Donor Mares Research Articles

The potential of embryo transfer in a German horse‐breeding programme

A reference horse-breeding programme with 13500 foals each year was modelled with ZPLAN+. This new software for the optimization of the structures in breeding programmes is based on ZPLAN. In two scenarios, the implementation of a rigorous selection of mares was implemented. In scenario I, the mare performance test was the point of selection, while in scenario II, further information on 20 competitions in two more years is available. These selected mares were used for embryo transfer (ET), partly in combination with multiple ovulation (MOET). The selection intensity and the number of foals out of (MO)ET were varied in both scenarios. It was expected that 250, 500 and 1000 mares are available for selecting 20, 50, 100 or 200 donor mares each year. The number of foals out of (MO)ET was varied between one and six foals per donor mare and year. Donor mares were used for ET for 4 years. It became clear that with high selection intensities of donor mares and high reproduction rates of them, the yearly genetic gain in a horse-breeding programme could increase over a large range. In scenario II, the additional information on 20 competitions increased the accuracy of the selection index to 0.85. With 200 selected donor mares of 1000 available mares and six foals per year, the genetic gain could almost be doubled compared to the reference scenario. The implementation of ET and a related higher usage of few selected mares entails rising costs and a reduction in the genetic variance. In the most extreme MOET scenario, the effective population size was reduced by 19% relative to the reference scenario. Only if the increase in genetic gain can be converted into higher return for the breeders, the implementation of (MO)ET schemes is a realistic and sensible option for horse-breeding programmes.

Breeding or Assisted Reproduction? Relevance of the Horse Model Applied to the Conservation of Endangered Equids

Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity among the different members of the genus Equus, the domestic horse can be used to optimize cryopreservation and embryo production protocols for future application in wild equids. We will give this hypothesis a scientific underpinning by listing successful applications of epididymal sperm freezing, embryo freezing, intracytoplasmic sperm injection, oocyte vitrification and somatic cell nuclear transfer in domestic horses. Some ART fertilization methods may be performed with semen of very low quality or with oocytes obtained after the death of the mare.

Preliminary studies on dynamics of the ovarian surface epithelium on the ovulation fossa in mares during the estrous cycle

was identified as Coccidiodes immitus, which is a human health concern. All cultures and other samples from themare were destroyed. Personnel involved with the case were evaluated for seroconversion (i.e. positive titer) and fortunately all were negative. The mare was re-bred and a second embryowas collected. However, fungal hyphaewere present in the search dish. The embryo was washed numerous times and transferred into a recipientmare. The recipientmarewas administered oral fluconazole (14mg/kg loading dose; 5mg/ kg maintenance dose) to inhibit fungal growth associated with transfer of the embryo. The recipient mare became pregnant and carried to term. The fungal organism in the embryo search dish was confirmed by PCR to be C. immitus. Since C. immitus has been reported to disseminate from a primary respiratory infection, chest radiographs and a bronchial-alveolar lavage were performed to determine if the lungs were the nidus of infection. No evidence of infection was identified in the respiratory tract. The donor mare was sent home with instructions to administer fluconazole orally for an additional 6 to 8 weeks. In conclusion, fungal endometritis due to C. immituswas diagnosed in an embryo donor mare. Two ET pregnancies were established in recipient mares. Caution should be taken by veterinarians performing fungal culture in geographical areas endemic for C. immitus as the organism is a potential human health hazard.

Data of three first seasons of embryo transfer from two clones of the same mare

underwent serial pregnancy ultrasound exams per rectum following transfer and 35-day pregnancy rates were recorded. Pregnancy rates were compared between recipient mares that ovulated 8-12 versus 1317 days postpartum. Study 2: Blastocysts (n 1⁄4 18) recovered from mares following insemination on their first PP ovulation were transferred transcervically into recipient mares that were either nonfoal-heat mares or nonlactating mares. The recipient mares underwent ultrasound examination per rectum and 35-day pregnancy rates were recorded. The Chi-square test was used to compare values. The results in Table I show that the 35-day pregnancy rate of recipient mares that had embryos transferred following their first postpartum ovulation was 65% (151 pregnancies/ 232 embryos transferred). There was no difference in pregnancy rates between recipient mares that ovulated 8-12 days versus 13-17 days after ovulation. The results in Table II show that the 35-day pregnancy rate of recipient mares was 83% (15 pregnancies/18 transferred foal heat embryos). In conclusion, mares on their foal heat can serve as embryo recipients. Furthermore, when embryos are recovered from donor mares inseminated on the first postpartum estrus, recipient pregnancy rates are high.

