Recipient Mares Research Articles

In-vitro equine embryo production: an adventure in multi-unit collaboration

The accessibility of assisted reproductive techniques related to oocyte collection and in vitro fertilization in the mare is increasingly becoming more widespread for private equine practitioners and their clients. The necessity of hauling a valuable mare long distances to the closest facility for transvaginal aspiration (TVA) of oocytes and on site intracytoplasmic sperm injection (ICSI) over a period of potential weeks before obtaining a pregnancy no longer exists. Instead, a mare’s primary veterinarian can manage the patient’s cycle and briefly transport her to a nearby facility where TVA can be performed. With TVA services being offered with increasing frequency in densely equine populated areas it is increasingly common for TVA to be offered as an outpatient procedure even if ICSI laboratories are not present at that location. The majority of reports associated with TVA and ICSI are associated with benchtop research and typically do not continue beyond blastocyst development to report pregnancy rates. A cumulative pregnancy rate after TVA and ICSI were performed on immature oocytes, with shipment of blastocysts to an off-site recipient herd has been reported at 72% with a 52% foaling rate.1 In 2017, in collaboration with an exclusively equine reproduction veterinary practice, we worked together to provide TVA services for client-owned mares. Mares were referred from the veterinary practice to our university setting where outpatient TVA procedures were performed; 330 follicles were aspirated and 212 oocytes were recovered (mare age range from 4-26 years) and shipped overnight at room temperature in an Equitainer® to an ICSI laboratory via FedEx™. A total of 43 (26.1%) blastocysts resulting from 165 ICSI procedures were obtained; 29 embryos were vitrified for future use and 14 embryos were transported to the referring reproductive veterinary practice via courier for fresh transfer to recipient mares on day 7 or 8 after ICSI. A 14 day pregnancy rate of 71.4% (10/14) and a 50% (7/14) pregnancy rate at heartbeat check were recorded. Three recipient mares have already successfully foaled and four are due to foal later in the spring of 2018. In light of these results sending a mare to a local practice specializing in TVA, as opposed to a distant, self-contained facility that has both TVA and ICSI capabilities has benefits for referring equine practitioners, their clients and patients. Being a key element in the management of the mare’s cycle prior to TVA has allowed the primary veterinarian to remain an integral partner in providing these specialty services. Shorter duration stays at referral facilities translates into less expense for the mare owner. Finally, as collaborators in this working partnership we have found working as a team to offer such services has enhanced the working relationship between practitioners and specialists.

Assisted reproductive techniques in mares.

A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm number or quality is low. Because of its ease of use for the mare owner and efficiency, oocyte recovery and ICSI is being used in some cases for management of normally fertile mares and stallions. Oocytes can be recovered from live mares by the referring veterinarian, and shipped overnight to a laboratory for ICSI, without any decrease in oocyte or embryo viability. In case of unexpected death of a mare, ovaries or oocytes can be transported to the ICSI laboratory for production of embryos. Embryos produced both in vitro and in vivo can be biopsied to determine their genetic makeup before they are transferred. Equine embryos can be vitrified successfully; collapse of the blastocoele cavity allows efficient vitrification of expanded blastocysts. In contrast, cryopreservation of unfertilized equine oocytes still has low success. Genetics of valuable animals can be preserved via nuclear transfer (cloning) and several commercial companies offer this service clinically.

Expression of glucose transporters in the endometrium and early conceptus membranes of the horse

IntroductionGlucose is the primary energy substrate for early conceptus development and, for the first 40 days of gestation, the equine conceptus depends solely on glucose available in the histotroph; thereafter, histotrophic glucose provision continues to support transport across the definitive placenta. MethodsTo investigate glucose provision routes during early equine pregnancy we examined expression of glucose transporters in conceptus membranes and endometrium recovered on days 7, 14, 21 and 28 after ovulation. To further differentiate the contributions of maternal progesterone priming and conceptus-endometrium crosstalk in regulating glucose transporter expression, day 8 embryos were transferred to recipient mares on day 8 (synchronous) or day 3 (asynchronous) after ovulation; conceptuses and endometrium were recovered 6 or 11 days later. ResultsThe glucose transporters SLC2A1, 2A3, 2A4, 2A8, 2A10 and 5A1 were expressed in equine endometrium. In conceptus membranes, expression of SLC2A1-3, 2A5, 2A8, 2A10, 5A1 and 5A11 increased from day 14, and SLC2A1 protein was highly abundant on the apical trophectodermal membrane and in the endoderm. Asynchronous embryo transfer (ET) resulted in reduced SLC2A1 expression in both the endometrium and conceptus membranes. DiscussionA wide range of glucose transporters are expressed in the pre-implantation equine conceptus and endometrium, presumably to ensure adequate glucose provision to the developing embryo. Endometrial expression of SLC2A1 appears to be regulated by a combination of progesterone-priming and conceptus signalling, and its delayed upregulation after asynchronous ET may contribute to the observed delay in conceptus development.

