Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

An oxymyoglobin liposome model was used to study the effects of a-tocopherol and/or ascorbate upon oxymyoglobin and lipid oxidation. Both oxidations were delayed (PcO.05) by increased concentrations of a-tocopherol alone and a-tocopherobascorbate relative to controls. The 14 )IM a-tocopheroV140 p,M ascorbate treatment resulted in greatest delay in both oxymyoglobin and lipid oxidation. Ascorbate alone delayed oxymyoglobin oxidation; effectiveness diminished as ascorbate increased. Ascorbate showed a prooxidant effect toward lipid oxidation which was unchanged in the presence of EDTA. Results support the hypothesis that a-tocopherol retards lipid oxidation directly and OxyMb oxidation indirectly.

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This study examined the onset of puberty, follicular dynamics, reproductive hormone profiles, and ability to maintain pregnancy in cloned heifers produced by somatic cell nuclear transfer. Four adult somatic cell-cloned heifers, derived from a 13-yr-old Holstein cow, were compared to 4 individual age- and weight-matched heifers produced by artificial insemination (AI). From 7 to 9 mo of age, jugular venous blood samples were collected twice weekly, and from 10 to 11 or 12 mo of age, blood sampling was carried out every other day. After the heifers reached puberty (defined as the first of 3 consecutive blood samples with peripheral plasma progesterone concentrations of >1 ng/ml), ultrasound examination of ovaries and jugular plasma sample collection were carried out daily for 1 estrous cycle. Cloned heifers reached puberty later than controls (mean +/- SEM, 314.7 +/- 9.6 vs. 272 +/- 4.4 days and 336.7 +/- 13 vs. 302.8 +/- 4.5 kg for clones and controls, respectively; P < 0.05). However, cloned and control heifers were not different in estrous cycle length, ovulatory follicle diameter, number of follicular waves, or profiles of hormonal changes (LH, FSH, estradiol, and progesterone). Three of the 4 clones and all 4 control heifers became pregnant after AI. These results demonstrate that clones from an aged adult have normal reproductive development.