Effect of different percoll procedures on sperm quality and in vitro production of bovine embryos / Efeito de diferentes protocolos de Percoll na qualidade espermática e na produção in vitro de embriões bovinos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Techniques for sperm selection are usually used for bovine in vitro embryo production (IVP) to improve semen quality after thawing. Among these techniques Percoll gradient has been the most used. The aim of this study was to evaluate the effect of different volumes of Percoll, time and force of centrifugation on sperm quality, embryo development and sex of IVP bovine embryos. Frozen-thawed semen from four bulls were submitted to three different Percoll procedures: T1 - 4 mL (final volume) of Percoll 45%:90%, centrifuged for 20 minutes at 700 g, T2 - 800 μL (final volume) of Percoll 45%:90% centrifuged for 20 minutes at 700 g and T3 - 800 μL (final volume) of Percoll 45%:90% centrifuged for 5 minutes at 5000 g. Semen was evaluated before and after Percoll treatment for sperm total motility, morphology, integrity of acrosome, membrane and chromatin. A total of 1194 oocytes were matured, fetilized and cultured in vitro, cleavage rate was valued on D2 and blastocyst rate on D7 after in vitro insemination. The blastocysts in D7 (n=360) were frozen for posterior sex determination. Data from embryo development and sperm characteristic were analyzed by ANOVA considering the effect of bull, treatment and interaction bull x treatment (P <0.05). The ratios were compared with the expected ratio 1:1 by  2 test (P<0.05). All Percoll methods significantly improved the proportion of motile spermatozoa. No effect of treatment was detected in any of the sperm characteristic (P>0.05), however bull-related differences were observed in all parameters tested. Similarly, cleavage and blastocyst rate were not affected by Percoll procedure (P>0.05). However, there was a difference among bulls (P<0.05). Sire 4 had very low embryos production (n=5), and was eliminated from the analysis. Higher rates of cleavage and blastocyst were observed for bull 1 (86 1.6 a % and 47 2.6 c%) and bull 2 (86 2.1a % and 39 6.6c %) showed than for bull 3 (65 5.9b % and 17 4.1d %). The proportion of male:female embryos was similar in all treatments for bulls 2 and 3. Only bull 1 in the T1 presented a greater percentage of male (61%) than female embryos (39%). When only treatments were considered, independent of bulls, no difference was found in proportion male:female (%) among T1 (55:45), T2 (45:55) and T3 (44:56). The results showed that the use of different speed, time of centrifuge and volume of Percoll gradient did not influence the rate of blastocyst and the male:female ratio. However, for bull 1 it was observed a higher production of male embryos when T1 was used. These results suggest the possibility of using a lower volume of Percoll for a shorter period of time, thus reducing the cost and time to perform the in vitro fertilization.

ASSUNTO(S)

sperm selections força e tempo de centrifugação ecologia embryo development sex ratio desenvolvimento embrionário proporção de sexo force and time of centrifugation seleção espermática

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