Effects of α-tocopherol and freezing rates on the quality and heterologous in vitro fertilization capacity of stallion sperm after cryopreservation.

The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C/min) and moderate (10 °C/min). After thawing, the viability (SYBR®-14 and propidium iodide [PI]), mitochondrial membrane potential (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolyl carbocyanine iodine) and membrane lipid peroxidation (C11-BODIPY(581/591)) of each sample were determined by flow cytometry. Moreover, the heterologous IVF rate was measured to evaluate the fertilization capacity of postthaw semen in the four different treatments. For both extenders, the viability was higher for spermatozoa cooled slowly (39.40 ± 2.17 vs. 17.59 ± 2.25-control; 31.96 ± 2.19 vs. 11.46 ± 1.34-Tocopherol; P < 0.05). The α-tocopherol extender improved (P < 0.05) postthaw lipid peroxidation (10.28 ± 0.70 vs. 15.40 ± 0.95-slow FR; 10.14 ± 0.40 vs. 13.48 ± 0.34-moderate FR); however, it did not improve viability and mitochondrial membrane potential. Regarding the IVF rate, in the moderate FR, α-tocopherol supplementation reported a higher percentage of IVF (20.50 ± 2.11; P < 0.05), comparing with the control (14.00 ± 1.84). Regarding the slow FR, no significance differences were observed for percentage of IVF between the two extenders and the FRs. However, it seems that the α-tocopherol supplementation improved the IVF rate. In conclusion, this research reported that bovine oocytes intact zona pellucida can be used to evaluate the quality of postthaw stallion semen and α-tocopherol supplementation in the stallion freezing extender might exert a protective effect against oxidative damage during heterologous IVF.

Effect of Conjugated Linoleic Acid on Boar Semen Quality After Long-term Refrigeration at 17°C.

In this study, the effect of conjugated linoleic acid (10 trans, 12 cis) (CLA) on refrigerated boar sperm quality parameters up to 14 days at 17°C was assessed. Semen was extended in Androhep and divided into four treatments supplemented with CLA (25, 50, 100 and 200 µm) and control group, then kept for 2 h at 22°C. Afterwards an aliquot of each treatment was removed, and mitochondrial activity, viability, lipid membrane peroxidation (LPO) and stability of the sperm plasma membrane were assessed by flow cytometry. The remaining extended semen was maintained at 17°C until 336 h, repeating the same analysis every 48 h. Regarding percentage of live spermatozoa, no statistical differences were observed among treatments up to 96 h. After this time, viability decreased significantly (p < 0.05) for CLA concentrations of 100 and 200 µm. Despite these results, there was an individual response to CLA. Although in the control group, the boar A presented better results when compared with the other boars, especially at concentrations of 50 and 100 µm boar B showed significantly higher results (p < 0.05). Supplementation with CLA improved (p < 0.05) LPO, but not the mitochondrial membrane potential of sperm. The highest two CLA concentrations showed to be toxic for sperm as all results were lower than the observed for the control. In conclusion, CLA at 50 µm seems to be an efficient concentration for reducing the oxidative stress, decreasing LPO, maintaining viability, membrane stability and mitochondrial potential on refrigerated boar spermatozoa.

Effect of alpha-tocopherol on bovine in vitro fertilization.

The objectives of this work are to determine if exogenous supplementation with alpha-tocopherol increases the in vitro fertilization (IVF) rate of bovine oocytes and improves viability of selected spermatozoa after 'swim-up'. The percentage of fertilized oocytes was significantly but negatively correlated (r = -0.941, p < 0.01) with the concentration of alpha-tocopherol. The control resulted in 95% of fertilized oocytes, which decreased as follows: 25 microM alpha-tocopherol (alpha25) 86%, 50 microM alpha-tocopherol (alpha50) 74%, 100 microM alpha-tocopherol (alpha100) 66% and 200 microM alpha-tocopherol (alpha200) 56%. Relatively to sperm viability after 'swim-up' with alpha-tocopherol supplementation, this antioxidant proved to have a beneficial effect as its concentration increased up to alpha50, decreasing for the concentrations of alpha100 and alpha200. Control resulted in 83% of live cells and 16% of dead cells; alpha25 resulted in 88% of live cells and 12% of dead cells; alpha50 resulted in 91% of live cells and 9% of dead cells; alpha100 resulted in 67% of live cells and 33% of dead cells; and finally alpha200 resulted in 57% of live cells and 42% of dead cells. In summary, the present study allows to conclude that, in our conditions, supplementation with the antioxidant alpha-tocopherol in IVF of bovine oocytes has a detrimental effect on fertilization rates. Nevertheless, exogenous supplementation with alpha-tocopherol at a concentration of 50 mM in the sperm-TALP media during the 'swim-up' technique has a significant beneficial effect on the selected spermatozoa viability.