progesterone by luteal cells. We postulate that, during NEB, the decreased concentration of IGF-I in serum impairs luteal development. If so, increasing IGF-I by injection of bST during the time of luteal development might alleviate this impaired luteal growth. Thus, objectives for this study are to determine if: 1) exogenous bST during luteal development will restore luteal weights of NEB heifers to normal, and 2) exogenous bST during luteal development will increase luteal weights in PEB heifers. MATERIALS AND METHODS Animals and Diets Thirty-two postpubertal Holstein heifers (z 13 months old) were group- housed indoors at the Michigan State University Dairy Research Center between January and July. Heifers were adapted to a feeding protocol in which they were fed all of their daily ration while locked in stanchions between 1000 and 1430 h each day. For the remainder of the day, animals could not eat but had free access to water and were free to allow social interaction and exhibition of estrus behavior. For at least two weeks before treatments started, heifers were fed a ration that supplied 140% of maintenance energy requirements. Heifers were allocated into two blocks by body weight (heavy and medium; 11 = 16). The heavy and medium groups began the experiment at different times so the average body weighs at the beginning of the treatments were similar for all heifers. Within each block heifers were assigned randomly to one of two dietary treatment groups (n = 8). At the start of the experiment, heifers were given two 24 25 injections of prostaglandin F2, (PGF,,; 5 CC.= 25 mg/head; Lutalyse®; Upjohn company, Kalamazoo, MI) eleven days apart to synchronize estrus. Beginning 2 d after the second PGFZ, injection half the heifers were switched to a low energy diet (270% of maintenance energy requirement) and the other half continued on the diet at 140% of maintenance energy requirements (Figure 2). Body condition scores were recorded at the beginning and the end of the experiment by two persons. Body weights were measured on three consecutive days weekly and averaged by week for each heifer. Diet ingredients were sampled and chemically analyzed (Northeast DHIA Forage Testing Laboratory, Ithaca, NY), and the ingredients and chemical composition are summarized in Table 1. Dietary ingredients were also sampled twice a week for the analysis of dry matter (DM). Samples (220 g) of alfalfa silage, corn silage, and silage mixture were used for weekly DM analysis. Samples were dehydrated at 100°C oven to achieve constant weight, and the DM percentage was calculated. Diets for each heifer were adjusted every week according to DM content of diets and individual body weight to ensure that each heifer was fed at the appropriate energy intake (see Appendix A). The orts were collected (if any) every day and weighed to calculate feed intake. 26 29286 1 EU ”£58 1 o 3 225.398 be 25.08; .N 95”....— I. 205; a. 2&0 5+ a 2&6 1+ a 2&0 i 2v .331; on cu ovmé :6 fl _ A _ L _ _ L L a a a a 58863:: £9. 5:: 58a ha ._ S u 3 EU 1% _--11-; 1. G_ n 53:. mmz A. a u 5 .55 .. 11-19530: mm 3 u 5 EU + ..... - a: 1 3 3:. mm; ii 9 E .5: 27 Table 1. Composition of diet (DM basis)“ PEB NEB Silage mixture, kg 3.7 2.6 Cracked corn, kg , 2.4 0.2 Protein supplement”, kg 0.5 0.3 Total intake, kg 6.6 3.1 Nutrient composition of total diet“ NEm, Mcal/kg DM 1.6 1.4 Crude protein % of DM . 15 16 Feed consumed, % of ME requirement 139 68 Energy Balance Meal/d 1.7 -2.2 “Daily average for four cycles. t’Contained soybean meal, trace-mineral and vitamin premixes and was formulated to provide 100% of protein, mineral and vitamin NRC requirement. cCalculated based on laboratory analyses. (Northeast DHIA Forage Testing Laboratory, Ithaca, NY) 28 Heifers were observed for estrus behavior for periods of 30 minutes three to five times a day. An animal was considered in estrus if she stood to be mounted by others for at least two seconds. Visual estrus was defined as d 0 of an estrous cycle. After 2.5 estrous cycles, prostaglandin F2, was injected. One day after prostaglandin