Post-thaw Sperm Plasma Membrane Integrity Research Articles

Effect of different egg yolk sources on dog semen quality following cryopreservation.

The aim of this study was to compare the cryoprotective effects of egg yolk from different avian species (hen, goose and quail) on post-thaw quality of dog semen. Total motility (TMOT) and progressive motility (PMOT) of frozen-thawed spermatozoa were not significantly differed among the extenders, but were higher in the quail-egg yolk based extender compared with extender containing hen or goose egg yolk. It was found that post-thaw sperm motion parameters, velocity VCL and ALH, were significantly higher in the quail-egg yolk based extender. No marked differences in post-thaw sperm plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) were observed among the extenders. In conclusion, the results of the present study suggest that goose or quail egg yolk is a suitable alternative to hen egg yolk for the cryopreservation of dog semen.

Cryopreservation of strip spawned sperm using programmable freezing technique in the blue mussel Mytilus galloprovincialis

In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, Mytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thawing temperature, sugar addition and sperm to oocyte ratio. The sperm quality was assessed by the fertilization rate or the integrity of sperm component and organelle. The highest post-thaw sperm fertilization rate was 91%, which was produced with sperm cryopreserved in 8% dimethyl sulfoxide at the cooling rate of -4°C/min from 2°C to -30°C before being plunged into liquid nitrogen for at least 12 h, thawed in a 20°C seawater bath and fertilized at sperm to egg ratio of 50 000:1. The addition of glucose, sucrose or trehalose to 8% dimethyl sulfoxide could not further improve fertilization rates. The fluorescent assessments showed that the post-thaw sperm plasma membrane integrity and acrosome integrity were significantly damaged in comparison with fresh sperm.

Improvement in non-programmable sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata

This study assessed the effects of the addition of vitamins (l-ascorbic acid), amino acids (glycine and taurine), and monosaccharides (glucose, fructose and galactose) on sperm cryopreservation using a non-programmable freezing technique in farmed greenlip abalone. The results showed that the addition of taurine, glycine or l-ascorbic acid significantly improved the post-thaw sperm motility, whereas the post-thaw sperm fertilization rates were improved by the addition of glycine or l-ascorbic acid. Flow cytometry analysis demonstrated that the addition of glycine significantly enhanced the post-thaw sperm plasma membrane integrity and acrosome integrity. Results from the investigation on monosaccharides demonstrated that glucose, fructose and galactose had a similar cryoprotective effect, resulting in a similar level of the post-thaw sperm fertilization rate, plasma membrane integrity, mitochondrial membrane potential and acrosome integrity. In this study, the highest post-thaw fertilization rate of 96% was achieved by using the cryoprotective mediums containing 6% DMSO, 1% glucose and 0.6% glycine.

Comparative viability of bovine sperm frozen on a cryomicroscope or in straws

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spermatozoa on the cryomicroscope. Semen was diluted in Test-yolk (20%) extender containing 7% glycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitrogen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicroscope. Thawed samples were diluted with Hepes buffered medium containing 0.1% bovine serum albumin (BSA) and stained with either carboxymethylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with propidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (stained with CMFDA or SYBR-14), which were classified as plasma membrane-intact and 2) spermatozoa with intense red fluorescence, (stained with PI), which were classified as plasma membrane-damaged. Samples frozen using the cryomicroscope contained 29 and 26 % plasma membrane-intact (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. Cryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane- intact sperm cells, while spermatozoa frozen in 0.25-ml straws resulted in 34 and 31% PMI sperm cells for CMFDA and SYBR-14, respectively. No significant difference was observed (P > 0.05) for PMI spermatozoa stained with either CMFDA or SYBR-14. In addition, the ability to recover spermatozoa after freezing on the cryomicroscope establishes the Linkam BCS 196 as a useful tool for the study of sperm cell cryopreservation.