Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The α-subunit possesses almost 3 times as much total carbohydrate as the β-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The α-subunit begins with the sequence NH₂-Phe-Pro (Gly or Pro) … and terminates with isoleucine. The β-subunit has the sequence NH₂-Ser-Pro-Gly …; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.     

Chemical and biological properties of the subunits of pregnant mare serum gonadotropin

The two subunits (α and β) of pregnant mare serum gonadotropin have been dissociated and partially characterized. Recombination of the biologically inactive subunits results in the restoration of both the follicle stimulating and leuteinizing activities of pregnant mare serum gonadotropin. In addition, the α subunit of pregnant mare serum gonadotropin can be combined with the β subunit of either ovine luteinizing hormone, human chorionic gonadotropin, or follicle stimulating hormone with generation of the specific activity expected of the β subunit.     

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits.

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The alpha-subunit possesses almost 3 times as much total carbohydrate as the beta-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The alpha-subunit begins with the sequence NH2-Phe-Pro (Gly or Pro) ... and terminates with isoleucine. The beta-subunit has the sequence NH2-Ser-Pro-Gly ...; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.     

Ovarian activity in progesterone treated mares following administration of pregnant mare serum gonadotropin

Twelve non-pregnant, non-lactating mares were randomly assigned to four treatment groups using a 2x2 factorial arrangement with three replicates per group. Mares were administered PGF(2alpha) (10 mg, IM) on days -14 and 0, followed by HCG (3000 IU, IM) on day 5. The following treatments were administered: Group A received PMSG on days 2 (4000 IU, IM) and 5 (1000 IU, IV); Group B received PMSG (4000 IU, IM) on day 2; Group C received PMSG (1000 IU, IV) on day; Group D received no PMSG. Mares received progesterone (25 mg, IM) on days 1 through 4. Reproductive tracts were recovered at necropsy on day 16 (10 days post-ovulation). Ovaries were weighed, CL number and weight determined, follicles counted and measured, and volume of follicular fluid quantified. Mean ovarian weight (g) and number of CL per mare, respectively were: Group A, 100.0+/-15.6, 1.7+/-.7; Group B, 128.6+/-40.4, 1.3+/-.7; Group C, 92.4+/-21.0, 2.0+/-.0; Group D, 93.3+/-12.3; .3+/-.3. Mean number of follicles >10 mm and total volume (ml) of follicular fluid per mare, respectively, were: Group A, 9.4+/-2.0, 21.8+/-10.9; Group B, 1.3+/-.3, 32.2+/-28.9; Group C, 4.3+/-1.8, 5.4+/-2.3; Group D, 6.0+/-4.5, 24.0+/-10.3. There was no difference (P>.05) in mean ovarian weight, CL number, CL weight, follicular fluid volume, number of follicles, or size of follicles between treatment groups. These results show no significant effect on ovarian activity in progesterone treated mares following administration of exogenous PMSG.     

Effect of long-term and short-term progestagen treatment on follicular development and pregnancy rate in cyclic ewes

The aim of this study was to evaluate the effect of the length of a progestagen treatment (12 d vs. 6 d) on follicular dynamics, estrus synchronization and pregnancy rate using medroxyprogesterone acetate (MAP) with or without an eCG dose at the end of MAP treatment. One hundred sixty Polwarth ewes were divided into four equal groups: long-term treated (LT, n=40); short-term treated (ST, n=40); long-term treated plus eCG (LTeCG, n=40); and short-term treated plus eCG (STeCG, n=40). Five ewes of each group were separated to undergo daily transrectal ultrasonography, and blood samples were taken for hormone determination. Until 96 h after sponge withdrawal the number of ewes in estrus was higher in both long-term-treated groups than in both short-term-treated groups but at the end of the observational period (144 h) no significant differences were found among groups. The pregnancy rate was higher in the ST group (87%) than in the other groups (LT, 63%; LTeCG, 67%; and STeCG, 58%; P< or =0.03). The ovulatory follicle emerged before sponge withdrawal in long-term-treated ewes (-3.8+/-0.4 d and -2.2+/-0.8 d for LT and LTeCG, respectively), whereas in short-term-treated ewes it emerges around sponge removal (0.4+/-1.1 d and 0.5+/-0.5 d for ST and STeCG, respectively; P< or =0.01). The ovulatory follicle in the LT group had a longer lifespan and attained a larger (P< or =0.05) maximum diameter than in the ST group. We conclude: a) that the lower pregnancy rate observed after long-term progestagen treatment was related to a slower follicular turnover that promoted the ovulation of persistent dominant follicles; (b) that short-term treatment resulted in a higher pregnancy rate probably due to the ovulation of newly recruited growing follicles; and (c) treatment with eCG had no advantage in association with long-term treatment and had a deleterious effect in combination with short-term treatment with MAP.     