F2, injection, bST injection was started. Within each dietary treatment group, half animals were randomly selected for daily subcutaneous recombinant bST injection (14 mg/day; courtesy of The Upjohn Company, Kalamazoo, MI ). On (1 10 of the fourth cycle (d 0 = estrus), CL of heifers were collected. All processes were approved by the University Committee on Animal Use and Care of Michigan State University. Energy Balance Dietary energy intake and body weight were used to estimate energy balance. For NEB heifers, it was calculated as in the Nutrient Requirements of Dairy Cattle (National Research Council; 1989): EB = NEm - NEm Ink he no. 29 Where NEmmm is the intake of net energy (NE) for maintenance and NEW is the NE“I requirement of the animal. NEminmkc was calculated as: NE 1...... = feedintake( kg dry matter) it NEm ( Mcal/ kg dry matter) where the NEm value of the feed was calculated based on laboratory analyses of each ingredient. NEmm is based on average weekly body weight (BW) and calculated as: NE = 0.086 X BW (kg)°'75 mm Because energy is used less efficiently for gaining weight than for maintenance, net energy intake that exceeded NEIn requirement was multiplied by 0.66 (the approximate ratio of NE for gain to NEIn for the diet). Thus, the EB in PEB heifer was calculated as: E8 = (NEmm. - NEm”) x 0.66 30 Collection of Blood Samples Beginning with the second estrous cycle, blood was sampled by venipuncture daily at 1400 h. After the injection of prostaglandin F 2,, in the third estrous cycle, blood samples were taken twice a day at 1000 and 1400 b. On day 9 of the fourth estrous cycle, blood was collected four times a day (0700, 1100, 1500, and 1900h). Blood samples were stored at 4°C overnight, and serum was harvested by centrifugation for 20 min at 800> F PEB NEB Pooled SEM Initial BW, kg 354 356 7 NS Final BW, kg 415 327 7 0.01 Weight change, g/d 820 -414 33 0.01 Final body condition score 3.8 2.1 0.1 0.01 39 350 11- DControl 300 .11- IbST 250 4*- 2001- 150 -~ 100 1- Concentration of NEFA (uM) PEB NEB Dietary Treatment Figure 4. Least squares means of the concentration of NEFA in serum at d 9 of the fourth estrous cycle. Data are from eight PEB- Control, seven PEB-bST, eight NEB-Control, and six NEB-bST heifers. Pooled standard error of the means among treatments was 3.38. Statistical analysis of mean comparisons: PEB-Control vs. NEB-Control; P < 0.01. PEB—Control vs. NEB-bST; P < 0.01. PEB-Control vs. PEB-bST; P = 0.63. NEB-Control vs. NEB-bST; P = 0.44. 4o Concentration of Somatotropin in Serum Concentration of somatotropin in serum on d 9 of the fourth estrous cycle was higher for NEB than PEB heifers (3.43 ng/ml vs. 1.06 ng/ml; P < 0.01). Exogenous bST increased (P < 0.01; Figure 5) concentration of somatotropin in serum 27 fold in PEB and 15 fold in NEB heifers compared to their respective controls. The interaction between dietary treatment and bST treatment was significant (P < 0.01). Concentration of Insulin-like Growth Factor-1 in Serum The mean concentration of IGF-I in serum from d 0 to d 10 of the fourth estrous cycle was 27% less (P < 0.01) in NEB than in PEB heifers. Exogenous bST increased (P < 0.01; Figure 6) the concentration of serum IGF-I in PEB and NEB heifers by 91 and 36%, respectively. The interaction between dietary treatment and exogenous bST was significant (P < 0.02). As we had planned, mean concentrations of IGF-I in serum were nearly identical for PEB-control and NEB-bST heifers (P = 0.99). 41 O‘ O r l D Control I bST M O r I A O L l b.) O r r N O r r fl O r r O Concentration of Somatotropin (ng/ml) PEB Dietary Treatment Figure 5. Least squares means of the mean concentration somatotropin at d 9 of the fourth estrous cycle. Data are from eight PEB-Control, seven PEB-bST, eight NEB-Control, and six NEB-bST heifers. Pooled standard error of the means among u’eatments was 2.5. Statistical analysis of mean comparisons: PEB-Control vs. NEB-Control; P =0.68. PEB-Control vs. NEB-bST; P < 0.01. PEB-Control vs. PEB-bST; P < 0.01. NEB-Control vs. NEB-bST; P < 0.01. 