Original Article Combination medium of cryoprotective agents containing L-glutamine and methyl-b-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm T Takeo and N Nakagata Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan Corresponding author: Naomi Nakagata. Email: nakagata@kumamoto-u.ac.jp Abstract Recently, a vast number of genetically-engineered mice have been created in various laboratories worldwide, all of which need to be effectively archived. The cryopreservation of mouse sperm provides a simple and economical means of storing the mice in mouse resource facilities. The current protocol for sperm cryopreservation using 18% raffinose pentahydrate and 3% skim milk (R18S3) has been adopted in most laboratories. In general, we can attain relatively high fertilization rates for frozen/ thawed sperm in many inbred and F1 hybrid strains. However, the sperm of C57BL/6J mice shows an extremely low fertility rate after freezing and thawing (0 20%). In this study, we attempted to improve the low fertility of frozen/thawed C57BL/6J mouse sperm. Our results showed that a combination of R18S3 containing L-glutamine and methyl-b-cyclodextrin (MBCD) in a preincubation medium dramatically increased the rate of fertilization (69.2 + 12.2%). Furthermore, the developmental potencies of two-cell embryos produced by frozen/thawed sperm to live young were normal (fresh: 46.0 + 8.2%, frozen/ thawed: 51.5 + 11.1%). In summary, we conclude that a new method of sperm cryopreservation and in vitro fertilization using modified R18S3 with L-glutamine and MBCD in a preincubation medium yields a high fertilization rate for frozen/thawed C57BL/6J strain sperm. Furthermore, the new method provides a reliable archiving and reproducing system for geneticallyengineered mice using sperm cryopreservation. Keywords: Mouse sperm cryopreservation, L-glutamine, sperm preincubation, methyl-b-cyclodextrin, in vitro fertilization Laboratory Animals 2010; 44: 132 137. DOI: 10.1258/la.2009.009074 Sperm cryopreservation is a useful tool for effectively archiving vast numbers of genetically-engineered mice, such as transgenes, targeted mutations and chemicallyinduced mutations, in a mouse repository and resource centre. 1 4 Now, the need for sperm cryopreservation is growing in recognition, so a great number of mice have been produced worldwide and some knockout mouse projects continue in Europe and America. 5 Most laboratories have adopted the protocol for sperm cryopreservation using cryoprotective agents (CPA) composed of 18% raffinose pentahydrate and 3% skim milk (R18S3). 6 In general, frozen/thawed sperm in many inbred and F1 hybrid strains show relatively high fertilization rates in in vitro fertilization (IVF). 6 8 However, the sperm from C57BL/6J mice, which is the main strain used for the production of transgenic mice, show a low level of fertility after freezing and thawing (0 20%). 6 10 Previously, we have demonstrated that methylb-cyclodextrin (MBCD) dramatically improved the fertility of frozen/thawed sperm in C57BL/6J mice by stimulating the cholesterol efflux from the plasma membrane. 11 MBCD is a methylated cyclic heptasaccharide consisting of a-(1-4)-glucopyranose units with a hydrophilic outer and lipophilic cavity at its centre, which can form inclusion complexes with many lipophilic agents by taking up a molecule. 12 It is well known that MBCD can remove cholesterol from the sperm membrane and induce sperm capacitation in mice. 11,13,14 On the other hand, we have found that cryopreserved C57BL/6J mouse sperm frequently suffer from cryo-induced cellular injuries during freezing and thawing. 10 Improving the conditions of sperm cryopreservation may further help to obtain a high and stable rate of fertilization from cryopreserved C57BL/6J mouse sperm. Laboratory Animals 2010; 44: 132 137
Takeo and Nakagata. Mouse sperm cryopreservation 133 There are some reports of L-glutamine being effective for sperm cryopreservation in several mammalian species. 15 20 L-glutamine is the most abundant free amino acid in the plasma and tissues and plays an important role in nitrogen metabolism and protein synthesis. 21 In sperm cryopreservation, the addition of L-glutamine into the cryopreservation solution prevents the sperm from suffering freezing and thawing stress and enhances the post-thaw motility of frozen/thawed sperm. However, the cryoprotective effects of R18S3 with L-glutamine on the mouse sperm are not clear. In this study, we tried to examine the combination effect of R18S3 containing L-glutamine and MBCD in a preincubation medium on the fertility of frozen/thawed sperm in C57BL/6J mice (experiment 1). In experiment 2, we examined the individual variability of the IVF rate in frozen/ thawed C57BL/6J mouse sperm. In experiment 3, we checked the development ability of two-cell embryos produced from frozen/thawed C57BL/6J mouse