Elsevier

Theriogenology

Volume 79, Issue 3, February 2013, Pages 508-516
Theriogenology

Research article
Cryopreservation of rabbit semen: Comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification

https://doi.org/10.1016/j.theriogenology.2012.11.008Get rights and content

Abstract

This study was designed to improve current freezing protocols for rabbit sperm by examining: (1) the toxicity of different permeable cryoprotectants (CPAs) used for standard vapor freezing (conventional freezing); (2) the feasibility of ultrarapid nonequilibrium freezing (vitrification) of sperm in the absence of permeating CPAs; and (3), the addition of bovine serum albumin (BSA), alone or with sucrose or trehalose as osmoprotectants. First, we evaluated the effects on sperm motility of the incubation time (5 to 60 minutes) with different final concentrations (5% to 20%) of glycerol, N-N-dimethylacetamide, dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, and methanol. N-N-dimethylacetamide (5%) and DMSO (5% and 10%) showed the least toxic effects; the use of 10% DMSO producing the best postthaw sperm motility and membrane integrity results (P < 0.05) after conventional freezing. For vitrification, semen was diluted in the absence of permeable CPAs and frozen by dropping semen directly in liquid nitrogen. However, this led to the low or null cryosurvival of sperm postvitrification (0.16 ± 0.4%, 1.8 ± 1.6%, and 94.5 ± 1.4% of motile, membrane-, and DNA-intact sperm cells, respectively). To assess the effects of albumin and osmoprotectants on sperm cryosurvival, sperm was conventionally frozen with 10% DMSO or vitrified in the absence of permeable CPAs without or with 0.5% BSA alone or combined with sucrose or trehalose (range, 0–0.25 M). In the conventional freezing procedure, the addition of BSA alone failed to improve sperm cryosurvival, however, in the presence of BSA plus either sucrose or trehalose, the postthaw motility (using 0.1 M sucrose or trehalose) and DNA integrity (using all additive concentrations) of sperm were significantly better (P < 0.05) than control. Higher numbers of motile and membrane-intact cells were observed when semen was vitrified with BSA alone or with BSA and sucrose (0.1 and 0.25 M) or BSA and trehalose (0.25 M) and a best recovery of DNA-intact sperm was recorded for BSA plus sucrose compared with semen vitrified without osmoprotectants (P < 0.05). Finally, the cryodiluent combinations BSA/sucrose and BSA/trehalose were compared in an insemination trial. Rabbit does were inseminated with fresh semen (N = 56), semen conventionally cryopreserved in the BSA-based cryodiluents containing 0.1 M sucrose or trehalose (N = 56 per group), or semen vitrified in the presence of 0.25 M sucrose or trehalose (N = 8 per group). Fertility rates and live born kids were similar for semen cryopreserved with BSA/sucrose (77% and 7.6) compared with fresh semen (84% and 8.1) and significantly higher than the figures recorded for the conventionally frozen semen in the BSA/trehalose group (52% and 6.1; P ≤ 0.05). In contrast, only one doe inseminated with semen vitrified in the presence of BSA/sucrose became pregnant, though no kids were delivered. The conclusions to be drawn from our study are: (1) incubation times and concentration toxicities established for the main permeable CPAs used for conventional freezing of rabbit sperm indicated that DMSO 10% was the least damaging; (2) CPA-free vitrification of rabbit semen led to a low or null sperm cryosurvival; and (3) enriching the freezing medium with BSA plus adequate amounts of sucrose or trehalose can improve the cryosurvival of rabbit sperm after conventional freezing or vitrification. In our working conditions, BSA/sucrose was more effective than BSA/trehalose at preserving the in vivo fertilization capacity of rabbit sperm cryopreserved using the standard procedure.

Introduction

The low survival of sperm after freezing is a major drawback for the widespread use of frozen semen in artificial insemination programs for livestock animals such as the rabbit, in which sperm cryopreservation has only been used for experimental purposes [1]. During cryopreservation, sperm cells undergo stress such as changes in the osmotic balance and temperatures during cooling, freezing, and rewarming. These changes lead to ice crystal formation, which is among the main biophysical factors which cause sperm death [2]. Cryoprotective agents (CPAs) that permeate the cell membrane are needed 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. However, the paradox is that CPAs themselves can have a toxic effect on sperm (membrane destabilization, protein and enzyme denaturation) and this effect is related directly to the concentration used and the time of cell exposure [3]. Added to the freezing medium, nonpermeating cryoprotective substances such as proteins, or amino acids and sugars, acting mainly as osmoprotectants, can mitigate the cryodamage caused by permeating CPAs. At similar concentrations, these substances are less toxic than permeable CPAs, inhibit ice growth and help the sperm to stabilize internal solute concentrations under osmotic stress, and this reduces the amount of penetrating CPAs needed [3].

