MATERIALS AND METHODS

Animals and management: The experimental work was carried out in a private Rabbit Production Farm in Zian location, Dakahlyia governorate, Egypt, during the period from August 2009 to March 2010. Total of 63 virgin New Zealand White (NZW) rabbit does (7-8 months of age and 3.5-3.85 kg body weight) as well as 18 fertile NZW rabbit bucks (10-13 months of age and 4-4.25 kg body weight) were used in the present study. All animals were healthy and clinically free of external parasites.

All does and bucks were housed in a naturally ventilated building and kept individually in flat deck cages (50x60x40 cm) only for does equipped with automatic water dispensers and internal nest boxes. Animals received a commercial diet containing 10.5 MJ digestible energy per kg, 18.5% crude protein and 12.5% crude fiber according to their physiological condition. Water was provided ad libitum all over the experimental period. Average of ambient temperature during the experimental period ranged from 15.4°C in December to 30.0°C in August, while relative humidity ranged from 65% in November to 81% in August.

Does and bucks were kept under the same management system. Cages and nest boxes were cleaned and disinfected regularly before each kindling. Light in the rabbit farms was controlled to allow 14-16 h per day during the period of the study. Urine and feces dropped from the cages on the floor were cleaned every day in the morning.

Experimental design: The experimental rabbit does were divided into similar three treatment groups (21 in each) according to their live body weight. Does in the 1^st group (control) were injected with 0.2 mL saline solution at mating (without treatment). Does in the 2^nd group (GnRH) were intramuscularly injected at mating with 0.2 ml/doe of GnRH analogue (Receptal, Intervet International B.V. Boxmeer-Holland, each ml of Receptal contained 0.0042 mg Buserelin acetate equivalent to 0.004 mg Buserelin). In the 3^rd group, vagina of each doe was stimulated mechanically immediately before mating by thermometer (IVMS). All treatments were applied at mating for six consecutive litters.

Reproductive management: On day of mating, receptive doe was transferred to the cage of buck and returned back to her own cage after mating. On day 27 of mating, the nest boxes were supplied with wooden straw to help the doe in preparing a worm comfortable nest for the kits of her litter. Does were mated 10-12 days after parturition of each litter. At the same time, does failed to kid from the previous mating in each treatment group were mated. After parturition, the size of the litter (total and live borns at birth) was recorded and rabbit kits were weaned up to the age of 35 days.

Blood samples and hormonal assay: Blood samples were taken from does in each group at 2, 3 and 4 h after mating. Blood samples were collected by puncture of the ear vein in heparinized clean test tubes and immediately centrifuged (4000 g) for 15 min. Then, plasma was separated and stored at -20°C until assay. Plasma concentrations of FSH and LH were determined in duplicate by radioimmunoassay (RIA), using AFP- 472176 and AFP-3120489 antibodies for FSH and LH, respectively, provided by the National Institute of Diabetes and Digestive and Kidney Diseases (Harbor UCLA Medical Center, CA, USA). The dilutions antibody used were 1:45,000 and 1:1,500,000 for FSH and LH, respectively. Hormones were iodinated with ¹²⁵I, using the chloramine-T- method (Greenwood et al., 1963). The assay sensitivity was 1.0 and 0.05 ng mL^-1 for FSH and LH, respectively. The intra-assay coefficients of variation, estimated from plasma pool measured 8 times in the same assay, were <8% for both hormones.

Data recorded: Within 12 h after kindling of each litter, total number of kits (live and dead) was calculated, dead kits were removed and then size and weight of live kits were recorded. Kindling rate (number of kindled does per number of mated doesx100), viability rate (number of live per total number of kitsx100) and length of gestation period (day) were calculated.

Statistical analysis: Data obtained in this study were statistically analyzed using computer program of SAS (2004). Factorial ANOVA (GLM) was used to perform the effect of treatment, litter number and their interaction different traits studied. Also, the effect of treatment, time post-mating and their interaction only for the 2^nd litter on concentration of FSH and LH in blood plasma was performed. Multiple rang test (Duncan, 1955) was employed to test statistical differences among means at (p<0.05). Only the significant interactions were included in the discussion.