42 —-D—PEB-Control 1600 +PEB-bST " - - a. - NEB-Control nt-NEB-bST a 1400-- E R 1200» t: v '7' La 1000« U — h- I n e 800 ‘r ............. - = ‘ ----. ------- 02 ‘.O a 600 3222:: ' ------- A ----- ‘0 """"" A °°°°° ‘A'--. a "'e °°°°° ‘ 5 A U 400T = e U 2004 Startof l:bSTTreatmerrt o 1 a t t r t a e 4 o 2 4 6 8 10 Days After Estrus Figure 6. Concentration of IGF-I in serum during the fourth estrous cycle. Data are from eight PEB-Control, seven PEB-bST, eight NEB- Control, and six NEB-bST heifers. Pooles standard error of the means among treatment was 170. d 0 = estrus Statistical analysis of mean comparisons for means from d0 to d1 0: PEB-Control vs. NEB-Control; P < 0.03. PEB-Control vs. NEB-bST; P = 0.99. PEB-Control vs. PEB-bST; P = 0.01. NEB-Control vs. NEB-bST; P < 0.09. 43 Concentration of Progesterone in Serum Mean concentrations of progesterone in serum from d 0 to d 10 and d 10 alone of the fourth estrous cycle were not affected by either dietary treatment or bST treatment (Table 3). The concentration of progesterone in serum was not correlated with the concentration of IGF-I in serum within treatments or across all heifers. Weights of Corpora Lutea Weight of the CL was decreased from 6.9 g in PEB to 4.2 g in NEB heifers (P < 0.01). Treatment with bST did not alter luteal weight in NEB or PEB heifers (Figure 7), and luteal weights of NEB-bST heifers were smaller than those of PEB-control heifers (P < 0.01). Weight of the CL was not correlated significantly with the concentration of IGF-I in serum (Figure 8). For PEB-control heifers, but not other groups, weight of the CL was correlated positively with mean concentration of progesterone in serum (r = 0.82, P = 0.01). Among all heifers, luteal weight was correlated negatively with the concentration of NEFA in serum (r = -0.32, P = 0.09). 44 Table 3. Least square means of serum progesterone, luteal progesterone, and luteal protein content - Treatment PEB- PEB- NEB- NEB- Pooled Control bST Control bST SEM Serum progesterone Meanfrodetole 1.2 1.1 1.2 1.1 0.1 (ng‘ ml") Day 10(ng/In1) 3.2 2.8 3 2.6 0.1 Luteal progesterone Concentration (Pg/g) 33.7 29.7 24.5 23.4 5.1 on tissue basis‘ Concentration (118/8) 0.14 0.14 0.1 l 0.1 0.01 on protein basisb Luteal protein content 241 216 224 231 6.3 (rig/mg wet tissue) ’1‘ issue basis is based on the weight of luteal tissue. PEB heifers tended (p < 0.09) to have higher luteal progesterone concentration than NEB heifers. ”Protein basis is based on luteal protein content. PEB heifers tended (p < 0.07 ) to have higher luteal progesterone concentration than NEB heifers. cLuteal content of progesterone is calculated as: luteal progesterone concentration (on tissue basis) x luteal weight. PEB-Control heifers have higher (p < 0.01) luteal progesterone content than NEB heifers in both control and bST heifers. 45 Cl Control 8 " IbST 1 .1 .. T 7 .L a) J 1 Weight of Corpus Luteum (g) ‘1" PEB Dietary Treatment Figure 7. Least squares means of the weight of corpus luteum at d 10 of the fourth estrous cycle. Data are from eight PEB-Control, seven PEB- bST, eight NEB-Control, and six NEB-bST heifers. Pooled standard error of the means among treatments was 0.27. Statistical analysis of mean comparisons: PEB-Control vs. NEB-Control; P < 0.01. PEB-Control vs. NEB-bST; P < 0.01. PEB-Control vs. PEB-bST; P = 0.69. NEB-Control vs. NEB-bST;P = 0.65. 46 10- O O 9.1 O 8 O . Weight of Corpus Luteum (g) 4 .1 3 -1 0 control 2 - O bST ----- Trendline (control) 1 d Trendline (bST) 0 . r r “1 0 500 1000 1500 2000 Mean Concentration of IGF-I (rig/ml) Figure 8. The correlation of the weight of corpus luteum with average concentration of IGF-I in serum from d 0 to d 10 of the fourth estrous cycle in control and bST-treated heifers. Data are from sixteen control and thirteen bST-treated heifers. The correlation coefficient of: control heifers is r = 0.27, P = 0.3. bST heifers is r = 0.48, P = 0.1. 