Meiosis Resumption of Canine Oocytes Cultured in the Isolated Oviduct

Abstract

The aim of this study was to investigate the effects of culture in isolated oviducts relative to meiotic maturation, the time required to resume meiosis and the viability of the canine oocytes. For this purpose, cumulus-oocyte complexes and isthmus-ampullar tracts of the oviducts were collected from bitches undergoing ovariohysterectomies and destined to two experiments of culture. In experiment 1, the oocytes were cultured for 24 or 30 h: (1) in 100 micro l drops under oil; (2) on the mucosal epithelium of the open oviducts; (3) in the ligated oviducts. In experiment 2, oocytes were cultured in the ligated oviduct for 24, 30 and 48 h. A group of control oocytes was not cultured (0 h). The results showed that within 30 h of culture, a higher proportion of oocytes (p < 0.001) resumed meiosis in the ligated oviduct (63.8%) than in drop (20.4%) or in the open oviduct (27.1%). Moreover, 24 and 30 h of culture assured higher proportions of meiosis resumption than 48 h (69.2 and 59.1% vs 35.8%, p < 0.005). Oocyte resumption of meiosis was mainly determined by oocytes at meiotic stages preceding metaphase I, while stages between metaphase I and II in the ligated oviduct ranged between 12.5 and 31.9%. The extension of the culture time up to 48 h in the oviduct increased oocyte degeneration significantly (59.3%, p < 0.0001) compared with 24 and 30 h (18.7 and 27.3%, respectively) and the oviductal epithelium showed nuclear picnosis and degeneration following culture. The present study suggests that the close physical interaction between the canine oocytes and the oviductal tract positively affects oocyte maturation, and meiosis is resumed within 30 h of culture. Moreover, the oocyte survival is better preserved within 30 h in the ligated oviduct compared with the conventional culture in drop or to the culture in the open oviduct, but the ligated oviduct does not assure viability of the oocytes up to 48 h of culture.

Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes

“…The incidence of 80% nuclear maturation (from the >2 mm diameter follicles) was easily >50% higher than rates reported when oocytes were not categorized on the basis of donor follicle size (Bogliolo et al, 2002;Bolamba et al, 2002;Otoi et al, 2002;Songsasen et al, 2002Songsasen et al, , 2003Luvoni et al, 2003;Rodrigues and Rodrigues, 2003;WiUingham-Rocky et al, 2003). The incidence of Mil (15%-30%) in smaller size follicles was quite comparable to a host of other reports for this species (Nickson et al, 1993;Yamada et al, 1993;Otoi et al, 2001;Willingham-Rocky et al, 2003).…”

“…It is generally accepted that lack of an effective procedure to in vitro mature oocytes is the major impediment to using embryo transfer in the dog (see reviews, Farstad, 2000;Luvoni, 2000). Since the first report on in vitro maturation of domestic dog oocytes in 1976 (Mahi and Yanagimachi, 1976) and despite significant efforts (Yamada et al, 1993;England, 1998, 1999;Saint-Dizier et al, 2001;Bogliolo et al, 2002;Bolamba et al, 2002;Otoi et al, 2002;Songsasen et al, 2002Songsasen et al, , 2003Luvoni et al, 2003;Rodrigues and Rodrigues, 2003;Willingham-Rocky et al, 2003), little progress has been made in developing a culture system that will consistently achieve nuclear maturation in canine oocytes. On average, only 15%• 20% of ovarian oocytes complete nuclear maturation after 48-72 hr of culture.…”

Microenvironnement tubaire: rôle dans la maturation des ovocytes canins in vivo et in vitro

“…Les oviductes peuvent être laissés ouverts ou ligaturés à leurs extrémités. L'avantage des ligatures est de conserver les sécrétions tubaires tout en permettant aux ovocytes de rester dans la lumière (Luvoni et al 2003). Toutefois, la difficulté est la récupération des embryons ou des ovocytes insérés (Rizos et al 2003) (figures 5a, 5b, 6).…”

“…Des explants d'oviductes de mouton ont également été utilisés avec succès pour cultiver des embryons bovins produits in vitro (Rizos et al 2002a). Chez la chienne, une seule étude rapporte des résultats obtenus par la technique des explants d'oviductes, prélevés chez des chiennes en anoestrus (Luvoni et al 2003). Ces auteurs recueillent 31,9 % d'ovocytes au stade métaphase I-métaphase II dans des oviductes ligaturés, contre 12,5 % dans des oviductes ouverts et 3,7 % dans un milieu de culture in vitro (M199).…”

Maturation ovocytaire in vitro chez la chienne

“…Aucune de ces techniques n'a augmenté significativement les taux d'ovocytes en métaphase II. Un système de culture dans un oviducte ligaturé a été décrit (LUVONI et al, 2003) et semble donner une augmentation significative des rendements, mais reste difficilement répé-table. D'autres conditions de culture restent encore à évaluer, en particulier en faisant varier la composition de l'atmosphère gazeuse par exemple, ce qui a donné de bons résultats chez les murins (EPPIG et WIGGLESWORTH, 1995).…”