Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Abstract

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 10 6 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Cryopreservation of jaguar (Panthera onca) sperm cells using different cryoprotectants and different thawing temperatures

“…Besides the successes in semen collection, cryopreservation still needs refinement, aiming to improve the quality of thawed sperm. As in other felids, the Tris-Egg-yolk extender is the method of choice for semen cryopreservation in jaguars, using 4 to 7.5% glycerol as the cryoprotectant ( Araujo et al, 2020a ; Paz et al, 2000 , 2007 ; Santos et al, 2022 ; Silva et al, 2020 ; Swanson et al, 1996 ). Although egg yolk is widely used, its instability and semen export restrictions necessitate the investigation of alternatives that have been used in other species, like soy lecithin and low-density lipoproteins ( Bencharif et al, 2008 ; Lambo et al, 2012 ).…”