47 Content of Protein in Corpus Luteum Negative energy balance did not alter the concentration of protein in CL (mg protein/g wet tissue; P = 0.38; Table 3). Protein content (mg/CL) of the CL was 45.4% less (P < 0.01) in control heifers in NEB than those in PEB. Exogenous bST did not alter the concentration or content of protein of CL. Concentration of Progesterone in Corpus Luteum Compared to PEB, NEB decreased the concentration of progesterone in luteal tissue on protein basis by 23%, but this decrease was not statistically significant (P = 0.19). Exogenous bST did not alter the concentration of luteal progesterone (Table 3). Among all heifers, the concentration of luteal progesterone did not correlate with the mean concentration of progesterone in serum or the mean concentration of IGF-I in serum (d 2 to d 10). The concentration of luteal progesterone was negatively correlated with the concentration of NEFA in serum (r = -0.45, P < 0.01). Content of Progesterone in Corpus Luteum The content of luteal progesterone was greater in PEB than NEB heifers (P < 0.01, Figure 9). Among all heifers, luteal progesterone content was 48 250 a- El Control I bST ? sub 1 I § Luteal Progesterone Content (ng) 8 PEB NEB Dietary Treatment Figure 9. Least squares means of luteal progesterone content at d 10 of the fourth estrous cycle. Data are from eight PEB-Control, seven PEB- bST, eight NEB-Control, and six NEB-bST heifers. Pooled standard error of the means among treatments was 12.56. Statistical analysis of mean comparisons: PEB-Control vs. NEB-Control; P < 0.01. PEB-Control vs. NEB-bST; P < 0.01. PEB-Control vs. PEB-bST; P = 0.40. NEB-Control vs. NEB-bST; P = 0.94. 49 correlated positively with the concentration of IGF-I in serum at d 10 of the fourth estrous cycle (r = 0.34; P = 0.07; Figure 10). The concentration of progesterone in serum at d 10 of the fourth estrous cycle was positively correlated with luteal progesterone content (r = 0.47, P < 0.01 ). 50 A 600 " u-I 0 Control (a 0 o bST g 500 . ----- Trendline (Control) 5; Trendline (bST) 5 3 400 , H m 0 gm 0 r... . 0 9 Ga 300 'e'i o g 200 d u-J 0.- O H E 100 4 0 fl = O U o 1 T U 0 500 1000 1500 Concentration of IGF-I (ng/ml/d) Figure 10. The correlation of content of luteal progesterone with average concentration of IGF-I in serum from d 2 to d 10 of the fourth estrous cycle in control and bST-treated heifers. Data are from sixteen control and thirteen bST-treated heifers. The correlation coefficient of: control heifers is r = 0.24, P = 0.37. bST heifers is r = 0.70, P < 0.01. DISCUSSION Weights of Corpora Lutea Our hypothesis was that NEB decreases luteal weight by decreasing the concentration of IGF-I in serum. To test this hypothesis, we assessed the relationship of concentrations of IGF-I in serum during the development of a CL with its weight on d 10 using diet and exogenous bST to manipulate the concentration of IGF-I. As expected, NEB heifers had less IGF-I in serum and smaller CL in confirmation of previous data (V andeHaar et al., 1995). Administration of bST elevated concentrations of IGF-I in NEB heifers to a level similar to that of PEB-control heifers. In PEB-bST heifers, the injection of bST increased the concentration of IGF-I to twice that of PEB-control heifers. However, the increased IGF-I in serum was not associated with increased weight of CL. Furthermore, weight of CL was not correlated with serum concentrations of IGF-I in heifers. Thus, our data suggest that injection of bST and changes in circulating concentrations of IGF-I during the development of a CL do not affect its weight. This is not consistent with the data of Chakravorty et a1. (1993) that 51 52 IGF-1 increases DNA synthesis in bovine luteal cells in vitro. Two possible reasons for this discrepancy are that: l) IGF-I may act through an autocrine or paracrine rather than endocrine pathway, and 2) IGF-binding proteins modulate the action of IGF-I in ovaries. In addition to endocrine actions, IGF-I may "act through autocrine and paracrine pathways. The presence of IGF-I and IGF-1 mRNA in luteal tissues suggests that IGF-I may act through these other pathways in the CL (Einspanier et al., 1990). However, in the present study, we observed no difference in the abrmdance of IGF-I mRNA in CL (data not shown; Shanna et al., 1994). This confrrrns the lack of effect of NEB on luteal IGF-I mRNA observed previously (V andeHaar et al., 1995). Furthermore, Spicer et al.