Vitrification of in vitro-derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures.

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. 'fresh') embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.

Comparative response of rams and bulls to long-term treatment with gonadotropin-releasing hormone analogs.

The objective was to compare the relative response between rams and bulls in characteristics of LH, FSH and testosterone (T) secretion, during and after long-term treatment with GnRH analogs. Animals were treated with GnRH agonist, GnRH antagonist, or vehicle (Control) for 28 days. Serial blood samples were collected on day 21 of treatment, and at several intervals after treatment. Injections of natural sequence GnRH were used to evaluate the capacity of the pituitary to release gonadotropins during and after treatment. Treatment with GnRH agonist increased basal LH and T concentrations in both rams and bulls, with a greater relative increase in bulls. Endogenous LH pulses and LH release after administration of GnRH were suppressed during treatment with GnRH agonist. Treatment with GnRH antagonist decreased mean hormone concentrations, LH and T pulse frequency, and the release of LH and T after exogenous GnRH, with greater relative effects in bulls. Rams previously treated with antagonist had a greater release of LH after administration of GnRH compared with control rams, while rams previously treated with agonist showed a reduced LH response. Bulls previously treated with agonist had reduced FSH concentrations and LH pulse amplitudes compared with control bulls while bulls previously treated with antagonist had greater T concentrations and pulse frequency. The present study was the first direct comparison between domestic species of the response in males to treatment with GnRH analogs. The findings demonstrated that differences do occur between rams and bulls in LH, FSH and testosterone secretion during and after treatment. Also, the consequences of treatment with either GnRH analog can persist for a considerable time after discontinuation of treatment.

Recent advances in bovine reproductive endocrinology and physiology and their impact on drug delivery system design for the control of the estrous cycle in cattle.

When methods of drug intervention are being developed to control estrous cycles, a thorough understanding of the endocrine and functional changes together with the reproductive behavior of the animals are essential. This review presents our current knowledge on reproductive endocrinology, physiology and behavior, and the methods of drug intervention to control estrous cycles. It also describes current efforts to develop advanced drug delivery systems that meet the animal scientist's demands to control the estrous cycle in cattle.

Ewe luteal function influenced by pulsatile administration of synthetic LHRH/FSHRH.

The influence of repetitive administration of synthetic LHRH/FSHRH or saline (S) on 17beta-estradiol (E2)-induced precocious luteal regression in the ewe was examined. Ewes were pre-treated on days 10 and 11 of the estrous cycle with either 750 mug E2 (total dose = 1.5 mg) in oil or with oil (O) alone. Treatment involved in delivery of 10 mug of synthetic LHRH/FSHRH in saline or an equal volume of saline only, administered at 2-h intervals beginning on day 12 of the estrous cycle and continuing through the succeeding 72 hours. During the period of LHRH administration, the serum LH patterns in the O-LHRH and E2-LHRH groups were characterized by rhythmic fluctuation, rising in response to LHRH and falling prior to the subsequent treatment injection. Throughout the course of the treatment period, the serum LH levels in the O-LHRH group were consistently higher than those in the E2-LHRH group. No increase in serum LH concentration was observed in the saline-treated animals. The mean luteal weight and mean luteal progesterone content at the end of the 72-h period were not significantly different between the O-S and E2-LHRH groups (543 +/- 88 vs. 455 +/- 126 mg and 13.1 +/- 6.2 vs 16.0 +/- 9.7 mug, respectively). Both luteal weight and progesterone content were increased (P less than .01) in the O-LHRH group (1089 +/- 87 mg and 47.5 +/- 3.1 mug) and significantly reduced (P less than .05) in the E2-S group (309 +/- 49 mg and 5.2 +/- 0.1 mug) compared with those of either the O-S group or the E2-LHRH group. Thus LHRH treatment increased mean luteal weight and mean luteal progesterone content while E2 pre-treatment depressed the same parameters. These data suggest that pulsatile administration of synthetic LHRH is able to elevate serum LH levels to an extent sufficient to counteract both natural luteolysis and premature luteal regression induced by E2 treatment.

Initiation of spermatogenesis and testicular growth in oestradiol-17 beta-implanted bull calves with pulsatile infusion of luteinizing hormone releasing hormone.

