eFSH in clinical equine practice

Equine follicle stimulating hormone (eFSH) has been used to induce follicular development in transitional mares and problem acyclic mares, as well as superovulate cycling mares. The most efficacious protocol is to administer 12.5 mg eFSH, intramuscularly, twice daily beginning 5 to 7 days after ovulation when the diameter of the largest follicle is 20 to 25 mm. Prostaglandins are to be administered on the second day of eFSH therapy. Treatment with eFSH is continued for 3 to 5 days until follicle(s) are >or=35 mm in diameter. The mare is subsequently allowed to 'coast' for 36 h, after which human chorionic gonadotropin is administered to induce ovulation.     

Association between numbers of ovarian follicles in the first follicle wave and superovulatory response in ewes

The aim of this study was to examine the variability in the number of ovarian follicles in sheep and to determine if the average number of follicles per day influences the response to superovulation and resulting embryo quality. Ewes (n = 83) were synchronized and the number of follicles (≥2 mm diameter) in the ovaries were counted daily between Days 0 and 4 of the oestrous cycle using transrectal ultrasonography. Fourteen to 21 days later, 47 ewes were randomly chosen from the group and were treated with an intravaginal progestagen pessary for 12 days and superovulated with 1500 IU eCG administered as a single injection 10 days after sponge insertion. Ewes were mated and reproductive tracts were recovered after slaughter on Day 6 of pregnancy. The number of corpora lutea was counted, uterine horns were flushed and the morphology and developmental stage of the recovered oocytes/embryos was assessed. The mean daily number (±S.D.) (≥2 mm diameter) of follicles per ewe was 8.5 ± 2.8 (ranging between 3 and 16). After superovulation animals with few follicles (Low group: <8 follicles/day; n = 21) had fewer (P < 0.005) corpora lutea, total structures (unfertilized oocytes and embryos), good quality and total embryos compared to animals with many follicles (High group: ≥8 follicles/day; n = 23). No difference was found in the proportion of good quality embryos (relative to the total number; Low 0.68 ± 0.11 versus High 0.79 ± 0.08; P = 0.21) between the two groups, or the recovery rate, the number of unfertilized oocytes or the number of poor quality embryos per animal. We conclude that ewes with a higher number of follicles (≥8) during the first follicular wave had a better superovulatory response (in terms of corpora lutea and high quality embryos) 2–3 weeks later; however, there was no relationship between the number of follicles and the proportion of good quality embryos per animal.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

Effects of FSH and LH on ovarian and follicular blood flow, follicular growth and oocyte developmental competence in young and old mares

Objectives of the experiment were to determine the effects of mare age and gonadotropin treatments on dominant follicle vascularity, ovarian blood flow and dominant follicle growth and to associate follicular vascularity with oocyte developmental capacity. Growing follicles >30mm from young (4-9 years) and old (>20 years) mares were assessed for blood flow using color Doppler ultrasonography before maturation induction with recombinant equine LH (eLH) and immediately prior to oocyte collection at 20-24h after eLH. Pulsed Doppler was used to obtain resistance indices of ovarian arteries ipsilateral to preovulatory follicles. For eFSH-treated estrous cycles, eFSH administration was started after detection of a cohort of follicles ≥20 to <25mm and continued until a follicle >30mm. Oocytes were harvested using transvaginal, ultrasonic-guided aspirations and cultured and injected with sperm at 40±1h after eLH. Presumptive zygotes were incubated, and rates of cleavage (≥2 cells) and blastocyst formation were obtained. Embryos were transferred nonsurgically into recipients' uteri, and pregnancy rates were assessed. Vascularity (number of color pixels per total pixels) was higher (P=0.003) in the follicles of old compared to young mares, with no significant interaction of eFSH or eLH. Effects of eFSH and time from eLH on follicle vascularity were not significant. The vascularity of follicles associated with oocytes that did compared to those that did not form blastocysts was greater (P=0.048), although follicular vascularity was less (P=0.02) for follicles associated with oocytes that did compared to those that did not develop into pregnancies. Resistance indices were not different for age, eFSH treatment, time after eLH administration and oocyte developmental potential. Growth of the dominant follicle was not associated with vascularity, although advanced age tended (P=0.09) to have a negative effect on follicle growth.     

