Journal of the American Association for Laboratory Animal Science Copyright 2009 by the American Association for Laboratory

Animal Science

Vol 48, No 1 January 2009 Pages 3943

Sperm Freezing and In Vitro Fertilization in Three Substrains ofC57BL/6Mice

Ling Liu,1,2 Lauryl M J Nutter,1,2 Napoleon Law,1,2 and Colin McKerlie1,2,* Traditional protocols for sperm recovery, cryopreservation, and in vitro fertilization (IVF) have been considerably less efficient for inbred mouse strains, including C57BL/6, than for hybrid and outbred strains. We report here that 3 changes to published and widely used protocols markedly improved fertilization rates for both fresh and frozenthawed sperm in 3 substrains of C57BL/6 mice (C57BL/6J, C57BL/6NCrl, and C57BL/6NTac). First, the traditional cyroprotective agent was modified by adding amino acids. Second, preincubation of sperm in a preincubation medium containing methyl--cyclodextrin and polyvinyl alcohol enabled collection of progressively motile sperm for IVF. Third, we evaluated 3 media for IVF: human tubal fluid (HTF), modified KrebsRinger bicarbonate medium (TYH), and minimal essential medium (MEM). HTF and TYH were modified by adding minimal essential amino acids. The methodology reported here increased the IVF rate of both fresh and frozenthawed sperm and enabled efficient isolation of capacitated viable sperm. Fertilization rates greater than 65% and 40% were obtained with the 3 tested substrains when fresh and frozenthawed sperm, respectively, were used for IVF. Higher fertilization rates were seen with frozenthawed sperm from C57BL/6NCrl and C57BL/6NTac mice than from C57BL/6J mice. Among all strains, fresh sperm from C57BL/6NTac mice gave the highest fertilization rate. Of 190 two-cell embryos, 63 (33.2%) developed to term after transfer to pseudopregnant recipient mice. The protocol we detail here provides reliable cryopreservation and recovery of live mice in 3 substrains of C57BL/6, making sperm cryopreservation and IVF a viable choice for preservation and distribution of mouse lines. Abbreviations: CPA, cyroprotective agent; mCPA, modified cryoprotective agent; HTF, human tubal fluid; IVF, in vitro fertilization; MEM, minimal essential medium; PM, preincubation medium; TYH, modified KrebsRinger bicarbonate medium.

An exponential increase in the number of mouse lines with induced mutations (including transgenes, targeted mutations, and chemically-induced mutations) from laboratories and commercial suppliers around the world has resulted in a concomitant increase in lines that need to be preserved or distributed for biomedical research.4,17,31,33 Yet, at the same time, the technical challenges and limitations associated with shipping live mice have increased dramatically due to increased airport security,2 ethical concerns, and other impediments, thereby limiting efficient distribution of important mouse models of human disease. Successful and efficient collection, cryopreservation, and reanimation of mouse sperm would be an ideal solution for preservation and distribution of these important in vivo models, whether for biomedical research or for drug discovery and development. However, the current methods for these processes have proven inefficient, difficult to perform, and rate-limiting. Mouse sperm is very sensitive to diverse stresses including mechanical, osmotic, and oxidative conditions.19-21,33 The raffinoseskim milk method25,28,35,36 currently predominates in many laboratories worldwide that routinely cryopreserve and store mouse sperm.22,23,38 However, high-efficiency reanimation appears to be restricted to mice with hybrid or outbred backgrounds and is considerably less effective for inbred mouse strains, including C57BL/6.35 This situation has a considerable effect on mouse models used for biomedical research, given that many transgenic and knockout lines are backcrossed onto inbred backgrounds, most frequently C57BL/6.35
Received: 19 May 2008. Revision requested: 16 Jul 2008. Accepted: 06 Aug 2008. 1Canadian Mouse Mutant Repository and 2Physiology and Experimental Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. *Corresponding author. Email: colin.mckerlie@phenogenomics.ca

In the present study, we focused on 3 aspects for improvement. First, the traditional cryoprotective agent containing a cryoprotectant (raffinose) and membrane protectant (skim milk) was modified to provide better cryoprotection during the freezing and thawing process. Second, frozenthawed sperm suspensions underwent a preincubation procedure to enrich for progressively motile sperm. Finally, we compared 3 fertilization media and 3 substrains by using established criteria to select an optimal in vitro fertilization (IVF) medium.

