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Serrano E.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca | Perez-Pe R.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca | Calleja L.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca | Guillen N.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca | And 4 more authors.
Gene | Year: 2013

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22. kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76-85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality. © 2013 Elsevier B.V.


Del Valle I.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca | Souter A.,University of Sydney | Maxwell W.M.C.,University of Sydney | Muino-Blanco T.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca | Cebrian-Perez J.A.,Instituto Universitario Of Investigacion En Ciencias Ambientales Of Aragon Iuca
Animal Reproduction Science | Year: 2013

The aim of this study was to assess biologically safer components as alternatives to egg yolk for the frozen storage of ram semen using casein, coconut or palm oil in either Salamon's diluent (S) or a swim-up medium (SU). Ejaculates were frozen as pellets and sperm motility (subjectively) and acrosome integrity (FITC-PNA/PI) by flow cytometry were assessed at 0, 3 and 6. h after thawing and incubation at 37. °C. Three experiments were done: different concentrations of palm oil (5%, 10% and 20%); casein added as emulsifier and protective agent; and differences between egg yolk, coconut and palm oil in S and SU. 20% of oil added to SU accounted for a lesser percentage (P<0.05) of motile cells compared to rest while no differences were found between different oil levels on viable cells. When casein was added to diluents containing 5% of palm oil, no differences were found between palm or casein (P>0.05). No differences were found when S and SU were compared neither as groups nor between S alone and containing coconut or palm oil; however, SU alone yielded less motility than SU 5% coconut. However, in both groups, S and SU, egg yolk accounted for the greatest values in both bases. These results indicate that none of biologically safer media components (casein, palm or coconut oil) used in this study maintained the function of ram spermatozoa after freeze-thawing better than S-containing egg yolk. The application of vegetable oils as substitutes for egg yolk in diluents for the cryopreservation of ram spermatozoa requires further research. © 2013 Elsevier B.V.

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