Tempo de equilíbrio, lipoproteínas de baixa densidade e colesterol na criopreservação do sêmen bovino

Abstract

Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. The aim of this study was to evaluate the association of Cholesterol (Cholesterol Loaded Cyclodextrin - CLC) and Low Density Lipoproteins (LDL) and possible interactions between these cryoprotective agents and equilibration time, during the process of cryopreservation, on post-thawing sperm motility, integrity, stability and lipid peroxidation of sperm membranes. Semen samples from 15 Nellore bulls, andrologicaly normal, 2-6 years old, were collected by artificial vagina and evaluated according to the standards of CBRA (1998). Each semen sample was divided into 4 aliquots and diluted at 32º C (30 x 106 sperms /mL) with the following extenders: 1) Tris-EggYolk (control); 2) LDL (8%); 3) LDL + CLC (8% LDL + CLC); 4) L-CLC (0,8% LDL + CLC). After dilution, the extended semen was cooled to room temperature (25ºC) and packaged in 0.5 mL straws. For cryopreservation three automated machines were used (TK-3000®), with the same cooling (- 0.25° C / min) and of freezing rates (- 20°C / min) for all treatments, varying only the equilibration time at 5ºC: 0h (T0), 4h (T4), and 6h (T6) for a total of twelve treatments. Thereafter, the straws were transferred to liquid nitrogen (−196 ◦C) for storage.Semen evaluations after thawing were performed with Computer-Assisted Semen Analysis (CASA) and flow cytometry for integrity of plasma and acrosomal membranes (PI/FITC-PSA/H33342), lipid peroxidation (PI/C11-BODIPY581/591/H33342) and sperm capacitation by plasma membrane stability (Merocianina 540/Yo-Pro1/H33342). The control treatments (0 h equilibration) had the lowest values for both total (MOT) and progressive motilities (PROG), as well as for percentage of sperm with intact plasma and acrosomal membranes (IPIA), for all extenders. The extender LDL (8%) provided the best cryoprotective action at all times, with the highest total and progressive motilities, better sperm movement characteristics (highest VAP, VSL and VCL), highest percentage of rapid cells (Rapid), the highest integrity of plasma and acrosomal membranes, and the highest proportion viable non-capacitated cells (Meroc (-)). The Egg-Yolk based extender provided the best protection against lipid peroxidation of sperm membranes. The extender L-CLC had the worst cryoprotective activity. Addition of cholesterol by pretreatment of semen with CLC did not allow a reduction in duration of equilibration time. Furthermore, the addition of CLC to purified LDL(8%) based extenders, reduced the sperm survival at T0 and T6. Equilibration for 4 h was suitable to achieve a high sperm survival, regardless of extender used, however, the Tris-Egg-Yolk and LDL extenders were benefited from a greater equilibriation time (6h). Overall, the best cryopreservation method was the combination of LDL (8%) extender and 6h of equilibration (LDL-T6). In conclusion, equilibration time is necessary in the process of cryopreservation of bovine semen for preservation of motility and integrity of sperm membranes, regardless of extender used. Furthermore, CLC alone or combined with low concentrations of LDL do not adequately protect sperm membranes from the detrimental effects of cryopreservation.