Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles > or = 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6). Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection.     

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.     

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.     

Intrauterine embryo reduction in the mare

The site of a hypothesized embryo reduction mechanism was studied. The number of normal-sized embryos and undersized embryos (>2 standard deviations below the mean embryo diameter in control mares) was determined at day 7 and 11 post-ovulation in single-ovulating control mares and in multiple-ovulating pituitary extract-treated mares in which all ovulations occurred on a single day. In two additional groups (control and treated), the embryonal enlargement was monitored by rectal palpation until day 49. An average of 0.7 normal-sized embryos per control mare at day 7 and at day 11 and 0.6 vesicles per control mare at day 49 was found. More normal-sized embryos (P<0.05) were recovered per extract-treated mare at day 7 (2.9 embryos/mare) than at day 11 (0.7), and fewer undersized embryos per extract-treated mare (P<0.05) were recovered at day 7 (0.1) than at day 11 (1.1). The total number of embryos (normal-sized plus undersized) per treated mare was greater (P<0.05) at day 7 and at day 11 than the number of palpated vesicles per treated mare at day 49. The number of mares with more than one normal-sized embryo in the day 7 treated group (10/14) was greater (P<0.05) than in the day 11 treated group (1/14) and was greater than the number of mares with more than one palpated vesicle in the day 49 treated group (2/14). Intrauterine reduction was therefore manifested between days 7 and 11 in multiple-ovulating mares, as demonstrated by the number of multiple normal-sized blastocysts recovered at day 7 and by the reduced number of normal-sized and the increased number of undersized blastocysts recovered at day 11.     

Fertilization rates in superovulated and spontaneously ovulating mares

Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Group 1 received 40 mg of equine pituitary extract (EPE; i.m.) daily beginning on Day 5 after ovulation; mares assigned to Group 2 served as untreated controls. All mares were given 10 mg PGF(2alpha) on Day 5 and Day 6, and 3,300 IU of human chorionic gonadotropin (hCG) were administered intravenously once mares developed 2 follicles >/=35 mm in diameter (Group 1) or 1 follicle >/=35 mm in diameter (Group 2). Mares in estrus were inseminated daily with 1 x 10(9) progressively motile spermatozoa once a >/=35 mm follicle was obtained. Two days after the last ovulation the ovaries and oviducts were removed. Ovaries were examined for ovulatory tracts to confirm ovulation, while the oviducts were trimmed and flushed with Dulbeccos PBS + 10% FCS to recover fertilized oocytes. All fertilized oocytes (embryos) recovered were cultured in vitro for 5 d using TCM-199 conditioned with equine oviductal cells. Ninety-two percent of the CL's from EPE mares resulted from ovulations compared with 94% for mares in the control group (P>0.05). The percentages of ovulations resulting in embryos were 57.1 and 62.5% for EPE-treated and control mares, respectively (P>0.05). Eighty-eight (Group 1) and 91% (Group 2) of the freshly ovulated oocytes recovered were fertilized (P>0.05). After 5 d of culture, 46.4 and 40.0% of the embryos from EPE-treated and control mares developed to the morula or early blastocyst stage (P>0.05). In summary, the CL's formed in superovulated mares were from ovulations not luteinizations. Although embryo recovery was less than expected, fertilization rates and embryo development were similar (P>0.05) between superovulated and control mares.     

Follicular dynamics in mares treated with an equine pituitary extract

The follicular dynamics of 112 mares treated with an equine pituitary extract were studied. Follicles >10 mm in diameter at day 15 post-ovulation appeared to represent the follicles which were induced with pituitary extract to grow and ovulate. This was shown by the greater number of >10 mm follicles in mares which subsequently had higher ovulation rates and by the subsequent decrease in number of small follicles (<20 mm) which corresponded with the increase in number of large follicles (>/=20 mm). The difference in diameter (mm) between the largest and second largest follicle on day 15 post-ovulation was greater (P<0.05) for extract-treated mares which subsequently had single ovulations than for extract-treated mares which subsequently had multiple ovulations (7.7 +/-1.5 vs 2.8 +/-0.6). The observed ratio of bilateral to unilateral multiple ovulations was not different (P>0.1) from the expected ratio which was calculated on the assumption that side of ovulation occurred independently (59:19 vs 62:16, observed vs expected).     

