Molecular phenotyping of domestic cat (Felis catus) testicular cells across postnatal development - A model for wild felids.

Molecular characterisation of testicular cells is a pivotal step towards a profound understanding of spermatogenesis and developing assisted reproductive techniques (ARTs) based on germline preservation. To enable the identification of testicular somatic and spermatogenic cell types in felids, we investigated the expression of five molecular markers at the protein level in testes from domestic cats (Felis catus) at different developmental phases (prepubertal, pubertal I and II, postpubertal I and II) classified by single-cell ploidy analysis. Our findings indicate a prominent co-labelling for two spermatogonial markers, UCHL1 and FOXO1, throughout postnatal testis development. Smaller subsets of UCHL1 or FOXO1 single-positive spermatogonia were also evident, with the FOXO1 single-positive spermatogonia predominantly observed in prepubertal testes. As expected, DDX4+ germ cells increased in numbers beginning in puberty, reaching a maximum at adulthood (post-pubertal phase), corresponding to the sequential appearance of labelled spermatogonia, spermatocytes and spermatids. Furthermore, we identified SOX9+ Sertoli cells and CYP17A1+ Leydig cells in all of the developmental groups. Importantly, testes of African lion (Panthera leo), Sumatran tiger (Panthera tigris sumatrae), Chinese leopard (Panthera pardus japonesis) and Sudan cheetah (Acinonyx jubatus soemmeringii) exhibited conserved labelling for UCHL1, FOXO1, DDX4, SOX9 and CYP17A1. The present study provides fundamental information about the identity of spermatogenic and somatic testicular cell types across felid development that will be useful for developing ART approaches to support endangered felid conservation.

Cooled storage of semen from livestock animals (Part II): Camelids, goats, and sheep.

This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques in cooled storage of semen from livestock animals such as camelids, goats, and sheep. Facing worldwide environmental changes and a trend towards more conscious and healthy eating behaviors, the development of a stable animal breeding industry is a significant challenge for the near future. In the present review, factors influencing semen handling in camelids, goats and sheep are described and relevant methods as well as current trends to improve liquid-storage of cooled semen are discussed, including extenders, additives, cooling rates, and storage temperatures. The species-specific physiology and resulting challenges are taken into consideration. While the main problem for camelid semen processing is the relatively greater viscosity as compared with that of some other animals, the deciding factor for successful artificial insemination (AI) in goats and sheep is the site (i.e., cervical or vaginal) of semen placement in the reproductive tract. Due to the type of cervical anatomy, the penetration of the cervix when using AI instruments is rather difficult. Furthermore, the seminal plasma of small ruminants affects the interaction with milk-based extenders and egg yolk which results in species-specific regimens for cooled liquid-preservation. Comparing all three species, the greatest pregnancy rates were obtained by AI with goat semen after cooled liquid-storage for several days.

Cell survival after cryopreservation of dissociated testicular cells from feline species.

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 µm and 20.5 ± 4 µm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.