Elicitation of release of luteinizing hormone by N-methyl-d,l-aspartic acid during three paradigms of suppressed secretion of luteinizing hormone in the female pig.

Two experiments were conducted to determine the minimal effective dose during lactation and site of action of N-methyl-d,l-aspartic acid (NMA) for elicitation of release of luteinizing hormone (LH) in female pigs. In the first experiment, three doses of NMA were given to lactating primiparous sows in which endogenous LH was suppressed by suckling of litters. In the second experiment, ovariectomized gilts were pretreated with estradiol benzoate or porcine antisera against GnRH to suppress LH and then given NMA to determine if it elicited secretion of LH directly at the anterior pituitary or through release of GnRH. In experiment 1, 3 lactating sows (17 +/- 1.5 d postpartum) were each given three doses of NMA (1.5, 3.0 and 5.0 mg/kg body weight [BW]; IV) on 3 consecutive days in a Latin Square design. Blood samples were collected every 10 min from -1 to 1 hr from injection of NMA. NMA at 1.5 and 3.0 mg/kg did not affect (p greater than .5) secretion of LH; however, 5 mg NMA/kg elicited a 114% increase (p less than .001) in circulating levels of LH during 1 hr after treatment. In experiment 2, 8 ovariectomized gilts were given either estradiol benzoate (EB; 10 micrograms/kg BW; IM n = 4) to suppress release of GnRH or porcine antiserum against GnRH (GnRH-Ab; titer 1:8,000; 1 ml/kg BW; IV; n = 4) to neutralize endogenous GnRH. Gilts infused with GnRH-Ab were given a second dose of antiserum 24 hr after the first. Gilts were then given NMA (10 mg/kg BW; IV) 33 hr after EB or initial GnRH-Ab. Blood samples were drawn every 6 hr from -12 to 24 hr from EB or GnRH-Ab treatments, and every 10 min from -2 to 2 hr from NMA. Serum LH declined (p less than .001) after EB (from 1.87 +/- .2 ng/ml at 12 hr before EB to 0.46 +/- .02 ng/ml during 24 hr after EB) and GnRH-Ab (from 1.97 +/- .1 to 0.59 +/- .02 ng/ml). In gilts treated with EB, the area under the curve (AUC) for the LH response (ng.ml-1.min) 1 hr after NMA (38.7 +/- 3) was significantly greater (p less than .01) than the 1 hr prior to NMA (21.3 +/- 1.5). Treatment with NMA had no effect (p greater than .5) on secretion of LH in gilts infused with GnRH-Ab.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth hormone release after N-methyl-D,L-aspartate in sheep: dose response and effect of an opioid antagonist

The objectives of our experiments were 1) to determine the effect of N-methyl-D,L-aspartate (NMA), an agonist of the neuroexcitatory amino acids aspartate and glutamate, on growth hormone (GH) release in ovariectomized ewes, and 2) to determine the effect of naloxone, an opioid antagonist, on the GH response to NMA. Jugular blood was collected via venipuncture at 12-min intervals for 2 h before and 2 h after i.v. injection of NMA. In Exp. 1, ewes received either 0, 6, 12 or 24 mg NMA/kg BW dissolved in .9% saline solution (n = 4 per treatment). Growth hormone concentrations were similar (P greater than .1) between groups prior to injection (9.8 +/- .7 ng/ml; mean +/- SEM) and were unaffected (P greater than .1) by saline treatment. In contrast, 6, 12 or 24 mg NMA/kg BW increased mean GH concentration by 210% (P less than .04), 273% (P less than .02) and 234% (P less than .02), respectively. In Exp. 2, ewes received NMA (6 mg/kg BW) 5 min after either saline (n = 4) or naloxone (1 mg/kg BW; n = 4) pretreatment. Serum GH concentrations averaged 7.0 +/- 1.1 ng/ml before pretreatment and increased similarly (238%; P greater than .1) in both groups following NMA. In summary, NMA increased GH concentrations in ovariectomized ewes by some mechanism that does not involve opioid receptors that are antagonized by naloxone.     

