Delayed treatment with GnRH agonist, hCG and progesterone and reduced embryonic mortality in buffaloes

The present study examined the effect of delayed treatment with tropic hormones and progesterone (P₄) on embryonic mortality in buffaloes. Buffaloes with a conceptus on Day 25 after AI were assigned to the following treatments: Control (n = 41), i.m. physiological saline; GnRH agonist (n = 36), i.m. 12 μg buserelin acetate; hCG (n = 33), i.m. 1500 IU hCG; P₄ (n = 38), i.m. 341 mg P₄ every 4 days on three occasions. Control buffaloes had an embryonic mortality of 41.4% (17/41) between Days 25 and 45, and this was reduced (P < 0.01) by treatment with GnRH agonist (11.1%, 4/36), hCG (9.0%, 3/33) and P₄ (13.1%, 5/38). On Day 45, buffaloes treated with hCG and which ovulated had greater (P < 0.05) concentrations of P₄ in whey (453 ± 41 pg/ml) than buffaloes in the same treatment that did not ovulate (297 ± 32 pg/ml). A similar but non-significant trend was observed for buffaloes treated with GnRH agonist. It was concluded from the findings that the treatment of buffaloes on Day 25 after AI with tropic hormones or P₄ is beneficial to processes associated with embryonic implantation.     

Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone

The effect of treatment with a GnRH agonist, hCG or progesterone (P(4)) on corpus luteum function and embryonic mortality was investigated in buffaloes inseminated during mid-winter. Italian Mediterranean buffaloes (n=309) were synchronized using the Ovsynch with timed-AI program and mated by AI at 16 h (Day 0) and 40 h after the second injection of GnRH. On Day 5, buffaloes were randomly assigned to four groups: Control (no treatment, n=69), GnRH agonist (buserelin acetate, 12.6 microg, n=73), hCG (1500 IU, n=75) and P(4) (PRID without E(2) for 10 days, n=77). Progesterone (pg/ml) was determined in milk whey on Days 5, 10, 15 and 20 and pregnancy diagnosis was undertaken on Day 26 by ultrasound and Day 40 by rectal palpation. Treatment with buserelin and hCG increased (p<0.05) P(4) on Day 15 compared with controls (456+/-27, 451+/-24 and 346+/-28 pg/ml, respectively). Buffaloes treated with a PRID had intermediate P(4) concentrations (380+/-23 pg/ml). Embryonic mortality between Days 26 and 40 (22.9%) and pregnancies at Day 40 (48.9%) did not differ between treatments. A higher (p<0.01) P(4) concentration was found on Day 20 in pregnant animals compared with non-pregnant and embryonic mortality buffaloes, which did not differ. In summary, buserelin and hCG increased P(4) concentrations on Day 15 but this was not associated with a reduced incidence of embryonic mortality in buffaloes during mid-winter.     

Pregnancy rates, LH and progesterone concentrations in mares treated with a GnRH agonist

The aim of the study was to investigate the effect of the GnRH agonist Buserelin given on day 10 after ovulation on pregnancy rate and concentrations of progesterone and LH. Altogether 191 warmblood mares were used for two trials. Fresh or frozen/thawed semen from 27 stallions was used for A.I. In trial A 171 mares received either Buserelin (Receptal, Hoechst, Germany, 40 microg/animal) or 10 ml 0.9% NaCl (placebo). On day 16 after A.I. pregnancy diagnosis was performed by ultrasound scanning of the uterus. For statistical analysis, data were analyzed by a mixed model, with four fixed factors (treatment, type of spermatozoa, A.I. number, reproductive status of the mare) and a random factor (stallion). Least Square Means (LSM) for pregnancy rate were 46.0% in GnRH agonist treated mares and 36.4% in the control group (P=0.22). In trial B 20 lactating and cycling mares were used for endocrine studies. Blood samples were recovered for analyses of progesterone and LH from days 0 to 11. The mean progesterone concentrations increased continuously from days 0 to 8 after ovulation in both groups (GnRH group: from 0.81+/-0.48 to 5.47+/-0.48 ng/ml, control group: from 0.63+/-0.68 to 5.83+/-0.68 ng/ml). Moreover, the progesterone concentrations from days 9 to 11 were not different between the GnRH and the control group. In contrast to this LH concentrations were markedly influenced by the GnRH agonist. On day 10 LH concentrations were significantly higher in GnRH agonist treated than in placebo treated animals. From the data obtained from individual animals it can be concluded that GnRH agonist, given during luteal phase may have different effect on luteal function.     