sperm using a novel sperm cryopreservation and IVF system. Material and method Animals C57BL/6J mice were purchased from CLEA Japan Inc (Tokyo, Japan) and used as sperm and oocyte donors. Female and male donors were an 8- to 10- and a 12- to 15-week-old, respectively. Mice used as recipients for the transfer of two-cell embryos were of the Jcl/ICR strain and were 8 to 16 weeks old. All animals were kept under a 12 h/12 h dark/light cycle (lights on: 07:00 to 19:00 h) at a constant temperature of 22 + 18C with free access to food and water. All animal experiments were carried out with the approval of the Animal Care and Use Committee of the Kumamoto University School of Medicine. Media Sperm cryopreservation solutions were used containing 18% raffinose pentahydrate and 3% skim milk (R18S3, 420 mosm) or R18S3 with 100 mmol/l L-glutamine (520 mosm), prepared according to the previously published method. 6 The amount of 1.8 g raffinose pentahydrate and 0.3 g skim milk were diluted in 10 ml of distilled water with or without 100 mmol/l glutamine and were then incubated in a water bath for 90 min at 608C. After incubation, the solution was centrifuged at 10,000 g for 60 min, and the supernatant was filtrated and stored at room temperature until use. A modified Krebs-Ringer bicarbonate solution (TYH) 22 with 4.0 mg/ml bovine serum albumin (BSA) or 0.75 mmol/l MBCD (Sigma, St Louis, MO, USA) with 1.0 mg/ml polyvinylalcohol (cold water soluble; Sigma) was used as a medium for sperm preincubation. 11 Human tubal fluid (HTF) 23 and modified Whitten s medium (mwm) 24 were used for IVF and the culture of two-cell embryos to the blastocyst stage. Sperm freezing and thawing The cryopreservation of sperm was performed as per our published method with a little modification. 6 After the male mice were sacrificed by cervical dislocation, two-tailed caudal epididymides were taken from one male mice (Figure 1a). Aliquots of 60 ml of the R18S3 with or without L-glutamine were placed on a 35 mm culture dish and covered with paraffin oil. Thereafter, a 60 ml aliquot of the same solution was added to the drop (final volume: 120 ml) to make a tall-semispherical drop. The five to six portions in tissues were cut using micro-spring scissors in the drop of CPA (Figure 1b). The dish was gently shaken every minute to disperse sperm from the organs at room temperature. After 3 min, the sperm suspension was divided into 10 aliquots of 10 ml on a culture dish. All specimens were put into a 0.25 ml plastic straw (IMV, Paris, France), and the straws were heat-sealed (Figure 1c). The straws were then cooled by putting them into the neck (liquid nitrogen gas layer) of a container for 10 min and plunging them directly into liquid nitrogen (Figure 1d). They were then stored in the liquid nitrogen (Figure 1e). After five days, the samples were removed from the liquid nitrogen and thawed in a water bath at 378C for 10 min (Figure 1f). In vitro fertilization The procedures used for preincubation and IVF using fresh or frozen/thawed sperm were essentially the same as those described previously. 11,13 Mature female mice were superovulated by an intraperitoneal injection of 7.5 IU of equine chorionic gonadotropin (ecg) (ASKA Pharmaceutical Co Ltd, Tokyo, Japan) followed by 7.5 IU of human chorionic gonadotropin (hcg) (ASKA Pharmaceutical Co Ltd) 48 h later. At 14 15 h after the injection, the mice were sacrificed by cervical dislocation and their oviducts were removed. The four to five cumulus-oocytes-complexes (COCs) obtained from the ampulla of the fallopian tube were introduced in a 90 ml drop of HTF medium covered with paraffin oil. An aliquot of 10 ml thawed suspension was added to the centre of the drop of preincubation medium (90 ml) covered with paraffin oil (Figure 1g). Two kinds of preincubation media, such as TYH with BSA or TYH with MBCD, were used as sperm preincubation in experiment 1, whereas TYH with MBCD was used as sperm preincubation in experiment 2. The thawed sperm were preincubated in the TYH with BSA for 60 min or in the TYH with MBCD for 30 min at 378C with 5% CO 2 in the air. After preincubation, an aliquot of 10 ml sperm suspension was collected from the peripheral part of the drop containing motile sperm using a wedge-shaped pipette tip (0.5 10 ml, Quality Scientific Plastics, Petaluma, CA, USA; Figure 1h). The sperm suspension was carefully transferred to an IVF drop containing COCs and incubated at 378C with 5% CO 2 in the air (final motile sperm concentration ¼ 200 400/mL; Figure 1i). After 5 to 6 h, the inseminated oocytes were washed three times in a drop of 100 ml HTF covered with paraffin oil and were then cultured at 378C with 5% CO 2 in the air. Twenty-four hours after insemination, the fertilization rates were calculated as the total number of two-cell embryos divided by the total number of inseminated oocytes 100.