Development of an endocrine challenge test to investigate subfertility in ewes

Simultaneous or sequential injection of 250 ng gonadotrophin releasing hormone (GnRH) and 25 micrograms oestradiol benzoate, with luteinizing hormone (LH) measurements at 0, +20 min (after GnRH) and +16 h (after oestradiol), enabled investigation of the positive feedback effects on the hypothalamus and pituitary. Control ewes had pretreatment LH values of 3.1 +/- 1.2 ng/ml with an increment of 3.2 +/- 2.3 ng/ml 20 min after GnRH. Subfertile ewes, in spite of elevated pretreatment LH concentrations (15.8 +/- 9.5 ng/ml) in eight out of 10 ewes, had increments of 1.4-84 ng/ml after GnRH. Control ewes had LH increments of 3-75 ng/ml 16 h after oestradiol. Subfertile ewes with pretreatment LH concentrations less than 15 ng/ml also responded to oestradiol whereas those with initial LH concentrations 16-40 ng/ml had no further LH increment. Subsequent administration of 1000 iu pregnant mares' serum gonadotrophin (PMSG), with measurement of LH and oestradiol at 0, +24, +30, +48, +54, and +72 h, allowed assessment of ovarian response and hypothalamus-pituitary function. Five control ewes were sampled up to 30 h post-PMSG and only 1 had oestradiol concentrations greater than 10 pg/ml. Sampling up to 72 h in another five control ewes resulted in oestradiol concentrations greater than 10 pg/ml. Increments in LH concentration greater than 3 ng/ml were recorded in control and subfertile ewes with oestradiol concentrations greater than 10 pg/ml. The use of these endocrine challenge tests enabled positive diagnosis of abnormality on 8 out of 10 occasions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estradiol-17 beta, naloxone and gonadotropin releasing hormone on postpartum secretion of luteinizing hormone in fall-lambing ewes

The interaction among exogenous estradiol-17 beta, naloxone and gonadotropin releasing hormone (GnRH) in the control of luteinizing hormone (LH) secretion was studied in intact postpartum ewes nursing their offspring. One-half of 30 fall-lambing ewes were implanted subcutaneously with an estradiol-17 beta containing Silastic capsule between postpartum d 1 and 12 which doubled their serum concentrations of estradiol (16.0 +/- .1 vs 8.4 +/- .1 pg/ml). Blood samples were collected from implanted and non-implanted ewes at 15-min intervals for 5 h on d 3, 8, 13, 20 and 28 postpartum. Pre-injection samples were collected for 1 h, and ewes were injected with saline, naloxone (NAL;1 mg/kg) or GnRH (100 micrograms/ewe). When averaged across all days and implant groups, serum LH in the three post-NAL samples was higher (P less than .05) than in the three pre-NAL samples (3.6 +/- 1.2 vs .6 +/- .2 ng/ml). Post-GnRH concentrations of serum LH were lower (P less than .05) in estradiol-implanted ewes than in non-implanted ewes on d 8 and 13, but there were no differences in any LH characteristics on d 20 and 28 after implant removal on d 12. In non-implanted ewes, serum LH responses to GnRH increased (P less than .05) eightfold from d 3 (3.8 +/- 1.4 ng/ml) to d 8 (31.6 +/- 1.4 ng/ml), remained elevated through d 20, but declined by d 28 (10.8 +/- 1.4 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of N-methyl-D,L-aspartate on LH,GH, and testosterone secretion in goat bucks maintained under long or short photoperiods

Photoperiod modulates reproduction in goats. We tested the hypothesis that the excitatory glutamatergic tone is reduced in the photoinhibited goat. The objectives of this study were to determine the effect of photoperiod and glutamatergic stimulation on LH, GH, and testosterone (T) secretion in goat bucks. Eight mature, intact bucks were used in two simultaneous 4 x 4 Latin square designs. Variables were two photoperiod regimens (short day; SD, 10 h light:14 h dark, n = 4; vs long day; LD, 16 h light:8 h dark, n = 4) and four doses of N-methyl-D-L-aspartate (NMA; 0, 1, 2 and 4 mg/kg BW, i.v.). Venous blood was obtained for 2 h before and after NMA injection, followed by GnRH injection and then a final 1 h of sampling. Injection of NMA increased (P < 0.002) LH secretion within 20 min. This increase was sustained for 120 min, but the response was most pronounced in LD goats. The increase in mean LH was associated with a concomitant dose-dependent increase in pulse frequency (P < 0.006). However, NMA treatment had no effect (P > 0.10) on LH pulse amplitude. The release of LH after injection of GnRH was not affected by photoperiod. Exposure of bucks to LD reduced T secretion relative to that of SD bucks (P < 0.01). However, GH secretion was enhanced in LD bucks (P< 0.001). The response of GH to NMA was dependent on photoperiod history. A highly significant immediate and sustained increase (P < 0.001) was observed in LD but not in SD bucks within 10 min. Overall, a dose-dependent increase (P < 0.01) in T secretion was stimulated by NMA in both LD and SD bucks. These results indicate that NMA receptors may be involved in the regulation of LH, GH, and testosterone secretion in the goat. Furthermore, length of day influences GH secretion in the goat and NMA receptor activation had divergent effects on the secretion of this hormone.     