Inhibition of hCG, alpha hCG and progesterone release from human placental tissue in vitro by a GnRH antagonist

A dramatic suppression of hCG, alpha hCG and progesterone release from midgestation, human placentas in vitro was effected when incubated with 1 microgram/ml of an antagonist to GnRH. This inhibition of hormonal release occurred rapidly and was partially restored by the addition of GnRH. Human chorionic somatomammotropin was also suppressed, but only two days following the decline of the other hormones. These data demonstrate that an antagonist to GnRH can rapidly inhibit human placental hormone release.     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

Ovulation and fertility after PRID,PRID + GnRH and GnRH in anestrous buffaloes

Two experiments, one in the winter (January) and the other in the summer (June), were undertaken to treat anestrous buffaloes at a farm. During experiment I, animals were treated with a progesterone releasing intra-vaginal device (PRID)^a, whereas during experiment II, animals were allocated to PRID + GnRH^b (125 μg) and GnRH-treated (250 μg). In both the experiments, animals were retained as untreated controls. All the treated animals were clinically examined four days post-treatment and inseminated two or three times at 24-hour intervals. The ovulation and fertility rates of PRID, PRID + GnRH and GnRH-treated animals were 25 and 12 %, 43 and 14 %, and 11 and 0 %, respectively. None of the controls exhibited estrus during the course of the study.     

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.     

Progesterone and follicular changes in postpartum noncyclic dairy cows after treatment with progesterone and estradiol or with progesterone, GnRH, PGF2alpha, and estradiol

A previous study showed that noncyclic dairy cows treated with 10 microg of GnRH and a progesterone-releasing CIDR insert on Day 0, 25 mg of PGF2alpha and CIDR removal on Day 7, followed by 1 mg estradiol benzoate on Day 9 for those cows that still had not shown estrus (CGPE program) had higher conception rate (47% vs. 29%) than cows treated only with CIDR and estradiol benzoate as above (CE program). This study was to investigate the mechanisms by which the CGPE program improved conception rate compared with the CE program. Sixteen noncyclic Holstein-Friesian cows were randomly assigned to 2 groups balanced for the size and growth pattern of the dominant follicles, which were determined by ultrasonography over a 3-d period. One group received the above CGPE treatment, and the other group received the CE treatment. Follicular and luteal development were monitored by daily ultrasonography. Blood samples were collected daily from Day -2 to Day 11, and thereafter milk samples were collected thrice weekly for a further 24 d. Blood and milk samples were analyzed for progesterone. The GnRH treatment induced ovulation in 7 of 8 cows, resulting in elevated (P<0.05) progesterone concentrations between Days 4 and 7 for cows in the CGPE group. All induced CL underwent luteolysis by 24 h after PGF2alpha treatment. Within 5 d of CIDR removal, 7 of 8 cows in both the CE and CGPE groups ovulated. The interval from emergence of the ovulatory follicle to ovulation was similar (P=0.32) but less (P<0.05) variable for the CGPE group (9.0+/-0.3 d) compared with the CE group (10.3+/-1.2 d). Progesterone concentration in milk samples was similar between the two groups up to 10 d after ovulation. In summary, the GnRH treatment induced ovulation or turnover of dominant follicles, induced a synchronized initiation of a new follicular wave, and increased the progesterone concentration from 4 d after treatment. These could be the reasons for the increased conception rate of cows treated with the CGPE program.     

