Leptin and its role in the central regulation of reproduction in cattle

Leptin, a 16kDa product of the adipose obese (ob) gene, has been shown to contribute to the regulation of energy metabolism, feeding behavior, and reproduction in several monogastric species, including humans. Recent reports have provided evidence that the leptin gene is functionally relevant in cattle and sheep, and may contribute to an array of important reproductive events, including puberty. Leptin gene expression and circulating leptin increase markedly during sexual maturation in heifers reaching puberty during late spring or early summer. In addition, serum leptin concentrations increased by over 30% from early winter to the summer solstice in mature cows, and also increased with significant changes in adiposity. However, only limited changes in circulating leptin have been observed during the estrous cycle. Short-term fasting of growing peripubertal heifers causes marked reductions in leptin gene expression and circulating leptin, concomitant with declines in LH pulse frequency, and serum concentrations of insulin and IGF-1. Although short-term fasting of mature cows in excellent body condition is without effects on LH pulse frequency, it has remarkably similar metabolic effects to those observed in heifers. Moreover, ICV administration of recombinant oleptin resulted in a marked hypersecretion of LH in fasted cows, and in vitro studies using both hypothalamic and anterior pituitary explants have provided evidence that this effect is at the pituitary level. Paradoxically, ICV administration of oleptin normalized circulating insulin in fasted cows but hleptin was without effect on insulin in estradiol-implanted wethers. Collectively, work in cattle and sheep indicates that leptin can modulate both the hypothalamic-pituitary axis and endocrine pancreas under defined nutritional conditions. Additional work to more fully characterize these roles is clearly warranted and could lead to the development of novel strategies for modifying reproductive potential in food-producing species.     

A study on ghrelin and LH secretion after short fasting and on ghrelin levels at perioestrual period in dairy cattle

In two experiments, we studied (a) the changes of LH secretion in heifers under different feeding schedules and (b) total ghrelin concentration at oestrus in cows and heifers. In experiment one, synchronized heifers were allocated in three groups (R, regularly fed controls; F, fasted; and F‐F fasted‐fed). One day after the completion of the oestrous induction protocol, group F and F‐F animals stayed without feed for 24 hr; thereafter, feed was provided to R and F‐F cattle; 2 hr later, GnRH was administered to all animals. Blood samples were collected for ghrelin, progesterone, LH and cortisol concentrations. Fasting caused increased ghrelin concentrations in groups F and F‐F, while in response to GnRH, LH surge was significantly attenuated in groups F and F‐F compared to R. In experiment 2, lactating cows and heifers were used. On day 9 of a synchronized cycle, PGF2α was administered, and blood samples were collected twice daily until the third day after oestrus and analysed for progesterone, estradiol, ghrelin, glucose and BHBA concentrations. No difference was recorded between groups in steroids and BHBA concentrations. In comparison to mid‐luteal values, ghrelin concentrations significantly increased at perioestrual period in cows, but not in heifers. This study provides evidence that starving‐induced elevated ghrelin concentrations can have suppressing effect on LH secretion, even after ghrelin's restoration to basal values and that during oestrus, ghrelin secretion is differently regulated in cows and heifers, likely being independent from oestradiol concentrations. Further research is required to identify the determining factors that drive the different regulation of ghrelin secretion in cows and heifers.     

Chronic administration of recombinant ovine leptin in growing beef heifers: effects on secretion of LH, metabolic hormones, and timing of puberty

Serum concentrations of leptin increase linearly from approximately 16 wk before until the week of pubertal ovulation in beef heifers. To test the hypothesis that exogenous leptin can hasten the onset of puberty in heifers, we examined the effects of chronic administration of recombinant ovine leptin (oleptin) on timing of puberty, pulsatile and GnRH-mediated release of LH, and plasma concentrations of GH, IGF-I, and insulin. Fourteen fall-born, prepubertal heifers (Brahman x Hereford, 12 to 13 mo; 304.7+/-4.12 kg) were used. Heifers were stratified by age and BW and assigned randomly to one of two groups (seven animals per group): 1) Control; heifers received s.c. injections of saline twice daily (0700 and 1900) for 40 d; and 2) Leptin; heifers received s.c. injections of oleptin (19.2 microg/kg) twice daily at 0700 and 1900 for 40 d. Blood samples were collected at 10-min intervals for 5 h on. d 0, 5, 10, 20, 30, and 40, and twice daily, just before each treatment injection, throughout the study. On d 41, heifers received i.v. injections of GnRH at 0 (0.0011 microg/kg) and 90 min (0.22 microg/kg), with additional sampling for 5.5 h to examine releasable pools of LH. Diets promoted a gain of 0.32+/-0.09 kg/d, which did not differ between groups. Plasma concentrations of leptin increased markedly in leptin-treated heifers and were greater (P < 0.001) than controls throughout (27.8+/-0.8 vs. 4.9+/-0.12 ng/mL). None of the heifers reached puberty during the experiment, but did so within 45 d of its termination. Mean concentrations of plasma LH, GH, IGF-I, and insulin were not affected by treatment, nor was there an overall effect on the frequency of LH pulses. However, a treatment x day interaction (P = 0.02) revealed that the frequency of LH pulses (pulses/ 5 h) was greater (P = 0.03) in controls (3.6+/-0.36) than in leptin-treated heifers (1.7+/- 0.28) on d 10. Characteristics of GnRH-induced release of LH were not affected by treatment. In summary, chronically administered leptin failed to induce puberty or alter endocrine characteristics in beef heifers nearing the time of expected puberty.     