Effects of season and estradiol-17 beta on luteinizing hormone release in ovariectomized sows.

This study examined the ability of estradiol-17 beta (E2) to suppress LH release in the sow during different months of the year. Six chronically ovariectomized sows were fitted with vena caval cannulas (d 0) and blood samples were collected at 6-h intervals for 6 d. Sows were treated s.c. with E2 capsules (24 mg of E2/275 kg of BW) at d 3. Additional blood samples were collected at 15-min intervals for 8 h on d 2 and 5. After each 8-h frequent sampling period, sows were treated i.v. with GnRH at .5 microgram/kg of BW, and blood samples were collected at 10-min intervals for 3 h. The protocol was repeated at monthly intervals for 13 mo. Luteinizing hormone concentrations were determined for all serum samples, and E2 concentrations were quantified in samples collected at 6-h intervals. Data were analyzed by split-block analyses of variance. Serum E2 concentrations increased (P less than .001) from 5.0 +/- .3 pg/ml before E2 treatment to 26.0 +/- .2 pg/ml after E2 treatment. The interval from GnRH administration to peak LH concentration was shorter (P less than .001) before E2 treatment than after E2 treatment (28.7 +/- 2.2 vs 71.0 +/- 2.2 min). It was evident that baseline LH, mean LH, pulse frequency, and pulse amplitude and LH release after GnRH administration failed to demonstrate seasonal changes. In summary, LH release was suppressed after treatment with E2 and was affected minimally by month of the year. In addition, E2 inhibitory effects of LH release included hypothalamic and anterior pituitary sites of action.     

Fasting-induced changes in ovulation rate, plasma leptin, gonadotropins, GH, IGF-I and insulin concentrations during oestrus in ewes

The aim of this experiment was to study the changes in the hormonal status and ovulation rate (OR) evoked by starvation during the follicular phase of the oestrous cycle in ewes. To achieve this goal, 12 female crossbreed sheep were synchronized and then half of them were fasted from the 12th to the 16th day of the oestrous cycle. On the 16th day, analysis of hormones and insulin-like growth factor-I (IGF-I) were performed in 10-min intervals. Then, on the 6th day of the following oestrous cycle, the OR in all ewes was determined by laparoscopy. Fasting reduced significantly (P < 0.05) the OR in ewes (1.25 +/- 0.50) in comparison with control (1.75 +/- 0.50). The drop in the OR was coincident with a significant (P < 0.001) decrease in the plasma concentration and pulse amplitude of leptin (0.29 +/- 0.08 ng/ml versus control 0.53 +/- 0.14 ng/ml), the plasma level of luteinizing hormone (LH) (0.19 +/- 0.06 IU/l versus 0.25 +/- 0.09 IU/l in control; P < 0.05) and the mean frequency of LH pulses (2.0/h versus 2.5/h in control). Fasting resulted also in a significant (P < 0.05) decrease in the plasma concentration and pulse amplitude of follicle stimulating hormone (FSH) in comparison with the control. Simultaneously, a significant (P < 0.001) drop in the IGF-I concentration in the fasted ewes (4.78 +/- 0.91 ng/ml) was found in comparison with control (7.63 +/- 1.85 ng/ml). Also the level of insulin were significantly (P < 0.001) lower in the fasted (178.99 +/- 39.08 pM/l respectively) than in the control sheep (302.66 +/- 49.01 pM/l respectively). Meanwhile, a double increase in the growth hormone (GH) pulses frequency and an augmentation in its plasma concentrations as a result of starvation was found. The obtained results shows that the acute fasting exerts an inhibitory effect on the ovulation rate in ewes coincident with suppression in leptin, FSH and LH secretion and changes in signalization mediated by GH.     

