Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.     

Folliculogenesis Is Not Fully Inhibited during GnRH Analogues Treatment in Mice Challenging Their Efficiency to Preserve the Ovarian Reserve during Chemotherapy in This Model

As many chemotherapy regimens induce follicular depletion, fertility preservation became a major concern in young cancer patients. By maintaining follicles at the resting stage, gonadotropin-releasing hormone analogues (GnRHa) were proposed as an ovarian-protective option during chemotherapy. However, their efficacy and mechanisms of action remain to be elucidated. Mice were dosed with cyclophosphamide (Cy, 100–500mg/kg i.p) to quantify follicular depletion and evaluate apoptosis at different times. We observed a dose-dependent depletion of the follicular reserve within 24 hours after Cy injection with a mean follicular loss of 45% at the dose of 200mg/kg. Apoptosis occurs in the granulosa cells of growing follicles within 12 hours after Cy treatment, while no apoptosis was detected in resting follicles suggesting that chemotherapy acutely affects both resting and growing follicles through different mechanisms. We further tested the ability of both GnRH agonist and antagonist to inhibit oestrus cycles, follicular growth and FSH secretion in mice and to protect ovarian reserve against chemotherapy. Although GnRHa were efficient to disrupt oestrus cycles, they failed to inhibit follicular development, irrespective of the doses and injection sites (sc or im). Around 20% of healthy growing follicles were still observed during GnRHa treatment and serum FSH levels were not reduced either by antagonist or agonist. GnRHa had no effect on Cy-induced follicular damages. Thus, we showed that GnRHa were not as efficient at inhibiting the pituitary-gonadal axis in mice as in human. Furthermore, the acute depletion of primordial follicles observed after chemotherapy does not support the hypothesis that the ovary may be protected by gonadotropin suppression.     

Synchronization of Oestrus in Heifers with Norethisterone

Sixty-five heifers in different stages of the oestrus cycle were fed norethisterone once daily for 17 days at doses of 0.2, 0.6 and 1.0 mg/kg body weight. During treatment swollen vulva, mucus discharge, open and reddened portio and udder enlargement were noted. Norethisterone was effective in suppressing oestrus and ovulation at doses of 0.6 and 1.0 mg/kg. Extremely good heat-synchronization followed the treatment and 98 % of the heifers came on heat within a two-day period. Fifty-five % conceived after the first insemination. In the group receiving 0.2 mg norethisterone per kg, three out of 13 heifers showed psychic heat during treatment. Poor heat synchronization and low conception rate were obtained in this group. A possible effect of cycle stage on heat synchronization was noted only in the 0.2 mg group where a better result was obtained when treatment began in the follicular phase.     

Effects of chronic treatment with a GnRH agonist on oestrous behaviour and on the secretion of LH and progesterone in the ewe

A study was carried out to investigate a novel approach to oestrus synchronization in the ewe by treatment with a gonadotrophin releasing hormone (GnRH) agonist. Groups of ewes were initially treated on Day 2, 10 or 14 of the oestrous cycle with 10 mug GnRH analogue (D-Ser(Bu(t)) 6 des Gly GnRH ethylamide) per ewe per day for 14 days. Behavioural oestrus was inhibited during GnRH agonist treatment and recurred from 8 to 38 days after the treatment in an unsynchronized manner. Luteal activity during treatment was not impaired but reduced progesterone concentrations occurred in cycles after the treatment. The rhythm of ovarian function, generally characterized by prolonged follicular development, was impaired. During the treatment and subsequent recovery period, integrity of pituitary function was examined by measuring luteinizing hormone (LH) after GnRH agonist was injected, and after stimulation test doses of 150 ng natural GnRH were administered. During treatment there was, with time, a decline in pituitary response to the agonist which suggested that pituitary release of LH was exhausted. After the 14-day treatment the stimulation test with GnRH revealed a gradual return to normal responsiveness although this was not complete three weeks after the treatment when compared to control ewes. This lowered pituitary activity could cause the impaired ovarian function.     