Testicular growth and secretory profiles of LH and testosterone were monitored in three bull calves implanted with oestradiol-17 beta and three bull calves implanted with oestradiol-17 beta and infused intravenously with LH releasing hormone (LH-RH; 500 ng/pulse per h, 30-s pulse) continuously between 34 and 42 weeks of age. Oestradiol-17 beta implants restricted testicular growth and spermatogenesis by interfering with the hypothalamo-pituitary-testicular endocrine axis. Initiation of pulsatile LH release by LH-RH pulse infusion was accompanied by a twofold increase in mean circulating levels of LH (3.4 v. 1.8 microgram/l) and a marked increase in serum testosterone (13.0 v. 0.4 microgram/l). Testicular diameter was enhanced significantly by week 4 of infusion and increased in a linear fashion up to and including week 8. Testicular weight (g) and total daily sperm production (X 10(9)) at 42 weeks of age were decreased in calves implanted with oestradiol-17 beta (105 +/- 14 (S.E.M.); 0.0) when compared with calves implanted with oestradiol-17 beta and infused with LH-RH (254 +/- 12; 1.2 +/- 0.3). Differences in testicular size and sperm production rates between LH-RH-infused and control bulls without implants (352 +/- 26; 3.3 +/- 0.9) were attributed to the 2-month delay between oestradiol-17 beta treatment and the initiation of LH-RH treatment. These results (1) confirm our earlier conclusion that oestradiol-17 beta can interfere with normal pubertal development in beef bulls and (2) provide additional support that pulsatile LH secretion is important for the initiation of testicular growth and spermatogenesis in pubertal bulls.

Individual differences in LH and FSH responses to orchidectomy and testosterone replacement therapy in rams.

Profiles of LH and FSH levels and pituitary LH responses to exogenous luteinizing hormone releasing hormone (LHRH) have been characterized in rams, castrated rams (wethers), and wethers implanted with testosterone. Rams were castrated when adult, and, at the time of castration, two groups of wethers were implanted with either four or eight testosterone capsules. Rams showed random pulses of LH and testosterone which were temporally related. The number of LH and testosterone pulses per 24 hours differed among rams, giving rise to large differences in the mean levels of these hormones. Mean FSH levels and pituitary LH responses to LHRH also differed among rams and were positively correlated to differences in LH levels. All three nonimplanted wethers showed a rhythmic pulsatile pattern of LH secretion and had elevated mean LH and FSH levels. There were, however, appreciable differences between wethers with regard to mean LH and FSH levels and pituitary LH responses to LHRH. Both four and eight testosterone capsules were effective in suppressing pulsatile LH secretion and mean LH and FSH levels in two out of three wethers. In a third animal within each of these groups, however, LH and FSH profiles and LH responses to LHRH were characteristic of nonimplanted wethers. These data suggest that individual rams have different inherent capacities to secrete gonadotropins which influence LH and FSH responses to castration and testosterone replacement therapy.

Acute effects of different intensities of weight lifting on serum testosterone.

The purpose of this investigation was to determine the effect of moderate weight lifting (MWL) and light weight lifting (LWL) on concentrations of serum testosterone in males. Baseline testosterone concentrations were determined via an indwelling catheter in the median cubital vein. An initial sample of blood was followed 7 min later by five samples taken at 4-min intervals. A final sample was taken 10 min after the last 4-min blood draw. Blood samples were obtained at similar times and intervals on the weight lifting days. The MWL consisted of four sets of six squats at 90-95% of a six-repetition maximum (RM), while the LWL consisted of four sets of 9 or 10 repetitions at 60-65% of the weight used for the sets during MWL. There was a significant increase in serum testosterone concentrations following the fourth set for both MWL and LWL when compared with baseline concentrations and both MWL and LWL testosterone concentrations returned to baseline levels at 10 min postexercise. These results indicate that MWL and LWL caused increases in serum testosterone that were greater than those associated with baseline levels. Postexercise responses for the MWL and LWL were similar.

Characterization of secretion of thymosin alpha 1 and thymosin beta 4 during prepuberty, estrus and pregnancy in the bovine female.

