# Morphological and Functional Evaluation of Kodkod (Leopardus guigna) Oocytes After In Vitro Maturation and Parthenogenetic Activation

## TL;DR

Researchers adapted techniques used for domestic cat oocytes to study kodkod oocyte maturation and embryo development in vitro, showing potential for conservation efforts.

## Contribution

The first successful adaptation of domestic cat oocyte protocols for in vitro maturation and embryo development in kodkod oocytes.

## Key findings

- Kodkod oocytes matured in vitro and developed to the blastocyst stage.
- Kodkod oocytes were smaller and had a thinner zona pellucida compared to domestic cat oocytes.
- Protocols for domestic cat oocytes can be adapted for kodkod in vitro embryo production.

## Abstract

The kodkod (Leopardus guigna) is a small wild felid native to Chile, whose population is currently in decline. Assisted reproductive technologies (ARTs) offer valuable tools for the genetic preservation of endangered species, including the kodkod. Conservation strategies for the kodkod have included cryobanking and the generation of cloned embryos through interspecific somatic cell nuclear transfer (iSCNT). However, the reproductive physiology and embryo development of this species are poorly understood. Here, we applied optimized protocols developed for domestic cat oocytes to evaluate the in vitro oocyte maturation (IVM) and in vitro culture (IVC) of embryos from kodkod. Immature cumulus–oocyte complexes (iCOCs) were collected from a female kodkod, and their morphology and developmental competence were evaluated. Immature kodkod oocytes were smaller than domestic cat oocytes. Kodkod oocytes were able to mature in vitro and develop to the blastocyst stage following chemical activation. Our work demonstrates that the protocols used to manage domestic cat oocytes can be successfully adapted for IVM and in vitro embryo production (IVP) in the kodkod. Our research provides a foundation for improving ARTs aimed at conserving this endangered felid and enhancing our understanding of its reproductive biology.

The kodkod (Leopardus guigna) is a vulnerable wild felid native to South America whose population is steadily declining. ARTs offer valuable tools for the preservation of its genetic diversity. Our study provides the first evaluation of the morphological and functional acquisition of competence in kodkod oocytes using protocols previously established for domestic cat oocytes. In total, 29 iCOCs were obtained from the ovaries of a single juvenile female kodkod that deceased in a wildlife rehabilitation center. Based on morphological criteria, 13 oocytes were selected for IVM and subsequently evaluated for developmental competence following parthenogenetic activation (PA) and in vitro culture (IVC). Kodkod oocytes appear to be smaller and have a thinner zona pellucida compared to those of domestic cat oocytes. These kodkod oocytes demonstrated the ability to mature in vitro, underwent cleavage, and developed in vitro to the blastocyst stage by day 9. Here, we show that protocols to manage domestic cat oocytes and embryos can support kodkod in vitro oocyte maturation, activation, and in vitro embryo development. However, given that the results were obtained from a single individual and the protocols were tested in a limited number of oocytes, further studies involving additional specimens are essential to validate these observations and refine ART applications for kodkod conservation.