Serum concentrations of luteinizing hormone, growth hormone, testosterone, estradiol, and leptin in boars treated with n-methyl-D,L-aspartate

Three experiments were conducted to determine the effects of n-methyl-D,L-aspartate (NMA), an agonist of the excitatory amino acid glutamate, on secretion of hormones in boars. In Exp. 1, boars (185.0+/-.3 d of age; mean +/- SE) received i.v. injections of either 0, 1.25, 2.5, 5, or 10 mg of NMA/kg BW. There were no effects of NMA (P>.1) on secretion of LH and testosterone. Treatment with NMA, however, increased (P<.01) circulating GH concentrations in a dose-dependent manner. In Exp. 2, boars (401 d of age) received an i.v. challenge of NMA at a dose of 10 mg/kg BW or .9% saline. Treatment with NMA, but not saline (P>.1), increased serum concentrations of LH (P<.01), GH (P <.01), and testosterone (P<.06). In Exp. 3, boars that were 152, 221, or 336 d of age were treated i.v. with NMA (10 mg/kg BW). Across ages, treatment with NMA increased circulating concentrations of LH (P<.07) and testosterone (P<.01). However, NMA increased (P<.01) mean GH concentrations in only the oldest boars. Treatment with NMA had no effect (P>.1) on circulating concentrations of estradiol or leptin; however, estradiol concentrations increased (P<.03) with age. In summary, NMA increased secretion of LH, GH, and testosterone in boars. However, endocrine responses to treatment with NMA may be influenced by age of the animal. Finally, NMA did not influence circulating concentrations of estradiol or leptin.     

N-methyl-d,l-aspartate modulation of pituitary hormone secretion in the pig: Role of opioid peptides

Sixteen ovariectomized (OVX) mature gilts, averaging 139.6 ± 3.1 kg body weight (BW) were assigned randomly to receive either progesterone (P, 0.85 mg/kg BW, n=8) or corn oil vehicle (OIL, n=8) injections im twice daily for 10 d. On the day of experiment, all gilts received either the EAA agonist, N-methyl-d,l-aspartate (NMA; 10 mg/kg BW, iv) alone or NMA plus the EOP antagonist, naloxone (NAL, 1 mg/kg BW, iv), resulting in the following groups of 4 gilts each: OIL-NMA, OIL-NMA-NAL, P-NMA and P-NMA-NAL. Blood samples were collected via jugular cannula every 15 min for 6 hr. All pigs received NMA 5 min following pretreatment with either 0.9% saline or NAL 2 hr after blood collection began and a GnRH challenge 3 hr after NMA. Administration of NMA suppressed (P<0.03) LH secretion in OIL-NMA gilts and treatment with NAL failed to reverse the suppressive effect of NMA on LH secretion in OIL-NMA-NAL gilts. Similar to OIL-NMA gilts, NMA decreased (P<0.03) mean serum LH concentrations in P-NMA gilts. However, in P-NMA-NAL gilts, serum LH concentrations were not changed following treatment. All gilts responded to GnRH with increased (P<0.01) LH secretion. Additionally, administration of NMA increased (P<0.01) growth hormone (GH) and prolactin (PRL) secretion in both OIL-NMA and P-NMA gilts, but this increase in GH and PRL secretion was attenuated (P<0.01) by pretreatment with NAL in OIL-NMA-NAL and P-NMA-NAL gilts. Serum cortisol concentrations increased (P<0.01) in all gilts and the magnitude of the cortisol response was not different among groups. In summary, results of the present study confirmed previous findings that NMA suppresses LH secretion in both oil- and P-treated OVX gilts, but we failed to provide definitive evidence that EOP are involved in the NMA-induced suppression of LH secretion. However, NMA may, in part, activate the EOP system which in turn increased GH and PRL secretion in the gilt.     

