In vitro production of meiotically competent oocytes from early antral follicles in sheep.

The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350-450 µm) and cultured in TCM199 medium supplemented with 0.15 µg/mL Zn sulfate, 10-4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 µM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction.

Effect of Strategic Supplementation of Dietary By-Pass Linseed Oil on Fertility and Milk Quality in Sarda Ewes.

The aim of the present study was to assess whether the strategic supplementation of bypass LO can enhance reproductive indexes­fertility, lambing rate, and prolificacy­in dairy Sarda ewes at the end of lactation. To assess whether LO supplementation leads to the adsorptions of PUFAs and their subsequent utilization by the body tissues, milk composition and fatty acid content were analyzed. Forty-eight ewes were assigned to the following groups: the control group (CT; N = 24), fed with a control diet without LO; and the treatment group (LO; N = 24), fed with a diet supplemented with LO (10.8 g/ewe/day). Both diets had similar crude protein and energy levels and were offered for 38 days (−21 to +17 days after artificial insemination). The trial included an adaptation period (7 days) followed by a regular supplementation (31 days) period. Estrus synchronization was induced in all the ewes using an intravaginal sponge and equine chorionic gonadotropin. Fifty-five hours after pessaries withdrawal, all ewes were inseminated using the cervical route and fresh semen. Cholesterol (p < 0.01), high-density lipoprotein (p < 0.001), and triglyceride (p < 0.05) levels in plasma were higher in the LO group. Plasmatic levels of non-esterified fatty acids were lower in the LO group after the end of the supplementation period (p < 0.05). Milk unsaturated fatty acids (UFAs), monounsaturated fatty acids (MUFAs), total polyunsaturated fatty acids (PUFAs), PUFAs omega 3 (PUFAs-ω3) and 6 (PUFAs-ω6), and trans fatty acids were higher in the LO group (p < 0.001), while saturated fatty acids (SFAs) were higher in the CT group during the supplementation period (p < 0.001). Three days after the end of the supplementation period, the content of milk UFAs (p < 0.05), PUFAs (p < 0.001), MUFAs, and PUFAs-ω6 (p < 0.01) were still higher in the LO group. whereas SFA was higher in the CT group (p < 0.01). There was no difference between groups in terms of ovulation rate, progesterone levels in plasma, fertility rate, prolificacy, and total reproductive wastage. However, the total area of luteal tissue was higher in the LO group (p < 0.01). Results obtained demonstrated that LO supplementation exerts a positive role in corpus luteum size at the onset of the peri-implantation period in Sarda dairy ewes. Additionally, the results obtained in the present study showed that the use of dietary bypass LO affects lipid metabolites in plasma and milk fatty acid profiles, demonstrating the ALA uptake by body tissues.

Optimizing injection time of GFP plasmid into sheep zygote.

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6-8 hpi, n: 120; 8-10 hpi, n: 122; 10-12 hpi, n: 110 and 12-14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8-10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8-10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.

Oct4 expression in in-vitro-produced sheep blastocysts and embryonic-stem-like cells.

Transcription factor Oct4 (octamer-binding transcription factor-4) is important in early embryonic development and differentiation. It is also required for maintenance of pluripotency of the inner cell mass, and is used as a staminality marker of embryonic stem cells. Changes in Oct4 expression during the different stages of early embryo development have been reported, and therefore we have conducted a quantitative study of Oct4 gene expression of sheep blastocysts in vitro, and of embryonic-stem-like cells at the undifferentiated stage and in the course of differentiation. To characterize embryonic-stem-like cells, alkaline phosphatase activity, stage-specific embryonic surface antigens SSEA-1, SSEA-3, SSEA-4 and three specific gene markers Nanog, Sox2 and Stat3 were assayed. cDNA produced by RT (reverse transcriptase)-PCR was synthesized and amplified by PCR; sequencing gave 98, 95 and 98% homology with the bovine sequences of Oct4, Nanog and Stat3 respectively. Using the ovine sequence of 290 bp, quantitative expression of Oct4 in the inner cell mass, trophoblast and embryonic-stem-like cells was performed by qRT-PCR (quantitative real-time PCR). Oct4 was expressed in the inner cell mass, trophoblast and embryonic-stem-like cells. Expression in the inner cell mass was significantly higher than in the trophoblast. This could be useful in defining the quality of embryos produced and makes it possible to use Oct4 to detect pluripotency. In addition, the different levels of Oct4 expression between undifferentiated and differentiating embryonic-stem-like cell cultures could be used to detect this gene as a staminality marker.

Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study.

Extending the preservation time of fresh semen is an important goal in artificial insemination programs particularly for ewes in natural oestrus, where insemination periods are longer than for ewes synchronized with hormonal treatments. The aim of this study was to evaluate the effect of the antioxidant TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) on the maintenance in long term storage of ram semen motility and fertility. Semen from Sarda breed rams was diluted in two extenders: sodium citrate buffer with TEMPOL and skimmed milk, used as control. Samples diluted with TEMPOL were cooled at either 15 degrees C or 22 degrees C, while those diluted with skimmed milk were cooled at 15 degrees C. Each sample was divided into four stocks, and stored for different times (5 min, 24, 48 and 72 h). Three aliquots were taken from each stock for every storage period. One was immediately evaluated under microscope; one was used for in vitro fertilization; one was incubated for 2 h in controlled humidified atmosphere (5% CO2, 7% O2 and 88% N2) at 39 degrees C, then evaluated for motility and utilized for in vitro fertilization. Ram semen diluted with media containing TEMPOL demonstrated increased motility, fertility and an improved protective effect when it was stored at 15 degrees C.