Induced puberty in prepuberal zebu heifers treated with norgestomet and pregnant mare serum gonadotropin

Three experiments were conducted to examine the progestogen plus PMSG treatment for its effectiveness in inducing synchronous puberty in prepuberal zebu heifers in three different seasons. In Experiment 1, ten Ongole heifers (age 21 months) were treated with Norgestomet implants for nine days and an intramuscular injection of 400 IU of PMSG two days before implant removal. Ten heifers (age 25 months) were kept as untreated controls. Animals were inseminated 12 h after detection of cyclic estrus (not bred at induced estrus) until all animals conceived. The proportion of treated animals showing estrous, ovulatory, and cyclic activity were 100%, 75% and 25% respectively, while the average age at first conception was significantly less (P < 0.05) than in the control group. In Experiment 2, 18 Ongole heifers (age 22 months) were divided into treatment and control groups. Fixed-time inseminations were done 48 and 72 h after implant removal and 12 h after being detected in heat at other times. Estrus was seen in all while 63% became pregnant (P < 0.05). At the end of the 100-day experiment, the percent pregnant were 33 and 63 in the control and treatment groups, respectively. In the third study, twenty-six Ongole heifers (age 22 months) were assigned to treatment and control groups. Eighty-eight percent of the animals exhibited estrus, 75% ovulated (P < 0.01) and 25% conceived to fixed-time insemination. The pregnancy rate at the end of the experiment was 10 and 56% (P < 0.01) respectively in control and treated groups. Estrous response and fertility were better in the cooler month (February) and the treatment imposed in the hotter month (May) resulted in a significantly higher (P < 0.05) age and body weight at conception.     

Synchronization of estrus with short- and long-term progestagen treatments and the use of GnRH prior to short-term progestagen treatment in ewes

The objective of the present study was to determine the efficacy of the synchronization of estrus using short- and long-term progestagen treatments in ewes at the onset of the breeding season, and to evaluate the effect of the exogenous GnRH administration immediately prior to short-term progestagen treatment on the reproductive performance. A total of 240 Tahirova cross-bred ewes, aged 18–24 months, and 40 rams, aged 2–4 years-old, were used in the trial. Ewes were divided equally into 3 groups (n = 80 per group). Intravaginal progestagen sponges containing FGA (30 mg) were inserted in the ewes for 7 d in the FGA1 (short-term) and GnRH treatment groups, and for 12 d in the FGA2 group (long-term). The ewes in the GnRH group received 10.5 μg busereline acetate i.m. at the time of sponge insertion. Tiaprost tromethamol (PGF_2α; 0.294 mg) and eCG (400 IU) were injected i.m. on the 6th day of progestagen treatment in the GnRH and FGA1 groups, and on the 11th day in the FGA2 group following sponge insertion. All ewes were hand-mated once at the detection of estrus. The estrous response, fertility rate, multiple birth rate and litter size recorded was 88.7, 87.3, 51.6% and 1.6 in the FGA1 group, 92.5, 71.6, 50.9% and 1.5 in the FGA2 group, and 96.2, 89.6, 71.0% and 1.8 in the GnRH group, respectively. No significant difference in estrous response between the groups was recorded, but the fertility rate in the FGA1 and GnRH groups was significantly (P < 0.05) higher than in the FGA2 group. The occurrence of multiple births and litter sizes were significantly (P < 0.05) higher in the GnRH group, compared to both the FGA1 and FGA2 groups, with the number of single lambs being significantly (P < 0.05) higher in the FGA1 (48.4%) and FGA2 (49.0%) groups than in the GnRH (29.0%) group. However, the differences recorded between any of the groups in terms of the number of twin and triplet lambs were insignificant. In conclusion, it can be said that estrous synchronization using the 12-d-FGA-eCG-PGF_2α regimen could be replaced with the 7-d-FGA-eCG-PGF_2α regime in sheep at the onset of the breeding season. However, the combination of GnRH with the latter regimen (7-d-GnRH-FGA-eCG-PGF_2α) increased the multiple birth rate and litter size in the ewes.     

