title: Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa.
abstract: The goal of the present experiment was to evaluate the suitability and effectiveness of single layer centrifugation (SLC), using the pig-specific colloid Androcoll-P, as a routine procedure for selecting boar spermatozoa for cryopreservation. The study focuses special attention on the effectiveness of SLC for processing a whole sperm rich ejaculate fraction and the fertilizing ability of frozen-thawed (FT) sperm selected using SLC prior to freezing. Thirteen sperm rich ejaculate fractions (one per boar) were split into three aliquots. Two aliquots of 15 and 150mL were SLC-processed (500×g for 20min) using 15 and 150mL (v/v) of Androcoll-P-Large and Androcoll-P-XL, respectively. The third aliquot remained un-processed as a control. The percentages of spermatozoa that were morphologically normal and showed rapid and progressive motility (assessed by CASA) spermatozoa were higher (P<0.01) and those with fragmented nuclear DNA (sperm chromatin dispersion test) were lower (P<0.01) after SLC than control semen samples, regardless of the Androcoll-P used. The recovery rates of total, motile, viable (flow cytometric evaluated after staining with H-42, PI and FITC-PNA) and morphologically normal spermatozoa ranged between 20 and 100% and those with intact nuclear DNA ranged between 60 and 100%, irrespective of the Androcoll-P used. Thereafter, the semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw percentages of sperm motility (both total motility and rapid progressive motility), viability and intact nuclear DNA were higher (P<0.05) in SLC-processed than in control semen samples, irrespective of the Androcoll-P used. SLC-processing also improved the in vitro fertilizing ability of FT-sperm (679 in vitro matured oocytes inseminated with a viable sperm:oocyte ratio of 300:1 and coincubated for 6h), measured as the percentage of penetrated oocytes and the mean number of swollen sperm heads and/or male pronuclei in penetrated oocytes. However, there was no effect of SLC-processing on the in vitro ability of putative zygotes to develop to blastocysts. Overall these results indicate that SLC-processing of boar ejaculates using Androcoll-P improves the quality and fertilizing ability of cryosurvival boar sperm. However, efforts should be made to ensure continued high recovery yields before considering the inclusion of SLC as a routine procedure in the cryopreservation protocol of boar ejaculates.
authors.affiliation: Department of Medicine and Animal Surgery, University of Murcia, Murcia, Spain.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Martinez-Alborcia
authors.forename: Jane M
authors.initials: JM
authors.lastname: Morrell
authors.forename: Maria A
authors.initials: MA
authors.lastname: Gil
authors.forename: Isabel
authors.initials: I
authors.lastname: Barranco
authors.forename: Carolina
authors.initials: C
authors.lastname: Maside
authors.forename: Diego V
authors.initials: DV
authors.lastname: Alkmin
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Parrilla
authors.forename: Emilio A
authors.initials: EA
authors.lastname: Martinez
authors.forename: Jordi
authors.initials: J
authors.lastname: Roca
mesh_terms: D000818:Animals; D002498:Centrifugation / Q000379:methods / Q000662:veterinary*; D003102:Colloids / Q000494:pharmacology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D008858:Microscopy, Phase-Contrast / Q000662:veterinary; D055101:Semen Analysis / Q000662:veterinary; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology*; D013552:Swine / Q000502:physiology*
chemical_list: D003102:Colloids
keywords: Boar; Colloid; Cryopreservation; In vitro fertilization; Sperm