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Reproduction (2007) 133 585-595
DOI: 10.1530/REP-06-0256
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse

Cengiz Yildiz1,2, Palma Ottaviani1,2, Napoleon Law1,2, Renise Ayearst1,2, Ling Liu1,2 and Colin McKerlie1,2

1 Integrative Biology Research Program, The Hospital For Sick Children, Toronto, Ontario, Canada M5G 1X82 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1X8

Correspondence should be addressed to C McKerlie, Integrative Biology Research Program, The Hospital For Sick Children, Toronto, Ontario, Canada M5G 1X8; Email: colin.mckerlie{at}sickkids.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen–thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milk + 0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14 + PI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P < 0.05–0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P < 0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P < 0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The mouse has become a significant research tool in genetic and molecular biology allowing the study of many models of human diseases based on transgenic or targeted mutations (Thornton et al. 1999, Crister & Mobraten 2000). The mouse is a convenient model because of its well-characterized genome and ease of genetic and experimental manipulation. Due to increasing demand for archiving through cryopreservation of newly emerging lines of outbred, hybrid, and inbred mutant mice, many laboratories have been working to better understand the freezing and thawing properties of mouse sperm in order to find more efficient methods for sperm storage and recovery. The primary problem in mouse sperm cryopreservation is that it becomes very sensitive to cold and osmotic effect (Critser & Mobraten 2000). The most commonly described adverse effect of freeze–thawing on mouse sperm is the dramatic and sharp decrease in sperm motility and plasma membrane integrity (Songsasen & Leibo 1997a, Sztein et al. 2000, 2001, Nishizono et al. 2004). Therefore, factors such as the type of cryoprotectant agents (CPA) used and genetic background of the mice all contribute to the successful cryopreservation of mouse sperm.

The CPA used in mouse sperm cryopreservation, whether permeating, nonpermeating, or combined is a key element. Polge et al.(1949) first reported that glycerol had a protective action on the survival of frozen–thawed spermatozoa of chicks and humans. The CPA provides a protective action as an energy source for spermatozoa (Watson 1979) and maintains the osmotic pressure by forming hydrogen bonds with membrane phospholipids and sugars reducing membrane damage and minimizing membrane destabilization during freezing and thawing (Strauss et al. 1986). The first successful CPA used with mouse sperm consisted of a combination of 3% skim milk and 18% raffinose (Tada et al. 1990, Takeshima et al. 1991). This CPA combination was further improved by Nakagata (1995) and Nakagata et al.(1992). The CPA produced normal offspring that were derived from cryopreserved oocytes fertilized in vitro by cryopreserved sperm (Nakagata 1993). Another successful CPA for mouse sperm consists of a mixture of raffinose, glycerol, and egg yolk (Songsasen & Leibo 1997a, 1997b). Other studies have used mono-, di- and tri-saccharides, polyols (e.g. glycerol, or DMSO), and macromolecules (e.g. polyvinylpyrrolidone, or metrizamide) alone or in combination with conventional CPAs (Storey et al. 1998, An et al. 2000, Sztein et al. 2001, Thampson et al. 2001, Koshimoto & Mazur 2002) to improve the efficiency of mouse sperm cryopreservation.

It is well established that the genetic background of outbred, hybrid, and inbred strains of mice also influence sperm-assessment parameters (Songsasen & Leibo 1997a, Sztein et al. 2000, Nishizono et al. 2004), in vitro fertilization rate (Kasai et al. 1978, Hoppe 1980, Niwa et al. 1980, Sztein et al. 2000), and in vitro embryo development rate (Dandekar & Glass 1978, Roudebush & Duralia 1996, Scott & Whittingham 1996, Sztein et al. 2000). Unfortunately, inbred strains, such as C57BL/6 and 129S that are among the most commonly used strains of mice for the production of mutant lines still pose a challenge to obtaining consistently high freeze–thaw sperm quality, in vitro fertilization rate, and live-birth rate compared to most outbred and hybrid strains (Songsasen & Leibo 1997a, Sztein et al. 2000, Nishizono et al. 2004).

The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA can have a significant negative impact on oocyte fertilization, embryo development rate, and live-birth rate. A significant correlation between the presence of nuclear DNA alterations in mature spermatozoa and poor sperm parameters or impaired reproductive efficiency is reported in both humans and animals (Ron-el et al. 1991, Hughes et al. 1996, Edwards & Beard 1999). To date, studies show that the cryopreservation process causes DNA damage to mammalian sperm in human (Royere et al. 1988, 1991, Donnelly et al. 2001, Hammadeh et al. 2001), boar (Fraser & Strzezek 2004), and ram (Peris et al. 2004).

