Vitrification of Domestic Cat Oocytes – Effect on Viability and Integrity of Subcellular Structures

Abstract

ContentsCryopreservation of female gametes is a crucial part of assisted reproduction techniques. The domestic cat is a model for both wild felids and human research. The aim of this study was to evaluate the effect of different vitrification protocols applied to feline oocytes at different maturational stages on the preservation of oocyte viability and integrity of subcellular structures. The vitrification solutions consisted of 20% ethylene glycol, 20% DMSO, 20% FCS, and 1.5 M Trehalose with and without 10% Ficoll PM-70. Markers for cell viability (PI/ FDA), cytoskeleton organization (Anti-a-Tubulin-FITC antibody, Phalloidin-TRITC), as well as nuclear configuration (DAPI) were used for evaluation of vitrified-warmed oocytes. Our results show that 52% and 41% of live mature and immature oocytes, respectively and until 32% of microtubules, 28% of nuclear configuration and 36% of microfilaments in the normal pattern can be obtained with protocol described in this paper. According to our data, Ficoll PM-70 essentially improves the oocytes survival upon vitrification. IntroductionAssisted reproduction techniques (ART) as in vitro fertilization and embryo transfer are important for wild animal conservation programs. Cryopreservation of domestic cat oocytes is also an important part of ART. Successful cryopreservation of female gametes would allowed to overcome time and space differences which are often an obstacle for research and field work of feline species.The domestic cat (Felis catus) is an easily obtainable biomedical model for studies on wild cat species and it has been demonstrated that many domestic cat ART protocols can be applied in wild felids (Pope 2000).Despite the fact that Tharasanit et al. (2011) andPope et al. (2012) reported a birth of live kittens obtained from vitrified cat oocytes, cryopreservation of immature or mature cat oocytes was not proven to be a reliable and repeatable tool so far. In the few published works, the cleavage rate of vitrified-warmed domestic cat oocytes ranged between 14% and 25% (Merlo et al. 2008;Comizzoli et al. 2009;Cocchia et al. 2010;Tharasanit et al. 2011). Cat oocyte cryopreservation is still considered to be an experimental technique (Luvoni 2006).The aim of this study was to evaluate the effect of different vitrification protocols applied to feline oocytes at different maturational stages on the preservation of oocyte viability and integrity of subcellular structures. The assessment by fluorescent markers of oocyte viability, cytoskeletal organization and nuclear configuration in vitrified and fresh oocytes was aimed to identify the damaging effects of cryopreservation.To achieve a very high cooling rate, which is necessary to prevent ice formation, the volume of the vitrification solution should be minimized by using devices specially designed for vitrification. In our study we focused on two of the most commonly used techniques -cryoloop and cryotop. Materials and MethodsAll chemicals were purchased from Sigma-Aldrich Chemie Gmbh (Munich, Germany), unless othe...

Desiccation and supra‐zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

“…In addition to protecting nuclear membrane integrity, desiccation approach also performed better at maintaining GV chromatin conformation than vitrification. Condensed chromatin observed in the cryopreserved samples has been described in previous reports (Falk et al, ; Mikołajewska et al, ; Royere, Hamamah, Nicolle, & Lansac, ). Although ice crystal formation is the usual culprit for cryoinjuries, several other factors during cryopreservation procedure could also impact DNA structure (Kopeika et al, ).…”

Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy

“…The debate on how vitrification procedures affect the microfilament network of the immature or mature oocytes has been reported in several studies [8,13,[17][18][19][20][21][38][39][40]. Consequences of vitrification/warming on the actin cytoskeleton depended on the CPAs used, the oocyte maturation stage and varied between species.…”

“…Several studies have shown that the exposure to cryoprotectants and the cold temperatures may lead to the disruption of microtubules and the change of the meiotic spindle morphology of mature oocytes [11][12][13][14][15][16]. Few reports focus on modifications caused by vitrification on the actin cytoskeleton [13,[17][18][19][20][21]. Actin is a major component of the cytoskeleton of the oocyte and it is present as the fibrous polymer, F-actin, in dynamic equilibrium with monomeric globular actin (G-actin).…”

Viabilidad de Ovocitos Vitrificados y Madurados in Vitro de Gata Doméstica Adulta (Felis catus) en Estación Reproductiva

“…La criopreservación de ovocitos se ha logrado con diversos grados de éxito en las pocas especies en que ha sido ampliamente estudiado, incluyendo el gato doméstico. Aunque han nacido gatitos después de una tasa de criopreservación controlada y transferencia de embriones derivados in vitro e in vivo, la criopreservación de ovocitos de gato sigue siendo un área urgente de mayor exploración (Cocchia et al, 2010;Pope et al, 2012) ya que todavía no se considera una herramienta confiable y repetible (Apparicio et al, 2013;Mikolajewska et al, 2012).…”