Mobility of twin embryonic vesicles in mares

The patterns of intrauterine mobility and fixation of multiple embryos were studied by ultrasonography in 33 mares with twins, six mares with more than two embryos, and 18 mares with singletons. For both single and multiple embryos, the embryonic vesicles showed a preference for the uterine body on days 11 (57% in body) and 12 (58%) and for the uterine horns on days 13 (40% in body), 14 (20%), 15 (15%), and 16 (1%). The preference for the uterine body was characteristic of vesicles that were 3 to 9 mm in diameter. Based on the number of individual embryos which were in different locations between two successive daily examinations, fixation (cessation of mobility) occurred for 97% of the embryos by day 16 and for all embryos by day 18. For 33 mares with twins, fixation involved one uterine horn in 23 mares and both horns in 10 mares (significantly different from equality). Location determinations were made every five minutes during two-hour trials on days 12, 13, or 14 in nine mares with singletons and ten mares with twins. Individual embryos of twin sets had mobility patterns similar to those of singletons. Summed over singletons and twins, the vesicles moved from one horn to another a mean of 0.9 times per two-hour trial (equivalent to 11 times per day). The smaller embryo of twin sets on the average spent more time in the uterine body, but this finding was attributed to their smaller diameter. The observed frequency with which both embryos of twin sets were simultaneously in a given segment of the uterus (28%) was greater (P<0.01) than the expected frequency if each embryo moved independently of the other (18%). Results indicated that 64% of the location changes of twin embryos occurred independently of one another, supporting the hypothesis that the embryonic vesicle plays an active role in its mobility.     

The outcome of twin pregnancies in mares

Brood-farm veterinarians were surveyed and theriogenology records were examined to obtain information on the natural outcome of rectally-diagnosed twin pregnancies. Four of the veterinarians estimated that 50% of mares with twin embryos had single foals. In the analyses of palpation records, 31 53 (58%) of the diagnosed twin pregnancies terminated in birth of one foal. However, the loss of one embryo without the loss of the other occurred significantly more often before day 40-42 (30 53 ; 57%) than after day 40-42 (1 16 ; 6%). Of the remaining 15 mares with diagnosed twins at day 40-42, 5 had two foals and 10 had no foals. The methods used for intervention when twins were diagnosed were unsatisfactory. Complete termination of pregnancy with a prostaglandin or an intrauterine flushing resulted in failure to reestablish a singleton pregnancy during the operational breeding season in 10 11 mares. Attempts to eliminate one embryo resulted in loss of both in 6 7 mares. The results indicated that, at the present time, nonintervention should be given more consideration as a method of handling twin pregnancies.     

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.     

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.     

Factors affecting spontaneous reduction of corpora lutea and twin embryos during the late embryonic/early fetal period in multiple-ovulating dairy cows

Spontaneous reduction of advanced twin embryos has been described in high-producing, Holstein-Fresian (Bos taurus) dairy herds. The first objective of the current study was to determine whether management and cow factors could have an effect on such a reduction in twin pregnancies during the early fetal period. Because loss of a corpus luteum was noted in cows suffering twin reduction, we expanded our study to include multiple-ovulating cows carrying singletons. Pregnancy was diagnosed and confirmed from Days 28 to 34 and 56 to 62 postinsemination. Sixty-nine (23.5%) of 293 pregnant cows with two corpora lutea carrying singletons and 132 (28.4%) of 464 twin pregnancies recorded on first pregnancy diagnosis subsequently lost one of the corpora lutea or one of the embryos, respectively. Thirty-four (25.8%) of the 132 twin pregnancies suffering embryo reduction lost one corpus luteum along with the embryo. Corpus luteum reduction always occurred in the ovary ipsilateral to the gravid horn suffering embryo reduction. Binary logistic regressions were performed considering corpus luteum and embryo reduction as dependent variables in single and twin pregnancies, respectively, and several management- and cow-related factors as independent variables. In cows carrying singletons, the risk of corpus luteum reduction was 14.3 (1/0.07) times lower for a given herd, whereas the interaction season by laterality significantly affected corpus luteum reduction such that in cows with two corpora lutea ipsilateral to the horn of pregnancy, the risk of reduction decreased during the winter period. In cows carrying twins, ipsilateral twin pregnancies were 3.45 (1/0.29) times more likely to undergo the loss of one embryo than bilateral twin pregnancies. As an overall conclusion, both corpora lutea and embryos were vulnerable to the effects of stress factors during the early fetal period in cows maintaining their pregnancies. A strong unilateral relationship between the corpus luteum and the conceptus was also observed.     

