Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure(® )and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure(® )and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure(®), but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure(® )method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure(® )treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure(® )method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure(® )could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos

Glutathione synthesis during in vitro maturation of ovine oocytes: effect of cysteamine and β-mercaptoethanol

Sheep oocytes were collected either by vacuum aspiration with a 20-gauge needle or by slicing ovaries obtained from a local slaughterhouse. The oocytes were in vitro matured, fertilized and cultured as described previously (Thompson et al, Biol. Reprod. 1995, 53:1385-1391). The rate of embryo development was recorded in sheep oocytes matured in IVM medium supplemented with 0, 50, 100, 200 uM cysteamine (exp.1) or with 0, 50, 100, 200 uM l beta-mercaptoethanol (exp.2). After IVM, GSH levels were measured in oocytes matured with 200 pM cysteamine or 50 uM beta-mercaptoethanol with or without 5 mM buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Logarithmic transformed data were analyzed by ANOVA and Tukey's test. It was demonstrated that 200 uM cysteamine stimulates embryo development (50.2 vs 33.7%) whereas beta-mercaptoethanol does not (31.8 vs 38.1%). Furthermore, GSH synthesis was stimulated in IVM oocytes in the presence of either cysteamine (6.7 vs 4.2 picomol/oocyte) or beta-mercaptoethanol (8.8 vs 6.5 picomol/oocyte). Moreover, the results obtained in the control groups _+ BSO suggests that intracellular GSH synthesis occurs

The Relation between Protein Malnutrition, Ethanol Consumption and Chromosomal Damage Induced by Cyclophosphamide in Bone Marrow Cells of Mice

The effect of protein malnutrition and alcohol consumption on the yield of chromosomal damage induced by cycloPhosphamide (CP) was studied. Chromosomal damage induced in bone marrow cells of BALB/c mice was established by scoring the frequency of dicentric chromosomes in C-banded slides. Results obtained showed that CP induced a significant increase of chromosomal damage in comparison with untreated mice. In addition, the yield of dicentric chromosomes was higher in mice fed with the hypoproteic diet. The animals which received ethanol in drinking water before treatment with CP exhibited the highest frequency of dicentric chromosomes, with no relation with the diet. Statistical analysis of these results showed the additive effect of diet and CP and are explained taking into account the metabolic pathways of CP as well as the decrease of enzymatic levels and the physiological condition in under-nourished mice.Facultad de Ciencias Veterinaria