Superovulation in mares

Embryo recovery from single ovulating mares is approximately 50 per cent per estrous cycle. Superovulation could be used to increase embryo recovery and provide extra embryos for embryo freezing. This review addresses some historical approaches to superovulation, as well as examines factors that affect the response of mares to equine FSH. eCG, GnRH and inhibin vaccines have been of limited success in stimulating multiple ovulation. Numerous studies have shown that injection of equine pituitary extract (EPE) will result in three to four ovulations per estrous cycle and two embryos. A purified, standardized EPE preparation (eFSH) also results in a similar response to EPE. Factors affecting the response to EPE and eFSH include day of initial treatment, size of largest follicle at initial treatment and frequency of injection. Embryos from single ovulating, untreated mares and eFSH-treated mares provide similar pregnancy rates upon nonsurgical transfer. Five to 7 days of eFSH treatment also has been shown to hasten the first ovulation of the breeding season. Potential problems after eFSH injections include anovulatory or luteinized follicles and overstimulation. Studies are needed to further evaluate the criteria for initiation of treatment and to determine how to increase ovulation rate without decreasing embryo recovery per ovulation.     

Embryo recovery from mares exposed to a year-to-year artificially prolonged daylength

The aim of the experiment was to determine the effect of a year-to-year prolonged daylength on the patterns of equine reproductive activity and results of embryo recovery. Experiments using Konik Polski mares were conducted over four reproduction seasons. Five mares were exposed to a regimen of artificially prolonged daylength (APD) and another five mares in a control group were kept under conditions of natural daylight. Both the control and experimental groups were examined for appearance of estrus, ovulation and also for the state of their coats. A single stallion was used for breeding all of the mares. The embryos were recovered nonsurgically 6 to 9 days after ovulation. All of the mares exposed to APD showed increased ovarian activity, which commenced earlier than in the control group. About 19% more ovulations were detected in the experimental group. The average number of ovulations per lighted mare per year was 15.3, while in the control group it was 12.4 ovulations (P<0.05). However, the embryo recovery rate and total number of embryos obtained from the mares exposed to APD did not exceed the number of embryos collected from the control mares (P<0.05). Modification of daylength had a visible effect on the mares by producing a change in their coats.     

Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares

Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasound examination at 24-h intervals. The ability of the inhibin antiserum to neutralize the bioactivity of equine inhibin was examined in vitro using a rat pituitary cell culture system. Suppression of secretion of FSH from cultured rat pituitary cells by equine follicular fluid was reversed by the addition of increasing doses of the inhibin antiserum, thereby indicating its bioactivity. Plasma levels of FSH and estradiol-17 beta were higher in mares treated with the inhibin antiserum. The ovulation rate was significantly higher in mares treated with antiserum (100 mL = 3.75 +/- 0.63; 200 mL = 4.50 +/- 0.65) than in control mares (1.25 +/- 0.25). These results demonstrate that inhibin is important in regulating FSH secretion and folliculogenesis in mares. They also show that neutralization of the bioactivity of inhibin may become a new method for the control of folliculogenesis and ovulation rate in mares.     

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.     

Ovarian follicles, ovulations and progesterone concentrations in aged versus young mares

The objectives of this study were: 1) to document age-related ovulation failure in mares and 2) to contrast the number of ovarian follicles, occurrence of ovulations, and postovulatory concentrations of progesterone in aged versus young mares. In Experiment 1, 4 of 10 aged (25- to 33-years-old) mares were anovulatory between July 1 and September 1, 1989. In Experiment 2, two of 25 aged (20- to 30-years-old) and none of 21 young (3- to 12-years-old) mares were anovulatory between February 1 and June 30, 1990. The average (+/- SEM) day of the first ovulation was later (P<0.05) for aged versus young mares (May 9 +/- 7.1 vs April 25 +/- 7.4 days, respectively). There tended (P<0.10) to be fewer 11- to 20-mm ovarian follicles in aged versus young mares (2.8 +/- 0.2 vs 5.3 +/- 0.1, respectively), but there was no difference (P>0.10) in the total number of ovarian follicles in aged versus young mares (21.0 +/- 0.3 vs 26.1 +/- 0.2, respectively) during the pooled periovulatory period of the first and second (single) ovulations. The number of ovulatory cycles during the study period was less (P=0.01) for aged versus young mares (2.2 +/- 0.3 vs 3.2 +/- 0.3). Plasma progesterone concentrations on Days 10 and 15 of the first ovulatory cycle were higher (P<0.05) in aged versus young mares.     