Spontaneous reduction of advanced twin embryos: its occurrence and clinical relevance in dairy cattle

Twin pregnancies represent a management problem in dairy cattle since the risk of pregnancy loss increases, and the profitability of the herd diminishes drastically as the frequency of twin births increases. The aim of this study was to monitor the development of 211 twin pregnancies in high producing dairy cows in order to determine the best time for an embryo reduction approach. Pregnancy was diagnosed by transrectal ultrasonography between 36 and 42 days after insemination. Animals were then subjected to weekly ultrasound examination until Day 90 of gestation or until pregnancy loss. Viability was determined by monitoring the embryonic/fetal heartbeat until Day 50 of pregnancy, and then by heartbeat or fetal movement detection. Eighty-six cows (40.8%) bore bilateral and 125 (59.2%) unilateral twin pregnancies. Embryo death was registered in one of the two embryos in 35 cows (16.6%), 33 of them at pregnancy diagnosis. Pregnancy loss occurred in 22 of these cows between 1 and 4 weeks later. Thus, 13 (6.2% of the total animals) cows, carrying one dead of the two embryos, maintained gestation. Total pregnancy loss before Day 90 of pregnancy (mean 69 +/- 14 days) was registered in 51 (24.2%) cows: 7 (8%) of bilateral pregnancies and 44 (35.2%) of unilateral pregnancies, and it was higher (P = 0.0001) for both right (32.4%, 24/74) and left (39.2%, 20/51) unilateral than for bilateral (8.1%, 7/86) twin pregnancies. The single embryo death rate was significantly (P = 0.02) lower for cows with bilateral twins (9.3%, 8/86) than for total cows with unilateral twins (21.6%, 27/125). By way of overall conclusion, embryo reduction can occur in dairy cattle, and the practical perspective remains that most embryonic mortality in twins (one of the two embryos) occurs around Days 35-40 of gestation, the period when pregnancy diagnosis is generally performed and when embryo reduction could be tried.     

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.     

Clinical implications of induced twin reduction in dairy cattle

Embryo reduction may prevent the negative effects of twinning in dairy cattle; however, the technique may carry an additional risk of pregnancy loss. The aim of this study was to evaluate the effect on pregnancy maintenance of embryo reduction by manual amnion rupture in unilateral and bilateral twin pregnant cows. A secondary objective was to examine the dynamics of endocrine factors following the treatment. On Day 35-41 of gestation 55 cows bearing two live twin embryos (28 bilateral, 27 unilateral) were randomly assigned to a twin reduction group (n = 27; cows fitted with a progesterone releasing intra-vaginal device for 21 days after manual amnion rupture) or control group (n = 28; untreated cows). Pregnancy loss before Day 90 was recorded in nine control and eleven twin reduction cows (32.1% vs 40.7%, respectively, p = 0.508). Logistic regression models indicated that laterality was the only variable significantly affecting pregnancy loss. The pregnancy loss risk was 8.7 times higher for unilateral than for bilateral twin pregnancies (59.3% vs 14.3%, respectively, P < 0.001) yet was similar in the unilateral control and unilateral twin reduction cows (62.3% vs 53.8%, respectively, P = 0.581). In contrast, four of 14 cows with bilateral twin pregnancies undergoing twin reduction lost their pregnancies while no losses were recorded in control cows with bilateral pregnancies (P = 0.049). A rise in plasma progesterone concentration was detected on the day following treatment in the twin reduction group and concentrations remained high within the first week of treatment. Plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations fell between Day 35-41 and Day 42-48, regardless of treatment. Our findings indicate that embryo reduction by manual amnion rupture did not carry an additional risk of pregnancy loss for unilateral twin pregnancies, whereas it increased the risk of pregnancy failure in bilateral twin pregnancies. However, benefits of preventing cows from delivering twins might also be considered when assessing the success of embryo reduction in bilateral twin pregnancies.     

Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro.

Late morulae and blastocysts produced in vitro were nonsurgically transferred to heifers by unilateral (n = 184) or bilateral (n = 94) transfer. Of the recipients, 58% had serum progesterone values greater than 1.4 ng ml-1 on day 21 and rectal palpation on day 35 showed that 50% (138 of 278) were pregnant. The embryonic mortality rate between days 21 and 35 was estimated to be about 14% and between days 36 and 90 to be about 12%. Of the animals, 8% aborted between days 91 and 250 of pregnancy. No difference was observed in pregnancy rates between unilateral transfer of one (47%) or two embryos (49%) and bilateral transfer (53%), or in the twinning rate between bilateral transfer (42%) and unilateral transfer of two embryos (33%). The pregnancy rate was 54% with embryos evaluated as morphologically excellent or good, 51% with fair embryos and 26% with poor ones. A higher pregnancy rate (60%) was obtained after embryo transfer when the synchrony between recipient and embryo was -1 day.     

A biological embryo-reduction mechanism for the elimination of excess embryos in mares

Only one embryo was found in each of 15 pregnant mares in which multiple, synchronous (0 or 1 day apart) ovulations were induced with a pituitary extract. In the brood-farm mares, fewer (P<.05) twin pregnancies were associated with synchronous, double ovulations (0 39 ), than with asynchronous, double ovulations (9 57 ). Higher (P<.05) pregnancy rates (number of mares pregnant, regardless of number of embryos) were obtained in pituitary extract-treated mares with multiple, synchronous ovulations (15 18 ) than in treated mares with single ovulations (6 14 ). Similarily, pregnancy rates were higher (P<.01) in brood-farm mares with double, synchronous ovulations (39 47 ) than in brood-farm mares with single ovulations (1320 2426 ). Our interpretation is that ova produced by synchronous, double ovulations are viable and fertilizable (indicated by higher pregnancy rates), but that one of the resulting embryos is eliminated (indicated by the absence of twins).     

One year old fillies can be successfully used as embryo donors

One year old fillies are able to conceive but, usually, not to give birth to a living foal. Although embryo transfer allows the production of foals from mature mares with repeated pregnancy losses, no reports are available on the use of one year old fillies as embryo donors. To evaluate this possibility, eleven 12-16 months old Haflinger mares were inseminated with fresh semen and subjected to embryo recovery. Some of the recovered embryos were non-surgically transferred into synchronized mature recipients. Pregnancies were terminated using PGF2alpha at day 25. Fillies' embryo recovery rate and their recipients' pregnancy rate at day 25 were compared with those achieved in two years old and mature mares of the same breed, subjected to the same management. Embryo recovery rate was 21/44 (47.7%), 12/16 (75%) and 22/26 (84.6%) (P>0.01) for one year old, two years old and mature mares, respectively. Five/7 (71.4%) one year old donors' embryos resulted in a pregnancy after transfer and 4/7 (57.1%) developed until day 25. Significant differences in pregnancy rates after transfer between donors' age groups were not observed; no short term side effects resulted from the use of fillies as embryo donors. This study showed that one year old mares employed as embryo donors produce embryos both morphologically normal and able to develop in recipient mares at least up to day 25 of pregnancy.     

Embryo recovery from exercised mares

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 °C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F_2α was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 °C to a mean of 39.9 °C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8 ± 0.5 compared with 41.5 ± 0.5 mm diameter; P < 0.05), and had an increased time from PGF_2α administration to subsequent ovulation (8.47 ± 0.337 compared with 9.27 ± 0.294 d; P < 0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P < 0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P = 0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.     

