Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Abstract

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.

Laser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups

“…Prior studies have evaluated various techniques to augment fertilization of V-W oocytes and demonstrated increased fertilization compared to non-treated V-W oocytes [7]–[11]. The use of oocytes zona-drilled after DAP213 vitrification in our study provided the highest % two-cell embryos reported to date using such techniques, to the best of our knowledge.…”

“…While 100% of oocytes that survive ICSI with frozen-thawed spermatozoa develop to form two-cell embryos, 78% of those injected do not survive, reducing the overall % two-cell embryos to 72.3% [9]. ZP digestion using acidic Tyrode’s solution resulted in 48.8% fertilization with cryopreserved spermatozoa, compared to 22.1% without digestion [7]. Meng et al reported a fertilization rate of 85.5% using piezo-actuated zona drilling and fresh outbred mouse spermatozoa following open pulled straw vitrification [8], while Wang et al used zona incision to obtain 69.9% fertilization of cryopreserved oocytes with frozen-thawed spermatozoa [10].…”

“…Spermatozoa are often unable to penetrate V-W mouse oocytes due to hardening of the zona pellucida (ZP) following premature release of cortical granules [7]–[10]. Previous studies have evaluated intracytoplasmic sperm injection (ICSI) [9], partial ZP digestion [7] and piezo-actuated zona drilling [8] or incision [10] for easier penetration, and found fertilization advantage.…”

“…Previous studies have evaluated intracytoplasmic sperm injection (ICSI) [9], partial ZP digestion [7] and piezo-actuated zona drilling [8] or incision [10] for easier penetration, and found fertilization advantage. One study also examined laser-assisted zona drilling prior to vitrification of mouse oocytes and demonstrated improved post-warming in vitro fertilization (IVF) using spermatozoa from a subfertile transgenic mouse [11].…”

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa

“…After cleavage evaluation embryos were washed in M2 and, in order to remove spermatozoa that could have remained attached, zona pellucida was removed by transferring the embryos into an acidic Tyrode´s solution (pH 3.1), [36]. Zona pellucida digestion was continuously observed under an inverted microscope and, when the zona was no longer visible, embryos were washed 3 times in M2 individually, snap frozen 290 and thawed on the day of PCR analysis as described in experiment 1 (2.1.4.…”