Follicular recruitment and ovulatory response to FSH treatment initiated on day 0 or day 3 postovulation in goats

The present study evaluates the effect of the presence of a large growing follicle at the onset of superovulatory treatment on follicular recruitment and ovulatory response in dairy goats. The treatment consisted of six equal doses of pFSH given every 12 h (total dose: 200 mg NIH-FSH-P1) which was initiated at Day 0 (Group D0) or Day 3 (Group D3) postovulation. Two half-doses of an analogue of prostaglandin F2alpha (delprostenate, 80 microg each) were administered together with the last two FSH doses to ensure luteolysis. A dose of a GnRH analogue (busereline acetate, 10.5 microg) was administered at the onset of estrus. Ovarian changes were evaluated twice a day by transrectal ultrasonography. Follicles were classified according to follicular diameter as small (3 to < 4 mm), medium (4 to < 5 mm) and large follicles (> or = 5 mm). The number of corpora lutea (CL) was recorded after laparotomy performed 6 days after estrus. The work was conducted in replicates. In the first trial, the does were assigned to either the D0 (n = 4) or D3 group (n = 4) and in the second replicate, each goat was assigned to the alternate group. No large follicles were recorded and the diameter of the largest follicle was 3.3 +/- 0.1 mm (mean +/- S.E.M.) at the initiation of the treatment in D0-treated goats. In contrast, a growing large follicle was present (6.7 +/- 0.4 mm, P < 0.01) when the treatment was initiated in D3-treated goats. In these goats, the number of small follicles increased 24 h after ovulation but then declined 48 h later, temporally correlated with the growth of the largest follicle of the first follicular wave. The number of small follicles recruited by the FSH treatment was significantly higher and occurred earlier in D0- than in D3-treated goats (9.0 +/- 1.3 versus 5.6 +/- 1.1 follicles; P < 0.05; and 24 h versus 48 h from the onset of the treatment, respectively). The number of large follicles at the onset of estrus was higher in D0- than in D3-treated goats (14.4 +/- 1.9 versus 10.3 +/- 1.3; P < 0.05). Consequently, the number of CL recorded 6 days after estrus were higher in D0- than in D3-treated goats (13.6 +/- 1.9 versus 10.4 +/- 1.9; P < 0.05, respectively). These results demonstrate that the presence of a dominant follicle at the time of initiation of super-stimulatory treatment is detrimental to ovulatory response. This study supports the advantages of the so-called Day 0 protocol, e.g. treatment starting soon after ovulation, when the emergence of the first follicular wave takes place and there are no dominant follicles.     

Effects of an LHRH antagonist on gonadotrophin and oestradiol secretion, follicular development, oestrus and ovulation in Holstein heifers

Mature cyclic Holstein heifers were given a luteolytic dose of cloprostenol followed by two i.v. injections, 12 h apart, of various doses of [Ac-D-Nal1, D-p-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10]-LHRH, beginning either at the time of first observation of behavioural oestrus, or 48 h after the cloprostenol injection. When treatment began at the first observation of oestrus, the time of ovulation, as determined by ultrasonic echography, was significantly delayed by total doses of 0.8 mg or more of the antagonist. When given at 48 and 60 h after cloprostenol injection, a total dose of 1.5 mg of the antagonist significantly delayed the growth of the ovulating follicle, the onset of oestrus, the preovulatory surges of oestradiol, LH and FSH, and ovulation. It is concluded that the LHRH antagonist can effectively suppress endogenous LH secretion and may therefore be useful in the study of follicular development, ovulation, and other events in the oestrous cycle of the cow.     

Hormonal induction of oestrus and pregnancy in anoestrous ferrets (Mustela putorius furo)

Three doses of FSH were tested for their ability to induce oestrus in ferrets. A dose of 0.25 mg, administered twice daily, induced oestrus and breeding in most females within 6-13 days. Addition of 5 i.u. hCG during the final stages of follicular development enhanced the percentage of females with implantation sites (85%), and 23% of the ferrets so treated gave birth to kits, none of which survived for more than 3 days. This may have been due to insufficient prolactin secretion and thus inadequate luteal maintenance and milk production, as these females were maintained on a short-day photoperiod.     

