Serum luteinizing hormone, growth hormone and insulin-like growth factor-I after releasing hormone challenge in prepubertal ewe lambs selected for twinning

Two studies evaluated hormonal markers as indicators of the onset of puberty in Debouillet sheep selected for twinning. In Trial 1, 29 ewe lambs (50 +/- 0.5 kg, 159 to 187 d of age) were given 10 microg GnRH (i.v.) on September 15 and blood was collected at 30 min intervals after the injection for 2 h. Additional samples were taken twice weekly and progesterone (P4) was measured. The day that serum P4 was greater than 1 ng/mL for 2 consecutive sampling days was classified as the day of puberty. Average day of puberty was October 12 (average age at puberty was 199 d) and ewes with values less or greater than the average were classified as early or late, respectively. Average weight at GnRH challenge was 50 kg and ewes weighing less or more were classified as light or heavy, respectively. Early ewes weighed more (P = 0.01) and reached puberty sooner (P = 0.01) than late ewes. Heavy lambs reached puberty earlier, weighed more at GnRH challenge, and had more LH area under the curve (AUC, P < 0.05) than light ewes. In Trial 2, we gave 27 ewe lambs (54 +/- 0.9 kg, 173 to 189 d of age) a single i.v. injection of 10 microg GnRH and 10 microg GHRH on September 17. Average day of puberty was October 13, average weight was 54 kg, and average age at puberty was 208 d. Categories were designated as described for Trial 1. Early lambs reached puberty sooner (P = 0.01) and weighed more (P = 0.01) than late lambs, but the puberty groups had similar LH AUC (P = 0.64) and GH AUC (P = 0.75), whereas IGF-I was greater (P = 0.01) in early puberty ewes than in late puberty ewes. Heavy lambs reached puberty earlier (P = 0.06), weighed more (P = 0.01), and tended (P = 0.11) to have more GH AUC than light ewes. No difference was observed in LH AUC or IGF-I between weight groups (P > 0.15). Results suggest that serum LH after GnRH is not a reliable indicator of the onset of puberty in ewe lambs selected for twinning, but heavier ewes tended to produce more GH after a GHRH challenge and reach puberty earlier than lighter ewe lambs.     

Induction of ovulation in the acyclic postpartum ewe following continuous, low-dose subcutaneous infusion of GnRH

Pituitary and ovarian responses to subcutaneous infusion of GnRH were investigated in acyclic, lactating Mule ewes during the breeding season. Thirty postpartum ewes were split into 3 equal groups; Group G received GnRH (250 ng/h) for 96 h; Group P + G was primed with progestagen for 10 d then received GnRH (250 ng/h) for 96 h; and Group P received progestagen priming and saline vehicle only. The infusions were delivered via osmotic minipumps inserted 26.6 +/- 0.45 d post partum (Day 0 of the study). Blood samples were collected for LH analysis every 15 min from 12 h before until 8 h after minipump insertion, then every 2 h for a further 112 h. Daily blood samples were collected for progesterone analysis on Days 1 to 10 following minipump insertion, then every third day for a further 25 d. In addition, the reproductive tract was examined by laparoscopy on Day -5 and Day +7 and estrous behavior was monitored between Day -4 and Day +7. Progestagen priming suppressed (P < 0.05) plasma LH levels (0.27 +/- 0.03 vs 0.46 +/- 0.06 ng/ml) during the preinfusion period, but the GnRH-induced LH release was similar for Group G and Group P + G. The LH surge began significantly (P < 0.05) earlier (32.0 +/- 3.0 vs 56.3 +/- 4.1 h) and was of greater magnitude (32.15 +/- 3.56 vs 18.84 +/- 4.13 ng/ml) in the unprimed than the primed ewes. None of the ewes infused with saline produced a preovulatory LH surge. The GnRH infusion induced ovulation in 10/10 unprimed and 7/9 progestagen-primed ewes, with no significant difference in ovulation rate (1.78 +/- 0.15 and 1.33 +/- 0.21, respectively). Ovulation was followed by normal luteal function in 4/10 Group-G ewes, while the remaining 6 ewes had short luteal phases. In contrast, each of the 7 Group-P + G ewes that ovulated secreted progesterone for at least 10 d, although elevated plasma progesterone levels were maintained in 3/7 unmated ewes for >35 d. Throughout the study only 2 ewes (both from Group P + G) displayed estrus. These data demonstrate that although a low dose, continuous infusion of GnRH can increase tonic LH concentrations sufficient to promote a preovulatory LH surge and induce ovulation, behavioral estrus and normal luteal function do not consistently follow ovulation in the progestagen-primed, postpartum ewe.     

