Ovariectomized steroid-treated mares as embryo transfer recipients and as a model to study the role of progestins in pregnancy maintenance

Embryo transfer into ovariectomized steroid-treated mares was used as a model to evaluate various progestin/estradiol treatments and to determine the level of progesterone necessary for the maintenance of pregnancy in mares. Once a donor mare was in estrus and had a >/=35 mm follicle, an ovariectomized recipient was selected and assigned to one of three groups: 1) 1 mg estradiol (E(2)) was injected subcutaneously daily until the donor mare ovulated; on the day of the donor mare's ovulation, daily intramuscular injections of 300 mg progesterone (P4) were commenced and continued until the end of the experiment (Day 35); 2) E(2) and P4 treatments were identical except E(2) was continued daily until Day 20; and 3) The same E(2) treatment as Group 1, 0.044 mg altrenogest per kilogram body weight were administered daily until Day 35. Embryos were recovered 7 d after the donor mare's ovulation and were transferred via surgical flank incision. Twenty additional embryos (controls) were transferred into intact recipients that ovulated 1 d before to 3 d after the donor. Pregnancy rates did not differ (P>0.05) among groups at Days 14 or 35. Pregnancy rates at Day 35 for mares administered injectable P4 (70%) were identical to those given altrenogest. Overall, pregnancy rates for ovariectomized-progestin treated recipients (28 of 40, 70%) were similar (>0.05) to that of intact mares (16 of 20, 80%). Dose of P4 was decreased in Groups 1 and 2 to 200 mg (Days 35 to 39), 100 mg (Days 40 to 44), 50 mg (Days 45 to 49) and 0 mg (>/=Day 50). Blood samples were collected once on Days 34, 35, 39, 40, 44, 45, 49 and 50 and assayed for P4. Dose of altrenogest was decreased to 0.022, 0.011, 0.0055 and 0 mg per kilogram body weight at Days 35 to 39, 40 to 44, 45 to 49 and >/=50. Number of mares in Groups 1 and 2 that lost their pregnancy while given 200, 100, 50 or 0 mg P4 was 0, 2, 8 and 4, respectively. Doses of 0.022, 0.011, 0.0055 and 0 mg altrenogest per kilogram body weight resulted in 0, 6, 4 and 3 mares aborting. Fetal death did not occur until concentrations of P4 decreased below 2.56 ng/ml 24 h after injection.     

Establishment and maintenance of pregnancy after embryo transfer in ovariectomized mares treated with progesterone

Pregnancy was established and maintained after embryo transfer in 3 ovariectomized mares treated with progesterone only. Four ovariectomized mares were used as recipients, and 7 transfers were performed. Progesterone in oil, 300 mg i.m. daily, was given starting 5 days before transfer of a 7-day embryo. If the mare was pregnant at 20 days, progesterone treatment was continued to 100 days of gestation. The 3 pregnant mares carried to term and delivered live foals with normal parturition, lactation and maternal behaviour. No differences were seen between pregnant and non-pregnant ovariectomized mares in jugular plasma concentrations of oestrogen, LH or FSH from day of transfer (Day 7) to Day 20. Pregnant ovariectomized mares showed a rise in LH, reflecting production of horse CG, starting at Day 36. Oestrogen values remained low until Day 50.     

Source of oestrogen in early pregnancy in the mare

Oestrogen secretion was determined by oestrogen conjugate (EC) analysis of urine in three groups of pregnant mares: Group I (N = 6), animals ovariectomized on Day 18-19 of gestation with pregnancy maintained by daily administration of an oral progestagen, altrenogest; Group II (N = 9), untreated, pregnant mares; Group III (N = 5) intact, pregnant mares treated daily with altrenogest. The mean EC concentrations in the ovariectomized mares in Group I increased in a constant linear manner from 17 ng/mg Cr on Day 20 to 291 ng/mg Cr on Day 70, with no apparent surge in oestrogen secretion around Day 39. Mean EC concentrations on Days 33, 39 and 44 were respectively 41, 48, and 73 ng/mg Cr. In the intact mares in Groups II and III (shown in parentheses), the mean urinary EC concentrations were 201 (171) ng/mg Cr between Days 20 and 33 of gestation, increased rapidly from 172 (77) ng/mg Cr on Day 33 to a peak of 1066 (895) ng/mg Cr on Day 39, followed by a decline to 637 (719) ng/mg Cr on Day 44. After Day 44, EC concentrations continued to increase in a linear manner to 1191 (842) ng/mg Cr on Day 70. The mean EC concentrations between Days 20 and 70 in Group I were significantly (P less than 0.05) lower than in mares in Groups II and III. EC concentrations in Group III mares were significantly lower (P less than 0.05) than in Group II mares between Days 28 and 34.(ABSTRACT TRUNCATED AT 250 WORDS)     