Endometrial histology and post-partum mares treated with progesterone and synthetic GnRH (AY-24,031).

Foal heat was significantly delayed in 15 Thoroughbred and Quarter-horse mares by 200 mg progesterone in oil from Days 5--14 post partum. Nine of these mares subsequently received daily i.v. injections of 2 mg of a synthetic GnRH preparation (AY-24,031) from Day 2 of the progesterone-delayed oestrus but this treatment did not significantly shorten oestrus or hasten ovulation. Uterine biopsies taken on Day 15 post partum from all the mares showed a mixed endometrial morphology having both oestrous and dioestrous characteristics. There was an increased proliferation of endometrial glands in these animals at the time of ovulation compared to control mares having a normal foal heat.     

Effects of treatment with a prostaglandin analogue on developmental dynamics and functionality of induced corpora lutea in goats

The aim of this study was to compare morphological and functional features of spontaneous and induced corpora lutea (CLs) in goats. Fourteen adult and cycling Anglo Nubian goats (Argentina) were randomly allocated to two groups: Group N (n = 7) included goats with natural spontaneous oestrus and Group PG (n = 7) included does in which oestrus was synchronized by the administration of two i.m. cloprostenol doses, 10 days apart. In both groups, oestrous behaviour was checked twice daily (Day of oestrus = Day 0) and daily transrectal ultrasonographies were performed for evaluating CLs and follicles dynamics through the complete subsequent oestrous cycle; the luteal activity was determined directly, in terms of progesterone (P4) secretion, and indirectly, by assessing effects of CL on follicular dynamics. All goats exhibited oestrous behaviour and ovulation without differences in ovulation rate (N: 1.67 ± 0.2, PG: 2.0 ± 0.1). The total luteal tissue area showed linear growth from Day 4 to Day 15 of oestrous cycle in all goats, but the developmental dynamics differed between groups, treated goats had larger area (P < 0.01). Plasma P4 concentrations also increased from Day 0 to Day 15 in all the does; however, from Day 5 to Day 15, treated does had a lower concentrations than the untreated group (P < 0.001). There were differences in the development of follicular waves between groups; assessment of size-distribution showed that treated group had a higher number of small and larger follicles (P < 0.05). The largest follicles recorded in treated goats had a higher maximum diameter both at the first (PG: 7.6 ± 0.8 mm; N: 4.9 ± 0.7 mm, P < 0.05) and second follicular waves (PG: 6.3 ± 1.4 mm; N: 5.0 ± 0.4 mm, P < 0.05) and a longer growth phase during the second wave (PG: 6.5 ± 1.7 days; N: 4.6 ± 0.7 days, P < 0.05), coincident with the period of maximal luteal secretion. In conclusion, synchronization of oestrus and ovulation by the administration of a prostaglandin analogue causes differences in developmental dynamics and functionality of induced corpora lutea when compared to natural spontaneous ovulation.     

Exogenous GnRH induces ovulation in seasonally anoestrous lactating goats (Capra hircus)

