Effect of centrifugation and electrical activation on male pronucleus formation and embryonic development of porcine oocytes reconstructed with intracytoplasmic sperm injection
Hwan Yul Yong A B C , Ji Young Hong D , Sun Il Pak D , Sung Keun Kang A E , Byeong Chun Lee A E , Eun Song Lee D and Woo Suk Hwang A B E FA Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
B School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea.
C Present address: Department of Animal Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA.
D Department of Veterinary Medicine, Kangwon National University, Chunchon 200-701, Korea.
E The Xenotransplantation Research Center, Seoul National University Hospital, Seoul 110-744, Korea.
F Corresponding author. Email: hwangws@snu.ac.kr
Reproduction, Fertility and Development 17(5) 557-563 https://doi.org/10.1071/RD04022
Submitted: 24 March 2004 Accepted: 6 December 2004 Published: 19 May 2005
Abstract
Oocyte centrifugation and electrical activation are commonly used in intracytoplasmic sperm injection (ICSI) of bovine and porcine oocytes, to facilitate visual identification of sperm release into the ooplasm and to support oocyte activation following injection with tail membrane-damaged sperm. The present study evaluated the necessity of these steps in porcine modified ICSI. In the first series of experiments, in vitro-matured gilt oocytes with or without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail first. Oocytes without centrifugation exhibited a significantly higher normal fertilisation rate, defined as male pronucleus (MPN) and female pronucleus (FPN) formation and the presence of two polar bodies, than centrifuged oocytes (40% v. 9%, respectively; P < 0.05). The rate of MPN formation was significantly higher in uncentrifuged oocytes compared with centrifuged oocytes (48% v. 17%, respectively; P < 0.05). The rates of survival, cleavage, blastocyst formation and total cell number in blastocysts did not differ between the two groups of oocytes. Next, the effect of electrical activation after ICSI on uncentrifuged oocytes injected with head membrane-damaged spermatozoa was determined. No significant differences were observed in the rate of MPN formation in sperm-injected oocytes regardless of electrical activation. However, the survival rates of sperm-injected or control oocytes without electrical activation were significantly higher than those of sperm-injected or control oocytes with electrical activation (88% and 84% v. 77% and 64%, respectively; P < 0.05). The cleavage rates of sperm-injected oocytes were significantly higher than those of control oocytes, regardless of electrical activation (77% and 81% v. 47% and 61% in sperm-injected and control oocytes with or without electrical activation, respectively; P < 0.05). Although development to blastocysts was similar in all experimental groups, the total cell numbers in blastocysts from control oocytes were significantly higher than those in sperm-injected oocytes, regardless of electrical activation (40 and 44 v. 22 and 26 in control and sperm-injected oocytes with or without electrical activation, respectively; P < 0.05). In conclusion, the present study clearly demonstrated that oocyte centrifugation before sperm injection is not beneficial to normal fertilisation and that electrical activation is not necessary in the modified porcine ICSI.
Acknowledgments
This study was supported by a grant from the Advanced Backbone IT Technology Development, Seoul, Korea (IMT2000-C1-1). The authors are grateful for a graduate fellowship provided by the Ministry of Education, Korea, through the BK21 programme. The authors thank Dr Barry D. Bavister, University of New Orleans, for his help with the manuscript.
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