Leptin: a metabolic signal affecting central regulation of reproduction in the pig

The discovery of the obesity gene and its product, leptin, it is now possible to examine the relationship between body fat and the neuroendocrine axis. A minimum percentage of body fat may be linked to onset of puberty and weaning-to-estrus interval in the pig. Adipose tissue is no longer considered as only a depot to store excess energy in the form of fat. Recent findings demonstrate that numerous genes, i.e., relaxin, interleukins and other cytokines and biologically active substances such as leptin, insulin-like growth factor-I (IGF-I), IGF-II and Agouti protein are produced by porcine adipose tissue, which could have a profound effect on appetite and the reproductive axis. Hypothalamic neurons are transsynaptically connected to porcine adipose tissue and may regulate adipose tissue function. In the pig nutritional signals such as leptin are detected by the central nervous system (CNS) and translated by the neuroendocrine system into signals, which regulate appetite, hypothalamic gonadotropin-releasing hormone (GnRH) release and subsequent luteinizing hormone (LH) secretion. Furthermore, leptin directly affects LH secretion from the pituitary gland independent of CNS input. Changes in body weight or nutritional status are characterized by altered adipocyte function a reduction in adipose tissue leptin expression, serum leptin concentrations and a concurrent decrease in LH secretion. During pubertal development serum leptin levels, hypothalamic leptin receptor mRNA and estrogen-induced leptin gene expression in fat increased with age and adiposity in the pig and this occurred at the time of expected puberty. In the lactating sow serum and milk leptin concentrations were positively correlated with backfat thickness and level of dietary energy fed during gestation as well as feed consumption. Although, these results identify leptin as a putative signal that links metabolic status and neuroendocrine control of reproduction, other adipocyte protein products may play an important role in regulating the reproductive axis in the pig.     

Role of leptin in modulating the hypothalamic-pituitary axis and luteinizing hormone secretion in the prepuberal gilt

Three experiments (EXP) were conducted to test the hypothesis that leptin modulates LH, GnRH, and neuropeptide Y (NPY) secretion. In EXP I, prepuberal gilts received intracerebroventricular (i.c.v.) leptin injections and blood samples were collected. In EXP II, anterior pituitary cells from prepuberal gilts in primary culture were challenged with 10(-14), 10(-13), 10(-12), 10(-11), 10(-10), 10(-9), 10(-8), 10(-7), or 10(-6) M leptin individually or in combinations with 10(-10), 10(-9), and 10(-8) M GnRH. In EXP III, hypothalamic-preoptic area (HYP-POA) explants were placed in perfusion system and exposed to 0 (n=5), 10(-12) M (n=4), 10(-10) M (n=4), 10(-8) M (n=4), or 10(-6) M (n=5) human recombinant leptin (LEP) for 30 min. In EXP I, serum LH concentrations were unaffected by leptin treatment. In EXP II, all doses of leptin increased LH secretion except for 10(-12) and 10(-7) M. Only 10(-7), or 10(-13) M leptin in combination with 10(-8) or 10(-9) M GnRH, respectively, suppressed LH secretion. In EXP III, prior to leptin, media GnRH concentrations were similar across treatments. Media GnRH concentrations increased after 10(-12), 10(-10), and 10(-8) M leptin compared to control. Leptin treatment failed to influence NPY secretion across treatments. These results indicate that components of the neuroendocrine axis that regulate GnRH and LH secretion are functional and leptin sensitive before the onset of puberty. Other neural peptides in addition to NPY may mediate the acute effects of leptin on the GnRH-LH system and lastly, the inability of i.c.v. leptin treatment to increase LH secretion may in part be related to stage of sexual maturation and associated change in negative feedback action of estradiol on LH secretion.     

Effects of nutrition and season on the onset of puberty in the beef heifer.