According to the freezing rate, sperm cryopreservation techniques can be divided into two main categories: slow freezing (conventional freezing) and ultrarapid freezing (vitrification or similar state to vitrification). Conventional freezing involves a step-wise reduction in temperature, but ice crystals that form within the cell can have extremely deleterious effects, such that a balance needs to be found between the beneficial and toxic effects of permeating CPAs. Alternatively, vitrification is a cryopreservation technique used mainly to freeze embryos and tissues. The vitrification process solidifies the sample into a glass-like state, avoiding the formation of both intra- and extracellular ice [4]. This is accomplished through the use of high cooling rates and high CPA concentrations [5]. Because of cytotoxic effects of high CPA concentrations on sperm, vitrification was initially considered inappropriate for freezing male gametes. However, research on sperm cryobiology has demonstrated that the DNA integrity and motility of human sperm is preserved after a vitrification-like state is achieved by ultrarapid nonequilibrium freezing in the absence of permeable CPAs (CPA-free vitrification) [6], [7], [8], [9], [10]. Moreover, the addition of nonpermeating substances such as proteins (mainly albumin) and/or osmoprotectants such as carbohydrates (mainly sucrose) to the vitrification solution has been reported to improve the cryosurvival of human, fish, and dog spermatozoa [11], [12], [13]. In general, research on the cryopreservation of rabbit sperm has focused on conventional slow freezing in liquid nitrogen vapor, and there is little information available in the literature regarding the use of ultrarapid freezing techniques to cryopreserve rabbit semen. Further, the few reports that do exist have described the complete loss of sperm motility upon thawing [14], [15]. In the conventional method of freezing of rabbit sperm, permeable CPAs are added to freezing solutions, alone or in combination with the nonpermeating agents, proteins or sucrose. In effect, several randomized trials have proposed protocols for freezing rabbit sperm by comparing different CPAs and/or the benefits of nonpermeating CPAs [16], [17], [18], [19]. However, none of these trials has been conclusive about the optimal nature and rate of CPA needed in freezing solutions and the survival of rabbit sperm after freezing/thawing procedures remains highly variable [1]. In addition, though vitrification with albumin and osmoprotectants in the absence of permeable CPAs has recently received much attention in other animal species, to the best of our knowledge the use of this procedure has not yet been explored in the rabbit.

The present study was designed to improve on the conventional freezing protocols used for rabbit sperm by investigating: (1) the toxicity of the different permeable CPAs used in standard vapor freezing (conventional freezing); (2) the feasibility of ultrarapid nonequilibrium freezing (vitrification) of sperm in the absence of permeating CPAs; and (3) the effects of adding bovine serum albumin (BSA), alone or with sucrose or trehalose as osmoprotectants. The effects of these factors were assessed in terms of sperm cryosurvival and subsequent fertility.

Section snippets

Chemicals

The LIVE/DEAD Sperm Viability Kit was purchased from Molecular Probes Inc. (Eugene, OR, USA). Acridine orange (AO) and all the other chemicals used in this study were purchased from Sigma Chemical Co. (Milan, Italy).

Animals and semen collection

The animals used for our study were 32 adult hybrid rabbit bucks and 184 does of the line pertaining to the Centro Genetica Martini reared at a private breeding facility (Azienda Gentile, Colle Sannita, BN, Italy). The rabbits were housed in individual flat-deck cages, subjected to

Effects on rabbit sperm of the exposure time and concentration of six conventional permeable CPAs

The semen quality variables determined in the fresh semen (Table 1) indicated the good initial quality of semen (93%, 85%, and 97% of motile, membrane-intact and DNA-intact sperm, respectively).

Exposure time and concentration toxicities of glycerol, DMSO, DMA, ethylene glycol, propylene glycol, and methanol are provided in Figure 1. The motility of fresh undiluted semen recorded at the different incubation times served as controls. We observed a significant effect of the CPA concentration and

Discussion

In this study, we first determined the optimal time of exposure and concentration of six permeable CPAs to avoid toxic effects on rabbit sperm, noting that DMSO 10% was the least damaging during conventional freezing. Moreover, we observed that the CPA-free vitrification of rabbit semen led to low or null sperm cryosurvival and that freezing media enriched with BSA combined with sucrose or trehalose can improve the cryosurvival of rabbit sperm after both conventional freezing and vitrification.

Acknowledgments

The authors thank Innocenzo Gentile and Michele Di Iorio for help with semen collection and artificial inseminations, and Ana Burton for editorial assistance.

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