Periparturient events in ovariectomized embryo transfer recipient mares

Ovariectomized mares treated with progesterone have established and maintained pregnancy after embryo transfer. This study evaluated the ability of ovariectomized embryo transfer recipients to successfully undergo parturition, raise a foal, and return to a useful reproductive status. Periparturient events in three ovariectomized embryo transfer recipient mares and three intact mares were compared. All mares foaled normally. Mammary scores were similar for both groups and all mares produced sufficient colostrum and milk to allow normal growth of healthy foals. Plasma progesterone levels decreased to < 5 ng/ml by Day 4 post partum in both groups. Progesterone concentrations continued to decrease and remained at <1 ng/ml in ovariectomized mares, but increased after the first postpartum ovulation (Day 9 to 15) in intact mares. Endometrial involution as determined by histological evaluation was complete in ovariectomized mares by Day 10 post partum and in intact mares by Day 11 post partum. As assessed by palpation per rectum and clearance of bacteria from the uterus, uterine involution was similar in all mares. The three ovariectomized mares subsequently received embryos by transcervical transfer and two of them established pregnancy. These results indicate that normal parturition, lactation, maternal behavior and uterine involution are independent of ovarian function.     

Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = −0.834, P < 0.05; day 35: r = −0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.     

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.     

Persistence of the luteal phase following ovulation during altrenogest treatment in mares

Two experiments were conducted to test the efficacy of altrenogest treatment in mares. The response to 15-d altrenogest treatment (Experiment 1) was characterized in 20 mares that were given 22 mg daily of altrenogest in oil (n = 10) or in gel (n = 10) from Day 10 to 25 after ovulation. In 17 mares, luteolysis occurred during altrenogest treatment (Day 17.7 +/- 0.5), while 2 mares retained their corpus luteum (CL), and 1 mare had a diestrous ovulation on Day 16, resulting in a prolonged luteal phase. Ten of the 17 mares in which the CL had spontaneously regressed returned to estrus after the end of treatment, and ovulated 5.7 +/- 0.8 d after the end of altrenogest treatment. Two of these 17 mares ovulated 2 and 3 d after the end of altrenogest treatment but ovulation was not accompanied by estrous behavior, and 5 mares ovulated during altrenogest treatment resulting in an interovulatory interval of 22.4 +/- 1.1 d (range: 20 to 25d). Five mares which ovulated during altrenogest treatment and 2 mares which ovulated during silent estrus after the end of altrenogest treatment failed to regress the CL around 14 d post ovulation, and had a prolonged luteal phase. In Experiment 2, the effect of altrenogest administered from luteolysis to ovulation on duration of the subsequent luteal period was analyzed. In 6 mares altrenogest was begun on Day 14 post ovulation and continued until the hCG-induced ovulation. The interval from ovulation during altrenogest treatment to spontaneous luteolysis was 45.6 +/- 2.4 d (range: 40 to 54d) in altrenogest-treated mares and was significantly longer than in 10 untreated control mares (14.5 +/- 0.3 d, range: 13 to 16d). The results suggest that the oil and gel altrenogest preparations are equally effective in modulating estrous behavior and time to estrus and ovulation. Altrenogest treatment started late in diestrus appears to result in a high incidence of ovulation during treatment and when luteolysis and ovulation occur during treatment; the subsequent luteal phase is frequently prolonged due to failure of regression of the CL.     

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.     

