32 a Cloned Foal Produced Using Oocytes Recovered by Transvaginal Aspiration of Immature Follicles

Abstract

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.