The purpose of this study was to examine seven different CPAs in a prototypic hybrid strain of mice to determine the optimal CPA for sperm cryopreservation. Using this optimal CPA, different molar concentrations and combinations were then used to assess its effects on progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in a number of outbred, hybrid and inbred strains of mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
Outbred male (CD-1), hybrid female (B6C3F1/CrIBR), and inbred male mice (DBA/2NCrIBR, 129S/SvPaslco, C57BL/6NCrIBR) were obtained from Charles River Canada (St-Constant QC, Canada). Hybrid B6129SF1 mice were obtained from Taconic (Germantown, NY, USA). Hybrid C3FeB6F1/J mice were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). All sperm-donor mice ranged from 12 to 15 weeks of age and were sacrificed by cervical dislocation. Females ranged from 4 to 5 weeks of age and were sacrificed in the same manner. During all experiments, mice were housed under controlled light conditions (12 h light:12 h darkness) at the Laboratory Animal Services facilities of The Hospital for Sick Children, maintained in pathogen free holding, and received standard mouse diet and water ad libitum. Their use and care at the Hospital were reviewed in advance and performed according to standards of the Canadian Council on Animal Care (CACC).

Reagents
Unless otherwise stated, all reagents were purchased from Sigma Chemical Company.

Summary of experimental design
Experiment 1 evaluated the effects of seven different cryoprotectants, sugars, and polyols, on sperm-assessment parameters. Fresh sperm samples were obtained from B6129SF1 mice and frozen using a programmable controlled-rate freezer. The results obtained from experiment 1 were analyzed and the two cryoprotectants that provided the best results were used for further evaluation in experiment 2.

Experiment 2 assessed the optimal CPA concentrations of 0.2 and 0.3 M raffinose alone and combined with permeating CPA in CD-1, C3B6F1, B6129SF1, C57BL/6, 129S, and DBA mice. Sperm obtained from donors was frozen using a manual freezing technique and tested for post-thaw progressive motility, plasma membrane integrity, and DNA integrity.

Experiment 3 studied the effects of the optimal CPA combinations identified in experiment 2 in C3B6F1 and C57BL/6 mice on unassisted in vitro fertility rate and in vitro embryo development rate to blastocyst.

Cryoprotectant agents
The seven CPA used in these studies were prepared according to the method described by Nakagata (2000). A measure of 0.2 M raffinose (D + raffinose pentahydrate) sugar was combined with either glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate (0.1 M and different amounts). Osmotic pressures were adjusted to 430–450 and 470–490 mOsm/l as shown in Table 1Go; 330–350 and 600–620 mOsm/l as in Tables 2Go and 3Go (Advanced Instruments, Inc., Model 3300, Norwood, Massachusetts, USA).


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  		Table 1 Post-thaw progressive motility and membrane integrity results for B6129SF1 mouse sperm using raffinose alone and or various combinations of permeating cryoprotectants under the same molar or same osmotic pressure (n = 6; mean ± S.E.).
 

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  		Table 2 Post-thaw progressive motility results for tested outbred, hybrid, and inbred strains of mice using alone or combined permeating and nonpermeating cryoprotectants (mean ± S.E.).
 

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  		Table 3 Post-thaw plasma membrane integrity results for tested outbred, hybrid, and inbred strains of mice using alone or combined permeating and nonpermeating cryoprotectants (mean ± S.E.).
 
The buffer used was Bacto Difco powder (Becton Dickinson, Franklin Lakes, NJ, USA) (w/v) dissolved in distilled water at 45 °C. The solution was centrifuged at 14 000 r.p.m. for 15 min at room temperature and the resulting supernatant was filtered through 0.45 µm Millipore filters (Carrigtwohill, Co. Cork, Ireland). The filtered solution was stored at –20 °C in 1.0 ml aliquots in sterile 1.5 ml polystyrene Eppendorf tubes (Eppendorf, Mississauga, Canada). Each aliquot was thawed and warmed to 37 °C prior to use.

Sperm freezing and thawing
Both epididymides and vasa deferentia from male mice were removed and transferred to a 35 mm petri dish (Falcon 351008, Becton-Dickinson, NJ, USA) containing 1 ml CPA pre-stored in an incubator at 37 °C, 5% CO2.They were then pierced eight to ten times using sterile forceps allowing spermatozoa to swim out. Each vas deferens was then gently compressed along its length to expel residual spermatozoa with fine-tipped sterile forceps. The CPA with spermatozoa was then incubated for 10 min at 37 °C. After incubation, debris and tissue was manually removed and the sperm-CPA sample was distributed as 100 µl aliquots into four cryovials (1.8-cc Nunc cryotubes; Nunc Cryotubes, Roskilde, Denmark) using wide pipette tips. Cooling rate was automatically controlled according to Songsasen & Leibo (1997b). The cryovials were placed into a controlled-rate freezer (Thermo Forma 7452, Marietta, Ohio, USA) at 20 °C. After 10 min in the freezer, the cryovials were cooled at 5 °C/min to + 4 °C and held for 3 min. The samples were cryopreserved without seeding. Sperm samples were cooled directly from + 4 °C at 20 °C/min to –100 °C and finally were plunged into liquid nitrogen (LN2). Sperm freezing for each CPA group was replicated six times by using B6129SF1 mice (Table 1Go).

