Effect of intramuscular injection of butaphosphan and vitamin B12 on the quality of fresh and cooled stallion semen

Abstract

hyperactivation patterns of equine cryopreserved sperm. Three ejaculates were collected from each of three stallions (n1⁄49). Semen was packaged in 0.5-mL straws to a final concentration of 200 x 106 sperm/mL in Botu-Crio extender (Botupharma Animal Biotechnology) and frozen Frozen-thawed semen was then centrifuged in bovine in vitro fertilization medium supplemented with sodium bicarbonate and bovine serum albumin for the capacitation of equine sperm. The pellet was incubated in the same medium added with different concentrations of L-arginine (0; 2; 5; 10; 20 mM) for 0, 60, 120 and 300 minutes at 38 C under 5 % CO2. CASA was used for motility assessment. Spermwas considered hyperactivated when VCL was 180 mm/s and ALH was 12 mm. Data was analyzed using ANOVA and means in each time were compared with Tukey test, using SAS software. Total motility was not altered during neither incubation time nor treatment. There was a reduction in total motility when 10mM of L-arginine was added compared to 0, 2 and 5 mM (10 mM, time 0: 33.5 4.4; time 60: 29.3 5.4; time 120: 26.5 4.8; time 300: 14.9 3.0). A reduction in progressive motility was noted using concentration of 5 mM of L-arginine and higher (5 mM, time 0: 10.1 1.6; time 60: 10.9 2.3; time 120: 9.6 2.1; time 300: 5.9 1.2), however the concentration of 20 mM caused the greater decrease on this parameter (time 0: 5.7 0.8; time 60: 7.0 1.9; time 120: 7.5 1.9; time 300: 4.8 1.1). Increasing doses of L-arginine lead to a decrease in VAP, VSL and LIN (P < 0.05). VCL was not affected by the interaction of treatment and incubation time. ALH was not altered in the different incubation times or different treatments. STR, RAP and BCF results were lower for the cells treated with 20 mM of L-arginine compared to concentrations of 0, 5 e 10 mM in times 0, 60, 120 and 300 minutes of incubation. There was an interaction between treatments and incubation time (P < 0.05) in the percentage of hyperactivated cells. However, only at time 0 therewas difference among treatments, and hyperactivated cells percentage decreased in concentrations of L-arginineof 10mM. It canbe concluded that total motility and hyperactivated cells were reduced at 10mM L-arginine concentration and that increasing doses of L-arginine are prejudicial to motility sperm patterns in cryopreserved equine sperm.