Comparison of equine pituitary extract and follicle stimulating hormone for superovulating mares

Sixty light-horse, nonlactating mares were used to compare the efficacy of equine pituitary extract versus follicle stimulating hormone (FSH-P) for inducing multiple ovulations. On Day 12 of diestrus, mares were assigned to receive 1) no treatment, controls; 2) subcutaneous injections of 750 Fevold rat units of equine pituitary extract once daily; or 3) intramuscular injection of 150 mg of FSH-P twice daily. Ultrasound was used twice daily to visualize follicular changes and ovulation. For mares in Groups 2 and 3, treatment was initiated when two or more follicles > 20 mm were detected, and it continued until all large follicles (> 30 mm) had ovulated or regressed. Five milligrams of prostaglandin F(2)alpha (PGF(2)) were administered to mares in Groups 2 and 3 on the first day of treatment. Human chorionic gonadotropin (3,300 IU) was given to all groups of mares during estrus when a 35-mm follicle was detected. Ovulation rate was greater (P < 0.05) for mares treated with pituitary extract (2.2) compared to FSH-P treatment (1.6) or no treatment (1.0). Thirteen of 18 mares treated with the extract had more than one ovulation versus only four of nine FSH-treated mares. Mares in the pituitary extract group were given injections for an average of 6.4 d compared to 6.8 d (13.7 injections) for FSH-treated mares. Intervals to estrus and ovulation from initial injection of extract were 2.9, 7.6; and 2.6, 9.2 d for FSH-treated mares. The mean number of medium-sized follicles (25 to 30 mm) was greater (P < 0.05) in extract-treated mares compared to the FSH-treated mares. Both extract and FSH increased (P < 0.05) the number of follicles > 30 mm and the size of the second largest follicle 1 and 2 d prior to ovulation when compared to controls. Overall, mares with multiple ovulations had more (P < 0.05) follicles 25 to 30 mm and > 30 mm on Day -6 through -1 (Day 0 = day of ovulation) than single ovulating mares. Those mares that had multiple ovulations had less (P < 0.05) size difference between the largest and second largest follicle when compared to single ovulating mares. In summary, FSH-P at the one dose studied was less effective than equine pituitary extract in inducing follicular activity and multiple ovulation in the mare.     

Efficiency of superovulation and in vivo embryo production in eFSH-treated donor mares after estrus synchronization with progesterone and estradiol-17β

Reliable methods of regulating estrus and stimulating superovulations in equine embryo transfer programs are desirable. Our objectives were to investigate the efficacy of a progesterone and estradiol-17β (P&E) estrus synchronization regimen in mares with and without subsequent equine follicle-stimulating hormone (eFSH) treatment and to examine the effects of eFSH on folliculogenesis and embryo production. Cycling mares were treated with P&E daily for 10 d. On the final P&E treatment day, prostaglandin F_2α was administered, and mares were randomly assigned to one of two treatment groups (n = 20 mares/group). In both groups, mares were examined daily by transrectal ultrasonography. In the eFSH group, twice-daily eFSH treatments were initiated at follicle diameter 20 to 25 mm and ceased at follicle ≥35 mm; human chorionic gonadotrophin (hCG) was administered after 36 h. In the control group, eFSH treatments were not given, but hCG was administered at follicle ≥35 mm. Mares were inseminated with fresh semen, and embryo recovery attempts were performed 8 d postovulation. Synchrony of ovulations within each group appeared to be similar. Six mares in the eFSH group failed to ovulate. The eFSH treatment resulted in higher (P < 0.05) numbers of preovulatory follicles and ovulations; however, embryo recovery rate did not increase (eFSH 1.0 ± 0.4 vs. control 0.95 ± 0.1 embryos/recovery attempt), and embryo per ovulation rate was significantly lower (36% vs. 73%). The eFSH-treated mares had significantly higher frequency of nonovulatory follicles (28% vs. 0) and higher periovulatory serum concentrations of estradiol-17β. Based on our findings, combined P&E and eFSH regimens cannot be recommended for cycling donor mares.     

Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares

The dynamics of ovarian follicular development depend on a timely interaction of gonadotropins and gonadal feedback in the mare. The development and efficacy of genetically cloned recombinant equine gonadotropins (reFSH and reLH) increase follicular activity and induce ovulation, respectively, but an optimum embryo recovery regimen in superovulated mares has not been established. The objective of this study was to determine if treatment with reFSH followed by reLH would increase the embryo per ovulation ratio and the number of embryos recovered after superovulation in mares. Sixteen estrous cycling mares of light horse breeds (4-12 years) were randomly assigned to one of two groups: Group 1; reFSH (0.65mg)/PBS (n=8) and Group 2; reFSH (0.65mg)/reLH (1.5mg) (n=8). On the day of a 22-25mm follicle post-ovulation mares were injected IV twice daily with reFSH for 3 days (PGF(2α) given IM on the second day of treatment) and once per day thereafter until a follicle or cohort of follicles reached 29mm after which either PBS or reLH was added and both groups injected IV twice daily until the presence of a 32mm follicles, when reFSH was discontinued. Thereafter, mares were injected three times daily IV with only PBS or reLH until a majority of follicles reached 35-38mm when treatment was discontinued. Mares were given hCG IV (2500IU) to induce ovulation and bred. Embryo recovery was performed on day 8 day post-treatment ovulation. Daily jugular blood samples were collected from the time of first ovulation until 8 days post-treatment ovulation. Blood samples were analyzed for LH, FSH, estradiol, progesterone and inhibin by validated RIA. Duration of treatment to a ≥35mm follicle(s) and number of ovulatory size follicles were similar between reFSH/reLH and reFSH/PBS treated mares. The number of ovulations was greater (P<0.01) in the reFSH/reLH group, while the number of anovulatory follicles was less (P<0.05) compared to the reFSH/PBS group. Number of total embryos recovered were greater in reFSH/reLH mares than in the reFSH/PBS mares (P≤0.01). The embryo per ovulation ratio tended to be greater (P=0.07) in the reFSH/reLH mares. Circulating concentrations of estradiol, inhibin, LH and progesterone were not statistically different between groups. Plasma concentrations of FSH were less (P<0.01) in the reFSH/reLH treated mares on days 0, 1, 4, 6, 7 and 8 post-treatment ovulation. In summary, reFSH with the addition of reLH, which is critical for final follicular and oocyte maturation, was effective in increasing the number of ovulations and embryos recovered, as well as reduce the number of anovulatory follicles, making this a more viable option than treatment with reFSH alone. Further evaluation is needed to determine the dose and regimen of reFSH/reLH to significantly increase the embryo per ovulation ratio.     

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles 相似文献   

Evaluation of three equine FSH superovulation protocols in mares

Superovulation could potentially increase embryo recovery for immediate transfer or cryopreservation. The objectives were to evaluate the effect of pretreatment with progesterone and estradiol (P+E) on follicular response to eFSH and compare doses of eFSH and ovulatory agents on follicular development and ovulation in mares. In Experiment 1, 40 mares were assigned to one of four treatment groups. Group 1 consisted of untreated controls. Group 2 mares were administered eFSH without pretreatment with P+E. Group 3 mares were administered P+E for 10 days starting in mid-diestrus followed by eFSH therapy. Group 4 mares were administered P+E for 10 days followed by eFSH therapy. All treated mares were administered 12.5mg eFSH twice daily and prostaglandins were given on the second day of eFSH therapy. Mares were bred with fresh semen the day of hCG administration and with cooled semen the following day. The numbers of preovulatory follicles and ovulations were lower for mares treated with P+E prior to eFSH treatment. Pretreatment with P+E in estrus also resulted in a lower embryo recovery rate per ovulation compared to the other two eFSH treatment groups. In Experiment 2, two doses of eFSH (12.5 and 6.25mg) and two ovulation-inducing agents (hCG and deslorelin) were evaluated. The number of preovulatory follicles was greater for mares given 12.5mg of eFSH compared to mares given 6.25mg. Number of ovulations was greatest for mares given 12.5mg of eFSH twice daily followed by administration of hCG. Embryo recovery per flush was similar among treatment groups, but the percent of embryos per ovulation was higher for mares given the low dose of eFSH. In summary, there was no advantage to giving P+E prior to eFSH treatment. In addition, even though the lower dose of eFSH resulted in fewer ovulations, embryo recovery per flush and embryo recovery per ovulation were similar or better for those given the lower dose of eFSH.     