Animals. C57BL/6 mice used in this experiment were obtained from Charles River Canada (C57BL/6NCrl, Montreal, Quebec, Canada), the Jackson Laboratory (C57BL/6J, Bar Harbor, ME), and Taconic Farms (C57BL/6NTac, Germantown, NY). Female mice were ordered at 4 to 5 wk of age and male mice at 5 to 6 wk of age. The mice were maintained under routine husbandry procedures in exclusion barrier facilities for 1 to 3 wk at the Toronto Centre for Phenogenomics (Toronto, Ontario, Canada). All animal use protocols were reviewed by the Animal Care Committee at The Hospital for Sick Children and conform to guidelines of the Canadian Council on Animal Care.5,6 All animals received ad libitum water and standard mouse diet and were maintained on a 12:12-h light:dark cycle. Media. Chemicals and mineral oil were obtained from Sigma (St Louis, MO). Water (resistance, 18.2 Ohm) was obtained in house from a water purification system (Millipore, Billerica, MA). Human tubal fluid (HTF) and modified KrebsRinger bicarbonate medium (TYH) were prepared on the day before use. Both HTF and TYH were modified by adding 1% minimal essential amino acids solution (50) and 0.5% nonessential
39

Materials and Methods

Vol 48, No 1 Journal of the American Association for Laboratory Animal Science January 2009

amino acid solution (100; Life Technologies, Carlsbad, CA). HTF, TYH, or minimal essential medium (MEM; Life Technologies) was used for IVF. Sperm preincubation medium (PM) was prepared by adding 1 mg/ml methyl--cyclodextrin and 1 mg/ ml polyvinyl alcohol to the IVF media. The basic cyroprotective agent (CPA; 18% raffinose and 3% skim milk) was prepared in the laboratory and stored at 4C for as long as 6 mo. Modified cryoprotective agent (mCPA) was made by adding 20 l of minimal essential amino acids solution (50) per milliliter CPA before use. Sperm collection and freezing. Male mice were euthanized by cervical dislocation, and the 2 caudal epididymides were removed aseptically and placed into 110 L CPA or mCPA in a 35-mm culture dish. The epididymides were minced 5 to 6 times by using forceps and scissors, and sperm was allowed to disperse by gently shaking the dish by hand for 3 to 5 min at room temperature. To load sperm for cryopreservation, a 1-mL syringe was attached to a labeled 0.25-mL Cassou straw followed by sequential aspiration of 50 mm IVF medium, 10 mm air, 10 L sperm suspension, and 10 mm air at room temperature. Both ends of the straw were sealed with an MP4 impulse sealer (JJ Elmer Corporation, St Louis, MO). Eight samples were prepared in sequence in the same manner. An additional sample of 10 L was used for sperm evaluation. Each of the 8 samples was placed into a freezing canister (made in the laboratory as described previously26) and floated on liquid nitrogen in a liquid nitrogen container. After 10 min, the freezing canister was submerged in liquid nitrogen. The samples were transferred to a storage container for long-term storage in the liquid phase of liquid nitrogen. Frozen sperm was stored in liquid nitrogen for at least 2 d before being thawed and used in subsequent experiments. Sperm thawing and preincubation. Each frozen straw was removed and immersed in a 37C water bath for 5 to 10 min. The straw was removed from the water bath and wiped dry, both ends of the straw were cut, and only the sperm suspension was expelled into the center of a flattened 90-L drop of PM and incubated for 40 to 45 min in humidified 5% CO2 in air at 37C. Progressively motile sperm were isolated by collecting sperm that had migrated to the periphery of the drop (horizontal collection). Collection of oocytes and IVF. Female mice were superovulated by 6 IU pregnant mare serum gonadotropin (Intervet Canada, Whitby, Ontario, Canada) IP, followed 46 to 48 h later by 6 IU human chorionic gonandotropin (Intervet Canada) IP, and euthanized 14 to 18 h thereafter. The oviducts were removed and dissected free from surrounding tissues using fine needles; 5 to10 cumulusoocyte masses were transferred into each well of a 4-well plate (Nunc Nunclon surface culture plate, Thermo Fisher Scientific, Rochester, NY). Each well contained 500 L IVF medium per well (TYH, HTF or MEM, modified as described earlier). With a 20-L micropipette, 10 to 20 L of the sperm suspension from the edge of the PM drop was transferred into fertilization wells containing oocytes (final sperm concentration of approximately 10 106/mL). The oocytes and sperm were incubated for 4 h in humidified 5% CO2 in air at 37C. The fertilized oocytes were washed twice with fresh IVF medium and then cultured for an additional 24 h. After the 24-h incubation, the fertilization rate was determined as the proportion of 2-cell stage embryos among all oocytes. The number of embryos at each developmental stage was counted. Sperm collection, freezethawing, and IVF were done 3 to 5 times for each substrain, using 1 or 2 sperm donors and 10 to 20 oocyte donors in each trial. Sperm and oocytes from the same substrain were
40

