1. Investigación

Despite the enormous efforts made to achieve effective tools that fight against Staphylococcus aureus, the results have not been successful. This failure may be due to the absence of truly representative experimental models. To overcome this deficiency, the present work describes and immunologically characterizes the infection for 28 days, in an experimental low-dose (300 colony-forming units) intradermal model of infection in rabbits, which reproduces the characteristic staphylococcal abscess. Surprisingly, when mutant strains in the genes involved in virulence (JΔagr, JΔcoaΔvwb, JΔhla, and JΔpsmα) were inoculated, no strong effect on the severity of lesions was observed, unlike other models that use high doses of bacteria. The inoculation of a human rabbitized (FdltBr) strain demonstrated its capacity to generate a similar inflammatory response to a wild-type rabbit strain and, therefore, validated this model for conducting these experimental studies with human strains. To conclude, this model proved reproducible and may be an option of choice to check both wild-type and mutant strains of different origins.

Staphylococcus aureus is an opportunistic multi-host pathogen that threatens both human and animal health. Animals can act as a reservoir of S. aureus for humans, but very little is known about wild animals’ epidemiological role. Therefore, in this study, we performed a genomic characterization of S. aureus isolates from wildlife, hunters, and their auxiliary hunting animals of Eastern Spain. Of 20 different species, 242 wild animals were examined, of which 28.1% were S. aureus carriers. The common genet, the Iberian ibex, and the European hedgehog were the species with the highest S. aureus carriage. We identified 30 different sequence types (STs), including lineages associated with wild animals such as ST49 and ST581, multispecies lineages such as ST130, ST398, and ST425, and lineages commonly isolated from humans, including ST1 and ST5. The hunters and the single positive ferret shared ST5, ST398, or ST425 with wild animals. In wildlife isolates, the highest resistance levels were found for penicillin (32.8%). For virulence factors, 26.2% of them carried superantigens, while 14.8% harbored the immune evasion cluster (IEC), which indicates probable human origin. Our findings suggest that wild animals are a reservoir of clinically relevant genes and lineages that could have the potential to be transmitted to humans. These data support the notion that wildlife surveillance is necessary to better understand the epidemiology of S. aureus as a pathogen that circulates among humans, animals, and the environment.

TheWorldHealth Organization (WHO) officially declared the novel coronavirus (COVID-19) as a pandemic on 11 March 2020, and educational institutions have had to modify most of their activities (face-to-face activities were suspended). This situation forced academic institutions to modify the evaluation format of students. The use of proctoring systems quickly became widespread, although some controversies arose. The twomain discussions regarding these systems are the integrity of the assessment and the capacity of the students to adapt to this new assessment method, without changes in theirs scores. To elucidate two controversies, we have analyzed the preferences and the scores obtained from a trial of 660 scores from 332 students of the third grade of Veterinary Medicine. The experiment involved three modalities of exam: an online format from home using the Respondus Lockdown Browser system (Modality 1), online in person using the Respondus Lockdown Browser system with the supervision of a teacher (Modality 2), or paper format in person with the supervision of a teacher (Modality 3). The results obtained showed that the students preferred Modality 1 (online at home with Respondus Lockdown Browser system). No statistical differences between the scores obtained by students were found between the three modalities analyzed. The proctoring system is a good method to adjudicate exams in higher education institutions, and the scores of students are similar to those obtained through traditional evaluation and control systems.

Variation in methylxanthines (theobromine and caffeine) and flavanols (catechin and epicatechin) was studied in a large set of cocoa powders (covering different origins, processing parameters and alkalization levels). The content of these compounds was established by high-performance liquid chromatography (HPLC), whose results showed that the alkalisation process lowered the content of all analytes, whose loss was more evident in flavanols. Therefore, the determination of these analytes in a huge set of samples allowed not only better knowledge of the concentration variability in natural commercial cocoas from different origins, but also the understanding of the effect that industrial alkalisation has on these contents. The feasibility of reflectance near-infrared spectroscopy (NIRS) combined with partial least square (PLS) to non-destructively predict these contents, was also evaluated. All the analytes were generally well predicted, with better predictions for methylxanthines (R2 P 0.882 for both analytes; RMSEP 0.020-0.061%, bias -0.027-0.006) than for flavanols (R2 P 0.818-0.863; RMSEP 6.63-15.87%, bias 1.942-3.056). Thus NIRS could be an alternative fast reliable method for the routine assessment of these analytes in the cocoa industry.

Hace 30 años, mi abuelo era cunicultor, tenía una granja con 200 conejas, trabajaba 8 horas al día y ganaba suficiente dinero para vivir con mucha tranquilidad. Junto a mi padre, fueron conscientes de que tener animales improductivos en la explotación no era rentable. Sin embargo, era muy difícil valorar individualmente a las conejas ya que no registraban la información de cada parto. Para evitar este problema se les ocurrió un sistema en el que marcaban una raya en el comedero de la jaula cada vez que una coneja no quedaba gestante después de la monta. De esta forma, cuando había 3 rayas marcadas en un comedero, reemplazaban la coneja. Con el paso de los años, el sector cunícola ha evolucionado y explotaciones de este tamaño ya no son rentables. Sin embargo, lo importante de esta historia es que, desde hace mucho tiempo, muchos cunicultores han sido conscientes de la necesidad del control de los animales para mejorar el beneficio económico de su explotación. De esta forma nacen los sistemas de gestión técnica con el objetivo de incrementar la rentabilidad de las explotaciones.

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.

Cocoa shell must be removed from the cocoa bean before or after the roasting process. In the case of a low efficient peeling process or the intentional addition of cocoa shell to cocoa products (i.e. cocoa powders) to increase the economic benefit, quality of the final product could be unpleasantly affected. In this scenario, the Codex Alimentarius on cocoa and chocolate has established that cocoa cake must not contain more than 5% of cocoa shell and germ (based on fat-free dry matter). Traditional analysis of cocoa shell is very laborious. Thus, the aim of this work is to develop a methodology based on near infrared (NIR) spectroscopy and multivariate analysis for the fast detection of cocoa shell in cocoa powders. For this aim, binary mixtures of cocoa powder and cocoa shell containing increasing proportions of cocoa shell (up to ca. 40% w/w based on fat-free dried matter) have been prepared. After acquiring NIR spectra (1100-2500 nm) of pure samples (cocoa powder and cocoa shell) and mixtures, qualitative and quantitative analysis were done. The qualitative analysis was performed by using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), finding that the model was able to correctly classify all samples containing less than 5% of cocoa shell. The quantitative analysis was performed by using a partial least squares (PLS) regression. The best PLS model was the one constructed using extended multiple signal correction plus orthogonal signal correction pre-treatment using the 6 main wavelengths selected according to the Variable Importance in Projection (VIP) scores. Determination coefficient of prediction and root mean square error of prediction values of 0.967 and 2.43, respectively, confirmed the goodness of the model. According to these results it is possible to conclude that NIR technology in combination with multivariate analysis is a good and fast tool to determine if a cocoa powder contains a cocoa shell content out of Codex Alimentarius specifications.

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.