Treatments resulting in pregnancy in nonovulating, hormone-treated oocyte recipient mares

Synchronization of follicle growth between oocyte donor and recipient mares is difficult. To avoid this, recipient mares in a clinical program were used during a period of low follicular activity, and were treated with estrogen before transfer and progesterone after transfer. Five pregnancies were established after oocyte transfer to nonovulating, hormone-treated recipient mares. One pregnancy was lost before 30 d gestation, and the other 4 foals were carried to term. One foal died at birth. Establishment and maintenance of pregnancy in these mares indicates that nonovulating, hormone-treated mares may offer an alternative to cyclic recipients in oocyte transfer programs.     

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.     

Non-surgical embryo transfer and live birth in a llama

The uteri of two donor female llamas were flushed non-surgically and one viable 7-day-old embryo was recovered. The embryo was transferred non-surgically within four hours into a recipient female llama whose estrous cycle was synchronized with the donor by injection of gonadotropin releasing hormone (GnRH). Pregnancy was confirmed 20 days after transfer, at which time the level of serum progesterone was determined and found to be consistent with that of gestation (>/=100 ng/100 ml). A normal and healthy male weighing 33 1bs was born on October 8, 1984, 326 days following transfer of the embryo to the recipient.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

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.     

Enhanced efficiency in the production of offspring from 4- to 8-week-old lambs

The rate of production of offspring from lambs following the transfer of embryos produced by in vitro maturation and fertilisation has been disappointing. This study was conducted on the rationale that gonadotropin treatment protocols used in the past failed to maximise oocyte quality. Consequently, three experiments were conducted in Merino lambs to examine: (1) the method of FSH administration (single versus multiple treatments), (2) the time of oocyte collection (48, 60 or 72 h after the first FSH treatment), (3) progesterone treatment and (4) eCG treatment and its timing on oocyte yield, cleavage rate and blastocyst formation rate. No factor significantly influenced oocyte yield, but all significantly influenced the rates of cleavage and/or blastocyst formation in one or more experiments. The preferred protocol consisted of the administration of 4x40 mg of FSH, no progesterone treatment and with 500 IU of eCG given at the time of the last FSH treatment. The optimal time of oocyte collection was 48 h after the first FSH treatment. A field evaluation, involving lambs from five generations, resulted in the production of 9.0-13.9 lambs per donor lamb; this protocol generated more lambs per donor lamb than protocols previously reported.     

Production of a mitochondrial-DNA identical cloned foal using oocytes recovered from immature follicles of selected mares

Cloned animals possess mitochondria derived from the host ooplast, which typically differ genetically from those of the donor. This is of special concern to horse breeders, as maternal lines are prized and athletic performance is a key factor in genetic value. To evaluate the feasibility of producing mitochondrial-identical cloned foals, we collected oocytes from immature follicles of two mares, BL and SM, maternally related to the donor stallion. In vitro matured, enucleated oocytes were treated with roscovitine-synchronized donor cells and blastocysts were transferred transcervically to recipient mares. In Mare BL, 10 aspiration sessions yielded 45 oocytes, of which 12 matured and seven were successfully recombined. One blastocyst was produced, which did not yield a pregnancy. In Mare SM, three aspiration sessions yielded 53 oocytes, of which 27 successfully recombined. These were assigned to either Scriptaid or Scriptaid plus Vitamin C treatments for the first 12 to 16 hours of embryo culture. Two blastocysts were produced from each treatment. One pregnancy was established after transfer from the Scriptaid treatment. This resulted in a viable foal whose genomic DNA and mitochondrial DNA matched to those of the donor animal. These results indicate that production of mitochondrial-identical cloned foals can be achieved using oocyte recovery from a very small number of selected mares. Despite mitochondrial homogeneity, the results varied with mare; Mare BL yielded both significantly fewer oocytes per aspiration session (P < 0.001) and significantly fewer reconstructed oocytes per oocyte recovered ( P < 0.001) than did Mare SM.     

Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle

Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate.     

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.     

Aberrant heteroplasmic transmission of mtDNA in cloned pigs arising from double nuclear transfer

Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.     