(1992) found no change in the concentration of IGF-I in follicular fluid during fasting in heifers, suggesting that local production of IGF-1 in a dominant follicle is not affected by underfeeding. Thus, the autocrine and paracrine IGF-I do not seem to mediate the effects of EB on the development of CL. A second possible explanation for why increased IGF-I failed to increase luteal weight is that IGF-binding proteins may modulate the action of IGF-I. The IGF binding protein-2 binds with IGF-I and inhibits DNA synthesis in cultured rat preovulatory granulosa cells, and IGF binding protein-3 extends the half-life 53 of IGF-I (Giudice, 1992). In this study, IGF binding protein-2 was increased in NEB heifers (data not shown), this increased IGF binding protein-2 might have further decreased DNA synthesis in granulosa and luteal cells and caused a smaller CL in NEB heifers. Injections of bST decreased the concentration of IGF binding protein-2 in both EB groups and increased IGF binding protein-3 in PEB heifers, but neither of these changes was associated with an increase in weight of CL. Thus, the differences in IGF binding protein-2 and IGF binding protein-3 in heifers did not seem to be the cause of the decreased luteal weight in NEB heifers. However, there are six types of IGF binding proteins found in follicular fluid (Giudice, 1992); perhaps another IGF binding protein may affect the action of IGF-I in ovary and help explain the difference between in vivo and in vitro studies. Another explanation for why increased IGF-I in response to bST in this study failed to increase luteal weight is that endocrine IGF-I may indirectly alter the development of the CL. Perhaps decreased IGF-I limits deve10pment of the dominant ovulatory follicle and thereby decreases the weight of the subsequent CL. In support of this, Terhune (1992) observed that NEB decreases the duration of the preovulatory period. NEB also decreases the persistency of dominant follicles and the maximum diameter of dominant follicles (Burns et 54 al., 1994). EB and IGF-1 are positively correlated and IGF-1 increases DNA synthesis in granulosa cells of the follicle (Monniaux and Pisselet, 1992). In our study, concentrations of IGF-I in the previous cycle were decreased in NEB heifers compared to PEB heifers, and the difference in IGF-I may have affected the development of the dominant ovulatory follicle. Treatment with bST increases the recruitment of small follicles and stimulates the growth of the preovulatory follicle in cattle (Gong et al., 1991; De La Sota et al., 1993), but, in the present study, injection of bST and subsequent increases in IGF-I did not occur until 2 d before ovulation, long after the ovulatory follicle had started its development. Since follicular cells are the parental cells of luteal cells, it is possible that decreased follicular development in NEB heifers decreased the development of the subsequent CL, and the increase in IGF-I beginning 2 d before ovulation was not enough to change it. Progesterone in Corpora Lutea and Serum Luteal Progesterone PEB heifers had greater concentration of progesterone in the CL than NEB heifers. The ratio of large to small luteal cells in mid-diestrus (d 7) was 55 decreased by NEB (Terhune, 1992). Large luteal cells secrete 20-fold greater progesterone than small luteal cells 0(oos and Hansel, 1981). Thus, the smaller ratio of large to small cells during NEB would result in less luteal progesterone. Our finding that progesterone content is reduced in the CL of NEB heifers is consistent with the previous study showing decreased progesterone content in CL during NEB (Terhune, 1992). In our study, the concentration of IGF-I was positively correlated with the luteal progesterone content, supporting the report that the concentration of IGF-1 in serum is positively correlated with the concentration of progesterone in