Age at puberty is a major determinant of lifetime reproductive efficiency of beef cows. Research conducted during the past 20 yr has documented the major endocrine events leading to first ovulation in heifers. The critical event seems to be a prepubertal increase in pulsatile LH secretion. Environment influences timing of puberty onset in beef heifers. Nutrition and season are two of the better-defined variables that have been studied. Age at puberty is related inversely to plane of nutrition. The effect of nutrition on sexual maturation involves effects on timing of the prepubertal increase in LH secretion and seems to involve the LH pulse generating system located in the hypothalamus. The precise mechanism by which nutrition influences pulsatile LH secretion has not been elucidated, but signals reflecting metabolic status seem to be involved. Seasonal conditions of the early (birth to 6 mo of age) and late (6 to 12 mo of age) postnatal periods also influence timing of puberty onset in the heifer. Autumn-born heifers attain puberty at younger ages than do spring-born heifers, and exposure to spring-summer temperatures and photoperiods during the second 6 mo of life reduces age at puberty regardless of season of birth. Photoperiod may be the major seasonal cue that influences puberty onset in cattle. Limited evidence suggests that melatonin, a pineal hormone, is involved with transducing photic stimuli into neuroendocrine signals that influence LH secretion. If the physiological mechanisms mediating the effects of nutrition and season on timing of puberty onset are determined, then management strategies for reducing age at puberty can be enhanced.     

Effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given GnRH

The effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given 100 microg of GnRH im were determined in three experiments. In Experiment 1, heifers were given GnRH 3, 6 or 9 days after ovulation; 8/9, 5/9 and 2/9 ovulated (P<0.02). Mean plasma concentrations of progesterone were lowest (P<0.01) and of LH were highest (P<0.03) in heifers treated 3 days after ovulation. In Experiment 2, heifers received no treatment (Control) or one or two previously used CIDR inserts (Low-P4 and High-P4 groups, respectively) on Day 4 (estrus=Day 0). On Day 5, the Low-P4 group received prostaglandin F(2alpha) (PGF) twice, 12 h apart and on Day 6, all heifers received GnRH. Compared to heifers in the Control and Low-P4 groups, heifers in the High-P4 group had higher (P<0.01) plasma progesterone concentrations on Day 6 (3.0+/-0.3, 3.0+/-0.3 and 5.7+/-0.4 ng/ml, respectively; mean+/-S.E.M.) and a lower (P<0.01) incidence of GnRH-induced ovulation (10/10, 9/10 and 3/10). In Experiment 3, 4-6 days after ovulation, 20 beef heifers and 20 suckled beef cows were given a once-used CIDR, the two largest follicles were ablated, and the cattle were allocated to receive either PGF (repeated 12h later) or no additional treatment (Low-P4 and High-P4, respectively). All cattle received GnRH 6-8 days after follicular ablation. There was no difference between heifers and cows for ovulatory response (77.7 and 78.9%, P<0.9) or the GnRH-induced LH surge (P<0.3). However, the Low-P4 group had a higher (P<0.01) ovulatory response (94.7% versus 61.1%) and a greater LH surge of longer duration (P<0.001). In conclusion, although high plasma progesterone concentrations reduced both GnRH-induced increases in plasma LH concentrations and ovulatory responses in beef cattle, the hypothesis that heifers were more sensitive than cows to the suppressive effects of progesterone was not supported.     

Effects of restriction of dietary energy intake during the prepubertal period on secretion of luteinizing hormone and responsiveness of the pituitary to luteinizing hormone-releasing hormone in heifers

The working hypothesis that a low plane of nutrition during the prepubertal period delays puberty in heifers by retarding the prepubertal increase in secretion of luteinizing hormone (LH) was investigated. Secretion of LH and the responsiveness of the pituitary to LH-releasing hormone (LHRH) were compared in heifers fed a growing diet (which allowed spontaneous occurrence of puberty; n = 12; control) or an energy deficient diet (which delayed puberty; n = 11; delayed) during the prepubertal period. The dietary treatments were initiated when the heifers were 299 +/- 14 (mean +/- SD) d of age (d 0 of the experiment) and continued until d 175 of the experiment (474 +/- 14 d of age). Weight gains were .79 +/- .05 (mean +/- SE) and .21 +/- .03 kg X head-1 X d-1 for control and delayed heifers, respectively. Puberty occurred on d 120 +/- 14 of the experiment (428 +/- 13 d of age) in control heifers, whereas none of the delayed heifers attained puberty during the feeding period. Serum concentration of LH and the frequency of LH pulses increased rapidly during the 175-d feeding period in control heifers. In delayed heifers, serum LH concentration increased less rapidly and no increase in pulse frequency was detected during the experimental period. Amplitude of LH pulses tended to be higher in control than delayed heifers. Responsiveness of LH secretion to LHRH was lower in delayed than control heifers. It is speculated that failure of secretion of LH to increase is the causative factor for delayed puberty when dietary energy is limited during the prepubertal period in heifers.     