pooled from donors in the same trial. Total sperm and oocyte donor numbers are reported for each strain. Embryo transfer. Two-cell embryos generated from frozen thawed sperm of each substrain were pooled and transferred to both sides of oviducts of 0.5-d pseudopregnant CD1 mice. Each recipient received 10 to 20 embryos. Embryos were allowed to develop to term, and the number of pups born was recorded. Statistics. Statistical analysis was performed (Prism version 5.0, GraphPad, San Diego, CA) using a 2 test to assess development to 2-cell embryos among strains or media. In no cases were fewer than 5 events observed in any category. Differences were considered to be significant when a P value less than 0.05 was achieved. Error bars on figures represent the SEM of the IVF rate.

Comparison of methods for sperm freezing and IVF. Sperm from 6 C57BL/6NCrl mice was isolated and frozen in CPA or mCPA. After thawing, sperm frozen with CPA were used for IVF directly, and sperm frozen with mCPA were preincubated for 45 min and then used for IVF of oocytes collected from 70 superovulated C57BL/6NCrl mice. Experiments were performed 6 times with MEM as the IVF medium. The fertilization rate was significantly (P < 0.01) higher when sperm were frozen in mCPA and subsequently preincubated than when frozen in traditional CPA and used without preincubation (Figure 1). Effect of sperm preincubation on the IVF rate of C57BL/6J sperm. Sperm from 2 C57BL/6J mice was frozen in mCPA. After thawing, sperm were preincubated for the specified time and used for IVF of oocytes pooled from 40 superovulated C57BL/6J mice. Experiments were done in duplicate by using TYH as the IVF medium. The fertilization rate was significantly (P < 0.01) higher when sperm was preincubated for 20 or 45 min compared with 60 min (Figure 2). The 20- and 45-min groups did not differ significantly. Effect of IVF media on the IVF rate of C57BL/6NCrl sperm. Fresh and frozenthawed sperm from 9 C57BL/6NCrl mice was preincubated in PM for 40 to 45 min and used to fertilize oocytes collected from 124 superovulated C57BL/6NCrl mice. Experiments were performed 2 to 6 times using MEM, TYH, or HTF as IVF media. The fertilization rate with frozenthawed sperm was significantly higher in MEM than TYH (P < 0.05) or HTF (P < 0.01); the rate was higher (P < 0.01) with TYH than HTF (Figure 3). No significant difference was seen between TYH and HTF with fresh sperm, but MEM supported significantly (P < 0.01) higher fertilization rates than did TYH and HTF when fresh sperm was used. Effect of IVF media on IVF rate of C57BL/6NTac sperm. Fresh and frozenthawed sperm from 6 C57BL/6NTac mice was preincubated in the indicated PM for 40 to 45 min and used for IVF of oocytes collected from 60 superovulated C57BL/6NTac mice. Experiments were done in triplicate with MEM and TYH as IVF media. Fertilization rates were significantly (P < 0.01) higher when MEM was used for IVF with fresh sperm than when TYH was used or when frozenthawed sperm was used (Figure 4). No significant difference was seen between other media conditions with either fresh or frozen sperm. Effect of IVF media on IVF rate of C57BL/6J sperm. Fresh and frozenthawed sperm from 6 C57BL/6J mice was preincubated in the specified PM for 40 to 45 min and used to fertilize oocytes collected from 60 superovulated C57BL/6J mice. Experiments were done in triplicate with MEM and TYH as IVF media. Fertilization rates were significantly (P < 0.01) higher in groups when fresh sperm was used than when frozen-thawed sperm

Results

Sperm freezing and IVF for B6 mice

Figure 1. Comparison of sperm freezing and IVF methods. The IVF rate for sperm cryopreserved in mCPA and preincubated in PM (new) is significantly (P < 0.01) higher than that for sperm cryopreserved in CPA and used for direct fertilization (old).

