P.G. Harms

Leptin acts at the bovine adenohypophysis to enhance basal and gonadotropin-releasing hormone-mediated release of luteinizing hormone : differential effects are dependent upon nutritional history

Abstract Recombinant ovine leptin (oleptin) stimulates an acute increase in the secretion of LH in fasted, but not in normal-fed, cows through an augmentation of the magnitude of individual pulses of LH. Herein, we tested the hypothesis that this effect could be accounted for by functional changes at the adenohypophyseal (AP) level. Eleven ovariectomized, estradiol-implanted cows were assigned to one of two dietary groups: normal-fed (n = 6) and fasted (fasted for 72 h; n = 5). After the animals were killed, the adenohypophyses were collected and AP explants were perifused with Krebs-Ringer bicarbonate buffer (KRB) for a total of 6.5 h, including a 2-h treatment at 2.5 h with KRB or increasing doses of oleptin and a challenge at 4.5 h with 50 ng of GnRH. To test for effects of leptin at the hypothalamic level, explants encompassing the medial basal hypothalamus-infundibular complex (HYP) were incubated in KRB alone (control) or in KRB containing 1000 ng of oleptin. Basal release of LH from AP explants treated with leptin was greater (P < 0.02) than that from control-treated explants in fasted, but not in normal-fed, cows. To the contrary, leptin-treated explants from normal-fed, but not from fasted, cows released more (P < 0.001) LH in response to GnRH than control-treated tissues. Neither fasting nor leptin affected (P > 0.1) the secretion of GnRH from HYP explants. These observations support the hypothesis that leptin modulates the secretion of LH in mature cows, to a large extent, by its direct actions at the AP. Differential manifestations of these effects are dependent upon nutritional history.

Relationship between body condition and levels of serum luteinizing hormone in postpartum mares

Abstract To study the endocrine environment of foaling mares at different levels of body condition, 12 mares were divided equally into two groups, one designed to foal at a condition score of 4.5 or less (thin) and the other to maintain a condition score of 6 or greater (control). Serum samples were collected twice weekly before foaling, and daily thereafter until 7 d following the second estrous cycle after parturition. Serum levels of luteinizing hormone (LH) were measured by Radioimmunoassay. In addition, mares were weighed, body condition was scored and body fat content was measured weekly. One consequence of low body condition at foaling in thin mares was a longer mean gestation length than in control mares. Also, the pattern of LH observed during the first and second estrous cycles after foaling was different for thin vs control mares. Serum LH concentrations for thin mares were greater during their second estrous cycle than during their first estrous cycle. Also, in thin mares, the interval from parturition to ovulation, as indicated by the day of maximum concentration of LH during estrus, was random. Control mares had equivalent concentrations of serum LH in both cycles, and they had predictable intervals from parturition to ovulation. LH patterns in thin mares were similar to those reported in mares during seasonal transition, whereas LH patterns of control mares were similar to those reported in mares during the middle of the breeding season.

GnRH in the infundibular stalk-median eminence is related to percentage body fat in carcasses of beef cows.

Mature Hereford cows (n = 28) were used to determine the effect of percentage body fat on secretion of LH and content of GnRH in the infundibular stalk-median eminence (ISME). Cows were fed to maintain, lose, or gain weight to achieve body condition scores (BCS; 1 = emaciated; 9 = obese) of 3 to 7. Then cows were fed to maintain weight and body condition. Before slaughter, estrus was synchronized using two injections of prostaglandin F2 alpha(PGF) 11 d apart. Five d after the second PGF injection, cows were given 100 micrograms of GnRH (im) and serum samples were obtained. LH was quantified using RIA. The anterior pituitary and ISME were obtained within 45 min of death. Anterior pituitary weight and LH concentration, total GnRH in the ISME, total carcass fat, and percentage carcass fat were determined. BCS of cows at the time of slaughter influenced percentage carcass fat (P less than .001), total GnRH in the ISME (P less than .02), and maximum LH after GnRH treatment (P less than .09), but did not influence pituitary weight or concentration of LH in the pituitary. Content of GnRH in the ISME averaged 76 +/- 12, 32 +/- 14, 27 +/- 13, and 24 +/- 13 ng for cows with BCS of 3, 5, 6, and 7, respectively. BCS was correlated (P less than .001) with percentage carcass fat (r = .94) and total fat in the carcass (r = .92). Total GnRH in the ISME was negatively correlated (P less than .005) with BCS (r = -.54), percentage carcass fat (r = -.55), and total carcass fat (r = -.49).(ABSTRACT TRUNCATED AT 250 WORDS)

Time-dependent loss of radioimmunoassayable levels of progesterone following ambient temperature incubation of heparinized bovine blood☆

