Análises bioquímicas do sêmen congelado bovino e interações com a reação acrossômica

Abstract

Proteins and ions have an important role in the binding of the spermatozoa to the ovum in the beginning of the fertilization. Alterations of some proteins and ions in the spermatozoa can contribute to the deficiency of the acrosome reaction and concequently to the fertility. The calcium has an integral role in the capacitation and acrosome reaction. Several proteins that constitute the spermatozoa were already mentioned as being binding proteins to the ovum, such as: D-manosidase, HIS-50 and HIS-100, galactosyltransferase, receptor tirosine kinase (ZRK), receptor galactosyl, PH20, sp56 and PLA2. In view of these aspects, the objective of this study was to analyze and to correlate the proteins of the bovine frozen semen of several breeds, the concentration of calcium ions of the seminal plasm and the activity of the enzyme phospholipase A2 of the spermatozoa with the acrosome reaction to find factors that influence in the process of bovine fertilization. The analysis of the eletrophoretics profiles of the proteins (total and membrane) of the spermatozoa and of the bovine seminal plasm revealed protein variability among bulls, qualitative and quantitative differences of proteins were identified in agreement with its molecular masses of 66 kDa (ZRK or HIS-50), 30-36 kDa (dimeric form of ZRK), 20 kDa (PH20) and 14-18 kDa (ZRK or PLA2) they can be proteins of great importance in the binding of the spermatozoa to the ovum and in the induction of the acrosome reaction. The technique developed by spectrophotometry with the use of Rhodamina 6G and egg lecitin detected activity of the fosfolipase A2 from 0,001 to 2,8 unit/min/ul. Its quantification revealed diversity among individuais. The concentration of calcium of the bovine seminal plasm revealed significant differences among bulls, indicating that genetic control should exist for the release of extracellular calcium. The correlation coefficient between concentration of calcium of the bovine seminal plasm and proteins of the semen with molecular masses of approximately 31, 27, 20, 18 and 16 kDa of thespermatozoa, as well as the proteins of 29, 18 and 16 kDa of the seminal plasm were significant, and could be acting in the activation of the extracellular calcium flow or inhibition, as it is the case of the proteins of 31 kDa of the spermatozoa and 16 kDa of the seminal plasm that presented negative correlation coefficients. The membrane protein of 15,7 kDa presented significant positive correlation (0,71) with the acrosome reaction, and this could probably be a ZRK an important protein in the binding of the spermatozoa to the ovum. The protein of 31,1 kDa presented a negative correlation coefficient (0,61). The protein of 15,7 kDa should be a factor of great importance, and could be used as a molecular marker for the fertility in the analysis of bovine semen. As already described, the PLA2 and the calcium possess great importance in the acrosome reaction; however, there was no signigicant correlation among them and the acrosome reaction, suggesting that the amounts of calcium and PLA2, although significantly different among bulls, were enough to induce the acrosome reaction, becoming secondary factors of the process of bovine fertilization. These results indicate that the bovine fertilization is a complex multifactorial process, to which the fertilization can not be limited to a genetic control, but also to the environment.