2. Universidad Cardenal Herrera-CEU

Precision nutrition in broilers requires tools capable of identifying amino acid imbalances individually or in groups, as well as knowledge on how more digestible proteins can be designed for innovative feeding programs adjusted to animals’ dynamic requirements. This work proposes two potential tools, combining traditional nutrition with biotechnological, metabolomic, computational and protein engineering knowledge, which can contribute to improving the precise amino acid nutrition of broilers in the future: (i) the use of serum uric nitrogen content as a rapid biomarker of amino acid imbalances, and (ii) the design and modeling of de novo proteins that are fully digestible and fit exactly to the animal’s requirements. Each application is illustrated with a case study. Case study 1 demonstrates that serum uric nitrogen can be a useful rapid indicator of individual or group amino acid deficiencies or imbalances when reducing dietary protein and adjusting the valine and arginine to lysine ratios in broilers. Case study 2 describes a stepwise approach to design an ideal protein, resulting in a potential amino acid sequence and structure prototype that is ideally adjusted to the requirements of the targeted animal, and is theoretically completely digestible. Both tools can open up new opportunities to form an integrated framework for precise amino acid nutrition in broilers, helping us to achieve more efficient, resilient, and sustainable production. This information can help to determine the exact ratio of amino acids that will improve the efficiency of the use of nitrogen by poultry.

The ideal protein concept refers to dietary protein with an amino acid profile that exactly meets an animal’s requirement. Low-quality protein levels in the diet have negative implications for productive and reproductive traits, and a protein oversupply is energetically costly and leads to an excessive N excretion, with potentially negative environmental impact. Urea Nitrogen (UN), which corresponds to the amount of nitrogen in the form of urea circulating in the bloodstream, is a metabolite that has been widely used to detect amino acid imbalances and deficiencies and protein requirements. This review aims to critically analyse how UN can contribute to accurately implementing the ideal protein concept in monogastric animals, particularly in pig, poultry, and rabbit nutrition (14,000 animals from 76 published trials). About 59, 37, and 4% of trials have been conducted in pigs, poultry, and rabbits, respectively. UN level was negatively correlated to main performance traits (Pearson Correlation Coefficient [PCC] of 􀀀0.98 and 􀀀0.76, for average daily gain and feed conversion ratio, respectively), and lower UN level was related to higher milk yield and concentration. High level of UN was positively correlated to N excretion (PCC = 0.99) and negatively correlated to protein retention (PCC = 􀀀0.99). Therefore, UN in blood seems to be a proper indicator of amino acid imbalance in monogastric animals. Great variability in the use of UN was observed in the literature, including uses as determination medium (blood, plasma, or serum), units, and feeding system used (ad libitum or restricted), among others. A standardization of the methods in each of the species, with the aim to harmonize comparison among works, is suggested. After review, UN measurement in plasma and, whenever possible, the utilization of the same nutritional methodology (ad libitum conditions or restriction with blood sampling after refeeding at standardised time) are recommended. More studies are necessary to know the potential of UN and other bioindicators for amino acid deficiencies evaluation to get closer to the ideal protein concept.

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.

As a consequence of the genetic selection process in growing rabbits, there are currently fastgrowing animals exhibiting an average daily gain that may exceed 45 g/d. The protein requirements of these animals, namely amino acid requirements, may differ from animals with low growth rates. The objective of this work was to evaluate growth performance, the coefficient of total tract apparent digestibility (CTTAD), the apparent ileal digestibility (AID) of amino acids and nutrient retention of fast-growing rabbits when they had access to a diet with high levels of amino acids and/or a diet formulated with current nutritional recommendations in a choice-feeding trial. To this end, two diets were formulated: the M diet following current nutritional recommendations for growing rabbits (including 8.1, 5.8 and 6.9 g/kg dry matter (DM) of total lysine, sulphur amino acids and threonine, respectively) and the H diet with higher levels of total lysine, sulphur amino acids and threonine (9.4, 6.6 and 7.8 g/kg DM, respectively). A total of 220 weaned rabbits, from a paternal line selected for the growth rate, had free access to the M diet, the H diet or both (MH) diets from 28 to 63 days of age. The CTTAD of DM, crude protein and gross energy from 49 to 53 days of age as well as the AID of amino acids of the diets at 63 days of age were determined. Protein, amino acids and energy retained in the empty body from 28 to 63 days of age were also registered. No significant differences in growth performance, CTTAD, AID and nutrient retention between dietary treatments were observed. However, animals fed the H diet showed a higher AID of cysteine (p < 0.05) and higher threonine retention (p < 0.05) compared to the M diet. As regards the choice-feeding trial, MH animals showed a higher feed intake of the M diet compared to the H diet (+8.61%; p < 0.001), and furthermore, more than 50% of the animals preferred the M diet throughout the experimental period (p < 0.05). Our results suggest that animals with high growth rates do not show significantly higher productive traits when fed the H diet compared to the M diet. As regards choice feeding, MH animals were capable of choosing their preferred diet, showing high intra-individual repeatability in preference for the M diet. It would be interesting to continue studying this behaviour of choice based on amino acid levels.

