Utilização de pentoxifilina e antioxidantes na criopreservação do sêmen bovino : parâmetros seminais e avaliação da fertilidade in vivo

Abstract

This study evaluated if the use of additives in the bovine semen extender reduces the damage caused by oxidative stress, and preserves sperm quality after thawing, and the addition of tocopherol in semen extender media for the purpose of preserving the fertilizing ability of semen to be used in fixed time artificial insemination (FTAI). In the first experiment 24 Nellore (Bos taurus indicus), with a mean age of 31 months, average live weight of 632Kg, reared under semi-intensive system, with good body condition (score 3 in the 1-5 scale). One ejaculate was collected from each bull by electrostimulation, and was diluted in extender TRIS-citrate-yolk-glycerol, divided into six parts, and then supplemented with: control (no additives), tocopherol (10 mmol/ml), tocopherol (10 mmol / ml) + pentoxifylline (1 mg/ml), ascorbate (0.45 mg / ml), ascorbate (0.45 mg / ml) + pentoxifylline (1 mg/ml); pentoxifylline (1mg/ml). The samples were cooled, frozen in liquid nitrogen and stored until the time of laboratorial analysis. After thawing, samples were evaluated for motility and motion characteristics, plasma membrane and acrosome integrity, mitochondrial activity and level of lipid peroxidation (TBARS). The semen extender supplementation did not affect (P> 0.05) mitochondrial activity, acrosomal integrity, and the concentration of TBARS. The addition of tocopherol + pentoxifylline reduced progressive motility compared to ascorbate and also sperm membrane integrity as compared to control and ascorbate (P ˂ 0.05). Already the addition of ascorbate + pentoxifylline was deleterious to linearity when compared with ascorbate treatment (P ˂ 0.05). In conclusion, the addition of ascorbate, tocopherol and pentoxifylline alone or in combination, was not effective in reducing the damage caused by oxidative stress in cryopreservation and post-thaw samples of bovine semen. In the second experiment three Nelore bulls (Bos taurus. Indicus), with a mean age of 48 months and average live weight of 750kg were used. Their ejaculates were diluted with extender TRIS-citrate-yolk-glycerol, divided into two parts, and then supplemented with: control (no additives) and tocopherol (10 mmol/ml). The semen was placed in 0.5 ml straws and the final concentration was fixed at 25 million sperm per straw. They were frozen in liquid nitrogen and stored until the time of FTAI. In the first lot we used 84 synchronized females Nelore (Bos taurus indicus) aged between 3 and 8 years and the second lot 44 females that not be pregnant in the first FTAI procedures. The cows were randomly divided into two groups: control (inseminated with semen cryopreserved without using additives) and treatment (inseminated with semen cryopreserved with added (10 mmol/tocoferol). The animals were kept during 30 days in paddocks of pasture Brachiaria humidícola until pregnancy diagnosis by ultrasound. The experimental design was completely randomized and pregnancy rates compared by chi-square test. Statistical analysis was performed using the SAS statistical package with a significance level of 5%. There were no effect (P> 0.05) in pregnancy rate using cryopreserved semen with added 10 mmol/ml tocopherol in the bovine semen extender compared to the control group (no additives) in the first synchronized group (n = 84, 38.5% vs 40%), in the second synchronized group (n = 44, 28% vs 31.6%) and the all animals (n = 128, 34.4% vs 37.5%). The addition of 10 mmol/ml of tocopherol in the semen extender did not improve the pregnancy rate after FTAI in the bovine species. In general effect was not observed in in vitro tests with the addition of antioxidants and pentoxifylline, and in vivo with the addition of tocopherol in the bovine semen extender.