Pituitary responsiveness to GnRH, hypothalamic content of GnRH and pituitary LH and FSH concentrations immediately preceding puberty in gilts

To determine whether pituitary concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) or hypothalamic content of gonadotropin releasing hormone (GnRH) change before puberty, 40 prepubertal gilts averaging 7 mo of age were slaughtered before or on the second, third or fourth day after relocation and boar exposure. Some gilts responded to relocation and boar exposure as indicated by swollen vulvae, turgid uteri and enlarged ovarian follicles at the time of slaughter. Pituitary concentrations of LH and FSH and hypothalamic content of GnRH were similar between gilts that responded to relocation and boar exposure and gilts that did not respond. In addition, boar exposure and relocation had no effect on pituitary concentrations of LH and FSH or on hypothalamic content of GnRH. To determine whether pituitary responsiveness to GnRH changes before puberty, a third experiment was conducted in which 72 gilts were injected with 400 micrograms of GnRH either before or on the second, third or fourth day after relocation and boar exposure. In gilts that subsequently responded (i.e., ovulated) as a result of relocation and boar exposure, pituitary responsiveness to GnRH was reduced as compared with gilts that failed to ovulate after relocation and boar exposure. Peak concentrations of serum LH after GnRH injection were 4.6 +/- 1.3 vs 9.8 +/- .8 ng/ml for responders vs nonresponders. Peak serum FSH after GnRH injection was also lower for responders than for nonresponders (29.5 +/- 4.2 vs 41.2 +/- 2.4 ng/ml). When compared with controls, relocation and boar exposure did not significantly affect GnRH-induced release of LH and FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of enclomiphene on estradiol-induced changes in LH secretion in ewes

Experiments were conducted to characterize the ability of the antiestrogen enclomiphene (ENC) to block the effects of estradiol on secretion of LH in ovariectomized ewes. To determine whether ENC could block an estradiol-induced LH surge, ewes (n = 4/group) were administered 10 to 250 mg ENC followed 30 min later by 25 micrograms estradiol. Ten or 25 mg ENC suppressed the estradiol-induced LH surge in one of four ewes, whereas 100- or 250-mg doses suppressed the LH surge in three and four of four ewes, respectively. In ewes that received a single treatment of 100 mg ENC plus 25 micrograms estradiol, serum concentrations of LH remained below 1 ng/ml for 3 wk. Compared with untreated ewes, the number of pituitary GnRH receptors was elevated (P less than .05) at 12 d and 28 d, but pituitary content of LH had decreased (P less than .05) by 28 d in ewes treated with 100 mg ENC. To determine whether ENC could block the inhibitory effects of estradiol on serum concentrations of LH, ewes received injections of .03, .1, 1 or 10 mg ENC every 4 d. Half the ewes treated with each dose also received estradiol implants. Injection of .03, .1 or 1 mg ENC alone did not affect serum concentrations of LH, whereas the 10-mg dose decreased serum concentrations of LH below 1 ng/ml by wk 1 of treatment. No dose prevented the inhibition of serum concentrations of LH caused by estradiol implants. In ovariectomized ewes, ENC was antagonistic to estradiol; it prevented the positive effects of estradiol required to induce an LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of castration on plasma luteinizing hormone concentrations in prepubertal boars

Mean concentrations and the occurrence of pulsatile release of luteinizing hormone (LH) were determined in 14-wk-old crossbred boars (50.5 +/- 1.5 kg) after bilateral or unilateral castration at 10 wk of age. Blood was collected at 10-min intervals for 5 h. Then gonadotropin releasing hormone (GnRH; 40 micrograms) was given and sampling was continued at 5-min intervals for 1 h. Compared with intact boars, bilateral castration increased (P less than .001) mean LH (982 +/- 56 vs 389 +/- 56 pg/ml), pulsatile releases of LH (7.0 +/- .6 vs 2.0 +/- .6 pulses/5 h) and LH pulse amplitude (617 +/- 29 vs 360 +/- 58 pg/ml). Unilaterally castrated boars did not differ from intact boars in any of the above measures of LH secretion. Testis weight increased more between 10 and 14 wk of age in the unilateral castrates than in the intact boars (432 +/- 42 vs 245 +/- 34%; P less than .05). Thus, compensatory hypertrophy occurred within 4 wk of castration. Plasma testosterone was lower for bilateral castrates than for intact animals (.1 +/- .8 vs 3.6 +/- .9 ng/ml; P less than .05) while unilateral castrates (3.8 +/- 1.0 ng/ml) and intact boars did not differ. Plasma estradiol concentrations in bilateral and unilateral castrates were not different from levels found in intact boars (1.8 +/- 1.8, 8.8 +/- 2.1 and 6.0 +/- 1.8 pg/ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in serum concentrations of luteinizing hormone and follicle-stimulating hormone following injection of gonadotropin-releasing hormone during pregnancy and after parturition in mares