Plasma FSH and LH in prepubertal Booroola ewe lambs

Basal plasma concentrations (four 30-min samples) and GnRH-induced release of gonadotrophins were measured every 15 days between 30 and 90 days and at 110 days of age in Merino ewe lambs from the prolific Booroola ('B') flock (n = 18-23), the medium prolificacy ('T') flock (n = 14-20), and the 'O' flock (n = 4-8) of low prolificacy. At ages of 30 and 45 days B ewe lambs had mean basal plasma FSH concentrations of 145 and 122 ng/ml which were significantly higher (P less than 0.01) than those seen in T (45 and 53 ng/ml), and O (39 and 38 ng/ml) flock ewes. Between 60 and 110 days of age there were no significant differences between genotypes. The increment in FSH concentrations above basal levels induced by the subcutaneous injection of 100 micrograms synthetic GnRH was only significantly (P less than 0.05) greater in B than T and O genotype ewe lambs at 110 days of age but not at other ages. The basal plasma FSH differences between the B, T and O genotypes at 30 and 45 days of age were not consistently related to the size of litter in which lambs were born. At 30 days of age the mean plasma LH concentration of B, T, and O flock lambs were 2.6 +/- 0.5, 1.2 +/- 0.6 and 0.7 +/- 0.8 ng/ml respectively. These differences were not significant. At later ages there were also no significant differences between the genotypes with respect to basal LH, and the increase in LH induced by exogenous GnRH was always similar for the three genotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of naloxone on gonadotrophin secretion at various stages of development in the ewe lamb

Spring-born crossbred ewe lambs were raised in a natural photoperiod and saline (N = 6) or naloxone (1 mg/kg) in saline (N = 6) was injected (i.m.) every 2 h for 6 h at 5, 10 and 15 weeks of age and for 8 h at 20, 25 and 30 weeks of age. Blood samples were taken every 12 min during treatment periods. Naloxone had no effect on time to first oestrus (controls 235 +/- 6 days, naloxone 242 +/- 7 days). Mean serum LH concentrations and LH pulse frequency were elevated by naloxone in ewe lambs at 20, 25, and 30 weeks of age (P less than 0.05). The only FSH response to naloxone was a depression of mean serum concentrations at 30 weeks of age (P less than 0.05). LH pulse amplitude was elevated at 5 weeks of age in all ewe lambs and declined thereafter to a nadir at 30 weeks of age in control, but not in naloxone-treated animals (P less than 0.05). LH pulse frequency was elevated at 10 weeks of age in control ewe lambs and in all animals at 30 weeks of age (P less than 0.05). FSH pulse frequency declined from 5 weeks of age in control ewe lambs (P less than 0.05), with very few pulses noted in 25- and 30-week-old animals. We conclude that (1) opioidergic suppression of LH, but not FSH, secretion developed at 20 weeks of age in the growing ewe lambs used in the present study, with no obvious change in suppression before the onset of first oestrus: (2) pulsatile FSH secretion occurred in the young ewe lamb but was lost as the lamb matured: (3) attainment of sexual maturity was preceded by an elevation in LH pulse frequency.     

The effects of GnRH analogue (buserelin) or hCG (Chorulon) on Day 12 of pregnancy on ovarian function, plasma hormone concentrations, conceptus growth and placentation in ewes and ewe lambs