Effect of uterine bacterial inflammation in donor mare plasmatic progesterone (PP4) at flushing time - is effluent analysis a helpful tool?

1.0 to 67.8 1.0 (L*), 5.8 0.5 to 14.9 0.5 (a*) and 17.4 1.0 to41.8 1.0 (b*)with specular componentexcluded.Data revealed highly significant differences (P < 0.0001) among individual uteri (ANOVA). Totalmeansof L*a*b*did not differ betweenuterine corpusandhorns for L* (57.9 0.7 vs. 59.1 0.5; P1⁄4 0.1522) and a* (11.5 0.3 vs. 10.9 0.2; P1⁄4 0.1313), but significantly differed for b* (23.9 0.7 vs. 25.9 0.5; P1⁄4 0.0206). Significant differences of color coordinates between uterine corpus andhorns occurred for L* in two (uterus 2 and 14), for a* in one (1) and for b* in 7 (1, 3, 5, 7,11,13 and 14) out of 23uteri. The coefficientofvariation ranged for L* from2.54 to 10.59 (uterus 1 and18), for a* from4.68 to 43.41 (19 and3) and for b* from 5.37 to 19.52 (20 and 23). Similarly, spectral reflectance curves also demonstrated small (Fig.1a) andhigh (Fig. 1b) variation in single uteri.

Embryo Transfer Efficiency of Quarter Horse Athletic Mares

This study was designed to compare embryo recovery rates and pregnancy rates of athletic and breeding Quarter Horse mares in a tropical warm climate. Thirty-nine barrel racing mares in training and 135 breeding mares as control donors were included. During the training period, the ambient temperature ranged from 31°C to 36°C and the average humidity from 70% to 90%. After the detection of a 35-mm follicle by ultrasound, ovulation was induced with 1 mg of deslorelin acetate (i.m), and insemination was performed 24 hours later with cooled and fresh semen from different fertile stallions. Embryos were collected on day 8 postovulation. The body temperature (rectal) was evaluated from eight athletic donor mares randomly selected from the same studied group. A total of 138 and 657 embryo collections were carried out on training and breeding mares, respectively, with a total of 105 (76%) and 466 (71%) embryos collected ( P > .05). Similarly, no differences ( P > .05) were observed for the pregnancy rates on day 15 (82/105, 78% vs. 370/466, 79%), and day 40 (73/105, 69% vs. 328/466, 70%) between the training and breeding donor mares. Just after training, the body temperature increased to an average of 39.4°C and the respiratory rate from 14.5 to 35.3 breaths per minute. The results of the present study showed that embryo production from appropriately trained donor mares in good condition were similar to non-athletic broodmares.

Relationship Between Sperm Quality Measures of Cooled-Shipped Semen and Fertility.

Features of fresh stallion semen such as sperm concentration, motility, morphology, and DNA quality are routinely evaluated as part of a breeding soundness evaluation and diagnostically to determine the cause of reduced fertility, however, there is limited information about the relationship between these values in cooled-stored semen and stallion fertility. The goals of this study were to determine the relationship of stallion sperm quality features after 24 hr of cooled-storage on fertility. Sperm concentration and viability were determined using a NucleoCounter SP-100, DNA quality by the Sperm Chromatin Structure Assay, sperm motility by phase-contrast microscopy and sperm morphology by differential interference microscopy. Stallion (n=135; age (years)- mean-11; range-3-25) seminal parameters were evaluated during the 2009 breeding season and donor mares (n=204) were bred with cooled-shipped semen at an embryo transfer facility (Royal Vista Southwest, Purcell, OK) either the same day as collection or 24hr after semen collection. Fertility was determined by embryo(s) recovery. The percentage of embryos recovered was higher (65 vs. 51%; P < 0.01) when mares were bred with semen collected from the stallion on the same day compared to mares bred approximately 24 hr after semen collection. Pregnancy data were then compared to each individual seminal parameter in order to determine the relationship between sperm quality and resulting embryo recovery (fertility). Sperm parameters influencing the success of embryo recovery rate were: total sperm motility (P < 0.01), progressive motility (P < 0.077), total sperm number (P < 0.002), total motile sperm (P < 0.003), total progressively motile sperm (P < 0.002), viability (P < 0.004), total viable sperm (P < 0.002), total progressively motile viable sperm (P < 0.0001), %COMP (P < 0.045), morphologically normal sperm (P < 0.001) and total morphologically normal sperm (P < 0.0001). In a large group of mares and stallions this study identifies sperm features that affect fertility as determined by embryo recovery. This information will help horse breeders and researchers determine those sperm quality factors that maximize fertility. (poster)

