Czech J. Anim. Sci., 2015, 60(6):241-249 | DOI: 10.17221/8237-CJAS

Male pronucleus formation after ICSI: effect of oocyte cysteine or sperm Triton X-100 treatmentsOriginal Paper

E. Garcia-Mengual^1,3, M.A. Silvestre^1,4, I. Salvador¹, A. Cebrian-Serrano^1,5, E. Garcia-Rosello²

¹ Centre for Animal Technology Research, Instituto Valenciano de Investigaciones Agrarias, Segorbe, Spain

² Faculty of Veterinary Science, CEU Cardenal Herrera University, Valencia, Spain

³ Reproductive Genetics Unit, Sistemas Genómicos S.L., Parque Tecnológico de Valencia, Paterna, Spain

⁴ Department of Functional Biology and Physical Anthropology, University of Valencia, Valencia, Spain

⁵ Neurogenetics group, Center of Excellence Hearing4All, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany

In pigs, intracytoplasmic sperm injection (ICSI) efficiency is still poor. The inadequate decondensation of the sperm chromatin, its transformation into the male pronucleus (MPN) together with the subsequent inability to activate the oocyte, seem to be the main causes of the low ICSI efficiency. In order to improve the MPN formation we took two different approaches. On the one hand, the in vitro culture (IVC) medium post-ICSI was supplemented with 1.71mM cysteine (CYS). Alternatively, the sperm membrane was digested with Triton X-100 (TX) before ICSI, to improve the exposure of the sperm chromatin to the oocyte cytoplasm. After 6 h post-ICSI, the activation rate was significantly higher in TX group (70.0%) compared with CYS and control groups (42.2% and 48.9%, respectively; P < 0.05). However, no significant differences between the three groups were observed in terms of the number of pronuclei, 2PN (oocytes with 2 pronuclei and no visible sperm), and 1PN + sperm (oocytes with 1 pronucleus and one sperm head). At 22 h post-ICSI, the activation rates were similar in TX, CYS, and control groups (73.1, 78.9, and 75.7%, respectively). In addition, we did not observe significant differences between TX, CYS, and control groups for the number of pronuclei, 2PN (52.6, 56.7, and 50%, respectively) or 1PN + sperm (21.1, 33.3, and 32.1%, respectively). While no cleavage was observed in the CYS group, no significant differences in the cleavage rate were observed between control (21.3%) and TX (10.5%) groups. In summary, and under our conditions, neither CYS supplement, nor sperm TX pre-treatment were able to improve MPN formation at 6 and 22 h post-ICSI. However, the sperm TX pre-treatment improved oocyte activation at 6 h post-ICSI, although 22 h post-ICSI such a beneficial effect did not persist.

Keywords: porcine; assisted reproductive technology; pronuclear formation

Published: June 30, 2015  Show citation