Recently we reported the birth of a viable foal produced by nuclear transfer (NT) using oocytes recovered from immature follicles of live mares by transvaginal ultrasound-guided aspiration (TVA; 2013 Theriogenology 79, 791–796). This procedure opens the door for production of mitochondrial DNA-identical cloned foals; typically, use of heteroplastic oocytes results in cloned offspring that have different mitochondrial DNA from that of the donor. We selected 2 mares (BL and SM) from the maternal line of the donor, a 23-year old stallion. Genetic analysis confirmed that the mares’ mitochondrial genotype was identical to that of the donor. Oocytes were obtained from the mares by TVA of all follicles ≥5 mm diameter, and were matured in vitro for 20 to 26 h. Donor fibroblasts were treated with 15 μM roscovitine for 24 h, then were directly injected into enucleated oocytes using a Piezo drill. Reconstructed oocytes were activated with 5 μM ionomycin for 4 min followed by injection with sperm extract, then incubation in 2 mM 6-dimethylaminopurine for 4 h. Oocytes from mare SM were assigned to treatment with either Scriptaid (500 nM) or Scriptaid plus vitamin C (50 μg mL–1) for 14 to 16 h, starting at the onset of 6-dimethylaminopurine exposure; mare BL did not provide sufficient oocytes for treatment grouping. Presumptive zygotes were cultured in vitro for 7 to 11 days and blastocysts were shipped for transfer to recipient mares, 1 embryo per mare. In mare BL, 10 aspiration sessions were conducted, 78 follicles were aspirated and 45 oocytes were collected, of which 4 were degenerating. After in vitro maturation, 12/40 (30%) oocytes were mature. Five of 12 oocytes lysed during manipulation; the remaining 7 were cultured and 1 blastocyst (14%) was obtained, which did not yield a pregnancy. In mare SM, 3 aspiration sessions were conducted and 53 oocytes were recovered from 81 follicles. After in vitro maturation, 31/53 (58%) were mature. Four oocytes were lysed during manipulation, 27 were cultured, and 4 blastocysts (15%) were produced, 2 from scriptaid treatment and 2 from scriptaid plus vitamin C. Transfer of these blastocysts yielded one pregnancy (scriptaid treatment); the mare delivered a healthy foal at 328 days of gestation. These results indicate that NT can be successful using low numbers of immature oocytes from selected mares. However, the individual mare may greatly affect the outcome in terms of oocyte number and quality; in this case, mare BL not only yielded fewer oocytes per aspiration session (4.5 v. 17.7 for mare SM; P < 0.001, t-test), but also fewer reconstructed oocytes per oocyte recovered (7/45 v. 27/53, respectively; P < 0.001, Fisher's exact test). Efficiency (14 to 15% blastocysts per reconstructed oocyte cultured; 1 foal from 5 embryos transferred) was similar to that achieved previously in our laboratory using heteroplastic oocytes.
This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, by Kit Knotts, and by Jack Waggoner.
Oocyte In Vitro Maturation with Crude Sperm Extract Protein of Bull’s Spermatozoa