Effects of Repeated Embryo Collection on Embryo Recovery Rate in Fertile Mares

In mares, repeated embryo collection in successive oestrous cycles is necessary if a greater number of foals should be produced. We investigated effects of repeated embryo collection in fertile donor mares on embryo recovery rates. In addition, an influence of the individual mare and season on embryo recovery rates was studied. In nine mares, a total of 153 embryo collections were performed during 30 months (17 ± 2.2 embryo collections per mare). The overall embryo recovery rate was 64% and did not differ among mares. Between successive embryo collection procedures, recovery rate varied significantly; however, no increase or decrease in the embryo recovery rate with increasing number of successive embryo collections was seen. In three mares, ovulation ceased from November to February. In the remaining six mares, embryo production was successfully continued throughout winter and no influence of the month on embryo recovery rates was detected.

Folliculogenesis, embryo parameters and post-transfer recipient pregnancy rate following equine follicle-stimulating hormone (eFSH) treatment in cycling donor mares

Induction of multiple ovulations, or superovulation, may potentially increase the efficiency of equine embryo transfer programs. Our objective was to investigate the effects of equine follicle-stimulating hormone (eFSH) treatment on the success rate of embryo transfer programs in mares. In the research facility of the University of Saskatchewan, Canada, we studied 12 donor mares and 37 recipient mares during the physiological breeding season. Donor mares were used in two consecutive oestrous cycles: the first served as the control cycle and in the second an eFSH regimen was applied (eFSH cycle). In the control cycle, mares were administered human chorionic gonadotropin (hCG) to induce ovulation when a follicle ≥35 mm in diameter was detected by transrectal ultrasonographic examination. In the second oestrous cycle, twice-daily eFSH treatment was initiated when a follicle ≥25 mm was detected and treatment ceased when a follicle ≥35 mm was present, at which time hCG was administered. All donor mares were artificially inseminated while in oestrus using fresh semen collected from a stallion of proven fertility. At 8 days post-ovulation, embryos were recovered transcervically and transferred individually to the uterus of a synchronised recipient mare. The eFSH treatment stimulated the ovary and resulted in greater numbers of ovulations and recovered embryos; however the recovered embryos tended to have a lower morphological grade than the control embryos, and the recipient pregnancy rate per transferred embryo was lower than anticipated. The numbers of recipient pregnancies and foals born that resulted from eFSH treatment were not different from the control.

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.

Effects of timing of induced luteolysis in embryo donor mares on reproductive performance and pregnancy rate in recipient mares

The objective was to evaluate the effects of giving prostaglandin F2α (PGF) to donor mares 48 h prior to embryo collection. Non-lactating donor mares (n = 20 estrous cycles in 10 mares), ranging from 2.5 to 10 y of age and 400 to 500 kg of body weight were used from September 2004 to February 2005 in the southern hemisphere (Brazil). Donor mares were randomly assigned in a cross-over design study. During a Treated cycle, 7.5 mg PGF was given 48 h prior to embryo collection, whereas in the Control cycle, 7.5 mg PGF was given at embryo collection. In Treated Cycles, serum progesterone concentrations decreased between the day of PGF treatment and the day of embryo collection (13.9 ± 5.4 and 0.5 ± 0.3 ng/mL, respectively; P < 0.05). In Treated versus Control cycles, the interovulatory interval was shorter (14.9 ± 0.9 vs 17.5 ± 1.1 d, P < 0.05). However, there was no significant difference between these groups for the interval from PGF to ovulation (average, 9.8 d), embryo recovery rate (average, 75%), embryo quality, uterine protein concentration, and pregnancy rate in recipient mares (average, 87% at 15 d after ovulation, with no pregnancy loss detected by 60 d). In conclusion, giving donor mares PGF 48 h prior to embryo collection reduced the average interovulatory interval by approximately 2.5 d, thereby potentially increasing the numbers of embryos that could be collected during a breeding season, with no deleterious effects on embryo recovery rate, embryo quality, or pregnancy rate in recipient mares.

