17 Identification of somatic cell populations in cryopreserved equine semen as a source of donor nuclei for somatic cloning: preliminary results

The isolation of somatic cells (SCs) from horse semen has the potential to be a valuable tool in the field of cloning. Nevertheless, there is currently no information available regarding the concentration and cytomorphological characteristics of SCs in long-term frozen equine semen. The main objective of our research was to gain such information. Cryopreserved 20-year-old (stallions, n = 2) and 10-year-old (stallions, n = 2) equine semen samples were obtained from a commercial supplier. The semen was thawed at 37°C for 30 s and then diluted in modified DMEM/F12 medium (1:5) and incubated at 37°C for a further 30 min. For each stallion, three straws of cryopreserved semen were used. SCs were isolated by micromanipulation and used in four experimental trials: (1) cytomorphometric study of SCs was done using ImageJ 53t free software; (2) cell concentration and cell viability of SCs were determined; (3) Annexin-V-FLUOS test was applied to evaluate cytoplasmic membrane integrity of SCs; and (4) enucleated MII equine oocytes were reconstituted with SCs using the free-zone cloning method. The Mann-Whitney U test was used for the comparison of numerical variables, and the chi-squared test was used for categorical variables. The analysis identified four distinct morphological groups based on the characteristics observed. The first group consisted of round SCs with smooth and granular cytoplasm. The second group included flattened squamous cells with irregular contours and small nuclei. The third group consisted of polygonal and round transitional epithelial cells that were either clustered or single. The median size of SCs was 16.36 μm in diameter, with a range between 6.91 and 57.52 μm. No significant differences were observed in SC concentration between samples cryopreserved for 20 and 10 years (1.74 ± 1.41 × 10⁶ vs. 0.91 ± 0.28 × 10⁶ SCs mL⁻¹ of semen, respectively) or in cell viability, with values of 17.2% (n = 47/273) versus 17.8% (n = 56/314), respectively. The Annexin-V-FLUOS test revealed that the cytoplasmic membrane integrity of SCs isolated from semen cryopreserved for 20 years was significantly compromised compared with samples cryopreserved for 10 years, 36.8% versus 12.1% (P < 0.05), respectively. The reconstruction of cytoplasts with SCs as nuclei donors was only successful in 10% of cases. Our findings indicate that a sufficient number of viable SCs can be collected using a simple method. However, we observed that the integrity of the membranes of SCs collected directly from frozen semen is compromised, affecting the reconstruction of equine embryos cloned by the cytoplast-karyoplast fusion method.

This study was supported by CONAHCYT (BRS, 2223801065) DCBS, UAM.