Serum leptin and its adipose gene expression during pubertal development,the estrous cycle,and different seasons in cattle

Circulating concentrations of leptin and IGF-I, leptin gene expression, and serum binding of [126I]ovine leptin in cattle during pubertal development, as well as leptin gene expression and circulating concentrations of leptin during the estrous cycle and different calendar seasons, were investigated. Multivariate regression analysis was utilized to evaluate temporal changes in BW, leptin mRNA, and serum concentrations of IGF-I and leptin normalized to the week of puberty (Exp. 1). Body weight accounted for most of the variation associated with the onset of puberty in the full regression model (R2 = 0.99; P < 0.01). However, serum leptin was closely related to changes in BW (r = 0.85; P < 0.02) and in the absence of BW was most predictive of pubertal onset (r2 = 0.73; P < 0.01). Mean concentrations of leptin increased (P < 0.0001) linearly from 16 wk before until the wk of pubertal ovulation in yearling heifers reaching sexual maturation from early spring to midsummer. Leptin mRNA transformed to a percent of the value at puberty increased (P < 0.02) as puberty approached, but serum leptin and leptin mRNA values were not well correlated. We found no evidence of leptin-binding proteins in serum of developing heifers. Combined mean serum concentrations of IGF-I (ng/mL) during periods III and IV (-9 wk to wk of puberty; 216.6 +/- 9) were 21% higher (P < 0.0001) than combined mean concentrations of IGF-I during periods I and II (-19 to wk of puberty; 193 +/- 10). In mature heifers and cows (Exp. 2), serum leptin tended to decrease (P = 0.10) during the late luteal/early follicular phase of the estrous cycle, which corresponded to a reduction (P < 0.03) in adipocyte leptin gene expression. In mature ovariectomized cows, serum concentrations of leptin increased (P < 0.001) by 34% from early winter to the summer solstice and remained unchanged throughout the remainder of the year (Exp. 3). Results from these studies indicate that marked increases in both circulating leptin and leptin gene expression occur in developing heifers during pubertal development and are associated with increases in serum IGF-I and BW. Seasonal effects on circulating leptin observed in mature cows from winter to summer could also plausibly account for a portion of the prepubertal rise in serum leptin observed in heifers.     

Induction of precocious puberty in heifers I: enhanced secretion of luteinizing hormone

In beef heifers weaned between 3 and 4 mo of age and fed a high-concentrate diet, approximately 50% reach puberty before 300 d of age (precocious puberty). The objectives of this experiment were 1) to determine whether precocious puberty could be induced experimentally by weaning heifers early and feeding a high-concentrate diet, and 2) to determine the dynamics of secretion of LH associated with precocious puberty. Crossbred Angus and Simmental heifer calves were weaned at 73 +/- 3 d of age and 115 +/- 3 kg of BW and fed a high-concentrate (60% corn; HI, n = 9) or control diet (30% corn; CONT, n = 9). Heifers were fed individually, and target BW gains were 1.50 and 0.75 kg/d for the HI and CONT treatments, respectively. Heifers were weighed every 2 wk. Blood samples were collected weekly and assayed for progesterone concentration to determine age at puberty. Serial blood samples were collected at 20-min intervals for 24 h at mean ages of 102, 130, 158, 172, 190, 203, 217, 231, and 259 d and assayed for LH concentration to evaluate the dynamics of secretion of LH. Heifers fed the HI diet exhibited greater BW gain (P < 0.01) than CONT heifers (1.27 +/- 0.05 vs. 0.85 +/- 0.05 kg/d, respectively). As a result, BW in the HI treatment was greater (P < 0.01) than in the CONT treatment by 188 d of age and remained different through the end of the experiment. Precocious puberty occurred in 8 of 9 heifers fed the HI diet and 0 of 9 heifers fed the CONT diet. Age at puberty was reduced in the HI (P < 0.01) compared with the CONT heifers (262 +/- 10 vs. 368 +/- 10 d of age, respectively). Body weight at puberty was also reduced in the HI (P < 0.05) compared with the CONT treatment (327 +/- 17 vs. 403 +/- 23 kg, respectively). Heifers attaining puberty during the experiment continued with subsequent luteal phases as evidenced by cyclic patterns of progesterone concentrations. Frequency of pulses of LH (pulses/24 h) increased with age (P < 0.01) for both treatments. Heifers in the HI treatment exhibited a greater number of pulses of LH (P < 0.01) than those in the CONT treatment by 190 d of age and in all subsequent collection periods (treatment x age, P < 0.05). Mean LH concentrations also increased with age (P < 0.01) for both treatments but did not differ between treatments. In conclusion, precocious puberty induced by early weaning and feeding of a high-concentrate diet is preceded by increasing frequency of pulses of LH.     