RESULTS AND DISCUSSION

Reproductive and litter size of doe rabbits
Effect of treatment: Results presented in Table 1 reveal that stimulation of ovulation by GnRH showed significantly (p<0.05) the higher kindling rate (71.4%), litter size per doe as a total (6.16/litter) and live kits (5.53/litter), viability rate (89.8%), kit weight (64.11 g) and litter weight (345.5 g as compared to 48.4%, 5.67 and 3.91/litter, 70.0%, 60.83 g and 1.76/litter in the controls, respectively). The differences between GnRH and IVMS groups in all traits weight were insignificant, but all values were higher for GnRH than IVMS treatment. In general, reproductive index was the highest (3.95) for GnRH-treated does, moderate in IVMS (3.46) and the lowest in the control (1.89).

Localization of GnRH within the hypothalamus has been well documented in rabbits (Arimura et al., 1973) and many investigators reported improvement in kindling of rabbit does as affected by exogenous GnRH.

Table 1:	Least square means and standard errors of means (SEM) of productive and reproductive traits of doe rabbits as affected by stimulation treatment and does parity
a, b and c: Means denoted within the same raw for group or parity having different superscripts are significantly different at p≤0.05. IVMS: Intravaginal mechanical stimulation, RI: Reproductive index. GPL: Gestation period length

In nearly similarity with the present results, Zapletal et al. (1990) reported that the total annual fertility rate was higher (73.02%, p<0.05) in rabbit group treated with lecirelin (GnRH) but with higher litter size as a total and live borns (9.82 and 9.70, respectively) than those obtained in the present study. It has been shown that GnRH mediates the hypothalamic control of pituitary gonadotropin secretion and biosynthesis (Dekel et al., 1988) to induce ovulation. However, the obtained improvement in kindling rate and litter size of rabbit does treated with GnRH as compared to the controls in this study was also due to the effects of GnRH on the ovary include stimulation of oocyte maturation (Dekel et al., 1988; Yang et al., 1995).

In addition, GnRH may interfere with the stimulatory effects of exogenous gonadotropin on follicular development, corpus luteum establishment and oocyte maturation (Zanagnolo et al., 1996). Some studies have shown that GnRH and its analogues also exert a direct effect on gonadal function, influencing rabbit oocyte maturation both in vivo and in vitro (Yang et al., 1995; Yoshimura et al., 1992). It has been suggested that GnRH induces oocyte maturation via activation of specific GnRH receptors on granulosa cells (Koves et al., 1989). Exposure to GnRH stimulates prostaglandin synthesis in preovulatory follicles (Wong and Richards, 1992). Increasing concentration of prostaglandin plays an important role in oocyte maturation (Calder et al., 2001). According to these studies, GnRH treatment increased number of kits per litter and decreased kit weight at birth (Table 1), which is in good agreement with the finding of Zapletal and Pavlik (2008), who reported that the conception rates ranged from 10.0 to 89.5% for different GnRH doses.

On the other hand, buserelin or triptorelin added to seminal doses (Viudes-de-Castro et al., 2007) have been shown to be valid methods for inducing ovulation in rabbit does submitted to artificial insemination. However, the hormonal doses needed were high and the kindling rates obtained were similar or lower than those for the control group. In agreement with the results of GnRH injection intramuscularly, Ondruska et al. (2008) found that supplementation of insemination dose at AI with GnRH-Lecirelinum (7.5 μg) increased kindling rate (72.09%) as compared to 62.74% in the control group (administered i.m. with 2.5 μg same synthetic GnRH immediately after AI). However, viability rate was not affected significantly by GnRH treatment (Ondruska et al., 2008). The present results are nearly similar for kindling rate with improving viability rate inspite the marked differences in level and location of GnRH treatment. In this respect, Quintela et al. (2004) used GnRH-buserelin in considerable higher concentrations for intravaginal use (10 up to 20 times) compared with intramuscular control (0.8 μg per female). Differences in kindling rate among treated groups were not statistically significant in their experiments. After intravaginally application of 20-times higher concentration of buserelin they obtained significantly higher prolificacy in experimental females compared with the control (11.7 young and 9.4 kits per litter, respectively). In conclusion, buserelin was evaluated to determine potential negative effects on the sperm characteristics and no detrimental effects were found on sperm motility, viability or acrosomal integrity during 24 h of sperm incubation at 16°C. According to the previous results and those obtained in our study, GnRH treatment to induce ovulation in rabbits is depending on type of analogue, does of GnRH and location of treatment.