Plasma progesterone levels in heparinized blood collected at 10 min intervals for 8 continuous hours from four nulliparous Holstein cows on day 3 (early luteal), day 10 or 11 (mid-luteal) and day 18 or 19 of the estrous cycle were found to decline over time when blood was incubated at ambient temperature. The loss was more obvious during the mid-luteal collection period than either the day 3 or day 18 or 19 periods in all cows. This appeared to be associated more with high progesterone levels on day 10 or 11 rather than with differences in the period of the estrous cycle. There was an average decrease in progesterone levels of 3.4, 1.0 and 1.5 ng/ml between samples having the shortest and longest incubation periods on day 10 or 11, day 3 and day 18 or 19, respectively. This apparent decrease in levels of progesterone from bovine blood indicates need in the future for careful consideration concerning the handling of bovine blood collected for subsequent radioimmunoassay (RIA) of progesterone. Further work to elucidate the mechanism which is responsible for the apparent loss is also warranted.

Effects of ethanol on hypothalamic luteinizing hormone-releasing hormone (LHRH) in the male rat an immunocytochemical study

SummaryThese studies were designed to determine if the acute alcohol-induced decreases in luteinizing hormone (LH) seen in previous studies using rats could be due to an inhibitory effect of ethanol (ETOH) on hypothalamic LHRH release. Thus, effects of multiple injections of ETOH on the relative amount of immunoreactive LHRH fibers in the hypothalamus and median eminence (ME) of castrate and intact male rats were determined immunocytochemically. Brains were removed following cardiac perfusion of 10% phosphate-buffered formalin. A block containing the hypothalamus with the ME was isolated from each brain, then postfixed in Bouins solution. Paraffin sections were rehydrated and stained for LHRH with the peroxidaseantiperoxidase technique using an antiserum to synthetic LHRH conjugated to bovine serum albumen. Differences visualized immunocytochemically between saline-treated intact and castrate rats indicated that the LHRH content of the ME was markedly depleted after castration. Conversely, castrate rats treated with ETOH showed only a slight reduction in immunoreactive LHRH fibers. In ETOH-treated intact animals, the LHRH fiber content of both the hypothalamus and ME appeared to be slightly greater than the saline-treated intact controls. Thus, these data support the hypothesis that ETOH diminishes LHRH release, and hence provides an explanation for the depressed plasma LH levels observed in ETOH-treated intact and castrate rats.

Effect of suckling manipulation on postpartum reproduction in primiparous Brahman-cross cows

Abstract The effect of suckling manipulation on postpartum reproductive performance was investigated in 106 primiparous Brahman-cross cows. Suckling manipulation treatments were 1) suckling ad libitum (SAL, n = 52) and 2) once-daily suckling (ODS, n = 54) beginning at 30 d after parturition. By 60 d after parturition, 57% of the ODS and 29% of the SAL groups, respectively (P

Immunocytochemical Evidence that Suckling Inhibits the Postovariectomy Depletion of Median Eminence Luteinizing Hormone Releasing Hormone

Suckling has been demonstrated to impair the release of pituitary luteinizing hormone (LH) and to prevent the dramatic increase in plasma LH observed following ovariectomy. In the present study, the effect of suckling (10 pups/animal) for either 1 or 3 weeks on the relative amount of luteinizing hormone releasing hormone (LHRH) present in the hypothalamus and preoptic area of ovariectomized and intact rats was examined using immunocytochemical methodology. Controls consisted of nonlactating animals which were either intact (diestrous) or ovariectomized for 1 or 3 weeks. Brains were removed following transcardial perfusion of phosphate-buffered formalin and Bouins fixative. After dehydration, clearing and paraffin embedding, the brains were sectioned and LHRH localized by an indirect immunoperoxidase technique. A positive reaction denoting the presence of immunoreactive LHRH was observed over axons and termini throughout the rostral to caudal extent of the median eminence (ME) and surrounding the organum vasculosum of the lamina terminalis (OVLT) in the preoptic area. Ovariectomy resulted in a progressive decline in the concentration of LHRH within the ME as evidenced by a reduction in the intensity of the staining reaction and in the number of axons over which the reaction was observed. In contrast, brains from ovariectomized rats which had been suckled appeared to have concentrations of LHRH in the ME equal to or greater than that of the diestrous controls. Similarly, the concentrations of LHRH In the ME of intact, suckled rats did not differ significantly from that of the diestrous controls. Neither ovariectomy nor suckling produced any observable change in the relative concentration of LHRH located near the OVLT. These data demonstrate that suckling prevents the depletion of LHRH from the ME following ovariectomy and provide evidence for mechanism by which the suckling stimulus may suppress plasma LH.