117 SERUM PROGESTERONE CONCENTRATION AND EMBRYO DIAMETER ON DAY 7 POST-OVULATION IN MARES

Monitoring serum progesterone (P4) concentrations has been a routine practice for determining phases of the oestrous cycle in mares. The use of maternal serum P4 concentration to estimate embryo size would be beneficial in determining the proper collection time for equine embryos destined for cryopreservation and transfer. Therefore, the objective of this study was to determine if embryo diameter was correlated with maternal serum P4 concentration on Day 7 post-ovulation. Light horse mares of various breeds (n = 40) were used in this study. The mares were in good body condition and ranged in age from 3 to 18 yrs. Oestrus detection was performed first with a stallion followed by reproductive tract examinations via transrectal ultrasonography to predict ovulation. Blood samples were collected via jugular venipuncture before artificial insemination (AI) (Day 0) and again on Day 7 post-ovulation. Blood samples were allowed to clot, then centrifuged for 10 min at 2600 × g and serum stored at –20°C until assayed. Each donor mare was inseminated with extended fresh semen every other day until ovulation. On Day 7 post-ovulation, a nonsurgical embryo collection was performed on inseminated mares by a single technician using a standard equine uterine flushing procedure. Only blood samples taken from mares in which an embryo was collected were analysed (n = 18). Blood samples were collected from similar noninseminated mares (n = 12) from the same research herd on the day of expected AI (Day 0) and again on Day 7 post-ovulation. Serum progesterone was determined using a P4 fluorometric enzyme immunoassay (FEIA; Tosoh Bioscience, Inc., South San Francisco, CA). Embryo diameter was measured including the zona pellucida using Micron Software (Westover Scientific). Mean P4 concentrations (±s.e.) for all inseminated donors producing an embryo were 0.49 ± 0.1 ng mL–1 at Day 0 and 12.9 ± 1.2 ng mL–1 on Day 7 post-ovulation. Mean P4 concentration of donors on Day 7 post-ovulation with embryo diameters of &lt;400 μm (n = 7) was not different from donors producing embryos &gt;400 μm (n = 10) and were 11.2 ± 1.7 ng mL–1 and 14.1 ± 1.7 ng mL–1, respectively. One mare produced viable twin embryos (284 and 373 μm in diameter) and had a P4 level of 35 ng mL–1 on Day 7 post-ovulation, and was not included in the data analysis. Mean P4 concentrations in the noninseminated mares were 0 0.68 ± 0.2 ng mL–1 on Day 0 and 12.3 ± 1.6 ng mL–1 on Day 7 post-ovulation and were not different from embryo-producing mares regardless of embryo size. From this preliminary study, that there is no evidence that embryo diameter is correlated with maternal progesterone concentrations on Day 7 post-ovulation.

32 A CLONED FOAL PRODUCED USING OOCYTES RECOVERED BY TRANSVAGINAL ASPIRATION OF IMMATURE FOLLICLES

Closure of all the horse slaughterhouses in the US has reduced the availability of equine oocytes in this country. We investigated the use of oocytes collected from immature follicles of live mares for cloning research. Because blastocyst development of equine cloned embryos is typically low (&lt;10%), we also investigated the effect of Scriptaid, a histone deacetylase inhibitor that increases blastocyst development, live birth rate, and neonatal health in cloned mice and pigs. Immature oocytes were transvaginally aspirated from all follicles ≥8 mm diameter in a herd of 11 mares. The oocytes were cultured in modified M199 for 24 to 26 h. Donor fibroblasts from a 27-year old stallion were treated with roscovitine for 24 h, then were directly injected into enucleated oocytes using the Piezo drill. Reconstructed oocytes were activated with ionomycin followed by injection of sperm extract and culture with 6- dimethylaminopurine (6-DMAP) for 4 h. Recombined oocytes in the Scriptaid treatment were cultured in the presence of Scriptaid, 250 nM, starting at the onset of 6-DMAP treatment and continuing for a total of 18 to 20 h. After embryo culture, blastocysts were shipped for transfer to recipient mares. Overall, each oocyte donor mare underwent aspiration up to 10 times; 653 follicles were aspirated and 271 oocytes were recovered. The in vitro maturation rate was 65% (172/263). After nuclear transfer procedures, 147 oocytes survived; 130 were used for the study. The blastocyst development rate was 2/47 (4%) in the control treatment and 1/83 (1%) in the Scriptaid treatment. All 3 blastocysts yielded pregnancies after transfer. Both control pregnancies were lost, 1 at 30 days and other at 9 months. The mare pregnant with the embryo from the Scriptaid treatment foaled at 326 days of gestation. The foal had medical issues at birth similar to those seen in some cloned foals previously, including maladjustment, patent urachus, and poor oxygenation. These issues were resolved with medical care; the foal is 3 months of age and healthy at the time of writing. These results indicate that immature oocytes obtained from a limited number of mares can be used successfully for nuclear transfer, providing the opportunity to control the mitochondrial identity of the host cytoplast. Scriptaid treatment did not improve the rate of blastocyst development or prevent health problems at birth; however, transfer of 1 embryo in this treatment produced a viable foal. More work is needed to determine the effect of histone deacetylase treatment on efficiency of cloning in the horse. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University, and by Ms. Kit Knotts. We thank Drs. Malgorzata Pozor, Margo Macpherson, and the Medicine team at the University of Florida for medical care of the foal.

