A viable foal obtained by equine somatic cell nuclear transfer using oocytes recovered from immature follicles of live mares.

The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares.

Evaluation of foal production following intracytoplasmic sperm injection and blastocyst culture of oocytes from ovaries collected immediately before euthanasia or after death of mares under field conditions.

OBJECTIVE: To evaluate the efficiency of foal production following intracytoplasmic sperm injection (ICSI) and blastocyst culture of oocytes from mares that died or were euthanized under field conditions. DESIGN: Prospective case series. ANIMALS: 16 mares (age, 3 to 19 years) that died or were euthanized for various causes. PROCEDURES: Ovaries were collected immediately before euthanasia (n = 10) or after death (6). Ovaries were transported to the laboratory for oocyte recovery (15 mares), or oocytes were recovered at a remote location and shipped to the laboratory (1). Oocytes underwent ICSI, and presumptive zygotes were cultured for 7 to 10 days. Blastocysts were shipped to embryo transfer facilities for transcervical transfer to recipient mares. RESULTS: Ovaries were processed 30 minutes to 12 hours (mean ± SD, 4.6 ± 3.3 hours) after mares' deaths. A mean of 14.1 ± 8.6 oocytes/mare were cultured, and 110 of 225 (49%) matured. Twenty-one blastocysts developed after ICSI and were transferred to recipient mares. Thirteen pregnancies were established; 10 healthy foals were produced from 6 donor mares. The number of blastocysts produced per mare and number of live foals produced per mare were significantly correlated with the number of oocytes recovered. CONCLUSIONS AND CLINICAL RELEVANCE: 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.

Physical and clinicopathologic findings in foals derived by use of somatic cell nuclear transfer: 14 cases (2004-2008).

OBJECTIVE: To describe the health status of foals derived by use of somatic cell nuclear transfer (NT) at a university laboratory. DESIGN: Retrospective case series. ANIMALS: 14 live-born NT-derived foals. PROCEDURES: Medical records from 2004 through 2008 were evaluated to identify all pregnancies resulting in live-born NT-derived foals. Information obtained included gestation length, birth weight, foaling complications, gross abnormalities of the fetal membranes, appearance of the umbilicus, mentation of the foal, limb deformities, and any other abnormalities detected in the neonatal period. Clinicopathologic data were also evaluated when available. Records of 4 recipient mares during gestation were included. RESULTS: Six foals were clinically normal for all evaluated variables. The most common abnormalities detected in the remaining 8 foals included maladjustment, enlarged umbilical remnant, and angular deformity of the forelimbs. Two foals died within 7 days after parturition; in the remaining foals, these conditions all resolved with medical or surgical management. Large offspring syndrome and gross abnormalities of the fetal membranes were not detected. The 12 surviving foals remained healthy. CONCLUSIONS AND CLINICAL RELEVANCE: Associated problems of calves resulting from use of NT have been reported, but there are few data on the outcome of foals resulting from adult somatic cell NT in horses. Although this population of foals had a lower perinatal mortality rate than has been reported for NT-derived calves, some NT-derived foals required aggressive supportive care. Birth of foals derived from NT should take place at a center equipped to handle critical care of neonates.

Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer.

We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase blastocyst development (0 and 4.5%) over a control treatment (injection of sperm extract after ionomycin exposure; 5.6%). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in five pregnancies, all lost before 70 days of gestation. In Experiment 3, cells from a second biopsy sample from the same horse produced significantly more blastocysts than did the original sample (4/44 vs. 0/58; p < 0.05). Transfer of these four blastocysts produced two viable foals. In Experiment 4, blastocyst development rates did not differ between oocytes in metaphase I or II at the time of nuclear transfer (16.7 and 3.0%, respectively). A healthy foal was produced from a blastocyst originating from a metaphase I oocyte.