Effects of hydroxyflutamide on rats treated with a superovulatory dose of pregnant mare serum gonadotropin

Immature female rats treated with superovulatory doses of pregnant mare serum gonadotropin (PMSG) were used to study the effects of the antiandrogen hydroxyflutamide on steroid production, particularly the biologically active androgens, in two experiments. In the first experiment, animals were given either 5 mg hydroxyflutamide or vehicle alone at 30 and 36 h following 40 IU PMSG. Compared with the vehicle group, hydroxyflutamide treatment significantly reduced the percentage of degenerate oocytes recovered from oviducts (p less than 0.05). Serum levels of testosterone and androstenedione, and their aromatized product 17 beta-estradiol, significantly decreased (p less than 0.05) in the hydroxyflutamide-treated group; however, nonaromatizable androgen, 5 alpha-dihydrotestosterone, was not affected. In the second experiment, ovaries obtained 48 h after stimulation with 4 or 40 IU PMSG were incubated with and without hydroxyflutamide (10(-5) M) and (or) testosterone (10(-7) M) to study [4-14C]pregnenolone metabolism to major steroids. In 40 IU stimulated ovaries, hydroxyflutamide significantly decreased the metabolism of pregnenolone to progesterone (p less than 0.01) and androstenedione (p less than 0.01), while the production of 17 beta-estradiol increased significantly (p less than 0.05); however, pregnenolone conversions to testosterone and 5 alpha-dihydrotestosterone were not affected. Testosterone completely reversed the hydroxyflutamide-induced alteration of pregnenolone metabolism. In contrast, there was no difference in the pregnenolone conversion patterns between untreated and hydroxyflutamide or hydroxyflutamide plus testosterone groups in 4 IU stimulated ovaries. Present results confirm our previous finding that hydroxyflutamide decreases the percentage of abnormal oocytes recovered from superovulating rats and indicates that this hydroxyflutamide effect may be partly mediated by altered ovarian steroidogenesis following inhibition of androgen binding in the ovary.     

The effect of pregnant mare serum gonadotropin on mouse oocytes as monitored at metaphase II

Ovulated oocytes were collected from random-bred, 7-12 week old ICR mice injected with 0, 3, or 6 i.u. pregnant mare serum gonadotropin (PMSG). Analyses of 872 metaphase figures from 87 females did not show a significant increase in chromosomal imbalance with PMSG treatment. A tendency toward ovum fragmentation was noted with an increase in PMSG dose.     

Effect of ram exposure at the end of progestagen treatment on estrus synchronisation and fertility during the breeding season in ewes

Two experiments were conducted to examine the effects of ram exposure during the breeding season, in combination with progestagen treatment on estrus synchronization, fertility the LH surge and ovulation in ewes. Experiment 1 was subdivided into experiments 1a and 1b. In all experiments cross-bred ewes were treated with an intravaginal sponge for 12-14 days and three days before sponge withdrawal ewes were divided into control (no further treatment; n=191, 103 and 50 for experiments 1a, 1b and 2, respectively) or ram exposed (three mature rams per 50 ewes were introduced; +Ram; n=187, 99 and 49 for experiments 1a, 1b and 2, respectively). At sponge withdrawal ewes in Experiments 1a and 2 received 500 IU eCG and rams were removed from all the +Ram groups. In Experiments 1a and 1b, raddled, entire rams were introduced to ewes 48 h after sponge withdrawal. The timing of mating was recorded and ewes were maintained until lambing. In Experiment 2, estrus behavior was determined every 4 h and the time of the LH surge and ovulation were determined from a subset of 10 ewes per group. In Experiment 1a, less +Ram ewes were bred by 48 h after ram introduction (control 98% versus +Ram 89%, P<0.001) and in Experiments 1a and 1b 14% fewer (P<0.05) of the ewes bred in the first 3 h after ram introduction lambed to that service. In Experiment 1a, ram exposed ewes had a lower litter size than control ewes (1.93+/-0.06 versus 1.70+/-0.06 lambs per ewe; P<0.05). In Experiment 2, rams advanced (P<0.05) estrus, the LH surge and ovulation by 2-6 h compared with control ewes. We speculate that exposure of ewes to rams increased LH secretion and that this in turn increased follicle development and the production of oestradiol that led to a more rapid onset of estrus, the LH surge and ovulation compared to control ewes. Unexpectedly, ewes that were bred had lower fertility in the +Ram groups than control groups.     