Prolonged In Vitro Maturation Time and Increased Intensity of Piezo Pulses During ICSI Enhance Cleavage and Embryo Development in the Horse

Asynchronous embryo transfer is an excellent tool to investigate how subtle differences in the uterine environment affect embryo development and survival. Progesterone secreted from the corpus luteum following ovulation is one of the main factors responsible for establishing endometrial receptivity for the pre-implantation embryo via complex alterations in the expression of genes involved in the secretion of the histotroph. The objective of this retrospective study was to determine whether the recipient's Day after ovulation and the number of CL at ET influence the pregnancy rates of IVP horse embryos. The study included 650 heterologous frozen ICSI horse embryo transfer cycles and evaluated the pregnancy and ongoing pregnancy rate. The ongoing pregnancy was significantly lower in recipient mares with ET performed 5 and 6 days after ovulation (47.4% and 37.5%, respectively) than in recipients with ET 4 days after ovulation (67.3%). Furthermore, Day 5 recipient mares (Day 0 = Day of ovulation) with 2 corpora lutea (CL) at the time of ET had lower ongoing pregnancy rate (36.1%) than Day 5 recipient mares with 1 CL (51.9%). In contrast, the presence of 2 CL was associated with a higher ongoing pregnancy rate (75.8%) in recipient mares with a less advanced uterine stage at the time of ET (Day 3 and 4 after ovulation), compared to recipients with only 1 CL at ET (62.7%). In conclusion, both the number of days after ovulation and the number of CL recorded in the recipient mare at ET influenced the ongoing clinical pregnancy rate. This study highlights the importance of establishing exactly when progesterone rises above a threshold (relative to the Day of ovulation) when trying to determine the optimal window for transferring an IVF/IVP embryo.

Effect of Duration of Embryo Holding on Recipient Mares’ Pregnancy Rate

Effect of Duration of Embryo Holding on Recipient Mares’ Pregnancy Rate

Enrofloxacin and Its Active Metabolite Ciprofloxacin Cross the Equine Placenta and Did not Cause Lesions in the Articular Surface of the Fetuses and Foals

Increased ambient temperatures has been identified to contribute to reproductive outcomes for several domesticated species, but its impact on equine reproduction has not been previously investigated. Therefore, this study aimed to examine the relationship between ambient climatic conditions, as measured by temperature-humidity index (THI) between day 7 and day 14 of gestation, and early embryonic loss (EEL) in recipient mares undergoing embryo transfer.The study examined records from 834 embryo transfers at an equine breeding facility in Victoria, Australia. Early embryonic loss was defined as a negative transrectal ultrasound on day 14 of gestation after an embryo was transferred on day 7. Both maximum THI on the day of transfer (day 7) and mean THI between day 7 and day 14 were investigated for association with the outcome of EEL using multivariable logistic regression, controlling for confounders including embryo age and quality, recipient mare quality and embryo transfer quality.EEL was observed in 21% of embryo transfers. A five-unit increase in maximum THI on the day of transfer was associated with an 18% increase in the odds of EEL (p = 0.01). Similarly, the odds of EEL increased by 25% for each five-unit increase in mean THI between day 7 and 14 (p = 0.003).As both single and cumulative episodes of high THI were associated with increased EEL in embryo transfer mares from this equine breeding facility, further studies are warranted to identify similar effects in a broader population, establish causality and evaluate possible mitigation strategies in anticipation of heat waves.