Effects of 17 beta-estradiol and diets varying in energy on secretion of luteinizing hormone in beef heifers

An experiment was conducted to test the hypothesis that 17 beta-estradiol (E2) would not suppress secretion of luteinizing hormone (LH) in heifers fed a diet limited in energy during the period before the onset of nutritionally induced anestrus. Sixteen of 20 heifers that had been exhibiting normal estrous cycles (20 mo of age, 409 +/- 6 kg body weight) were ovariectomized, and half of them were assigned at random to receive an E2 implant. The ovariectomized heifers were assigned at random to receive diets that contained low (L; 5.8 Mcal X animal-1 X d-1, n = 8) or high levels of energy (H; 20.0 Mcal X animal-1 X d-1, n = 8) for 100 d. The other four heifers remained intact and were fed the L-diet. The intact heifers were utilized to determine the status of reproductive function in animals fed the L-diet. Heifers lost body weight rapidly after initiation of feeding the L-diet. Heifers fed the L-diet then stabilized at a lighter weight until the latter part of the experiment. One of the four intact heifers fed the L-diet became anestrus near the end of the study. Mean concentrations of LH in blood serum increased linearly (P less than .05) in ovariectomized heifers fed the L- and H-diet. Mean concentration of LH in heifers fed the H-diet that were implanted with E2 was similar to ovariectomized heifers fed the H-diet that received no E2. Mean LH in serum of ovariectomized heifers implanted with E2 fed the L-diet was suppressed and remained low throughout the study. Frequency of pulses of LH in ovariectomized heifers fed the L-diet was less (P less than .01) than that in ovariectomized heifers fed the H-diet. Estradiol decreased the number of pulses of LH in heifers fed the L-diet. We conclude that dietary energy restriction in beef heifers has a direct action on the hypothalamo-pituitary axis to lower the number of pulses of LH in the absence of ovarian steroids. However, ovarian E2 appears to suppress further secretion of LH in heifers fed limited levels of dietary energy before the onset of nutritional anestrus occurs, therefore, our working hypothesis is rejected.     

Luteinizing hormone,growth hormone,insulin-like growth factor-i,insulin and metabolites before puberty in heifers fed to gain at two rates

Fall born Angus x Hereford heifers were allotted to treatments at 9 mo of age to achieve the following growth rates: 1) fed to gain 1.36 kg/d (n = 10; HGAIN); and 2) fed to gain 0.23 kg/d for 16 wk, then fed to gain 1.36 kg/d (n = 9; LHGAIN). Growth hormone (GH), insulin-like growth factor-1 (IGF-I), insulin, glucose, nonesterified fatty acids (NEFA), and progesterone were quantified in twice weekly blood samples until onset of puberty. Body weight, hip height, and pelvic area were recorded every 28 d. Frequent blood samples (n = 8 heifers/treatment) were collected every 14 d, commencing on day 29 of treatment until onset of puberty to evaluate secretion of luteinizing hormone (LH) and GH. The HGAIN heifers were younger (369 d; P < 0.001), were shorter at the hip (115 cm; P < 0.05) and had smaller pelvic area (140 cm²; P < 0.10), but body weight (321 kg) did not differ at puberty compared with LHGAIN heifers (460 d; 119 cm; 155 cm²; 347 kg, respectively). The HGAIN heifers had greater (P < 0.05) concentrations of LH, IGF-I, and insulin in serum and glucose in plasma during the first 84 d of treatment than LHGAIN heifers, whereas LHGAIN heifers had greater (P < 0.05) concentrations of GH in serum and NEFA in plasma than HGAIN heifers. On Day 68 of treatment, HGAIN heifers had less mean GH (P < 0.01) and greater (P < 0.05) LH pulse frequency than LHGAIN heifers, whereas LH pulse amplitude and mean LH did not differ (P > 0.10) between treatments. Treatment did not influence secretion of LH and GH at 1 and 3 wk before puberty. Mean GH concentrations in serum and GH pulse amplitude in all heifers were greater (P < 0.05) 2 to 9 d (12.9 and 40.7 ng/ml, respectively) than 16 to 23 d (10.4 and 20.0 ng/ml, respectively) before puberty. Nutrient restriction decreased LH pulse frequency and delayed puberty in beef heifers. Furthermore, dramatic changes in mean concentration and amplitude of GH pulses just before puberty in beef heifers may have a role in pubertal development.     

Release of luteinizing hormone after administration of naloxone in pre- and peripuberal heifers.

