**5. Concluding remarks**

In this chapter described in detail the effects of the various components of cryodiluent that are used for rat sperm cryopreservation. We found that lactate, ATP and dbcAMP conferred freezability on rat sperm and enhanced oocyte fertilization by frozen-thawed sperm. In addition, if the rat sperm can be controlled flagellar movement in a way that changed by energy-yielding substrates such as lactate, it would be possible to enhance their fertilizing ability of rat sperm for *in vitro* fertilization by using a both of the fresh and frozen-thawed sperm (Fig. 7). However, there is still a lack of information for physiological trigger

Fig. 7. Representative patterns of rat sperm movement which were extended in the presence (A) or absence (C and D) of lactate in raffinose-mKRB egg yolk solution. In B, sperm was incubated at 37°C for 3 h in the lactate containing solution. Panels a to l indicate the turn. Scale bars = 50μm

Cryopreservation of Rat Sperm 175

Cook, S.P., & Babcock, D.F. (1983). Activation of Ca2+ permeability by cAMP is

Cummins, J.M., & Woodall, P.F. (1986). On mammalian sperm dimensions. *J Reprod Fertil.*

Degasperi, G.R., Velho, J.A., Zecchin, K.G., Souza, C.T., Velloso, L.A., Borecký, J., Castilho,

Fujinoki, M., Kawamura, T., Toda, T., Ohtake, H., Ishimoda-Takag,i T., Shimizu, N.,

Gallina, F.G., Deburgos, N.M.G., Burgos, C., Coronel, C.E., & Blanco, A. (1994). The lactate–

Geurts, A.M., Cost, G.J., Freyvert, Y., Zeitler, B., Miller, J.C., Choi, V.M., Jenkins, S.S., Wood,

Gibbons, I.R. (1963). Studies on the protein components of cilia from Tetrahymena

Gibbs, R.A., Weinstock, G.M., Metzker, M.L. et al., (2004). Genome sequence of the Brown Norway rat yields insights into mammalian evolution. *Nature* 428, pp. 493–521. Hagiwara, M., Choi, J.H., Devireddy, R.V., Roberts, K.P., Wolkers, W.F., Makhlouf, A., &

Harris, S.E., Gopichandran, N., Picton, H.M., Leese, H.J., & Orsi, N.M. (2005). Nutrient

Holt, W.V., Head, M.F., & North, R.D. (1992). Freeze-induced membrane damage in ram

Izsvak, Z., Fröhlich, J., Grabundzija, I., Shirley, J.R., Powell, H.M., Chapman, K.M., Ivics, Z.,

Kinukawa, M., Oda, S., Shirakura, Y., Okabe, M., Ohmuro, J., Baba, S.A., Nagata, M., &

cancer: a central role for Ca2+. *J Bioenerg Biomembr.* 38, pp. 1–10.

Garbers, D.L. (2001). Ion channels. Swimming with sperm. *Nature* 413, pp. 581–582.

pyriformis. *Proc Natl Acad Sci USA.* 50, pp. 1002–1010.

predicts post-thaw motility. *Biol Reprod*.1, pp. 700–706 Hamra, F.K. (2010). Gene targeting: Enter the rat. *Nature* 467, pp. 211–213.

cryomicroscopy. *Biol Reprod*. 46, pp. 1086–1094.

spermatogonial stem cells. *Nat Methods*. 7, pp. 443–445.

Jacob, H.J. (1999). Functional genomic and rat model. *Genome Res.* 9, pp. 1013–1016.

*Theriogenology* 64, pp. 992–1006.

22408–22413.

75, pp. 153–175.

137, pp. 509–520.

515–519.

433.

coordinated through the pHi increase induced by speract. *J Biol Chem.* 268, pp.

R.F., & Vercesi, A.E. (2006). Role of mitochondria in the immune response to

Yamaoka, S., & Okuno, M. (2004). Identification of 36 kDa phosphoprotein in fibrous sheath of hamster spermatozoa. *Comp Biochem Physiol B Biochem Mol Biol.*

pyruvate shuttle in spermatozoa: operation in vitro. *Arch Biochem Biophys*. 308, pp.

A., Cui, X., Meng, X., Vincent, A., Lam, S., Michalkiewicz, M., Schilling, R., Foeckler, J., Kalloway, S., Weiler, H., Ménoret, S., Anegon, I., Davis, G.D., Zhang, L., Rebar, E.J., Gregory, P.D., Urnov, F.D., Jacob, H.J., & Buelow, R. (2009). Knockout rats via embryo microinjection of zinc-finger nucleases. *Science* 325, pp.