High Prevalence of Subclinical Endometritis in Problem Mares– Effect of Activation and Treatment on Fertility

In the present study, 2.228 cycles of 180 Polo Argentino donor mares from an embryo transfer program in Argentina were examined to evaluate the effects of: (1) Interval from Prostaglandin F2alpha analog treatment to ovulation (ITO) on embryo recovery rate (ERR); (2) ITO on number of embryos per flushing (EPF); (3) ITO on multiple ovulation (MO) rate; (4) ITO from donor mare on pregnancy rate (PR) in recipient mares. Mares were inseminated with fresh semen from 31 fertile stallions in the induced estrus. Embryo flushing was performed 7–8 days postovulation. Following embryo flushing, donor mares were treated with prostaglandin F2alpha analog (cloprostenol 250 μg). The ERR increased along with the ITO (P = .01), with the lowest ERR (30.7%) for mares with an ITO of <4 days, and the highest (78.3%) in mares with an ITO of 10 days. The ITO from the donor mare in which the embryo was recovered did not have a significant effect on PR: ITO <6, 6 to 10, and >10 days were 74.6, 81.4, and 77.3%, respectively. The number of EPF and MO rate increased gradually along with the ITO (P < .05). In conclusion, the results of this study indicated that the ITO is positively correlated with the embryo recovery and the multiple ovulation rate.

Equine Embryo and Early Fetus Development in Recipient Mules and Mares

Equine Embryo and Early Fetus Development in Recipient Mules and Mares

Is Aspiration of Blastocoelic Fluid Essential to Vitrify Large Equine Embryos?

Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey embryo viability in vivo, vitrified IVP blastocysts were shipped to an embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in embryo holding medium at an embryo transfer facility before transfer to recipient mares; fresh IVP embryos were shipped and transferred as controls. In Exp. 1, embryos increased significantly in diameter after culture (P < 0.01), with no difference among treatments. There was no difference (P > 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5–2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4–87.5%) or heartbeat (57.1%–85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP equine embryos.

Retrospective Study on the Influence of Recipient Mare's Height on Foal Height of Twin Embryos, From Birth to Three Years of Age

It is not clear whether a high level of high-density lipoprotein cholesterol (HDL-C) is associated with lower risk of atherosclerosis. It is likely that HDL-C is a double-edged sword for atherosclerosis.The purpose of this study was to evaluate the relationship between HDL-C levels and endothelial function in men.This was a cross-sectional study. We evaluated flow-mediated vasodilation (FMD) and serum levels of HDL-C in 5842 men aged 18 to 92 years who were not receiving lipid-lowering therapy. All participants were divided into four groups by HDL-C level: low HDL-C (<40 mg/dL), moderate HDL-C (40–59 mg/dL), high HDL-C (60–79 md/dL), and extremely high HDL-C (≥80 mg/dL). We were not able to evaluate the amount of alcohol intake because there was limited information on the amount of alcohol drinking in our database.FMD values were significantly smaller in the low group and the extremely high group than in the high group (P = .001 and P = .016, respectively). There was no significant difference in FMD between the low group and the extremely high group. Multiple logistic regression analysis revealed that extremely high HDL-C, but not low HDL-C, was independently associated with the lowest quartile of FMD (odds ratio: 1.39, 95% confidence interval: 1.09–1.77; P = .009).An extremely high level of HDL-C in men (8.1% of this population) was associated with a significant reduction in FMD.

The Use of Endoscopic Biopsy to Compare Histological Finds of Stallions With and Without Seminal Vesiculitis

This study compared hormone treatments given to mares during anestrus, spring transition, and different stages of the estrous cycle, by assessing uterine features and pregnancy rates after embryo transfer (ET). Embryo recipient mares (n = 160) were equally arranged as follows: G1-spontaneous ovulation (control), G2-anestrus, G3-spring transition, G4-early estrus, G5-estrus, G6-diestrus, G7-early diestrus treated with a dose of dinoprost, and G8-early diestrus treated with two doses of dinoprost. At treatment initiation (Day-4), G2-7 were given dinoprost and estradiol-17β, thereafter, estradiol-17β was repeated on Days-3,-2, and -1. On Day0, mares received long-acting altrenogest. Then, each mare had one ET performed from Day + 3 to Day + 8 after altrenogest. Immediately before the ET, mares received a boost of altrenogest and had uterine features assessed. Pregnant mares on each of the checks (by 7, 30, 60, and 120d after ET) were maintained on weekly injections of LA-P4 until 120d. G8 received similar management, but dinoprost was repeated on Day-3. G1-G6 and G8 displayed uterine edema and satisfactory pregnancy rates ≥65%. Repeating dinoprost to G8 likely ensured proper luteolysis and response to estrogen as determined by higher uterine edema scores and pregnancy rates than G7 (p < .05). Our results were consistent with previous studies and other successful commercial ET programs (except G7), thus, demonstrating the usefulness of the hormone treatments described herein to synchronize embryo recipient mares with donor mares. Thus, we foresee that other groups may use the strategies described herein for the management of embryo recipient mares.