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation; (3) administration of EPE twice daily in successively decreasing doses. In the first experiment, 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily; (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of >35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation, or two doses of a GnRH-a agonist upon detection of a follicle >35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles >35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF2, and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF2alpha, but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles > or = 35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose, improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Steroid concentrations in follicular fluid aspirated repeatedly from transitional and cyclic mares

The objectives of the present study were to determine follicular progesterone (P4) and estradiol-17beta (E2) in transitional mares and to compare follicular steroid concentrations between transitional and cyclic mares. Follicles > 8 mm were aspirated under transvaginal ultrasound-guidance 4 times at 3 to 4 day intervals (T1-T4) in Norwegian pony mares during vernal transition. During the breeding season, follicular aspirations were conducted in each mare on Day 6, Day 14 and Day 18 after ovulation of 3 separate estrous cycles (Day of ovulation = Day 0). Plasma and follicular fluids were analyzed for P4 and E2 with ELISA and RIA, respectively. Plasma P4 concentrations remained below 1 ng/mL throughout T1-T4, while the follicular P4 concentrations increased significantly to cyclic levels after the first transitional aspiration. Plasma E2 concentrations similarly remained at low levels during the course of the transitional aspirations, while the follicular E2 concentrations increased gradually over the 4 aspirations to cyclic concentrations. The mares ovulated on average 9.8 +/- 1.6 (mean +/- SEM) days after the last transitional aspiration, and 16.6 +/- 0.2, 11.3 +/- 1.5 and 23.2 +/- 4.4 days after aspirations conducted on Day 6, 14 and 18, respectively. The present study demonstrates that in the transitional mare newly developing follicles exhibit biosynthesis of P4 and E2. Furthermore, an increase in follicular steroid concentrations is not necessarily reflected in the peripheral steroid concentrations.     

Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition

The objective was to compare the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early spring transition. Starting January 30th, mares kept under ambient light were examined by transrectal ultrasonography. When a follicle ≥25 mm was detected, mares were assigned to one of two treatment groups, using a sequential alternating treatment design. In the eFSH group, mares (n = 18) were treated twice daily with eFSH (12.5 mg im) until they achieved a follicle ≥35 mm; hCG was given 36 h later. In the deslorelin group, mares (n = 18) were treated twice daily with deslorelin (63 μg im) until a follicle ≥35 mm was detected, and then they were given hCG. Estrous mares were inseminated with fresh semen. Eight days after ovulation, embryo recovery attempts were performed. In each group, 14/18 (78%) mares ovulated following the eFSH or deslorelin treatment regimes. The mean (95% CI) interval from treatment initiation to ovulation was 8.2 d (7.3, 8.9) and 7.2 d (6.2, 8.1) in the eFSH and deslorelin groups, respectively. In the eFSH group, the number of ovulations was significantly higher (mean ± S.E.M.; 3.4 ± 0.4 vs. 1.1 ± 0.1 ovulations), and more embryos were recovered (2.6 ± 0.5 vs. 0.4 ± 0.2 embryos/recovery attempt). We concluded that eFSH and deslorelin treatment regimes were equally effective in inducing ovulation in early transitional mares, within a predictable time of treatment; however, the eFSH regime increased the number of ovulations and embryos recovered per mare.     

Superovulation using recombinant human FSH and ultrasound-guided transabdominal follicular aspiration in baboon (Papio anubis)

The response of baboon females to a modified human ovarian stimulation protocol incorporating start of pituitary suppression in the luteal phase of the cycle with a GnRH agonist (GnRHa) and recombinant human FSH (rhFSH) was studied. A long-acting GnRHa implant supplying goserelin acetate was administered s.c. to six adult female baboons experiencing regular menstrual cycles (33–34 days) on days 22–24 of the cycle. Follicular development was monitored by transabdominal ultrasonography and serum levels of E2 and progesterone (P4) and rhFSH were determined by ELISA. Menses occurred 9–10 days after GnRHa administration. Daily i.m. administration of 75 IU rhFSH commenced 9–10 days after menses and continued for 9–10 days. When most follicles were ≥5 mm diameter and serum E2 had reached its maximum level, 2000 IU hCG was administered i.m. to induce follicle maturation. Transabdominal ultrasound-guided follicular aspiration of follicles ≥2 mm diameter was performed 30–34 h after hCG administration.

One baboon did not show an adequate response to rhFSH stimulation. This animal did not receive further treatment and no data for it are presented. The number of follicles aspirated was 21±4 and 17.2±3.8 oocytes were recovered per animal with an average recovery rate of 82% (86/105). The number of oocytes collected from five animals were 14, 21, 16, 15, and 20 (n=86). Most of the oocytes recovered were in metaphase II and 3 h after recovery 91% (78/86) were considered suitable for in vitro fertilization. It was concluded that recombinant human FSH can successfully induce follicular recruitment and oocyte maturation in baboon females during pituitary suppression with a GnRHa     

Production of embryos by assisted reproduction in the horse

In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound-guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured in vitro. Recovery rates of immature oocytes from mares in vivo are lower than those for cattle. In addition, work on oocytes recovered from horse ovaries post-mortem has shown that horse oocytes from smaller (< 20 mm diameter) viable follicles may not yet be meiotically competent. Methods for in vitro fertilization and for obtaining adequate numbers of competent immature oocytes from the mare must be developed before in vitro embryo production can become a useful clinical and research procedure in the horse.     