High concentrations of estrogens in the peripheral circulation during late gestation inhibit synthesis of LH and markedly reduce pituitary content of LH at the end of pregnancy in most domestic species. Because blood concentrations of estrogen peak shortly before mid-gestation in the mare and then gradually decrease until parturition, we hypothesized that pituitary content of LH may increase during late gestation. To test this hypothesis 10 horse mares were challenged with a maximally stimulatory dose (2 micrograms/kg) of GnRH on d 240 and 320 of gestation and d 3 after parturition. A separate group of four mares were treated with GnRH on d 2 or 3 estrus. Blood samples were collected at -2, -1, 0, .25, .5, .75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7 and 8 h relative to injection of GnRH and serum was analyzed for concentration of LH and FSH. Basal serum concentration and total quantity of LH released after GnRH stimulation (assessed by determining the area under the response curve) were not different on d 240 and 320 of gestation or on d 3 after parturition (12.5 +/- 3.5, 5.7 +/- 1.5 and 29.1 +/- 12.1 ng.min/ml, respectively) and were less (P less than .05) than on d 3 of estrus (311.0 +/- 54.0 ng.min/ml). There was little difference in the basal serum concentration of FSH at any of the time points examined. In contrast, GnRH-induced release of FSH continually decreased (P less than .05) from d 240 of gestation (559.8 +/- 88.9 ng.min/ml) to d 3 of estrus (51.8 +/- 6.2 ng.min/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synchronization of Ovulation and Fixed Time Intrauterine Insemination in Ewes

A novel method for oestrus-ovulation synchronization in sheep followed by fixed time insemination is presented herewith. Mature dry ewes (n = 28) of Karagouniko breed being at an unknown stage of the oestrous cycle, were used during the middle of breeding season. The treatment protocol consisted of an initial administration of a GnRH analogue followed 5 days later by a prostaglandin F2alpha injection. Thirty-six hours later a second GnRH injection was administered to synchronize ovulation, and laparoscopic intrauterine insemination was performed 12-14 h later. Three days after insemination, fertile rams were introduced into the flock twice daily and oestrus-mating detection was carried out. For progesterone (P(4)) determination, blood samples were collected on alternate days, starting 2 days before the first GnRH injection and continuing for 17 days after insemination. An additional sample was taken on the day of insemination. Pregnancy diagnosis was carried out by trans-abdominal ultrasonography. Fourteen ewes (50%) conceived at insemination and maintained pregnancy; from the remainder 14 ewes 10 became pregnant at natural service, while four, although they mated at least two to three times, failed to conceive. In response to the first GnRH, P(4) concentration increased at higher levels in ewes that conceived at AI compared with those that failed to conceive (47.54 and 22.44%, respectively; p < 0.05). Significant differences (p < 0.05) in mean P(4) concentration between pregnant and non-pregnant animals were detected 1 day before AI (0.17 +/- 0.06 and 0.26 +/- 0.14 ng/ml, respectively) on the day of AI (0.15 +/- 0.04 and 0.24 +/- 0.08 ng/ml, respectively) as well as 9 and 11 days thereafter (0.48 +/- 0.12 and 0.38 +/- 0.12 ng/ml; 0.68 +/- 0.14 and 0.50 +/- 0.18 ng/ml, respectively). These results indicate that using the proposed protocol, an acceptable conception rate can be achieved which could be further improved by modifying the time intervals between interventions.     

N-methyl-d,l-aspartate stimulates growth hormone and prolactin but inhibits luteinizing hormone secretion in the pig.