Evaluation of environmental effects on reproductive characteristics of Mangalarga Marchador mares in a commercial embryo transfer program

The objective of this study was to evaluate the environmental effects on embryo recovery rate and pregnancy rate of Mangalarga Marchador mares. The reproductive characteristics of donor and recipient mares were evaluated during five years in Brazilian tropical environment. The mares were used throughout the year and seasons were classified as: October to April (breeding season – BS); May (autumn transition out of the breeding season - ATBS); June to August (non-breeding season – nBS); and September (vernal transition into the breeding season – VTBS). Daily temperature rainfall and hours of daylight (photoperiod) were measured during all months and years of evaluation. The embryo recovery rate (ERR) and the pregnancy rate (PR) were observed and frequencies were calculated. The effect of environmental variables, day of flushing, and hormonal treatments (estradiol benzoate and progesterone) were determined for the reproductive measures using the Chi-square and Kruskal-Wallis tests. Significant effects were noted of the year, season and temperature on ERR (P < 0.05), but no significant effects were observed of the environmental parameters (year, season, hormone treatment, rainfall and photoperiod) on PR (P > 0.05). The day of uterine flush affected ERR (P < 0.05) but did not affect PR (P > 0.05). In addition, hormone treatment also supported favorable results of PR in recipient mares during nBS. The conclusion is that mares of this breed can be used in reproduction all year long, with good pregnancy rates, in Brazil’s tropical environment. The hormone treatment also supported favorable pregnancy rates in recipient mares during the non-breeding season. It seems that mares can have good pregnancy rates throughout the year in Brazil´s tropical environment.

Horse embryo diameter is influenced by the embryonic age but not by the type of semen used to inseminate donor mares

The diameter of embryos recovered from mares on Day 8 after ovulation varies greatly, from as little as 130 μm to as much as 2500 μm. Several factors have been proposed to affect embryo size at recovery, one of which is the type of semen (frozen vs fresh or cooled-transported) used to inseminate the mare. In addition, it has been shown that smaller embryos (<300 μm) recovered on Day 8 are less likely than larger embryos to result in successful pregnancy after transfer. However, whether the actual age of the embryo (interval from fertilization to flushing) in relation to its size also influences the post-transfer viability is unclear. The aims of this study were: a) To determine the effect of semen type (frozen-thawed vs cooled-transported) on embryo diameter after pre-ovulatory insemination; and b) To establish the relationship between embryonic age, embryo size and likelihood of pregnancy and pregnancy loss following transfer. A total of 179 embryos were recovered from mares inseminated with: frozen semen post-ovulation 8 days previously (G1; n = 35); cooled-transported semen pre-ovulation 8.5 days previously (G2; n = 95); frozen semen pre-ovulation 8.5 days previously (G3; n = 30); and frozen semen post-ovulation 9 days previously (G4; n = 19). The effect of embryonic age, type of semen, donor mare and its age, number of ovulations and embryos per flush on embryo diameter was tested using a general linear model of variance. In addition, the proportions and survivals of small embryos (<300 μm) in each group were compared with those of respective larger embryos by Fisher's exact test. Embryonic age (P < 0.001) and age of the donor mare (P = 0.07), but no other factor, influenced embryo diameter. The proportion of small embryos was 42.9, 10.5, 10.0 and 10.5% for Groups 1, 2, 3 and 4, respectively. The pregnancy status of recipient mares 35 days post-transfer for small embryos from Group 1 (12/15; 80.0%) was not different (P > 0.1) from that of recipients of small embryos from Groups 2 to 4 combined (8/15; 53.3%).

Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses.

