**3. Principal ways of cryoconservation of rabbit genetic resources**

To keep rabbit genetic resources, we can distinguish three principal ways according to the nature of frozen biological material (Figure 2).

#### **3.1 Germinal cells**

180 Current Frontiers in Cryopreservation



Most of Type II populations mainly have a scientific interest with high experimental values. Some populations could be promoted in biomedical research and by the pharmaceutical

Type III regroups all the populations of rabbits selected for meat production (female lines

Some strains are today completely extinct and subsist only under embryo-frozen form waiting for a possible perspective of reuse or selection scheme reorientation (Dutch

Other populations are still selected and the frozen biologic material represents a selection control to measure genetic progress precisely. Moreover, for security, several populations commercially spread are subject to regular cryoconservation in order to save the selection core from a sanitary risk (Inra 2066, commercial lines of Hypharm and Hycole societies…)

> Rabbit « Orylag® castor (type III) bred for its "Rex" fur

Rabbit "Sauteurs d'Alfort" (type II). They move on forlegs when they are stressed

Type II concerns animals with one or more specific character:

and male lines) or fur production with high economic value.

Rabbit « Brun Marron de Lorraine » (type I), endangered breed

**3. Principal ways of cryoconservation of rabbit genetic resources** 

To keep rabbit genetic resources, we can distinguish three principal ways according to the

hymalayan, Orylag, INRA 1077 female strains…).

Fig. 1. Examples of different categories of rabbits

nature of frozen biological material (Figure 2).

**2.2 "Type II" material** 

created)

industry.

**2.3 "Type III" material** 

Rabbit « Fauve de Bourgogne » (type I) adapted to a traditionnal breeding system

mortality rate-diverging lines).

Based on the conservation of germinal cells, this way helps to conserve the gene pool of particular male or female individuals.

Mature spermatozoids (sperm collected in artificial vaginas) or immature (epididymal spermatozoids, spermatogonies in gonadic tissue) helps to save the male line. The freezing of an individual's semen (n generation) helps to obtain progeny (n+1 generation) after thawing and artificial insemination (AI) of females.

Semen samples of bad quality can be promoted by new technologies of *in vitro* fertilization (FIV) or intracytoplasmic sperm injection (ICSI), which then permits *in vitro* embryo production and progeny delivering after embryo transfer in synchronized recipient female (Daniel *et al*, 2007). Nowadays, semen freezing is not a reliable or repeatable method yet, and results after AI with rabbit frozen semen are still too inconstant to plan a routine utilization of this technique (Vicente *et al*, 1996; Moce *et al*, 2003). Only half of the sampled males can produce semen with freezable quality and approximately 50% of females give birth after insemination of thawed semen with a large variability [15%~80%]. However, this is the only available method to preserve precious males semen, mainly for type I and II.

Mature oocytes (picked up in oviduct 15 hours after ovulation) or immature oocytes (present in follicles of ovarian tissue) permits to save genetic resources by female way and to preserve cytoplasmic heredity. Mature oocytes freezing of a female individu (n generation) would permit to obtain progenies (n+1 generation) after FIV or ICSI, but no young rabbit has been obtained from thawed oocytes yet (Salvetti *et al*, 2010). But, recently, young rabbits obtained from frozen ovarian tissue are born from females transplanted by orthotopic autograft (Almodin *et al*., 2004; Neto *et al*., 2007). In emergency situations (sanitary problems, injured animals), ovarian cortex freezing, even if this method is not yet completely under control (Neto *et al.,* 2008), can be proposed to save the heredity pool of an important female of type II.

Fig. 2. Biotechnologies of reproduction applied to the cryopreservation of the rabbit genetic resources

**females % donor Nb of frozen** 

**stimulation** 1226 77% 8633 9.2

**Total 2815 73% 32466** 

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

animals of type II or to small effective populations of type I.

method is not totally under control (Mehaisen *et al.,* 2006).

**(FSH)** 1589 71% 23833 21.2

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

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

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

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

**controlled** 277 82% 2746 1118 41%

**transfer** 87 56% 846 176 21% **Total 364 76% 3592 1294 36%** 

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),

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

**Nb of thawed embryos** 

**Nb of borned pups**

**Embryo developmental rate** 

**% of delivering female** 

**embryos** 

**Nb of embryos per donor** 

**Treatment Nb of treated** 

**4.2 Embryos recovering and freezing** 

**No ovarian** 

**Superovulation**

slaughter of females.

**4.3 Embryo transfer and births** 

**Nb of recipients** 

(**table 2**).

**Transfer conditions** 

**Standard** 

**On Field** 