This study was conducted to investigate regulation of LH release by opioid peptides during puberal development in beef heifers. Fourteen heifers were randomly assigned to receive naloxone (opioid antagonist) i.v. at dosages of either 1 mg.kg BW-1.wk-1 (Dose 1) or .25 mg.kg BW-1.wk-1 (Dose 2) for 13 wk or until puberty. Blood was sampled (one sample every 15 min) 6 h before (prenaloxone) and 2 h after naloxone administration. Two hours after naloxone administration, GnRH (10 ng/kg BW) was administered and blood was sampled for 1 h. Nine heifers attained puberty during the study. There were no differences between naloxone dosage groups for any measured variables. Therefore, heifers were grouped dependent on the attainment of puberty. Prenaloxone concentrations of serum LH and LH pulse frequency were normal for prepuberal heifers. Serum LH concentrations increased within 30 min after naloxone 135 of 139 times it was administered (P less than .05). Serum LH concentrations during the hour after naloxone were higher (P less than .05) than those during the hour before naloxone in both puberal and nonpuberal heifers. In puberal heifers, serum LH pulse height during the hour after naloxone was greater (P less than .02) at 5 wk before puberty and lower (P less than .02) the week before puberty than at other times during the trial. There was no effect of week on serum LH pulse height after naloxone in heifers that failed to attain puberty during the study. Response of LH to GnRH was similar between groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of dose, frequency, and duration of infused gonadotropin-releasing hormone on secretion of luteinizing hormone and follicle-stimulating hormone in nutritionally anestrous beef cows.

Nutritionally induced anovulatory cows were ovariectomized and used to determine the relationships between dose, frequency, and duration of exogenous gonadotropin-releasing hormone (GnRH) pulses and amplitude, frequency, and concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum. In Experiment 1, cows were given pulses of saline (control) or 2 micrograms of GnRH infused i.v. during a 0.1-, 1.25-, 5-, 10-, or 20-min period. Concentrations of LH and FSH during 35 min after GnRH infusion were greater than in control cows (P < 0.01), and FSH concentrations were greater when GnRH infusions were for 10 min or less compared with 20 min. In Experiment 2, the effect of GnRH pulse frequency and dose on LH and FSH concentrations, pulse frequency, and pulse amplitude were determined. Exogenous GnRH (0, 2, or 4 micrograms) was infused in 5 min at frequencies of once every hour or once every 4th hr for 3 d. There was a dose of GnRH x frequency x day effect on LH and FSH concentrations (P < 0.01), indicating that gonadotropes are sensitive to changes in pulse frequency, dose, and time of exposure to GnRH. There were more LH pulses when GnRH was infused every hour, compared with an infusion every 4th hr (P < 0.04). Amplitudes of LH pulses were greater with increased GnRH dose (P < 0.05), and there was a frequency x dose x day effect on FSH pulse amplitude (P < 0.0006). We conclude that LH and FSH secretion in the bovine is differentially regulated by frequency and dose of GnRH infusions.     

Leptin receptor expression and its change in association with the normalization of EGF profile after seminal plasma treatment in repeat breeder dairy cows

Factors associated with high milk production levels have been linked to alterations in the endometrial epidermal growth factor (EGF) profile, a cause of reduced fertility in dairy cows. Therefore, we examined the leptin system that connects nutritional status and reproduction in dairy cattle related to reduced fertility in repeat breeder cows. Plasma leptin concentrations were measured in 18 heifers, 20 high-yielding control cows, and 26 repeat breeder cows, showing an altered EGF profile. Then, all repeat breeder cows were infused with seminal plasma (SP) into the vagina at the next estrus to normalize the EGF profile, while heifers and control cows were infused with vehicle alone. All animals were examined for EGF profiles. Eighteen repeat breeder cows, nine heifers, and nine control cows were also determined for leptin receptor (Ob-R) expression levels in the estrous cycle before and after the infusion. SP normalized the EGF profile in 53.8% of the repeat breeder cows. Leptin concentrations were similar in all groups, regardless of the treatment results for the EGF profile. In contrast, Ob-R levels in repeat breeder and control cows were similar and higher than those in heifers before SP treatment. Ob-R in repeat breeders showing a normal EGF profile after treatment decreased to an intermediate level between heifers and control cows and may provide a clue to take measures against repeat breeding in dairy cows.     

Hypothalamic control of gonadotropin secretion by LHRH,FSHRF, NO,cytokines, and leptin

