2. Universidad Cardenal Herrera-CEU

European wild rabbit (Oryctolagus cuniculus) has been defined as a keystone species in the Mediterranean ecosystem. Rabbits have been classed as “endangered” by the IUCN within their native range. In this sense, animal nutrition may play a fundamental and limiting role in the conservation of wild species. The overarching goal of ecological nutrition is to unravel the extensive web of nutritional links that direct animals in their interactions with their ecological environments. The main aim of this work was to evaluate the effect of different feed intake, geographic location, animal sex, and reproductive stage on glucose, non-esterified fatty acids (NEFA), and plasmatic urea nitrogen (PUN), albumin, glutamate, and total protein metabolites. Additionally, we examined the potential of these metabolites as biomarkers. Full stomach contents and blood samples were collected from European wild rabbits (n = 89) for the analysis of the metabolites described above. Our work shows that the levels of these metabolites are affected by the sex of the animals, as well as by their reproductive stage (glucose, NEFA and albumin). There were signs of better optimisation of resources by females than by other groups of animals. These data may be interesting in the study of nutritional components that could be affecting physiological state of this species.

European wild rabbit (Oryctolagus cuniculus) populations have drastically reduced, and recently, rabbits have been classed as “endangered” by the IUCN. This animal plays an important ecological role in Mediterranean ecosystems and its introduction could significantly contribute to ecological restoration. Rabbits have high nutrient requirements that apparently cannot be covered in all ecosystems, and there are clues that nutrition can limit their abundance and density. On the other hand, some studies reflect the effects of food availability on the metabolomic status of other animal species, but there are no specific studies on this keystone species. The main aim of this work is to find biomarkers to assess the previous levels of ingestion of European rabbits (Oryctolagus cuniculus). To address this gap, gastric content and blood samples were collected from European rabbits (n = 99) in a Mediterranean area for the analysis of glucose, non-esterified fatty acids (NEFA), plasmatic urea nitrogen (PUN), albumin, glutamate and total protein metabolites. Depending on their previous feed intake (gastric content and the ratio between the gastric content and the weight of the animal), the animals were divided into two groups (lower and normal previous feed intake). Our work shows that the metabolomic profiles of the animals were affected. Levels of glucose (+82%; p = 0.0003), NEFA (􀀀61%; p = 0.0040) and PUN (+139%; p < 0.001) were different in the animals with lower previous feed intake than the animals with normal previous feed intake. This work summarises that metabolic phenotype can be interesting when seeking to discover the limiting nutrients and food availability in diets that could affect the ecological fitness and conservation of European wild rabbits. It is important to mention that in this work, only the effects on six different metabolites have been analysed and more studies are necessary to complement the knowledge of possible metabolites that indicate the level of ingestion in this species and others. These (and new) biomarkers could be used as a tool to provide information about individual or population characteristics that other physiological parameters cannot detect, improving the conservation physiology field.

The main purpose of this study was to find several early factors affecting stayability in rabbit females. To reach this goal, 203 females were used from their first artificial insemination to their sixth parturition. Throughout that period, 48 traits were recorded, considered to be performance, metabolic and immunological indicators. These traits were initially recorded in females’ first reproductive cycle. Later, removed females due to death or culling and those that were non-removed were identified. A first analysis was used to explore whether it was possible to classify females between those reaching and those not reaching up to the mean lifespan of a rabbit female (the fifth reproductive) cycle using information from the first reproductive cycle. The analysis results showed that 97% of the non-removed females were classified correctly, whereas only 60% of the removed females were classified as animals to be removed. The reason for this difference lies in the model’s characteristics, which was designed using early traits and was able to classify only the cases in which females would be removed due to performance, metabolic or immunologic imbalances in their early lives. Our results suggest that the model defines the necessary conditions, but not the sufficient ones, for females to remain alive in the herd. The aim of a second analysis was to find out the main early differences between the non-removed and removed females. The live weights records taken in the first cycle indicated that the females removed in their first cycle were lighter, while those removed in their second cycle were heavier with longer stayability (−203 and þ202 g on average, respectively; P < 0.05). Non-removed females showed higher glucose and lower beta-hydroxybutyrate concentrations in the first cycle than the removed females (þ4.8 and −10.7%, respectively; P < 0.05). The average lymphocytes B counts in the first cycle were 22.7% higher in the non-removed females group (P < 0.05). The females removed in the first reproductive cycle presented a higher granulocytes/lymphocytes ratio in this cycle than those that at least reached the second cycle (4.81 v. 1.66; P < 0.001). Consequently, non-removed females at sixth parturition offered adequate body development and energy levels, less immunological stress and a more mature immune function in the first reproductive cycle. The females that deviated from this pattern were at higher risk of being removed from the herd.

To achieve functional but also productive females, we hypothesised that it is possible to modulate acquisition and allocation of animals from different genetic types by varying the main energy source of the diet. To test this hypothesis, we used 203 rabbit females belonging to 3 genetic types: H (n=66), a maternal line characterised by hyper-prolificacy; LP (n=67), a maternal line characterised by functional hyper longevity; R (n=79), a paternal line characterised by growth rate. Females were fed with 2 isoenergetic and isoprotein diets differing in energy source: animal fat (AF) enhancing milk yield; cereal starch (CS) promoting body reserves recovery. Feed intake, weight, perirenal fat thickness (PFT), milk yield and blood traits were controlled during 5 consecutive reproductive cycles. Females fed with CS presented higher PFT (+0.2mm, P<0.05) and those fed AF had higher milk yield (+11.7%, P<0.05). However, the effect of energy source varied with the genetic type and time. For example, R females presented a decrease in PFT at late lactation (􀀐4.3%; P<0.05) significantly higher than that observed for H and LP lines (on av. 􀀐0.1%; P>0.05), particularly for those fed with AF. Moreover, LP females fed with AF progressively increased PFT across the RC, whereas those fed with CS increased PFT during early lactation (+7.3%; P<0.05), but partially mobilised it during late lactation (-2.8%; P<0.05). Independently of the diet offered, LP females reached weaning with similar PFT. H females fed with either of the two diets followed a similar trajectory throughout the RC. For milk yield, the effect of energy source was almost constant during the whole experiment, except for the first reproductive cycle of females from the maternal lines (H and LP). These females yielded +34.1% (P<0.05) when fed with CS during this period. Results from this work indicate that the resource acquisition capacity and allocation pattern of rabbit females is different for each genetic type. Moreover, it seems that by varying the main energy source of the diet it is possible to modulate acquisition and allocation of resources of the different genetic types. However, the response of each one depends on its priorities over time.