Conditions of embryonic development in the ewe after modification of the hormone balance of the dam

Embryos were recovered in vivo from donor ewes at day 4 and transferred into superovulated unmated recipient ewes given an injection of PMSG (1600 IU) at day 13.5 of the preceding cycle. The recipient ewes were slaughtered at either 5 (group 1) or 8 (group 2) days after transfer. The recovered blastocysts were transferred back into the original donor ewes and pregnancy was allowed to continue until term. In order to observe the effect of the two transfers on blastocyst viability, the recipient ewes were not superovulated in group 3. Only one transfer was carried out at day 4 in group 4, and then pregnancy was allowed to continue in the superovulated recipient ewes.From day 3 to day 8, 12 or 20 (groups 1, 2 + 3 and 4, respectively), the peripheral blood of recipient ewes was sampled once a day for progesterone assay and four times a day for estradiol-17β assay.At 9 or 12 days, 50, 62 and 68% of the transferred embryos were recovered in groups 1, 2 and 3, respectively. These rates were not statistically different from the pregnancy rate in group 4 (64%). After the second transfer, 43, 54 and 40% of the blastocysts developed into lambs (groups 1, 2 and 3, respectively). There was no statistical difference between these results. However, as we noted in previous studies, in spite of the changes in the uterine medium caused by superovulation and which accelerated blastocyst development, the uterus of superovulated ewes could assume pregnancy.The first transfer decreased the number of pregnant ewes to 65% and the second transfer lowered the number of blastocysts giving lambs to 50%. The level of progesterone varied considerably in recipient ewes giving lambs. When the level of progesterone was low at D4, one embryonic mortality was recorded. The level of estradiol-17β showed large variations and seemed to have no relation to blastocyst survival.     

Embryo-uterine interaction in implantation

Pregnant donor (day 3) and non-pregnant recipient rats were hypophysectomized and injected daily for 6 days with 2 mg of progesterone. A single dose of 20 ng of estradiol-17β in saline was administered via a tail vein to either the donor, the recipient, or to both animals; blastocysts were transferred 60 to 90 minutes after the latter injection. Twenty-four hours later uterine implantation sites were delineated by injection of Chicago Blue-B dye. The results indicate that both the blastocyst and the uterus must be exposed to estrogen to obtain normal implantation rates. While 43.2% of the embryos implanted when both the donor and the recipient received estrogen, only 6.3% implanted when only the recipient was injected with estrogen. No implantations were found in animals in which only the embryos had been exposed to estrogen, suggesting that if this steroid was synthesized by the embryo it was insufficient to induce implantation in the rat.     

Regulation of expression of microvillus membrane proteins by estrogen in baboon fetal ovarian oocytes

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estrogen. Ezrin (total and phosphorylated) and SLC9A3R1 expression was localized to the surface of oocytes at mid and late gestation in estrogen-replete baboons and to the cytoplasm in late gestation after estrogen suppression. These results are the first to show that the fetal baboon oocyte expressed ezrin, SLC9A3R1, and alpha-actinin, and that these proteins were localized to the oocyte surface consistent with their role in microvilli development in epithelial cells. The current study also showed that the developmental increase in oocyte expression of alpha-actinin is regulated by estrogen and correlated with the estrogen-dependent increase in oocyte microvilli demonstrated previously. Therefore, we propose that development of oocyte microvilli requires expression of alpha-actinin and that expression of alpha-actinin and localization of ezrin-phosphate and SLC9A3R1 to the oocyte membrane are regulated by estrogen.     

Variation in the responsiveness of female rats to ovarian hormones as a function of preceding hormonal deprivation

Ovariectomized female rats were tested for the display of lordosis behavior 30 days after gonadectomy. They were then tested 7, 14, 21 and 81 days later following estrogen and progesterone treatment. Finally, on Day 88 of the experiment the animals were tested after either estrogen and progesterone treatment or after progesterone alone. The response to estrogen and progesterone treatment was found to be limited on the first test and on the fifth test which occurred after 2 mo without hormone treatment. When hormone treatment was repeated at seven day intervals (Tests 2–4) the tendency to show lordosis increased markedly. On the final test the animals given both hormones showed lordosis, while those which received only progesterone did not. The data suggest that the response to estrogen decreases after estrogen deprivation.     

