Vitrification of Immature Bovine Oocytes by the Microdrop Method

Abstract

Abstract. This study was conducted to determine the optimal vitrification conditions for immature bovine oocytes using the microdrop method. In experiment 1, the optimal pre-equilibration period for microdrop vitrification was examined. The maturation rate of vitrified oocytes with a 3 min first preequilibration period (41.1%) was higher than that of vitrified oocytes with a 0 min first preequilibration period (21.4%), and the values of those with a 1 (33.9%) or 5 min (27.4%) first preequilibration period were intermediate. The value for a 1 min second pre-equilibration period (44.4%) was significantly higher (P<0.05) than those for a 0.5 (28.6%) and 2 min (21.4%) second preequilibration period. In experiment 2, the distribution of microtubules in matured oocytes was investigated. There was no difference among the first pre-equilibration times in terms of the rates of normal spindles in vitrified oocytes. However, this value was significantly higher (P<0.05) in the 1 min group (52.8%) compared with the 0.5 (16.7%) and 2 min groups (12.3%). In experiment 3, we investigated the developmental capacity of immature bovine oocytes vitrified under optimal preequilibration conditions (3 min and 1 min for the first and second pre-equilibrations, respectively). Although the total fertilization rates were significantly lower (P<0.05) in the vitrified oocytes (65.6%) compared with the control oocytes (92.4%), there was no difference in the rate of normal fertilization (2PN) between the vitrified (78.6%) and control (82.0%) oocytes. Cleavage and blastocyst rates were significantly lower (P<0.05) in vitrified oocytes (55.7 and 2.3%) than in control oocytes (84.4 and 34.7%). Thus, these results indicated that immature bovine oocytes can survive after microdrop vitrification and subsequently can be cultured to mature oocytes capable of undergoing fertilization in vitro and developing into blastocysts.

Aumento na sobrevivência após vitrificação de oócitos bovinos imaturos em recipientes com maior condutividade e nitrogênio super-resfriado

“…As taxas de clivagem observadas entre os grupos vitrificados foram semelhantes, sendo MV (31,8%), PB (32,64%), OPS (31,43%) e PM (43%). Estas taxas se assemelham às obtidas em trabalhos previamente desenvolvidos no laboratório, ou seja, 46 a 49% [24] e 33,9 a 41,2% [20], sendo ainda comparáveis às obtidas por outros autores após a vitrificação de oócitos bovinos imaturos (55,7%) [6] ou maturados (49,1% [17] e 50% [22]). A clivagem é um bom indicativo de sobrevivência após a criopreservação, e as taxas obtidas neste estudo e nos demais trabalhos atestam a viabilidade do processo.…”

“…A criopreservação de oócitos possibilita o estabelecimento de bancos genéticos, mantendo a biodiversidade animal; flexibiliza a produção in vitro, proporcionando o avanço do melhoramento genético; e disponibiliza material para pesquisas básicas [6]. Entretanto, o alto teor lipídico, o baixo coeficiente de permeabilidade, a alta relação volume/superfície, o grande tamanho, as células do cumulus e o complexo citoesqueleto dos oócitos interferem negativamente na sua sobrevivência à criopreservação [9,10].…”

“…A metodologia mais adequada para criopreservar oócitos é a vitrificação [23], que, por sua alta velocidade de resfriamento, permite uma rápida passagem pela zona crítica de temperatura, na qual ocorrem as lesões às células [12]. As chamadas metodologias abertas, como as microdrops [6,7], electron microscope grids [15], open pulled straws (OPS) [22], micropipetas de vidro (MV) [16,18], o cryoloop [8] e o nylon mesh [1] proporcionam alta taxa de resfriamento. O emprego de vácuo, para eliminar o vapor de nitrogênio e seu efeito isolante, poderia ampliar ainda mais esta velocidade.…”

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

“…This method was first proposed by Landa and Tepla (1990) for mouse embryos. It was then successfully used for bovine embryos (Riha et al, 1991), zygotes (Yang and Leibo, 1999), both mature oocytes (Papis et al, 2000) and immature oocytes (Kim et al, 2007), and pig embryos (Misumi et al, 2003). However, no further application of this technology has been published, probably because of the difficulties encountered when handling the embryos.…”

Live birth after transfer of vitrified embryos from superovulated goats

“…Thus, succeeding efforts were directed at the *Corresponding author: Marlon B. Ocampo, Reproductive Biotechnology Unit, Philippine Carabao Center, Science City of Muñoz, Nueva Ecija, Philippines; E-mail: ocampomarlon29@yahoo.com; Tel. No: 09098868938 improvement of the vitrification procedure for embryo cryopreservation using the electron microscope grids (Martino et al, 1996), open pulled straws (Vajta et al, 1998), cryoloop (Lane et al, 1999a), cryotop (Kuwayama and Kato, 2000), microdrop method ( Kim et al, 2007;Ocampo et al, 2014), gel loading tip (Tominaga and Hamada, 2001) and a paper container (Kim et al, 2012) with the ultimate aim of preventing injury from intracellular ice formation. One remarkable improvement was the use of a simplified method using a vitrification solution based on ethylene glycol (Kasai et al, 1990;Yushiati and Holtz, 1990;Miyake et al, 1993;Zhu et al, 1993;Guignot et al, 2006) that allow rapid permeation of the cell within 2 min of treatment at 20°C before directly plunging the sample into liquid nitrogen.…”