ABSTRACT
This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)
O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)
Subject(s)
Animals , Male , Semen Preservation/veterinary , Sperm Capacitation , Sheep , Cryopreservation/veterinaryABSTRACT
OBJECTIVES: To evaluate the effects of sperm motility stimulants on the hyperactivation (HA), acrosomal reaction (AR) and sperm penetration assay (SPA) in fresh and frozen-thawed spermatozoa from fertile men. METHODS: We treated the semen samples obtained from 20 normospermic men (fresh semens from 10 and cryopreserved ones from 10) with pentoxiphylline (PF) and 2-deoxyadenosine (2-DXA) to evaluate the change of the patterns of motility using the computerized motility analyzer. The semen samples treated with motility stimulants were incubated in the medium with calcium ionophore A23187 for the examination of the proportion of acrosome lost spermatozoa. Finally we performed SPA in both groups for the evaluation of fertilizing capacity after stimulant treatments. RESULTS: In both fresh and cryopreserved semen samples, the addition of PF and 2-DXA significantly altered the patterns of motility (ALH, VCL, HA) known to have association with sperm quality without increasing the number of sperms with progressive motility and velocity. A23187 induced AR was also augmented by the treatment with PF and 2-DZA. Although the treatment with PF did not increase the mean rates of egg penetration significantly, in selected cases in the cryopreserved semen group, the improvement of the motility pattern was impressive. CONCLUSION: PF and 2-DXA can improve the quality of sperm function in both fresh and frozen-thawed semen from normal fertile men and may increase the sperm penetration rate of zona-free hamster eggs in selected samples of the frozen-thawed semen. The results suggest that PF and 2-DXA pretreatment can be used in the clinical practice for intrauterine insemination (IUI) program with frozen-thawed sperms as well as with samples from men with abnormal semen parameters. In addition, it may be a cost- effective therapy to try IUI combined with such a pretreatment for the couples planned to enter into the ART program.