Body condition and protein supplementation positively affect periovulatory ovarian activity by non LH-mediated pathways in goats

Effects of rumen undegradable intake protein (UIP) supplementation on ovarian activity and serum insulin, GH, and LH were evaluated in goats having low or high body condition (BC). Goats with either low BC (n = 16, 28.7 ± 0.8 kg BW, BC = 2.1 ± 0.3) or high BC (n = 16, 38.4 ± 0.8 kg, BC = 3.2 ± 0.3) received, during 40-days, one of the two protein supplementation levels: without UIP or with UIP (120 g goat⁻¹ d⁻¹). Oestrus was synchronized with two i.m. doses of PGF₂, and jugular blood samples were collected from 36 to 42 h after the second prostaglandin injection at 15 min intervals. Serum concentrations of insulin, LH, and GH were measured The number of preovulatory follicles and the number of corpora lutea (CL) were evaluated by transrectal ultrasonography at 1 and 4 days after the second prostaglandin dose, respectively. Does with higher BC had more CL than those in the lower condition group (2.8 ± 0.2 versus 1.8 ± 0.2, P < 0.05). Similarly, goats receiving UIP supplementation had more follicles (2.6 ± 0.2 versus 1.9 ± 0.2, P < 0.05) and tended to have more CL (2.6 ± 0.2 versus 2.0 ± 0.2, P = 0.05) than does not receiving UIP. Neither BCS nor UIP supplementation affected serum GH or LH concentrations, pulsatility, or area under the curve. High BC does produced more insulin (1.92 ± 0.17 versus 0.81 ± 0.17 ng/mL, P < 0.01 ng/mL) than lower BC goats; the same for UIP-supplemented (1.69 ± 0.18 versus 1.04 ± 0.18, P < 0.05). Results suggest that the increased ovarian activity observed in both UIP-supplemented and higher BC goats was not the result of changes in LH or GH, suggesting effects at a local level, through changes in insulin in a non-GnRH-gonadotrophin dependent manner.     

Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.     

The effect of follicle-stimulating hormone on follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary

The effect of FSH on goat follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor (IGF)-I were studied through both in vivo and in vitro experiments. The FSH treatment was begun on Day 9 after estrus and consisted of injections twice a day for 3 days in decreasing doses (7.5–7.5–5.0–5.0–2.5–2.5 mg). Does in both treatment and control groups were slaughtered for ovaries on Day 12. Granulosa cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Expression of IGF-I and IGF-II mRNA was determined by RT–PCR, while concentrations of progesterone (P4), estradiol (E2), IGF-I and IGF-II were measured by radioimmunoassay (RIA). Following parameters increased significantly (P<0.05) after the FSH treatment: follicle number (5.0±1.5 versus 9.0±2.0 per ovary), the level of E2 (0.1±0.1 ng/ml versus 0.7±0.2 ng/ml), the E2/P4 ratio (0.7±0.4 versus 4.7±3.0) and the concentrations of IGF-I (0.5±0.2 ng/ml versus 119.4±15.1 ng/ml) and IGF-II (0.12±0.03 ng/ml versus 40.9±18.7 ng/ml) in follicular fluid of the medium sized (3–5 mm) follicles and in the ovarian cortex the relative quantity of IGF-I mRNA (0.37±0.17 versus 0.90±0.12 Max OD). In contrast, the ratio of apoptotic granulosa cells in these follicles was reduced significantly (0.53±0.1 versus 0.10±0.01, P<0.05). In large (>5 mm) follicles, however, only the follicle number (2.3±0.7 versus 7.0±1.5 per ovary) and the level of IGF-I (38.4±11.0 ng/ml versus 87.3±13.9 ng/ml) increased significantly (P<0.05), whereas other values did not change. In vitro culture of granulosa cells showed that FSH significantly (P<0.05) enhanced IGF-I production (12.7±2.1 ng/ml versus 26.±21.9 ng/ml) by these cells, and both FSH and IGF-I reduced the ratios of apoptotic cells (from 0.7±0.07 to 0.3±0.1 and 0.2±0.04, respectively) and the effect was additive when both were used together. H89, the PKA pathway inhibitor, blocked the effect of FSH on granulosa cell apoptosis and IGF-I production in vitro. These results indicated that FSH mainly enhanced the development of medium sized follicles in the goat by suppressing the apoptosis of granulosa cells via increasing production of IGF-I and steroids, possibly through the PKA pathway.     

