Copyright © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved. Vol. 10, No. 1, January-March 2018  Original Article 41 Male Pronuclear Formation and Embryo Development Following Intracytoplasmic Injection of Ovine Pretreated Sperm Abolfazl Shirazi 1,2* , Arefeh Golestanfar 2 , Masomeh Bashiri 2 , Ebrahim Ahmadi 2 , and Naser Shams-Esfandabadi 2 1. Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 2. Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran Abstract Background: Failure of Male Pronucleus (MPN) formation is a major concern in the success of Intracytoplasmic Sperm Injection (ICSI) in some species. Despite the con- ducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some com- pounds. Methods: In experiment 1, sperm were treated with either 2-mercaptoethanol (2ME), glutathione (GSH), or DTT (dithiothreitol) in combination with Heparin (Hep). Fol- lowing DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head decondensation was applied for the second and third experiments. In experiment 2, in vitro matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract (SE) co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm. Results: The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control. Conclusion: In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+ GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control. Keywords: Intracytoplasmic sperm injection (ICSI), Male, Ovine, Pronucleus Introduction Intracytoplasmic Sperm Injection (ICSI) as an inte- gral part of assisted reproduction has become increas- ingly popular in human male patients with certain in- fertility problems 1 . In animal species, this technique has several applications such as avoidance of the poly- spermy problem (e.g. porcine) 2 , extending the sperm vector system for transgenic animal production, and preserving the endangered species. ICSI also provides an opportunity for research into cell cycle control and mechanisms involved in sperm-induced oocyte activa- tion 3 . A problem commonly encountered in ICSI tech- nique is the low rate of viability of the microinjected oocytes and inconsistent level of male pronuclear for- mation after microinjection. The inadequate sperm chromatin decondensation and its transformation into the Male Pronucleus (MPN) together with a failure to activate the oocyte seem to be the major causes behind the poor ICSI efficiency in some species 4,5 . In this context, while ICSI alone in some species such as mice, humans, hamsters, and rabbits is suffi- cient to activate the oocytes for further embryonic de- velopment 6-10 , in other species such as cattle, sheep, pig 11,12 , buffalo 13 , and horse 14,15 , an additional par- thenogenetic activation is necessary to activate the oo- cytes after ICSI 16 . The nuclei of mammalian sperma- tozoa are genetically inactivated and structurally stabi- lized by association of sperm DNA with protamines. * Corresponding author: Abolfazl Shirazi, Ph.D., Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran Tel: +98 21 22404144 Fax: +98 21 22404145 E-mail:    shiraziabbas@yahoo.com Received: 24 Jan 2017 Accepted: 15 Apr 2017 Avicenna J Med Biotech 2018; 10(1): 41-48