In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

Superovulatory responses to eCG in llamas (Lama glama )

Llamas are copulation-induced single-ovulators, and multiple ovulation and embryo transfer (MOET) methods have not yet been developed for this species. Superovulatory responses to eCG given during an induced (Group A) or simulated (Group B) luteal phase were investigated using ultrasound to observe ovarian follicles and corpora lutea (CLs) and plasma progesterone was used to assess luteal function. Embryos were recovered nonsurgically. Group A (n = 19): donors were given 8 microg, im GnRH analogue (Day 0) to induce ovulation of a mature follicle, 1000 IU, im eCG (Day 7), and 250 microg PGF(2alpha) analogue (Day 9). Group B (n = 17): donors were given a subcutaneous progestagen implant (3 mg Norgestomet) at Days 0 to 7) and 1000 IU, im eCG (Day 5). When most (>65%) of the follicles in both Groups A and B had matured at 5 to 11 d post eCG, the donors were given 8 microg, im GnRH and mated once (n = 26) or twice within a 24-h interval (n = 10); embryos were recovered 6 to 9 d post ovulation. More follicles and corpora lutea were induced in Group B than in Group A, but a similar mean number of embryos were recovered (1.3 vs 1.6), and a similar proportion of donors yielded multiple embryos (35 vs 32%). The embryo recovery rate was similar for Groups A and B (39 and 37%), but it was higher (P < 0.001) with 2 (72%) rather than 1 (22%) mating, and it was negatively correlated with CL number (P < 0.05). Overall, 80% of the llamas had a precocious CL and elevated plasma progesterone concentrations when multiple follicles reached maturity. This was associated with increased subsequent superovulation and embryo recovery (P < 0.01). Peak plasma progesterone was positively correlated with the CL number (P < 0.05). From these results we conclude that superovulation may be achieved with eCG given during either an induced or a simulated luteal phase, that embryo recovery is improved following 2 matings rather than 1, and that MOET may indeed be feasible for use in the llama.     

Effect of prior FSH treatment on the estrus and ovulation responses to eCG in prepubertal gilts

The objective of this study was to determine the effect of pre-treatment of prepubertal gilts with FSH on the estrus and ovulatory responses to eCG injection at two ages. A total of 149 prepubertal Hypor gilts were selected at 150 days (n=76) or 180 days (n=73) of age and assigned to injection of 400 IU eCG plus 200 IU hCG (PG600), 600IU eCG alone (Folligon), pre-treatment with 72 mg FSH (Folltropin) administered as 6 x 12 mg injections at 12 h intervals with 600 IU Folligon 12h after last FSH injection, or non-injected controls. To facilitate detection of estrus, gilts were exposed to a mature boar for 15 min daily for 7 days. To determine ovulatory responses, blood samples were obtained on the day of injection and 10 days later and assayed for progesterone content. Following treatment at 150 days, one control gilt (5.3%) was deemed estrus but ovulation did not occur. Compared to treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus response (52.6% compared with. 26.3%) and increased (P<0.01) the ovulatory response (89.5% compared with. 47.4%). The estrous response in gilts pretreated with Folltropin was intermediate (42.1%) but the ovulatory response (47.4%) was the same as for Folligon alone. Following treatment at 180 days, two control gilts (10.5%) were deemed estrus and ovulation did occur in these gilts. There was no difference between hormone-treated groups for estrus or ovulatory responses, although the ovulatory response of PG600-treated gilts tended (P=0.1) to be greater than for the Folligon-treated group (89.5% compared with 66.7%), with Folltropin-pretreated gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory responses of gilts were greater for PG600 than for Folligon and that while responses to PG600 were not affected by gilt age, for the combined Folligon groups, estrous response (P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.     

Effects of estradiol-17β and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2–3 months postpartum were used to investigate the effect of addition of estradiol-17β and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production.The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 μg cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17β given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.The mean number of CL were 0.91 ± 0.66 and 9.08 ± 5.0 for Groups 1 and 2, respectively. The average recovery rate based on CL counts at slaughter was 60% in Group 2. No embryos were recovered from the two animals in Group 1. Seventy-nine percent of the collected ova were fertilized and more than 60% of them had developed into hatched blastocysts. The percentages of buffalo with excellent and good estrus were 41.6 and 91.6% for Groups 1 and 2, respectively.These results showed that the supplementation of estradiol-17β and the hCG treatment significantly improved the level of ovarian stimulation in swamp buffalo.     

