Reduced fertility of cryopreserved semen can be associated with decreased mitochondrial activity

Mitochondrial activity appears to be an important characteristic associated with sperm quality and fertilization capacity. The objective of this study was to determine the influence of sperm mitochondrial activity with fertility rate in different batches from high, medium and low fertility and its relationship with other semen quality parameters. Samples were classified in high fertility (HF), above 56% of pregnancy rate, medium fertility (MF) between 45 and 55% pregnancy rate and low fertility (LF) below 44% pregnancy rate. This study included sixteen straws (n=16) of different bulls, nine (n=9) with high fertility (HF) and seven (n=7) of medium fertility (MF), that were used in a timed-AI program, under the same standard management conditions and as minimum 100 AI per batches. No batch showed low fertility. Pregnancy diagnosis was made with a B-mode sonogram 30-45 days after insemination. Two semen straws from the same batch were thawed at 37oC for 30 seconds in water bath and were assessed by conventional sperm analyses (motility, vigor, concentration and morphology), computer-assisted sperm analysis (total and progressive motility) by Sperm Class Analyzer software (SCA, Microptics, Barcelona, Spain) and flow cytometric analyses to estimate the percentage of cells with plasma membrane integrity (PMI), acrosomal integrity with intact plasma membrane (AIMI), high mitochondrial membrane potential with intact plasma membrane (MIHP), proportion of sperm with a ratio of high: low mitochondrial potential (HP/LP) and mitochondrial ROS generation (mROS). Samples for flow cytometry analyses were diluted (5×106 spermatozoa/mL) in a Tyrode's medium (TALP). For simultaneous assessment of AIMI, a sample of 150 µL of semen diluted was incubated per 8 min at 37 °C, in the dark, with 1 µL peanut agglutinin conjugated with fluorescein isothiocyanate (FITC-PNA; L-7381 Sigma Chemical Co.), 1 μL of propidium iodide (PI, 0.5 mg/mL; P4170, Sigma) and 1 μL of Syto-59 (750 mM; S11341-Thermo Fisher). For simultaneous assessment of MIHP and HP/LP, a sample of semen diluted (150 µL) was incubated per 8 min at 37°C in the dark with 1 µL of 5,5′,6,6′tetracloro 1,1′,3,3′tetraetilbenzimidazolilcarbocianin iodide (JC-1, 153 μM, Life Technologies), 1 μL of PI (0.5 mg/mL) and 1 μL of Syto-59 (750 mM). For mitochondrial ROS generation samples were incubated 1 µL Mitosox red (2 µM, MSR-M36008, Thermo Fisher), 2 µL de SYTO-59® and 1 µL de YOPRO (25 nM, Y3603, Thermo Fisher) per 30 min at 37 °C in the dark. Flow cytometry assessments were conducted using a conventional flow cytometer Accuri™ C6 (BD Pharmingen™). Data were analyzed with R software (version 3.3.1.) by analysis of variance. Preliminary results were expressed as mean and standard error of mean (s.e.m.). The pregnancy rates (%) were 62.55±5.36% for HF and 51.85±3.23% for MF (p 0.05) observed to total motility between HF (66.91±8.8%) and MF (68.7±17.57%), or for progressive motility (HF= 49.95±12.5%; MF = 46.5±16.5%). Flow cytometric tests had not statistical differences (P>0.05) between HF and MF, to PMI (49.14±10.02 and 44.94±9.5%) and AIMI (48.76±9.9 and 43.60±8.1%) respectively, however, numerically MIHP was higher for HF (23.83±12.98%) than MF (16.88±9.85%) and the HL/PL proportion decreased to MF (1.86±1.58%) when compared to HF (2.38±1.64%). The median fluorescence intensity of MSR were 451.50±343.88% to HF and 540.57±223.38 to MF (P>0.05). The decrease of MIHP and HP/LP ratio, together with and the increase of mROS in the group of MF, with no decrease in sperm motility, suggested that the decrease mitochondrial activity could be affecting the fertilization capacity. In conclusion, the sperm mitochondrial activity could be influencing the sperm fertilization. Then, analyses of sperm mitochondrial functions may be useful characteristics to consider predicting bull fertility; however, further study should be conducted to test this hypothesis.