Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations
Rocío Fernández-Gago A , Manuel Álvarez-Rodríguez B , Marta E. Alonso C , J. Ramiro González A , Beatriz Alegre A , Juan C. Domínguez A D and Felipe Martínez-Pastor D E F
+ Author Affiliations
- Author Affiliations
A Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
B IKE (Department of Clinical and Experimental Medicine), Linköping University, SE-581 83 Linköping, Sweden.
C Department of Animal Production, University of León, 24071 León, Spain.
D INDEGSAL (Institute for Animal Health and Cattle Development), University of León, 24071 León, Spain.
E Molecular Biology (Cell Biology), University of León, 24071 León, Spain.
F Corresponding author. Email: felipe.martinez@unileon.es
Reproduction, Fertility and Development 29(8) 1576-1584 https://doi.org/10.1071/RD15530
Submitted: 14 December 2015 Accepted: 18 July 2016 Published: 22 August 2016
Abstract
Seminal plasma could have positive effects on boar semen after thawing. In the present study we investigated changes in the motility and chromatin structure in spermatozoa over 4 h incubation (37°C) of boar semen thawed in the presence of 0%, 10% or 50% seminal plasma from good-fertility boars. Cryopreserved doses were used from seven males, three of which were identified as susceptible to post-thawing chromatin alterations. Motility was analysed by computer-aided sperm analysis every hour, and data were used in a two-step clustering, yielding three subpopulations of spermatozoa (slow non-linear, fast non-linear, fast linear). Chromatin structure was analysed using a sperm chromatin structure assay and flow cytometry to determine the DNA fragmentation index (%DFI) as a percentage, the standard deviation of the DFI (SD-DFI) and the percentage of high DNA stainability (%HDS), indicating chromatin compaction. Thawing without seminal plasma resulted in a rapid loss of motility, whereas seminal plasma helped maintain motility throughout the incubation period and preserved the subpopulation comprising fast and linear spermatozoa. The incidence of chromatin alterations was very low in samples from non-susceptible males, whereas samples from males susceptible to post-thawing chromatin alterations exhibited marked alterations in %DFI and %HDS. Seminal plasma partly prevented these alterations in samples from susceptible males. Overall, 50% seminal plasma was the most efficient concentration to protect motility and chromatin. Some changes were concomitant with physiological events reported previously (e.g., semen thawed with 50% seminal plasma increased the production of reactive oxygen species and yielded higher fertility after AI). Thawing in the presence of seminal plasma could be particularly useful in the case of samples susceptible to post-thawing chromatin damage.
Additional keywords: cryopreservation, flow cytometry, sperm physiology.
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