Gonadotropin secretion by the pituitary gland is under the control of luteinizing hormone-releasing hormone (LHRH) and the putative follicle stimulating hormone-releasing factor (FSHRF). Lamprey III LHRH is a potent FSHRF in the rat and seems to be resident in the FSH controlling area of the rat hypothalamus. It is an analog of mammalian LHRH and may be the long sought FSHRF. Gonadal steroids feedback at hypothalamic and pituitary levels to either inhibit or stimulate the release of LH and FSH, which is also affected by inhibin and activin secreted by the gonads. Important control is exercised by acetylcholine, norepinephrine (NE), dopamine, serotonin, melatonin, and glutamic acid (GA). Furthermore, LH and FSH also act at the hypothalamic level to alter secretion of gonadotropins. More recently, growth factors have been shown to have an important role. Many peptides act to inhibit or increase release of LH and the sign of their action is often reversed by estrogen. A number of cytokines act at the hypothalamic level to suppress acutely the release of LH but not FSH. NE, GA, and oxytocin stimulate LHRH release by activation of neural nitric oxide synthase (nNOS). The pathway is as follows: oxytocin and/ or GA activate NE neurons in the medial basal hypothalamus (MBH) that activate NOergic neurons by alpha₁ (α₁) receptors. The NO released diffuses into LHRH terminals and induces LHRH release by activation of guanylate cyclase (GC) and cyclooxygenase. NO not only controls release of LHRH bound for the pituitary, but also that which induces mating by actions in the brain stem. An exciting recent development has been the discovery of the adipocyte hormone, leptin, a cytokine related to tumor necrosis factor (TNF) α. In the male rat, leptin exhibits a high potency to stimulate FSH and LH release from hemipituitaries incubated in vitro, and increases the release of LHRH from MBH explants. LHRH and leptin release LH by activation of NOS in the gonadotropes. The NO released activates GC that releases cyclic GMP, which induces LH release. Leptin induces LH release in conscious, ovariectomized estrogen-primed female rats, presumably by stimulating LHRH release. At the effective dose of estrogen to activate LH release, FSH release is inhibited. Leptin may play an important role in induction of puberty and control of LHRH release in the adult as well.     

Influence of prepubertal ovariectomy and estradiol replacement therapy on secretion of luteinizing hormone before and after pubertal age in heifers

Secretion of luteinizing hormone (LH) and effects of estradiol were evaluated during and after the prepubertal decline in negative feedback of estradiol on secretion of LH. Prepubertal heifers (269 ± 4 days of age; n=10) were ovariectomized on February 6, 1981 (Day 0). Five ovariectomized heifers were administered a subcutaneous implant on Day 0 which provided physiological serum concentrations of estradiol (OVX-E₂). The remaining 5 heifers were not implanted (OVX). A second estradiol implant was administered to OVX-E₂ heifers on Day 164 (n=3) or Day 206 (n=2) of the study. Blood samples were collected sequentially (every 12 min for 8 hr) at approximately two week intervals from Days 0 to 232 of the experiment. The experimental period spanned from approximately 100 days before (269 days of age) to 100 days after (501 days of age) the expected age at puberty. Mean serum concentration of LH and frequency of LH pulses increased rapidly from Days 0 to 36 in OVX heifers and were followed by a further gradual rise in pulse frequency (Day 50 to 232) and a reciprocal decline in mean LH and pulse amplitude. The rapid post-ovariectomy increase in secretion of LH was blocked by estradiol in OVX-E₂ heifers. All characteristics (mean, frequency and amplitude) of secretion of LH increased gradually during the experimental period in OVX-E₂ heifers (Days 0 to 232). Mean concentration and amplitude of pulses were higher in OVX-E₂ than in OVX heifers by Days 148 and 134, respectively. These differences were maintained for the remainder of the experimental period. No acute effects of the second estradiol implant on secretion of LH were observed in OVX-E₂ heifers. Results of this study indicate that long-term changes in secretion of LH occur following prepubertal ovariectomy in heifers and suggest that the previously documented prepubertal decline in negative feedback of estradiol on secretion of LH is followed by a period of positive feedback after pubertal age is surpassed.     

Effect of short-term fasting on plasma concentrations of leptin and other hormones and metabolites in dairy cattle

We determined the effects of short-term fasting and refeeding on temporal changes in plasma concentrations of leptin, insulin, insulin-like growth factor- 1 (IGF-1), growth hormone (GH), glucose, and nonesterified fatty acids (NEFA), in early lactating cows, non-lactating pregnant cows, and postpubertal heifers. In experiment 1, Holstein cows in early lactation were either fed ad libitum (Control, n=5) or feed deprived for 48 h (Fasted, n=6). Plasma leptin, insulin, and glucose concentrations rapidly declined (P<0.05) within 6h, and IGF-1 by 12h, but all these variables sharply returned to control levels (P>0.10) within 2h of refeeding. Plasma NEFA and GH concentrations were elevated (P<0.05) by 4 and 36 h of fasting and returned to control levels (P>0.10) by 8 and 24h after refeeding, respectively. In experiment 2, four ruminally cannulated pregnant non-lactating Holstein cows were used in a cross-over design and were fasted for 48 h (Fasted) or fasted with partial evacuation of rumen contents (Fasted-Evac). The plasma variables measured did not differ (P>0.10) between Fasted and Fasted-Evac cows. Plasma leptin, insulin, and IGF-1 concentrations were reduced by 10, 6, and 24h of fasting, respectively, in Fasted-Evac cows; and these variables were reduced by 24h in Fasted cows (P<0.05). Plasma glucose levels were reduced (P<0.05) by 48 h of fasting in both groups of fasted animals. Plasma NEFA and GH levels were increased (P<0.05) by 12 and 48 h of fasting, respectively. In experiment 3, postpubertal Holstein heifers were either fed ad libitum (Control, n=4) or feed deprived for 72 h (Fasted, n=5). Concentrations of leptin, insulin, IGF-1, and glucose in plasma were reduced (P<0.05) by 24, 10, 24, and 48 h of fasting, respectively. Plasma NEFA concentrations increased (P<0.05) by 4h, of fasting while GH levels were not significantly (P>0.10) affected by fasting. Collectively, our data provide evidence that plasma leptin concentrations are reduced with short-term fasting and rebound on refeeding in dairy cattle with the response dependent on the physiological state of the animals. Compared to the rapid induction of hypoleptinemia with fasting of early lactation cows, the fasting-induced hypoleptinemia was delayed in non-lactating cows and postpubertal heifers.     