A specific sheep LH radioimmunoassay was validated for the measurement of goat LH, and used to monitor luteal-phase LH episodes and the preavulatory LH surge in progestagen sponge-synchronized cycling goats. No luteal-phase LH episodes were detected during 12 h of frequent (15-min) blood sampling in 2 goats. A preovulatory LH surge was recorded in 5/5 goats, with a mean amplitude of 45.4 +/- 7.2 ng/ml and a mean time of onset of 38.4 +/- 1.2 h after removal of a progestagen-impregnated sponge. In anoestrous goats, single i.v. injections of 1000 and 2000 ng GnRH induced LH episodes with a mean amplitude of 2.04 +/- 0.11 and 3.67 +/- 0.06 ng/ml respectively, but injections of 250 or 500 ng did not consistently elevate LH concentrations. Progestagen-primed, seasonally anoestrous lactating goats were treated with repeated injections of 1500 ng GnRH (every 2 h for 52 or 78 h) in May 1985 or 1986. All 10 had kidded in March of the same year, and were consequently at peak lactation at the time of GnRH treatment. A preovulatory LH surge was detected in 9 goats with a mean time of onset of 59.5 +/- 2.9 h (1985) or 39.6 +/- 3.3 h (1986) after vaginal sponge removal. All animals displayed oestrus and ovulated, and 9 of the goats were mated: in 5 of these animals pregnancies were successfully carried to term. The results show episodic LH release in response to GnRH and indicate that ovulation can be induced in seasonally anoestrous goats, even at peak lactation, and normal pregnancies may result.     

The effect of a GnRH antagonist pre-treatment,in the superovulation of goats

The effect of a short pre-treatment with a GnRH antagonist (Cetrorelix acetate) and the induced superovulation response was evaluated in goats. A total of 16 nanny goats were allocated to two groups. The goats in the antagonist treatment group (n = 8) received 6 doses of the GnRH antagonist, once daily, as a pre-treatment to superovulation. The does in the control group were provided with saline only. Natural mating was implemented using three fertile bucks. Large follicles on the ovaries were detected via ultrasound at 3.5 MHz. All goats were laparotomized and the CL's and cystic follicles visually recorded. Embryos were recovered via flushing and recorded 6 days after sponge removal. The number of large follicles induced and embryos produced were significantly increased by treatment. However, no differences were noted regarding the numbers of cystic follicles, indicating a lack of negative side-effects following the use of GnRH. In general, the number of large follicles and embryos were significantly (P < 0.05) higher in the treated group. It was demonstrated that a GnRH antagonist treatment can improve the superovulatory response by 64% and embryo recovery rate by 90% in goats.     

Pattern and manipulation of follicular development in Bos indicus cattle

Bos indicus cattle are widespread in tropical regions due to their adaptation to these environments. Although data on reproductive performance have indicated both inferior and superior results for B. indicus cattle, there is little doubt that B. indicus cattle are superior than Bos taurus cattle when they are both kept in tropical or subtropical environments, where stressors like hot temperatures, humidity, ectoparasites and low quality forages are greater. Reproductive endocrinology and oestrus behaviour of the B. indicus cattle have been studied for over 30 years; however, the application of technologies such as real time ultrasonography and Heat-Watch systems has expanded our knowledge on the ovarian follicular-wave dynamics during the oestrous cycle and the time of ovulation. Ovarian follicular dynamics in B. indicus cattle is characterised by the occurrence of two, three or sometimes four waves of follicular development. While dominance is similar to that in B. taurus cattle, maximum diameters of the dominant follicle and CL are smaller than those reported in B. taurus and are probably due to a lower capacity for LH secretion than in B. taurus. Duration of oestrus is approximately 10 h and the interval from oestrus to ovulation is about 27 h. However, the variability in response to prostaglandin F2alpha (PGF) treatments and the difficulty for oestrus detection in B. indicus cattle have limited the widespread application of artificial insemination (AI) and emphasizes the need for treatments that control follicular development and ovulation. Follicular-wave development in B. indicus cattle can be controlled mechanically by ultrasound-guided follicle ablation, or hormonally by treatments with GnRH or oestradiol and progestogen/progesterone in combination. Treatments with GnRH plus PGF and a second GnRH (synchronization protocol known as Ovsynch) or oestradiol benzoate (known as GPE) have resulted in acceptable pregnancy rates after fixed-time AI (FTAI) in cycling cows, but results were lower in heifers and cows in postpartum anoestrus. Alternatively, treatments with oestradiol and progestogen/progesterone releasing devices resulted in synchronous emergence of a new follicular wave, and a second oestradiol or GnRH treatment after device removal resulted in synchronous ovulation and acceptable pregnancy rates to FTAI. Furthermore, oestradiol and progesterone treatments combined with eCG (given at the time of device removal) increased pregnancy rates in suckled B. indicus cows and may be useful for the treatment of cows in postpartum anoestrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in B. indicus cattle by offering the possibility of planning AI programs without the necessity of oestrus detection and without sacrificing the overall results.     

