**4.1 Embryos production**

As rabbit is naturally prolific species, two methods can be used to produce embryos:


Overall, 73% of the treated and collected females are embryos donors and produce an average of 9.2 embryos without ovarian stimulation or with a slight eCG stimulation, and two to three times more (21.2 embryos) after a superovulation treatment (**table 1**). The embryo recovering rates (number of collected embryos/number of corpus luteum) are between 70% and 80% and those of freezable embryos (number of frozen embryos/number of collected embryos) are from 75% to 85%.


Table 1. Method of embryo production (activity from 1998 to 2011)

#### **4.2 Embryos recovering and freezing**

182 Current Frontiers in Cryopreservation

Embryo freezing, allowing the preservation of both male and female ways, is the main way to recreate quickly a population from thawed embryo transfer. Most of the time, embryos are frozen at compacted morula stage (65h~72h *post coïtum*) even if freezing at earlier stage (4 cells stage) is possible. They can be produced *in vivo* from superovulated females or not, or *in vitro* after *In Vitro* Fertilization and *in vitro* culture. Embryos freezing from a planned mating ( at n generation) permits to obtain progenies (at n+1 generation) after thawing and

This way has the main advantage of being easy to do, which permits to sample tissues quickly and simply on a large number of individuals. But the main difficulty is the recreation of animals after somatic cloning, still not well controlled. The cells can be isolated from different tissues: skin, cartilage, bones, blood… These cells, present in a large number in each organism, would allow the reconstitution of an individual of the same generation

More recently, a new opportunity came with induced pluripotent stem cells, which are able to differentiate in many tissues. These cells are potential vectors for genetic traits transmission and could be used after thawing as an important core source to regenerate

The embryo is the favored biological material to conserve most of rabbit populations, by both male and female ways, and then preserve cytoplasmic heredity. This method has been widely proved for 15 years with more than 32 000 produced embryos but can only be


Overall, 73% of the treated and collected females are embryos donors and produce an average of 9.2 embryos without ovarian stimulation or with a slight eCG stimulation, and two to three times more (21.2 embryos) after a superovulation treatment (**table 1**). The embryo recovering rates (number of collected embryos/number of corpus luteum) are between 70% and 80% and those of freezable embryos (number of frozen embryos/number

As rabbit is naturally prolific species, two methods can be used to produce embryos:

than the tissues giver after somatic cloning (Chesne *et al.,* 2002).

many individuals after nuclear transfer (Honda *et al*, 2010).

applied to living and fertile animals.

of collected embryos) are from 75% to 85%.

**4.1 Embryos production** 

**4. Frozen embryo, the main way for rabbit cryoconservation** 

donor female ( Kauffman *et al*, 1998; Salvetti *et al*., 2007).

**3.2 Embryonic cells** 

transfer in recipient females.

**3.3 Somatic cells** 

Slaughter the females with uterine tract washing is the most effective method to collect rabbit embryos. This method is simple and easy to implement directly on the breeding place of the animals. Cryoconservation of a population, about 40 to 50 females is feasible in one single day. The method is to keep for large population of type I and III because of systematic slaughter of females.

In addition, a new method of embryos collection by endoscopy allows renewing about four times the operation on the same female, preserving the female's integrity (Garcia *et al*., 1991; Besenfelder *et al*., 1998). A lot heavier to implement, it is reserved to rare and precious animals of type II or to small effective populations of type I.

Embryos were frozen in the same cryoprotective solution containing 1.5 M DMSO and by the same slow freezing process, even more vitrification could provide good results but this method is not totally under control (Mehaisen *et al.,* 2006).

#### **4.3 Embryo transfer and births**

After thawing of a part of these embryos, the embryo transfer results vary according to the environmental conditions defined by the recipient female genotype and breeding conditions (**table 2**).