Bischof, J.C. (2009). Cellular biophysics during freezing of rat and mouse sperm

concentrations in murine follicular fluid and the female reproductive tract.

spermatozoa is manifested after thawing: observations with experimental

& Hamra, F.K. (2010). Generating knockout rats by transposon mutagenesis in

Aoki, F. (2006). Roles of cAMP in regulating microtubule sliding and flagellar bending in demembranated hamster spermatozoa. *FEBS Lett*. 580, pp. 1515–1520.

regarding how rat sperm switches on their flagellar movement at the real-time of fertilization, such as "hyper-activation" or "ultra-activation" (Yamashiro et al., 2009). Further, we believe that the *IVF* medium developed in our study is effective for the *in vitro* production of embryos from cryopreserved rat sperm. These results not only indicate that the cryopreservation of rat sperm with the present method can be applied to reproductive technologies but also indicate that exogenous lactate, rather than glucose and pyruvate, exerts a mediating effect on energy-dependent synthetic processes.

In conclusion, this is one of the very few information in the field that actually test the components of a cryodiluent in a logical manner for rat sperm cryopreservation. From this point of view, the chapter is likely to be important not only for freezing of rat sperm but also for freezing of sperm of other species in general.

#### **6. References**


regarding how rat sperm switches on their flagellar movement at the real-time of fertilization, such as "hyper-activation" or "ultra-activation" (Yamashiro et al., 2009). Further, we believe that the *IVF* medium developed in our study is effective for the *in vitro* production of embryos from cryopreserved rat sperm. These results not only indicate that the cryopreservation of rat sperm with the present method can be applied to reproductive technologies but also indicate that exogenous lactate, rather than glucose and pyruvate,

In conclusion, this is one of the very few information in the field that actually test the components of a cryodiluent in a logical manner for rat sperm cryopreservation. From this point of view, the chapter is likely to be important not only for freezing of rat sperm but also

Agca, Y., & Critser, J.K. (2002). Cryopreservation of spermatozoa in assisted reproduction.

Aitman, T.J., Critser, J.K., Cuppen, E., Dominiczak, A., Fernandez-Suarez, X.M., Flint, J.,

Aoki, F., Sakai, S., & Kohmoto, K. (1999). Regulation of flagellar bending by cAMP and Ca2+

Brokaw, C.J. (1972). Flagellar movement: a sliding filament model. *Science* 178, pp. 455–

Brooks, G.A., Dubouchaud, H., Brown, M., Sicurello, J.P., & Butz, C.E. (1999). Role of

Bunch, D.O., Welch, J.E., Magyar, P.L., Eddy, E.M., & O'Brien, D.A. (1998). Glyceraldehyde

Canzian, C. (1997). Phylogenetics of the laboratory rat rattus norvegicus. *Genome Res.* 7, pp.

Cao, W., Haig-Ladewig, L., Gerton, G.L. & Moss, S.B. (2006). Adenylate kinases 1 and 2 are

Cardullo, R.A. & Baltz, J.M. (1991). Metabolic regulation in mammalian sperm:

Chularatnatol, M. (1982). Motility initiation of quiescent spermatozoa from rat caudal

mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular

3-phosphate dehydrogenase-S protein distribution during mouse spermatogenesis.

part of the accessory structures in the mouse sperm flagellum. *Biol Reprod.* 75, pp.

Mitochondrial volume determines sperm length and flagellar beat frequency. *Cell* 

epididymis: effects of pH, viscosity, osmolality and inhibitors, *Int J Androl.* 5, pp.

Gauguier, D., Geurts, A.M., Gould, M., Harris, P.C., Holmdahl, R., Hubner, N., Izsvák, Z., Jacob, H.J., Kuramoto, T., Kwitek, A.E., Marrone, A., Mashimo, T., Moreno, C., Mullins, J., Mullins, L., Olsson, T., Pravenec, M., Riley, L., Saar, K., Serikawa, T., Shull, J.D., Szpirer, C., Twigger, S.N., Voigt, B., & Worley, K. (2008). Progress and prospects in rat genetics: a community view. *Nat Genet.* 40, pp. 516–

exerts a mediating effect on energy-dependent synthetic processes.

in hamster sperm. *Mol Reprod Dev.* 53, pp. 77–83.

lactate shuttle. *Proc Natl Acad Sci USA.* 96, pp. 1129–1134. Brooks. G,A. (2002). Lactate shuttles in nature. *Biochem Soc Trans.* 30, pp. 258–264.

for freezing of sperm of other species in general.

Semin Reprod Med. 20, pp.15–23.

*Biol Reprod.* 58, pp. 834–841.

*Motil Cytoskeleton.* 19, pp. 180–188.

**6. References** 

522.

462.

262–267.

492–500.

425–436.