Effects of norgestomet, altrenogest, and/or estradiol on follicular and hormonal characteristics of late transitional mares

Two experiments were conducted to investigate the effect of norgestomet and altrenogest, alone or in combination with estradiol, on late transitional mares. In the first experiment, 32 mares were assigned to four treatment groups: controls (C), those treated with 1.5 mg of norgestomet (N1), 3.0 mg norgestomet (N2) or 26 mg altrenogest (AT). Treatments were initiated during the months of April and May and given daily for 15 d. During treatment, altrenogest suppressed estrous behavior and diameter of the largest follicle, whereas norgestomet had no effect at either dose. The rise in serum luteinizing hormone (LH) levels following the withdrawal of altrenogest treatment was significantly greater than that for the other three groups. In the second experiment, 24 late transitional mares were assigned to three treatments: controls (C), those receiving 26 mg altrenogest (AT) daily, or 26 mg altrenogest plus 10 mg estradiol (AE) daily for 16 d. Both altrenogest treatments suppressed estrous behavior and follicular growth compared with controls. However, suppression of follicular activity was significantly greater for the combined steroid treatment. Following treatment, the interval to ovulation and estrus was longer for the combined steroid group. We concluded that: 1) norgestomet at a dose up to 3.0 mg per day had no effect on follicular activity, estrous behavior or serum LH levels in late transitional mares, 2) estradiol combined with altrenogest had greater suppressive activity on follicular growth than altrenogest alone, and 3) the greater suppression by the combined steroid treatment had no advantage over altrenogest alone on induction of estrus and ovulation in late transitional mares.     

Improvement of pregnancy rate in Japanese Black cows by administration of hCG to recipients of transferred frozen-thawed embryos

Japanese Black primiparous and multiparous beef cows (n = 120) were selected as recipients and randomly divided into three groups (A, B, and C) of 40 recipients each. Group A received an intramuscular (i.m.) treatment of 1500 IU human chorionic gonadotropin (hCG) on day 1 (day 0 = onset of estrus), while Group B received an i.m. treatment of hCG on day 6. Group C received an i.m. treatment of 5 ml saline on day 6 as a control. On day 7, frozen-thawed embryo transfer was conducted in all groups, and pregnancy was diagnosed by palpated per rectum 40-50 days after the transfer. Twelve recipients were randomly selected from each group. Plasma progesterone (P) and estradiol-17beta (E2) concentrations were determined in these recipients on days 6, 7 and 14, and at the time of pregnancy diagnosis, and their ovaries were examined for a corpus luteum and follicles by palpated per rectum. The pregnancy rate in Group B was higher (67.5%. P < 0.05) than the rate in Group C (45.0%) and in Group A (42.5%). The plasma P concentration on day 14 tended to be higher although not significantly in Group B than in Groups C and A. At the time of pregnancy diagnosis, the blood P concentration of pregnant recipients in Group B was higher (P < 0.05) than that of those in Groups C and A. The plasma E2 concentrations on days 7 and 14 were lower (P < 0.05) in Group B than in Groups C and A. These results showed that administration of hCG 6 days after estrus improved the pregnancy rate for non-surgical frozen embryo transfer 7 days after estrus by enhancing luteal function and depressing E2 secretion.     

Reproductive function of mares given daily injections of prostaglandin F2alpha beginning at day 42 of pregnancy

Mares at Day 42 of pregnancy received daily intramuscular (i.m.) injection of 5 mg of prostaglandin F2alpha (PGF(2alpha)) until the beginning of the first (Group I, n = 3) or second estrous cycle (Group II, n = 2). All mares aborted 3 to 4 d after the first injection; they displayed estrus 2 to 6 d after this injection. As determined by palpation per rectum and serum progesterone levels, each estrus was accompanied by an ovulation. Endometrial cups did not regress after PGF(2alpha) treatment since serum samples from the mares contained pregnant mare serum gonadotropin (PMSG) for at least 30 d after first injection, as determined by mare immunopregnancy test. After the first estrus, two of three mares in Group I displayed a prolonged diestrus (> 25 d). In contrast, the first estrous cycle was short (8 to 12 d) for mares in Group II. Serum progesterone levels in the first 6 d postovulation were lower (P < 0.05) for Group II than for Group I, indicating that formation of the corpus luteum was impaired by daily injections of PGF(2). Results indicate that 1) daily injections of PGF(2alpha) can induce abortion in mares at Day 42 of pregnancy, 2) abortion is followed by estrus and ovulation, 3) the endometrial cups do not regress as a result of this treatment, and 4) daily injections of PGF(2) can impair early corpus luteum development.     