134 Laboratory Animals Volume 44 April 2010 Figure 1 Schematic descriptions of mouse sperm cryopreservation and in vitro fertilization (IVF) protocol. Sperm cryopreservation was performed by following procedures (a e). (a) One pair of cauda epididymides was collected from a male mouse. (b) The cauda epididymides were transferred into a 120 ml drop of cryoprotective agent (CPA) and cut into five portions as indicated by arrowheads. (c) Prepared sperm suspension was divided into 10 aliquots and packed into toa 0.25 ml plastic straw. (d) Plastic straws containing 10 ml sperm suspension were cooled in the liquid nitrogen (LN 2 ) vapour for 10 min and were then plunged directly into LN 2. (e) These samples were then stored in the LN 2 before use. Thereafter, IVF using frozen/thawed sperm was performed following steps (f i). (f) The stored samples were retrieved from LN 2 and immediately soaked and warmed in the water bath at 378C for 10 min (g) Aliquots of 10 ml sperm suspension were loaded and preincubated in a 90 ml drop of TYH with methyl-b-cyclodextrin (MBCD) for 30 min at 378C. (h) An aliquot of 10 ml sperm suspension was collected from the peripheral part of the drop containing motile sperm using a wedge-shaped pipette tip in the direction shown by the arrow. (i) Sperm suspension was added to 90 ml human tubal fluid (HTF) containing cumulus-oocytes-complexes (COCs) and coincubated for 5 6 h. Twenty-four hours after insemination, two-cell embryos were obtained at 378C with 5% CO 2 in the air Embryo culture and transfer After IVF using fresh or frozen/thawed sperm, the fertilized oocytes that had developed to the two-cell stage within 24 h of insemination were divided into two groups (experiment 3). One group of two-cell embryos was transferred and washed three times in a 100 ml drop of mwm and further cultured for 72 h. At this time, the development rates of blastocyst stage embryos were calculated by the number of blastocyst stage embryos divided by the number of two-cell embryos 100. In the other group, 20 of the
Takeo and Nakagata. Mouse sperm cryopreservation 135 two-cell embryos were transferred into the oviducts of each pseudopregnant Jcl/ICR female on the day a vaginal plug was found (day 1 of pseudopregnancy). After 19 days, the number of offspring was recorded. Statistical analysis Statistical analysis was performed using Prism version 3.0 (GraphPad, San Diego, CA, USA). Data are given as the mean + SD. Comparison of the differences between the means for each treatment were carried out using analysis of variance after arcsine transformation of the percentage data. Differences between the means were considered to be significant when P, 0.05 was achieved. Results Experiment 1. In vitro fertilization of frozen/thawed C57BL/6J mouse sperm using various media for cryopreservation and preincubation L-glutamine in R18S3 improved the fertilizing ability of frozen/thawed sperm when compared with R18S3 only (Table 1). In addition, preincubated sperm in TYH with MBCD massively increased the fertilization rate when compared with TYH. The highest rate was obtained from a combination of sperm freezing with R18S3 containing L-glutamine and preincubation in TYH with MBCD. Experiment 2. Individual variability of fertilization rates in frozen/thawed C57BL/6J mouse sperm Using a novel protocol for the IVF using frozen/thawed C57BL/6J sperm, the fertilization rates were relatively high for each mouse (Table 2). Fertilization rates of frozen/ thawed sperm showed to be a little lower than those of fresh control; however, relatively high and stable rates were obtained for the cryopreserved sperm from individual males using the novel protocol combined with R18S3 containing L-glutamine and MBCD in a preincubation medium. Experiment 3. In vitro and in vivo development of embryos derived from fresh and frozen/thawed C57BL/6J mouse sperm The rate at which two-cell embryos derived from frozen/ thawed sperm developed into blastocysts or live young is Table 2 Fertilization rate of fresh and