1. Introduction
In the last four decades, rabbit production underwent a noticeable change from a traditional and familiar organization to industrial and intensive systems. Consequently, genetic selection programs and new breeding management systems were established, improving the production of the new hybrid lines used. This has led to an increase in female nutritional requirements [
1,
2], health problems [
3,
4,
5,
6,
7] and welfare necessity [
8,
9,
10,
11]. Nutritional strategies in reproductive females need to be global and consider both the short-term productive factors (litter size, milk production, or fertility) as well as the long-term factors (body condition or health). Therefore, the expected improvement in nutritional management should be based on an accurate analysis of the requirements of the doe, its evolution during successive reproductive cycles and the identification of crucial moments in the life of rabbit does to optimize productivity and longevity.
Two of the key points of the reproductive success of rabbit does are their birth weight and maturity at the first artificial insemination (AI). There is an optimal threshold for birth weight (>57 g) to optimize the initial reproductive performance, which is associated with an increase in the live weight and fat reserves at the first AI [
12,
13,
14]. The latter are the traits used to define the maturity of rabbit does at the first AI, and both are also related to the nutritional rearing strategy and the time of AI [
13,
15]. However, there is no clear recommendation indicating the weight and/or body condition of the nulliparous rabbit doe at the onset of its reproductive life [
16]. In the current management systems, does are inseminated at a fixed age with minor or no consideration of their weight and chemical body composition. As in other species, there must exist a balance between ensuring at least the minimal degree of body development needed to guarantee a successful reproductive life, and minimizing the unproductive rearing period. Accordingly, the study of the body chemical composition of rabbit does seems to be a useful tool not only to improve feeding, but also for general rabbit doe management [
17]. The final aim is to extend the lifespan of rabbit does, which is limited by the relatively high early mortality and culling rate in intensive production systems [
5].
A new non-destructive technique to estimate rabbit doe body chemical composition (moisture, ash, protein, fat, and energy content) based on the bioelectrical impedance measurement, live weight and physiological status of rabbit does, was developed by Pereda [
18]. This method is easier and cheaper than TOBEC [
19], and in both methods, the variations of gut contents are included in the error term. Furthermore, it allows the prediction of total fat and energy content (not only the perirenal fat content, as it is with the case with the ultra-sound technique [
20]), as well as the body protein, which is not usually estimated with other methodologies to evaluate body condition.
The aim of this work was to establish the relationship between chemical body composition (at AI and parturition), determined using bioelectrical impedance, and both fertility and the percentage of kits born alive during the first three inseminations, and to identify the most important moments to record the body chemical composition.
4. Discussion
A general problem observed in rabbit does is their low fertility rate in the second parturition [
31]. It is usually explained by the energy deficiency observed before the second insemination [
16]. In this period, it might be difficult for the rabbit doe to meet the requirements for both pregnancy and growth due to the limited feed intake [
1,
32,
33,
34,
35]. However, when the energy supply was increased, it was dedicated to milk production with no limitation of reserve mobilization [
36,
37]. In this way, Pascual [
13] hypothesized that this negative energetic balance would be a natural adaptation to optimize evolutionary success. In this context, it is interesting to study the relationship between the factors related to body chemical composition and their influence on fertility and kit survival at birth, in order to identify the threshold to be met by rabbit does at the beginning of their reproductive life.
The evolution of the live weight and chemical composition of rabbit does from their first AI onwards indicated that they were still growing when inseminated the first two times, which agreed with recent data [
38]. Live body weight, body energy and fat were closely and positively correlated, which was similar to the correlation between perirenal fat thickness and body energy content reported previously [
39,
40]. In contrast, body protein had a minor or no correlation with the latter traits, but a negative one with live weight from the second parturition onwards. These results agree with rabbit doe maturation in this period, which would depend on maturity at the first AI and on reproductive success. Once maturity is reached (or nearly reached), the changes in body weight might be mainly associated with fat mobilization and/or deposition. This would agree with the moderated and positive correlation between live weight and body condition score [
41].