Does exogenous progesterone and oestradiol treatment from the mid-luteal phase induce follicular cysts in goats?

The purpose of the present study was to investigate the effects of exogenous ovarian steroid treatment, which is known to induce follicular cyst experimentally in cows, on ovarian activity in goats. Eleven female Shiba goats with the length of the normal oestrous cycle (approximately 21 days) received subcutaneously either 1 ml of ethanol (control group, n=4) or 4 mg of progesterone and 2mg of oestradiol (treatment group, n=7) daily for 7 days beginning on day 14 of the oestrous cycle (day 0=ovulation). Ultrasonographic images of the ovary and blood samples were collected daily to monitor the ovarian activity. Ovulation was observed before 1 day after the end of treatment in the control group. In the treatment group, no detectable structures of follicles or corpus luteum (static ovarian condition) were found for 6.0+/-1.4 days (mean+/-S.D.) after the end of treatment. Then, detectable follicles appeared and ovulation was observed in all animals of the treatment group. There was no significant difference in the maximum diameter of the ovulatory follicle between the control and treatment group (4.7+/-0.4mm versus 5.1+/-0.7 mm). The large non-ovulatory follicles, which grew more than 10mm in diameter were observed after the static ovarian condition in one goat of the treatment group, whereas no turnover of the cystic follicular structures was found. The length of the inter-ovulatory intervals in the treatment group was significantly longer than that in the control group (38.4+/-7.4 days versus 20.3+/-0.5 days, P<0.05). The present results demonstrated that the exogenous treatment of progesterone and oestradiol, which was adapted from the follicular cyst model in cows, did not induce follicular cysts in goats, suggesting that there is/are different mechanism(s) mediating the occurrence of follicular cysts between cows and goats.     

Two days of pulsatile GnRH infusion beginning 4 days before weaning in sows initiates a wave of follicular growth that is not sustained after weaning

Intervals to estrus and ovulation in weaned sows depend partially on the diameter of ovarian follicles at weaning. The objective was to determine if follicular diameter in sows could be increased by a 48h period of GnRH infusion before weaning and whether this pre-weaning growth would advance follicular development after weaning. The posterior vena cava was cannulated in eight sows at 10+/-1 day after farrowing. Sows were randomly assigned to receive intravenous treatment with either 2mL of GnRH (1microg/mL; n=4) or 2mL of saline (n=4) every 0.5h for 48h beginning 94h before weaning. The average follicular diameter and the number of follicles within diameter classes were determined daily by ultrasonography. Serum LH concentrations increased on the first infusion day but serum LH was equal to control on the last infusion day (P<0.077). The GnRH infusion increased the average diameter of ovarian follicles (P<0.001). Serum estradiol increased (P<0.001) and serum FSH decreased (P<0.016) coincident with GnRH-induced follicular development but these changes were reversed within 24h after the end of the infusion period. Follicles that grew in response to GnRH regressed and were replaced by a new population of follicles within 4 days after weaning. Within the experimental model for the present study, a GnRH infusion increased follicular growth in lactating sows but follicles could not be sustained beyond the end of GnRH infusion.     