Pituitary and ovarian responses of post-partum acyclic beef cows to continuous long-term GnRH and GnRH agonist treatment

Post-partum acyclic beef cows received continuous long-term treatment with GnRH (200 or 400 ng/kg body wt/h) or the GnRH agonist buserelin (5.5 or 11 ng/kg body wt/h) using s.c. osmotic minipumps which were designed to remain active for 28 days. All treatments increased circulating LH concentrations whereas FSH remained unchanged. Ovulation and corpus luteum (CL) formation as judged by progesterone concentrations greater than or equal to 1 ng/ml occurred in 0/5 control, 4/5 200 ng GnRH, 4/4 400 ng GnRH, 4/5 5.5 ng buserelin and 3/5 11 ng buserelin cows. The outstanding features of the progesterone profiles were the synchrony, both within and across groups, in values greater than or equal to 1 ng/ml around Day 6, and the fact that most CL were short-lived (4-6 days). Only 3 cows, one each from the 400 ng GnRH, 5.5 ng buserelin and 11 ng buserelin groups, showed evidence of extended CL function. Cows failed to show a second ovulation which was anticipated around Day 10 and this could have been due to insufficient FSH to stimulate early follicular development, or the absence of an endogenously driven LH surge. The highest LH concentrations for the respective groups were observed on Days 2 and 6 and by Day 10 LH was declining, although concentrations did remain higher than in controls up to Day 20.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of hCG and GnRH for inducing ovulation in the jenny

Knowledge about management of ovulation in the donkey is limited compared to that in the horse. This experiment was designed to evaluate the efficacy of injecting single doses of lecirelin (a GnRH-analogue) or of hCG to induce ovulation in the jenny and to determine whether effects are dependent upon follicular diameter at time of injection. Ovarian activity and follicular growth were monitored by rectal ultrasonography. Jennies were randomly allotted to the following groups: Group GnRH, treated with 100 microg lecirelin; Group hCG, treated with 2500 IU hCG; Group C, untreated and monitored for spontaneous ovulation. Animals were also categorized into subgroups depending upon follicular diameter: 30-35 mm (GnRH-1, hCG-1 and C-1) or 36-40 mm (GnRH-2, hCG-2 and C-2). Jennies in the two hormone treatment groups did not differ significantly for time from treatment to ovulation, but there was a significant reduction in time to ovulation as follicle size at treatment increased. Jennies treated with either lecirelin or hCG had significantly smaller follicle size at ovulation than jennies in the Control groups that underwent spontaneous ovulation. Treatment groups did not differ significantly in the proportion of jennies that ovulated within 48 h of injection or between 25 and 48 h following injection. These results highlight the usefulness of lecirelin for induction and synchronization of ovulation in the jenny, particularly since it would avoid the risk of reduced hCG response in reproductive management programs in which that hormone was repeatedly used.     

Prenatal testosterone treatment alters LH and testosterone responsiveness to GnRH agonist in male sheep

Although evidence is accumulating that prenatal testosterone (T) compromises reproductive function in the female, the effects of excess T in utero on the postnatal development of male reproductive function has not been studied. The aim of this study was to assess the influence of prenatal T excess on age-related changes in pituitary and gonadal responsiveness to GnRH in the male sheep. We used the GnRH agonist, leuprolide (10 microg/kg), as a pharmacologic challenge at 5, 10, 20 and 30 weeks of age. These time points correspond to early and late juvenile periods and the prepubertal and postpubertal periods of sexual development, respectively. LH and T were measured in blood samples collected before and after GnRH agonist administration. The area under the response curve (AUC) of LH increased progressively in both controls and prenatal T-treated males from 5 to 20 weeks of age (P<0.01). The LH responses in prenatal T-treated males were lower at 20 and 30 weeks of age compared to controls (P<0.05). AUC-T increased progressively in control males from 5 through 30 weeks of age and prenatal T-treated males from 5 to 20 weeks of age. The T response in prenatal T-treated males was higher at 20 weeks compared to controls of same age but similar to controls and prenatal T-treated males at 30 weeks of age (P <0.05). Our findings suggest that prenatal T treatment advances the developmental trajectory of gonadal responsiveness to GnRH in male offspring.     

Endocrine, morphological, and cytological effects of a depot GnRH agonist in bovine

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.     

