Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

ABSTRACT

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (11, 12, 13, 14) of diluent I (Tris-based and egg yolk) under the condition of 11 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (11, 12, 13, 14) of diluent I under the condition of 12 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups one-step dilution method and two-step dilution method. The two-step dilution method includes two groups 12, 11 and 13, 12, and the one-step dilution method includes two groups 15 and 111. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 12 group and significantly higher (p < 0.05) than those in the 11 and 14 groups under the condition of 11 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 13 group were significantly higher (p < 0.05) than those of the other groups under the condition of 12 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (13, 12) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (13, 12) was significantly lower (p < 0.05) than that in the one-step groups (15 and 111). Therefore, a two-step dilution (13, 12) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.