Table 2. Pups production after transfer of thawed embryos (activity from 1998 to 2011)

The optimal environmental conditions are defined by the recipient from a mother female line placed in a control environment in an aboveground breeding (16 hours of light per day),

Almodin, C.G., Minguetti-Camara, V.C., Meister, H., Ferreira, J.O., Franco, R.L., Cavalcante,

Besenfelder, U., Strouhal, C., Brem, G. (1998). A method for endoscopic embryo collection and transfer in the rabbit. *Zentralbl Veterinarmed A*, Vol.45, No.9, pp: 577-579, Bolet, G., Monnerot, M., Arnal, C., Arnold, J., Belle, D., Bergoglio, G., Besenfelder, U., Bosze,

Chesne, P., Adenot, P.G., Viglietta, C., Baratte, M., Boulanger, L., Renard, J.P. (2002). Cloned

Daniel, N., Chesne, P., Baratte, M., Renard, J.P. (2007). Lapins produits par injection intra-

Garcia-Ximenez, F., Vicente, J.S., Santacreu, M.A. (1991). Embryo transfer in lactating rabbit

Honda, A., Hirose, M., Hatori, M., Matoba, S., Miyoshi, H., Inoue, K., Ogura, A. (2010).

Kauffman, R.D., Schmidt, P.M., Rall, W.F., Hoeg, J.M. (1998). Superovulation of rabbits with

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Mocé, E., Vicente, J.S., Lavara, R. (2003). Effect of freezing-thawing protocols on the

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following radiotherapy. *Hum Reprod,* Vol.19, No.6, pp:1287-1293,

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*Journées de la Recherche Cunicole*, Le Mans, France, pp.33-36,

does by laparoscopy. *Anim Reprod Sci*, Vol.24, pp:343-346,

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treatment in rabbit does. *Theriogenology*, Vol.65, pp:1279-1291,

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*Theriogenology*, Vol.60, pp:115-123,

A.A., Radaelli, M.R., Bahls, A.S., Moron, A.F., Murta, C.G. (2004). Recovery of fertility after grafting of cryopreserved germinative tissue in female rabbits

S., Boucher, S., Brun, J.M., Chanteloup, N., Ducourouble, M.C., Durand-Tardif, M., Esteves, P.J., Ferrand, N., Hewitt, G., Joly, T., Koehl, P.F., Laube, M., Lechevestrier, S., Lopez, M., Masoero, G., Piccinin, R., Queney, G., Saleil, G., Surridge, A., Van Der Loo, W., Vanhommerig, J., Vicente, J.S., Virag, G., Zimmermann, J.M. (2000). Evaluation and conservation of european rabbit genetic resources : first results and

rabbits produced by nuclear transfer from adult somatic cells. *Nat Biotechnol*,

cytoplasmique de spermatozoïdes (ICSI) stockés à température ambiante. *12èmes*

Generation of Induced Pluripotent Stem Cells in Rabbits : Potential experimental models for human regenerative medicine. *J. Biol. Chem.* Vol.285, pp :31362-31369, Joly, T., Rochambeau, H. de., Renard, J.P. (1998). Etablissement d'une cryobanque

d'embryons pour la conservation ex situ de la diversité génétique chez le lapin :

FSH alters *in vivo* development of vitrified morulae. *Theriogenology*, Vol. 50, pp:

*vivo* viability of vitrified and non-vitrified embryos derived from eCG and FSH

performance of semen from three rabbit lines after artificial insemination.

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freezing parameters on the morphology and viability of preantral follicles after cryopreservation of doe rabbit ovarian tissue. *Fertil. Steril*. Vol.89, No.3, pp:1348-

**7. References** 

while uncontrolled conditions are defined by a recipient placed in an uncontrolled environment (sanitary, light, temperature) in a traditional farm's breeding conditions.

Globally, 76% of the recipients give birth to young rabbits after thawed embryo transfer with an embryo development rate of 36%. It is particularly important to control the recipient's genotype and the breeding conditions in order to guarantee the population recreation after cryoconservation.

The efficiency of the cryoconservation method has been concretely proved after transfer of embryos stored for more than 15 years in liquid nitrogen. In November 2006, 69 Brun Marron de Lorraine young rabbits got born after thawing and transfer of 134 embryos in recipients. The young rabbits born from frozen embryos of 1992 were presented to public at the international agriculture show in Paris in March 2007, in collaboration with fancy breeders and the FFC (Salvetti *et al.,* 2007).