IntroductionCell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts.Materials and methodsZona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination (AI) recovered by uterine flushing and transferred to recipient mares were used as controls (AI group).ResultsBlastocyst development was higher in the MSC group than in the AF group (18.1% vs 10.9%, respectively; p<0.05). However, pregnancy rates and delivery rates were similar in both cloning groups, although they were lower than in the AI group (pregnancy rates: 17.7% [41/232] for MSC, 12.5% [37/297] for AF and 80.7% [71/88] for AI; delivery rates: 56.8% [21/37], 41.5% [17/41] and 90.1% [64/71], respectively). Remarkably, the gestation length of the AF group was significantly longer than the control (361.7±10.9 vs 333.9±8.7 days), in contrast to the MSC group (340.6±8.89 days). Of the total deliveries, 95.2% (20/21) of the MSC-foals were viable, compared to 52.9% (9/17) of the AF-foals (p<0.05). In addition, the AF-foals had more physiological abnormalities at birth than the MSC-foals; 90.5% (19/21) of the MSC-delivered foals were completely normal and healthy, compared to 35.3% (6/17) in the AF group. The abnormalities included flexural or angular limb deformities, umbilical cord enlargement, placental alterations and signs of syndrome of neonatal maladjustment, which were treated in most cases.ConclusionIn summary, we obtained 29 viable cloned foals and found that MSCs are suitable donor cells in horse cloning. Even more, these cells could be more efficiently reprogrammed compared to fibroblasts.

Embryo transfer in the mare was introduced 30 years ago.

Its application improves the reproduction rate, creates experimental models for the study of physiology and pathology of reproduction, while it promotes genetic improvement of the species. The method includes the following stages: 1) Selection of the donor and recipient mares. 2) Induction of multiple ovulation in the donor-mare. A reliable method that could be applied commercially has not been developed yet. 3) Synchronization of donor and recipient mares' estrus. Recipients are usually used during the breeding season, but can be used during anestrus or after surgical removal of their ovaries as well. Non-synchronized recipients may also be used after proper treatment. 4) Artificial insemination of the donor mare. 5) Collection, evaluation and washing of the embryo. Embryo is usually collected by non-surgical and rarely surgical methods. Embryo evaluation involves the assessment of its developmental stage in relation with the day of selection and its quality evaluation. Embryo washing is accomplished by its successive passage through three different dilutions of culture medium. 6) Embryo transfer in the uterus of the recipient mare. Surgical (midventral laparotomy, flank laporotomy, laparoscopic and transvaginal method) or nonsurgical (unguarded and guarded methods) transcervical methods are used.

Amino acid transporter expression in the endometrium and conceptus membranes during early equine pregnancy.

Maternally derived amino acids (AA) are essential for early conceptus development, and specific transporters enhance histotrophic AA content during early ruminant pregnancy. In the present study we investigated AA transporter expression in early equine conceptuses and endometrium, during normal pregnancy and after induction of embryo-uterus asynchrony. 'Normal' conceptuses and endometrium were recovered on Days 7, 14, 21 and 28 after ovulation. To investigate asynchrony, Day 8 embryos were transferred to recipient mares on Day 8 or Day 3, and conceptuses were recovered 6 or 11 days later. Endometrial expression of AA transporters solute carrier family 38 member 2 (SLC38A2), solute carrier family 1 members 4 and 5 (SLC1A4 and SLC1A5) increased during early pregnancy, whereas solute carrier family 7 member 8 (SLC7A8), solute carrier family 43 member 2 (SLC43A2) and solute carrier family 7 member 1 (SLC7A1) SLC7A8, SLC43A2 and SLC7A1 expression decreased and the expression of solute carrier family 1 member 1(SLC1A1) and solute carrier family 7 member 2 (SLC7A2) was unaffected. In conceptus membranes, most transporters studied were upregulated, either after Day 14 (solute carrier family 7 member 5 - SLC7A5, SLC38A2, SLC1A4, SLC1A5 and SLC7A1) or Day 21 (SLC43A2 and SLC7A2). Asynchronous ET indicated that endometrial SLC1A5, SLC1A1 and SLC7A8 are primarily regulated by conceptus factors and/or longer exposure to progesterone. In conclusion, AA transporters are expressed in early equine conceptus membranes and endometrium in specific spatiotemporal patterns. Because conceptuses express a wider range of transporters than the endometrium, we speculate that the equine yolk sac has recruited AA transporters to ensure adequate nutrient provision during an unusually long preimplantation period.