Cryopreservation of Rat Sperm 177

Ruiz-Pesini, E., Díez-Sánchez, C., López-Pérez, M.J., & Enríquez, J.A. (2007). The role of the

Seita, Y., Sugio, S., Ito, J., & Kashiwazaki, N. (2009a). Generation of live rats produced by

Seita, Y., Ito, J., & Kashiwazaki, N. (2009b). Removal of acrosomal membrane from sperm

Si, W., Benson, J.D., Men, H., & Critser, J.K. (2006). Osmotic tolerance limits and effects of

Suarez, S.S., Marquez, B., Harris, T.P., & Schimenti, J.C. (2007). Different regulatory systems

Tong, C., Li, P., Wu, N.L., Yan, Y., & Ying, Q.L. (2010). Production of p53 gene knockout

Varisli, O., Uguz, C., Agca, C., & Agca, Y. (2009a). Various physical stress factors on rat

Varisli, O., Uguz, C., Agca, C., & Agca, Y. (2009b). Effect of chilling on the motility and

Vasudevan, K., Sztein, J.M. Treatment of sperm with extracellular adenosine 5'-triphosphate

Warner, F.D., & Mitchell, D.R. (1980). Dynein: the mechanochemical coupling adenosine

Yamashiro, H., Han, Y.J., Sugawara, A., Tomioka, I., Hoshino, Y., & Sato, E. (2007).

Yamashiro, H., Toyomizu, M., Kadowaki, A., Takeda, Z., Nakazato, F., Toyama, N.,

Yamashiro, H., Toyomizu, M., Kikusato, M., Toyama, N., Sugimura, S., Hoshino, Y., Abe, H.,

Yamashiro, H., Toyomizu, M., Toyama, N., Aono, N., Sakurai, M., Hiradate, Y., Yokoo,

is this a purely glycolytic process? *Curr Top Dev Biol.* 77, pp. 3–19.

injection. *J Reprod Dev*, 2009, 55, pp. 475–479.

*Reprod Fertil Suppl.* 65, pp. 331–334.

Lab Anim Sci. 48, pp. 499–505.

fertility. (2011). *Theriogenology* 76, pp. 729-736.

and oxidative activity. *Am J Appl Sci.* 6, pp. 1854–1859.

integrity of rat sperm. *Cryobiology* 53, pp. 336–348.

510.

213

75–86.

pp. 1–43.

294.

155–161.

mitochondrion in sperm function: is there a place for oxidative phosphorylation or

in vitro fertilization using cryopreserved spermatozoa. *Biol Reprod.* 80, pp. 503–

head improves development of rat zygotes derived from intracytoplasmic sperm

cryoprotectants on the motility, plasma membrane integrity and acrosomal

operate in the midpiece and principal piece of the mammalian sperm flagellum. *Soc* 

rats by homologous recombination in embryonic stem cells. *Nature* 467, pp. 211–

sperm motility and integrity of acrosome and plasma membrane. J Androl. 30, pp.

acrosomal integrity of rat sperm in the presence of various extenders. J Am Assoc

improves the in vitro fertility rate of inbred and genetically modified mice with low

triphosphatase of microtubule-based sliding filament mechanisms. *Int Rev Cytol.* 66,

Freezability of rat epididymal sperm induced by raffinose in modified Krebs– Ringer bicarbonate (mKRB) based extender solution. Cryobiology 55, pp. 285–

Kobayashi, J., & Sato, E. (2009).Oxidation of exogenous lactate by lactate dehydrogenase C in the midpiece of rat epididymal sperm is essential for motility

Moisyadi, S., & Sato, E. (2010a). Lactate and adenosine triphosphate in the extender enhance the cryosurvival of rat epididymal sperm. *J Am Assoc Lab Anim Sci*. 49, pp.

M., Moisyadi, S., & Sato, E. (2010b). Extracellular ATP and dibutyryl cAMP


Lindemann, C.B. (1978). A cAMP-induced increase in the motility of demembranated bull

Lindemann, C.B., & Gibbons, I.R. (1975). Adenosine triphosphate-induced motility and

Litvin, T.N., Kamenetsky, M., Zarifyan, A., Buck, J., & Levin, L.R. (2003). Kinetic properties

Luria, A., Rubinstein, S., Lax, Y., & Breitbart, H. (2002). Extracellular adenosine triphosphate

Millette, C.F., Spear, P.G., Gall, W.E., & Edelman, G.M. (1973). Chemical dissection of

Montamat, E.E., Vermouth, N.T., & Blanco, A. (1988). Subcellular localization of branched-

Mukai, C. & Okuno, M. (2004). Glycolysis plays a major role for adenosine triphosphate

Nakatsukasa, E., Inomata, T., Ikeda, T., Shino, M., & Kashiwazaki, N. (2001). Generation of

Nakatsukasa, E., Kashiwazaki, N., Takizawa, A., Shino, M., Kitada, K., Serikawa, T.,

Odet, F., Gabel, S.A., Williams, J., London, .RE., Goldberg, E., & Eddy, E.M. (2011). Lactate

Parks, J.E., & Lynch, D.V. (1992). Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes, *Cryobiology* 29, pp. 255–266.