Calcium/Calmodulin-Dependent Protein Kinase II Involvement in Release of Gonadotropin-Releasing Hormone

Involvement of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in regulation of GnRH release was tested by determining the effect of CaM kinase II antagonists (KN-62 or KN-93) on GnRH release from rat or cattle infundibular (stalk median eminence) explants. Preincubation of male rat infundibular explants for 30 min with KN-62 (0.5, 1, 5 or 10 µM) 1.5 h prior to the addition of 59.3 mM (high) K+ resulted in a dose-dependent suppression of GnRH release. A longer pretreatment period (2 h) of rat infundibular explants with KN-62 (1 or 10 µM) appeared to enhance the suppressive effect of the CaM kinase II antagonist. Exposure (2 h) of rat infundibular explants to 10 µM, but not 0.1 µM KN-93, resulted in a complete inhibition of high K+-induced GnRH release. Exposure of steer infundibular explant halves to KN-62 (50 or 100 µM) or KN-93 (50 µM) inhibited high K+-induced GnRH release. Likewise, treatment of heifer infundibular explant halves with KN-93 (50 µM) abolished high K+-induced GnRH release. The period of exposure required for KN-62 to elicit its effect was relatively short since exposure of KN-62 (100 µM) for only 91–150 min of incubation was sufficient to block high K+-induced GnRH release from steer infundibular explant halves. In conclusion, these results: (1) support the hypothesis that CaM kinase II is involved in GnRH release from the rat and cattle infundibulum, (2) demonstrate that the effect of CaM kinase II on GnRH release from cattle infundibula is independent of reproductive state, (3) confirm previous reports supporting Ca2+ and CaM involvement in GnRH release from rat and cattle infundibula and (4) establish that infundibular explants incubated in vitro are useful for studying selected mechanisms regulating hypothalamic neurohormone release from neuron terminals.

Milk yield and composition in the multiparous mare fed to obesity

Summary Eighteen multiparous Quarter Horse mares were utilized in a study to determine whether nutritionally-induced obesity, initiated during gestation, would adversely affect milk yield and milk composition. Mares were assigned to one of two treatment groups during gestation: 1) control (n=11): fed to maintain a moderate degree of body fatness (mean postpartum condition score ± SE = 6.1 ± 0.2) and 2) obese (n=7): fed to achieve (prepartum) and then maintain (postpartum) an extremely high degree of body fatness (mean postpartum condition score ± SE = 8.8 ± 0.2). Foal growth was monitored at weekly intervals until the study was terminated on Day 63 of lactation. Foals were not allowed access to supplemental feed. A weigh-suckle-weigh technique was used to estimate milk yield on Days 30 and 60 of lactation. Samples for laboratory analysis of milk composition were collected on Days 31 and 61 of lactation. The 63-d total gain in body weight, heart girth, and third metacarpal bone circumference was greater (p 0.05). Milk yield tended to be greater in control as compared to obese mares (14.4 vs 13.0 kg/d, SE=0.5, p

Ontogeny of ovarian inhibition of pulsatile luteinizing hormone secretion in postnatal holstein heifers

The hypotheses that pulsatile secretion of luteinizing hormone (LH) increases in heifers between 3 and 9 weeks of age and that ovarian inhibition of pulsatile LH release is first established between 3 and 9 weeks of age were tested in Holstein heifers. Heifers were bilaterally ovariectomized at 3 (n=5), 6 (n=4) or 9 (n=4) weeks of age. Blood samples were collected via indwelling jugular cannulae at 10-min intervals for 8 hr 1 week before and 1 and 4 weeks after ovariectomy. During the preovariectomy sampling period, no LH pulses were detected in the 3- or 6-week-old heifers, while one LH pulse occurred in three of four heifers in the 9 week age group. Both mean plasma LH concentration and number of LH pulses increased (P<0.01) by 1 week after ovariectomy only in the 6 and 9 week age groups. Between 1 and 4 weeks following ovariectomy, mean LH concentration increased (P<0.01) in all age groups, while number of LH pulses increased (P<0.01) only in the 3 week group. When the data from the three age groups were combined, amplitude of LH pulses increased (P<0.01) between 1 and 4 weeks following ovariectomy. Mean plasma levels of corticoids did not differ among age groups within sampling periods. n nWe conclude that ovarian inhibition of pulsatile LH secretion is established by 6 weeks of age in Holstein heifers and that the initial increase in mean LH concentration following ovariectomy is mainly due to an increased frequency of LH pulses and the subsequent increase is due to a greater amplitude of LH pulses. Furthermore, the delayed development of pulsatile LH secretion following ovariectomy in the 3-week-old heifers provides a basis for further studies to determine the integrity of the hypothalamic-pituitary axis in postnatal heifers.