The sperm-CPA samples were separately distributed as 100 µl aliquots into eight labeled 1.8-cc cryovials by using wide pipette tips.

For manual rate freezing, a styrofoam box was used measuring 20.5 cm (length) x 20.5 cm (width) x 20 cm (depth). It was filled to 2.5 cm from the bottom with LN2. Cryovials containing sperm-CPA were placed into a rack at 2 cm above the surface of LN2 exposing them to LN2 vapor (approximately –120 °C) for 10 min at a cooling rate of 20–40 °C/min. Sperm-CPA samples were then plunged directly into LN2 for storage. One frozen–thawed sperm sample was analyzed for sperm-assessment parameters and recorded prior to thawing and in vitro fertilization. Sperm freezing for each CPA group wasreplicated between 6 and 15 times using different mice (Tables 2Go and 3Go). Samples were rapidly thawed by transferring them from LN2 into a 37 °C water bath for 2 min until the ice melted.

Sperm analysis
Progressive motility
Progressive motility of sperm samples was determined using a Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) according to World Health Organization (WHO 1999) guidelines. Frozen sperm samples were diluted 1:4 or 1:6 with Human Tubal Fluid (HTF; Quinn et al. 1985) depending on sperm cell concentration. After 15-min incubation of frozen sperm-CPA with HTF in a 37 °C water bath, 10 µl of sperm-CPA sample was placed into a heated (37 °C) Makler counting chamber and evaluated for progressive motility of sperm. The number of motile and nonmotile spermatozoa was quantified by counting at least six predetermined fields (sequences) with a minimum of 200 spermatozoa. The routine sperm analysis was performed using an inverted phase contrast microscope (Zeiss, Axiovert 100; Zeiss, Oberkochen, Germany) with 10 x magnification.

Plasma membrane integrity
Cell plasma membrane integrity was evaluated by the fluorescent staining method for sperm viability (Live/Dead Sperm Viability Kit, Molecular Probes Inc., Eugene, OR, USA) according to manufacturer’s guidelines. This vital stain contains two nucleic acid dyes: (1) SYBR-14 that permeates intact plasma membrane causing viable spermatozoa to fluoresce green and (2) propidium iodide that permeates only damaged plasma membrane, causing nonviable spermatozoa to fluoresce red. Stain was prepared according to Songsasen & Leibo (1997b). After staining, 10 µl of sperm sample was placed on a microscope slide covered with a cover slip and observed under a fluorescence microscope (Zeiss Model IM35, filter set 487709; excitation filter 450–490 nm). For each mouse, 200 spermatozoa were counted on each slide.

DNA analysis
DNA analysis was performed as described by Larson et al.(2000) and Traina et al.(2003). Briefly, fresh sperm cells were expelled from the isolated vas deferens into 1 ml of TNA (0.15 M NaCl, 0.01 M Tris–HCl, 0.001 M EDTA, pH 7.4) using watchmaker’s forceps. After 15-min incubation, sperm samples were filtered through a 70 µm nylon mesh and were counted to determine sperm concentration. From each fresh sperm sample, 1–2 x 106 cells were taken to a 15 ml conical tube and a pellet was prepared after centrifugation at 300 g for 4 min. Frozen sperm samples were archived in LN2 for 12 weeks prior to DNA analysis. Frozen–thawed sperm aliquots (0.1 ml) were mixed with 1 ml TNA and incubated for 10 min in an incubator at 37 °C, 5% CO2, then the solution was centrifuged at 300 g for 4 min, the pellet re-suspended in 0.2 ml TNA, and then mixed with 0.4 ml acid–detergent solution (0.1% Triton X-100, 0.15 M NaCl, and 0,08 N HCL, pH 1.2). After 30 s, the cells were stained by adding 1.2 ml of a solution containing 6 µg/ml of Acridine Orange (AO; Molecular Probes Inc.) in staining buffer (0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, 0.15 M NaCl, pH 6). When excited with blue light AO intercalated into double-stranded DNA fluoresces green. AO associated with single-stranded DNA emits red fluorescence. Abnormal sperm chromatin structure was measured by flow cytometry (FCM), on a cell-by-cell basis, and expressed as green (native DNA) to red (denatured, single-stranded DNA) AO fluorescence intensity shift using an index called {alpha}T. Alpha-T represents the ratio of red to total (red + green) fluorescence. Normal, native chromatin remains structurally sound and produces a narrow {alpha}T distribution. DNA in sperm with abnormal chromatin structure has increased red fluorescence, thereby producing a broader {alpha}T distribution. Cells were analyzed using the FACSCalibur flow cytometer (Becton Dickinson, Serial no. E0939, Biosciences, NJ, USA). Green fluorescence was measured between 515 and 545 nm band pass filter and red fluorescence using an LP670 nm long pass filter. The two colors were separated by a 560 nm dichroic filter. Data was stored in list-mode at 10 bit resolution. A total of 5 x 104 gated events were accumulated for each measurement. A gate was set on FL1 vs FL3 to exclude debris. Flowjo software (Version 4.0, Tree Star, Inc. Ashland, OR, USA) was used to analyze values of {alpha}T. Fresh sperm from B6129SF1 mice sonicated for 5 min using a sonicator (power rate 5, pulsar duty cycle 75%/s; Ultrasonic Processor XL Sonicator, Hert Systems, Farmingdale, NY, USA) adjusted to increase DNA fragmentation was used as positive control for the sperm chromatin structure assay (SCSA) system (Fig. 1Go).