Control of FSH, follicular development and estrus synchronization in the mare with steroid-free follicular fluid

Twenty-two pony mares were used in a project designed to determine the effectiveness of different treatments in controlling FSH, follicular development and synchronization of estrus and ovulation. Mares in Group 1 (n=8) received daily oral altrenogest (0.044 mg/kg); those in Group 2 (n=7) received daily altrenogest (0.044 g/kg) and, during the last 4 days of treatment they received steroid-free follicular fluid, (15 cc) intravenously (I.V.) two times a day; Mares in Group 3 (n=7) received daily intramuscular (I.M.) injections of progesterone (80 mg) and estradiol valerate (7 mg). All treatments lasted for 10 days, at the end of which prostaglandin (PgF(2)alpha, 10 mg) was administered. Sexual behavior, follicular development and FSH concentrations were monitor daily. Concentrations of FSH in Group 2 mares, were not significantly different (P>0.05) from those of Group 1 until the mares in Group 2 were treated with follicular fluid (P<0.05). Concentrations of FSH in Group 3 mares, were significantly lower than those of Groups 1 and 2 (P<0.05) until the mares in Group 2 were treated with steroid-free follicular fluid. At this point there was no significant difference between groups 2 and 3 (P>0.05). Steroid-free follicular fluid appears to induce atresia in larger follicles (>11 mm), and the initiation of new follicular wave. The combination of progesterone and estradiol valerate appears to delay follicular growth and not to induce atresia, since larger follicles (>11 mm) continued to grow after treatment. Both treatments (groups 2 and 3) resulted in ovulations within 5 days period. The treatment in Group 1 did not have any effect on FSH or follicular development and ovulations were dispersed through a 9-day period. We concluded that steroid-free follicular fluid offers a new possibility to synchronize ovulation in the mare by controlling FSH and follicular development.     

Treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis

The objective was to determine if long-term treatment with trimethoprim sulfamethoxazole (antimicrobial), pentoxifylline (anti-inflammatory/anti-cytokine) and altrenogest (synthetic progestin), would improve pregnancy outcome in mares with experimentally induced placentitis. Seventeen normal, pregnant pony mares were enrolled in the study at 280-295 d of pregnancy. Placentitis was induced in all mares by intra-cervical inoculation of Streptococcus equi subsp. zooepidemicus (10⁷ CFU). Five mares served as infected, untreated control animals (Group UNTREAT). Twelve mares (Group TREAT) were infected and given trimethoprim sulfamethoxazole (30 mg/kg, PO, q 12h), pentoxifylline (8.5 mg/kg, PO, q 12h) and altrenogest (0.088 mg/kg, PO, q 24h) from the onset of clinical signs to delivery of a live foal or abortion. Blood samples were cultured from all foals at delivery and fetal stomach and thoracic contents were obtained for culture from dead fetuses. More mares in Group TREAT delivered viable foals (10/12; 83%; P < 0.05) than mares in Group UNTREAT (0/5; 0%). Ten of 12 foals (83%) in Group TREAT had negative blood cultures at birth. All foals in Group UNTREAT (5/5; 100%) had positive cultures from one or more samples (blood, stomach contents, and thoracic fluid). Bacteria were recovered from uterine culture samples in both groups. Streptococcus equi subsp. zooepidemicus was the predominant organism recovered from fetal/foal or mare culture samples. The authors inferred that administration of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest may improve the viability of foals from mares with experimentally induced placentitis.     

Evaluating recipient and embryo factors that affect pregnancy rates of embryo transfer in beef cattle

The objectives of this experiment were to determine the effects of corpus luteum characteristics, progesterone concentration, donor-recipient synchrony, embryo quality, type, and developmental stage on pregnancy rates after embryo transfer. We synchronized 763 potential recipients for estrus using one of two synchronization protocols: two doses of PGF2alpha (25 mg i.m.) given 11 d apart (Location 1); and, a single norgestomet implant for 7 d with one dose of PGF2alpha (25 mg i.m.) 24 h before implant removal (Location 2). At embryo transfer, ovaries were examined by rectal palpation and ultrasonography. Of the 526 recipients presented for embryo transfer, 122 received a fresh embryo and 326 received a frozen embryo. Pregnancy rates were greater (P < 0.05) with fresh embryos (83%) than frozen-thawed embryos (69%). Pregnancy rates were not affected by embryo grade, embryo stage, donor-recipient synchrony, or the palpated integrity of the CL. Corpus luteum diameter and luteal tissue volume increased as days post-estrus for the recipients increased. However, pregnancy rates did not differ among recipients receiving embryos 6.5 to 8.5 days after estrus (P > 0.1). There was a significant, positive simple correlation between CL diameter or luteal tissue volume and plasma progesterone concentration (r = 0.15, P < 0.01 and r = 0.18, P < 0.01, respectively). There were no significant differences in mean CL diameter, luteal volume or plasma progesterone concentration among recipients that did or did not become pregnant after embryo transfer. We conclude that suitability of a potential embryo transfer recipient is determined by observed estrus and a palpable corpus luteum, regardless of size or quality.     