50 FIRST EQUINE CLONE BORN IN ARGENTINA BY SOMATIC CELL NUCLEAR TRANSFER FROM A POLO ARGENTINO MARE

Births of the first cloned equids were reported in 2003 (Woods et al. 2003 Science 301(5636), 1063; Galli et al. 2003 Nature 424 (6949), 635) but no equine clones have been reported in Latin America. The aim of this work was to inform the birth of the first equine clone in Argentina as a result of interdisciplinary teamwork. Equine oocytes were obtained from slaughterhouse ovaries and were transported at 38°C for 15 h in TCM-199 buffered with 20 mM HEPES and supplemented with 1 mM glutamine, 0.19 mM sodium pyruvate, 2 μg mL–1 LH, 5 μg mL–1 FSH, 100 ng mL–1 EGF, 100 ng mL–1 IGF-I, and 10% FCS. After 16 to 18 h of maturation, oocytes were denuded and enucleated. Frozen–thawed somatic cells from 1 of 2 aged (23 and 20 years old) Polo Argentino breed donor mares were inserted into the perivitelline space of each enucleated oocyte and fusion was induced. Activation was performed by exposure to Ionomycin (5 μM) for 4 min, followed by 4 h culture in a combination of 6-DMAP and cycloheximide in SOFaas culture media (Choi et al. 2001 Reproduction 122, 177–183). Embryos were cultured in 50 μL SOFm droplets for 7 to 8 days and then were transferred non-surgically to anovulatory recipient mares treated with 1.5 mg oestrogen, followed by long acting progesterone (BioRelease P4 300 LA, BET Pharm, KY). The production rates of the first cell line were: fusion 301/553 (54.4%), cleavage 163/218 (53.4%), blastocysts produced 20/163 (12.2%), blastocysts transferred 18/20 (90%), pregnancies 1/18 (5.5%) and no foal born. The production rates of the second cell line were: fusion 252/553 (45.6%), cleavage 55/252 (21.8%), blastocysts produced 8/55 (14.5%), blastocysts transferred 7/8 (87.5%), pregnancies 4/7 (57%) and 1 filly was born. DNA microsatellite analysis confirmed that the filly was a clone from the original donor cell line. The filly, born naturally on Day 329 of gestation, suffered premature placental separation and weighed 12 kg. Despite the intensive care provided, the filly’s respiratory, nervous, and cardiovascular functions deteriorated rapidly and she died 14 h postpartum. Fetal membranes weighed 5.7 kg (47.5% of the filly’s weight) and showed marked oedema of the chorioalantois and umbilical cord (which also showed a torsion of 9 twists). Histopathologically, the chorioalantois showed scant and poorly developed villi and also connective tissue oedema. Death could have been due to marked immaturity, as a consequence of placental insufficiency and umbilical cord torsion.

Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares

Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 °C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 ( HSPA1A) mRNA were determined by real-time RT-PCR in resulting blastocysts, and were compared to those for embryos derived in vivo from control or exercised mares. Exposure of oocytes to heat at the onset of in vitro maturation did not affect any measured end point. However, exposure to 42 °C late in maturation culture reduced rates of oocyte nuclear maturation for both the 2 h (43/105 (43%) compared to control 70/103 (68%); P < 0.01), and 4 h (47/106 (44%) compared to control 60/103 (59%); P < 0.05) groups. Additionally, late heat exposure reduced development to morulae and blastocyst stages after intracytoplasmic sperm injection (ICSI; 18/89 (20%) compared to control 43/128 (34%); P < 0.05). Seven days after oocyte heat exposure, resultant blastocysts had a higher abundance of HSPA1A gene transcripts, relative to those for 18S rRNA. In vitro-produced embryos and lower-quality in vivo-produced embryos had significantly higher relative HSPA1A mRNA (lower 18S rRNA) concentrations than did higher-quality in vivo-produced embryos. The authors concluded that equine oocytes were sensitive to heat during late in vitro maturation, and responded to thermal shock with an increased ratio of HSPA1A:18S gene expression that was measurable in the resulting blastocyst. Embryos produced in vitro (including controls) had increased levels of HSPA1A mRNA relative to 18S rRNA compared to in vivo-produced embryos, suggesting a response to environmental insult.