The objective of the present study was to characterize secretion of thymosin alpha 1 (A1) and thymosin beta 4 (B4) during different stages of ovarian function and pregnancy in bovine females. One hundred and thirty-five prepubertal heifers averaging 9 mo of age at the time the study was initiated were used. Estrous detection was conducted twice daily using intact bulls fitted with marking harnesses. Blood samples were collected at first behavioral estrus and at the time of rectal palpation for pregnancy. Heifers were weighed at the beginning of the study and every 56 days and additional blood samples were collected at those times. Plasma was harvested and radioimmunoassays were conducted to quantify concentrations of thymosin A1 and B4. Mean concentrations of thymosin A1 and thymosin B4 were determined during prepuberty, estrus during which conception did not occur, estrus during which conception did occur, pregnancy and in heifers that failed to attain puberty. Mean plasma concentrations of thymosin A1 and B4 were greater at estrus during which conception occurred than at any other period (P less than .01). Regressions of days prepubertal and days post conception on thymosin A1 and B4 were analyzed. During the 100 days prior to puberty, thymosin A1 and B4 did not change in the heifers that ultimately conceived. However, in the heifers that were never determined to be pregnant by rectal palpation, concentrations of both thymosin A1 and B4 increased linearly during prepuberty (P less than .01). A decline in thymosin A1 and B4 concentrations occurred in a quadratic fashion from conception through the first 100 d of pregnancy (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Opioid and 17 beta-estradiol regulation of LH and FSH secretion during sexual maturation in heifers.

The objective of the present study was to examine the involvement of opioid neuropeptides and E2 in regulating circulating concentrations of gonadotropins during sexual maturation in the bovine female. Prepubertal (immature) and postpubertal (mature) bovine females were used. Mean concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in circulation before and after administration of naloxone were determined in ovariectomized heifers administered E2 and ovariectomized heifers not administered E2. A linear decline (P less than 0.01) in opioid suppression of LH and FSH occurred during the experimental period in immature heifers receiving E2. This decline in opioid suppression of LH and FSH occurred during the same period of time that intact control heifers were initiating estrous cycles at puberty. Little change of opioid suppression of LH and FSH occurred during the experimental period in immature heifers not receiving E2 and mature heifers receiving E2. Our research indicates that opioid neuropeptides and E2 act together to regulate LH and FSH secretion during sexual maturation in the bovine female.

Response to luteinizing hormone releasing hormone in postpubertal boars with large testes.

The objective of the present study was to determine if postpubertal boars (12-13 months of age; 156 +/- 8 kg) with large testes had altered hypothalamic control of secretion of luteinizing hormone (LH). Seven boars with the highest estimated 150 d, paired testis weights from a line selected for large testes (769 +/- 60 g = mean weight of excised testes) and 8 boars from a control group (control, 544 +/- 20 g) were tethered in stalls and fitted with indwelling jugular catheters. Males were bled when they were intact, 14 days after castration and during administration of sodium pentobarbital anesthetic (subsequent to castration) to block secretion of endogenous LH-releasing hormone (LHRH). Blood samples were collected at 12-min intervals for 6 hr before and 1 hr after intravenous injection of LHRH in intact and castrated males. During anesthesia, LHRH was administered 4 times at 1-hr intervals and blood samples were collected every 6 min. All samples were analyzed for concentrations of LH and pooled samples were analyzed for concentrations of 17-beta estradiol (E2) and testosterone (T). In intact and castrated males, mean concentrations of LH, frequency and amplitude of pulses of LH, and concentrations of E2 and T were not different between boars of the two groups (P greater than .10). Response to exogenous LHRH was less (P less than .05) in intact males with large testes than in corresponding males from the control group (P less than .05). Fourteen days after castration, males that had larger testes before castration had less of a response to LHRH than males from the control group (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating concentrations of 17-estradiol influence pattern of LH in circulation of cows.

