To determine the most optimal stage for antioxidant supplementation of culture medium to improve developmental competence, cryotolerance and DNA-fragmentation of bovine embryos. Presumptive zygotes were first cultured in presence or absence of β-mercaptoethanol (β-ME), for 8 days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 min and post-warming embryos along with their corresponding non-vitrified embryos were cultured for two further days in presence or absence of (100 µM) βME. For vitrified and non-vitrified embryos, the best effect was found when βME was added from day 1 of in vitro culture in continuation with post-warming culture period. Day 1–8 supplementation significantly increased the rates of cleavage, day 7 and day 8 blastocyst production. For non-vitrified embryos, βME addition during day 1–8 and/or 9–10 of embryo culture improved both hatching rate and quality of hatched embryos. For vitrified embryos, however, the percentage of DNA-fragmentation (18.5%) was significantly higher (p ≤ 0.05) than that of embryos developed in absence of βME but supplemented with βME during post-warming period (13.5%). Exogenous antioxidant increases the chance of embryos, even those of fair-quality, to develop to blastocyst. However, antioxidant inclusion during in vitro embryo development is not sufficient to maintain the redox state of these embryos during the critical period of post-warming embryo culture, and therefore, there should be a surplus source of exogenous antioxidant during post-warming embryo culture.

King oyster mushroom (Pleurotus eryngii) contains many medicinal and nutritional values, excellent flavors, and a long shelf life. Cultivation of edible mushroom on agricultural and lignocellulosic waste like wheat straw and wood chips gives a high yield and nutritional contains. In present study, the effects of lignocellulosic organic wastes, such as wood chips, wheat and barley straw, sugar beet pulp, sawdust and maize stem residue as basal substrate and wheat and rice bran, soybean powder and their combinations were used as supplements. We analyzed mushroom production gap, mushroom fresh weight, moisture content, dry matter, and protein content of fruit body traits. A factorial experiment was conducted in a completely randomized design with four replications. Supplementation of wheat bran in wood chips increased the mushroom production gap and dry matter content. The highest mushroom fresh weight and moisture content were achieved on barley straw and sugar beet pulp substrate complemented with rice bran, respectively. In addition, protein content ranged from 4.64 % (barley straw + wheat bran and wood chips + soybean powder + rice bran treatments) to 13.66 % (wheat straw + wheat bran + soybean powder treatment). The quality of P. eryngii was significantly affected by substrate ingredients. The type of substrate as well as the type and quantity of supplement appeared to have a substantial effect on prolonging the delayed-release nutrients.

Background: Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. Results: Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 μg supercoiled plasmid DNA and 0.3 × 10⁶ cells in 100 μl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. Conclusions: The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.