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors

The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF(2α) was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen-thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means±standard error. The FSH/eCG group had higher superstimulatory (33.8±3.9 compared to 23.8±2.6 follicles; P=0.03) and SOV (16.8±2.9 compared to 10.8±2.1 CL; P=0.10) responses. Although the number of total ova/embryos was not different between groups (8.2±1.8 compared to 5.9±1.4 for FSH/eCG and FSH groups, respectively; P=0.25), the number (5.8±1.3 compared to 2.6±0.7; P=0.02) and percentage (75.6±5.7 compared to 53.2±9.7%; P=0.05) of transferable embryos was greater for the FSH/eCG females. Therefore, there was improvement in follicular superstimulatory and SOV responses and embryo quality in FSH/eCG-treated females.     

Use of progestagen-gonadotrophin treatments in estrus synchronization of sheep

The main objective of this study was to investigate the effectiveness of certain progestagen-gonadotrophin treatments on synchronization of estrus in sheep. In Experiment I, 30 Chios ewes were treated at the beginning of the breeding season with medroxyprogesterone acetate (MAP) intravaginal sponges for 12 days and a single i.m. treatment of either FSH (Group 1,10 IU, n = 8; Group 2, 5 IU, n = 8; Group 3, 2.5 IU, n = 8) or eCG (Group 4, 400 IU, n = 6) at the time of sponge removal. Ten days after sponge removal laparotomy was performed to record ovarian response. Clinical estrus was observed in more (though not at a significant level) FSH treated than eCG treated sheep (62.5% versus 33.3%). Administration of 400 IU eCG resulted in the highest mean number of CL perewe ovulating (2.8 +/- 0.2), with administration of 10 IU FSH producing the next best results (2.1 +/- 0.3). Statistically significant differences in the mean number of CL per ewe ovulating were found only between ewes in Group 3 (1.7 +/- 0.4) and Group 4 (2.8 +/- 0.2) (P < 0.05). In Experiment II, 53 Chios and 30 Berrichon ewes were treated during the mid-breeding season with MAP intravaginal sponges for 12 days and a single i.m. treatment of either 10 IU FSH (27 Chios and 16 Berrichon ewes) or 400 IU eCG (26 Chios and 14 Berrichon ewes), at the time of sponge removal. Ewes that were in estrus on Days 2-4 and 19-23 after sponge removal were mated to fertile rams. No significant differences were recorded between treatment or breed groups in the proportions of ewes observed in estrus after treatment. In the Berrichon breed, FSH administration resulted in higher lambing rates (93.8% versus 57.1%, P < 0.05) and higher mean number of lambs born per ewe exposed to rams (1.4 +/- 0.2 versus 0.8 +/- 0.2, P < 0.05) than that of eCG. After treatment with eCG, the mean number of lambs born per ewe exposed to rams was higher in the Chios than the Berrichon breed (1.4 +/- 0.2 versus 0.8 +/- 0.2, P < 0.05). After treatment with FSH, the lambing rate was higher in the Berrichon than the Chios breed (93.8% versus 63.0%, P < 0.05). In conclusion, a single FSH treatment (5 or 10 IU) at the end of progestagen treatment appears to be more effective than eCG for the induction of synchronized estrus in sheep at the beginning of the breeding season, with no cases of abnormal ovarian response observed. During the mid-breeding season FSH (10 IU) appears to be equally as effective as eCG (400 IU) in respect of lambing rate and mean number of lambs born per ewe.     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.     

Superovulation of dairy cows with purified FSH supplemented with defined amounts of LH