The effects of n-methyl-d,l-aspartate (NMA), a neuroexcitatory amino acid agonist, on luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) secretion in gilts treated with ovarian steroids was studied. Mature gilts which had displayed one or more estrous cycles of 18 to 22 d were ovariectomized and assigned to one of three treatments administered i.m.: corn oil vehicle (V; n = 6); 10 micrograms estradiol-17 b/kg BW given 33 hr before NMA (E; n = 6); .85 mg progesterone/kg BW given twice daily for 6 d prior to NMA (P4; n = 6). Blood was collected via jugular cannulae every 15 min for 6 hr. Pigs received 10 mg NMA/kg BW i.v. 2 hr after blood collection began and a combined synthetic [Ala15]-h GH releasing factor (1-29)-NH2 (GRF; 1 micrograms/kg BW) and gonadotropin releasing hormone (GnRH; .2 micrograms/kg BW) challenge given i.v. 3 hr after NMA. NMA did not alter LH secretion in E gilts. However, NMA decreased (P < .02) serum LH concentrations in V and P4 gilts. Serum LH concentrations increased (P < .01) after GnRH in all gilts. NMA did not alter PRL secretion in P4 pigs, but increased (P < .01) serum PRL concentrations in V and E animals. Treatment with NMA increased (P < .01) GH secretion in all animals while the GRF challenge increased (P < .01) serum GH concentrations in all animals except in V treated pigs. NMA increased (P < .05) cortisol secretion in all treatment groups. These results indicate that NMA inhibits LH secretion and is a secretagogue of PRL, GH and cortisol secretion with ovarian steroids modulating the LH and PRL response to NMA.     

Study of enteral versus parenteral application of the gonadotropin releasing hormone agonist Gonadorelin[6-D-Phe] (D-Phe6-LHRH) on LH secretion in Goettinger miniature pigs

With respect to the assessment of residue situation and as a part of preclinical trials to determine the biological activities of potential gonadotropin releasing hormone (GnRH) residues in porcine organisms the GnRH agonist Gonadorelin[6-D-Phe] (D-Phe(6)-LHRH) was administered either enterally or intramuscularly (i.m.) to female Goettinger miniature pigs in order to evaluate the GnRH-induced luteinizing hormone (LH) surge. Gilts received an (i) enteral application of 10 mg D-Phe(6)-LHRH via a probang (enteral group, n=7), (ii) i.m. injection of 0.1 mg D-Phe(6)-LHRH (parenteral group, n=5), or (iii) saline injection (control group, n=4). The GnRH and saline applications were repeated every second day with up to seven repetitions. Blood samples were collected via previously fitted jugular catheters immediately before injections, over an 8 h period in 1 h intervals beginning 2 h after injections, and at 24, 26, 28 and 30 h after applications. Enteral application of D-Phe(6)-LHRH induced an LH surge in 23 of 30 treatments. All gilts in the parenteral group exhibited LH release after each D-Phe(6)-LHRH application (P<0.05), whereas no LH surges were observed after saline injection in the control group. A significant (P<0.05) LH rise to mean maximum LH concentrations of 3.25 +/- 0.43 and 3.05 +/- 0.26 ng/ml occurred in both the enteral and parenteral groups, but there was no difference in the time interval after GnRH (2.6 +/- 0.3 vs. 2.3 +/- 0.3 h) and the mean duration of LH peak (6.5 +/- 0.4 and 6.8 +/- 0.3 h) between the treatment groups. In conclusion, (i) enteral application of 10 mg D-Phe(6)-LHRH induced LH release in a physiological range from the pituitary of female minipigs, and (ii) neither an accumulative effect nor a cumulative LH response were found after repeated GnRH application. Furthermore, (iii) in regard to consumer protection and gonadotropin secretion, D-Phe(6)-LHRH residues can be excluded from having long-term effects.     

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.     