The objective of the research was to determine the relationship between circulating 17 beta-estradiol (E2) and secretion of luteinizing hormone (LH) in cows. A second objective was to determine if response to E2 was influenced by interval between ovariectomy and the start of E2 treatment. Thirty-one nulliparous cows 3 yr of age were randomly assigned to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age (long term), and the other 15 cows were ovariectomized at 36 mo of age (short term). At the time of ovariectomy of cows in the short term group, 11 cows in the short term group and 12 cows in the long term group were implanted subcutaneously with 1, 2 or 4 polydimethylsiloxane capsules containing E2. The other eight cows served as non-implanted controls (n = 4-short term, n = 4-long term). All cows were fitted with jugular vein catheters on day 29 of treatment, and on day 30 blood samples were collected at 12-min intervals for 6 hr. At the end of 6 hr, luteinizing hormone-releasing hormone (LHRH) was administered and blood sampling continued at 12-min intervals for an additional hour. Serum was analyzed for LH and E2. Variables of LH secretion analyzed were mean concentration, frequency of pulses, amplitude of pulses and maximum concentration after LHRH. There were no significant interactions for any of the variables of LH among cows ovariectomized for the long and short term. There was a significant linear increase in mean concentration of LH with increased circulating concentration of E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Duration of ovulation suppression with subcutaneous silicone implants containing norgestomet in Bos indicus heifers and cows.

The aim of this study is to determine the duration of suppressing oestrus or ovulation in Bos indicus heifers and cows using norgestomet (N) incorporated into a silicone implant. Twelve heifers and 18 cows undergoing oestrous cycles were allocated to one of two treatment groups (6 heifers and 9 cows per group). Animals were treated with a single subcutaneous (s.c.) silicone implant containing 3 mg of N or two identical silicone implants (6 mg of N in two silicone implants) on day 0 of the study. An analogue of prostaglandin F2-alpha was administered intramuscularly, to all animals on days 0 and 6 to induce regression of corpora lutea. Implants were removed from all animals on day 21. Animals were observed for signs of behavioural oestrus while implants were in situ and for 6 d following implant removal. Blood samples were collected on alternate days from day 0 to 20 and again on day 21 and analysed for plasma progesterone (P4). Transrectal ultrasonography was performed on days 0, 6, 10, 14, 18, 21, 24, 26,27, and along with concentration of P4 in plasma was used to confirm ovulation during the treatment period. The cumulative percentages of animals ovulating during the 21 days implants were in situ were 6.7% (3 mg of N in one silicone implant) and 0% (6 mg of N in two silicone implants)Intervals from implant removal to oestrus (mean +/- S.E.M.) did not differ significantly among animals treated with either 3 or 6 mg of N in silicone implants (46.8 +/- 7.0 vs. 55.4 +/- 7.1 h, P = 0.398). Variances in interval from implant removal to oestrus among animals treated with 3 or 6 mg of N in silicone implants were also homogeneous (P = 0.942). The mean diameter of the ovulatory follicle on day 21 was larger in the animals treated with 3 mg of N compared to animals treated with 6 mg of N in two silicone implants ( 13.1 +/- 0.8 vs. 9.7 +/- 0.7 mm, P = 0.005). We conclude that treatment with 3 mg of N in a silicone implant will suppress ovulation in Bos indicus heifers and most cows for 21 d. Six mg of N in two silicone implants will suppress ovulation for 21 d in both cows and heifers.

Effects of short-term treatment with progesterone superimposed on 11 or 17 days of norgestomet treatment on the interval to oestrus and fertility in Bos indicus heifers.

The aims of this study were to determine: (1) if short-term treatment of Bos indicus heifers with progesterone (P4) while implanted with a s.c. norgestomet implant for 17 days would influence the time interval to oestrus and increase fertility of the synchronised oestrus, and (2) whether the response to treatment with P4 would differ between heifers treated with a norgestomet implant for 17 vs. 11 days when short-term treatment with P4 is applied 3 days prior to implant removal. B. indicus heifers at two separate sites (A and B) were allocated to three groups at each site. Heifers in two groups (NG and NGP4 groups) were given a single s.c. norgestomet implant on the first day of treatment (day 0) while heifers in a third group (NGP4PG group) were implanted on day 6. A single P4 releasing Controlled Internal Drug Release device (CIDR) was inserted on day 14 in heifers in the NGP4 and NGP4PG groups and was removed 23.5 +/- 0.07 h later (day 15). Heifers in the NGP4PG group were administered an analogue of prostaglandin F2 alpha (PGF2 alpha) at the time of CIDR removal to regress corpora lutea. Implants were removed from all heifers on the same day (day 17) and a 400 IU of equine chorionic gonadotrophin (ECG) was administered s.c. Animals were artificially inseminated 11.1 +/- 0.17 h after detection of oestrus, using frozen semen from one bull at site A and one of five bulls at site B. Inseminations were carried out by one of two technicians. Treatment with P4 delayed oestrus and reduced the synchrony of oestrus at site A (hours to oestrus +/- SD: NG group, 39.0 +/- 13.7; NGP4 group, 66.3 +/- 24.4; NGP4PG group, 58.9 +/- 20.5 h; P < 0.05) but not at site B (41.4 +/- 15.2, 42.5 +/- 10.1, 45.4 +/- 10.3 h; P > 0.05). Pregnancy rates 6 weeks after insemination were found to be significantly associated with bull (P < 0.001), treatment group (P = 0.013) and insemination technician (P = 0.033). Pregnancy rates were greater in the heifers in the NGP4 group than heifers in the NG group [50.3% (78/155) vs. 36.4% (60/165); odds ratio = 1.83, 95% CI = 1.14 to 2.96] and similar between heifers in the NGP4 and NGP4PG groups [50.3% (78/155) vs. 51.1% (63/117); odds ratio = 1.06, 95% CI = 0.67 to 1.69]. It was concluded that acute treatment with P4 can improve pregnancy rates in B. indicus heifers treated for 17 days with norgestomet implants. Reducing the duration of norgestomet treatment to 11 days and administration of PGF2 alpha at the time of ending treatment with a CIDR device resulted in no differences in fertility, mean intervals to oestrus or synchrony of oestrus.