This study investigated the effects of a purified follicle stimulating hormone (FSH) preparation supplemented with three different amounts of bovine luteinizing hormone (bLH) and a commercially available FSH with a high LH contamination on superovulatory response, plasma LH and milk progesterone levels in dairy cows. A total of 112 lactating Holstein-Friesian crossbred dairy cows were used for these experiments; the cows were randomly assigned to treatment groups consisting of purified porcine FSH (pFSH) supplemented with bLH. Group 1 was given 0.052 IU LH 40 mg armour units (AU) FSH (n = 6); Group 2 was given 0.069 IU LH (n = 32); Group 3 received 0.423 IU LH (n = 34); while Group 4 cows (n = 36) were superovulated with a commercially available FSH-P((R)). This compound appeared to contain 8.5 IU LH 40 mg AU FSH according to bioassay measurement. All animals received a total of 40 mg AU FSH at a constant dose twice daily over a 4-d period. Levels of milk progesterone and plasma LH were determined during the course of superovulatory treatment. The Group 1 treatment did not reveal multiple follicular growth, and no embryos were obtained. Superovulation of Group 3 cows resulted in significantly (P<0.05) more corpora lutea (CL; 12.6+/-1.1) and fertilized ova (5.1+/-1.3) compared with Groups 2 and 4 (10.1+/-0.9 and 2.6+/-0.6, 9.0+/-0.9 and 2.7+/-0.5, respectively). Due to a high percentage of degenerated embryos (33%) Group 3 yielded only one more transferable embryo than Groups 2 and 4. Among groups, LH levels differed in the period prior to induction of luteolysis and were similar thereafter. The progesterone pattern following FSH LH administration reflected the amount of LH supplementation. Milk progesterone levels on the day prior to embryo collection were correlated to the number of CLs and recovered embryos. It is concluded that under the conditions of our experiment superovulation with 0.423 IU LH 40 mg AU FSH may yield a significantly improved superovulatory response in dairy cows. It is further suggested that LH supplementation exerts its effects mainly on follicular and oocyte maturation during the period prior to luteolysis.     

Follicular growth, ovulation and embryo recovery in dairy cows given FSH at the beginning or middle of the estrous cycle

Variability in the superovulation response is an important problem for the embryo transfer industry. The objective of this study was to determine whether FSH treatment at the beginning of the cycle would improve the ovulation rate and embryo yield in dairy cows. Twenty-eight postpartum cyclic dairy cows were allocated at random to 4 treatment groups (A, B, C and D). Group A cows (n = 10) received FSH (35 mg) at a decreasing dose, starting on Day 9 (Day 0 = day of estrus) for 5 days followed by PGF(2alpha) (35 mg) on Day 12. Cows assigned to Groups B, C and D (n = 6 cows each, respectively) were given 35 mg FSH at a decreasing dose from Days 2 to 6 followed by PGF(2alpha) on Day 7. Group C and D cows received PRID inserts from Day 3 to Day 7. Cows in Group D additionally received 1000 IU hCG 60 hours after PGF(2alpha) treatment. Ovaries were scanned daily using a real time ultrasound scanner from the beginning of FSH treatment until embryo recovery, to monitor follicular development, ovulation and the number of unovulated follicles. Embryos were recovered from the uterus by a nonsurgical flushing technique 7 days after breeding. There were no differences (P>0.01) in the number of follicles > 10 mm at 48 hours after PGF(2alpha) treatment among the 4 groups. The mean numbers of follicles were 10.6 +/- 1.2, 9.3 +/- 1.3, 12.2 +/- 1.3 and 15.0 +/- 2.9 for Groups A, B, C and D, respectively. A significantly (P<0.001) higher number of ovulations was observed and a larger number of embryos was recovered in Group A than in the other groups. The results of this study indicate that superovulation with FSH at the beginning of the cycle causes sufficient follicular development but results in very low ovulation and embryo recovery rates.     

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.     

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.     

Endocrine and superovulatory responses in heifers pretreated with FSH or bovine follicular fluid