Figure 1
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  		Figure 1 SCSA data with cytogram and histogram ({alpha}-t) showing the effects of sonication in unsonicated B6129SF1 fresh mouse sperm (control sample; A, B, and C) and with sonication (D, E, and F). Cells (gated cells : comp-{alpha}T) outside the main population have a larger ratio of red:green fluorescence, indicative of abnormal chromatin structure.

 
In vitro fertilization
In vitro fertilization was performed as modified by Sztein et al.(2000). Four to five-week-old B6C3F1 hybrid mice were superovulated by i.p. injection of 0.5 IU of pregnant mare serum gonadotrophin (PMSG) followed by 0.5 IU of human chorionic gonadotrophin (hCG) 48 h later. Female mice were sacrificed by cervical dislocation 14 h after the hCG injection and their oviducts removed into a plastic culture dish containing 2 ml of HTF medium. The cumulus–oocyte complexes were released from the ampulla into the HTF by rupturing with fine sterile forceps, and then placed into a 250 µl HTF fertilization drop. Dishes with HTF drops for fertilization and culture were overlaid with mineral oil for sealing, and then placed overnight in a modular incubator chamber at 37 °C in a humidified atmosphere of 5% CO2. IVF drop for C3B6F1 sperm + B6C3F1 oocyte combinations was used per three oocyte donors with a low concentration, 5 x 105 of progressively motile sperm per milliliter of fresh or frozen sperm.

Frozen sperm samples were thawed in a 37 °C water bath for 3 min and centrifuged at 3000 g (Mikro 20, D-78532, Tuttlingen, Germany) for 4 min. The supernatant was discarded and replaced with 50 µl of HTF. The sperm samples were re-suspended by gently tapping of the tube and then incubated for 20 min at 37 °C to allow a minimal ‘swim up’. A 40 µl aliquot after incubation was taken for the IVF assay from each sperm sample. The fertilization drop was maintained for 4 h at 5% CO2 in an incubator and then the oocytes were washed with HTF to eliminate excess sperm. The inseminated oocytes were cultured overnight in a 100 µl drop of HTF. The next morning, the number of two-cell embryos was scored, and transferred to a 100 µl drop of KSOM/AA medium (Ho et al. 1995) for in vitro embryo development to the blastocyst stage. This IVF procedure was replicated three or four times for fresh and frozen sperm using different males for each strain of mice evaluated.

Zona-thinned oocytes for in vitro fertilization
To test the in vitro fertilization rate and in vitro embryo development rate to blastocyst of C57BL/6 sperm IVF zona-thinned B6C3F1 oocytes were used. A minimum count of 1 x 106 progressively motile sperm per milliliter was used in combination with C57BL/6 fresh or frozen sperm + B6C3F1 oocytes. To account for any parthenogenetic effects on oocytes in the zona-thinned group, cleaved parthenogenetic oocytes were identified and removed from culture according to Nagy et al.(2002).

The oocyte–cumulus complexes were collected from superovulated C3B6F1 mice. Cumulus cells were removed by using 0.05 mg/ml hyaluronidase (Sigma; H-3506) for 5 min in HTF medium. After washing the oocytes with HTF, zona thinning was performed using zona tyrode solution containing 0.8 g NaCl, 0.02 g KCl, 0.024 g CaCl22H2O, 0.301 g MgCl2 6H2O, 0.1 g Glucose, 0.4 g polyvinylpyrolidone (PVP) in 100 ml water (pH 3.0). Oocytes were exposed for 45 s to the acid tyrode solution and washed three times with fresh HTF media prior to use.