Ovarian follicular response to different hormonal stimulation treatments in Canindé goats

The objective of the present study was to evaluate the effect of different hormonal stimulation treatments on the antral follicular population of naturalized Canindé goats. Adult goats (n=17) having estrous cycles at regular intervals were treated with intra-vaginal sponges containing 60 mg medroxyprogesterone acetate for 11 days, combined with an application of 50 μg d-cloprostenol on the Day 8 of treatment. For ovarian stimulation, goats were distributed into the following experimental groups: (a) multiple doses (MD), with a total of 120 mg NIH-FSH P1 in five intramuscular injections (30/30; 20/20 and 20 mg) at 12-h intervals; (b) three doses (TD), with a total of 120 mg NIH-FSH P1 in three intramuscular injections (60; 40 and 20 mg) at 24 h intervals; (c) one dose (OD), which consisted of the use of 70 mg NIH-FSH P1 combined with 200 IU eCG administered intramuscularly 36 h before sponge removal. In the MD and TD groups, FSH injections were begun on the Day 8 of progestagen treatment. The ovaries of all animals were observed by transrectal real time ultrasonography (TRU) during the follicular stimulation protocols. All follicles ≥2 mm were counted, measured and classified according to greatest diameter. The ultrasonographic assessment of the ovaries provided for well-defined ovarian structures. At the time of insertion of the sponges (Day 0), significant differences were observed (P<0.05) for the mean number of large follicles between the treated groups. Meanwhile, on Day 11, the three treatments did not differ (P<0.05), regardless of the follicular category. The diameter of small follicles was similar in MD, TD and OD during the whole period of the study. In the TD group, diameter of the large follicles was less (P<0.05) on Day 10 when compared to MD and OD. However, these differences were not observed on Day 11. In conclusion, the three treatments produced comparable distribution of the follicular populations. However, the single dose treatment can be preferred because of its simplicity and efficacious follicular response.     

A new method for controlling the precise time of occurrence of the preovulatory gonadotropin surge in superovulated goats

In goats treated to induce superovulation, insemination at a predetermined time after the end of progestagen treatment leads to a low fertilization rate. To solve this problem we developed a new treatment based on the control of the occurrence of the endogenous LH peak with a GnRH antagonist (Antarelix). The first experiment was designed to determine the dose of LH required to mimic a spontaneous LH preovulatory discharge; the injection of 3 mg, i.v. of pLH induced a peak of the same amplitude and duration as the spontaneous peak. Subsequently, in the second experiment, we compared 2 doses of Antarelix (0.5 and 1 mg, sc) administered 12 h after sponge removal (9 goats/treatment group). The dose of 0.5 mg was selected for further experiments because it was effective in the inhibition of the endogenous LH peak and had no detrimental effect on the quality of embryos. In the final experiment, 48 goats received the new treatment and were inseminated (intrauterine) only once 16 h after LH injection; 41 were flushed and produced 5.3 +/- 4.5 (m +/- SD) transferable embryos. The developmental stage and the number of cells/embryo were within the range that has been reported for embryos produced with conventional treatments. In conclusion, with the described method, it is possible to inseminate goats at a predetermined time without decreasing the number of transferable embryos. This technique will encourage the development of embryo transfer within genetic programs, and it will be a valuable tool for the production of zygotes for gene transfer.     

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)     

Effect of maternal treatment with altrenogest on age at puberty, hormone concentrations, pituitary response to exogenous GnRH, oestrous cycle characteristics and fertility of fillies