This study examined the effects of altered serum FSH concentration on subsequent ovarian response to superovulation. Synchronized heifers were assigned randomly on Day 1 of the cycle (estrus = Day 0) to three pretreatment groups that consisted of 6-d of saline (7ml, s.c., b.i.d.; Group I), FSH-P (0.5 mg, i.m., b.i.d.; Group II) or charcoal-extracted bovine follicular fluid (BFF; 7 ml, s.c., b.i.d.; Group III) injections. Superovulation was initiated on Day 7 and consisted of FSH-P in decreasing dosages over 4 d (4,3,2,1 mg; i.m., b.i.d.), with cloprostenol (500 mug) on the morning of the third day. A second replicate with 14 heifers was conducted using the same protocol but twice the pretreatment dosage of FSH-P (1 mg) and BFF (14 ml). Endogenous plasma FSH decreased during BFF and FSH-P pretreatments compared to controls (P < 0.02). Endogenous FSH concentrations in both primed groups (II and III) were similar to control values (Group I) 12 h after the start of superovulation. Basal LH concentrations were not different between pretreatment groups. The interval from cloprostenol treatment to the preovulatory LH surge in Group III was 21.3 and 23.9 h longer (P < 0.0001) than it was in Groups I and II. The postovulation progesterone rise was delayed in Group III. The number of corpora lutea (CL) was lowest in the BFF-primed group (4.2 +/- 0.8) compared with the FSH-primed (7.4 +/- 1.3) and the control (12.0 +/- 1.8; P < 0.003) groups. In the FSH-primed group (0.68 +/- 0.06 cm(3)), CL volumes were larger than in the control group (0.45 +/- 0.03 cm(3)), whereas in the BFF-primed group (0.27 +/- 0.02 cm(3)) CL volumes were smaller compared with the control group (P < 0.0001). Mean FSH concentrations for 48 h preceding superovulation and the number of CL per cow were positively correlated (r = 0.55; P < 0.004; n = 26). We concluded that both FSH-P and BFF pretreatments decreased the superovulatory response of heifers to FSH-P. The mechanism for this would appear to be associated with reduced endogenous FSH prior to the start of superovulation.     

Effect of estradiol supplementation on superovulation in Swamp buffalo

The effect of estradiol-17beta (E(2)) supplementation on superovulation with (PMSG) or (FSH) was investigated in Swamp buffalo. Sixty-eight buffalo were treated in seven groups. Group 1 served as control and was superovulated by standard PMSG or FSH treatment used in routine bovine embryo transfer protocols. Group 2 was superovulated by standard PMSG regimen plus two injections of E(2) at a 48 h interval beginning one day before the onset of gonadotropin treatment (short-term supplementation) for a total dosage of 2.5 mg E(2); Groups 3 and 4 received the same regimen as Group 2, but in doses of 5.0 and 7.5 mg E(2), respectively. Group 5 received the standard FSH regimen (40% LH). Group 6 received short-term E(2) (7.5 mg) supplementation of FSH-p. Group 7 was superovulated by standard FSH regimen (40% LH) plus three injections of E(2) at 48-72 h intervals beginning five days before the onset of gonadotropin treatment (long-term supplementation) for a total dosage of 7.5 mg E(2). The number of corpora lutea (CL) and follicles >/= 8 mm in diameter were recorded by palpation per rectum and after slaughter. The mean numbers of CL and follicles were 0.99, 5.8, 8.0, 10.6, 4.0, 3.9, 8.1 and 0.25, 6.8, 6.2, 6.2, 1.6, 0.0, 4.1 for Groups 1, 2, 3, 4, 5, 6, 7, respectively. In Group 7, the rates of nonsurgical and postmortem embryo recovery were 46 and 90.4%, respectively and 54.4% of the collected ova were fertilized. These results indicate the possibility of producing viable embryos in buffalo by using E(2) supplementation for the gonadotropin treatment.     

Hormonal control of estrous cyclicity and attempted superovulation in wood bison (Bison bison athabascae)

The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05).     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Comparison of three protocols for superovulation of brown brocket deer (Mazama gouazoubira)

This study aims to evaluate the ovulation rate and the presence of functional corpora lutea after treatment by three different protocols designed to cause superovulation in brown brocket deer. Six female received an intravaginal device containing 0.33 g of progesterone (CIDR®) for 8 days, followed by 0.5 mg injection of estradiol benzoate at the time of insertion and 265 µg of cloprostenol at the time of removal. Afterwards, the hinds were divided into three groups (n = 2): Treatment A received injection of 600 IU eCG on Day 4 after CIDR® insertion; Treatment B received injection of 300 IU eCG at the same time; and Treatment C received injection of 250 IU FSH dissolved in PVP, also on Day 4 post‐insertion. The treatments were crossed over with 44–48 day intervals after CIDR® removal, such that all the deer were submitted to all three treatments. The mean ovulation rate (Treatment A = 3.40 ± 0.68, Treatment B = 1.40 ± 0.24, Treatment C = 0.80 ± 0.49), total ovarian stimulation (Treatment A = 4.80 ± 1.02, Treatment B = 1.80 ± 0.37, Treatment C = 1.40 ± 0.60), and mean CL diameter (Treatment A = 7.33 ± 0.76 mm, Treatment B = 3.94 ± 0.19 mm, Treatment C = 2.18 ± 0.49 mm) in Treatment A were significantly higher than the mean ovulation rates, total ovarian stimulation, and mean CL diameter in Treatments B and C. The mean fecal progesterone metabolites at the luteal phase in Treatment A (6,277.94±2,232.47 ng/g feces) was significantly different from Treatment C (1,374.82±401.77 ng/g feces). Thus, although fertility was not evaluated directly, Treatment A proved capable of induce superovulation in the species Mazama gouazoubira, presenting the greatest mean ovulation rates, with the formation of functional corpora lutea. lutea. Zoo Biol 31:642‐655, 2012. © 2011 Wiley Periodicals, Inc.     

