Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes.

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

Vitrification with glutamine improves maturation rate of vitrified / warmed immature bovine oocytes.

The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 µg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. L-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation.

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency.

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A3 (CMA3) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA3 staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA3-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA3-positive sperm cells compared to the others. CMA3 is a simple and useful tool for detecting sperm protamine deficiency in bulls.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows.

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha -lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position - 1689 (g. A > G) and + 15 (g. A > G) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide - 46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A - 1689, A - 46, and A + 15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5'-flanking and the 5'-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Effect of chemical or electrical activation of bovine oocytes on blastocyst development and quality.

Activation of in vitro-matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 microM CA, 5 min; n = 88), CA + BSA (5 microM CA, 5 min; BSA, 5 min; n = 90), IO (5 microM IO, 5 min; n = 91), IO + BSA (5 microM IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 micros; n = 120). Blastocyst rates were higher (p < 0.05) for CA (54.4%), IO (51.4%) and EL (54.5%) than for IO + BSA (18.3%). Treatment CA + BSA (39.8%) did not differ from the others. There was no difference (p > 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.

Effects of serum deprivation and cycloheximide on cell cycle of low and high passage porcine fetal fibroblasts.

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 +/- 0.65) when compared with non-starved cells (71.44 +/- 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.