Effect of sperm and cryoprotectant concentration on equine semen freezability

Pucci, B.; Papa, Federico; Sanches, F.M.; Nobrega, J.N.; Crespilho, André Maciel; Pasquini, D.F.; Campos, Jorge; Melo, Cely Marini; Dell’Aqua, C.P.F.; Alvarenga, Marco Antônio; Dell’Aqua, J.A.

doi:10.1016/j.anireprosci.2008.05.119

Cited by 4 publications

(2 citation statements)

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“…Specifically in poor freezing stallions, the use of ButoCrio containing a combination of glycerol and methylformamide provided better post thaw motility as opposed to semen frozen with glycerol only [ 33 ]. Similarly to our study, shorter cooling times of 20–30 minutes to 5–8°C prior to freezing showed significantly improved post thaw sperm quality and better fertility in stallions with poor freezing spermatozoa [ 33 , 34 ].…”

Section: Discussionsupporting

confidence: 83%

Cryopreservation in rhinoceros—Setting a new benchmark for sperm cryosurvival

2018

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At times when rhinoceros are fiercely poached, when some rhinoceros species are closer than ever to extinction, and when the scientific community is in debate over the use of advanced cell technologies as a remaining resort it is time to simplify and improve existing assisted reproduction techniques to enhance breeding and genetic diversity in the living populations under our care. Semen cryopreservation has been performed in all captive rhinoceros species with limited degree of success. Here we tested three freezing extenders, containing different cryoprotectants and various freezing rates for the cryopreservation of rhinoceros sperm from 14 bulls. In experiment I, semen from 9 bulls was used to determine the most suitable diluent, cryoprotectant and freezing rate for the successful cryopreservation of rhinoceros sperm. In experiment II, semen from 5 bulls was used to assess whether the removal of seminal plasma could further improve post thaw sperm quality following cryopreservation with conditions identified in Experiment I. Semen was diluted with Berliner Cryomedia, ButoCrio® or INRA Freeze®, packaged in 0.5 mL straws and frozen 3, 4, and 5 cm over liquid nitrogen (LN) vapour or directly in a dryshipper. It was found that semen extended with ButoCrio® (containing glycerol and methylformamide) and frozen 3cm over LN vapour provided the best protection to rhinoceros spermatozoa during cryopreservation. When pooled over treatments, total and progressive post thaw motility was 75.3 ± 4.2% and 68.5 ± 5.7%, respectively marking a new benchmark for the cryopreservation of rhinoceros sperm. Post thaw total and progressive motility, viability and acrosome integrity of semen diluted in ButoCrio® was significantly higher than semen extended in Berliner Cryomedia or INRA Freeze®. The removal of seminal plasma did not improve post thaw sperm survival (p > 0.05). In conclusion, the cryosurvival of rhinoceros spermatozoa was significantly improved when using a mixture of glycerol and methylformamide in combination with a fast freezing rate at 3 cm. These results describe a new protocol for the improved cryosurvival of rhinoceros spermatozoa and will enable a more successful preservation of genetic diversity between males, especially in donors whose spermatozoa may already be compromised prior to or during collection. The successful reduction of glycerol concentration in favour of methylformamide as a cryoprotectant could be a novel suggestion for the improvement of cryopreservation techniques in other wildlife species.

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Section: Discussionsupporting

confidence: 83%

Cryopreservation in rhinoceros—Setting a new benchmark for sperm cryosurvival

2018

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“…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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confidence: 99%

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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