Debora A Anzalone1, Domenico Iuso1, Marta Czernik1, Grazyna Ptak1,2, Pasqualino Loi3. 1. Faculty of Veterinary Medicine, University of Teramo, R. Balzarini Street 1, Campus Coste Sant'Agostino, 64100, Teramo, Italy. 2. National Research Institute of Animal Production, 1, Krakowska Street, 32-083, Balice n/Krakow, Poland. 3. Faculty of Veterinary Medicine, University of Teramo, R. Balzarini Street 1, Campus Coste Sant'Agostino, 64100, Teramo, Italy. ploi@unite.it.
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 injection (ICSI). METHODS: Prior to in vitro fertilization (IVF) or ICSI, the oocytes 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 oocytes, 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 mechanically damaged substantially improves embryonic development until the blastocyst stage.
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 injection (ICSI). METHODS: Prior to in vitro fertilization (IVF) or ICSI, the oocytes 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 oocytes, 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 mechanically damaged substantially improves embryonic development until the blastocyst stage.