Statistical analyses
Statistical analyses were performed using Graphpad Prism version 3.0 software (Graphpad software, San Diago, CA, USA). Post-thaw sperm assessment parameter results for DNA integrity, invitro fertilization rate, and invitro embryo development rate to blastocyst were evaluated for significance using repeated-measures one-way ANOVA (Tables 1Go–6GoGoGoGoGo). The Dunnett multiple-comparison test was used to compare differences among groups studied.


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  		Table 4 Post-thaw DNA integrity results for mouse sperm using combined permeating and nonpermeating cryoprotectants with skim milk on selected strains (n = 8; mean ± S.E.).
 

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  		Table 5 In vitro fertilization rate and embryo development rate to blastocyst for C3B6F1 mouse sperm cryopreserved with different cryoprotectants.
 

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  		Table 6 In vitro fertilization rate and embryo development rate to blastocyt for C57BL/6 mouse sperm cryopreserved with different cryoprotectants.
 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Experiment 1
The greatest progressive motility of B6129SF1 sperm was obtained with the combination of 0.2 M raffinose + 0.1 M fructose (430–450 mOsm) compared to 0.3 M raffinose alone (P < 0.05). However, there were no significant differences in sperm membrane integrity between the groups with CPA and the negative control. The combination of 0.2 M raffinose + 0.1 M glycerol significantly improved progressive motility and plasma membrane integrity compared to other polyols, such as prophylenglycol or ethylenglycol, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate with osmotic pressure between 430 and 490 mOsm (P < 0.05, Table 1Go). All combinations of CPA in all groups after thawing improved progressive motility and membrane integrity when compared with the negative control group (no CPA; P < 0.05; Table 1Go).

Experiment 2
Raffinose alone, and raffinose combined with fructose and glycerol were evaluated by using sperm from several strains of mice selected for this study (Tables 2Go and 3Go). The optimal molar concentrations were determined to be 0.2 and 0.3 M raffinose in outbred, hybrid, and inbred strains of mice.

The results of comparing different combinations and concentrations of CPA are presented in Tables 2Go and 3Go. Combination of 0.3 M raffinose + 0.1 M fructose had the highest progressive motility rates when compared with 0.3 M raffinose alone and 0.3 M raffinose + 0.1 M glycerol groups in CD-1, C3B6F1, and B6129SF1 mice (P < 0.05–0.01). There were no significant differences in rates of DNA fragmentation in frozen–thawed sperm from CD-1, C3B6F1, B6129SF1, and DBA mice when compared with the CPA control groups (Table 4Go). A 0.2 M concentration of raffinose in CPA solution had a significant beneficial impact on the progressive motility and membrane integrity of sperm from C57BL6 and 129S mice when compared with 0.3 M raffinose alone after freezing and thawing. Additionally, 0.2 M raffinose + 0.1 M glycerol and 0.2 M raffinose + 0.1 M fructose in C57BL/6 and 129S mice provided for the highest progressive motility when compared with 0.3 M raffinose alone (P < 0.001), but did not significantly affect membrane integrity. Post-thaw C57BL/6 and 129S inbred sperm had a higher DNA fragmentation rate when compared with CD-1, C3B6F1, B6129SF1, and inbred DBA mice (Table 4Go). Interestingly, both 0.2 M raffinose + 0.1 M fructose and 0.2 M raffinose + 0.1 M glycerol combination in C57BL/6 or 129S mice resulted in more robust sperm cell DNA integrity when compared with raffinose alone (P < 0.01). In other words, supplementation of CPA solution with glycerol or fructose showed a protective effect for DNA integrity against freezing damage in C57BL/6 and 129S mice (P < 0.01). The combination of 0.3 M raffinose + skim milk resulted in increased values for progressive motility, membrane integrity, and a lower rate of DNA fragmentation after freezing in DBA mice (P > 0.05). Although DBA was one of the inbred strains, it showed the highest resistance to DNA damage during freezing when compared with C57BL/6 and 129S inbred mice. In addition, resistance to DNA damage during freezing in the strains of outbred and hybrid mice tested were significantly greater than C57BL/6 and 129S inbred mice (P < 0.01). The cryopreservation process increased DNA fragmentation in all strains of outbred, hybrid, and inbred mice when compared with the fresh control sperm (P < 0.01; Table 4Go).