Influence of varied progestogen treatments on ovarian follicle status and subsequent ovarian superstimulatory responses in cows.

The influence of ovarian follicle status and follicle dominance on the response to superstimulatory treatment with FSH was examined in cows. In Experiment 1, oestrus was synchronised using Crestar and on Days 4-6 of the ensuing oestrous cycle cows were assigned to: Group NO (n = 9), control, endogenous CL and no treatment; Group N1 (n = 15), injected with a luteolytic dose of cloprostenol (500 micrograms) and implanted with one implant (3 mg) of the synthetic progestogen, norgestomet; Group N8 (n = 18), injected with 500 micrograms cloprostenol and implanted with eight (24 mg) implants of norgestomet. On Days 9-11, seven implants were removed from six cows in Group N8 and these cows, plus eight Group N1 and all Group N0 cows, were superstimulated with porcine FSH (Folltropin-V) over 4 days (360 mg total dose). The remaining implants were removed from cows in Groups N1 and N8 on Days 11-13, and all cows received 500 micrograms cloprostenol. Numbers and sizes of ovarian follicles, and CL, were recorded by trans-rectal ultrasonography; the largest follicle > 10 mm in diameter was considered morphologically dominant (DF). On Days 9-11, the proportions of cows with a DF were: Group N0, 3/9; Group N1, 14/15; Group N8, 0/18. Total follicles on the 4th day of FSH treatment were greater (P < 0.05) for cows in Group N1 (21.6 +/- 4.2) compared with Group N0 (10.9 +/- 2.4), with cows in Group N8 (13.2 +/- 0.9) not different from the other two groups. Subsequent numbers of CL were lower (P < 0.05) for cows in Group N1 (5.0 +/- 1.3) compared with Group N0 (9.4 +/- 2.0), with cows in Group N8 (8.5 +/- 1.0) not different from the other two groups. In Experiment 2, oestrus was synchronised in cows and on Days 4-6, cows were assigned to: Group C0 (n = 7), control, endogenous CL and no treatment; Group C3 (n = 6), received three CIDR-B intra-vaginal devices that delivered progesterone. On Days 9-11, two CIDR-B were removed from cows in Group C3 and all cows treated with FSH as in Experiment 1. The remaining CIDR-B was removed from cows in Group C3 on Days 11-13 and all cows injected with 500 micrograms cloprostenol. Proportions of cows with a DF on Days 9-11 and diameter of largest follicle were: Group C0, 6/7 and 12.6 +/- 0.9 mm; Group C3, 2/6 and 9.6 +/- 0.8 mm. Numbers of CL on Day 8 after oestrus were: Group C0, 20.0 +/- 7.1; Group C3, 14.8 +/- 4.8 (P > 0.05). Exposure to low dose norgestomet allowed development of a persistent dominant follicle, resulting in a reduced response to superstimulation with FSH. High dose progestogen restricted follicle growth without apparent effects on responses to superstimulation.

