Silk protein sericin pretreatment enhances osmotic tolerance and post-thaw sperm quality but reduces the ability of sperm cells to undergo in vitro induced acrosome reaction in rabbit

Artificial insemination provides an excellent opportunity to spread desired genes and determining high productivity in domestic animals. Cryopreservation and long term storage of sperm cells expands potential application of artificial insemination technology throughout continents and countries. Although sperm freezing technology has a long history, nearly 50 years, still lots of sperm cells die during freezing and thawing process. This basically occurs due to the damages which take place in various cellular components including plasma membrane, DNA, mitochondria and acrosome etc. The sperm cells of different animal species are highly specific in terms of plasma membrane structure and biochemical composition. Therefore, sperm cells may behave differently to freezing extenders and the protocols.

Rabbit sperm membranes are very peculiar for high cholesterol/phospholipid ratio, similar to human spermatozoa and opposing to most of the domestic ruminants. Cryopreservation mostly results in poor post-thaw semen quality in the rabbit, therefore artificial insemination with cryopreserved semen is utilized only for experimental or genetic resource bank purposes [37]. On the other hand, commercial rabbit industries rely on artificial insemination by fresh or chilled semen, stored for no more than three days [49] and more preferably, within few hours after semen collection [15]. Thus, protecting the fertility of stored semen remains a major target for the rabbit meat industry. Innovative attempts to find a suitable freezing extender might improve the functional status of stored rabbit semen and prolong fertility.

Cryopreservation of rabbit sperm cells in the extenders including Me₂SO (TCG-sucrose-Me₂SO) provides reasonable post-thaw motility and high fertility after vaginal inseminations [58]. Egg yolk stabilizes sperm membranes during cooling or freezing and is used frequently in the extender formulations, however, its combination with Me₂SO gives poor freezing results [50]. Therefore, further research is needed to develop a suitable substitute of egg yolk to combine with Me₂SO in rabbit semen extenders.

The silkworm Bombyx mori synthesizes silk treads which cover 25–30% of the cocoon weight. Sericin is a structural component and surrounds two fibroin filaments of the silk fiber [13]. Sericin, a glycoprotein which is synthesized by alternative splicing of at least three (Ser 1, Ser 2, and Ser 3) genes, which enable the cells to synthesize proteins by putting exons together in different ways [55]. Furthermore, it is also produced by solvent extraction methods from the silk threads with a molecular weight of 10–310 kD.

Sericin has a strong property to stick and cover the cell membrane because it contains positively charged residues like arginine or lysine [52]. Therefore, it is recently used to replace serum in the culture and cryopreservation media with several favorable results [9]. Likewise, protective impact of sericin supplementation against freezing injuries in different somatic cell lines was reported by Sasaki et al. [51]. In sperm cells, the effect of sericin pretreatment on sperm function i.e., capacitation, acrosome reaction and fertility status, and its influence during cooling or freezing is not yet documented except for three articles dealing with the impact of sericin on goat [47], buffalo [31] and horse sperm cells [38] during freezing. Thus, the aims of the present study were to investigate the effect of sericin pretreatment on the (1) osmotic tolerance of sperm cells; (2) ability of spermatozoa to undergo acrosome reaction in the presence of inducing agents; (3) post-thaw sperm quality following freezing after sericin pretreatment and; (4) fertility levels after artificial inseminations.