Evidence for endogenous opioid modulation of serum luteinizing hormone and prolactin in the steer

Opioid modulation of LH and prolactin (PRL) concentrations in Angus steers was investigated. In Exp. 1, morphine sulfate (M) was administered at either 1, 2 or 3 mg/kg BW (n = 4) as an i.v. injection. Blood samples were obtained at 15-min intervals for 4 h pre- and post-treatment for serum hormone analyses. Mean serum LH concentration and number of LH secretory pulses decreased (P less than .1) for 2 h after M (4.1 to nadir of 2.4 ng/ml, and .33 vs. .21 pulses/h; pre- vs post-treatment). Luteinizing hormone pulse amplitude decreased (P less than .01; 7.3 vs 2.6 ng/ml; pre- vs post-treatment) during the 2 h following M. Prolactin concentrations increased 126.6%, 170.6% and 187.6% following 1, 2 and 3 mg M/kg BW, respectively (P less than .05, 1 vs 2; P less than .01, 1 vs 3). In Exp. 2, either saline solution (S, n = 6) or M (.31 mg/kg BW, i.v. injection followed by .15 mg/(kg.h) infusion; n = 6) was given for 7 h. Concentration of LH was unaffected. Response of LH to naloxone was determined in Exp. 3. Blood samples were obtained for 2 h pre- and post-administration of either naloxone (1 mg/kg BW, i.v. injection; n = 5) or S (n = 5). Response of LH at 15, 30 and 45 min posttreatment was greater (P less than .05) in naloxone- compared with S-treated steers. In summary, M had no significant effect on serum LH concentration or LH pulse frequency, but it decreased pulse amplitude and increased serum PRL concentrations. In contrast, naloxone increased LH secretion. These observations taken together indicate a physiological role for opioid modulation of LH and PRL secretion in the steer.     

Administration of single short-acting doses of GnRH antagonist modifies pituitary and follicular function in sheep

Current study determined the effect of two different single subcutaneous doses (1.5 and 3 mg) of GnRH antagonist (GnRHa) on pituitary and follicular function in non-lactating cyclic ewes. Both doses abolished the pulsatile secretion of luteinizing hormone (LH) for at least 3 days and decreased mean LH concentration during 6 days (0.64 +/- 0.09 for control and 0.54 +/- 0.05, P < 0.005, and 0.46 +/- 0.02, P < 0.00001, for 1.5 and 3 mg, respectively). Supply of GnRHa decreased the number of large dominant follicles, so the total number of smaller follicles, 2-3 mm in size, increased in both treated groups from day 0, reaching its maximum at day 2 in ewes treated with 1.5 mg (19.83+/-1.05 versus 5.83 +/- 0.50 in the control, P < 0.005) and at day 4 in sheep treated with 3mg (18.67 +/- 0.65 versus 5.50 +/- 0.65 in the control, P < 0.0001). However, the analysis of follicular function in terms of inhibin A indicated a possible effect of the higher dose of GnRHa on follicular function. The pattern of inhibin secretion in the group treated with 3mg of GnRHa decreased after the first 48 h, reaching its lowest value on day 4.5 (182.59 +/- 3.75 to 140.28 +/- 9.91 pg/ml, P < 0.05) concentration significant lower than control sheep (171.93 +/- 6.21 pg/ml, P +/- 0.01) or treated with 1.5 mg (168.04 +/- 7.16 pg/ml, P+ /- 0.05). Hence, the use of 1.5 mg would be more suitable to induce the presence of a high number of follicles able to grow to preovulatory sizes.     

Effects of pulsatile injection of GnRH into 6- to 14-wk-old Holstein bulls

With the goal of hastening puberty, we evaluated the effects of dose of gonadotropin releasing hormone (GnRH) during pulsatile injection on luteinizing hormone (LH) secretion in bulls 6, 10 or 14 wk old, and of pulsatile administration of GnRH every 2 h to bulls from 6 to 12 wk of age on reproductive development. Based on response to the last three of 12 bihourly injections of 20, 200 or 2,000 ng GnRH/kg, only the two higher doses of GnRH induced secretion of LH at 6 wk. At all ages, 200 ng GnRH/kg induced maximal discharges of LH. Based on comparisons between seven treated bulls and their identical twins, bihourly injections of GnRH starting on d 42 elicited discharge of LH for less than or equal to 4 d in progeny of one sire and greater than 28 d but less than 42 d in progeny of another sire. After 14 d of treatment, both elicited and spontaneous discharges of LH were smaller in all treated bulls. Within 2 d after cessation of GnRH injections on d 84, LH discharges were similar in frequency and amplitude in treated and control twins. Testicular and body growth were similar in treated and control bulls, but puberty was delayed (P less than .05) in bulls in which exogenous GnRH suppressed endogenous discharges of LH.