senrrn (Spicer et al., 1990), although in neither case was the correlation very strong. In addition, IGF-I increases the production of progesterone by granulosa and luteal cells in vitro (Schams et al., 1988; Monniaux and Pisselet, 1992; McArdale and Holtorf, 1989) although some studies using shorter culture times have not shown the increase (Schams etal., 1988; Ealy, 1990). Thus, these studies generally support the idea that IGF-1 may mediate the effect of EB on luteal progesterone content. However, in our study, treatment with bST during the luteal phase, and subsequent increases in IGF-I in serum, did not affect the concentration or content of progesterone in the CL. Thus, we conclude that IGF-I in serum during luteal phase does not affect the synthesis of progesterone in the CL. Rather, as we found for luteal growth, 56 concentrations of IGF-I during growth of follicles before ovulation may be more important for progesterone synthesis by the CL. Serum Progesterone Energy balance did not altered mean concenu'ation of progesterone in serum from d 2 to d 10 nor the concentration of progesterone on d 10 alone. In previous studies using similar experimental protocols, Villa-Godoy et al. (1988) observed a decrease in progesterone in serum of NEB compared to PEB heifers but another observed no change (Terhune, 1992). We found that the luteal progesterone content was decreased in NEB heifers compared to PEB heifers. Furthermore, the concentration of progesterone in serum on d 10 was not closely correlated to that in CL; within group, serum and luteal progesterone were positively correlated in PEB but not NEB heifers (r = 0.65, P = 0.01; r = -0.02, P = 0.93). Possible reasons that NEB decreased luteal but not serum progesterone are: l) NEB heifers had less reserves of body fat compared to PEB heifers, and 2) NEB heifers had less hepatic blood flow than PEB heifers. First, because progesterone is a steroid hormone, it distributes in adipose tissue. During luteal phase, fat tissue contains ~36 ng/g of progesterone (Tsujioka, 1992), which is 10 times more than the concentration of progesterone 57 in blood. Assuming that blood is about 10% of the body and that adipose tissue is at least 10%, then more than 90% of progesterone in the body will be found in adipose tissue. Thus, most progesterone in the body is in adipose tissue and only 5-10% of progesterone is in blood. Furthermore, NEB heifers had lower final body condition scores than PEB heifers, indicating that NEB heifers had less adipose tissue for distribution of progesterone than did PEB heifers. Thus the clearance of progesterone from blood likely was slower in NEB than PEB heifers. A slower clearance rate for progesterone from serum of NEB heifers would increase the concentration of progesterone in serum and might mask the effects of a decrease in progesterone synthesis by CL. Thus, the different reserves of body fat in the two EB groups likely minimized the concentration of progesterone in serum. Second, liver is a primary site of clearance of progesterone (Bedford et al., 1974; Freetly and Ferrell, 1994). In vivo studies indicated that increased feed intake increases liver oxygen consumption and hepatic blood flow ( Burrin et al., 1989; Parr et al., 1993; Freetly and Ferrell, 1994). Parr et a1.( 1993) reported that increased feed intake increased the metabolic clearance rate of progesterone in liver, although Freetly and Ferrell (1994) did not find the change in metabolic clearance rate of progesterone. In present study, feed intakes of NEB heifers 58 were half of those of PEB heifers; consequently the hepatic blood flow and metabolic clearance rate of progesterone may have been slower in NEB than PEB heifers. A decrease in metabolic clearance rate may have disguised the decreased synthesis of progesterone of NEB heifers. In lactating cows, NEB is associated with decreased serum progesterone (Spicer et al., 1990). However, in lactating cows, NEB during early lactation is more often the result of high milk production than low feed intake compared to a cow in late