Regulation of LH beta subunit mRNA in immature female rats during GnRH agonist treatment

In order to determine the changes in the expression of LH beta messenger ribonucleic acid (mRNA) during GnRH agonist (GnRHa) treatment (0.94 mg/28 days), the concentration of the mRNA of LH beta was assessed together with the serum LH concentration, pituitary LH content and LH response to GnRH at various times during long-acting GnRHa treatment in immature female rats. The serum LH concentration was increased at hour 1, gradually decreased starting at approximately hour 3 and had returned to the control level on day 28. Pituitary LH began to decrease at hour 3. The concentrations of LH beta mRNA were not significantly different from those in the control group from hour 1 to hour 18, but were lower from day 3 to day 28. Serum LH response to native GnRH (1 micrograms) began to be inhibited on day 7. These results indicate that the short term treatment with GnRHa stimulates the release of preformed LH rather than synthesis of LH beta mRNA and that the long term treatment inhibited the expression of LH beta mRNA in a time dependent manner.     

Follicular Development and Ovulation in Sows: Effect of hCG and GnRH Treatment

Follicular growth, chronology of ovulation and embryo morphology were compared in sows ovulating spontaneously and sows, in which the ovulation was attempted induced by hCG or GnRH. Indwelling catheters were placed on day 1 (weaning = day 0) in the ear veins of 18 sows, which were then randomly divided into 3 groups: a control group (N = 6), a group (N = 6) given 750 iu hCG (Physex®) im 76h after weaning (hCG group) and a group (N = 6) given 500 µg GnRH (Fertagyl®) im 76h (N = 3) or 100h after weaning (N = 3) (GnRH group). Follicular diameter and time of ovulation were monitored by ultrasonography every 4h from day 3 until ovulation or development of cysts by means of a sector scanner fitted with a 5.0/7.5 MHz multiangle probe. Heat detection was performed every 8h from day 3 until ovulation. On day 13, the sows were slaughtered, the number of corpora luteae (CL) was counted, and embryos were flushed from the uteri. The control group showed clear heat symptoms, and on day 3, the follicles were typically 3–7 mm and grew up to 7–10 mm over 2 days, where they remained for approximately 24h until ovulation took place 41h ± 9h after first sign of standing heat. The hCG group exhibited no signs of heat, and the follicles only reached 5–8 mm in diameter at time of ovulation, which occurred 40h ± lh after hCG-injection. The GnRH group exhibited inconsistent signs of heat, and the follicles reached a maximum size of 7–12 mm in diameter where they remained for more than 24h. Only 2 sows in this group ovulated within 84–92h after the GnRH injection, and development of bursa cysts and cystic follicles was a common finding. The average number of CL was 18.2 ±5.7 per sow (N = 16, range: 3–27) with no significant difference between the groups. Total embryo recovery was 79 ± 13 % with no significant difference between groups. The embryo diversity calculated as standard deviation of the maximum diameter was higher in the hCG group as compared with the control group. It is concluded that (1) transrectal ultrasonography can be used in sows for accurate assessment of follicular growth and ovulation; (2) the use of hCG results in lack of heat symptoms and reduced follicle size at the time of ovulation when injected 76h after weaning; (3) administration of a single injection of GnRH, if given before the first signs of heat, results in inconsistent heat symptoms and no or late ovulations.     

Neutralization of pregnancy-preventing activity of cabergoline, a dopamine agonist with progesterone

Cabergoline (Cab), a dopamine agonist, inhibits the production and/or release of prolactin and reduces progesterone biosynthesis. Cab at doses of 20 microg/kg/d and higher administered on Days 1, 2 and 3 of pregnancy prevented implantation of fertilized ova in all treated female rats. The pregnancy-preventing activity of Cab is attributed to a deficiency of progesterone since estradiol levels remain unchanged. To validate the hypothesis that progesterone deficiency is the cause of the infertility, exogenous progesterone was administered to Cab-treated pregnant female rats and their fertility assessed. Progesterone at a dose of 5 mg/kg/d sc was administered on Days 1 to 7 of pregnancy to female rats treated with Cab (20 and 60 microg/kg/d) on Days 1, 2 and 3 of pregnancy. All progesterone-treated animals became pregnant, and all mated female rats treated with Cab (60 microg/kg/d) and progesterone (5 mg/kg/d) on Days 1 to 7 inclusive became pregnant. In conclusion, progesterone can neutralize the pregnancy preventing activity of Cab. Pregnant female rats treated with Cab can be used to assay for progestagenic activity of compounds.     