Experiment 3
The combination of 0.3 M raffinose + 0.1 M fructose had a higher in vitro fertilization rate than 0.3 M raffinose alone when compared with C3B6F1 oocytes fertilized with C3B6F1 fresh sperm (P < 0.05; Table 5Go). However, there were no significant differences in embryo development rate to blastocyst between fresh C3B6F1 hybrid sperm using 0.3 M raffinose alone or 0.3 M raffinose + 0.1 M fructose. For the inbred C57BL/6 strain, the results from experiment 3 further showed the efficiency of 0.2 M raffinose + 0.1 M glycerol combination in improving the in vitro fertilization rate and in vitro embryo development rate to blastocyst using C3B6F1 oocytes and C57BL/6 sperm (Table 6Go). A measure of 0.3 M raffinose alone resulted in lower in vitro fertilization rate when compared with fresh C56BL/6 sperm or 0.2 M raffinose + 0.1 M glycerol combination (P < 0.05). On the other hand, the combination of 0.1 M glycerol + 0.2 M raffinose significantly improved in vitro fertilization rate for frozen C57BL/6 sperm (P < 0.05). In addition, this CPA combination had a lower DNA fragmentation rate that correlated with an increased in vitro embryo development rate to blastocyst when compared with 0.3 M raffinose alone for frozen C57BL/6 sperm.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Investigation into the effects of variable combinations and concentrations of cryoprotectants on sperm quality is critical to generate more efficient techniques for mouse sperm cryopreservation.

The results of the first set of experiments from this study show that the type (chemical structure) of cryoprotectant and combination of permeating and nonpermeating cryoprotectants are important factors that effect the progressive motility and plasma membrane integrity of B6129S1 hybrid mouse sperm during freezing and thawing. Supplementation of glycerol or fructose to raffinose increased post-freezing progressive motility when compared with raffinose alone or combined permeating cryoprotectants (i.e. glucose or ethylene glycol) in B6129SF1 mice. The beneficial effects of using combined permeating and nonpermeating cryoprotectants in this study concur with previous reports in the literature that showed combinations of 18% raffinose and 1.75% glycerol (Tada et al. 1990), 10% raffinose and 5% glycerol (Yokoyama et al. 1990), and 6% glycerol plus trehalose (Storey et al. 1998) provided for the highest motility and plasma membrane integrity rates after sperm freezing and thawing.

The values for sperm progressive motility and membrane integrity reported in the second set of experiments from this study vary according to the molar concentration of CPA and mouse strain used. The use of raffinose alone or combined with permeating fructose or glycerol provided improved progressive motility and plasma membrane integrity when compared with molar concentration of raffinose (0.2 or 0.3 M). Koshimoto & Mazur (2002) pointed out that combinations of raffinose, sucrose, and glucose in PBS did not improve normalized motility when compared with raffinose alone under the same osmotic pressure (410–427 mOsm) or fixed at 18% (w/v) in outbred ICR mouse sperm. A similar finding is presented in Tables 1Go and 2Go of the present study for the response of raffinose + glucose or fructose combination in B6129SF1 and CD-1 strains. However, Koshimoto & Mazur (2002) also suggested that there is no special protection conferred by a particular sugar, and that protection against damage during freezing and thawing depends on the mass concentration of the sugar rather than its molar concentration. On the contrary, according to our results presented in Table 2Go, even though fructose (MW = 180.2) has the same molecular weight as glucose, it significantly increased post-thaw progressive motility. The beneficial effects and improved sperm-assessment parameters produced as a result of varying sugar types and CPA combination (i.e glycerol) have also been reported for ram (Molinia et al. 1994), bovine (Garcia & Graham 1989), dog (Yildiz et al. 2000), and human sperm (Critser et al. 1988, McGonagle et al. 2002). The effectiveness of fructose on sperm motility in CD-1, C3B6F1, B6129SF1, C57BL/6, and 129S mice can be explained by its relatively higher permeation rate across the cell plasma membrane and rapid metabolic processing in the cell when compared with other monosaccharides, such as glucose or galactose (Schwartz 1997).

