Conversely, PI stained only nonmotile sperm that had lost their membrane integrity. Microscopic examination revealed that SYBR-14 stained the nuclei of living sperm bright green as determined by simultaneous examination of fluorescence and motility. ![]() SYBR-14, a newly developed fluorescent nucleic acid stain, maximally absorbs at 488 nm and emits at 518 nm when bound to DNA. The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using the stains SYBR-14 and propidium iodide (PI). The results of this study indicated that components of the long-term extenders have different effects on the sperm functionality and prolonged semen longevity by delaying the processes associated with sperm ageing during liquid storage. Furthermore, the percentage of spermatozoa with NAR acrosomes decreased during prolonged storage, being markedly lower in DC-diluted semen compared with semen diluted with either AeG or SCP extender. There were not any marked differences in sperm ATP content between the extenders, regardless of the storage time. Marked differences in the proportions of spermatozoa with high MMP were observed between the extenders, particularly on Day 10 of storage. Among the four analyzed extenders, AeG and SCP showed the best performance in terms of TMOT and PMI on Days 5, 7 and 10 of storage. In all extenders the metabolic activity and membrane integrity of the stored spermatozoa decreased continuously over time. Furthermore, the storage time had a significant effect (P < 0.05) on the sperm metabolic activity and membrane integrity during semen storage. Extender type was a significant (P < 0.05) source of variation in all the analyzed sperm parameters, except for ATP content. Parameters of the analyzed sperm metabolic activity included total motility (TMOT), progressive motility (PMOT), high mitochondrial membrane potential (MMP) and ATP content, whereas those of the membrane integrity included plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome. Boar semen was diluted with Androhep EnduraGuard (AeG), DILU-Cell (DC), SafeCell Plus (SCP) and Vitasem LD (VLD) extenders and stored for 10 days at 17 degrees C. This study was aimed to analyze the metabolic activity and membrane integrity of boar spermatozoa following storage in long-term semen extenders. It can be suggested that the type of extender and storage temperature, used prior to freezing, have a significant effect on the sperm cryo-tolerance. The results of this study indicated that protection against cryo-induced sperm damage was most prominent in the AHP-extended semen. Significantly higher (P < 0.05) percentages of membrane-intact frozen-thawed spermatozoa were observed in both the AHP and ASP extenders compared with those stored in the TCP extender. Furthermore, samples stored in AHP extender at 17 ☌ or 10 ☌, prior to freezing, exhibited significantly higher (P 0.05) were observed in ALH values among the extenders, regardless of the storage period. There were no marked significant differences (P > 0.05) either in the sperm motion parameters or the percentage of spermatozoa with intact membrane between samples stored at 17 ☌ and 10 ☌ prior to freezing, irrespective of the extender type. ANOVA results showed that differences in extender type and storage temperature (extender type × storage period) did not have any significant effect (P > 0.05) on the analyzed sperm parameters following freezing-thawing. ![]() Sperm plasma membrane integrity was assessed by dual staining with SYBR-14 and propidium iodide (SYBR-14/PI assay). Sperm motion parameters, analysed by the computer-assisted sperm analysis (CASA) system (HTR-IVOS 12.3, Hamilton Thorne Biosciences), included total motility (TMOT), progressive motility (PMOT), velocity average pathway (VAP), velocity straight line (VSL), velocity curvilinear (VCL) and mean amplitude of lateral head displacement (ALH). After each storage period the extended semen was subjected to quality assessment and then frozen in an extender consisting of 5% lyophilized lipoprotein fractions of ostrich egg yolk (LPFo), 11% lactose, 3% glycerol and 0.5% Equex STM. The extended semen was stored for 2h at 17 ☌ and then for 24h at 10 ☌ prior to freezing. ![]() Sperm rich fractions, collected from 3 boars, were diluted in 4 extenders: Androhep® Plus (AHP), Androstar® Plus, ASP (Minitübe, Germany), Safecell® Plus, SCP (IMV Technologies, France) and TRIXcell® Plus, TCP (IMV Technologies, France). This study investigated the effects of cooling of boar semen in different extenders at 17 ☌ or 10 ☌ on motion parameters and plasma membrane integrity of spermatozoa following freezing-thawing. The success of boar semen cryopreservation is dependent on many factors, such as the type of extender and storage temperature used prior to freezing.
0 Comments
Leave a Reply. |