Effects of a single injection of hCG or GnRH agonist on day 12 post mating on fetal growth and reproductive performance of sheep

Reproductive performance and fetal growth were determined when hCG (150 i.u. Pregnyl; n=44), GnRH (4 microg synthetic GnRH agonist, buserelin, Receptal; n=43) or saline (control, n=45) was administered (i.m.) to ewes on day 12 post mating during the breeding season. A total of 12 ewes was slaughtered on day 45 of pregnancy (four from each treatment group). Non-return rate and lambing rate were higher for ewes in the hCG (0.89 and 84%) and GnRH treated groups (0.86 and 79%) than for ewes in the control (0.69 and 62%) group (P<0.05). The ewes in the hCG and GnRH groups also had more twins (P<0.05). Birth weights of these twin lambs in the hCG and GnRH groups were heavier than those in the control group (P<0.05), but this difference had disappeared at weaning 60 days later. Lamb mortality was similar among treatment groups resulting in a higher number of lambs weaned in the hCG and GnRH groups. The ovarian weights and the number of corpora lutea (CL) and luteal weights of ewes slaughtered on day 45 of pregnancy were greater (P<0.05) in the hCG and GnRH treated groups than those measured in the control group. Administration of hCG on day 12 post mating increased gravid uterus weight, crown-rump-length (CRL), chorioallantois weight and total cotyledon weight (P<0.05) of conceptuses recovered on day 45 of pregnancy compared to the control group. The weights of caruncules (P<0.05) and placenta (P<0.01) were higher in conceptuses of both the hCG and GnRH groups. The weights of fetuses in the hCG group were higher than those in both the GnRH and control groups (P<0.05). In conclusion, both hCG and GnRH administration improved reproductive performance of ewes when administered on day 12 post mating. However, hCG and GnRH appeared to act differently on embryo survival because only hCG administration increased fetal growth.     

Studies on GnRH agonist suppression of estrogen production in patients with endometriosis

The chronic administration of GnRH agonists to women results in the reversible suppression of estrogen production by the ovary. In the present study, the mechanism of the GnRH agonist suppression of estrogen production was investigated in patients with endometriosis. During the treatment with intranasal buserelin spray, the concentration of serum estradiol-17 beta (E2) was suppressed to near-castrate levels. Despite this marked suppression of serum E2, immunoreactive LH and FSH levels in serum were not changed. On the other hand, serum bioactive LH was markedly reduced. It was also observed during the treatment that the pituitary LH pulse disappeared and pituitary response to exogenous GnRH was significantly suppressed. In contrast, ovarian response to human menopausal gonadotropin (hMG) was not altered during the treatment. These findings suggest that the GnRH agonist suppression of estrogen production in the patients with endometriosis is through both suppression of the secretion of biologically active LH and the reduction of the LH pulse, but not through a direct inhibitory effect on ovarian estrogen biosynthesis.     

Induction of fertile oestrus in the bitch using Deslorelin, a GnRH agonist

Oestrus induction in various canine breeds was attempted in 32 bitches. A group of 8 bitches were treated 80–160 d following their previous oestrus (G1) whereas a second group of 24 bitches (G2) were implanted 200–590 d following their previous oestrus. The treatment for each bitch consisted in one Deslorelin implant (Suprelorin®4,7mg, Virbac, France), inserted subcutaneously in the post-umbilical region. Ovulation, pregnancy rate and litter size were recorded. All bitches came in heat 4.3 ± 1.4 d after implantation (2–7 d). Ovulation was reported in 62.5% in G1 and 87.5% in G2. One bitch refused mating and since no AI was performed, she was not considered for further analysis. Pregnancy was obtained in 25% in G1 versus 78.3% in G2. Mean litter size was 6.7 ± 3.5 puppies (1–14). Luteal failure was suspected in 3 bitches, two that remained non-pregnant and one which aborted 58 d post-ovulation since the owner refused progesterone supplementation. Deslorelin implants can therefore be considered as a valuable alternative to induce fertile oestrus in bitches in anoestrus. Follow-up of the luteal phase is recommended, since some bitches might encounter luteal failure.