The objectives of this study were to determine the effect of GnRH analogue (buserelin) or human chorionic gonadotrophin (hCG, Chorulon) treatment on Day 12 of pregnancy on ovarian function, plasma hormone concentrations, conceptus growth and placentation in ewes and ewe lambs. After oestrus synchronization with progestagen sponges and eCG, all the animals were mated with fertile rams. Both ewes and ewe lambs (20 per treatment group) were given either normal saline or 4 microg GnRH or 200 IU hCG on Day 12 post-mating. Pre- and post-treatment plasma hormone concentrations were determined in seven pregnant animals per treatment group in samples collected 1h before and 0, 2, 4, 6, 8, 24, 48 and 72 h after treatment. Overall mean progesterone concentrations were higher (P<0.001) in ewes as compared with ewe lambs in saline-treated controls. GnRH or hCG treatment increased (P<0.001) mean plasma progesterone concentrations in both age groups, however, post-treatment concentrations were significantly (P<0.05) higher in ewes than in ewe lambs. Oestradiol concentrations were similar in the two control groups. In ewes, but not in ewe lambs, both GnRH and hCG treatments significantly (P<0.05) increased the mean oestradiol concentrations above pre-treatment levels. Moreover, post-treatment oestradiol concentrations in GnRH- and hCG-treated animals were significantly (P<0.05) higher than those in the saline-treated controls. LH release in response to GnRH treatment was greater (P<0.05) in ewes than in ewe lambs, whereas FSH release in ewes was less (P<0.05) than that of ewe lambs. The effects of GnRH or hCG on conceptus growth and placentation was determined at slaughter on Day 25. In ewes, GnRH treatment increased (P<0.05) luteal weight, amniotic sac width and length, and crown-rump length compared with controls, but had no effect on these parameters in ewe lambs. In ewes, hCG treatment also enhanced (P<0.05) luteal weight, amniotic sac width and length, crown-rump length, embryo weight and number of placentomes as compared with controls. In ewe lambs, there was no difference (P<0.05) between hCG and control groups in luteal weight, embryo weight and amniotic sac width but crown-rump length, amniotic sac length and the number of placentomes forming the placenta were greater (P<0.05). In conclusion, GnRH or hCG treatment on Day 12 of pregnancy can increase ovarian function, conceptus growth and placental attachment in ewes. However, these treatments were less effective in ewe lambs.     

Secretory dynamics of bioactive and immunoactive LH during the oestrous cycle of the sheep

Blood samples were collected every 15 min for 6 h during the follicular (1 day before oestrus), and early (Days +1 to +3), mid- (Days +4 to +8), and full (Days +9 to +14) luteal phases of the oestrous cycle. Serum concentrations of immunoactive LH were measured by radioimmunoassay. The biological activity of serum LH was determined by an in-vitro bioassay that uses LH-induced testosterone production from mouse interstitial cells as an endpoint. Only ovine and bovine LH and hCG had appreciable activity in this bioassay. The temporal pattern of secretion of bioactive LH paralleled the secretory pattern of immunoactive LH at all stages of the ovine oestrous cycle. However, the secretory pattern itself varied regularly through the oestrous cycle. The frequency of secretory excursions of LH was highest during the follicular phase (6.2 +/- 0.9 pulses/6 h) and was progressively reduced through the luteal phase (1.1 +/- 0.1 pulses/6 h during full luteal phase). Conversely, amplitude of secretory excursions of immunoactive LH was low during the follicular phase (0.79 +/- 0.08 ng/ml) and significantly (P less than 0.05) increased during the mid- and full luteal phases (1.49 +/- 0.10 and 2.37 +/- 0.20 ng/ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotrope function in ovariectomized ewes actively immunized against gonadotropin-releasing hormone (GnRH)

The gonadotrope cells of the ovine anterior pituitary were insulated from hypothalamic inputs by imposing an immunologic barrier generated by active immunization of ovariectomized ewes against gonadotropin-releasing hormone (GnRH) conjugated to keyhole limpet hemocyanin (KLH) through a p-aminophenylacetic acid bridge. All GnRH-KLH animals immunized developed titers of anti-GnRH that exceeded 1:5000. The antisera were specific for GnRH and cross-reacted with GnRH agonists modified in position 10 to an extent that was less than 0.01%. Ewes actively immunized against GnRH-KLH displayed levels of basal and GnRH agonist-induced gonadotropin secretion that were markedly lower (p less than 0.05) than comparable parameters in ewes actively immunized against KLH. In contrast, basal and thyrotropin-releasing hormone (TRH)-induced prolactin (PRL) secretion were not compromised by active immunization. Immunization against the GnRH-KLH conjugate, but not KLH alone, prevented expression of the positive feedback response to exogenous estradiol (E2). Pituitary stores of immunoactive luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly (p less than 0.001) reduced in ewes immunized against GnRH-KLH but stores of PRL were not affected by such immunization. Further, the biopotency of the residual LH stores in tissue of animals from the anti-GnRH group was significantly (p less than 0.05) lower than LH biopotency in anti-KLH animals. Serum levels of LH in anti-GnRH ewes were restored by circhoral administration of a GnRH agonist that did not cross-react with the antisera generated. Pulsatile delivery of GnRH agonist in anti-GnRH ewes significantly (p less than 0.05) elevated serum LH within 48 h and reestablished LH levels comparable to anti-KLH ewes within 6 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive characteristics of lambs actively immunized early in life with inhibin-enriched preparations from follicular fluid of cows

