Abstract
The aim of the present study was to evaluate the effect of three different freezing curves on male goat’s sperm viability and acrosome integrity. Semen was collected on three adult male goats in February (Azores, Portugal, latitude 38°39′N) using an artificial vagina and a female with induced heat. Three collection sessions were spaced of approximately 5 days. The study followed a factorial design of repeated measurements. After volume, sperm concentration and mass motility evaluation, live and dead spermatozoa (spz) were counted and acrosome integrity was evaluated by Flow Cytometry (FCM). In the meantime semen was frozen using three curves differing in the second cooling ramp rate (curve A = 25°C/min, curve B = 35°C/min and curve C = 45°C/min). After thawing, the percentage of live/dead sperm cells and the acrosome integrity were determined by FCM, using respectively Propidium Iodide (PI) and Pisum Sativum Agglutinin conjugated to Fluorescein Isothiocinate (FITC-PSA). By combining these two fluorochromes, the thawed semen was divided into: Live sperm with intact acrosome, unstained PI and unstained FITC-PSA (LL); live sperm with damaged acrosome, unstained PI and stained FITC-PSA (LR); dead sperm with intact acrosome, stained PI and unstained FITC-PSA (UL); dead sperm with damaged acrosome, stained PI and stained FITC-PSA (UR). Although different freezing curves did not significantly influence live sperm rate with intact (19.14±0.86% spz curve A, 17.72±0.86% spz curve B and 18.18±0.86% spz curve C) or damaged (0.44±0.04 % spz curve A, 0.38±0.04% spz curve B and 0.49±0.04% spz curve C) acrosome, a significant (P<0.05) effect of animals was observed in the four parameters studied. Regarding the percentage of LL spermatozoa, buck 1 presented values lower (P<0.05) by about 50% than bucks 2 and 3. Data also were affected significantly by the day of collection. These results strongly suggest that although goat sperm cells are not influenced by the pattern of cooling curves investigated, a considerable variability in the response of individual animals as well as the collection date may be expected.
Highlights
The success of mammalian semen cryopreservation depends upon many factors (Purdy, 2006), including interactions among cryoprotectants, type of extender, cooling rate, thawing rate and packaging, as well as variation among individual animals (Cotter et al, 2005)
The post-thawing semen evaluation was performed by flow cytometry with Propidium Iodide (PI) and Pisum Sativum Agglutinin conjugated to Fluorescein Isothiocinate (FITC-PSA), as described by Franco et al (2013), determining the living and dead spermatozoa with intact or damaged acrosome
The mean active sperm ratio was 79.02±5.40% and the sperm viability assessed by flow cytometry was (42.36±5.66% of live epz)
Summary
The success of mammalian semen cryopreservation depends upon many factors (Purdy, 2006), including interactions among cryoprotectants, type of extender, cooling rate, thawing rate and packaging, as well as variation among individual animals (Cotter et al, 2005). These particular characteristics of male goat semen requires an extended attention to maximize the post-thawing sperm viability
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