The choice of sugar in the CPA formulation in the second set of experiments presented here had a significant impact on post-thaw mouse sperm motility in a strain-dependent manner. CD-1, C3B6F1, B6129SF1, and DBA sperm were frozen successfully using 0.3 M raffinose alone or 0.3 M raffinose + 0.1 M fructose combination (Table 2Go). It has been previously reported that the same molar concentration of CPA (i.e. 0.3 M raffinose) is not effective and does not equally preserve the sperm from some strains of mice, such as C57BL/6 and 129S when compared with hybrid and other inbred mice (Songsasen & Leibo 1997a, Sztein et al. 2000, Sztein et al. 2001, Nishizono et al. 2004). Also C57BL/6 and 129S sperm were frozen successfully using 0.2 M raffinose + 0.1 M fructose or glycerol combination (Table 2Go). It is also reasonable to assume that there can also be differences in sperm head or biochemical composition of sperm head membrane between C57BL/6 and 129S and other outbred, hybrid, and inbred strains of mice. If a suitable CPA’s molar concentration is not used, the biochemical membrane properties or differences in sperm head dimension may possibly induce cell death caused by intracellular or extracellular ice crystallization during freezing; resulting in inadequate dehydration and intracellular high-solute concentration. Also in the second set of experiments presented here, even though 0.3 M raffinose alone had a lower osmolarity (470–490 mOsm) than 0.3 M raffinose–0.1 M fructose combination (600–620 mOsm), this combination of sugar showed statistically higher progressive motility than 470–490 mOsm group in CD-1, C3B6F1 and B6129SF1 mice. Willoughby et al.(1996) reported a critical osmolarity of 500 mOsm in anisotonic PBS for mouse sperm. However, Agca et al.(2002) recently reported that egg yolk and skim milk significantly protected sperm motility and extended its osmotic tolerance limit (150–600 mOsm) when compared with NaCl, choline, or sucrose alone in ICR and B6C3F1 mice. Similar results using higher osmotic pressures were also found in this study.

DNA integrity of sperm is essential for accurate transmission of paternal genetic information. Normal condensation and stabilization of sperm chromatin in the nucleus followed by decondensation after sperm penetration and injection into the cytoplasm of the oocyte are pre-requisites for fertilization (Flaherty et al. 1995). The effects of cryopreservation on the integrity of the sperm nucleus are not well characterized. It is reported that the cryopreservation process of freezing and thawing can increase inappropriate chromatin condensation in human (Royere et al. 1988, Royere et al. 1991, Donnelly et al. 2001, Hammadeh et al. 2001), boar (Fraser & Strzezek 2004), horse (Linfor & Meyers 2002), and ram (Peris at al. 2004) sperm. Recently, Chohan et al.(2004) noted that normal chromatin packaging significantly decreases after the freeze–thawing procedure in human sperm. Likewise, the cryopreservation process in our studies significantly increased the rate of abnormal sperm DNA in all tested strains compared to the rate in fresh control sperm (Table 4Go). However, different from the results presented here, Jiang et al.(2005) reported that nuclear DNA of fresh or cryopreserved sperm using isotonic CZB-HEPES solution did not affect the level of DNA fragmentation in outbred Kunming mice. The discrepancy between our findings and this report may result from using isotonic CZB-HEPES solution as a cryoprotectant and a different strain of mice. The second set of experiments reported here showed that the sperm DNA integrity was significantly increased by the cryopreservation process in C57BL/6 and 129S inbred mice, but was not affected by the freezing process in CD-1, C3B6F1, B6129SF1, and DBA strains of mice. This clearly implies that there are significant differences in resistance and susceptibility to DNA fragmentation between strains of outbred, hybrid, and inbred mice. In addition, supplementation of fructose or glycerol to raffinose not only protected progressive motility and membrane integrity, but also DNA integrity of frozen–thawed C57BL6 and 129S sperm when compared with raffinose alone. It is well known that cryoprotectants are essential for the survival of cells during cryopreservation and the most commonly used cryoprotectans are sugars and glycerol. Strauss et al.(1986) and Anchordoguy et al.(1987) showed that sugars have cryoprotective properties because they lower the Van der Waals interactions at the membrane hydrocarbon chains that enhance trans-membrane transfer (especially phospholipids). The exact mechanism of action for glycerol in protecting cells from freeze–thaw damage is not completely understood. However, it is known that glycerol is osmotically active and is slow to permeate membranes resulting in cell volume changes from both the addition of glycerol and water loss during the freeze–thaw cycle (Schneider & Mazur 1984, Hammerstedt et al. 1990). Glycerol induces interdigitation of the two bilayer leaflets, altering membrane fluidity by increasing the order of the interior fatty acyl chains (Boggs & Rangaraj 1985). The beneficial effects of fructose and glycerol on DNA integrity in our study may be related to the different interactions on the DNA defense mechanisms of fructose or glycerol during the cryopreservation process.