A comparison between intravaginal progestagen and melatonin implant treatments on the reproductive efficiency of ewes

A total of 548 Manchega ewes were used to compare progestagen impregnated intravaginal sponges (12 days) plus eCG with melatonin implant (100 days) treatments, as possible stimulants of reproductive performance during the seasonal anoestrus and the lactation period. The use of both methods of oestrous manipulation induced a significant improvement in conception rate (78% for progestagen and eCG; 78% for melatonin versus 65% for the control) and fecundity (1.50 for progestagen and eCG; 1.55 for melatonin versus 1.26 for the control), when compared to the control group. Likewise, the lambing rate was also increased (1.17 for progestagen and eCG; 1.21 for melatonin versus 0.82 for control). A total of 35 and 39 extra lambs/100 ewes mated were produced, with an increase in productivity (percentage of extra lambs) of 43% and 48%, for ewes treated with progestagen sponges and melatonin, respectively, compared to the control group. The interval between ram introduction and parturition was significantly decreased, with matings in February–April (early seasonal anoestrus), compared to matings in March–May (seasonal anoestrus) in the group treated with progestagen sponges. An increase in the interval between parturition and the following mating was also recorded. As the body condition improved, the number of lambing ewes and fecundity were significantly improved in all groups. All the fertility results obtained with the progestagen sponges and melatonin treatments were similar. It could be concluded that progestagen sponges plus eCG and melatonin implants improved the reproductive performance of ewes, in seasonal anoestrus and during the lactation period, to a similar extent.     

Effect of exogenous melatonin and plane of nutrition after weaning on estrous activity, endocrine status and ovulation rate in Salz ewes lambing in the seasonal anestrus

Forty-nine Spanish Salz ewes lambing in the second fortnight of March (20 March +/- 1.5 d) were used to determine the effects of exogenous melatonin and postweaning nutrition on endocrine status, date of first estrus and ovulation rate. Experimental design was a factorial defined by 2 postweaning planes of nutrition, 1.80 (high) and 1.35 (low) times the maintenance requirements, and treatment with a single 18-mg subcutaneous implant of melatonin (M) 32 d after lambing or no treatment control (C). Mean weaning to first estrus interval was shorter in treated than in control ewes (50.8 +/- 4.2 vs 87.6 +/- 6.3 d; P < 0.01). Considering both the treated and control animals together, the ratio between mean night and daytime plasma melatonin levels was significantly correlated with the implant insertion-first estrus interval on Day 5 (0.67; P < 0.01) and Day 35 (0.63; P < 0.05) after implantation. Melatonin implants induced a significant increase of mean LH concentrations at Days 14 and 33 after implantation (P < 0.01) without any significant influence of plane of nutrition. Ovulation rate was higher for treated than control ewes in the second estrus (P < 0.05). An interaction between plane of nutrition and exogenous melatonin on ovulation rate at the second cycle after weaning was detected (P < 0.01), being close to the significance in the first, fourth and fifth cycles (P < 0.1). These results suggest that exogenous melatonin in April may be an effective way of advancing the breeding season and enhancing ovulation rate associated with a low rather than a high plane of nutrition.     

Effects of pregnant mare serum gonadotropin (eCG) on follicle development and granulosa-cell apoptosis in the pig

