RESUMO
In the present study, four experimental groups were used: fresh embryos, cultured during in vitro maturation and in vitro culture in media supplemented with bovine serum albumin (BSA) (fresh BSA) or fetal bovine serum (FBS) (fresh FBS); and two groups of cryopreserved and thawed embryos, produced under the same conditions (frozen BSA and frozen FBS). Experiment 1 evaluated the protein source effect on embryo development and response to cryopreservation. At day 7, half of the expanded blastocysts (Bx) from each group were cryopreserved and warmed and the other half were used as controls. After warming, embryos were incubated under the same conditions for 48 hours, and the hatching rate was measured at 24 and 48 hours. The total and the apoptotic cell numbers were measured in a subset of Bx after 24 hours. Experiment 2 used the Bx of experiment 1 to compare the expression of KRT8, PLAC8, FOSL1, HSP1A1, and HSPA5 genes in hatched blastocysts at 24 and 48 hours for all groups. The FBS group showed a higher percentage (p < 0.05) of embryos (42.8% vs. 27.9%) and higher rates of Bx (75.0% vs. 63.8%) on day 7, compared with the BSA group. At 24 hours postwarming, the fresh FBS group showed the highest hatching rate (p < 0.05) in comparison with other treatments. However, at 48 hours, the hatching rate was similar (p > 0.05) among groups: fresh FBS (68.1% ± 23.3%), fresh BSA (70.0% ± 31.0%), frozen FBS (39.2 ± 27.1), and frozen BSA (38.2 ± 23.9). After 24 hours, frozen BSA showed a higher number of cells compared with frozen FBS (p < 0.05). The expression of the PLAC8 gene was higher (p < 0.05) in fresh BSA embryos compared with frozen FBS embryos at 24 hours. In the present study, BSA replacement reduced embryo development, but did not affect the response to cryopreservation. However, upregulation of the PLAC8 gene suggests that embryos cultured in BSA might have better quality to support further development.
Assuntos
Blastocisto/citologia , Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Soroalbumina Bovina/farmacologia , Animais , Apoptose , Bovinos , Meios de Cultura/química , Feminino , Fertilização in vitro , Congelamento , Regulação da Expressão Gênica no Desenvolvimento , Hibridização GenéticaRESUMO
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.