The differences in chemical body composition and live weight between the first AI and the first parturition seemed to be related to the success in the first insemination, which was not influenced by body condition or live weight. This effect was reported by other authors [
2,
34,
42]. It may be explained by the specific situation of non-pregnant does, which would use the entire intake for body protein and fat accretion, and accordingly, energy accretion, towards the completion of the final step to reach their maturity, where the fat deposition is much higher than the protein deposition (24 vs. 6% increment, respectively). Meanwhile, pregnant does have to supply gestation requirements that are especially important during the last 10 days of gestation, and which can impair not only fat content [
17,
43,
44], but also protein balance [
45], with respect to non-pregnant does. It must be taken into account that rabbit does inseminated later show a higher feed intake capacity [
46] and lower growth requirements. Furthermore, rabbit does reduce feed intake in the days before parturition, contributing to the impairment of their nutrient balance [
43].
The impairment of fertility in the second insemination reflects the specific situation of primiparous rabbit does, which suffer a negative energetic balance during their first pregnancy and lactation, compared to non-pregnant does, which seems to negatively influence reproductive performance and especially fertility [
2,
32,
34], although this could be considered a natural adaptation, as commented before [
13]. Live body weight at the moments of first AI, first parturition and second AI did not affect the fertility rate in the second parturition. This agrees with the findings of Rommers et al. [
46], who did not observe any effect of body weight at the first AI on fertility rate in the first two parturitions. Nevertheless, the combination of a high live weight and high weight gain between the first two AI was also related to better fertility. Rabbit does that lose weight between the first two AI, regardless their initial body weight, were unable to present an acceptable reproductive performance in the second cycle. In this period, live weight was positively correlated with body fat and energy content, but not with protein, as does are finishing their protein accretion. The relationship between the fertility in the second AI and the chemical body composition at the first AI confirms the importance of the rearing management of rabbit does. Diets used [
2,
14,
42,
47] and the time of first insemination [
15,
48,
49,
50] influenced body composition, which consequently affected fertility. Therefore, at the end of the rearing period, reproductive does should reach an optimal body condition (minimal body protein, fat and energy content), assuring an adequate feed intake and body development, which enable high fertility rates during first parturitions [
16]. In this sense, Pascual et al. [
13] stated that body data at the first AI are a sign of doe soma and might be related to its productive potential. In this context, the supplementation of reproductive sows with certain daily amounts of amino acids enabled an adequate retention of nitrogen that led to an acceptable reproductive performance [
51]. In this study, the threshold in the body composition at the first AI to avoid a sharp reduction in fertility in the second insemination might be set at 18% protein and 20% fat, but few does met this condition (12 and 7%, respectively). Other studies where the time of first insemination was delayed (to 18.4 or 19.5 weeks of age) rendered an increase in the body protein at that moment, as well as higher fertility values (83–87%) that were also associated with a higher fat content [
38], although this was not always the case [
52]. When the latter two studies were considered together, the does that were successful in the first five AI were lighter and had less body fat than the average (although their mean and range were similar to the current study), and the same body protein (although it was in the upper threshold that was previously mentioned: 17.9%) [
53], suggesting the potential relevance of body protein at the beginning of the productive life. Another difference between these extraordinary does and the average population was their higher fat mobilization between the second AI and the first weaning (this was recovered between weaning and the third AI) [
52]. These results partially differed from those of Theilgaard et al. [
54], who indicated that there was no positive effect of perirenal fat at the first AI on reproductive life. In fact, a higher risk of culling was associated with high fat mobilization, although does that were too lean also seemed to increase their risk of culling. Similarly, Castellini et al. [
50], using perirenal fat, found that does that were too fat and too lean (at AI) showed the poorest fertility. Recent results confirmed the negative effect of fatness at the first AI on the risk of being culled and litter size [
14]. The disagreement among these studies and the current one might be related to the different fatness range (probably the absence of does that are too fat at the first AI in our work: maximal fat content at the first AI: 22.1%;
Table 2), which might be associated with the time of the first AI (in the latter studies, nulliparous does were inseminated later than in the current one). Body fat and energy at the second AI was also related to fertility in the second AI, which might also reflect the observed positive influence of initial body condition on fertility at the second AI.
