Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation

https://doi.org/10.1016/j.anireprosci.2012.06.010Get rights and content

Abstract

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose–egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37 °C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P < 0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing–thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P < 0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition.

Introduction

Recently it has achieved great advances in the use of frozen–thawed semen in artificial insemination of swine (Okazaki et al., 2009, Casas et al., 2010, García et al., 2010). But there are still many weaknesses in the sperm cryopreservation process that should be improved before frozen–thawed boar semen can be applied with the same efficiency of liquid semen in routine AI programmes in commercial pig farms. One important improvement should be adjustments in the composition of the freezing extenders. The basic ingredients of current sperm freezing extenders are the same as those used 35 years ago, including the cryoprotectant agents glycerol, egg yolk and sugars (Johnson et al., 2000, Barbas and Mascarenhas, 2009).

The beneficial effect that sugars have on boar sperm freezing has been reported in several works (Pursel et al., 1972, Yi et al., 2002, De Los Reyes et al., 2002, Corcuera et al., 2007, Malo et al., 2010). Nevertheless, the cryoprotective effect of these compounds is not fully defined. Sugars are not able to diffuse across the plasma membrane, creating an osmotic pressure that induces cell dehydration and a lower incidence of intracellular ice formation (Fuller, 2004), and some authors declare that the cryoprotective effects of the sugars on sperm cells may differ according to the molecular weight of the sugars (Anchordoguy et al., 1987, Molinia et al., 1994), and the sugars having higher molecular weights exhibited a greater cryoprotective effect (Pursel et al., 1972).

Nowadays lactose is the sugar most commonly used for boar sperm cryopreservation, due to the good results it provides (Roca et al., 2004, Fraser et al., 2007, Sancho et al., 2007, De Mercado et al., 2010, Buranaamnuay et al., 2011). However, recent studies showed that the sugars, like trehalose, with the same molecular weight than lactose but with different composition in monosaccharides, had different protective effect against the freezing process (Hu et al., 2008, Hu et al., 2009, Gutiérrez-Pérez et al., 2009, Malo et al., 2010). These results suggest that the molecular weight, the composition or structure of these sugars could influence their cryoprotectant ability.

Bearing these results in mind, the objective of this study was to compare the cryoprotective effects of different sugars on boar sperm, according to their composition.

Section snippets

Reagents and media

All chemicals were of analytical grade. Unless otherwise stated, all media components were purchased from Sigma Chemical Co. (St. Louis, MO, USA), and were made with purified water (18  cm; Automatic GR Wasserlab, Spain). Orvus ES Paste (OEP) is marketed as Equex STM (Nova Chemical Sales Inc., Scituate, MA, USA).

The basic medium used for semen extension was Beltsville Thawing Solution (BTS, composed of 205 mM glucose, 20.39 mM NaCl, 5.4 mM KCl, 15.01 mM NaHCO3, and 3.35 mM EDTA; pH 7.2 and 310 ± 5 

Experiment 1: Determining the cryoprotective effect of different monosaccharides

The results of this experiment show that the control (lactose) is the sugar which has higher post-thawing quality. Between monosaccharides results obtained for most of the parameters were very similar (Table 1, Table 2), but the egg yolk–glucose extender provided higher percentages of total motile sperm 30 min after thawing (P > 0.05) than the galactose or fructose extenders (Table 2). In addition, glucose was the only monosaccharide that matches in some of the results (plasma membrane integrity

Discussion

In this work is possible to observe that glucose was the monosaccharide providing higher cryoprotective effect. Thus, although the sperm quality of the samples frozen with glucose extender was lower than the quality obtained with the lactose extender, in many parameters did not differ significantly between both extenders. Indeed, glucose is used as a cryoprotectant in some boar freezing extenders (De Los Reyes et al., 2002, De Mercado et al., 2009). But even so, results of the experiment 1 may

Acknowledgments

This research was made possible by the financial support of project 2010-1282 Plan de Investigación del ITACyL. C. Tomás was supported by a scholarship from Ministerio de Ciencia e Innovación (Ref. BES-2007-17036; Madrid, Spain), and E. Mocé was supported by funds from Ministerio de Ciencia e Innovación from Subprograma Ramón y Cajal and the European Social Fund (Ref. RYC-2010-06162; Madrid, Spain).

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