Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine

Abstract

To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 ml modified Medium 199-suspended spermatozoa at 2.5 3 10 5 sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 6 2.2) than 8 h (207.6 6 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells, as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 mmol adenosine/l for 90 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

Pre-fertilization zona pellucida hardening by different cross-linkers affects IVF in pigs and cattle and improves embryo production in pigs

“…oocytes. A comparative study was carried out in order to investigate whether there are differences in effectiveness of the mechanisms that inhibit polyspermic penetration, because it is well known that the incidence of polyspermic fertilization is much lower in bovine IVF (Iwata et al 2008, Wang et al 2007) than in porcine IVF (Hancock 1959, Funahashi & Romar 2004, Hao et al 2006. Furthermore, a standard protocol incorporating cross-linkers in IVF, particularly due to the ability of cross-linkers to induce ZP hardening, may facilitate other problems associated with IVF including resistance to infection by viruses (Bielanski et al 2003), such as the transmission of bovine viral diarrhoea virus in abattoirderived in vitro produced embryos (Perry 2007).…”

“…After coincubation time, putative zygotes were washed twice with fresh TALPp by gentle aspiration through a glass pipette and allowed to continue in culture at 38.5 8C under 5% CO 2 until fixation (at 22 h post-insemination -hpi -in experiment 3 and at 168 hpi in experiment 4, where blastocyst formation was evaluated). The rationale of this methodology is based on using an optimized IVF system for porcine embryo production (Funahashi & Romar 2004).…”

“…The regulation of the binding of spermatozoa, the establishment of species specifity, the protection of the zygote from lethal microorganisms or parasites, and the block to polyspermy at fertilization are among its classical roles. However, in domestic animals, this latter role of the ZP under in vitro conditions is not completely understood , Wang et al 2003, Funahashi & Romar 2004, Hao et al 2006. In the mouse, the exocytosis of cortical granules, as a consequence of the fusion between a sperm and the oocyte, results in modification of the ZP (hardening), thereby avoiding binding and penetration of additional sperm (Ducibella et al 1990, Vincent et al 1990.…”

Methylxanthines and Reproduction

“…However, a limited exposure of gametes to caffeine significantly reduced the mean number of sperm cells that penetrated into the oocyte and asynchrony in the morphology of sperm nuclei in polyspermic oocytes (Funahashi and Romar 2004). Other epidemiology studies evaluated the timing and amount of caffeine intake by women and men undergoing IVF and gamete intra-Fallopian transfer (GIFT) on oocyte retrieval, sperm parameters, fertilization, multiple gestations, miscarriage, and live births.…”

Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro

“…Recently, the author and a colleague found that a transient co-culture of porcine oocytes with spermatozoa in the presence of caffeine followed by an additional culture in the absence of sperm cells and caffeine effectively decreased the incidence of polyspermic penetration without any reduction in the penetration rate (Funahashi & Romar 2004). Since this new in vitro fertilization (IVF) system can separate the process of sperm capacitation and zona binding from the following process of sperm penetration under different conditions, this system may be useful in analyzing further details of the effects of bME during the IVF period.…”