Evaluation of two oestrus synchronization regimens in eFSH-treated donor mares

Reliable methods for regulating oestrus and superovulation in equine embryo transfer (ET) programs are desirable. The objective in this study was to compare two oestrus synchronization methods combined with equine follicle-stimulating hormone (eFSH) treatment in an ET program. In the progesterone and estradiol-17β (P&E) group, mares (n=12) were given progesterone and estradiol-17β, daily for 10 days, followed by prostaglandin (PG)F(2α) on the last day. In the PG group, mares (n=12) were given PGF(2α) 5 days post-ovulation. In both groups donor mares were allocated to eFSH therapy, and were subsequently bred. Embryo recovery and transfer were performed routinely. The interval to ovulation (mean ± SEM, range) was not statistically different between donor mares in the P&E group (10.2±0.3, 9-12 days) and donor mares in the PG group (8.7±0.7, 4-12 days). Among donor mares, the synchrony of ovulations was higher following the P&E regimen (P<0.05); however, there was a tendency (P<0.06) for fewer ovulations than in the PG group (1.5±0.3 vs. 2.5±0.4 ovulations, respectively). Embryo recovery (0.9±0.3 vs. 1.4±0.3 embryo/recovery) and recipient pregnancy rate per transferred embryo (4/9, 44% vs. 4/15, 27%) were similar. It was concluded that the P&E regimen was more reliable for synchronization of oestrus in eFSH-treated mares but the fewer ovulations may curtail any advantage of this regimen.

158 SUCCESSFUL TRANSFER OF FRESH AND VITRIFIED BIOPSIED EQUINE EMBRYOS

In horses, determination of certain genetic traits/alleles (e.g. HYPP, SCID, sex, or color) before transfer of embryo often would be advantageous due to the costs of these pregnancies. An attractive option is pre-implantation genetic diagnosis, but to date, few biopsied equine embryos have resulted in pregnancies. In the current experiment, 10 embryos ranging in diameter from 160-575 μm were biopsied. To obtain embryos, donor mares were monitored using transrectal ultrasonography. When a follicle &gt;35 mm in diameter was observed, 2500 IU of hCG or 1.5 mg of deslorelin acetate was administered, and mares were inseminated daily until ovulation was detected. Embryos were recovered nonsurgically on Days 6.5-7 (Day 0 = ovulation). Trophoblast biopsies were collected in a 30-μL droplet of Syngro Holding Media (Bioniche, Belleville, Ontario, Canada) using a Piezo drill and beveled injection pipette (7-10 μm outer diameter). Biopsy samples ranged from 4-10 cells by visual assessment. After removal of the embryo, the droplet containing the biopsied cells was moved into an Eppendorf tube and centrifuged at 11 000g for 10 min. Supernatant was removed leaving 5 μL of sample, which was snap frozen for later genetic testing. Embryos were immediately transferred nonsurgically into the uteri of recipient mares synchronized to ovulate 0 to 2 days after the donor. Pregnancy examinations were performed via ultrasonography on Days 11, 12, 14, 16, 20, and 25. Pregnancies were confirmed on Day 16 for 6 of the 10 transfers. Five embryos (175-240 μm) had confirmed heartbeats on Day 25; one recipient mare lost her pregnancy (160 μm) between Days 16 and 20. The two largest embryos, 390 and 575 μm, did not result in pregnancies. As a preliminary experiment, 7 embryos (150-300 μm) were vitrified according to Eldridge-Panuska et al. (2005) after biopsying and later warmed and transferred directly. Three (all 200 μm) Day 16 pregnancies resulted from the transfer of vitrified, biopsied embryos. Two pregnancies were maintained to a heartbeat; one pregnancy resulted in the formation of an empty trophoblastic vesicle. All pregnancies were terminated on or after Day 25 to collect embryos for further genetic testing. For pre-implantation genetic testing, a duplex-nested PCR was developed for amplification of the DNA from the biopsied cells using primers for sex chromosome-linked zinc finger protein genes (ZFX/ZFY; 445 bp), and 2 pairs of primers for equine-specific sex-determining region on the Y-chromosome (SRY; 217 bp, 121 bp). Experiments on XX and XY genomic DNA from white blood cells revealed accurate genetic testing on as little as 9 pg of DNA, which equals 1 cell. Preliminary results revealed successful sex genotyping on biopsied material, and current experiments are underway to confirm and expand on these results.