Effect of exogenous progesterone on pregnancy rates after surgical embryo transfer in mares

A completely randomized experimental design was used to investigate the effect of supplemental progesterone on pregnancy rates of recipient mares. Every other recipient mare received daily 200 mg progesterone in oil beginning the day of surgical embryo transfer and lasting until either Day 120 of pregnancy or until pregnancy failure was confirmed by ultrasound. Progesterone supplementation did not affect pregnancy rate (P > 0.05). Overall, embryos that did not result in pregnancy were of greater mean diameter than embryos that resulted in pregnancy (P < 0.05). Pregnancy rates tended (P < 0.1) to be greater in recipients that were detected to be ovulating the same day or prior to that of the donor and that had been supplemented with progesterone (75 %) as opposed to untreated control mares of the same synchrony group (40 %). Progesterone supplementation did not affect the incidence of embryonic loss; however, there was a slightly higher loss of pregnancies between Day 15 and 30 in treated versus untreated recipients. There was no effect (P > 0.05) of treatment on pregnancy rate for embryos recovered from fertile versus subfertile donor mares. However, overall, there tended (P < 0.1) to be fewer pregnancies with embryos recovered from subfertile (50 %) as compared to fertile donors (75 %). It was concluded that supplemental progesterone at the dosage and frequency described was not beneficial in improving pregnancy rates in cyclic recipient mares after surgical embryo transfer.     

Identifying Maternal Constraints on Fetal Growth and Subsequent Perinatal Outcomes Using a Multiple Embryo Implantation Model

IntroductionAlthough the majority of singleton births after in vitro fertilization (IVF) are uncomplicated, studies have suggested that IVF pregnancies may be independently associated with low birth weight (LBW), preterm birth (PTB), and perinatal mortality. These outcomes complicate multiple gestations as expected, but have also been reported in singletons. A multiple embryo implantation model allows for assessment of the early in utero environment, and therefore, assessment of any maternal constraints on developing fetuses. We question whether adverse perinatal outcomes associated with assisted reproductive techniques (ART) occur as a result of maternal physiologic adaptations.ResultsA total of 17,415 cycles were analyzed. The average maternal age was 36.9 (±5.0) years. An overall fertilization rate of 73.4% generated approximately 48,708 good quality cleavage-stage embryos. In most patients (92.8%), an average of 3 embryos were transferred. The clinical pregnancy rate was 39.2% (n = 6,281). The overall occurrence of multiple gestations was 38.2% (n = 2,608) consisting of 2,038 twin, 511 triplet, and 59 quadruplet pregnancies. Of these multiple gestations, 18.6% of twin, 54.2% of triplet and 76.3% of quadruplet gestations spontaneously reduced. Failure of the implanted embryo to progress was not related to maternal age. Singleton newborns resulting from multiple implantation sites had lower birth weights (P<0.01) and shorter gestational ages (P<0.01) than those from a single implanted embryo. The number of embryos transferred did not affect the gestational length of singleton newborns. Although the birth weights of singletons from multiple implantation sites (virtual singletons) were lower than true singletons, the birth weight of virtual singletons were comparable to the birth weights of true twin, triplet, and quadruplet live births. Multiple logistic regression revealed that virtual singletons were an independent risk factor for PTB (odds ratio: 4.55, 95% CI 2.23–9.29) and LBW (odds ratio: 3.61, 95% CI 1.78–7.32), even after controlling for the number of oocytes, stimulation protocol type, sperm source, total gonadotropins administered, age, embryo quality, and day of embryo transfer.ConclusionsOur study highlights that embryonic implantation sites during early gestation set the growth profile of each embryo, dictating later growth patterns. Specifically, spontaneous reduction of an embryo after multiple embryo implantations can confer greater perinatal risk in the form of LBW and PTB to the surviving fetus. Our findings suggest that maternal constraints or physiologic adaptations maybe one of the mechanisms mediating adverse perinatal outcomes when multiple embryo implantation occurs.     

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.     

Improvement of in vitro fertilisation after treatment with buserelin,an agonist of luteinising hormone releasing hormone