frozen/thawed sperm in individual C57BL/6J mice Sperm Mouse no. shown in Table 3. In vitro, 81% of two-cell embryos developed into blastocysts from the frozen/thawed group. In vivo, 52% of transferred embryos were born from the frozen/thawed group. There was no significant difference in the developmental ability of two-cell embryos of the fresh and frozen/thawed groups. Discussion inseminated eggs two-cell embryos (%) Fresh 1 70 60 (85.7) 2 79 67 (84.8) 3 101 76 (75.2) 4 93 69 (74.2) 5 87 69 (79.3) Total 430 341 (79.3 + 5.3) Frozen/ 6 94 65 (69.1) thawed 7 46 30 (65.2) 8 77 27 (35.1) 9 59 39 (66.1) 10 71 48 (67.6) 11 56 35 (62.5) 12 50 26 (52.0) 13 82 36 (43.9) 14 74 32 (43.2) 15 61 50 (82.0) Total 670 388 (57.9 + 14.5) In vitro fertilization was performed using fresh and frozen/thawed sperm Each percentage values in the lowest row of fresh and frozen/thawed group represent mean + SD (fresh sperm: n ¼ 5, frozen/thawed sperm: n ¼ 10) Values are significantly different compared with fresh controls at P, 0.05 In the present study, we demonstrated that the application of R18S3 with L-glutamine and TYH with MBCD in sperm cryopreservation and preincubation improves the fertilizing ability of frozen/thawed C57BL/6J mouse sperm (Table 1). A combination of R18S3 and L-glutamine enhances the fertilization rate when compared with R18S3 only. Furthermore, MBCD used for sperm preincubation dramatically increases fertilization rate, while a high, stable rate was obtained by treating the sperm with R18S3 with L-glutamine and TYH with MBCD in sperm cryopreservation and preincubation. Our previous findings demonstrated that the treatment of frozen/thawed C57BL/6J mouse sperm with MBCD lead to a dramatic activation in their fertility in vitro. 11 The period Table 1 Effects of sperm freezing media with L-glutamine and preincubation media with methyl-b-cyclodextrin (MBCD) on the ability of frozen/ thawed sperm to fertilize in vitro Media Sperm freezing Preincubation inseminated eggs two-cell embryos (%) R18S3 TYH 224 40 (17.9 + 8.0) TYH þ MBCD 247 143 (57.9 + 11.6) R18S3 þ L-glutamine TYH 227 66 (29.1 + 5.8) TYH þ MBCD 266 184 (69.2 + 12.2) Sperm cryopreservation was performed using a cryopreservation media of R18S3 or R18S3 containing L-glutamine Frozen/thawed sperm were preincubated in TYH or in TYH with MBCD before in vitro fertilization Each percentage value represents mean + SD (n ¼ 5). Values are significantly different compared with controls of R18S3 and TYH group at P, 0.05, P, 0.01
136 Laboratory Animals Volume 44 April 2010 Table 3 Sperm In vitro and in vivo development of two-cell embryos produced from fresh and frozen/thawed sperm In vitro development In vivo development inseminated eggs two-cell embryos (%) examined twocell embryos blastocysts at 96 h (%) two-cell embryo transferred recipient mouse live young (%) Fresh 430 341 (79.3 + 5.3) 241 202 (83.8 + 1.6) 100 5 46 (46.0 + 8.2) Frozen/thawed 670 388 (57.9 + 14.5) 188 161 (85.6 + 8.6) 200 10 103 (51.5 + 11.1) Two-cell embryos derived from fresh and frozen/thawed sperm were divided two groups: one group was incubated in the modified Whitten s medium (mwm) for 72 h or the other group was transferred into oviducts of pseudopregnant mice (20 embryos/female) Development rates were calculated by number of blastocysts/number of examined two-cell embryos 100 (in vitro development) or number of live young/number of two-cell embryos transferred 100 (in vivo development) Each percentage value represents mean + SD (fresh sperm: n ¼ 5, frozen/thawed sperm: n ¼ 10) Values are significantly different compared with fresh controls at P, 0.05 of preincubation affects sperm fertility, so different preincubation conditions were for BSA and MBCD to gain the highest possible fertilization rate. The highest rate of fertilization using BSA was obtained at 60 120 min, whereas that of MBCD was acquired at 30 min. Therefore, in this study, we adapted the preincubation period for these media in experiment 1. As a result, MBCD has high potential for capacitating sperm and can greatly enhance the fertilization rate compared with BSA. On the other hand, most of the C57BL/6J mouse sperm remain in a cryo-induced damaged state after freezing and thawing. 