Abstract
The aim of this study was to assess post-thaw sperm quality following initial sperm selection using density gradient centrifugation (DGC) prior to cryopreservation. Ejaculates from four mature Charolais cross Kedah-Kelantan bulls were collected using artificial vagina at IBVK Pahang, Malaysia. The ejaculates were aliquoted into 3 groups: non-cryopreserved group (NC); control group of cryopreserved sperm without DGC (ND) and treatment group of sperm undergoing DGC sperm selection before cryopreservation (CDGC). Prior to analysis, samples from both cryopreserved groups were thawed at 37 °C for 30 sec. All samples were analysed for kinematics parameters, viability and compromise in DNA integrity (evaluated as DNA Fragmentation Index, DFI). All kinematics parameters were analysed using computer aided sperm analysis (CASA). Results indicated significant (p < 0.05) kinematics parameter changes for all parameters of velocity (VCL, VSL, VAP) and progression (WOB, LIN, ALH and BCF). Unfortunately, changes in spermatozoa straightness were insignificant (STR) F(2, 68) = 1.004, p = 0.371. Spermatozoa viability had increased by 26.2% (p < 0.01) following the treatment. DFI revealed the treatment group recorded a significant reduction in DFI value (0.17% fragmented DNA). In conclusion, DGC sperm selection prior to cryopreservation reduced the effects of cryodamage and showed an improvement in post-thaw sperm quality, thus reducing the occurrence of asthenozoospermia in populations of frozen-thawed cross-bred bovine spermatozoa.
Highlights
Previous accounts of shortened lifespan in frozen-thawed sperm samples have been reported across many species (Cerolini et al, 2001; Katila, 2001; O’Connell et al, 2002; Breininger et al, 2005; Knox et al, 2015; Mari et al, 2015) and have affected the outcome of breeding programs utilising these samples.The induced cryodamage following cryopreservative procedures have known to render sperm immotile with increased DNA fragmentation index (DFI)
The percentage of retrieved motile sperm cells after cryopreservation remained constant with the inclusion of density gradient centrifugation (DGC)
This was proven from motility analysis using computer aided sperm analysis (CASA) that showed significant increase in overall motility rates, motility patterns, and viability rates following cryopreservation (Table 1)
Summary
Previous accounts of shortened lifespan in frozen-thawed sperm samples have been reported across many species (Cerolini et al, 2001; Katila, 2001; O’Connell et al, 2002; Breininger et al, 2005; Knox et al, 2015; Mari et al, 2015) and have affected the outcome of breeding programs utilising these samples.The induced cryodamage following cryopreservative procedures have known to render sperm immotile with increased DNA fragmentation index (DFI). Previous accounts of shortened lifespan in frozen-thawed sperm samples have been reported across many species (Cerolini et al, 2001; Katila, 2001; O’Connell et al, 2002; Breininger et al, 2005; Knox et al, 2015; Mari et al, 2015) and have affected the outcome of breeding programs utilising these samples. Findings suggest that compromise in the quality of cryopreserved sperm is likely due to synergistic effect of environmental and cyrobiological factors (Bailey et al, 2000). Previous studies have established that using sperm from heterogeneous populations for cryopreservation would later result in deteriorated post-thaw values for sperm kinematics and viability (Verstegen et al, 2002; Murase et al, 2010). In order to obtain a homogenous population from post-thaw sperm samples, it is advisable for a sperm selection method to be included as part of the cryopreservation routine. Several methods of selection are available such as swim-up procedure, migration-sedimentation technique, glass wool filtration and density gradient centrifugation (DGC)
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