Active immunization of Merino and crossbred ewe lambs early in life with a partly purified inhibin derived from bovine follicular fluid (bFF) advanced their puberty. This occurred whether the lambs were immunized from 3 weeks of age or from 9 weeks of age or just 3 times between 3 and 9 weeks. However, the effect was more obvious with multiple injections starting at 3 weeks of age. The ovulation rate of the immunized lambs was significantly higher than that of the control ewes. When lambs were subjected to multiple immunizations the increased ovulation rate persisted in Merinos for at least 1 year after immunization ceased. The lambs injected 3 times only (at 3, 6 and 9 weeks of age) did not show any increase in ovulation rate. Plasma concentrations of FSH and LH measured in blood samples taken 1 week after immunization were not significantly different between the treatment groups. Daily sperm output in the urine and testis diameter were measured in ram lambs from the same breeds and flocks as the ewe lambs. Immunization with the inhibin preparation from 3 weeks of age resulted in a significant increase in daily sperm output (4255 +/- 701 vs 2344 +/- 344 x 10(6), P less than 0.01) and testis diameter (5.21 +/- 0.3 vs 4.33 +/- 0.2 cm, P less than 0.05) in Merino rams examined in the non-breeding season when the rams were aged 23 months. Although the same trend was seen in the following year the differences were not significant. Immunization from 9 weeks of age had no effect. A similar increase in daily sperm output was seen (at 20 and 25 months of age) in crossbred rams immunized from 3 weeks of age but the difference was not significant. Plasma concentrations of FSH (but not LH) in samples from rams immunized from 3 weeks of age were significantly higher than those of control rams at 7 and 60 weeks of age in Merino rams and at 23 and 31 weeks of age in crossbred rams. Plasma FSH concentrations at 21 and 26 weeks of age in the Merino rams immunized from 3 weeks of age were higher than those of control lambs, and these increases preceded a significant (P less than 0.05) increase in testicular diameter at 30 weeks of age (4.99 +/- 0.2 vs 4.37 +/- 0.2 cm). These results suggest that active immunization early in life with an inhibin-enriched fraction from bFF advances puberty in ewe lambs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pituitary response to GnRH and ovariectomy in Booroola-Awassi and Awassi ewe lambs

Booroola-Awassi ewe lambs were heterozygous (F+) for a major gene F, influencing their ovulation rate, while Awassi lambs were non-carriers (++). Basal plasma FSH concentration (mean +/- s.e.m.) in Booroola-Awassi ewe lambs at 4 weeks of age was significantly higher than in Awassi lambs of the same age (5.06 +/- 0.60 and 2.04 +/- 0.32 ng/ml respectively; P less than 0.001). After GnRH administration, FSH increased from 3.89 +/- 1.10 to 10.58 +/- 1.30 ng/ml in Booroola-Awassi (N = 6) and from 1.87 +/- 0.29 to 4.64 +/- 0.33 ng/ml in Awassi (N = 6) ewe lambs (P less than 0.05). Ovariectomy caused an increase in plasma FSH in Booroola-Awassi (N = 4) and Awassi (N = 4) ewe lambs. At 1 week after ovariectomy plasma FSH increased from 5.96 +/- 1.02 to 7.06 +/- 1.05 ng/ml in F+ and from 1.67 +/- 1.06 to 5.21 +/- 0.66 ng/ml in ++ ewe lambs, suggesting a stronger negative feed-back effect exerted by the ovaries of Awassi lambs. At 15 weeks after ovariectomy FSH values were similar in Booroola-Awassi (18.28 +/- 1.96 ng/ml) and Awassi (16.07 +/- 0.70 ng/ml) lambs. Although the overall pattern of pituitary response to ovariectomy was similar in the F+ and ++ ewe lambs, Booroola-Awassi lambs had small ovaries (132.5 +/- 24.9 mg) and follicular development did not proceed beyond the preantral stage in 3/4 animals, and Awassi lambs had large ovaries (600.0 +/- 233.9 mg) (P less than 0.05) with many preantral and antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Naloxone evokes large-amplitude GnRH pulses in luteal-phase ewes