The obtained results clearly demonstrated that the GnRH analogue (buserelin) i.m. injected was extremely efficient, significantly increasing kindling rate and viability rate of kits at birth for 6 successive litters in comparison with IVMS and control.

Effect of parity: Results in Table 1 show that only viability rate of kits and litter weight at birth significantly (p<0.05) affected by parity. However, kindling rate, litter size (total and live), kit weight and gestation period were not affected significantly by parity. Generally, most of the studied traits were the highest at the 2^nd parity and the lowest at the 3^rd one.

Inspite the insignificant differences in kindling rate at different parities, rabbit does showed pronounced increase in kindling rate from 66.7 % at the 1^st parity to 71.4% at the 2^nd one and decreased to 57.1% at the 3^rd parity, then insignificantly increased with progress of parity up to 68.3%, at the 6^th parity. The noted increase in kindling rate and litter size at the 2^nd compared with 1^st parity may be due to linkage between pseudo-pregnant does and parity. In this respect, Theau-Clement et al. (2008) recorded increasing the percentage of pseudo-pregnant does in primiparous (31.2%) than in secondiparous does (12.2%, p<0.001).

Fig. 1:	Interaction between treatment and parity for number of live kits at birth

Fig. 2:	Interaction between treatment and parity for viability rate at birth

Yet, the components of litter size have been studied by genetic (Argente et al., 1997) and phenotypic (Santacreu et al., 1992) points of view.

Analysis of variance revealed that the effect of interaction between treatment and parity was significant (p<0.05) only on number of live kits/litter and viability rate, reflecting nearly similar trend of change (p<0.05) in GnRH and IVMS groups as compared to control group (Fig. 1, 2).

At the 1^st and 2^nd parities, number of live kits (Fig. 1) was significantly (p<0.05) greatest (6.09 and 6.14/litter), followed by IVMS (4.90 and 5.00/litter), and the lowest in control group trend (4.19 and 4.33/litter). At the 3^rd parity, live kits decreased in GnRH and IVMS groups to the lowest values (4.67 and 3.90/litter, respectively) versus slight increase to the highest values (4.52/litter) in control group, but the differences were not significant. At the 4^th parity, live kits showed marked increase to 5.67/litter in GnRH and to 5.81/litter in IVMS groups, being significantly higher than 2.95/litter in control group. At the 5^th and 6^th parities, live kits were significantly (p<0.05) higher in both GnRH (5.19 and 5.43/litter) and IVMS (5.28 and 5.57/litter) groups than in control group (3.67 and 3.81 /litter).

Regarding viability rate of kits, results illustrated in Fig. 2 show that at the 1^st parity, viability rate was 84.69, 86.69 and 89.39% in control, GnRH and IVMS groups, respectively and the differences were not significant. Then, it showed similar trend to that of number of live/litter at subsequent parities. Viability rate was significantly (p<0.05) the highest at the 2^nd, 3^rd and 4^th parities in GnRH group (99.21, 75.55 and 93.43%, respectively) and at the 5^th and 6^th parities in IVMS group (89.32 and 90.43%, respectively). Yet, kits of the control group showed significantly (p<0.05) the lowest viability at all parities, except for the 3^rd one.

