Effect of Centrifugation Treatment before Vitrification on the Viability of Porcine Mature Oocytes and Zygotes Produced In Vitro

Abstract

Abstract. The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 × g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes. Key words: Centrifugation, Oocyte, Pig, Vitrification, Zygote (J. Reprod. Dev. 54: [149][150][151][152][153][154][155] 2008) he oocytes and early embryos of cattle and pigs are known to contain large numbers of cytoplasmic lipid droplets, which have an important role in their energy metabolism [1]. Nevertheless this characteristic has a huge impact on their availability for certain methods in animal biotechnology. For instance, it is known that a large number of lipid droplets causes the sensitivity of porcine embryos to low temperatures, which makes them very difficult to cryopreserve [2]. Also, the high lipid content that makes the cytoplasm of oocytes and early embryos dark blocks the visibility of organelles, which is essential for certain techniques such as microinjection of DNA into pronuclei [3].Polarization of lipid droplets by centrifugation is an efficient method to increase visibility of pronuclei [4]. Besides its importance in the production of transgenic cattle and pigs, centrifugation can be also used to confirm the onset of oocyte activation by the existence of pronuclei [5] or to separate monospermic zygotes from polyspermic ones [6]. Moreover, after centrifugation treatment, polarized ...

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

“…To our knowledge, these are the first piglets obtained from vitrified zygotes. Recently, our group reported for the first time the use of a modified SSV system for the successful cryopreservation of porcine zygotes (selected by the presence of a second polar body), which developed in vitro to the blastocyst stage; however, only a low percentage (5%) of these vitrified zygotes reached the blastocyst stage [43]. Here, we report an improved (approximately 15%) rate of in vitro blastocyst formation after vitrification of zygotes.…”

“…Nevertheless, for decades there had been no reports of the cryopreservation of zygotes in livestock species, such as cattle and pigs. Recently, our group was the first to report the in vitro production of blastocysts from IVP porcine zygotes cryopreserved by solid surface vitrification (SSV) after visualization of the pronuclei by centrifugation [43]. The question remains, however, as to whether vitrified zygotes can develop to term, because the cryopreservation procedure might alter important processes such as DNA synthesis, which occurs at the pronuclear stage [44].…”

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art

“…Other measures, such as induction of osmotic stress (by exposure to NaCl) has shown to improve developmental competence after vitrification (Lin et al 2008). Centrifugation (lipid depot relocation) for vitrification appears detrimental for in vitro-matured oocytes, but not in zygotes or later stages (Somfai et al 2008) Vitrification of in vivo-developed, ZP-intact pig embryos, where lipids were polarized by centrifugation of the blastomeres, by delipation and/or treatment with cytochalasin for cytoskeleton stabilization, has resulted after rewarming and ET, in piglets (Dobrinsky 1997, Dobrinsky et al 2000, Kobayashi et al 1998, Berthelot et al 2000, Cameron et al 2000. Blastocysts were also developed by in vitro fertilization (IVF) of follicular oocytes vitrified as cumulus-oocyte complexes from offal porcine follicles (Somfai et al 2010).…”

“…Use of alternative methods such as vitrification instead of slow cooling led to better survival (see Massip 2001) including the birth of offspring (Berthelot et al 2000). However, large variation was seen among methods, sources and laboratories (Holm et al 1999, Cuello et al 2007, Somfai et al 2008, Ogawa et al 2010, including the method used for intrauterine deposition (Rodriguez-Martinez 2007b.…”

Oocyte and embryo cryopreservation – state of art and recent developments in domestic animals

“…Otoi et al (1997) found that although centrifugation has a negative effect on bovine oocytes, its use is advantageous in the context of cryopreservation. A recent study with pig oocytes and embryos (Somfai et al, 2008) showed that centrifugation (10 000 g for 20 min) reduced the rate of surviving vitrified oocytes, although the proportion of parthenogenetic divisions was similar in the group of centrifuged and non-centrifuged oocytes (42 and 47%, respectively). Meanwhile, although zygote centrifugation slightly improves their survival after cryopreservation, it does not increase the developmental competence of surviving zygotes.…”