Figure 3. Effect of IVF media on IVF rate of C57BL/6NCrl sperm. The IVF rates for sperm used with various PM and IVF media are shown. Different lowercase letters indicate significantly different IVF rates (2 test; a versus b, P < 0.01; a versus c, P < 0.01; b versus c, P < 0.05; b versus d, P < 0.01).

Figure 2. Effect of sperm preincubation time on IVF rate of C57BL/6J sperm. The IVF rate for sperm incubated in PM for the specified time is shown. Different lowercase letters indicate significantly different IVF rates (2 test; a versus b, P < 0.01).

was used (Figure 5). No significant difference was seen between TYH and MEM with either fresh or frozenthawed sperm. Effect of C57BL/6 substrain on IVF rate. Fresh and frozen thawed sperm from 4 C57BL/6J, 6 C57BL/6NCrl, or 3 C57BL/6NTac mice were separately preincubated for 40 to 45 min in PM and used for IVF of oocytes collected from 60 C57BL/6J, 70 C57BL/6NCrl, or 30 C57BL/6NTac superovulated mice, respectively. Experiments were repeated 2 to 3 times in MEM. Fertilization rates with fresh sperm were significantly (P < 0.05) higher in the C57BL/6NTac group compared with C57BL/6NCrl and C57BL/6J groups (Figure 6). No significant difference was observed between C57BL/6NCrl and C57BL/6J groups when fertilized with fresh sperm. Significantly (P < 0.05) fewer oocytes developed to 2-cell stage embryos in C57BL/6J mice compared with C57BL/6NCrl mice when frozenthawed sperm was used. No significant differences were found between C57BL/6NCrl and C57BL/6NTac groups when frozenthawed sperm was used. Liveborn mice from frozen sperm. In total, 190 two-cell embryos were transferred to pseudopregnant recipient mice (10 to 20 embryos per recipient), and 63 (33.2%) live pups were born 19 to 20 d later.

Figure 4. Effect of IVF media on IVF rate of C57BL/6NTac sperm. The IVF rates for sperm used with various PM and IVF media are shown. Different lowercase letters indicate significantly different IVF rates (2 test; a versus b, P < 0.01).

The archiving of mouse strains by sperm cryopreservation has great potential because it is simple, rapid, and inexpensive.

Discussion

However, for some of the most commonly used inbred strains, including C57BL/6, the progressive motility of thawed sperm (less than 10%)1,3 and subsequent fertilization rate (0% to 12%) is too low3 to provide efficient cryopreservation and predictable recovery of liveborn offspring. The traditional approach uses a CPA containing 18% raffinose and 3% skim milk and does not provide sufficient cryoprotection for C57BL/6 mouse sperm. Raffinose is a nonpenetrating trisaccharide incapable of moving across cell membranes. Its role as a plasma membraneimpermeant uncharged molecule in the medium is to provide the hypertonicity needed for cell desiccation prior to freezing. Skim milk is a macromolecule that acts as a buffer and diluent to stabilize or assist in stabilizing the cell membrane.34 The cell membrane of spermatozoa differs from that of other cell types and contains high concentrations of polyunsaturated fatty acids that are highly susceptible to damage by free radicals generated during freezing and thawing.18,24 Supplementation with any free-radical scavenger, such as amino acids, likely would be compatible with enhanced viability of sperm by effectively removing or inactivating free radicals. In addition, supplementation of media with amino acids may both stimulate the growth and prolong the viability of cells in culture. L-arginine plays an important role in stimulating sperm motility in humans, rabbits, and goats in vitro by preventing sperm membrane lipid peroxi41

Vol 48, No 1 Journal of the American Association for Laboratory Animal Science January 2009

Figure 5. Effect of IVF media on IVF rate of C57BL/6J sperm. The IVF rates for sperm used with various PM and IVF media are shown. Different lowercase letters indicate significantly different IVF rates (2 test; a versus b, P < 0.01).