Based on the previous results of parity effect, GnRH treatment or IVMS at mating for 6 successive litters had beneficial effects on fertility and viability of kits at birth.

Concentration of gonadotrophins: Results in Table 2 show that stimulation of ovulation by GnRH led to significant (p<0.05) increase in concentration of FSH and LH in blood plasma to 16.71 and 16.57 ng mL^-1 as compared to 5.79 and 9.23 ng mL^-1 in the control, respectively. However, IVMS significantly (p<0.05) increased only FSH concentration as compared to the control (14.02 vs. 5.79 ng mL^-1).

Table 2:	Effect of type of stimulation and sampling time after mating on concentration of FSH and LH in blood plasma of rabbit does
a, b and c: Means denoted within the same column for each effect having different superscripts are significantly different at p<0.05. IVMS: Intravaginal mechanical stimulation

As affected by mating time, FSH concentration significantly decreased 4 h post-mating, however, LH concentration showed significant decrease 3 h post-mating. At 2, 3 and 4 h post-mating, FSH concentration was 14.04, 12.90 and 9.58 ng mL^-1, while LH concentration was 16.0, 10.38 and 9.63 ng mL^-1, respectively.

The insignificant interaction between treatment and mating time on concentration of both FSH and LH was reflected in the highest concentration in GnRH treatment 2 h post-mating.

In this study the changes occurring in plasma LH and FSH concentrations 2, 3 and 4 h post- mating may explain ovulation rate and litter size. The reproductive hormone profile of FSH and LH in rabbit does have been studied at mating by Ubilla et al. (1992, 2000 a,b). The FSH may influence the follicular growth; meanwhile LH may determine the number of breakage of pre-ovulatory follicles. According our results, there was association between FSH and LH peaks. Similar findings were reported by Muelas et al. (2008) and Rodriguez et al. (1989).

Many authors observed that the highest LH and FSH concentrations reached 60-90 min after exogenous administration of GnRH, decreasing to basal concentrations 4-5 h later (Mills and Gerardot, 1984; Rodriguez et al., 1989; Rebollar et al., 1997).

In agreement with our results concerning the FSH, Muelas et al. (2008) did not find change in FSH concentration 48 h before or after mating and 2 h after mating. Also, Ubilla et al. (2000b) did not find any difference in FSH concentrations between 48 h before and 7 h after AI. However, the present trend of change in LH was comparable with that observed on rabbits by Muelas et al. (2008), who found significant differences among LH plasma concentrations measured 48 h before or after the mating (5.1 and 5.4 ng mL^-1, respectively) vs. 2 h after the mating (29.0 ng mL^-1). Similar trend was observed by Ubilla et al. (2000a). In accordance with the marked increase in LH concentration in GnRH-treated does, Rodriguez et al. (1989) showed that LH level sharply increased after 15 min of GnRH challenge, being 200-1000%, depending on sexual receptivity of does and GnRH dose. However, Muelas et al. (2008) found that the LH levels were 4-6 fold higher 2 h after the mating in does with high ovulation rate, which may explain the highest litter size of GnRH-treated does as compared to the control group. In addition, This may explain the higher ovarian stimulation and pituitary responsiveness after GnRH biostimulation. The pronounced reduction in litter size with advancing parity may be related to that the response of circulating FSH to active immunization against GnRH. In several species, active immunization against GnRH consistently decreased LH secretion to undetectable levels (Garza et al., 1986; Ondruska et al., 2008).

CONCLUSION

Based on the foregoing results, stimulation of ovulation in rabbit does by 0.2 mL GnRH (receptal)/doe at mating for 6 consecutive litters can be successfully used to improve fertility of rabbits and their litter weight. Also, intra-vaginal mechanical stimulation at mating could have some advantages on rabbit fertility by avoiding the intramuscular application of GnRH analogues and probably reducing the sanitary risk derived from incorrect use of needles. Further studies are necessary for determining an optimal level of GnRH analogue at different times of mating in relation with the physiological status of rabbit does at the moment of mating.