Ewes were sampled during the mid-late luteal phase of the oestrous cycle. Hypophysial portal and jugular venous blood samples were collected at 5-10 min intervals for a minimum of 3 h, before i.v. infusions of saline (12 ml/h; N = 6) or naloxone (40 mg/h; N = 6) for 2 h. During the 2-h saline infusion 2/6 sheep exhibited a GnRH/LH pulse; 3/6 saline infused ewes did not show a pulse during the 6-8-h portal blood sampling period. In contrast, large amplitude GnRH/LH pulses were observed during naloxone treatment in 5/6 ewes. The mean (+/- s.e.m.) amplitude of the LH secretory episodes during the naloxone infusion (1.07 +/- 0.11 ng/ml) was significantly (P less than 0.05) greater than that before the infusion in the same sheep (0.54 +/- 0.15 ng/ml). Naloxone significantly (P less than 0.005) increased the mean GnRH pulse amplitude in the 5/6 responding ewes from a pre-infusion value of 0.99 +/- 0.22 pg/min to 4.39 +/- 1.10 pg/min during infusion. This episodic GnRH secretory rate during naloxone treatment was also significantly (P less than 0.05) greater than in the saline-infused sheep (1.53 +/- 0.28 pg/min). Plasma FSH and prolactin concentrations did not change in response to the opiate antagonist. Perturbation of the endogenous opioid peptide system in the ewe by naloxone therefore increases the secretion of hypothalamic GnRH into the hypophysial portal vasculature. The response is characterized by a large-amplitude GnRH pulse which, in turn, causes a large-amplitude pulse of LH to be released by the pituitary gland.     

Effect of melatonin on pulsatile release of luteinizing hormone in female lambs.

This study investigated the effect of melatonin treatment of ewe lambs on LH pulsatility in an attempt to examine the mechanism whereby melatonin advances the onset of puberty. Six ewe lambs were given intravaginal melatonin implants at 12.8 weeks of age. Another six lambs received empty implants. All lambs were serially blood sampled every 15 minutes for six hours on several occasions prior to the onset of puberty. One week after implantation LH pulse frequency and mean LH levels were higher in treated lambs than the control lambs (pulse frequency 0.13/h vs 0.03/h; mean LH levels 2.0 +/- 0.2 ng/ml vs 1.3 +/- 0.1 ng/ml; p less than 0.05). Melatonin treatment failed to alter pulse frequency after the initial increase. Puberty was advanced by 3 weeks in the treated group. In the second experiment six lambs received melatonin implants at 13 weeks of age and another six lambs served as control. In this experiment blood samples were taken intensively during the first few weeks after treatment. Results of this study show that mean plasma LH levels and LH pulse frequency were again higher during the first week after implantation. This transient increase in LH release may be part of the mechanism initiating the eventual advancement of puberty although the significance of this increase is questionable. In both experiments the LH response to estradiol injection was monitored at various times after treatment, but no effects of melatonin were found, although the magnitude of the response increased with age.     

Melatonin and the pituitary response to GnRH in prepubertal and adult ewes

GnRH-induced LH-release was studied in female lambs from 5 weeks of age until puberty and in adult anoestrous ewes. In pre-pubertal animals LH was released rapidly after GnRH treatment but after puberty the responses became slower and more sustained although the peak concentration did not change. In neither pre-pubertal nor adult sheep did prior treatment with melatonin influence LH release after GnRH treatment.     

Plasma LH and FSH responses and ovarian activity in prepubertal heifers treated with repeated injections of low doses of GnRH for 72 h

Twelve 5-month-old Hereford X Friesian heifers were injected i.v. with 2.0 micrograms GnRH at 2-h intervals for 72 h. Blood samples were collected at 15-min intervals from 24 h before the start until 8 h after the end of the GnRH treatment period. Over the 24-h pretreatment period, mean LH concentrations ranged from 0.4 to 2.2 ng/ml and FSH concentrations from 14.1 to 157.4 ng/ml; LH episodes (2-6 episodes/24 h) were evident in all animals. Each injection of GnRH resulted in a distinct episode-like response in LH, but not FSH. Mean LH, but not FSH, concentrations were significantly increased by GnRH treatment. The GnRH-induced LH episodes were of greater magnitude than naturally-occurring episodes (mean maximum concentration 6.7 +/- 0.5 and 4.9 +/- 0.6 ng/ml respectively). Preovulatory LH surges occurred between 17.0 and 58.8 h after the start of treatment in 9/12 heifers, with a coincident FSH surge in 8 of these animals. This was not followed by normal luteal function. There were no apparent correlations between pretreatment hormone concentrations, and either the pituitary response to GnRH or the occurrence of preovulatory gonadotrophin release.     

