The effects of permeating cryoprotectants on intracellular free-calcium concentrations and developmental potential of in vitro-matured feline oocytes Jason R. Herrick A,D , Chunmin Wang B,C and Zoltan Machaty B A National Foundation for Fertility Research, 10290 RidgeGate Cr, Lone Tree, CO 80124, USA. B Department of Animal Sciences, Purdue University, Lilly Hall, 915 West State St, West Lafayette, IN 47907, USA. C Current Address: Vivere Health-Houston Surgery Center and IVF Laboratory, 2500 Fondren Rd, Suite 350, Houston, TX 77063, USA. D Corresponding author. Email: jherrick@fertilityresearch.org Abstract. Embryos produced from vitrified feline oocytes have resulted in pregnancies, but the efficiency of oocyte vitrification in cats is still low. Our objectives were to evaluate the effects of exposing feline oocytes to ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO) on changes in intracellular free-calcium concentrations ([Ca 2þ ] i ), the time needed for enzymatic digestion of the zona pellucida (ZP), the incidence of parthenogenetic activation and degeneration and embryonic development following in vitro fertilisation (IVF). All of the chemicals tested altered [Ca 2þ ] i , but changes in [Ca 2þ ] i , resistance of the ZP to enzymatic digestion and the incidence of parthenogenetic activation (,5% for all treatments) were not affected (P . 0.05) by extracellular Ca 2þ . Exposure to EG (.44.1%) and DMSO (19.7%) increased (P , 0.05) oocyte degeneration compared with control oocytes and oocytes exposed to PrOH (#2.5%). Following exposure to a combination of PrOH and DMSO (10% v/v each), blastocyst development (per cleaved embryo; 52.1%) was similar (P . 0.05) to control oocytes (64.4%). When oocytes were vitrified with PrOH and DMSO, 28.3% of surviving (intact plasma membrane) oocytes cleaved following IVF, but no blastocyst developed. When a non-permeating cryoprotectant (galactose, 0.25 M) was added to the vitrification medium, 47.7% of surviving oocytes cleaved and 14.3% developed to the blastocyst stage. Additional keywords: cat, DMSO, intracellular calcium, propanediol, vitrification. Received 3 July 2014, accepted 26 August 2014, published online 11 September 2014 Introduction Management of ex situ populations of endangered felids has become an important component of the conservation efforts for these species. Unfortunately, most of these captive populations are not sustainable due to a limited number of individuals, inadequate genetic diversity and poor reproductive success. Assisted reproductive technologies (ARTs), like in vitro ferti- lisation (IVF) and embryo transfer (ET), could play an important role in ensuring the sustainability of captive populations by overcoming behavioural incompatibility and reducing the need to transport animals between zoos (Swanson 2006). Combining these procedures with gamete and embryo cryopreservation enhances the potential role of ARTs by allowing an individual’s genetics to be preserved well beyond the animal’s lifespan and facilitating transport of those samples over long distances (Swanson 2006). Following cryopreservation, motility and fertilising ability of spermatozoa from numerous species of felids are sufficient for IVF and artificial insemination (AI), providing a reliable means of preserving the male genome (Swanson et al. 1996; Stoops et al. 2007; Herrick et al. 2010). However, an efficient procedure to preserve the genome of female felids has remained elusive. It is possible to preserve a female’s genetics by collect- ing oocytes, producing embryos through IVF and cryopreser- ving the resulting embryos, but this approach has several limitations. First, population managers must select a suitable male at the time of oocyte collection from those in the current population or those males whose spermatozoa have already been cryopreserved. Second, only a handful of zoological institutions have the personnel and laboratories to conduct ARTs. There- fore, IVF is often conducted in improvised laboratories equipped with portable versions of all necessary equipment (Swanson 2003). The ability to successfully cryopreserve feline oocytes would allow oocytes to be stored for extended periods of time until a suitable male is identified and a sperm sample is available. Similarly, oocytes could be cryopreserved within minutes of recovery and transported to a specialised laboratory for IVF where the chances of success are greatly improved. CSIRO PUBLISHING Reproduction, Fertility and Development, 2016, 28, 599–607 http://dx.doi.org/10.1071/RD14233 Journal compilation Ó CSIRO 2016 www.publish.csiro.au/journals/rfd