Synchronization of cyclic and acyclic embryo recipient mares with donor mares

This study compared hormone treatments given to mares during anestrus, spring transition, and different stages of the estrous cycle, by assessing uterine features and pregnancy rates after embryo transfer (ET). Embryo recipient mares (n = 160) were equally arranged as follows: G1-spontaneous ovulation (control), G2-anestrus, G3-spring transition, G4-early estrus, G5-estrus, G6-diestrus, G7-early diestrus treated with a dose of dinoprost, and G8-early diestrus treated with two doses of dinoprost. At treatment initiation (Day-4), G2-7 were given dinoprost and estradiol-17β, thereafter, estradiol-17β was repeated on Days-3,-2, and -1. On Day0, mares received long-acting altrenogest. Then, each mare had one ET performed from Day + 3 to Day + 8 after altrenogest. Immediately before the ET, mares received a boost of altrenogest and had uterine features assessed. Pregnant mares on each of the checks (by 7, 30, 60, and 120d after ET) were maintained on weekly injections of LA-P4 until 120d. G8 received similar management, but dinoprost was repeated on Day-3. G1-G6 and G8 displayed uterine edema and satisfactory pregnancy rates ≥65%. Repeating dinoprost to G8 likely ensured proper luteolysis and response to estrogen as determined by higher uterine edema scores and pregnancy rates than G7 (p < .05). Our results were consistent with previous studies and other successful commercial ET programs (except G7), thus, demonstrating the usefulness of the hormone treatments described herein to synchronize embryo recipient mares with donor mares. Thus, we foresee that other groups may use the strategies described herein for the management of embryo recipient mares.

37 Healthy Foals Produced Using Bone Marrow-Mesenchymal Stem Cells as Nuclear Donors in Horse Cloning

Somatic cell nuclear transfer efficiency is based on the capacity of the donor cell to be reset and reprogrammed to an embryonic state. So, the less differentiated the donor cells are, the more easily they could be reprogrammed by a recipient cytoplasm. Failures on appropriate nuclear reprogramming frequently lead to abnormalities associated with the placenta, umbilical cord, birthweight, and limbs. In the present study, we evaluated the efficiency of bone marrow mesenchymal stem cells (BM-MSC) compared with adult fibroblasts (AF) as nuclear donors in horse cloning and evaluated both in vitro and in vivo development of the embryos generated. Moreover, we focused on comparing the health of the foals generated and on the presence of anatomical abnormalities in foals produced from the different treatments. Embryos produced by AI, recovered by uterine flushing, and transferred to recipient mares were used as controls. All variables were analysed by Fisher test (P &lt; 0.05). The cloning procedure was performed according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Both cleavage and blastocyst rates were higher when MSC were used as nuclear donors (P &lt; 0.05). Cleavage rates were 85.6% (3875/4527) v. 90.2% (3095/3432) and blastocyst rates were 10.9% (492/4527) and 18.1% (622/3432) for AF and MSC groups, respectively. In the AF group, 476 blastocysts were transferred to recipient mares (232 transfers), and in the MSC group, 594 blastocysts were transferred 297 transfers). In the AI control group, 88 embryos were transferred. Pregnancies were diagnosed by transrectal ultrasonography 15 days after embryo transfer in all the groups. Pregnancy rates were similar between both cloning groups (41/232, 17.7% and 37/297, 12.5%for AF and MSC, respectively), but higher in the AI group (71/88, 80.7%). However, significant differences were observed in the birth of viable offsprings among the cloning groups. Despite similar rates of foal delivery (AF, 17/41, 41.5%; MSC, 21/37, 56.7%), a higher proportion of viable foals were obtained from the MSC group (20/37, 54.1%) compared with the AF group (9/41, 22%; P &lt; 0.05). Surprisingly, as in the AI group (63/63, 100%), all of the viable foals obtained using MSC (20/20, 100%) were considered normal and did not show abnormalities associated with cloning. In contrast, in the AF group, only 4/9 (44.4%) were considered normal foals. The defects present in the other 5 foals were related to flexural and angular limb deformities and umbilical cord malformations. These were corrected rapidly with standard treatments or, in the case of the umbilical cords, minor surgery. This study shows for the first time that BM-MSC can be used as nuclear donors in horse cloning and that the foals obtained are as healthy as those produced by AI, showing no abnormalities related to deficiencies in nuclear reprogramming.