Clinical report: Recovery of a degenerating 14-day embryo in the uterine flush of a mare 7 days after ovulation

A 15-mm diameter degenerating embryonic vesicle and a normal, 200-u early blastocyst were recovered in a uterine flush of a mare 7 d after ovulation. From its size, the degenerating vesicle appeared to be 13 to 14 d of age. The mare had been bred during a previous cycle and then treated with prostaglandin 9 days after ovulation. The advanced vesicle that was recovered suggests that a conceptus from the previous cycle continued to grow for about 5 d after prostaglandin administration, and remained in the uterus during estrus, when plasma progesterone concentrations were below 1 ng/ml. From the estimated age of the conceptus, its development stopped at about the time the mare was inseminated. Had this conceptus survived through estrus and insemination, superfetation would have occurred.     

Failure of luteolysis leads to prolonged gestation in a bitch: a case report

A healthy, 5-yr-old rough collie bitch exhibited prolonged gestation, characterized by intrauterine fetal death and plasma progesterone concentrations (PPC) above 6 nmol/L/d until Day 65 or 66 of cytologic diestrus, which then only dropped following treatment with dinoprost tromethamine. The bitch showed no signs of parturition prior to laparotomy and hysterotomy on Day 63, which revealed 5 corpora lutea (CL) on the left ovary and 1 on the right ovary; 5 full-term, dead conceptuses without obvious autolysis but with 4 of the 5 placentas detached; and a tightly closed cervix. All conceptuses were in the left uterine horn. Treatment with dinoprost tromethamine was initiated 1 d after hysterotomy and was continued for IO d (120 microg/kg twice daily for a total dose of 240 microg kg/d). The discharge of lochia through the cervix and lactation started 3 d after the onset of treatment. Plasma progesterone concentration was 14.4, 16.2 and 1.6 nmol/L/d on the day of hysterotomy and at 1 and 3 d after the onset of prostaglandin therapy, respectively. The reason for the failure of luteolysis to occur is not known.     

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.     

Effect of diethylstilboestrol on the relationship between LH, PMSG and progesterone during pregnancy in the mare.

Two studies were conducted to determine the relationship between LH and progesterone and between PMSG and progesterone during pregnancy in mares. In the first, samples of jugular blood were collected daily from 7 mares from the first day of oestrus until Day 28 of pregnancy, and in the second, samples were collected weekly from 14 mares from Day 35 of gestation until parturition. In an attempt to prolong secretion of progesterone from accessory corpora lutea, 7 of these 14 mares were injected with increasing doses (2--10 mg) of diethylstilboestrol (DES) between Days 84 and 142 of gestation. The remaining 7 mares received injections of vehicle. Concentrations of LH, PMSG and progesterone in serum were determined by radioimmunoassay. From the onset of oestrus until Day 4 of gestation, serum concentrations of LH and progesterone were negatively correlated (r = 0.67, P less than 0.01), but from Days 5 to 28 a positive correlation (r = 0.80, P less than 0.01) was noted. Likewise, serum concentrations of PMSG and progesterone were highly correlated between Days 35 and 196 in mares injected with DES (r = 0.72, P less than 0.01) and the vehicle (r = 0.75, P less than 0.01). Injections of DES did not influence serum concentrations of LH, PMSG or progesterone, or affect the length of gestation. It was concluded that DES does not influence the maintenance of pregnancy in the mare.     

LHRH - Induced ovulation and fertility of anestrous goats

Seventeen female mature anestrous does were used to study the effect of luteinizing hormone-releasing hormone (LHRH) on ovulation (Experiment I) and on fertility rate and blood estrogen/progesterone concentrations (Experiment II). Laparotomy after day 8 of treatment with a single injection of LHRH (300 ug) and estradiol cypionate (0.2 mg/kg) revealed evidence of ovulation in two out of three and a developed follicle in the third. Similar treatment to six does in Experiment II, when followed by natural mating with a fertile buck, produced pregnancy in two does, pseudopregnancy in two and no effect (nonpregnancy) in the remaining two animals. The pregnant does had normal parturition after 148 to 150 days of gestation. In pregnant does, blood progesterone levels first showed a gradual increase until day 130 (10.07 ng/ml) and then declined sharply at 48 hours before parturition. Estrogen concentration, on the other hand, failed to increase until day 80, and thereafter it reached a peak (1800 pg/ml) at 24 hours prior to parturition; the level declined sharply at 24 hours after parturition. In pseudopregnant does, progesterone levels remained in close proximity with those of pregnant does until day 90, when they started to decline.