Effect of 48 h treatment with 17 beta-oestradiol or progesterone on follicular wave emergence and synchrony of ovulation in Bos indicus cows when administered at the end of a period of progesterone treatment.

The objective of this study was to determine the effect of treatment with additional progesterone (P4) or 17 beta-oestradiol (E2) at the end of a period of P4 treatment on ovarian follicular development, ovulation time, and plasma gonadotrophin and steroid hormone concentrations of Bos indicus cows. Initially, two injections of PGF2 alpha were given 14 days apart, and at the time of the second injection (Day 0) all cows received a single P4-releasing controlled internal drug release (CIDR) device that was removed 10 days later. Control cows (Group 1, n = 8) received no other treatment. On Day 8, cows in Group 2 (n = 8) and Group 3 (n = 8) received either a s.c. implant containing E2, or a second CIDR device, respectively. All CIDR devices and E2 implants were removed at a similar time on Day 10. Treatment with E2 or P4 delayed mean (+/- SD) time of ovulation (113.1 +/- 25.6 h, 153.4 +/- 44.5 h and 150.8 +/- 25.1 h for Groups 1, 2 and 3, respectively; P < 0.05) and the mean time (+/- SD) of the luteinising hormone (LH) peak (87.4 +/- 24.5 h, 124.3 +/- 45.0 h and 122.3 +/- 25.04 h for Groups 1, 2 and 3, respectively; P < 0.05). Both treatments delayed the mean (+/- SD) day of emergence of the ovulatory follicle (7.7 +/- 3.6 days, 11.3 +/- 1.7 days and 11.1 +/- 1.5 days for Groups 1, 2 and 3, respectively; P < 0.05), and reduced the variability in the day of emergence of the ovulatory follicle (P < 0.05) compared with the control cows. Variability in age and duration of dominance of the ovulatory follicle was greater in control animals compared with treated animals (P < 0.05). Treatment with E2 on Days 9 and 10 did not alter mean concentrations of gonadotrophins in the cows in Group 2 compared with control cows (P > 0.05), whereas treatment of cows with an additional CIDR device resulted in greater mean concentrations of FSH and lesser concentrations of LH on Day 9 (P < 0.05) compared with cows in Groups 1 and 2. By Day 10 mean concentrations of gonadotrophins were similar among cows in all three groups. Concentrations of E2 were less in cows in Group 3 compared with cows in Groups 1 and 2 from Day 9 to Day 11 (P < 0.05). We conclude that treatment with either E2 or P4 can influence the pattern of ovarian follicular development and ovulation in cattle; however, the mechanism of action of the two treatments may differ. Atretogenic treatments for ovarian follicles applied at the end of a period of progesterone treatment did not improve synchrony of ovulation.

Treatment with 17beta-oestradiol does not influence age and weight at puberty in Bos indicus heifers.

The working hypothesis was that treatment of heifers with 17beta-oestradiol (E2) during specific periods of prepuberty would reduce the response of the hypothalamic-pituitary axis to E2 negative feedback and induce an earlier onset of puberty. The effects of chronic treatment with exogenous E2 administered at specific maturational phases on the age and weight at puberty were studied in 96 prepubertal Brahman (3/4-7/8 Bos indicus) heifers (187.0 +/- 3.3 days of age, mean +/- SEM), weighing 149.9 +/- 2.5 kg. Heifers were randomly assigned to one of six groups (n = 16 per group). Groups 2-6 received E2 implants (Compudose 200) for 90-day periods starting at 10, 13, 16, 19 and 22 months of age, while animals in group 1 remained untreated. Implants were placed subcutaneously at the base of the ear. Blood was collected for progesterone (P4) determination by radioimmunoassay (RIA) and the animals were weighed at monthly intervals from 6 to 15 months then weekly from 15 to 28 months of age. Puberty was defined by concentrations of P > 1 ng/ml in plasma and identification of a corpus luteum (CL) by transrectal ultrasonography (Aloka 210DX:7.5 MHZ probe). Treatment with exogenous E2 at any of the ages/treatment intervals evaluated in this study did not reduce age or weight at puberty (P > 0.7). The mean age and weight at puberty of control heifers was 735.3 +/- 19.7 days (range: 597-861) and 299.2 +/- 10.2 kg (range: 233-382), respectively, which is greater than the age and weight at puberty of 481 days and 246 kg, that was previously reported for B. indicus heifers [Post, T.B., Reich, M.M., 1980. Puberty in tropical breeds of heifers as monitored by plasmaprogesterone. Proceedings of the Australian Society of Animal Production 13, 61-62.]. The large variation in age and weight at puberty that was observed in the present study among heifers might indicate an individual animal effect to E2 treatment among some of the treated animals. The lengthy interval from birth to puberty observed in this study, as compared to other studies, reflects the effects of other factors such as genotype, environmental or nutritional influences on puberty.

