**Cryopreservation of Rat Sperm**

Hideaki Yamashiro1 and Eimei Sato2

*1 Laboratory of Animal Genetics and Reproduction, Faculty of Agriculture, Niigata University 2 Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University Japan* 

#### **1. Introduction**

164 Current Frontiers in Cryopreservation

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The laboratory rat, *Rattus norvegicus*, was the first mammalian species domesticated for scientific research, which work dating back to before 1850. From this auspicious beginning, the rat has become the most widely studied experimental animal model for biomedical research (Jacob, 1999). Since the development of the first inbred rat strain by King 1909, over 500 inbred rat strains have been developed for a wide range of biochemical and physiological phenotypes and different disease models (Aitman et al., 2008, Canzian. 1997). In the last decade, there has been an extraordinary increase in rat genomic resources (Gibbs et al., 2004, Pennisi. 2004), and the advent of knock-out technology allow the insertion or deletion of individual genes into the rat by advances in stem-cell technology (Geurts et al., 2009, Izsvak et al., 2010, Tong et al., 2010). Thus, a wide array of research opportunities now open up, especially in studies involving the laboratory rat (Hamra. 2010). However, protocol for sperm cryopreservation and oocytes fertilized *in vitro* by using cryopreserved sperm are still under development for preservation of most rat strains. Therefore, greater use of the cryopreservation of rat sperm may provide an essential resource to preserve and increase the number of valuable genetic strains for research and application.

In this chapter, we will introduce several of these approaches to cryopreserving the rat sperm. It will be valuable for developing new freezing extender for cryopreservation of rat sperm, and might be applied to other reproductive technologies in this species for preservation of valuable rat strains.

#### **2. Reporting studies of rat sperm cryopreservation**

The first live-born rat derived from frozen-thawed sperm were successfully reported artificial insemination (AI) by using frozen-thawed rat sperm, and also reported cryopreservation of several strains of rat sperm, including those from mutant and transgenic rats (Nakatsukasa et al., 2001, Nakatsukasa et al., 2003). More recent publication by same group confirmed that cryopreserved rat sperm can be revitalized and result in the birth of live offspring through embryo transfers after in vitro fertilization (*IVF*) (Seita et al., 2009a). Although the authors mentioned by another publication that intracytoplasmic sperm injection (ICSI) is the only way to routinely obtain offspring routinely derived from oocytes

Cryopreservation of Rat Sperm 167

thaw survival of sperm were obtained when raffinose in mKRB was used with Equex STM,

Fig. 1. Motility of frozen-thawed rat sperm which were frozen in different sugars dissolved in an mKRB egg yolk, and then subjected to a thermal resistance test at 37°C. Values are the mean ±SEM (n=3). a-b Different superscripts within the same column denote significant

Mammalian sperm provide energy for metabolic requirements by both mitochondrial oxidative respiration and glycolysis, and sperm motility is driven by the flagellum and is dependent on the availability of an adequate and continued supply of ATP (Cummins and Woodall, 1985). ATP is used by the dynein ATPases that function as the flagellar motors and in protein kinase A-mediated signal transduction pathways to regulate motility throughout the tail (Cao et al., 2006). In the mouse sperm, Mukai and Okuno (2004) reported that glycolysis has an important role in providing the ATP required for mouse sperm motility than mitochondrial respiration throughout the length of the flagellum. Bunch et al. (1998) also suggested that mouse sperm utilize glycolysis to generate ATP in the principal piece of the tail. While, Odet et al., (2011) demonstrated that lactate dehydrogenase (LDH) is responsible for the maintenance of energy metabolism in progressive and hyperactivated in mouse sperm. In rat sperm, Gallina et al. (1994) was studied the operation of shuttle functions for the ATP reconstituted systems in the mitochondria present in the middle place of rat, mouse and rabbit. They showed that the redox couple lactate/pyruvate and lactate dehydrogenase are active with rat and rabbit mitochondria, and it does not work with mouse. From these examining the energy metabolism of sperm, the glycolytic activity is exclusively responsible for the generation of mouse sperm metabolism, and mitochondrial metabolism seems to interacted with motility activity of rat sperm. These findings in conjunction with the present study indicate that successful cryopreservation of rat sperm in

and egg yolk (Fig.1).

differences (*P* < 0.05).

fertilized in vitro using fresh and cryopreserved sperm (Seita et al., 2009b). Hagiwara et al, (2009) also pointed out that only one group has reported successful cryopreservation of rat sperm, subsequently used for AI and yielding live offspring. This result has not yet been repeated by other investigators and labs, and further investigation of the ability to yield viable rat sperm after cryopreservation is urgently needed. Further, each of cryopreservation procedures has not been completed for several mammalian species to date, the basic science and technology required to do so is rapidly becoming available and this should be completed for a number of species, in the future (Agca and Critser, 2002). Those of the information will also allow further improvements in cryopreservation of rat sperm from various mammalian species.