The effect of eCG on follicular development and granulosa-cell apoptosis in sexually mature and immature gilts and on granulosa-cell apoptosis in vitro were studied. The sexually mature gilts were treated with eCG on Day 11 of the estrous cycle, and effects were analyzed at different times after treatment with untreated animals at corresponding stages of the cycle as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), hematoxylin and eosin staining, and DNA ladder. The proportion of apoptotic cells in atretic follicles (39%) was significantly higher (P<0.01) than that in healthy follicles (9%). At 24h after eCG treatment in mature gilts, the total number of follicles visible on the ovarian surface (57 per ovary), the number of small (<3mm) follicles (31.5 per ovary) and the number of medium-sized (3-5mm) follicles (23 per ovary) were significantly higher (P<0.05) than those of control animals (28, 20 and 6.5 per ovary, respectively), and declined gradually thereafter to below the level of control animals. The number of large (>or=5mm) follicles began to show a marked increase at 72h after eCG (8.5 versus 2.5, P<0.05). At 24h after eCG treatment, the proportions of apoptotic cells in small (7.2%) and medium-sized follicles (7.4%) were markedly lower (P<0.01) than those in controls (21.5 and 21%, respectively) and increased gradually thereafter to approach the level in controls. The percentage of apoptotic cells in large follicles (10% at 24h post-eCG) did not change significantly. Before eCG treatment, there were markedly fewer follicles of all types on ovaries of immature gilts than of mature gilts (9 versus 25 per ovary) and the proportion of apoptotic cells in small and medium follicles was high (25 and 34%, respectively). After eCG treatment, the changes in follicle number and proportion of apoptotic cells in the immature gilts followed a similar pattern to that of the mature gilts. Equine chorion gonadotropin inhibited apoptosis of granulosa cells cultured either in vitro or in intact follicles in a dose-dependent manner. Thus, follicular atresia in the pig, as in other animals, was characterized by apoptosis of large numbers of granulosa cells, and eCG promoted follicular development by inhibition of granulosa-cell apoptosis.     

Ovulation of aged follicles does not affect embryo quality or fertility after a 14-day progestagen estrus synchronization protocol in ewes

The aim was to examine the effect of ovulation of aged follicles on embryo quality and fertility in ewes. In Experiment 1, ewes (n = 39) received a prostaglandin analogue on Day 6 of the cycle and then received either a progestagen sponge from Day 6 to 20 after estrus (Single sponge) or a progestagen sponge on Day 6 that was replaced on Day 11 and 16 and removed on Day 20 (Multiple sponges). In a subgroup of ewes, the growth of ovarian follicles was characterised using ultrasonography. Fertile rams were introduced 48 hours after sponge withdrawal; we slaughtered the ewes on Day 5 of pregnancy and recovered the embryos. The mean age of the ovulatory follicles was greater in ewes that received a single sponge compared with multiple sponges (8.7+/-0.8 days, range 4 to 14, versus 4.5+/-0.7 days, range 3 to 6; P<0.05). However, the groups did not differ (P>0.05) in ovulation rate (2.4+/-0.3 corporal lutea per ewe) or the proportion of good quality embryos recovered (71 to 82%; developed to the early morula stage or further). In Experiment 2, ewes (570 in total) received treatments similar to those in Experiment 1 but were kept until lambing. Ewes that received a single sponge came into heat earlier (P<0.05) than those that received multiple sponges, but > or = 97% of ewes in all groups (P>0.05) were bred by 48 to 72 hours after ram introduction. There was no difference (P>0.05) between groups for the proportion of ewes that lambed to first service (80 to 86%) or the number of lambs per ewe (1.94+/-0.08 lambs). We conclude that when luteolysis occurs at the beginning of progestagen synchronisation, ewes will ovulate aged follicles, but that compared to shorter duration follicles, these follicles produce oocytes that are equally competent to be fertilised and develop into good quality embryos and full-term lambs.     

Effect of type of intravaginal progestagen treatment of estrous response and reproductive performance of ewes

A comparison was made of the relative effectiveness of sponge pessaries impregnated with 40mg flourogestone acetate (FGA) or 60mg medroxyprogesterone acetate (MAP) to induce a synchronized estrus in ewes. Ewes were treated with sponge pessaries for 14 days and 500 IU pregnant mares' serum gonadotropin was injected i.m. at the time of sponge removal. The degree and pattern of mating response of ewes were similar, irrespective of the treatment used, approximately 92% of the ewes being marked by the ram by 72h after sponge removal. No significant differences in fertility or litter size were observed between the treatment groups. Ewes treated with FGA sponges had a fertility of 53% and litter size of 2.3 after mating at the synchronized estrus. The corresponding values for ewes treated with MAP sponges were 57% and 2.1. Use of MAP sponges was associated with a 17.8% sponge loss during treatment compared with 1% sponge loss in ewes treated with FGA sponges. Such losses could compromise the use of MAP sponges by reducing their overall efficacy.