Work in progress: A simple technique that may improve the rate of embryo recovery on uterine flushing in mares

Embryo recovery rates from uterine flushings of normal mares on Day 7 or later after ovulation currently range from 55% to 80%. In contrast, pregnancy rates at 14 d in experimental mares are often higher. There appears to be a discrepancy between pregnancy rates and recovery rates of embryos on uterine flushing, indicating that some embryos are not recovered from the uterus on flushing. Per rectum ultrasound examination of the uterus of mares during flushing suggested that in some mares, the infused fluid may accumulate in the uterine body and not extend to contact the entire uterus, even after massage of the filled uterus per rectum. To increase embryo recovery rates, the flusing technique was altered to allow 3 min contact time of the flush fluid with the uterus during each of three flushes. It was thought that during this time, if the embryo was not directly contacted by the infused fluid, mobility of the embryo might cause it to move into the fluid, and thus be collected. This technique was used in 20 flushes on 14 mares, from 7 to 11 d after ovulation. Embryos were recovered on 18 of the 20 flushes. A total of 21 embryos was recovered, for an embryo recovery rate of 105%. The recovery rate from mares with single ovulations was 13/15 (87%); the recovery rate from mares with multiple ovulations was 8/5 (160%). These rates appear to be higher than those obtained previously in our laboratory and those reported by other workers in the field. These results indicate that further investigation into the efficacy of this procedure is warranted.     

Spontaneous multiple ovulation in the mare and its effect on the incidence of twin embryo collections

Records from 183 nonlactating mares that experienced spontaneous multiple ovulation were examined to determine if: 1) double ovulations are as likely to be unilateral as bilateral; 2) the interval between two ovulations is shorter when the ovulations are unilateral than when they are bilateral; 3) the mean diameter of the two follicles on the day prior to ovulation is less when the ovulations are synchronous and unilateral; 4) for both unilateral and bilateral ovulation, twin embryos are more likely to be detected when double ovulations are asynchronous; and 5) for both synchronous and asynchronous ovulations, twin embryos are more likely to be detected when the ovulations are bilateral. Mares were teased daily with a stallion and follicular development was assessed daily during estrus by ultrasonography. Mares were inseminated daily during estrus and embryo recovery attempts were performed 6 to 7 d post ovulation. Double ovulations occurred as frequently from the same, as from opposite ovaries. The interval between the double ovulations was not shorter (P > 0.05) in unilateral versus bilateral ovulations. In addition, size of the largest and second largest preovulatory follicles was not altered (P > 0.05) by type of ovulation (bilateral vs unilateral) or synchrony of ovulation. Synchrony of ovulations had no affect (P > 0.05) on the incidence of twin embryos recovered. However, more (P < 0.05) twin embryos were recovered from bilateral ovulators compared to unilateral ovulators.     

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.     

The long-term effect of active immunization against inhibin in goats

This experiment addresses the long-term effect of active immunization of goats against a recombinant ovine inhibin α subunit (roIHN-α). In late anestrus 100 μg of roINH-α was administered to 40 pluriparous Boer goat does, followed, 4 weeks later, by a booster injection. Weekly blood samples were drawn to monitor the inhibin binding capacity with the aid of a radio-tracer binding assay. From the onset until 48 h after the end of each estrus, follicular development and ovulation rate were monitored at 24 h intervals by transrectal ultrasonography. Beginning in August and continuing into January, does were mated at every other estrus, and submitted to transcervical embryo collection. Seven months after the first immunization, the does were mated again and permitted to carry to term. All immunized does produced inhibin antibodies, an elevated titre being first detected 2 weeks after primary immunization. Maximum titres were reached after 6 weeks, i.e. 2 weeks after the booster injection. Thereafter, in the course of the following 32 weeks, the titre subsided gradually. The does started cycling by mid-August. At that stage the average number of follicles more than 4 mm in diameter, ovulations and total embryos and ova recovered were 14.7 (±2.3), 5.3 (±0.7) and 4.4 (±1.0), respectively. A steady decline followed and in January the corresponding means were: 5.2 (±0.6) follicles, 3.1 (±0.6) ovulations and 1.2 (±0.4) embryos and ova recovered. When mated toward the end of the breeding season, 85% of the does became pregnant to the first mating and 73% went to term. Healthy kids were born, the average litter size being 2.2 (±0.1). In conclusion, immunization of goats against a recombinant inhibin α-subunit proved to be a practicable means of producing embryos for transfer purposes. After about half a year, when the inhibin antibody titre has subsided, it is possible to return the does to the breeding flock without risking complications with normal breeding activity.     

