Post-thaw Plasma Membrane Integrity Research Articles

Studies on Fresh and Cryopreserved Seminal Attributes in Beetal Bucks Outside their Breeding Tract

The objectives of the present research were to assess fresh and cryopreserved. seminal characteristics of breeding Beetal buck semen outside of its breeding tract. Semen was collected twice in a week from twelve Beetal bucks for 6 weeks. The mean value of ejaculate volume(ml), mass motility(o-5 scale), progressive motility(%), sperm viability (%), intact acrosome(%), sperm abnormality(%) and plasma membrane integrity(%) were ranged from 0.73 ± 0.08 ml to 1.17 ± 0.27 ml; 4.17+ to 4.67+, 80.83 ± 1.54 to 87.50 ± 2.14; 83.83 ± 0.95 to 90.33 ± 0.80; 87.00 ± 0.86 to 94.67 ± 0.33; 6.83 ± 0.31 to 10.33 ± 0.67 and 59.33 ± 1.52 to 66.67 ± 1.17, respectively. The mean value of post thaw sperm motility, post thaw sperm viability, post thaw acrosome integrity, post thaw sperm abnormalities and post thaw plasma membrane integrity were ranged from 52.50± 1.12 to 66.67 ± 1.67; 60.00 ± 1.24 to 69.67 ± 1.05; 66.00 ± 0.77 to 78.83 ± 2.15; 9.00 ± 0.45 to 12.83 ± 0.60 and 41.50 ± 1.18 to 53.50 ± 1.23, respectively. It was determined that, even outside of the breeding tract, the foundational characteristics of Beetal bucks are within the range defined for other Indian breeds of goat.

Mitochondria and membrane cryoinjury in micropatterned cells: Effects of cell–cell interactions

The maintenance of cell membrane integrity is an absolute minimum criterion for the selection of a successful cryopreservation process; however, it is often used as the sole determinant of cell “viability”. Membrane stresses and strains that develop with cell volume fluctuations are only one component of the overall cellular response to freezing. Damage to organelles resulting from excessive concentration of intracellular solutes and/or the alternation of molecular signalling events may affect post-thaw outcomes. As the low temperature response of cells is affected by the presence of cell–cell interactions, the cryopreservation of tissues and tissue model systems would benefit from a more detailed understanding of the sites and mechanisms of cryoinjury. The purpose of this study was to examine the relationship between mitochondria and plasma membrane damage in frozen micropatterned cells and to identify the role of cell–cell interactions. Madin Darby Canine Kidney cells (MDCK) were micropatterned using a polydimethylsiloxane (PDMS) elastomeric stamp to create non-adhesive regions of agarose on untreated glass substrates. Five different cell arrangements were used to examine the effect of cell–cell contact: single cells, cell doublets, linear arrangement of cells, randomly arranged cells and confluent monolayers. Cells were cooled in a programmable alcohol bath at 1 °C/min to −40 °C after extracellular ice nucleation at −5 °C. Post-thaw plasma membrane integrity and mitochondria depolarization were determined using trypan blue and the lipophilic, cyanine derivative JC-1, respectively. alamarBlue was used to assess the post-thaw metabolic activity of the cell arrangements. We found that the incidence of plasma membrane damage and mitochondria integrity increased with decreasing temperature and was dependent on the degree of cell–cell interaction. Mitochondria damage was evident in cells that displayed intact plasma membranes, however this injury could be reversed in the micropatterned cells that are exposed to suprazero temperatures. The results from this study suggest that the exclusive use of membrane integrity as a measure of cell “viability” does not consider subcellular injury that may contribute to delayed recovery and/or cell death following low temperature exposures.

Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa

The effect of a prolonged holding time (HT) during cooling on plasma membrane integrity (PMI), motility and in vitro oocyte penetration ability of boar spermatozoa frozen-thawed in different types of package was investigated. Boar semen was frozen in a split-sample design using 3 different HTs (3, 10 and 20 h) during cooling and three different types of freezing package: Maxi-straws, Medium-straws and FlatPacks. Assessment of PMI (SYBR-14 and propidium iodide, fluorescence microscopy) and sperm motility (visually and with CASA) was done during cooling (at 32°C, 15°C, 5°C) and post-thaw (PT). The in vitro oocyte penetration ability of the spermatozoa was tested only PT, using a homologous in vitro penetration assay (hIVP). During cooling the HTs used had no significant (p<0.05) effect on either PMI or percentage of motile spermatozoa. Post-thaw PMI was significantly higher (p<0.05) for 10 h and 20 h HT compared with 3 h, and the percentage of motile spermatozoa decreased significantly with 20 h HT as opposed to 3 h and 10 h. Regarding the freezing packages, the FlatPacks and Maxi-straws yielded significantly more PMI than did the Medium-straws (p<0.05). Post-thaw motility was significantly higher for FlatPacks than for straws, in terms of both percentage motile spermatozoa, and sperm velocity and lateral head displacement (LHD). The hIVP did not show any significant differences among the HTs, although FlatPacks yielded a significantly higher penetration rate and more spermatozoa per penetrated oocyte (p<0.05) than did the straws. Changes in motility patterns, toward a more circular motility during cooling and PT, could be noticed where individual spermatozoa showed a capacitation-like motility pattern. The changes were more obvious with 10-h and 20-h HTs than with 3-h HT.

Post-thaw plasma membrane integrity of bull spermatozoa separated with a sephadex ion-exchange column

Previous experiments have established that filtration of bovine semen through a Sephadex ion-exchange column improves its quality before and after freezing. The present study was conducted to determin the post-thaw membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column and to determine the kind of protection to spermatozoa is provided by glycerol during freezing and thawing. Semen from Holstein bulls diluted in TEST-yolk extender (with and without glycerol) was filtered through a Sephadex ion-exchange column and frozen in liquid nitrogen (−196 °C). After thawing, there were more normal acrosomes in filtered spermatozoa than nonfiltered (P < 0.01). Post-thaw plasma membrane integrity and swelling ability in a hypoosmotic solution revealed that the filtered spermatozoa had a stronger (P < 0.005) plasma membranes than the nonfiltered. Filtered spermatozoa demonstrated higher zona-free hamster oocyte penetration than the nonfiltered (30.5 vs 11.5%; P < 0.0005). Spermatozoa extended in TEST-yolk without glycerol had the lowest (P < 0.001) normal acrosomes, intact plasma membranes and swelling ability. Plasma membrane over the post-acrosomal region of the head and post-midpiece region of the tail was more sensitive to damages caused by freezing and thawing than acrosomal and midpiece regions of spermatozoa. Glycerol in the extender provided significant (P < 0.05) protection to the sensitive regions of filtered and nonfiltered spermatozoa during freezing and thawing. Filtered plus glycerolated spermatozoa had the highest (P < 0.01) normal acrosomes, intact plasma membranes and swelling ability. In conclusion, the pre-freezing filtration of bovine semen harvested the spermatozoa possessing stronger plasma membranes which enabled them to endure freezing and thawing stresses. The addition of glycerol to the extender protected the post-acrosomal region of the head and post-midpiece region of the tail of spermatozoa from freezing and thawing shocks.