Suppression of plasma FSH concentrations with bovine follicular fluid blocks ovulation in GnRH-treated seasonally anoestrous ewes

The specific requirement for FSH in the final stages of preovulatory follicle development was assessed in seasonally anoestrous ewes given 2-h injections of GnRH (250 ng/injection), with (N = 10) or without (N = 10) concurrent treatment with bovine follicular fluid (bFF: 2 ml given i.v. at 8-h intervals). Treatment with bFF significantly (P less than 0.01) suppressed plasma FSH concentrations, but, at least for the first 30 h of treatment, did not influence the magnitude of GnRH-induced LH episodes (mean max. conc. 3.00 +/- 0.39 and 3.63 +/- 0.51 ng/ml for bFF-treated and control ewes, respectively). Of 10 animals treated with GnRH for 72 h, 5/5 control ewes showed oestrus and ovulated whereas 0/5 bFF-treated ewes showed oestrus or ovulated in response to GnRH treatment. There was, however, a transient (13.2 +/- 1.0 h) increase in plasma LH concentrations in the ewes given bFF (mean max. conc. 4.64 +/- 1.57 ng/ml), which was coincident with the preovulatory LH surge recorded in animals given GnRH alone. In 10 GnRH-treated ewes slaughtered after 32 h of treatment, the mean diameter of the largest antral follicle was significantly (P less than 0.001) greater in control ewes (5.92 +/- 0.17 mm) than in animals that were also given bFF (3.94 +/- 0.14 mm). In addition, the incidence of atresia in the 3 largest antral follicles present at this time was greater in bFF-treated ewes. These results show that, when plasma FSH concentrations are suppressed by administration of bFF, although the magnitude of GnRH-induced LH episodes is unchanged, preovulatory follicular development is impaired and ovulation does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ability of ram introduction to induce LH secretion, estrus and ovulation in fall-born ewe lambs during anestrus.

The ability of ram introduction (RI) and progesterone pre-treatment to induce increases in LH secretion and ovulation, and the ability of progesterone pre-treatment with or without estrogen to induce estrus and ovulation in fall-born ewe lambs during seasonal anestrus was investigated. In early July, lambs of mixed breeds (41.8+/-0.6 kg and 250.7+/-1.3 days of age) were assigned to receive no treatment (C, n=7), to be introduced to rams (7:1 ewe:ram ratio; R, n=7), to be treated with progesterone (a used CIDR device) for 5 days (P, n=5), to be treated with progesterone and introduced to rams at CIDR removal (PR, n=11), or to receive the latter treatment plus an injection of estradiol benzoate (25 microg, E2beta i.m.) 24 h after CIDR withdrawal/RI (PER, n=11). Blood samples were collected from all lambs every 4h for 60 h beginning at RI/CIDR withdrawal (0 h), to characterize the LH surge profile and in groups R and C every 15 min for 8 h between 12 and 20 h for determination of LH pulse frequencies. Ultrasonographic examinations of the ovaries were conducted at 0, 36 and 60 h. In ram-exposed groups lambs were also observed for raddle marks every 4h from 0 to 60 h. The LH pulse frequency (pulses/8 h) was higher in group R (P<0.01; 7.7+/- 0.5) than group C lambs (2.7+/- 0.8). More lambs in groups exposed to rams than in the C or P groups showed an LH surge (P<0.05; 0, 100, 0, 72.7 and 100%, for C, R, P, PR and PER groups, respectively). Time from RI/CIDR removal to initiation of the LH surge was greater in lambs in the PR (43.5+/- 3.8h) than in the R (32.6+/- 4.6h; P=0.08) or PER (33+/- 1.2h; P<0.01). Diameter of the largest follicle at 0 h (3.2+/- 0.2mm) was not different among groups. Growth rate of the largest follicle between 0 and 36 h was greater (P<0.05) in RI than in C or P groups. Diameter of the largest follicle at 36 h was larger (P<0.05) in lambs in R (5.6+/- 0.2mm) and PR (5.1+/- 0.5mm) than C (4.0+/- 0.6mm) or P (3.8+/- 0.4mm) groups, and in R than PER (4.3+/- 0.4mm) treatment groups. Only lambs in the RI groups ovulated. Among RI groups the percentage of lambs ovulating was greater in the R (P<0.05; 85.7%) than PR (33.3%) groups with an intermediate response observed in lambs in treatment group PER (71.4%). The estrous response in progesterone pre-treated groups was greater (P<0.05) in lambs also treated with estrogen (PER; 81.8%), than in lambs introduced to rams alone (PR; 45.5%). In conclusion, ram introduction by itself, but not progesterone treatment alone, induces increases in LH pulse frequency, follicular development, and ovulation in fall-born ewe lambs during seasonal anestrus, further, P4 pre-treatment and RI when combined with E2 results in a high estrous response.     

