Effect of different cryopreservation extenders added with antioxidants on semen quality and in vitro embryo production efficiency in cattle.

To evaluate the addition of antioxidants in extenders on post-thaw bovine semen quality and in vitro embryo production efficiency. Six semen samples were collected from five Holstein bulls. In the experiment I, the samples were diluted with AndroMed® and Bovimix® and added antioxidants glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM). In the experiment II, the best treatments obtained in experiment I were used for in vitro fecundation. Glutathione did not improve sperm viability. Melatonin had a negative effect on semen characteristics. Andromed® showed better results in sperm kinetics parameters. Bovimix® was more efficient in maintaining cell integrity parameters. Significant correlation was found between sperm kinetics parameters and between cell integrity parameters. For in vitro embryo production, after oocyte selection, maturation, fertilization and cultivation were performed using the four treatments previously evaluated. Andromed® was more efficient in the cleavage rate, no effect of the addition of glutathione. However, the addition of 2.5 mM glutathione in the Bovimix® improved the cleavage rate. There was a significant moderate correlation between cleavage rate and sperm kinetic characteristics. Glutathione did not improve sperm viability. Melatonin reduced the maintenance of sperm characteristics. Andromed® was more efficient in in vitro embryo production and no effect of glutathione was found in this extender. Addition of 2.5 mM glutathione in the Bovimix® extender provided a higher cleavage rate.

Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF).

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time.

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.