lactation. Some serum progesterone ends up in milk. Thus, spontaneous NEB in lactating cows may be associated with increase clearance of progesterone fi'om blood, whereas NEB may have caused decrease progesterone clearance in the present experiment in which heifers were fed a restricted diet. Differences in clearance would explain why serum progesterone is decreased during NEB in cows, but was not decreased by NEB in this experiment. Treatment with bST did not affect the concentration of progesterone in serum or in the CL in this study. This seemingly conflicts with reports that long- term administration of bST increases the concentration of progesterone in serum of lactating cows (Gallo and Block, 1989; Schemm et al., 1990) but agrees with the study of Gong et al. (1993) that administration of bST beginning ~14 d 59 before ovulation did not increase the concentration of progesterone in the subsequent estrous cycle in heifers. Two obvious differences among these studies are the timing of bST administration and the physiological state of animals. Therefore, treatment with bST during luteal phase does not affect luteal development of heifers. Somatotropin and Insulin-like Growth Factor-1 In the present study, concentrations of ST in serum were higher and concentrations of IGF-I were lower in heifers in NEB than those in PEB, as expected based on previous reports (Ronge et al., 1988; Rutter et al., 1989; VandeHaar et al., 1995). Administration of 14 myd of bST increased concentrations of ST in serum of PEB and NEB heifers, but the increase was greater in heifers in NEB than in PEB. Because heifers in NEB weighed less than those in PEB at the end of the study, the reason for the greater serum ST response in NEB heifers was likely in part that they were injected with a higher dose of bST per kg of body weight ( 0.03 vs. 0.04 mg/kg/day for PEB vs. NEB; respectively). Also, during underfeeding, the abundance of mRNA for ST receptors and the number of high-affinity binding sites for ST in liver are lower (VandeHaar et al., 1995; Breier etal., 1988); decreased ST receptors in liver 60 might decrease the metabolic clearance rate of ST from blood and thereby increase concentrations of ST in NEB heifers compared to PEB heifers. Despite their greater concentration of ST, NEB-control heifers had less IGF- I in serum than PEB-control heifers, as previously shown (VandeHaar et al., 1995; Breier and Gluckrnan, 1991). Furthermore, exogenous bST increased the concentration of IGF-I in serum less in heifers in NEB than in heifers in PEB. This decreased sensitivity to ST in NEB heifers suggests that ST and IGF-1 in serum are partly but not entirely uncoupled during NEB. Heifers in NEB have less hepatic mRNA for ST receptors than those in PEB, so the mechanism for this uncoupling may be partly through fewer ST receptors (V andeHaar et al., 1995; Breier and Gluckman, 1991). In addition, heifers in NEB have less IGF-I mRNA in liver than those in PEB (V andeHaar et al., 1995). Thus, in spite of more ST in serum, NEB-bST heifers had less IGF-I in serum than PEB-bST animals likely because of decreased binding of ST in liver and decreased synthesis of IGF-I in liver. Another possrble reason that bST increases IGF-I more in PEB heifers than in NEB heifers is that the concentrations of IGF-binding proteins are different in the two EB groups. Administration of bST decreased the level of serum IGF binding protein-2 in all heifers but increased IGF binding protein-3 only in PEB 61 heifers (data not shown; Sharma et al., 1994). The increased IGF binding protein-3 of PEB heifers in response to bST likely extended the half-life of IGF-I (Giudice. 