Hypothalamic deafferentation in prepuberal beef heifers: Effects of gonadotropin-releasing hormone and estradiol benzoate on luteinizing hormone secretion

Hypothalamic control of luteinizing hormone (LH) secretion was investigated in crossbred beef heifer calves by comparing anterior (AHD), posterior (PHD), and complete (CHD) hypothalamic deafferentation with sham operated controls (SOC). Heifers (n = 16) were fitted with an indwelling jugular catheter for 6 days before cranial surgery, and assigned randomly to treatments. Blood for radioimmunoassay of LH was collected sequentially at 15-min intervals during an 8-h period on days ? 1 before and day 6 after hypothalamic deafferentation or sham operation. On the day of surgery, blood samples were collected sequentially at 15-min intervals 2 h before induction of anesthesia and throughout surgery and early recovery. Seven months after hypothalamic deafferentation, all experimental and sham operated heifers were ovariectomized and treated with vegetable oil (i.m.) plus saline (i.v.), vegetable oil plus gonadotropin releasing hormone (GnRH), estradiol benzoate (EB, 1 mg) in vegetable oil. After ovariectomy basal plasma concentrations of LH increased (P < 0.01) compared with the low circulating hormone levels before ovariectomy. The amplitude of LH response to GnRH was greater (P < 0.01) in CHD and PHD when compared with SOC and AHD heifers. Injection of EB failed to induce a LH surge in CHD and PHD 900–1100 min later when compared with the robust response seen in SOC and AHD heifers. Injection of EB plus GnRH elicited LH release in all deafferentated and sham operated heifers. These results indicate a transient change in LH secretion after AHD or CHD in prepuberal heifers with intact ovaries. After OVX, the integrity of the neural connection of the posterior hypothalamus is required for EB-induced LH release in beef heifers.     

The effects of 3 gonadorelin products on luteinizing hormone release,ovulation, and follicular wave emergence in cattle

The objective was to determine luteinizing hormone (LH) secretion and follicular dynamics in cattle following administration of 3 gonadorelin formulations that are commercially available in Canada. In experiment 1, nonlactating Holstein cows (n = 4 per group) were randomly assigned to receive 100 micrograms gonadorelin diacetate tetrahydrate, intramuscularly (C; Cystorelin, or FE; Fertagyl). Blood samples (for LH analysis) were collected 0, 1, 2, and 4 hours after treatment. In experiment 2, nonlactating Holstein cows (n = 10 per group) were randomly allocated to receive 100 micrograms gonadorelin, intramuscularly as follows: 2 mL of C; 1 mL of FE; or 2 mL of Factrel (FA, gonadorelin hydrochloride). Gonadorelin treatment was done on days 6 or 7 after ovulation and blood samples for LH analysis were collected at 0, 1, 2, 4, and 6 hours after treatment. Ovaries were examined by ultrasonography, twice daily, to detect ovulation. A replicate was conducted using only C (n = 10) or FE (n = 10); blood samples were collected at 0, 1, 2, 3, and 4 hours. In experiment 3, beef heifers (n = 10 per group) were randomly assigned to receive 1 of 3 GnRH gonadorelin treatments (as in the first phase of experiment 2) on days 6 or 7 after ovulation and blood samples were collected at 0, 0.5, 1, 1.5, 2, and 4 hours. In experiments 2 and 3, both mean and mean peak plasma LH concentrations were higher (P < 0.05) in cattle treated with C. The proportion of dominant follicles that ovulated was higher (P < 0.02) in Holstein cows treated with C than in those treated with FE or FA (18/19, 11/19, and 4/7, respectively), but there was no significant difference among the products in beef heifers (6/10, 6/10, and 4/10, respectively). No significant differences were found in the interval from treatment to the emergence of the next follicular wave. In summary, C induced a greater LH release and this resulted in a higher ovulatory rate in Holstein cows but not in beef heifers.