Gonadotropin-releasing hormone at estrus: luteinizing hormone, estradiol, and progesterone during the periestrual and postinsemination periods in dairy cattle

Administering gonadotropin-releasing hormone (GnRH) improved conception rates in our previous studies. Our objective was to determine if the effect of GnRH was mediated through serum luteinizing hormone (LH) and/or by altered secretion of serum progesterone (P) and estradiol-17 beta (E) during the periestrual and post-insemination periods. Cattle were given either GnRH (n = 54) or saline (n = 55) at 72 h and inseminated artificially (AI) 80 h after the second of two injections of either prostaglandin F2 alpha or its analog, cloprostenol. Progesterone and E were measured in blood serum collected during 3 wk after AI (estrus) from 60 females. Blood was collected for LH determinations via indwelling jugular cannulae from 14 cows and 11 heifers. Collections were taken every 4 h from 32 to 108 h after the second PGF injection (PGF-2) (periestrual period) and at more frequent intervals during 240 min after administration of GnRH (n = 18) or saline (n = 7). Ten females had a spontaneous preovulatory LH surge before GnRH treatment (GnRH-spontaneous), whereas GnRH induced the preovulatory LH surge in six females. A spontaneous LH surge appeared to be initiated in two heifers at or near the time of GnRH treatment (spontaneous and/or induced). The remaining seven cows had spontaneous LH surges with no subsequent change in LH after saline treatment. Serum P during the 21 days after estrus was lower (p less than 0.05) in both pregnant and nonpregnant (open) cattle treated previously with GnRH compared with saline. Serum P during the first week after estrus was greater (p less than 0.01) and increased (p less than 0.05) more rapidly in saline controls and in GnRH-spontaneous cattle than in those exhibiting GnRH-induced or GnRH-spontaneous and/or-induced surges of LH. Conception rate of cattle receiving GnRH was higher (p = 0.06) than that of saline-treated controls. These data suggest that GnRH treatment at insemination initiated the preovulatory LH surge in some cattle, but serum P in both pregnant and open cows was compromised during the luteal phase after GnRH treatment. Improved fertility may be associated with delayed or slowly rising concentrations of serum progesterone after ovulation.     

Effect of clomiphene citrate on pituitary responsiveness to gonadotropin releasing hormone in rams and wethers

The objective of this study was to determine the effect of clomiphene citrate (clomid) on pituitary responsiveness to gonadotropin releasing hormone (GnRH) in rams and wethers. Doses of 200 mg clomid per ram and 1 mug GnRH per 50 kg body weight were used in studies on 12 rams and 4 wethers. The experimental design involved bleeding each animal at 15-minute intervals for 6.5 hours. At the end of the first hour, GnRH was injected IV. The second GnRH challenge was administered 0.5 hours after an injection of clomid or vehicle (4.5% sorbitol solution) which was given on the third hour. The relative response to clomid or vehicle was calculated as the mean increase in concentration of LH during the two-hour period after the second GnRH injection. Each treatment (clomid and vehicle) was given to all animals with a 14-day recovery period between treatment days. The relative response for the rams receiving vehicle (1.80 +/- 0.65) was greater (P < 0.05) than the response during clomid treatment (0.34 +/- 0.22). This suppression of LH response by clomid was observed in 10 of the 12 rams. In contrast to the rams, the concentrations of LH in wethers after the second GnRH injection were lower than those observed after the first GnRH injection. Similar to the rams, the relative response following clomid treatment of wethers (0.04 +/- 0.04) was less than the relative response (P > 0.05) following vehicle (0.40 +/- 0.16). The results suggest that clomid at this dosage inhibits GnRH-induced release of LH from the pituitary of rams but not of wethers.     

