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