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

The objective was to compare the reproductive performances associated with the first (Cycle-1), second (Cycle-2), and mid-season (MS-Cycle) ovulations of the breeding season in donor mares that were treated with equine-FSH (eFSH) in the early vernal transition. Mares (n = 15) kept under ambient light were examined ultrasonographically per-rectum starting January 30. When an ovarian follicle ≥25 mm in diameter was detected, twice daily eFSH treatments were initiated. The eFSH treatments ceased when a follicle ≥35 mm was detected, and 36 h later hCG was administered. Thereafter, mares were artificially inseminated every 48 h until ovulation (Day 0). Trans-cervical embryo recovery attempts were performed on Day 8, and subsequently PGF2α was administered. Equine FSH was not administered in the subsequent estrous cycles. In Cycle-2 and in the MS-Cycle, hCG was administered when a follicle ≥35 mm was detected; breeding, embryo recovery, and PGF2α administration, were similar to Cycle-1. Mares had an untreated estrous cycle (no treatment or breeding) between Cycle-2 and the MS-Cycle. All mares developed follicle(s) ≥35 mm after 4.9 ± 0.6 days of eFSH treatment, and subsequently ovulations occurred; mean (95% CI) interval from treatment initiation to ovulation was 7.9 (6.5–9.3) days. The number of preovulatory follicles (≥30 mm) at the time of hCG administration (Cycle-1: 2.2 ± 0.3 compared with Cycle-2: 1.0 ± 0 compared with MS-Cycle: 1.1 ± 0.1 follicles), and the number of ovulations (2.5 ± 0.4 compared with 1.0 ± 0 compared with 1.1 ± 0.1 ovulations) were greater (p < 0.05) in Cycle-1. Nevertheless, mean embryo numbers did not differ among cycles (0.8 ± 0.2 compared with 0.5 ± 0.1 compared with 0.5 ± 0.1 embryo/mare). On average, embryo morphology grade was less (p < 0.05) in Cycle-1 as compared to non-eFSH cycles (combined Cycle-2 and MS-Cycle). This impaired embryo quality could be due to a seasonal effect, or negative effect of the eFSH treatment, which was possibly related to alterations in the hormonal environment (estradiol-17β and progesterone). A prolonged IOI (>21 days) was recorded in 7 of 15 mares following the Cycle-1 ovulation, but not subsequently. In conclusion, eFSH treatment of vernal transitional donor mares stimulated ovulation within only few days of treatment, and the following embryo recovery rate was at least as good as in the subsequent estrous cycles; however, on average, embryos were morphologically impaired. In subsequent estrous cycles in the breeding season, ovulations, embryo recovery rates, and embryo variables did not appear to be negatively affected; however, the first inter-ovulatory interval of the breeding season was prolonged in approximately half of the mares.     

Embryo-initiated oviductal transport in mares.

The hypothesis that equine embryos initiate oviductal transport in mares was tested by placing day 6 uterine embryos in the oviducts of day 2 (n = 10) or day 5 (n = 10) recipient mares and attempting to collect the embryos from the uterus 48 h later. To determine whether the surgical transfer procedure initiated oviductal transport, medium alone was placed in the oviducts of day 2 (n = 10) inseminated mares (sham transfer), and uterine embryo collections were attempted 48 h later. Embryos were transported through the oviduct of day 2 recipients by day 4 (instead of day 5 to 6) in six of ten mares, which was not significantly less (P greater than 0.1) than in day 5 recipients (9 of 10). Oviductal transport was not primarily initiated by the surgical transfer procedure, since oviductal transport occurred in only one sham transfer. There was no significant difference (P greater than 0.1) in the diameter of embryos placed in the oviducts of day 2 and day 5 recipient mares (180 +/- 13.8 versus 187 +/- 11.3 microns, respectively). However, embryos collected from the uterus were significantly smaller (P less than 0.05) in day 2 than in day 5 recipients (375 +/- 85.4 versus 659 +/- 43.6 microns, respectively). One uterine embryo had shed its zona pellucida before being placed in, and transported through, the oviduct of the recipient mare.     

The effects of once or twice daily injections of pFSH on superovulatory response in heifers

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.     

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.