Efficacy of intermittent or continuous administration of GnRH in inducing ovulation in early and late seasonal anoestrus in the Père David's deer hind (Elaphurus davidianus)

Père David's deer hinds were treated with GnRH, administered as intermittent i.v. injections (2.0 micrograms/injection at 2-h intervals) for 4 days, or as a continuous s.c. infusion (1.0 micrograms/h) for 14 days. These treatments were given early (February-March) and late (May-June) in the period of seasonal anoestrus. The administration of repeated injections of GnRH increased mean LH concentrations from pretreatment values of 0.54 +/- 0.09 to 2.10 +/- 0.25 ng/ml over the first 8 h of treatment in early anoestrus, and from 0.62 +/- 0.11 to 2.73 +/- 0.49 ng/ml in late anoestrus. The mean amplitude of GnRH-induced LH episodes was greater (P less than 0.01) in late (4.03 +/- 0.28 ng/ml) than in early (3.12 +/- 0.26 ng/ml) anoestrus, but within each replicate (early or late anoestrus), neither mean LH episode amplitude nor mean plasma LH concentrations differed significantly between the four periods of intensive blood sampling. On the basis of their progesterone profiles, 6/12 hinds had ovulated in response to treatment with injections of GnRH (1/6 in early anoestrus and 5/6 in late anoestrus), and oestrus and a preovulatory LH surge were recorded in all of these animals. Oestrus and a preovulatory LH surge were also recorded in one other animal treated in early anoestrus in which progesterone concentrations remained low. The mean times of onset of oestrus (91.0 +/- 1.00 and 62.4 +/- 0.98 h) and of the preovulatory LH surge (85.8 +/- 3.76 and 59.4 +/- 0.25 h) both occurred significantly earlier (P less than 0.001) in animals treated in late anoestrus. Continuous infusion of GnRH to seasonally anoestrous hinds resulted in an increase in mean plasma LH concentrations, but this response did not differ significantly between early (2.15 +/- 0.28 ng/ml) and late (2.48 +/- 0.26 ng/ml) anoestrus. Ovulation, based on progesterone profiles, occurred in 2/7 hinds in early anoestrus and in 4/6 hinds in late anoestrus. Oestrus was detected in all except one of these hinds. The mean time of onset of oestrus occurred earlier in animals treated in late anoestrus (66.2 +/- 0.32 h and 46.7 +/- 0.67 h, P less than 0.01). The administration of GnRH, given either intermittently or continuously, will induce ovulation in a proportion of seasonally anoestrous Père David's deer. However, more animals ovulate in response to this treatment in late than in early anoestrus (75% compared with 23%).     

Response of prepubertal ewes primed with monensin or progesterone to administration of FSH

Prepubertal ewe lambs were treated with FSH after progesterone priming for 12 days (Group P), monensin supplementation for 14 days (Group M) or a standard diet (Group C). Serial blood samples were taken for LH and progesterone assay, and ovariectomy was performed on half of each group 38-52 h after start of treatment to assess ovarian function, follicular steroid production in vitro and the concentration of gonadotrophin binding sites in follicles. The remaining ewe lambs were ovariectomized 8 days after FSH treatment to determine whether functional corpora lutea were present. FSH treatment was followed by a preovulatory LH surge which occurred significantly later (P less than 0.05) and was better synchronized in ewes in Groups P and M than in those in Group C. At 13-15 h after the LH surge significantly more large follicles were present on ovaries from Group P and M ewes than in Group C. Follicles greater than 5 mm diameter from ewes in Groups P and M produced significantly less oestrogen and testosterone and more dihydrotestosterone, and had significantly more hCG binding sites, than did similar-sized follicles from Group C animals. Ovariectomy on Day 8 after the completion of FSH treatment showed that ewes in Groups P and M had significantly greater numbers of functional corpora lutea. These results indicate that, in prepubertal ewes, progesterone priming and monensin supplementation may delay the preovulatory LH surge, allowing follicles developing after FSH treatment more time to mature before ovulation. This may result in better luteinization of ruptured follicles in these ewes, with the formation of functional corpora lutea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.