Effect of organic or inorganic trace mineral supplementation on follicular response, ovulation, and embryo production in superovulated Angus heifers

We determined whether source of trace mineral supplementation prior to embryo collection affected embryo production and quality. Angus half-sibling heifers (n = 20) originating from a common herd were assigned to three treatment groups using a 3 × 3 latin square design replicated in time (3×) and space (6× complete and 1× incomplete): (1) heifers received no added mineral to their diet (control; n = 53); (2) heifers received a commercially available organic mineral supplement (organic; n = 52); or (3) heifers received an all inorganic mineral supplement (inorganic; n = 55). All heifers had ad libitum access to hay and were fed a supplement containing corn and soybean meal. Treatments were initiated 23 days prior to embryo recovery. Heifers were given a 45-day adaptation period of no mineral supplementation before initiating a new treatment. Ovarian structures were evaluated using transrectal ultrasonography to determine the presence and number of follicles and CL on each ovary. The mean number of recovered ova/embryos was similar among treatments (4.1 ± 0.7, 3.8 ± 0.7, and 3.3 ± 0.7 for control, inorganic, and organic treatments, respectively), the number of unfertilized oocytes was greater (P < 0.05) for inorganic (2.3 ± 0.5) and control (1.6 ± 0.5) treated heifers than organic (0.4 ± 0.4) treated heifers. No differences among treatments existed for the number of degenerate or transferable embryos, but individual heifer influenced the total number of embryos/ova, unfertilized ova, and transferable embryos recovered. We conclude that heifer accounted for the greatest differences in embryo production and quality. Source of trace mineral supplementation did not significantly alter embryo number or quality in superovulated purebred Angus heifers fed a well-balanced diet, meeting all trace mineral requirements.     

Control of postpartum anestrous with an intra-vaginal progesterone device plus eCG or calf removal for 120 h in suckled crossbred cows managed in a pasture-based system

To control postpartum anestrus and reduce calving to conception interval, 167 crossbred non-pregnant cows that were 90–130 days postpartum were allotted randomly to one of the following treatments: PH (n = 59), intra-vaginal sponge with 250 mg of medroxyprogesterone acetate (MAP) for 7 days plus 50 mg of MAP and 5 mg 17-β estradiol (17β-E) in the first day of treatment (day −8), 500 UI eCG (day −3) and 1.5 mg 17β-E in 24 h after sponge removal (day 0); CR (n = 57), temporary calf removal for 120 h; CG (n = 51), control group without treatment. Estrus rate differed among treatments (P < 0.01) being greater in PH (78.2%), followed by CR (52.0%) and CG (22.9%). A greater proportion of cows in the PH (80.0%) and CR (54%) groups had ovulations when compared to CG (35.4%). Intervals to first estrus were 13.5 ± 6.3 days, 26.1 ± 6.4 days and 52.5 ± 7.5 days for the PH, CR and CG groups, respectively. First insemination conception was similar in the three groups. Postpartum intervals to first breeding (PFS) and to conception (PCI) were longer in CG than PH and CR groups (P < 0.05; P < 0.01). The PH and CR groups had a similar PFS but PCI was different (P < 0.02). Accumulated pregnancy rate at 30 and 60 but not at 90 days were different (30 days: P < 0.09; P < 0.01; P < 0.09; 60 days: P < 0.06; P < 0.01; P < 0.03) among treatments. After 90 days post-treatment, 9%, 18% and 33% of cows from the PH, CR and CG groups had not conceived. Similarly, 5.4%, 6.0% and 12.5% of cows from the PH, CR and CG groups, respectively, were culled from the herd because of lack of pregnancy after 180 days post treatment. In the group of cows evaluated by ultrasonography, only those cows having larger ovaries and dominant follicles had ovulations. It was concluded that the hormonal treatment was more efficient in inducing a fertile estrus and reducing calving to conception interval followed by the calf removal for 120 h. Each method can be considered as an important tool to reduce the postpartum anestrous period in dual purpose herds when AI is conduct in the tropics.