1. Investigación

Mycobacteriosis is an important disease that affects captive and wild aquatic fish. Syngnathids are susceptible to infection by non-tuberculous mycobacteria. The aim of this study was to describe clinical signs, and macroscopic and histological lesions in 25 syngnathids and the molecular characterization of the causative mycobacteria. Clinical presentation ranged from sudden death to non-specific signs, including anorexia, poor body condition, weight loss and marked dyspnea with increased respiratory effort and rate. Gross lesions were mostly ulcers on the tail and small white nodules in the liver, coelomic cavity and inside the eye. The most affected organs were gills, liver, intestine and coelomic mesentery. Microscopic lesions consisted of areas of multifocal to diffuse granulomatous inflammation and bacterial emboli with numerous intralesional acid-fast bacilli. Epithelioid cells, multinucleated giant cells, lymphocytes and fibrous connective tissue, which are commonly observed in granulomatous inflammation, were not observed here. In the real-time PCR, M. fortuitum, M. chelonae and M. marinum common primers, Mycobacterium spp. were detected in 4, 7 and 14 individuals, respectively. In addition, this is the first description of mycobacteriosis found in Syngnathus acus.

Although the effects of sperm microbiota and sperm quality have been described previously, recent studies provide evidence that female genital modifications triggered by seminal components could be of significant importance to identify some disturbances associated with fertility. So, sperm microbiota could play a key role in sperm quality, contributing to fertilisation. To understand how sperm microbiota diversity is influenced by the host genetics, the symbiotic bacteria in four inbred lines raised in the same animal facility and their effects on sperm quality and fertility were analysed. Forty healthy rabbits from four selected Spanish commercial lines were used in this research (three based on litter performance, designated A, V and LP, and one selected for daily body weight gain, called R). Significant variations in the seminal concentration, morphology and some motion parameters were found among inbred lines, but sperm motility and viability were similar among inbred lines. After mating, inbred lines selected for litter size had the same fertility rate, significantly higher than inbred line selected for body weight (82±3.3%, 79±3.5% and 89±4.5% versus 61±3.7%, for the A, V and LP vs R lines, respectively, p<0.05). Bacteria belonging to Proteobacteria, Firmicutes, Fusobacteria and Bacteroidetes were identified in sperm microbiota. At genus level, the bacterial community composition in the sperm microbiota was influenced by host genetics. A total of 35, 16, 34, and 51 genera were accurately detected in the A, V, LP, and R lines, respectively. Moreover, Enhydrobacter, Ferruginibacter, Myroides Paracoccus, Rheinheimera, Tepidiphilus, Tetradesmus obliquus and Thauera genera were present only in the inbred lines selected for litter size. Moreover, the discriminant analysis revealed Lysinibacillus and Flavobacterium genera as potential biomarkers for fertility. Thus, these two genera may play a key role in fertility. Our results demonstrated the 3 existence of a rabbit inbred line-specific variation in bacterial occurrence in sperm microbiota. Moreover, fertility differentials among inbred lines that were not predicted by routine semen analysis could be partly explained by the symbiotic state of the semen microbiota.

La importancia del estudio de la fisiología reproductiva del conejo (Oryctolagus cuniculus) viene dada por un lado, por su carácter como animal de laboratorio de interés en biomedicina y, por otro como especie de interés zootécnico. En este trabajo se han tratado de caracterizar a nivel reproductivo dos líneas de conejo seleccionadas por diferente criterio, estudiando diferentes parámetros. Los resultados obtenidos han mostrado diferencias en varios de estos parámetros entre las dos líneas, entre los que se encuentran la inducción de la ovulación, el número de embriones implantados a los 12 días de gestación y el tamaño de camada. Con el fin de estudiar algunas de las posibles causas de estas diferencias en el número de embriones implantados, en la segunda parte del trabajo se ha analizado la expresión génica de algunos genes relacionados con el fenómeno de implantación embrionaria en blastocistos de 5 y 6 días, como son el Oct4, la Integrina, los Interferones γ y ω, el Factor de Crecimiento del Endotelio Vascular (VEGF), el receptor 3 del Factor de Crecimiento Epidérmico (erbB3) y el Factor β de Crecimiento Transformante (TGF-β).

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.