Experimental design
The research was performed from April to July of 2020 at the Universidad Autónoma Agraria Antonio Narro premises. Three Charoláis breed bulls were used. A semen collection from each bull was evaluated and divided into six portions. Three of these portions received a-tocopherol and the rest were used as control. For freezing, all six portions from each bull were divided into three groups, each with two samples: one with α-tocopherol and the other without. Afterwards, they were frozen by exposure to nitrogen vapor during 8, 11 and 15 min respectively. At the end, half the portions had α-tocopherol (E+8 min, E+11 min, E+15 min) and the rest without antioxidant (8 min, 11 min, 15 min). The straws were kept submerged in liquid nitrogen until their analysis 60 days later.
Experimental animals and semen collection
Three bulls in the age of 4 years were used for semen collection. They were kept under the same housing conditions and nutrition with water ad libitum. Semen collection were performed using artificial vagina and the initial evaluation was performed immediately using a Zeiss® optic microscope (Carl Zeiss Microlmaging GmbH, series No. 3108027112). Motility was microscopically (400×) evaluated using a preheated (37°C) slide. Sperm concentration was determined with a Spermacue® digital counter (Minitube of America, Inc. Series No. 1020204133. EE. UU.). Samples with a motility ≥85 %, volume of ≥10 mL and a concentration of ≥900×106 SPZ • mL
-1 were processed. Semen collections that complied with the stablished parameters, were mixed with a diluent 1:1 (v/v) proportion until an 80×10
6 SPZ • mL
-1 concentration was achieved.
Diluent preparation with or without a-tocopherol
The diluent used was Optidyl® (Biovet, Francia) brand. It was diluted at a 1:1.5 mL proportion with distilled water. The resulting dilution was divided into two parts. The first portion was used as an antioxidant group to which 5 mg • mL
-1 of α-tocopherol
(Membrillo-Ortega et al., 2011) were added, the other portion only contained the diluent and distilled water. Once the dilutions were performed, they were heated to 37°C for mixing with the semen. Afterwards, both portions (with and without antioxidant) were cooled to 4°C for 4 h and, immediately placed in 0.5 mL straws with a sperm concentration of 40×10
6 SPZ • mL
-1 per straw and sealed with polyvinyl alcohol. Subsequently, they proceeded to freezing.
Freezing and thawing of straws
Vapor freezing was performed by the polystyrene foam box method using liquid nitrogen
(Nasiri et al., 2012). The freezing process was the same for all samples, with the difference that, for each group, the exposure time to nitrogen vapors changed (8, 11,15 min). All the straws were pooled and frozen in separate groups (8, 11 and 15 min). For thawing, semen straws were immersed in a water bath at 37°C for 40 seconds.
DNA fragmentation
This was determined with the sperm chromatin dispersion test (SCD) given by Fernández
et al. (2003). The adaptation of the technique to bovine semen was in the control sample, in which, to cause damage to the chromatin, the use of peroxidation by DNase was changed. For this, 100 µL of the semen sample was placed in an Eppendorf tube and centrifuged at 12,800 G for 10 min. The excess liquid was then removed, replacing it with 100 µL of the enzymatic solution, then it was stirred and allowed to stand for 30 min, at which time the steps described for the technique were followed.
Evaluation of motility and mobility
Motility was evaluated on slides preheated to 37°C. To avoid ambiguities in the subjective evaluation, the evaluation criteria were categorized according to the WHO (2010) manual. Three types of movements were recorded: progressive motility (PM), non-progressive (NP) and no motility (NM). With this procedure a confidence interval of at least 95% was reached. The same counting method was used for percentage viability and DNA fragmentation. Viability was evaluated with the eosin-nigrosine (E/N) staining technique (WHO, 2010;
Nasiri et al., 2012).
Statistical analysis
The statistical package SPSS (IBM Corp. Released, 2017) was used. For the time and antioxidant variables effect, a Kolmogorov-smirnov normality test was performed. Variables that presented a normal distribution were analyzed by and ANOVA and Tukey tests. For data that did not have a normal distribution, a Kruskal-Wallis tests was used. For the α-tocopherol effect upon each variable a Student-t and Mann-Whitney U (non-normally distributed data) tests were used. Results are presented as mean ± standard error. A
p≤0.05 was considered as statistically significant.