Staphylococcal mastitis is a major health problem in humans and livestock that leads to economic loss running in millions. This process is currently one of the main reasons for culling adult rabbit does. Surprisingly, the two most prevalent S. aureus lineages isolated from non-differentiable natural clinical mastitis in rabbits (ST121 and ST96) generate different immune responses. This study aimed to genetically compare both types of strains to search for possible dissimilarities to explain differences in immune response, and to check whether they showed similar virulence in in vitro tests as in experimental intramammary in vivo infection. The main differences were observed in the enterotoxin gene cluster (egc) and the immune-evasion-cluster (IEC) genes. While isolate ST121 harboured all six egc cluster members (seg, sei, selm, seln, selo, selu), isolate ST96 lacked the egc cluster. Strain ST96 carried a phage integrase Sa3 (Sa3int), compatible with a phage integrated into the hlb gene (β-haemolysin-converting bacteriophages) with IEC type F, while isolate ST121 lacked IEC genes and the hlb gene was intact. Moreover, the in vitro tests confirmed a different virulence capacity between strains as ST121 showed greater cytotoxicity for erythrocytes, polymorphonuclear leukocytes and macrophages than strain ST96. Differences were also found 7 days after experimental intramammary infection with 100 colony-forming units. The animals inoculated with strain ST121 developed more severe gross and histological mastitis, higher counts of macrophages in tissue and of all the cell populations in peripheral blood, and a significantly larger total number of bacteria than those infected by strain ST96.

Genetic selection and nutrition management have played a central role in the development of commercial rabbitry industry over the last few decades, being able to affect productive and immunological traits of the animals. However, the implication of different energy sources in animals from diverse genetic lines achieving such evolutionary success remains still unknown. Therefore, in this work, 203 female rabbits housed and bred in the same conditions were used from their first artificial insemination until their fifth weaning. The animals belonged to three different genetic types diverging greatly on breeding goals (H line, hyper-prolific (n=66); LP line, robust (n=67) and R line, selected for growth rate (n=67), and were assigned to two experimental diets, promoting major differences in energy source (cereal starch or animal fat)). The aims of this work were to: (1) characterize and describe blood leucocyte populations of three lines of rabbit does in different physiological stages during their reproductive period: first artificial insemination, first weaning, second parturition and fifth weaning; and (2) study the possible influence of two different experimental diets on the leucocyte populations in peripheral blood. Flow cytometry analyses were performed on blood samples taken from females at each different sampling stade. Lymphocyte populations at both weanings were characterized by significantly lower counts of total, CD5+ and CD8+ lymphocytes (–19.8, –21.7 and –44.6%; P<0.05), and higher counts of monocytes and granulocytes (+49.2 and +26.2%; P<0.05) than in the other stages. Females had higher blood counts of lymphocytes B, CD8+ and CD25+ and lower counts of CD4+ at first than at fifth weaning (+55.6, +85.8, +57.5, –14.5%; P<0.05). G/L ratio was higher at both weanings (P<0.05), and CD4+/CD8+ ratio increased progressively from the 1AI to the 5 W (P<0.001). Regarding the effect of genetic type in blood leucocyte counts, LP animals presented the highest counts for total, B, CD5+ and CD8+ lymphocytes (+16.7, +31.8, +24.5 and +38.7; P<0.05), but R rabbits showed the highest counts for monocytes and granulocytes (+25.3 and +27.6; P<0.05). The type of diet given during the reproductive life did not affect the leucocyte population counts. These results indicate that there are detectable variations in the leucocyte profile depending on the reproductive stage of the animal (parturition, weaning or none of them). Moreover, foundation for reproductive longevity criteria allows animals to be more capable of adapting to the challenges of the reproductive cycle from an immunological viewpoint.

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.

The specialization process associated with genetic selection could be associated with functional disorders, affecting the reproductive success of females (‘fitness’). We hypothesized that by modulating energy acquisition and allocation of females we could balance productivity and reproductive success. To test this hypothesis, we used 203 rabbit females belonging to three genetic types: H (n=66) maternal line specialised in prolificacy, LP (n=67) generalist maternal line, R (n=70) paternal line specialised in growth rate. We fed each genetic type with two diets specifically designed to promote milk yield (AF) or body reserves recovery (CS). We controlled females between their first and fifth reproductive cycles, recording traits related with productivity and fitness of females. H females fed CS had on average 11.2􀁲0.43 kits with an individual weight of 54􀁲1.2 g at birth and 525􀁲11 g at weaning. Their conception rate when multiparous was 44% and their survival rate at the end of the experiment 30%. When they were fed AF, the individual weight of kits was 3.8 g heavier (P<0.05) at birth and 38 g heavier at weaning (P<0.05), the conception rate when multiparous increased 23 percentage points (P<0.05) and the survival rate at the end of the experiment 25 percentage points (P<0.05). LP females fed CS had on average 10.8􀁲0.43 kits with an individual weight of 52􀁲1.2 g at birth and 578􀁲11 g at weaning. Their conception rate when multiparous was 79% and their survival rate at the end of the experiment 75%. When they were fed AF, it only increased individual weight of kits at weaning (+39 g; P<0.05). R females fed CS had on average 8.4􀁲0.43 kits with an individual weight of 60􀁲1.2 g at birth and 568􀁲11 g at weaning. Their conception rate when multiparous was 60% and their survival rate at the end of the experiment 37%. When they were fed AF, they presented 1.4 kits less at birth (P<0.05) but heavier at birth (+4.9 g; P<0.05) and at weaning (+37 g; P<0.05). Therefore, we observed that genetic types prioritised different fitness 3 components and that diets could affected them. In this sense, seems that more specialised genetic types, were more sensitive to diets than the more generalist type.