Treatment with buserelin, an agonist of luteinising hormone releasing hormone, and human menopausal gonadotrophin was compared with the conventional treatment of clomiphene citrate and human menopausal gonadotrophin in the outcome of in vitro fertilisation. Seventy seven infertile women had 83 cycles of treatment with buserelin and human menopausal gonadotrophin, and concurrently another 328 infertile women were treated with clomiphene citrate and human menopausal gonadotrophin. Seven (8%) cycles were cancelled owing to inadequate superovulation or ovarian hyperstimulation in the women receiving buserelin and 103 (31%) were cancelled because of poor follicular development in those receiving clomiphene citrate. The mean number of oocytes recovered was significantly higher with buserelin (9·5 (SD 4·5) v 5·5 (2·2)) as was the mean number of embryos obtained (4·3 (2·4) v 2·9 (1·7)). Significantly more women who had an embryo transfer became clinically pregnant after treatment with buserelin (53% (30/57) v 30% (48/159), or 36% v 14% of treatment cycles). Altogether 33% (10) of pregnancies in women treated with buserelin were multiple compared with 23% (11) in those treated conventionally. Of the 17 completed pregnancies in women treated with buserelin, 11 resulted in the birth of live babies (eight singletons, two sets of twins, and one set of triplets) and six failed, five before 12 weeks'' gestation and one at 22 weeks. The 13 continuing pregnancies (32 weeks) were eight singletons, two sets of twins, and three sets of triplets. Of the 48 completed pregnancies in women treated with clomiphene citrate, 35 resulted in the birth of live babies (26 singletons, five sets of twins and four sets of triplets) and 13 failed, eleven before 12 weeks'' gestation and two by 27 weeks.Buserelin increased the chance of pregnancy after in vitro fertilisation compared with conventional treatment, but the risk of multiple pregnancy may be increased.     

Some observations on early embryonic death in mares

Early embryonic death (EED) was studied in 354 pregnant mares during two breeding seasons. Pregnant mares from six independent research projects were examined at 15, 20, 25, 30, 35, 40 and 50 days post-ovulation using a real-time ultrasound scanner. Mares that had previously been treated with anabolic steroids tended (P<0.10) toward a higher incidence of EED than untreated mares (21.1% vs 0%). Of the 59 pregnancies obtained from embryo transfer in 1982, 10 had undergone EED by day 50 (16.9%) compared to 10.1% for embryo transfer recipients in 1983. Overall, there was a 13.3% incidence of EED in embryo transfer recipients. This percentage was nearly identical to 13.4% incidence of EED for mares inseminated with fresh semen. In 1982, mares inseminated with frozen-thawed semen tended (P<0.10) to have a higher incidence of EED compared to those inseminated with fresh semen (34.6% vs 15.8%). However, in 1983 there was no difference (P>0.05) in the incidence of EED between mares inseminated with frozen-thawed and fresh semen (16.7% vs 12.5%). Further, combined over both years, there was no difference (P>0.10) in the incidence of EED (24.2% vs 14.4%). There was a 38.9% incidence of EED in infertile mares.Of the 354 pregnant mares, the overall incidence in EED through day 50 postovulation was 17.3%. The majority of EED (77.1%) occurred prior to day 35 post-ovulation. During the period 15 to 35 days post-ovulation, a greater (P<0.05) incidence of EED occurred between days 15 to 20 (26.2%) and 30 to 35 (29.5%) post-ovulation.     

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).     

Effect of reproductive status on twinning and on side of ovulation and embryo attachment in mares

Brood-farm records were used to test several hypotheses concerning twinning and inequalities in the side of ovulation and embryo attachment. Ovulation occurred more frequently (P<0.05) from the left ovary (61% versus 39%) in maiden mares, but with equal frequency from either ovary in lactating and barren mares. The embryo attached more often (P<0.05) to the left horn in lactating mares (60%) and to the right horn in barren (59%) and maiden (67%) mares. Double ovulations and twin embryos were diagnosed more frequently (P<0.05) in barren mares (11% and 6%, respectively) than in lactating mares (5% and 1%). Pregnancy rates for double ovulations were affected (P<0.01) by the length of the interval between the two ovulations (0 days, 83%; 2 days, 87%; 4 days, 67%; 6 days, 54%; 8 days or more, 38%). Pregnancy rates were higher (P<0.01) for double ovulations 0 or 2 days apart (84%) than for single ovulations (54%). Twins were diagnosed more frequently (P<0.05) when double ovulations were two or more days apart (9 32 ) than when the ovulations were synchronous (0 19 ). These results support the conclusion that an embryo-reduction mechanism exists in mares for the elimination of excess embryos and indicate that the mechanism loses its effectiveness when the embryos originate from asynchronous ovulations.     

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.