Seasonal fluctuations in plasma concentrations of luteinizing hormone and progesterone in Brahman (Bos indicus) and Hereford-Shorthorn (Bos taurus) cows grazing pastures at two stocking rates in a subtropical environment.

The effects of day length and grazing intensity on seasonal fluctuations in plasma concentrations of luteinizing hormone (LH) and cyclic ovarian activity were determined in Brahman (Bos indicus) and Hereford-Shorthorn (Bos taurus) cows maintained at two stocking rates in a subtropical environment. Contemporary groups of ovariectomised cows were monitored for fluctuations in plasma concentrations of LH. Equal numbers (n = 5) of entire and ovariectomised Brahman and Hereford-Shorthorn cows were assigned to a pasture with a greater or lesser stocking rate. Over a 15-month period, live weight was recorded weekly, and a blood sample was taken at the same time for measurement of plasma LH in entire and ovariectomised cows, and plasma progesterone in entire cows. Plasma concentrations of progesterone were used as an index of cyclic luteal function (time of cessation or onset of oestrous cycles). Regression coefficients were calculated to determine the least-order regression coefficient (LORC; range 1st to 10 order) for which time of year explained at least 50% (r2 > 0.05) of changes in live weight, plasma LH, or plasma progesterone; regression coefficients of 4th and 5th order indicated seasonally-related changes in these variables. For all cows, live weight was greatest in late summer to early autumn and lowest in winter. Changes in live weight were more closely related to seasonal changes in pasture availability for cows on pastures at a greater stocking rate (LORC 4th-5th) than for cows on pastures at a lesser stocking rate (LORC 1st-3rd). Cyclic ovarian activity ceased in four Hereford-Shorthorn cows on pastures at a greater stocking rate in late autumn to early winter, and onset of oestrous cycles did not occur in all of these cows until late spring. Oestrous cycles were not detected in one of five cows in the other groups during different periods of the study; however, there were no apparent patterns to cessation of oestrous cycles in these groups. There were no seasonally-related changes in plasma LH in entire cows, at either stocking rate (LORC 10th; r2 = 0.16 to 0.41). In contrast, distinct seasonal fluctuations in plasma LH occurred in ovariectomised cows, with increases in spring and winter. Environmental cues induced greater fluctuations in plasma LH in ovariectomised cows at a greater stocking rate (LORC 3rd-5th; r2 = 0.71 to 0.72) compared with ovariectomised cows at a lesser stocking rate (LORC 3rd-5th; r2 = 0.53 to 0.58). The findings demonstrated that marked seasonal changes in reproductive activity of the hypothalamic pituitary axis can occur in cows with B. indicus and B. taurus genotypes in a subtropical environment; however, changes in plasma concentrations of LH are only apparent in ovariectomised cows. Concentrations of plasma LH in ovariectomised Brahman and Hereford-Shorthorn cows increased during winter, when pasture availability was limiting and cyclic luteal function ceased in four of five Hereford-Shorthorn cows.

Gonadotropin secretion and development of ovarian follicles during oestrous cycles in heifers treated with luteinizing hormone releasing hormone antagonist.