Induction of sexual activity in female camels during the nonbreeding season

Sixteen anestrous adult female camels (Camelus dromedarius ) in good health and with inactive ovaries were selected from the herd during the month of June (non-breeding season). The camels were randomly divided into 4 equal groups. To induce ovarian activity, camels in Groups I,II and III were given an intramuscular injection of 250 mg hydroxyprogesterone hexanoate followed by 1000 IU eCG on days 2 and 3 of treatment. The camels were mated on Day 5 after the last eCG injection. Ovulation in Groups II and III was induced by intravenous administration of 3000 IU hCG and 40 mcg GnRH, respectively. Group IV was administered saline and served as the control. Periodic examinations per rectum were performed to explore the status of the ovaries. Blood samples were collected at 8 different stages and sera were analyzed for estradiol 17-B and progesterone using specific RIA kits. All camels in the control and treated groups were mated successfully. Levels of estradiol 17-B did not exhibit any particular trend. Blood progesterone levels suggested ovulation in 2 camels (50%) in Group I and in 3 camels (75%) in each of Groups of II and III. This was confirmed by presence of CL in the ovary during per rectum examination. No camel ovulated in the control group. One camel conceived in each of Groups I and III.     

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.     

Gonadotropin-independent mechanisms participate in ovarian responses to realimentation in feed-restricted prepubertal gilts.

Short-term feed restriction in prepubertal gilts suppresses episodic LH secretion in the absence of changes in body weight or composition. To assess non-gonadotropin-mediated effects of realimentation at the ovarian level, 52 gilts were assigned to six treatments after 7 days (Days 1-7) of maintenance feeding (approximately 30% ad libitum). Groups R12 and R9 were maintenance-fed Days 8-12 or Days 8-9, respectively; A12 and A9 were fed to appetite Days 8-12 or Days 8-9, respectively. Groups R9P and A9P were fed as groups R9 and A9 were but received 750 IU eCG at 1500 h on Day 8. Groups R12 and A12 were ovariectomized at 1500 h on Day 12, and all other groups were ovariectomized at 1500 h on Day 9. All gilts received oral progestogen (15 mg allyl trenbolone) from Day 1 to ovariectomy, to antagonize the usual increases in endogenous gonadotropins that follow realimentation. Blood samples were obtained at 10-min intervals during selected windows during the experiment. Ovarian follicles were analyzed for development and steroidogenesis, and plasma samples were analyzed by RIA to determine concentrations of LH, FSH, insulin, and insulin-like growth factor-1 (IGF-1). Allyl trenbolone abolished pulsatile LH secretion, and realimentation did not stimulate LH or FSH secretion, with the exception of FSH secretion on Day 8 in A9 gilts. Postprandial insulin concentrations on Day 9 were greater after feeding to appetite (A9, A9P, and A12) than after feed restriction (R9, R9P, and R12). Pre- and postprandial IGF-1 concentrations were higher in re-fed gilts on Day 9 (A9 and A12) and Day 12 (A12) than in feed-restricted gilts. Follicular diameter, fluid volume, and basal granulosa cell estradiol synthesis per follicle were greater in A12 gilts than in R12 gilts, although there was no difference between A9 and R9 gilts. There was no effect of realimentation on follicular fluid concentrations of estradiol or testosterone, or on androgen-driven granulosa cell estradiol synthesis. Treatment with eCG increased follicular diameter, fluid volume, basal and androgen-driven estradiol synthesis, and fluid estradiol concentrations without interaction with feeding level. In conclusion, in the absence of LH elevations, realimentation over 5 days exerts effects at the ovary, increasing follicular growth and estradiol synthesis. These effects may be mediated by insulin, IGF-1, or unmeasured growth factors and would be expected to synergize with increases in endogenous gonadotropin that follow realimentation.