Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post‐thaw sperm quality in stallions

Abstract

Although glycerol is the cryoprotectant most commonly used in stallions, it has also a considerable toxicity for equine sperm. It was the aim of this study to analyse the quality of frozen-thawed stallion semen after complete or partial replacement of glycerol in the freezing extender by alternative cryoprotectants. We hypothesized that partial or total replacement of glycerol by cryoprotectants occurring in cold-resistant frog, insect or plant species results in similar or better semen quality after freezing-thawing. As basic medium, the commercial Ghent basic extender was used and either supplemented with glucose and urea, trehalose and proline, or trehalose and betaine. Based on a series of preliminary experiments, semen was frozen in either commercial Ghent cryopreservation extender (Ghent control), Ghent glucose-urea extender or a Ghent combined extender (glucose-urea, trehalose-betaine and trehalose-proline; volume ratio of 2:1:2) in a computer-controlled rate freezer. After freezing-thawing, semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential and chromatin condensation. No differences between Ghent control and Ghent glucose-urea extender were seen, while all endpoints except DNA integrity were negatively affected in Ghent combined extender (e.g., progressive motility: Ghent 49.2 ± 3.7, Ghent glucose-urea 46.5 ± 4.6, Ghent combined 24.4 ± 2.8%; p < .001). In conclusion, glycerol concentration in a commercial freezing extender for equine spermatozoa can be successfully reduced when urea as an additive cryoprotectant is added and the glucose concentration is elevated. However, total glycerol replacement with urea, betaine, proline and trehalose was less successful.

Donkey semen cryopreservation: Alternatives with permeable, non‐permeable cryoprotectants and seminal plasma

“…Similarly, Diaz‐Jimenez et al (2019) found that sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm. In the same way, De Oliveira, Budik, et al (2017) reported that the complete removal of glycerol and replacement by a combination of urea, proline and betaine in freezing extender for stallions, resulted in lower post‐thaw semen quality than Gent control, a commercial extender (Minitube, glycerol concentration 5%).Yet, they demonstrate that equine semen can be successfully frozen after reduction of glycerol content to a final concentration of 1.25% when compensated by addition of two other alternative cryoprotectants namely glucose and urea (De Oliveira, Teixeira, et al, 2017).…”

Comparison of the characteristics of chinchilla epidydimal semen after collection, storage at 5°C and cryopreservation

“…Contrary, chilling allowed for preservation of membrane and acrosome integrity, but not of mitochondrial potential. In other species, a gradual loss of mitochondrial activity was observed during cold storage (De Oliveira, Budik, & Aurich, ; Kadirvel, Kumar, & Kumaresan, ). Although a number of reports indicate that sperm mitochondria are sensitive to damage during freezing and thawing (Kadirvel et al., ; Peña, Johannisson, Wallgren, & Rodriguez Martinez, ; Prochowska et al., ) in this study loss of mitochondrial potential after cryopreservation was small and not significant.…”

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

“…Immediately after collection, semen was diluted 1:1 in skim milk‐based antibiotic‐free extender (EquiPro Minitub Ibérica, Spain), and subjective motility was assessed by examining 10 μl of semen placed in a pre‐warmed (37°C) slide covered by a coverslip in a light microscope with a built‐in stage warmer, at ×200 magnification. After these evaluations, the ejaculate was split into two fractions to be frozen in two different extenders—Gent (Minitub Ibérica, Spain), a 5% glycerol‐based extender (De Oliveira et al., ) or BotuCrio ® (Nidacon, Sweden), a 4% methylformamide and 1% glycerol‐based extender (Cordova et al., ), using two different freezing methods for each extender—cryopreservation in liquid nitrogen vapours inside a Styrofoam box or in a programmable freezer (Figure ). After thawing, in vitro evaluation of several parameters of membrane integrity, mitochondria potential, and semen kinetic was carried out.…”

“…After these evaluations, the ejaculate was split into two fractions to be frozen in two different extenders-Gent (Minitub Ibérica, Spain), a 5% glycerol-based extender (De Oliveira et al, 2017) or BotuCrio ® (Nidacon, Sweden), a 4% methylformamide and 1% glycerol-based extender (Cordova et al, 2014), using two different freezing methods for each extender-cryopreservation in liquid nitrogen vapours inside a Styrofoam box or in a programmable freezer ( Figure 1). After thawing, in vitro evaluation of several parameters of membrane integrity, mitochondria potential, and semen kinetic was carried out.…”

“…Sperm motility, membrane integrity, mitochondrial membrane potential and viability decrease after freezing‐thawing due, in part, to the damage of sperm plasma and mitochondrial membranes produced by osmotic shock (De Oliveira, Budik, & Aurich, ). The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box using liquid nitrogen vapour or in a programmable freezer, utilizing a 5% glycerol‐based extender (Gent) (De Oliveira et al., ) and (BotuCrio ® ), an extender that combines methylformamide (4%) and glycerol in a low (1%) concentration (Cordova et al., ). Post‐thaw spermatozoa kinetic was evaluated using a computer‐assisted sperm analyser system (CASA), and semen viability and mitochondria potential were assessed through flow cytometric assays.…”