Melatonin treatment of embryo donor and recipient ewes during anestrus affects their endocrine status, but not ovulation rate, embryo survival or pregnancy

Thirty-two Border Leicester x Scottish Blackface ewes that lambed in March were individually penned with their lambs from April 16th and given daily an oral dose of 3 mg melatonin at 1500 h (Group M). A further 32 acted as controls (Group C). Within each group half were used as embryo donors (Group D) following superovulation and half received embryos (Group R) following an induced estrus. Prior to weaning on 21 May ewes received ad libitum a complete diet providing 9 megajoules (MJ) of metabolizable energy and 125 g/kg crude protein. Thereafter each received 1.6 kg of the diet daily. In early June each ewe received an intravaginal device (300 mg progesterone) inserted for 12 d. Donors were superovulated with 4 i.m. injections of porcine FSH 12 h apart, commencing 24 h before progesterone withdrawal. Ovulation in recipients was induced with 800 IU PMSG injected i.m. at progesterone removal. Donor ewes were inseminated 52 h after progesterone withdrawal. Embryos were collected 4 d later and transferred to recipients. Melatonin suppressed plasma prolactin (P < 0.001) and advanced estrus (P < 0.05) and timing of the LH peak (P < 0.05). These events also occurred earlier in donors than in recipients (P < 0.01). Mean (+/- SEM) ovulation rates for melatonin-treated and control donors were 5.5 +/- 0.71 and 4.7 +/- 0.66, respectively (NS). Corresponding recipient values were 3.3 +/- 0.40 and 3.4 +/- 0.39 (NS). Mean (+/- SEM) embryo yields were 2.9 +/- 0.64 and 2.6 +/- 0.73 for melatonin-treated (n = 15) and control (n = 16) donors, respectively, and for the 12 ewes per treatment that supplied embryos, corresponding numbers classified as viable were 2.7 +/- 0.47 and 2.3 +/- 0.61 (NS). Following transfer, 57% of embryos developed to lambs when both donor and recipient received melatonin, 86% when only the donor received melatonin, 91% when only the recipient received melatonin, and 67% when neither received melatonin (NS). Thus, embryo survival following transfer was not improved by treating recipients with melatonin. Gestation length and lamb birthweights were unaffected by melatonin. Unlike nonpregnant control ewes, melatonin-treated recipients that failed to remain pregnant sustained estrous cyclicity following embryo transfer.     

Effect of ovariectomy on pregnancy in mares.

Pony mares were bilaterally ovariectomized at different stages of pregnancy between Days 25 and 210. Abortion or fetal resorption occurred within 2 to 6 days after operations in all 14 mares ovariectomized between Days 25 and 45 and after an interval of 10 to 15 days in 9 of 20 other ovariectomized between 50 and 70 days. All 12 mares ovariectomized on either 140 or 210 days carried their foals to normal term. The termination of early pregnancy was preceded by a loss of uterine tone and of a palpable uterine bulge. The mean length of gestation in all mares in which pregnancy was not interrupted by ovariectomy was not significantly different from that in a group of contemporary control mares. Plasma progestagen concentrations dropped to less than 2 ng/ml after ovariectomy, whether or not pregnancy was maintained. Mares ovariectomized on Day 25 and injected with 100 mg progesterone daily for 10 or 20 days remained pregnant during treatment but showed a loss of uterine tone and the fetal bulge disappeared within 4 to 6 days after the end of treatment. Non-pregnant ovariectomized or intact seasonally anoestrous mares injected i.m. with 50 or 100 mg progesterone daily for 8 weeks showed changes in uterine tone, length and thickness similar to those occurring in mares during early pregnancy.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

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.     

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.     

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.