Quevedo et al. [
55] suggested that the success of AI 11 days after parturition was conditioned by the rabbit doe’s body condition at parturition rather than at insemination. This fact was not observed in this work, probably due to the fact that in our work, we measured body condition at parturition three days before birth (to avoid disturbing the doe) instead of after birth. This prevented the recording of an important proportion of fat mobilization, which was described by Savietto et al. [
17]. Once confirmed, the BIA measurement did not alter the doe immediately after parturition (unpublished results); subsequent studies recorded it just after parturition of the doe [
38,
52].
The reproductive success at the beginning of reproductive life also influenced the fertility of the third AI. In fact, rabbits that are reproductively successful during the first two parturitions were more vulnerable, and consequently, their reproductive performance was impaired in the third AI if they did not have the opportunity to recover. In this sense, Castellini et al. [
50,
56] proposed the delaying of the second AI after weaning in order to allow rabbit does to recover properly from the first gestation lactation and continue their growth. Otherwise, reproductive success at the beginning of the reproductive life of rabbit does, when they cannot recover their body reserves, might worsen the rabbit doe’s productivity and shorten its life span [
13].
The absence of effects of the breeding system and farm may be explained by the synchronization of rabbit does at the moment of AI. Rebollar et al. [
57] did not register a rhythm effect on fertility in the second parturition when using controlled lactation as the does’ synchronization tool. However, in experiments without any synchronization method [
56,
58], it was concluded that the reproductive rhythm was related to fertility rate. Anyway, it must be stressed that the current study was not designed specifically to study the effect of breeding system on fertility rate. Besides, an influence of the breeding systems on the body condition could not be ruled out and further studies would be required to figure out the nature of their relationship.
There were also a positive influence of body protein and energy at the first AI on the percentage of kits born alive at the first parturition. Rommers et al. [
15,
59] also related a higher protein content at the first insemination, and lower fat content, with a trend towards increasing numbers of kits born alive and percentages of kits born alive at the first parturition. They observed that restricted nulliparous rabbit does inseminated at 17.5 weeks, compared with those fed ad libitum and inseminated at 14.5 weeks, showed higher protein and lower fat content with a similar live weight, and tended to increase the number of kits born alive and the percentage of kits born alive at the first parturition. In primiparous rabbit does, a better body condition (higher body protein, lipid, and energy contents) was also related with changes in metabolic signals (increase in serum protein and leptin concentrations) that might influence ovarian follicle and gamete quality and might be associated with an improved reproductive outcome [
60]. Similarly, in sows, ovarian activity and oocyte quality were influenced by the body protein content [
61,
62]. Another explanation may involve body protein being related to fetal survival, as the increase in litter size has been related to a higher fetal survival, independently of ovulation rate [
63].
Live weight and body fat content were also positively correlated with body energy content at the first AI, but negatively with body protein, and had no effect on the percentage of kits born alive. In contrast, Rommers et al. [
46] reported that heavier females at the first AI (>4.0 kg) decreased the percentage of kits born alive, although this was combined with an improvement in the litter size at the first parturition. They related it to the development of the reproductive apparatus (larger uterine horns and more
corpora lutea in the ovaries). Moreover, these results did not agree with those of Quevedo et al. [
44], where rabbit does with higher perirenal fat thickness at 3 months of age tended to increase their percentage of kits born alive at the first parturition.
The reduction in the percentage of kits born alive in the second parturition, and the increase in the number of kits born alive, agreed with the results reported by Rommers et al. [
46], who also observed, at the second parturition, a higher number of kits born alive and a lower percentage of kits born alive with respect to the first parturition. The different percentage of kits born alive observed in the two farms might be due to the different hybrids used and/or the different environmental management conditions in each farm. No effect of the breeding system or farm on percentage of kits born alive at the second parturition was detected. Weight and body composition at the first AI were not related to this trait. However, higher body protein at the second AI increased the percentage of kits born alive. This result is similar to that recorded at the first AI (on the percentage of kits born alive at the first parturition) and again suggests a positive role of nitrogen content on conception success and fetus viability.