Figure 6. Comparison of IVF rate among 3 C57BL/6 substrains. The IVF rates for fresh and frozen-thawed sperm from different C57BL/6 substrains are shown. Different letters indicate significantly different IVF rates (2 test; a versus b, P < 0.05; A versus B, P < 0.05). Conditions indicated by uppercase letters were not compared with those indicated by lowercase letters.

dation.32 In the present studies, essential amino acids solution, including L-arginine, was added to the traditional CPA solution to create mCPA. The use of this mCPA significantly increased the fertilization rate of sperm frozen in mCPA compared with the traditional CPA solution (Figure 1). Why frozen-thawed C57BL/6 sperm has difficulty penetrating the intact zona pellucida during IVF is unclear. This inability may be related to the low percentage of progressive motility in frozenthawed samples26,27 or to the presence of numerous dead sperm during IVF.29 Traditionally, IVF is conducted by directly adding frozenthawed sperm suspensions into the fertilization medium or by collecting motile sperm by the vertical swim-up method.16 Subsequent fertilization rates for inbred strains have been extremely variable and low (0% to17%),3,10 possibly due to damage to the frozen-thawed sperm, resulting in poor recovery of motile cells or the presence of an overwhelming and large number of dead sperm.29,35 Removing cell debris and dead sperm from sperm suspensions greatly improved IVF.3 In the present study, the PM drop was flattened and the thawed sperm suspension was loaded in the central area of the PM drop. Motile sperm swam out of the drop horizontally, instead of up, to form a wedge-shaped zone. This zone was filled only with progressively motile sperm. Collection of these
42

sperm from the periphery of the PM drop enables recovery of large numbers of progressively motile sperm isolated from the dead sperm in frozenthawed samples. An improved IVF rate of frozenthawed C57BL/6 (Japanese substrain) mouse sperm was reported recently with a similar preincubation of sperm for 30 to 60 min.37 Fertilization occurs in mammals after sperm undergo various biochemical and physiologic changes in the female reproductive tract, a process termed capacitation.7 Capacitated sperm can then undergo the acrosomal reaction, a morphologic alteration consisting of a series of point fusions between the outer acrosomal membrane and the overlying plasma membrane.40 Cholesterol loss from the surface membrane of sperm is one of the molecular events of capacitation.11 Removal or loss of cholesterol from the plasma membranes of sperm can be mediated by cholesterol acceptors present in the fluid of the female reproductive tract, such as albumin12,14 and sterol-binding protein.13 Polyvinyl alcohol has been used as a substitute for bovine serum albumin to facilitate sperm capacitation in vitro.8 Cyclodextrins, cyclic oligosaccharides consisting of 6, 7, or 8 glucopyranose units, originate from enzymatic degradation of starch through the action of cyclodextrin glucanotransferase.15 Cyclodextrins function as carrier molecules and dissolve lipophiles in their hydrophobic core.30 Among the 3 kinds of cyclodextrins, -cyclodextrin has a suitable structure to encase natural and synthetic molecules such as hormones, vitamins, and lipid components.39 -Cyclodextrin first was reported to enable and induce mouse sperm capacitation by removing cholesterol from the sperm membrane. The concentration of -cyclodextrin was optimized to 0.75 mM with fresh sperm in outbred mice (Japanese substrain).9 More recently, improved IVF with frozenthawed C57BL/6J (Japanese substrain) mouse sperm occurred when sperm was preincubated in 0.75 to 1.5 mM of -cyclodextrin for 30 to 60 min. The onset of cholesterol efflux from the frozenthawed sperm treated with -cyclodextrin occurred earlier than it did in fresh sperm. The increase in fertilization rate was associated with the efflux of cholesterol from sperm.37 In the present study, fertilization rates in all 3 C57BL/6 substrains increased significantly when sperm were incubated in PM including -cyclodextrin at 1 mg/ml (approximately 1 mM) for 45 min before being used for IVF. These data indicate that mouse sperm were capacitated more efficiently after preincubation and are consistent with previous reports.9,37 More than 10 media have been used for IVF in the mouse over the past decade. The most frequent media used for inbred strains of mice have been HTF, TYH, and MEM.7,34,35 Our results show that MEM supported comparable or improved fertilization rates for either fresh or frozenthawed sperm (Figures 3 through 5) from all 3 C57BL/6 substrains evaluated. Fresh sperm supported higher fertilization rates than did frozenthawed sperm, regardless of the substrain of C57BL/6 mice (Figure 6). Frozenthawed sperm yielded a lower fertilization rate in C57BL/6J mice than in C57BL/6NCrl mice; fertilization rates in C57BL/6NTac mice were similar to those of the C57BL/6NCrl substrain. In general, the fertilization rates of all 3 substrains exceeded 40%. Embryo quality was confirmed through the production of liveborn pups after transfer of IVFderived 2-cell embryos to pseudopregnant recipient mice at a rate comparable to that previously reported.3,35 The data presented here show that the inclusion of minimal essential amino acids in a sperm cryoprotective agent significantly increases the fertilization rate obtained by using frozenthawed sperm and unassisted IVF. This improvement may be due to free-radical scavenging action of amino acids or

