Effects of single-layer centrifugation in Percoll Plus® before freezing on the morphological and functional sperm characteristics evaluated in vitro and in vivo after TAI.

Abstract

This study was designed to verify the effects of sperm selection, before freezing, through use of single layer centrifugation with Percoll Plus® (SLCPP), at different concentrations of colloid, either with or without sperm pre-dilution, as well as different concentrations of sperm per dose, evaluating the morphological and functional in vitro bovine sperm characteristics, post-thawing, and rates of IVF and TAI. Semen of four Nelore bulls, kept at artificial insemination station, will be collected by artificial vagina. In exp. 1, the ejaculate of each bull (6 replicates) will be mix and divided equally (1.000 x 106 espermatozoa) between groups for SLCPP, at concentrations of 90 and 70%, without previous dilution (WD), i.e., 1 x109 fresh semen or diluted semen (D), at a final concentration of 250 x 106/mL (in 4 mL of based freezing medium), consisting the groups 90WD, 70WD, 90D and 70D, respectively. In exp. 2, inseminating doses at concentrations of 30, 20 and 15 x 106 of spermatozoa will be compared, after freezing, from samples previously obtained through SLCPP (best result of exp.1). In exp. 3, two groups, control (without SLCPP) and other subjected to SLCPP will be compared, taken the best result of exp. 2. In all experiments the spermatic selection will be done by putting the fresh semen on the top of a 9-ml column of Percoll Plus®, set in 15 ml- centrifuged tube, following centrifugation at 700 x g/13 min, before freezing. Afterwards, the samples will be frozen and, subsequently, thawed for comparative analysis of motility and movement (CASA), sperm morphology and sperm morphological and functional alterations by using fluorescent probes in fluxo citometry (plasma membrane integrity -PM), acrossomal integrity, mitochondrial function, intracellular ROS, lipidic peroxidation, membrane stability, tyrosine phosphorylation and sperm chromatin structure assay, as well as rates of in vitro fertilization (72 h post-insemination) and of conception after TAI. ANOVA and Tukey test will be employed to analyse the results of the variables related to sperm morfology, CASA, fluorescente probes and IVF. Logistic regression will be applied to evaluate data of conception rate and Pearson correlation to compare the different sperm variables. P<0.05 will be taken as significant. (AU)