10 In this study, we tried to overcome the problem by modifying the R18S3 with the addition of L-glutamine. As a result, we confirmed the cryoprotective effect of L-glutamine for C57BL/6J mouse sperm and obtained a high, stable rate of fertilization via a novel system of mouse sperm cryopreservation and IVF using R18S3 with L-glutamine and MBCD in TYH (Tables 1 and 2). There are many reports stating that L-glutamine acts a cryoprotectant agent for sperm freezing and thawing in several mammalian species. 15 20 The addition of L-glutamine to CPA maintains post-thaw motility well and reduces plasma membrane damage to sperm. Moreover, L-glutamine protects small unilamellar vesicles composed of 75% palmitoluoleoyl phosphatidylcholine and 25% phosphatidylserine against damage during freezing. 25 The cryoprotective effects of L-glutamine on the lipid layer membrane depend on the ionic interaction between the positive charged amine group of amino acids and the negatively charged phospholipids. Previously, Liu et al. 26 showed that R18S3 with amino acids increased the fertility of frozen/thawed sperm in three substrains of C57BL/6J mice. L-glutamine is one of the main amino acids used. Considering these results, the distinctive effect of L-glutamine may add to the conventional CPA composed of raffinose pentahydrate and skim milk, resulting in an excellent capacity to protect sperm against freezing and thawing stress. However, detailed mechanisms regarding the cryoprotective effect of L-glutamine on mouse sperm are not fully clear, so further studies will be required. Recently, there have been some reports of mouse sperm cryopreservation using modified R18S3. 27,28 Yildiz et al. 27 reported membrane permeating polyols such as glycerol and fructose have protective effects for sperm cryopreservation when added to the modified R18S3. Ostermeier et al. 28 showed that a reducing reagent of alphamonothioglycerol added to R18S3 protects the frozen/ thawed sperm. These reports demonstrated that modified R18S3 tends to enhance the fertilizing ability of frozen/ thawed C57BL/6J mouse sperm. However, they did not investigate the individual variability of fertilization rate in each male. In this study, we compared the fertilizing ability of frozen/thawed C57BL/6J mouse sperm in 10 males. As a result, we obtained stable and relatively high rates of fertilization (between 35.1% and 82.0%) in all males. Therefore, our novel system will become practical and available for sperm cryopreservation and IVF of numerous geneticallyengineered mice strains with a C57BL/6J background. In conclusion, we suggest that L-glutamine is a useful compound for cryopreserving sperm taken from C57BL/6J mice. Moreover, we suggest that a novel sperm cryopreservation and IVF system using modified R18S3 with L-glutamine and TYH with MBCD can provide a reliable archiving and producing system for genetically-engineered mice using sperm cryopreservation. ACKNOWLEDGEMENTS We wish to thank T Kondo, K Fukumoto, Y Nakagawa, Y Takeshita and Y Nakamuta for excellent technical assistance and the Center for Animal Resources and Development, Kumamoto University for its important contributions to the experiments. REFERENCES 1 Thornton CE, Brown SD, Glenister PH. Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses. Mamm Genome 1999;10:987 92 2 Critser JK, Mobraaten LE. Cryopreservation of murine spermatozoa. ILAR J 2000;41:197 206 3 Landel CP. Archiving mouse strains by cryopreservation. Lab Anim (NY) 2005;34:50 7 4 Sakuraba Y, Sezutsu H, Takahasi KR, et al. Molecular characterization of ENU mouse mutagenesis and archives. Biochem Biophys Res Commun 2005;336:609 16 5 Davisson M. FIMRe: Federation of International Mouse Resources: global networking of resource centers. Mamm Genome 2006;17:363 4
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