GAMETE BIOLOGY Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development Debora A. Anzalone 1 & Domenico Iuso 1 & Marta Czernik 1 & Grazyna Ptak 1,2 & Pasqualino Loi 1 Received: 27 January 2016 /Accepted: 23 March 2016 /Published online: 8 April 2016 # Springer Science+Business Media New York 2016 Abstract Purpose This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm in- jection (ICSI). Methods Prior to in vitro fertilization (IVF) or ICSI, the oo- cytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oo- cytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS). Results No differences were observed in the zygote/cleavage/ blastocyst rate between chemically activated and non- activated oocytes (50 vs. 45 %, 11.6 vs. 10.1 %; 1.8 vs. 1.1 %, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45 %, p < 0.01; 15.5 vs. 1.1 %, p < 0.0001, respectively). Moreover, the percentage of PPS- derived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2 %). The ICSI blastocysts’ development was also improved with PPS pre- treated with ionomycin (15.6 %), but was completely impeded with PPS pre-treated with Triton X-100 (0 %). Conclusion Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechani- cally damaged substantially improves embryonic develop- ment until the blastocyst stage. Keywords ICSI . Ovine sperm . Plasma membrane . Acrosome Introduction The intracytoplasmic sperm injection (ICSI) bypasses funda- mental physiological steps such as oocyte-sperm interaction and fusion. ICSI is well-established in human and mouse but, for unknown reasons, is less efficient and not yet standardized in other species, small ruminants in particular [5,6,16,35]. Oocyte/spermatozoon fusion at fertilization ensures the loss of the acrosomal vesicle and plasma membrane, whereas Capsule Intracytoplasmic sperm injection (ICSI) with spermatozoa whose plasma membrane and acrosome have been mechanically dam- aged substantially improves sheep embryonic development until the blas- tocyst stage. * Pasqualino Loi ploi@unite.it Debora A. Anzalone debora.anzalone@gmail.com Domenico Iuso dome.iuso@gmail.com Marta Czernik czernik.ma@gmail.com Grazyna Ptak gptak@unite.it 1 Faculty of Veterinary Medicine, University of Teramo, R. Balzarini Street 1, Campus Coste Sant’Agostino, 64100 Teramo, Italy 2 Present address: National Research Institute of Animal Production, 1, Krakowska Street, 32-083 Balice n/Krakow, Poland J Assist Reprod Genet (2016) 33:757–763 DOI 10.1007/s10815-016-0709-1