172 PREGNANCY RATES AND EARLY PREGNANCY LOSSES IN POSTPARTUM EQUINE EMBRYO RECIPIENTS

In commercial equine embryo transfer programs, the cost of recipient mares represents one of the major economical items that increases the final cost of the pregnancies (Douglas 1985; Losinno and Alvarenga 2006). Due to the risks to newborn foal, it is uncommon to use foaling recipients in the large equine embryo transfer programs in Argentina. The objective of this study was to evaluate pregnancy and early embryonic loss rates between foaling and non-foaling recipients in a commercial embryo transfer program. A total of 173 embryos were collected from Arabian donor mares during 3 consecutive breeding seasons, 2006-2009. Donor mare’s ages ranged from 2 to 22 years, and donors were inseminated with either fresh or frozen semen from 8 stallions 2-17 years old and of proved fertility. Uterine flushing was performed on Day 8 to 9 post-ovulation by standard techniques (Douglas 1985) and recovered embryos were transferred nonsurgically. Recipient mares 3-12 years old were selected clinically by rectal palpation and ultrasound. All embryos were transferred to synchronized recipient mares (-1 to +1 according to donor’s ovulation day) and randomly assigned to non-foaling (n = 84) or foaling (n = 89) recipient groups. For the analysis, we first considered pregnancy and early pregnancy loss rates in both groups, and second, the foaling mares were divided in 3 groups according to the days between foaling and transfer: 1) before 30 days postpartum (n = 10), 2) between 31 and 60 days postpartum (n = 30), and 3) after 61 days postpartum (n = 49). Pregnancy diagnosis was performed by ultrasonography on Days 14, 30, and 60 post-ovulation of the donor. Results were evaluated by chi-square test. No statistical differences (P &gt; 0.05) were observed in pregnancy rates between non-foaling and foaling recipients (76.2% and 80.9%, respectively) and in post-foaling groups (80% for recipients transferred before 30 days postpartum, 73.3% for recipients transferred between 31 to 60 days postpartum, and 85.7% for recipients transferred after 61 days postpartum). No statistical differences (P &gt; 0.05) were found in pregnancy loss rates between recipients transferred before 30 and after 61 days postpartum (25% v. 4.8%, respectively) and between recipients transferred before 30 days postpartum and the non-foaling group (25% v. 10.9%, respectively). Pregnancy loss rates for recipients transferred after 31 and 61 days postpartum (9.1% and 4.8%, respectively) were not significantly different with the non-foaling group, and both are within previously reported values. Our results show that postpartum recipients can be used 30 days after normal foaling in a commercial embryo transfer program with similar pregnancy and early pregnancy loss rates and can be a valuable tool maximizing the use of the recipient mares during consecutive breeding seasons.

