Use of different types of egg yolk and insulin concentrations in cryopreservation of ram sperm

The objective of this study was to evaluate the viability of ram sperm, submitted to the diluting and cryopreservation process, using two different types of egg yolks diluents and three insulin concentrations. One ejaculate from six Santa Ines rams, was divided into treatments T1 (Lactose yolk chicken x Insulin NPH - 0μIU/mL, 100μIU/mL and 200μIU/mL) and T2 (Lactose yolk duck x Insulin NPH - 0μIU/mL, 100μIU/mL and 200μIU/mL), previously evaluated according to CBRA 1998 parameters (Manual for breeding soundness examination and evaluation of animal semen. Belo Horizonte, 1996. 65p. Prepared according to the CBRA/MA n. 017/1998 agreement). Subsequently, the semen was packaged and then frozen using an automated system for cryopreservation (TK - 3000® model), through equal stability times. After thawing, the semen was evaluated for motility, vigor, rapid thermoresistance test (RTT), hypoosmotic swelling (HOST) and Trypan Blue/Giemsa staining tests. It was a randomized block design with a 2x3 factorial (two types of yolk x three concentrations of insulin). For parametric analysis, the Tukey test was used, while for the non-parametric analysis the Freidman test was done (P 0.05) between the periods within extenders and insulin concentrations, and being grouped with mean T1: 35 and 3.61% and T2: 28 and 4.44% for times 0 and 30 minutes, respectively. The same was found in HOST, with average values of 7.83% and 6.33% in T1 and T2, respectively. In the evaluation with Trypan blue/Giemsa stain, the integrity of the membrane was similar (P>0.05) for insulin concentrations and types of egg yolk used. The rate of acrosome reaction evaluated by Giemsa, showed no significant difference (P>0.05) for both alive with and without acrosome for insulin concentrations and types of egg yolk used. However, the dead sperm cells with and without acrosome showed significant difference (P<0.05) when 100μIU/mL of insulin was used in both extenders. Thus, it was observed that among the dead sperm, those with higher amount of intact acrosomes were from the duck egg yolk extender supplemented with 100 μIU/mL of insulin, obtaining 77.83% of sperm with acrosome compared to 44.83% of sperm acrosome with chicken egg yolk extender treatment. These results indicate that the duck egg yolk can be used in cryopreservation of ram semen and the supplementation of 100μIU/mL of insulin in duck egg yolk extender can promote fewer acrosome reactions during cryopreservation.