1992) and, in turn, increased the concentration of IGF-I in serum of PEB-bST heifers. In NEB heifers, this increase in IGF-binding protein-3 did not occur and therefore the increase in IGF-I was not as great as in PEB heifers. F urthennore, serum IGF binding protein-2 was greater in NEB-control than PEB-control heifers (data not shown; Shanna et al., 1994), and IGF binding protein-2 may facilitate the transport of IGF-I to target tissues (Giudice, 1992). This increased IGF binding protein-2 in NEB heifers might have increased the uptake of IGF-I by target cells and thereby increased the metabolic clearance rate of IGF-I and decreased the concentration of IGF-I in serum. Conversely, the decreased IGF binding protein-2 of bST-treated groups might have decreased the metabolic clearance rate of IGF-I and increased the concentration of IGF-I. CONCLUSIONS Administration of bST increased the concentration of IGF-I in serum of both PEB and NEB heifers although the increase was less in NEB heifers. This indicates that, during NEB, the stimulation of IGF-I by ST was partly but not entirely uncoupled. The main objective of this thesis was to determine the effect of bST injection during luteal phase on grth of the CL in heifers. Results from the present study indicate that administration of bST beginning 2 days before ovulation does not affect luteal development in NEB or PEB heifers. Furthermore, although bST injections increased concentrations of IGF-I of NEB heifers to those of PEB control heifers, weight of the CL was not restored to normal nor was the content of progesterone in the CL affected, indicating that IGF-I in serum during the luteal phase has no effect on luteal development. Thus, my original hypothesis that serum IGF-I mediates the effect of EB on luteal development is rejected. 62 APPENDICES 3.0 0 $0.0 0.0 200 0.2 00 0N0 0 $00 0.0 000 0.0. 000 Rd 0 9000 0.0 000 0.0 _0_ 00.0 0 30.0 0.0 000 .0- 02 0.0.0 0 9\..00 0.0 000 0 .0. v2 2.0 0 .20 0.0 20 0.0- 03 2.0 0 $00 0.0 $0 0.0.- 02 2 .0 0 30.0 0.0 000 0.2 02 052 2 .0 1 $02 0.5 000 N0 00— 20.0 3 A.002 0.0. 000 0.0 00— 00.0 3 0002 0.0 2v _.0 V0— 000 3 9002 00 000 0.0 03 00.0 E 900.2 0.0. 2v 0.0 NS 00.0 E O\902 00 N00 0.0 02 :0 3 9002 0.0. 000 0.0 02 00.0 E °\e02 0.0. 20 0.0 02 050 EsoE< com m< 256:2 com m< A00: €00.00 3035 Ev: F60 03680 2352 000:0 Eoth: om 9820 Ev: 3m Ev: 03 9830.5 0_ 65> 8.0 0900 8 00.5 908022 3.5 «co—:25 om EmZ 0 65> 0o 00:20 3m 35:2 .327. 0503.. 0.25 2:. .< 50:250. 63 64 Appendix B. The radioimmumoassay of IGF-I A. Iodination of IGF-I 1. Prepare a lO-ml sephadex G-50 (coarse: medium = 2: 1) column that uses glass wool to regulate the flow of degassed phosphate buffer (0.25% BSA; 0.03 M phosphate; 0.02 M sodium azide, and pH 7.5) for about 1 ml/5 min. 2. Run 1 ml of 8% BSA through the column. 3. Prepare fresh solutions (keep in dark) of: a. 5 mg/ml sodium metabisulfite in elution buffer (0.03 M sodium phosphate; 0.01 M EDTA; 0.02% protamine sulfate; 0.02% sodium azide; 0.05% Tween-20; 0.25% BSA; pH 7.5). b. 1 mg/ml chlorarnine T in assay buffer (0.03 M sodium phosphate; 0.01 M EDTA; 0.02% protarnine sulfate; 0.02% sodium azide; 0.05% Tween-20; pH 7.5). 4. Add in a epp. tube (500 pl) : a. 2 pl IGF-l in 10 p1 0.1 N HCl and 35 pl 0.5 M PBS. b. 1 mCi ”’1 in 1-10 pl volume and mix. 65 c. 25 pl chlorarnine T (use Hamilton syringe), mix and allow exact 15 seconds for reaction. d. Add 50 pl sodium metabisulfite (use Hamilton syringe), mix and allow exact 20 seconds for reaction. e. Add 100 pl transfer solutions (0.5 M PBS; pH 7.4). 5. Apply the mixture to the column bed carefully. 6. Run plenty of elution buffer and collect 30 fractions (0.5 ml/tube) in 12 x 75 mm tubes. 7. Take 10 pl from each fraction in 12 X 75 tube, capped, and count for 0.1 min. 8. Save the fraction comprising the first peak. B. Extraction of serum I GF -1 from binding proteins 1. Take 50 pl serum, add 12.5 pl 2.4 molar formic acid, 250 pl absolute ETOH and vortex and incubated in room temperature for 30 min. 2. Centrifuge for 30 min at 4°C and 600 g. 3. Remove 50 p1 supernatant and dilute it into 1.0 ml total volume with neutralizing buffer (0.11 M NazHPO4; 0.154 M NaCl; 0.01 M NazEDTA; 0.015 M Sodium azide; 0.5% Tween-20). 66 C. Radioimmunoassay of IGF-I 1. Label 12 x 75 mm tubes at the following order: Total count (TC) Non-specific binding (NSB) Standard curve Samples which standard curve and samples have total volume of 200 pl. 2. 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