165 OVARIAN RESPONSE AND EMBRYO RECOVERY RATES OF OLD MARES TREATED WITH A LOW DOSE OF EQUINE PITUITARY EXTRACT

Mares older than 15 years have low embryo recovery rates, mostly because of disturbances in follicle or oocyte maturation and early embryonic development. A recent publication showed that circulating LH level is lower in older mares (Ginther et al. 2009 Theriogenology 5, 780-788). Equine pituitary extract (EPE) is rich in LH and consequently is able to increase circulating LH levels after injection into mares. The present study aimed to evaluate the effect of low doses of EPE on follicular growth, ovulation rate, and embryo recovery rates in older mares. Embryo donors (n = 20) were Quarter Horses from a commercial embryo center that ranged in age from 15 to 20 years. Donors were used during 3 consecutive cycles. The first and third cycles were used as control cycles, and the EPE treatment was performed on the second cycle. During estrus in each of the 3 cycles, mares were inseminated with cooled or fresh semen from the same stallion and received an injection of deslorelin (1 mg i.m.) upon detection of at least 1 follicle of 35 to 40 mm in diameter. Embryo flushes were performed on Day 8 post-ovulation and were followed by administration of prostaglandin. On the treated cycle, EPE injections (7 mg twice daily i.m.) were started on Day 8 after ovulation until a preovulatory follicle was observed. The percentage of mares with more than one ovulation was compared using Fisher’s test. The mean number of ovulations and embryos, as well as follicular diameter, was compared using Student’s t-test. The percentage of mares with more than one ovulation as well as the number of ovulations per cycle were higher (P &lt; 0.05) in the EPE-treated cycle (65% of mares and 1.8 ovulations, respectively) than in the control cycles before (5% and 1.0) and after treatment (5% and 1.0). Also, the mean number of embryos recovered per cycle was higher (P &lt; 0.05) in EPE-treated cycles (1.0) than in nontreated cycles (0.42). However, the embryo recovery rate per ovulation was similar (P &gt; 0.05) between nontreated (0.4 embryo) and treated cycles (0.6 embryo). Pregnancy rates of the transferred embryos were also similar between embryos recovered on treated (14/20; 70%) and nontreated cycles (9/17; 55%). The daily growth of the dominant follicle was not altered by the EPE treatment. However, embryos recovered during treated cycles were more advanced in development than controls, with more embryos (P &lt; 0.05) classified as morula or early blastocysts when recoverd during nontreated (58%) v. EPE-treated cycles (0%). Based on the results of the present experiment, we can conclude that EPE treatment was able to increase the reproductive efficiency of older embryo donor mares. The improvement in embryo recovery rates on EPE-treated cycles seemed to be more related to the increase in the number of ovulations per cycle than with an improvement in embryo viability.

181 CHANGES IN THE BODY CONDITION STATUS OF DONOR MARES THROUGHOUT THE BREEDING SEASON AND THEIR RELATIONSHIP WITH REPRODUCTION TRAITS

The objective was to investigate the relationship between changes in body condition on reproductive traits of donor mares in an embryo collection program. Fifty-six non-pregnant mares, 3 to 18 years old, were studied from August 2008 through April 2009. Body condition score (BC; 1-9 scale, thin to obese) was estimated monthly. Body weight and tail and rib s.c. fat depths (SF) were measured monthly. SF was determined by ultrasound. Dominant follicle diameter (DF) was measured daily during the follicular phase. Mares were artificially inseminated and embryo transfers were conducted 6 d after ovulation. Analyses were performed with SAS® (SAS Institute Inc., Cary, NC, USA). Age classes were as follows: age 1, 3 to 6.9 (n = 17); age 2, 7 to 12.9 (n = 25), and age 3, ≥13 years (n = 14). Months in experiment were T1 to T6. BC classes were as follows: B1, &lt;6.5; B2, 6.5 to 6.9; B3, 7.0 to 7.5; and B4, &gt;7.5. Embryo recovery rate was compared by chi square. Effects of month and age on BC, weight, and SF were analyzed by a mixed model. Age, BC, and month fixed effects on DF diameter were analyzed by ANOVA with mare within class as the error term. Means were compared by contrasts. BC was higher (P = 0.02) in older mares (6.78 ± 0.2 and 6.93 ± 0.2 in age 1 and age 2 v. 7.25 ± 0.2 in age 3) and increased (P = 0.009) with time (from 6.79 ± 0.20 to 7.42 ± 0.26 for T1 to T6, respectively). Weight (kg) was not associated with age but increased (P = 0.03) throughout the experiment. Tail fat increased with age (P = 0.04) and decreased with increasing months on experiment (P = 0.02). DF diameter increased daily through the follicular phase (P &lt; 0.0001) and was smaller (P &lt; 0.05) in mares with lower body condition (33.80 ± 0.31 in BC1 v. 34.17 ± 0.31 mm in BC4). DF diameter decreased (P &lt; 0.01) with month on study. Embryo recovery rate was not affected by age or BC but was lower (P = 0.003) in mares that ovulated follicles smaller than 40 mm compared to mares ovulating follicles greater than 40 mm. BC above 6.5 was associated with higher embryo recovery rate, which was, in turn, correlated positively with DF diameter. Thus, our results support the use of BC scoring to predict performance in equine embryo transfer programs. Body condition scoring was not a good indicator of SF under the current experimental conditions because SF decreased during the breeding season. BC scoring may indicate lean tissue gain, which may be a better indicator of reproductive efficiency in mares. FAPEMIG, CAPES, Haras El Far.