Foals were produced from mares after death or euthanasia under field conditions. Proportions of foals born overall (10 foals/16 mares) and mares from which ≥ 1 foal was produced (6/16) were greater than those reported following recovery and oviductal transfer of oocytes to inseminated recipients after death of donor mares under field conditions.

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SummaryReasons for performing study: Transvaginal ultrasound-guided follicle aspiration (TVA) is performed clinically but there is little information available on complications associated with this procedure. Objectives: It is possible that TVA is associated with damage to the ovary and may induce peritonitis or peritoneal adhesions. This study was conducted to determine the effect of repeated TVA on mare health and ovarian status. Methods: Thirty-two mares were used for oocyte recovery via repeated TVA over a 3 year period; different mares were used each year. In Year 1, ovarian status was monitored in 11 mares by transrectal palpation and ultrasonography. In Year 2, 6 of 11 mares underwent abdominocentesis and were examined by laparoscopy after one TVA and again after multiple TVAs. In Year 3, 10 mares underwent multiple TVAs with either a 15 or a 12 gauge needle and the ovaries were removed for examination. Results: Four hundred and twenty-seven aspiration sessions (390 via TVA and 37 via needle placement through the flank) and 3202 follicle punctures (3161 TVA and 41 flank) were performed. One mare developed an ovarian abscess. Transient rectal bleeding was evident after 16% of TVA sessions. No adhesions were found on laparoscopic or gross examination of ovaries and there were minimal changes on histological evaluation. Conclusions: Follicle aspiration carries a small possibility (<0.5%) of ovarian abscess formation. There is a possibility of rectal abrasion or puncture but little gross or histological damage to the ovary. Potential relevance: These results provide a basis for using prophylactic administration of antibiotics after TVA and for advising mare owners of the rare but potential complications associated with the procedure.

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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 (<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&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.

Transvaginal ultrasound-guided follicle aspiration (TVA) is the most effective way to recover multiple immature oocytes from live mares. Because of the tight attachment of the equine immature oocyte to the follicle wall, TVA in this species is time consuming, taking up to 1 h per horse. Thus, it may be difficult to search follicular aspirates immediately after recovery. In 2009 in a series of 6 replicates, we observed a blastocyst development rate of 32% (13/41) after intracytoplasmic sperm injection of oocytes collected by TVA and held for ∼1.5 h at ambient temperature (26 to 32°C) before isolation from aspirated fluid (unpublished data). Therefore, in the present study we compared the effects of immediate oocyte isolation v. holding the follicular aspirate before oocyte isolation on oocyte maturation and blastocyst rates. Ten mares were used for this study; TVA was performed on each mare every 14 days, for 4 aspiration sessions per mare. Collected aspiration fluid was either processed immediately or held for 2 h at 32°C before processing. At each aspiration, one ovary was randomly assigned to each treatment [immediate (Imm) or 2-h holding (2-H)]. Follicle aspiration was performed as previously described (Jacobson et al. 2010 Theriogenology 73, 1116–1126) using M199 with Hank’s salts to flush the follicle lumen up to 8 times per follicle. Oocytes were recovered from the aspirates by filtration. Overall, 325 follicles were aspirated and 140 oocytes were obtained (43% recovery). The proportion of degenerating oocytes was not significantly different between treatments (1/68 and 0/72 for Imm and 2-H, respectively). Oocytes were held overnight in modified M199 as previously described (Choi et al. 2006 Theriogenology 66, 955–963) before maturation culture. After 30 h of maturation culture, there was no significant difference in maturation rates between treatments [75% (50/67) and 65% (46/71) for Imm and 2-H, respectively]. Fertilization was performed by intracytoplasmic sperm injection, and injected oocytes were cultured 7 to 10 days, as previously described (Choi et al. 2006). The rates of blastocyst development per injected oocyte were 23% (11/47) for Imm and 16% (7/44) for 2-H; these were not significantly different (P > 0.3). The reason for the discrepancy in blastocyst rates for held oocytes in this study (16%) compared with our 2009 observations (32%) is unclear; this could be a factor of the time held (∼1.5 v. 2 h), the temperature (26–32°C v. 32°C), or minor changes in protocol between the 2 years. From these results, we conclude that holding oocytes in the follicular aspirate for up to 2 h following collection may be performed when necessary without significantly affecting the rate of subsequent blastocyst development. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

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