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Influence of Sperm Pretreatment on the Efficiency of Intracytoplasmic Sperm Injection in Pigs

PEDRO N. MOREIRA

From the ^* University of Murcia, Department of Veterinary Physiology, Faculty of Veterinary Science, Murcia, Spain; and the Department of Animal Reproduction, Nacional Institute of Agricultural Research and Technology (INIA), Madrid, Spain.

Correspondence to: Pilar Coy, Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain (e-mail: pcoy{at}um.es).

The purpose of this study was to determine the influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection (ICSI) in pigs. This was done by examining the effect of 1) the conservation method (fresh vs frozen); 2) the sperm treatment preinjection (resuspension in Dulbecco phosphate-buffered saline (DPBS) vs selection by a Percoll gradient); and 3) the acrosomal and live or dead status of the spermatozoa (by incubation with or without calcium ionophore, 1 µM and 5 µM). In vitro matured porcine oocytes were injected with treated spermatozoa according to each experiment. All the experiments were done with non–artificially activated oocytes. The percentages of activation and cleavage were higher (68% vs 43% and 63% vs 43%, respectively, P < .05) in oocytes injected with fresh vs frozen spermatozoa. The DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P < .05). Moreover, a boar effect was observed in the percentage of developing blastocysts. None of the studied parameters was affected by the acrosomal or the live or dead status of the spermatozoa injected. In conclusion, the use of fresh semen is recommended for porcine ICSI, as well as careful selection of the boar; Percoll treatment is only recommended for poor-quality samples or for removing toxic agents, and no exogenous form of activation or induction of the acrosome reaction is necessary for porcine oocytes to develop a male pronucleus and cleave up to the 2-cell stage after ICSI, although experimental conditions to reach the blastocyst stage need to be investigated further.

     Key words: Capacitation, sperm cryopreservation, acrosome reaction, early development

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