213 The efficacy of sericin in feline IVF and parthenogenesis and its potential in jaguar cloning

Conserving endangered felidae species is critical owing to habitat loss and human activities that increase the risk of inbreeding. Assisted reproductive technologies (ARTs), including IVF and interspecies somatic cell nuclear transfer (iSCNT), are essential for preserving genetic diversity and enhancing population numbers. In this context, sericin (SER), a protein derived from silk, has shown promising outcomes improving ARTs due to its cryoprotective, antioxidant, and subsequent accelerated blastocyst formation effects for SER in contrast to bovine serum albumin (BSA), as we previously demonstrated in our laboratory. In accordance with these positive results in livestock, our group saw the opportunity to explore SER efficacy in felidae ARTs applications. This study aimed to evaluate the effect of SER on IVF, parthenogenetic activation (PA), and iSCNT in jaguar (Panthera onca) as a proof of concept. We used the same experimental groups based on our previous results (Velasquez-Vasquez et al., 2024 Reprod. Dev. Fert. 36, 166–167) for IVM: (1) 0.3% BSA, (2) 0.3% BSA + 1% SER (B+S), (3) 1% SER, and (4) without protein source (WPS). For IVF, oocytes were co-incubated with 4 × 10⁶ sperm mL⁻¹ at 38.5°C in a humidified atmosphere of 21% O₂ and 5% CO₂ for 18–20 h. Presumptive zygotes were cultured in 50-µL drops of modified Tyrode’s medium (MTM) in 5% CO₂, 5% O₂, and 90% N₂. For PA, oocytes underwent a 1-min incubation in 1.5 mg mL⁻¹ pronase in TALP-H to remove the zona pellucida (ZP). ZP-free oocytes were treated with 1 µL mL⁻¹ ionomycin in TALP-H, followed by a 3-h incubation in 2 mM 6-DMAP. All groups were cultured in MTM for 7 days at 38.5°C, 5% CO₂, and 5% O₂ in a humidified atmosphere. Cleavage was assessed 72 h post-fertilization or PA, and the blastocyst stage was evaluated on Day 7. Data were analyzed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc.) with differences considered significant at P < 0.05. For the iSCNT proof of concept, we used an established protocol for feline species (Moro et al. 2015 Reproduction 150, 1–10). For the IVF, cleavage rates were BSA (77/137, 56.2%), B+S (64/140, 45.7%), SER (89/140, 63.6%), and WPS (59/145, 40.7%); for PA, cleavage rates were BSA (41/70, 58.6%), B+S (33/70, 47.1%), SER (42/68, 61.8%), and WPS (28/69, 40.6%). We observed that cleavage rates decreased with both the B+S and WPS, with no significant differences between SER and BSA. In IVF, the blastocyst rates were BSA (35/137, 25.5%), B+S (33/140, 23.6%), SER (44/140, 31.4%), and WPS (22/145, 15.2%); for PA, the blastocyst rates were BSA (20/70, 28.6%), B+S (16/70, 22.9%), SER (22/68, 32.4%), and WPS (9/69, 13%). The BSA and SER groups showed a significative difference compared with the WPS group. Finally, as a proof of concept, we obtained three jaguar cloned blastocysts from 77 enucleated cat oocytes subjected to SER-supplemented IVM medium (3/77, 4%). SER proved to be as effective as BSA in supporting cleavage and blastocyst development in feline IVF and PA. Our results suggest that SER could replace BSA in maturation medium, and it shows potential in ARTs for conserving species such as the jaguar.