Puberty was studied using 15 fillies of Quarter Horse phenotype. Fillies were from dams treated daily from Days 20 to 325 of gestation with: (1) 2 ml neobee oil per 50 kg body weight (controls); or (2) 2 ml altrenogest (2.2 mg/ml) per 50 kg body weight. The clitoris was measured at birth and approximately every 12 weeks until 84 weeks of age. Blood samples were collected from 9 fillies (5 treated, 4 controls) every 4 days over a 28-day period at 8-week intervals from 4 to 68 weeks of age; sampling continued every 4 days after 72 weeks of age until first oestrus. Blood samples were collected daily during oestrus (greater than or equal to 35 mm follicle) and on Days 4, 6, 10, and 14 after ovulation for the first 2 oestrous cycles. GnRH challenges (5 micrograms/kg) were administered every 8 weeks from 32 to 96 weeks of age. Puberty was defined as the first oestrus with ovulation. Beginning 1 February 1987, fillies were teased daily and their ovaries were examined by ultrasonography every 3 days (daily during oestrus). Fillies were inseminated with 500 x 10(6) motile spermatozoa from one stallion. Pregnancy was diagnosed by ultrasonography on Days 11, 12, 15 and every 5 days until Day 50 after ovulation. Prenatal altrenogest treatment caused clitoral enlargement (P less than 0.05) and increased serum concentrations of LH from 1 to 7 months of age. The amount of LH released in response to exogenous GnRH was greater (P less than 0.05) in treated fillies at 32, 64, and 72 weeks of age. Treated fillies had higher serum concentrations of FSH from 1 to 4 months (P less than 0.05), but FSH was lower (P less than 0.05) in treated fillies before and during first oestrus. Serum concentrations of LH and FSH peaked transiently at 10 months and LH was depressed from 64 to 88 weeks and began to rise 14 days before first oestrus. Concentrations of FSH began to decline 14 days before first oestrus. The median age at puberty was 90 weeks. Durations of oestrus, dioestrus, and the oestrous cycle were not different between groups and were similar to those for adult mares. First cycle pregnancy rates and overall rates were 100 and 82% and 100 and 91.7% for control and treated fillies, respectively (P greater than 0.05). Maternal treatment with altrenogest did alter gonadotrophin secretion before puberty, but had no effect on functional reproductive performance in fillies.     

In vivo and in vitro studies of the role of the adrenergic system and follicular wall contractility in the pathogenesis and resolution of bovine follicular cysts

Bovine follicular cysts (FCs) are a common cause of economic loss in modern dairy herds. Their aetiopathogenesis is not completely understood, even though an inadequate hypothalamic release of GnRH at the time of ovulation is considered to be their main cause. Much evidence, however, suggests a role for adrenergic innervation in ovarian functions, such as follicular development, steroid hormone secretion, and follicular contractility, the latter being an event important for ovulation. Moreover, in humans, polycystic ovary syndrome, a disease very similar to bovine follicular cysts, is characterised by increased density of adrenergic nerves. Given these premises, the aim of our study was to evaluate the effectiveness and mode of action of a novel strategy for the treatment of bovine follicular cysts. In the in vivo experiment, 170 Friesian cows diagnosed with follicular cysts were assigned to four groups (groups A, B, C, and D) to assess the effects of epidural administration of a β-adrenergic antagonist (carazolol) alone or in combination with a GnRH analogue (lecirelin). The four groups underwent the following treatments: Group A was administered lecirelin in combination with carazolol; Group B was administered carazolol; Group C was administered lecirelin; and Group D was administered only normal saline solution. In the in vitro experiment, strips of the walls of cystic follicles recovered post-mortem were suspended in an organ bath, connected to an isometric force transducer and exposed to increasing doses of epinephrine or to the same treatment after exposure to carazolol for 15 min (n = 10). The amplitude and frequency of the contractile activity were recorded. None of the control cows was observed in oestrus or was submitted to AI. The combination of lecirelin and carazolol induced a significant increase in the number of cows in oestrus (88%) compared to lecirelin alone or to carazolol alone (P < 0.05 and P < 0.01, respectively). The combination of lecirelin and carazolol and lecirelin alone were significantly more efficacious than carazolol alone (P < 0.01 and P < 0.05, respectively). In the in vitro experiment, epinephrine increased the amplitude of the contractions of the strips in a dose-dependent manner. This response was significantly enhanced in strips pre-treated with carazolol. The treatments had no effect on the frequency of contractions. In conclusion, our work demonstrates that the epidural administration of a GnRH analogue and a β-adrenergic blocker is an effective means of treating cows with cystic ovarian disease. Moreover, it confirms, from a clinical point of view, that alterations of the adrenergic system and of the contractility of the follicular wall can be considered aetiopathogenic factors involved in the development of FCs. The results of this study lay the basis for a new therapeutic approach to FCs.     

Effect of GnRH injection timing in the production of pronuclear-stage zygotes used for DNA microinjection

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE and 47 adult standard goats (1-5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 microg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 microg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p < 0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p = 0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.