Effects of freezing extenders and cryoprotectants on sperm motility and viability post-thaw

Ferreira, Heder Nunes; Johnson, Amy E.; Silva, M.A. Coutinho da

doi:10.1016/j.anireprosci.2008.05.097

Cited by 2 publications

(4 citation statements)

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“…There are a number of works dealing with the search for alternative cryoprotectants not only for stallion ejaculate, but also for the cryopreservation of sperm in other animal species and humans (Futino et al 2010;Swelum et al 2011). Investigated substances are specifically compounds of glycol: ethylene glycol, propylene glycol, diethylene glycol, dimethyl sulphoxide (Chenier et al 1998;Moore et al 2006;Hoffmann et al 2010) or various amides: formamide, methylformamide, dimethylformamide, acetamide or methylacetamide (Gomes et al 2002;Vidament et al 2002;Squires et al 2004;Alvarenga et al 2005;Ferreira et al 2008). Some of these alternative cryoprotectives are combined with glycerol and varying concentrations of each cryoprotective both individually and in combination have been described (Vidament et al 2002;Melo et al 2007;Pucci et al 2008;Hoffmann et al 2011;Alvarez et al 2014;Wu et al 2015;Moff ett et al 2016).…”

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Effects of glycerol concentration on the motility of equine spermatozoa after thawing

et al. 2017

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The aim of this study was to evaluate the effect of different glycerol concentrations on stallion sperm motility after thawing. For statistical analysis 228 ejaculates were used. The semen was filtrated to remove gel fraction; macroscopic and microscopic evaluation was done. After evaluation the ejaculates were centrifuged, the supernatant was removed and the spermatozoa were re-suspended in French diluent with different concentrations of glycerol (2.0; 2.5; 4.0 and 6.0%). The choice of concentration of glycerol for a particular ejaculate was completely random. The spermatozoa were packed into 0.5 ml straws and placed for 2 h in a fridge (4 °C). Then the straws were placed in liquid nitrogen vapor (-80 to -100 °C) and after 10 min plunged into liquid nitrogen and stored at -196 °C for at least 48 h. The selected straws were individually thawed in a 38 °C water bath for 30 s prior to post-freezing analysis. Two progressive motilities using phase contrast microscopy (magnification × 400) were recorded: motility II immediately after thawing and motility III after 2 h incubation in a 38 °C water bath. The Spearmen/Kendall rang correlation test was selected to prove whether there is a correlation between the selected indices (glycerol concentration and motility II and motility III). Nonparametric multiple group analysis (Steel-Dwass test) was applied for finding the differences between groups. The Spearman/Kendall rang correlation proved a relationship between motility II and glycerol concentration. It can be stated that in this study the best glycerol concentration for freezing equine spermatozoa is with a concentration of 4.0% glycerol.

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Effects of glycerol concentration on the motility of equine spermatozoa after thawing

et al. 2017

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“…It was initially hypothesized that Botu-Crio ® would be more efficient, based on its composition that includes cryoprotectants of amine groups, thus displaying lower molecular weight, which allows faster penetrance on sperm cells during cryopreservation and ultimately leads to greater sperm membrane protection. According to Ferreira and co-workers [12], the Botu-Crio ® is composed of intracellular cryoprotectants, such as the amine derived ones, and may be associated with low glycerol concentrations, probably by using 4% methylformamide and 1% glycerol. Therefore, it differs sharply from other diluents that are formulated with 3% glycerol or more, and that should be avoided for cryopreservation of semen from LFA stallions.…”

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confidence: 99%

“…Semen cryotolerance in horses depends upon genetic factors and cryopreservation protocol [2,38], such as diluent and cryoprotectant choice [2,28]. For these reasons, the effect of individual stallions has been extensively investigated throughout the years, aiming to identify factors associated to semen cryotolerance in horses, especially for stallions classified as holding low semen freezability potential [12,15,18,32,33,39].…”

Section: Introductionmentioning

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“…Diluent and cryoprotectant choice for horse semen cryopreservation is under intense investigation [12,18,30,38], aiming to reduce toxicity and osmotic stress during cryopreservation [12,14,26]. Several parameters are used to measure horse semen cryotolerance, such as sperm motility [1,2], sperm membrane integrity and acrosome and mitochondria viability [8,30], zona pellucida binding assays [27,36], and incidence of DNA fragmentation [20,32].…”

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confidence: 99%

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Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

Ferreira

Ferreira-Silva

Rocha

et al. 2019

Acta Scientiae. Vet.

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Background: fertility rates using horse frozen-thawed semen remain lower than in other livestock species. This fact further suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is a substantial influence of genetic factors and diluent choice upon horse cryopreservation outcome. Collectively, these genetic and technical properties of horse semen could be explored to identify factors or conditions that may increase semen viability after freeze-thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: frozen-thawed semen was evaluated for motility, membrane integrity, and sperm DNA fragmentation using the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h. Discussion: frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Sperm DNA fragmentation was quite low for both groups, as described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions, since it is caused by multiple origins such as genetic factors. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability.

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Effects of freezing extenders and cryoprotectants on sperm motility and viability post-thaw

Cited by 2 publications

References 1 publication

Effects of glycerol concentration on the motility of equine spermatozoa after thawing

Effects of glycerol concentration on the motility of equine spermatozoa after thawing

Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

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