Research articleCryopreservation 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
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.
References (34)
- et al.
Rabbit sperm cryopreservation: a review
Anim Reprod Sci
(2009) Fundamental aspects of sperm cryobiology: the importance of species and individual differences
Theriogenology
(2000)- et al.
Improving cryopreservation systems
Theriogenology
(2006) - et al.
Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions
Cryobiology
(2011) - et al.
Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success
Reprod Biomed Online
(2003) - et al.
Clean technique for cryoprotectant-free vitrification of human spermatozoa
Reprod Biomed Online
(2005) - et al.
Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness
Anim Reprod Sci
(2011) - et al.
Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose
Theriogenology
(1997) - et al.
In vitro survival and lipid peroxidation status of rabbit spermatozoa after both chilled and frozen storage in lycopene enriched extenders
Livestock Science
(2012) - et al.
The cryoprotectant used, its concentration and the equilibration time are critical for the successful cryopreservation of rabbit sperm: DIMETHylacetamide versus dimethylsulfoxide
Theriogenology
(2012)