Sperm freezing and IVF for B6 mice

the known activation effect of L-arginine on spermatozoa and other cell types. Furthermore, our data show that IVF rates for both fresh and frozenthawed sperm are improved after preincubation and horizontal collection of motile sperm in media containing -cyclodextrin and polyvinyl alcohol, agents thought to contribute to sperm capacitation in vitro. Compared with C57BL/6J mice, C57BL/6NCrl and C57BL/6NTac mice supported higher fertilization rates when frozenthawed sperm was used. C57BL/6NTac mice produced higher fertilization rates when fresh sperm was used. In conclusion, we detail a protocol that provides reliable cryopreservation and recovered fertility rates of C57BL/6 sperm that make sperm cryopreservation a viable and efficient alternative for preservation and distribution of valuable mouse lines.
1. An TZ, Iwakiri M, Edashige K, Sakurai T, Kasai M. 2000. Factors affecting the survival of frozenthawed mouse spermatozoa. Cryobiology 40:237249. 2. Awasthi PR, French J, Sztien R, Bedigian R, Sharp JJ, Lloyd KC. 2003. Frozen sperm as an alternative to shipping live mice. Contemp Top Lab Anim Sci 42:811. 3. Bath ML. 2003. Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm. Biol Reprod 68:1923. 4. Brown SDM, Nolan PM. 1998. Mouse mutagenesis systematic studies of mammalian gene fuction. Hum Mol Genet 7:1627 1633. 5. Canadian Council on Animal Care. 1993. Guide to the care and use of experimental animals, vol. 1, 2nd edn. Ottawa (Canada): Canadian Council on Animal Care. Available at http://www.ccac. ca/en/CCAC_Programs/Guidelines_Policies/PDFs/ExperimentalAnimals_GDL.pdf. 6. Canadian Council on Animal Care. 1984. Guide to the care and use of experimental animals, Vol. 2. Ottawa (Canada): Canadian Council on Animal Care. Available at http://www.ccac.ca/en/ CCAC_Programs/Guidelines_Policies/PDFs/ExperimentalAnimalsV2_GDL.pdf. 7. Chang MC. 1951. Fertilizing capacity of spermatozoa deposited into fallopian tubes. Nature 168:697698. 8. Choi YH, Landim-Alvarenga FC, Seidel GE Jr, Aquires EL. 2003. Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes. J Anim Sci 81:20802087. 9. Choi YH, Toyoda Y. 1998. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol Reprod 59:13281333. 10. Cister JK, Mobraaten LE. 2000. Cryopreservation of murine spermatozoa. ILAR J 41:197-206. 11. Davis BK. 1981. Timing of fertilization in mammals: sperm cholesterol:phospholipid ratio as a determinant of the capacitation interval. Proc Natl Acad Sci USA 78:75607564. 12. Davis BK, Byrne R, Bedigian K. 1980. Studies on the mechanism of capacitation: albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells. Proc Natl Acad Sci USA 77:15461550. 13. Ehrenwald E, Foote RH, Parks JE. 1990. Bovine oviductal fluid components and their potential role in sperm cholesterol efflux. Mol Reprod Dev 25:195204. 14. Go KJ, Wolf DP. 1985. Albumin-mediated changes in sperm sterol content during capacitation. Biol Reprod 32:145153. 15 . Haga K, Harata K, Nakamura A, Yamane K. 1994. Crystallization and preliminary X-ray studies of cyclodextrin glucanotransferase from alkalophilic Bacillus spp. J Mol Biol 237:163164. 16. Hinting A, Lunardhi H. 2001. Better sperm selection for intracytoplasmic sperm injection with the side migration technique. Andrologia 33:343346. 17. Hrabe de Angelis M, Balling R. 1998. Large-scale ENU screens in the mouse: genetics meets genomics. Mutat Res 400:2532. 18. Kasimanickam R, Kasimanickam V, Thatcher CD, Nebel RL, Cassell BG. 2007. Relationships among lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in dairy bulls. Theriogenology 67:10041012.

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