Efeito da proteína anticongelante sobre a qualidade dos espermatozoides criopreservados de caprinos

Abstract

This study aimed to investigate the effect of antifreeze protein type III (AFP III) added to cryopreservation extender of goat sperm on post-thawing sperm viability, with the objective of improving semen cryopreservation protocols. In the first experiment, 16 testicular pairs were collected in a slaughterhouse and transported at approximately 5 °C in a thermal box around 10 hours, until processing in the laboratory. Epididymis spermatozoa were recovered by washing and evaluated with phase contrast microscope. Then cryopreserved in Egg Yolk based extender , supplemented with antifreeze protein type III (0; 1; 10; 100 μg/mL), using automated system. After thawing (37 C /30 sec), spermatic kinetics were evaluated by CASA automated system, acrosome plasma membrane integrity, mitochondrial membrane potential and intracellular ROS production by flow cytometry. No difference (P ≥ 0.05) was seen between the experimental groups for the parameters of spermatic kinetics, mitochondrial membrane potential and ROS production. However, the integrity of plasma membrane and acrosome of frozen sperm with 100 μg/mL of AFP III was lower (P < 0.05) when compared to control group. The addition of AFP III to tris-egg yolk extender, used in the freezing of sperm obtained from the epididymis of goats, does not favor preservation of goat sperm recovered from epididymis. In addition, when at high concentration (100 μg/mL) it compromises the integrity of the plasma and acrosome membrane of these cells. The second experiment was divided into two parts: Experiment I and II, where the effect of AFP III on extenders based on Tris- egg yolk and skimmed milk was investigated, respectively. The semen of four Saanen goats (6 replicates) was used for pool formation and used in Egg Tris-yolk Diluent (Experiment I) and Skim Milk Based Diluent (Experiment II), both supplemented with AFP III concentrations, obtaining the following sample groups: 0; 1; 10; 100 μg/mL. After dilution (200 x 106 sperm/mL), the samples were filled in 0.25 mL straws and frozen (-196 ºC). For each group, two straws were thawed (37 ºC for 30 s) and grouped for in vitro analysis of spermatic kinetics, through CASA; and plasma membrane integrity (IMP) and acrosome integrity (IAC) by epifluorescence microscopy. Two straws were thawed for ultrastructural analysis of spermatozoa by scanning electron microscopy. No difference (P ≥ 0.05) was seen between experimental groups from the two experiments for the parameters of kinetics, plasma membrane integrity and acrosome. Except for the progressive motility that the concentration of 1 and 100 μg/mL differed (P < 0.05) from each other when skim milk Based Diluent was used. The results of the ultrastructural evaluation showed that regardless the diluent used, AFP III damaged the plasma membrane of the spermatic cells in a dose-dependent manner. Based on the results of the present study, it is concluded that AFP III in the concentrations used, in extenders based on egg tris-yolk and skimmed milk, does not improve seminal quality.