The hypothesis tested was that reduced LHRH stimulation of the anterior pituitary would lead to attenuated development of ovarian follicles as a result of reduced gonadotropin secretion during oestrous cycles of cattle. Twenty heifers were randomly assigned to be treated ( n = 5/treatment) with an antagonist to LHRH (LHRH-Ant) 1) from Day 2 to 7 (Day 0 = behavioural oestrus), 2) Day 7 to 12, 3) Day 12 to 17, 4) or serve as untreated control animals. LHRH-Ant suppressed LH pulses of heifers in all treatment groups from treatment initiation through Day 17 as compared with untreated control heifers [Peters et al., 1994. Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers. Biol. Reprod., 51 (1994) 1248-1254]. Circulating concentration of FSH from Day 8 to 10 of the oestrous cycle did not increase in heifers treated with LHRH-Ant from Day 2 to 7 or Day 7 to 12; however, there was increased (P < 0.05) FSH from Day 8 to 10 of the oestrous cycle in heifers treated with LHRH-Ant from Day 12 to 17 and control heifers. Compared with control heifers, heifers treated with LHRH-Ant from the Day 2 to 7 had suppressed (P < 0.05) size and persistence of the first and second dominant ovarian follicles. Heifers treated with LHRH-Ant from Day 7 to 12 had suppressed size (P < 0.05 and tended (P < 0.10) to have a shorter persistence of the second dominant ovarian follicle compared with control heifers. Heifers treated with LHRH-Ant from Day 12 to 17 had a similar (P > 0.10) size and persistence of dominant ovarian follicles but had reduced (P < 0.10) numbers of large follicles compared with control heifers. Heifers treated with LHRH-Ant from Day 2 to 7 had lower (P < 0.01) concentrations of 17 beta-oestradiol during the treatment period and tended (P < 0.10) to have lower concentrations of 17 beta-oestradiol from Day 7 to 12 of the oestrous cycle compared with control heifers. Heifers treated with LHRH-Ant from Day 7 to 12 or Day 12 to 17 had similar (P > 0.10) circulating LH concentrations of l7 beta-oestradiol compared with control heifers. Reduced LHRH stimulation of the pituitary from Day 2 to 12 of the oestrous cycle and the resulting modulation in circulating LH and FSH led to suppressed ovarian follicular development and oestradiol secretion. After Day 12 of the oestrous cycle, reduced LHRH stimulation of the anterior pituitary did not lead to altered ovarian follicular development to the extent as reduced LHRH stimulation before Day 12 although pulsatile release of LH was similarly suppressed by treatment with the LHRH-Ant.

Regulation of frequency of luteinizing hormone pulses by magnitude of acute change in circulating concentration of progesterone of female cattle.

The hypothesis was the greater the magnitude of acute increase in circulating concentration of progesterone of female cattle, the greater the acute inhibitory effect on frequency of pulsatile LH release. From Day 0 to 4 of the treatment period, females without functional corpora lutea were treated with varying doses of progesterone to result in varying concentrations of progesterone within the typical physiological range in blood. From Day 4 to 7, cattle were treated with a single dose of progesterone to achieve a similar circulating concentration of progesterone among all females in the study. Therefore, from Day 0 to 4 relative to Day 4 to 7 of the treatment period, females had a: (1) large (3.1 ng/ml), (2) moderate (2.5 ng/ml), or (3) small (0.5 ng/ml) increase in concentration of progesterone in blood. Frequency of LH pulses was greater (P <0.10) in females with the greatest magnitude of change in concentration of progesterone during the first 24 h following the change in concentration as compared with females with the moderate or small of change in concentration of progesterone suggesting our working hypothesis should be rejected. The greater the magnitude of acute change in concentration of progesterone, however, the longer time required for re-initiation of release of LH pulses of the amplitude of pulses that preceded the change in concentration of progesterone.

Concentrations of gonadotropins, estradiol and progesterone in sows selected on an index of ovulation rate and embryo survival.

The objective of this study was to determine concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4) and 17beta-estradiol (E2) in sows from a line selected on an index which emphasized ovulation rate (Select) and from a control line. A further classification of the sows in each line was made according to the estimated number of ovulations during an estrous cycle. Sows in the Select line were ranked into a high (HI) or low group (LI) when their estimated number of ovulations were 25 or more and 14 to 15, respectively. Sows of the control line were classified into groups as high (HC) or low (LC) when the estimated values for ovulation rate were 14-15 and 8-9 ovulations, respectively. Blood samples were collected every 12 h during a complete estrous cycle and samples were analyzed for concentrations of FSH and LH. Samples collected every 24 h were assayed for P4 and E2. Mean concentrations of FSH, LH, P4 and E2 did not differ (P > 0.10) between lines or between HI and LI or HC and LC groups. Selection of pigs for ovulation rate and embryonal survival did not affect concentrations of FSH, LH, P4 and E2 in sows during the estrous cycle.