Effects of Ovary Storage Time and Temperature on DNA Fragmentation and Development of Porcine Oocytes

Abstract

Abstract. The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (≥15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.

HEPES buffer in ovary-transportation medium influences developmental competence of cattle oocytes

“…These findings are in agreement with those reported by Love et al (2003) and Yuge et al (2003), but contrast those of Wongsrikeao et al (2005) and Evecen et al (2010). Some disparities might be caused by long storage time, which was studied by these investigators.…”

“…Lack of suitable buffers resulted in pH decreases, which increased fragmented DNA nuclei significantly. It was suggested that the plasma membrane of oocytes was highly permeable to H + ions without any regulatory mechanism (Wongsrikeao et al, 2005). If external pH was maintained by increasing buffering capacity, the expulsion of protons would continue for a prolonged period, thus reducing the drop in intracellular pH buffers (Salehi et al, 2004), which has biological importance in determining the net electric charge of amino acids, which affects cell membrane permeability (Patel et al, 1973).…”

“…The differences in the quality of the oocytes may be associated with the stresses imposed during in vitro production (IVP), which begins from ovary removal. Practical problems often encountered are the effects of transportation, preservation medium and temperature (Yuge et al, 2003;Pedersen et al, 2004;Wongsrikeao et al, 2005). The 0.9% saline solution and the PBS are used extensively as ovary, cumulus oocyte complexes (COCs), zygote and embryo transportation media in goats (Silva et al, 2004;Rodriguez-Dorta et al, 2007), sheep (Smith et al, 1994;Wani et al, 2000;Shi et al, 2009), buffaloes (Amer et al, 2008;Ubaidullah et al, 2009), cows (Niemann, 1991;Thibodeaux et al, 1992;Martino et al, 1996;Schernthaner et al, 1997;Yang, 2002), sows (Mattioli et al, 1988;Westhof et al, 1991;Whitaker & Knight, 2004) and other animals (Nureddin et al, 1990;Jewgenow et al, 1998).…”

“…The effects of physiological saline and PBS use on oocyte quality and developmental competence for ovary-transportation medium at different temperatures (4 ºC, 15 ºC, 25 ºC, 33 ºC, and 35 ºC) have been investigated extensively (Wongsrikeao et al, 2005;Kim et al, 2006;Wang et al, 2011). However, there is little information about partially complex media use, such as KSOM and CZB, on the developmental competence of rescued COCs.…”

In vitro maturation and degeneration of domestic cat oocytes collected from ovaries stored at various temperatures

“…Storage at a warmer temperature conditions increased the percentage of porcine oocytes with DNA fragmented nuclei by altering the follicular pH and resulted in a lower rate of oocyte in vitro maturation. Wongsrikeao et al (2005) observed that storage of porcine ovaries at 4°C to 35°C for 6 h decreased the pH of follicular fluid and induced DNA frag- Within a column, groups with the same letter are not significant different (P < 0.05) *indicates that groups are not highly significantly different (P < 0.01) . Porcine oocytes have been described to be very sensitive to chilling, leading to a reduction in membrane integrity in oocytes stored < 15°C (Didion et al, 1990;Yuge et al, 2003).…”