Poole, R.C., & Halestrap, A.P. (1993). Transport of lactate and other monocarboxylates across mammalian plasma membranes. *Am J Physiol.* 264(4Pt 1), pp. C761–C782. Ren, D., Navarro, B., Perez, G., Jackson, A.C., Hsu, S.Q., Shi, Q., Tilly, J.L., & Clapham, D.E.

Rodriguez-Miranda, E., Buffone, M.G., Edwards, S.E., Ord, T.S., Lin, K., Sammel, M.D.,

cryopreserved at -196 degrees C, *Reproduction* 122, pp. 463–467.

Pennisi, E. (2004). Genetics DNA reveals diatom's complexity. *Science* 306, pp. 31.

mammalian spermatozoa. *J Cell Biol.* 58, pp. 662–675.

spermatozoa. *Biochem J.* 255, pp. 1053–1056.

sliding of filaments in mammalian sperm extracted with Triton X-100. *J Cell Biol*. 65,

of 'soluble' adenylyl cyclase: synergism between calcium and bicarbonate. *J Biol* 

stimulates acrosomal exocytosis in bovine spermatozoa via P2 purinoceptor. *Biol* 

chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse

supplementation in mouse sperm flagellar movement. *Biol Reprod.* 71, pp. 540–

live rat offspring by intrauterine insemination with epididymal spermatozoa

Halamata, Y., Kobayashi, E., Takahashi, R., Ueda, M., Nalashima, K., & Nakagata, N. (2003). Cryopreservation of spermatozoa from closed colonies, and inbred, spontaneous mutant, and transgenic strains of rats, *Comp. Med.* 53, pp.

Dehydrogenase C (LDHC) and Energy Metabolism in Mouse Sperm. *Biol Reprod.*

(2001). A sperm ion channel required for sperm motility and male fertility. *Nature*

Gerton, G.L., Moss, S.B., & William, C.J. (2008). Extracellular adenosine 5′ triphosphate alters motility and improves the fertilizing capability of mouse sperm.

sperm models. *Cell* 13, pp. 9–18.

*Chem*. 278, pp. 15922–15926.

*Reprod.* 66, pp. 429–437.

pp. 147–162.

547.

639–641.

85, pp. 556-564

413, pp. 603–609.

*Biol Reprod*. 79, pp. 164–171.


*UPSP ICE 'Interactions between Cells and their Environment', Team Cryobio* 

After the ratification of the international convention on the biodiversity in Rio, a National Cryobank was created in France in 1999 to preserve the genetic resources of domestic animals (www.cryobanque.org). Particular attention was carried on Oryctolagus cuniculus species with the extension of the national cryobanking to the rabbit (Joly et al., 1998).

Cryopreservation corresponds of all the steps of collection and long term storage of animal

Cryopreservation is not a museology action to freeze the products of the past. Contrary to that, it corresponds to the practical implementation of new biotechnologies of reproduction. It includes also all the technical means to maintain the evolutionary potential of population. So, the French National Cryobank was created to secure the biological material stored at - 196°C in liquid nitrogen. It constitutes a real tool to serve all stakeholders to manage the animal diversity as part of the National Charter supported by the Genetic Resources Office

The archaeological origins of rabbit are located in Spain (Bolet et al., 2000). Actually, rabbit is widely spread all around the world and can be considered according situations as wild animal, domestic animal, pets and laboratory models. The rabbit populations can be classified into 3 categories according to their genetic originality, their specific uses and the motivations of breeders involved in the *in situ* management of populations (figure 1).

Type I regroups the breeds identified according to an official standard, as well as for large breeds (Butterfly, Champagne Argente, Fauve de Bourgogne...) and endangered breeds with less than 100 females (Brun Marron de Lorraine ...). These breeds are reared by fancy breeders and animals are presented regularly in local or regional meetings supported by FFC the federation of French fancy rabbit breeders (www.ffc.asso.fr). These breeds must be

Nowadays, this tool is very useful for the management of animal diversity in France.

populations, preserved as live cells and able to generate live animals.

preserved for their patrimonial values and socio cultural interests.

**1. Introduction** 

(www.fondationbiodiversite.fr).

**2.1 "Type I" material** 

**2. Genetic diversity in rabbit species** 

Thierry Joly, Vanessa Neto and Pascal Salvetti *Université de Lyon, France, VetAgro Sup – Isaralyon,* 

*France* 

enhance the freezability rat epididymal sperm. *J Am Assoc Lab Anim Sci*. 49, pp. 167–172. **9**

Thierry Joly, Vanessa Neto and Pascal Salvetti *Université de Lyon, France, VetAgro Sup – Isaralyon, UPSP ICE 'Interactions between Cells and their Environment', Team Cryobio France* 