29 Co-Incubation of Equine Cloned Embryos with Sialic Acid: Effect on Pregnancy Rate

In vitro-produced equine embryos have certain morphological characteristics that differ from embryos produced in vivo. One of them is the absence or inadequate formation of the embryo capsule. This capsule is composed of mucin-like glycoproteins produced by the trophectoderm with high proportion of sialic acid, which confers anti-adhesive properties. This characteristic is necessary for the intrauterine embryo migration process to occur, which is vital and fundamental for pregnancy recognition. For this reason, inadequate formation of the glycoprotein capsule could result in a lower pregnancy rate. In this study, we aimed to evaluate the effect of cloned equine embryo co-incubation with sialic acid on blastocyst and pregnancy rates. To achieve this, equine oocytes obtained from slaughterhouse ovaries were matured in TCM-199 HEPES medium with 2% fetal bovine serum, 2% antibiotics, and 1 μg mL−1 FSH, incubated at 39°C for 24 h. Matured oocytes were denuded with pronase, enucleated, and fused to donor bone marrow mesenchymal cells according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Chemical activation was induced using 8.7 μM ionomycin for 4 min and embryos were incubated with 1 mM 6-DMAP and 5 mg mL−1 cycloheximide for 4 h. Afterwards embryos were cultured in microwells for 8 days in DMEM-F12 medium. On Day 6, cloned equine embryos were exposed to 5 μM sialic acid for 48 h (SA group). On Day 8, blastocysts were transferred to recipient mares 5 days post-ovulation and pregnancy was confirmed 15 days post-transfer by transrectal ultrasound. Embryo clones generated without sialic acid exposure were used as a control (C) group. Fisher test was used to analyse both blastocyst and pregnancy rates. Blastocyst rates were 14% (46/328) and 15% (62/413) and pregnancy rates were 30.4% (7/23) and 19.4% (6/31) for SA and C groups, respectively. No statistical differences were observed between groups for the analysed parameters, even though pregnancy rates tended to be higher in the SA group. This effect could be a consequence of higher concentrations of the glycoprotein involved in the formation of the embryo capsule.

30 Vitrification of Equine Embryos: Application in a Commercial Cloning Program

During a commercial horse cloning program, a critical point is the availability and maintenance of suitable recipient mares for a large quantity of embryo transfers. Usually, pregnancy rates and viable births off the breeding season decrease significantly, whereas the rate of in vitro embryo production remains constant. For this reason, an efficient vitrification system allows continuous embryo production throughout the year with the advantage of doing the transfers only during the breeding season. The vitrification technique evaluated in this study was the one described by Kuwayama et al. (2007 Theriogenology 67, 73-80). By using this method, we compared post-warming recovery efficiency, pregnancy rates, and viable foaling rates in 2 experimental groups: cloned blastocysts vitrified off-season and transferred in breeding season (VC, n = 337), and non-vitrified cloned blastocysts also transferred in breading season (no-VC, n = 516). To achieve this, equine oocytes were collected from slaughterhouse ovaries, matured, enucleated, and fused to a donor cell according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). The reconstructed embryos (RE) were cultured in a well-of-the-well system by adding 3 RE per well for 7 to 8 days to reach the blastocyst stage, at which they were vitrified as mentioned above. During the breeding season, blastocysts were warmed and transferred in couples in a single cycling receptive mare. Pregnancies were confirmed by transrectal ultrasonography 15 days post-transfer. All variables were analysed by Fisher test (P &lt; 0.05). The warming recovery rate was 91% (308/337) for cloned blastocysts. In addition, pregnancy and viable birth rates were similar for the VC and no-VC groups: 15.6% (24/154) v. 16.7% (43/258) for pregnancy rates, respectively, and 37.5% (9/24) v. 37.2% (16/43) for foaling rates, respectively. In summary, 9 viable cloned foals were obtained with off-season embryos warmed and transferred during the breeding season, showing that vitrification did not affect embryo quality. Hence, the proposed strategy provides the ability to maximize production efficiency of equine clones by generating a large number of pregnancies without stopping in vitro embryo production at any time of the year.