The impact of the chemical formulation and combinations of CPA on unassisted in vitro fertility rate and in vitro embryo development rate to blastocyst was evaluated by using C3B6F1 and C57BL/6 sperm donors in the third set of experiments reported here (Tables 5Go and 6Go). Our findings show that both inbred and hybrid fresh sperm had higher in vitro fertility rates and embryo development rates to blastocyst when compared with frozen–thawed sperm. These findings are consistent with previous reports on in vitro fertilization and in vitro embryo development rate to blastocyst of inbred strains previously reported (Niwa et al. 1980, Songsasen & Leibo 1997a, Sztein et al. 2000). The combination of 0.3 M raffinose + 0.1 M fructose with IVF produced higher in vitro fertility rates than 0.3 M raffinose alone and a lower in vitro fertility rate than C3B6F1 fresh sperm. However, there were no significant differences in embryo development rate to blastocyst between fresh sperm, 0.3 M raffinose alone, or 0.3 M raffinose + 0.1 M fructose. Progressive motility is one of the most important criteria for fertilization potential of sperm (WHO 1999). Thus, higher fertilization results reported here that were obtained from the combination of 0.3 M raffinose + 0.1 M fructose in C3B6F1 mice may be related to their greater post-thaw progressive motility rate when compared with 0.3 M raffinose alone (Tables 2Go and 5Go). A similar correlation can be assumed between the combination of 0.2 M raffinose + 0.1 M glycerol due to the higher post-thaw progressive motility rate observed when compared with 0.3 M raffinose alone in C57BL/6 mice (Tables 2Go and 6Go). Recently, Nishizono et al.(2004) reported that using 0.3 M raffinose-skim milk combination significantly increased the occurrence of abnormal or dysmporphic acrosome after the cryopreservation process in C57BL/6 sperm, and reported a strong correlation between dysmorphic spermatozoa and fertilization rates. Unlike this report, our in vitro fertilization rate for C57BL/6 mice using a combination of 0.2 M raffinose + 0.1 M glycerol can be related to the better protective effect of raffinose–glycerol–skim milk combination on sperm membrane compared to 0.3 M raffinose–skim milk. Glycerol has previously been shown to be effective in protection against freezing membrane injuries in commonly used species of livestock (Parks & Graham 1992, Curry 1995) and a combination of sucrose and glycerol successfully protects embryos from various mammalian species and also bull sperm (Honadel & Killian 1988, De Leeuw et al. 1993) during cryopreservation. Classic cryobiology studies suggest the use of glycerol or any permeating additive to more efficiently protect cells from freezing injuries through colligative or solution effects (Mazur 1970). Therefore, using a CPA combination of glycerol and raffinose in C57BL/6 spermatozoa may have a synergistic effect on the protection of sperm cell membranes (i.e. the acrosome), resulting in higher in vitro fertilization rates. The combination of 0.1 M glycerol + 0.2 M raffinose in the present studies protected sperm DNA integrity and significantly improved in vitro embryo development rate to blastocyst when compared with 0.3 M raffinose alone in C57BL/6 mice (Table 6Go). Decreases in fertilization rate and poor embryo implantation have been shown to be related to sperm DNA fragmentation (Hughes et al. 1996, Edwards & Beard 1999). Likewise, Larson et al.(2000) and Tesarik et al.(2004) noted that chromatin abnormalities decrease late embryonic development after ICSI treatment with human sperm. Similarly, in the results presented here, higher rates of DNA fragmentation that were obtained using 0.3 M raffinose significantly decreased embryo development rate to blastocyst when compared with fresh sperm and the combination of 0.1 M glycerol + 0.2 M raffinose in C57BL6 mice (Table 6Go). These results suggest that the chemical formulation of CPA is important and varies in its ability to protect the DNA material of sperm during cryopreservation, and that increased levels of DNA fragmentation significantly decreases embryo development rate to blastocyst in C57BL6 mice. Recently, Seli et al.(2004) also reported a negative correlation between DNA fragmentation and blastocyst development for human sperm.

In summary, the results obtained from the experiments reported here support conclusion that: (1) the type of monosaccaride sugar or polyols used in CPAs are an important factor for mouse sperm cryopreservation, (2) molar concentrations of nonpermeating raffinose sugar in CPA solution have a significant impact on sperm-assessment parameters in C57BL6 and 129S mice, (3) the cryopreservation process increases DNA fragmentation in all tested outbred, hybrid, and inbred strains of mice when compared with fresh control, (4) the resistance of sperm DNA to damage during freezing and thawing of inbred strains appears to be strain dependent, (5) C57BL/6 and 129S mice are the most sensitive strains to sperm DNA fragmentation, (6) supplementation of CPA solution with either of glycerol or fructose shows a significant protective effect for DNA integrity during cryopreservation in C57BL/6 and 129S mice, (7) supplementation of fructose or glycerol to raffinose significantly improves in vitro fertilization rate in C3B6F1 and C57BL/6 mice, and (8) the rate of sperm DNA fragmentation significantly decreases in vitro embryo development rate to blastocyst in C57BL/6 mice.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This research was supported by Genome Canada through the Ontario Genomics Institute and Genome Prairie; Canadian Institutes of Health Research grants to C M; Canada Foundation for Innovation and Ontario Innovation Trust grants to C M; and Charles River Canada. C M is a recipient of the Ontario Ministry of Innovation’s Early Researcher Award. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 6 October 2006
First decision 10 November 2006
Accepted 8 December 2006


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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