Elsevier

Cryobiology

Volume 71, Issue 1, August 2015, Pages 85-90
Cryobiology

Effects of vitrification on ram spermatozoa using free-egg yolk extenders

https://doi.org/10.1016/j.cryobiol.2015.05.004Get rights and content

Abstract

The present study aimed to examine the behavior of ram spermatozoa subjected to a vitrification process in free-egg yolk diluents in relation with conventional diluents and cryopreservation protocol used in this species. Previously it was investigated the toxicity of cryoprotectants, sucrose and glycerol, based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.15 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) compared to a commercial extender (Biladyl® with 20% egg yolk and 7% glyerol). Cryoprotectants which reported less toxicity were chosen to perform the vitrification and results were compared with the conventional cryopreservation. Semen from three rams was collected by electroejaculation. The sperm evaluation was carried out at 0, 2 and 4 h through the incubation time at 37 °C for the experiment of toxicity and, at thawing when cryopreservation was performed. The sperm quality throughout the incubation time always resulted lower (P  0.05) for the free-egg yolk diluents in relation to Biladyl® (control), obtaining the lowest values of sperm quality with the highest concentrations of sucrose and glycerol. The vitrification was carried out with combinations of sucrose and glycerol (sucrose at 0.03 and 0.05 M with 3% and 7% of glycerol, respectively) and with Biladyl® (at different sperm concentrations). The vitrification decreased drastically (P  0.05) the sperm quality when combinations of sucrose and glycerol were used. Nevertheless, the sperm samples vitrified with Biladyl® at the lowest sperm concentration showed acceptable values of viability, acrosome integrity and DFI, although the sperm motility was strongly decreased. In conclusion, the use of vitrification with diluents based on combinations of sucrose and glycerol did not work for semen cryopreservation of ram. Promising results were obtained when diluents with egg yolk were used in the vitrification procedure, although more studies are necessary to improve this technique and the use of diluents without egg yolk.

Introduction

The conventional cryopreservation of spermatozoa is used in small ruminants as part of breeding or conservation programs. It is known that this technique, which involves cooling to 5 °C, equilibration at this temperature and freezing using vapors of liquid nitrogen, causes important chemical–physical damage to the intracellular structures originated by changes in the osmotic balance and temperatures during the different steps. Thus, ice crystals are formed and as consequence, sperm membranes are affected, an increase of lipid peroxidation is produced, sperm motility rates and mitochondrial activity are decreased and processes associated with cell death are induced [31]. Cryoprotective agents which permeate the cell membrane has been routinely used to increase membrane fluidity and partially dehydrating the cell, lowering the freezing point, and thus reducing the number and size of intracellular ice crystals formed. Nevertheless, these cryoprotectants themselves can have a toxic effect on spermatozoa being this effect related to the concentration used and the time of cell exposure [39]. In addition, egg yolk has been usually included as non-permeable cryoprotector into sperm freezing extenders. The main disadvantage is that egg yolk is a non-defined substance which leads to variability between batches. Moreover, it has an animal origin and it could be a way to transmit diseases.

During the last years, several studies have been carried out in order to avoid the use of cryoprotectans with animal origin. Thus, extenders based on soybean or extenders containing sugars with high molecular weight combined with permeable cryoprotectants such as glycerol has been studied for conventional sperm freezing in different species [2], [6], [16], [17], [18], [27], [34]. Moreover, some investigations have been aimed to evaluate the effect of using permeable cryoprotectants [36], sucrose [37], or even a cryoprotectants-free technique joined to other preservation methods such as vitrification [11], [13], [36].

The vitrification process has been proposed as a novel technique widely used for embryo storage, but it has not been applied to routine sperm cryopreservation due to deleterious osmotic effect of high concentration of permeable cryoprotectans. This technique is based on the ultra-rapid freezing of the cell by direct immersion in liquid nitrogen. The main advantage of this method is to prevent the formation of ice crystals and the possibility to use free-egg yolk extenders. Also, the procedure is faster, simpler in the application and, more cost effective than the conventional cryopreservation. As disadvantage, vitrification needs a high concentration of cryoprotectant agents, and sperm cells are very sensitive to these agents [10]. Another limitation of this methodology is that so far, vitrification has been performed using small volumes and open systems which not prevent direct contact with the liquid nitrogen [10], [31]. Nevertheless, in recent studies in human, the spermatozoa have been vitrified in large volume in straws obtaining good results [15], [38].

Taking into account the lack of knowledge about optimum extenders to vitrify ram spermatozoa and about the impact of sperm vitrification in this species, the present study was designed to investigate: (1) the toxicity of sucrose and glycerol based on different concentrations (sucrose at 0.03 M, 0.05 M, 0.015 M and 0.25 M; and glycerol at 3%, 7%, 14% and 18%) under incubation conditions; and (2) the effect of the vitrification using the best combination of the previous cryoprotectans. The effects of these factors were assessed in terms of sperm quality through the incubation or at thawing, depending on objectives.

Section snippets

Experimental design

Fig. 1 shows the experimental design followed in the present study, including the main experiments.

Experiment 1 (Fig. 1A) was carried out to evaluate the effects that different concentrations of sucrose and glycerol (sucrose: 0.03, 0.05, 0.15 and 0.25 M; glycerol: 3%, 7%, 14% and 18%) added to a SOF-based media have on sperm quality after 0, 2 and 4 h of incubation at 37 °C. The values of sperm quality obtained from the use of these extenders were compared with that obtained from the use of the

Experiment 1: Effect of cytotoxicity of sucrose and glycerol at different concentrations on sperm quality after incubation period at 37 °C

Samples extended in Biladyl® showed higher sperm quality (P  0.05) than the other treatments evaluated including the control using only SOF (Fig. 2A and B; Tables 1 and 2 in Supplementary material). There was no effect of the dilution (P > 0.05), showing B50 and B5 similar values of sperm quality.

Adding sucrose or glycerol had a significant negative effect (P  0.05) on sperm quality for all sperm parameters assessed, except for DFI which was unaffected. Moreover, higher sucrose or glycerol

Discussion

Up to date, sperm cryopreservation in small ruminants has been performed by the conventional technique which involves three phases: cooling to 5 °C, equilibration at this temperature for a variable time and freezing on liquid nitrogen vapors. For this, extenders based on different concentrations of egg yolk and glycerol have been widely used. Conventional techniques have been well investigated and it has been shown that intra- or extra-cellular ice crystal formation and osmotic changes may occur

Acknowledgments

We want to acknowledge Jose María Luján for his help during sperm samples collection. M Ramón is supported by the DOC-INIA program.

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    Statement of funding: This work has been funded by the Castilla-La Mancha University (Gl20152913).

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