Biología reproductiva del macho aplicada al desarrollo de un banco de recursos genéticos en el oso pardo (Ursus arctos)

Laburpena

Genetic resource banks are a priority tool for the conservation of endangered species or populations such as the Cantabrian brown bear population. Optimizing this conservation strategy means using protocols adapted to the species, which require prior knowledge of its reproductive biology. However, the difficulties of observing and handling brown bears in the wild, together with their complex physiology, have led to a significant lack of knowledge on the matter. This doctoral dissertation provides relevant information about the social structure of male brown bears living under semi-free conditions, the influence of their annual reproductive biological rhythm on their behaviour and physiology, and the relationship between seminal quality and seasonality, as well as individual male factors (age, testosterone levels, testicular volume and social status). In the first experience, the dominance hierarchy of the 27 males older than three years in the brown bear population from Cabárceno Nature Park and the factors associated with dominance status were studied. The study of the dominance relationship between all pairs of individuals (dyads) showed that in the vast majority of aggressive interactions in which winner and loser can be determined (decisive encounters) the same male of each dyad was always the winner. This shows a very stable social hierarchy during the two years of the study. Attempts to define a linear hierarchical order for the population revealed the absence of an appropriate method of analysis, mainly due to the lack of information about decisive encounters in more than two thirds of the population dyads. The solution found was to allow situations of equality in dominance between individuals, using an optimized version of the I & SI hierarchical ordering algorithm developed by Schmid & de Vries (2013), to be able to describe groups of individuals in which their dominance status is interchangeable. The use of the Python language allowed to speed up the analysis of large volumes of data. The resulting order revealed three dominance classes: the dominant class with 3 individuals, the intermediate class with 8 individuals and a subordinate class with 16 individuals in which the majority of subadult individuals were included. These dominance classes could be explained based on morphometric (body weight, measurements of canine teeth, feet and hands), endocrine (testosterone levels) and ethological (interruption of mating) parameters. The most representative characteristics of dominant males were higher body weight and display of interruption of mating behaviour towards other bears. In the second experience, the annual biological rhythm of brown bear males was defined based on reproductive criteria and it was described in terms of ethological (aggressive, mounting and play behaviours and number of bears in the study area), morphological (body weight and testicular volume) and blood (complete hemogram, total protein, cholesterol, glucose,testosterone and cortisol levels) parameters. The reproductive season occurred between March and August and had a central reproductive period in the second half of April and May characterized by a peak in reproductive activity and serum testosterone levels (>5 ng/mL). Thus, the natural year is divided into five temporal units called periods: PreB (Pre-Breeding; January and February), EB (Early Breeding; March and the first fortnight of April), PB (Peak Breeding; the second half of April and May), LB (Late Breeding; June, July and August) and PostB (Post-Breeding; September to December). There were changes between periods in all types of behaviours, all morphological parameters and in most blood parameters. Males had higher body weight in PB than during the rest of the reproductive season (p < 0,01). During PB, males showed the highest rate of sexual behaviour (p < 0,01) and testosterone concentration (p < 0,01) and, due to the intense competition for females, also the highest rates of agonistic behaviour (p < 0,01) and the lowest rates of play behaviour (p < 0,01) of the year. If compared to LB, PB also showed higher testicular volume (p < 0,05), higher leukocyte, neutrophil and monocyte concentrations (p < 0,05) and lower eosinophil (p < 0,01) concentrations. Although lymphopenia was not observed, the rest of the findings regarding the white blood cells match the indicators of a stress leukogram during PB. However, serum cortisol, also considered to increase under stress conditions, did not present changes during the whole year. The components of the red and platelet series did not change during the reproductive season. The lowest levels of cholesterol during the breeding season were observed during PB (p < 0,05) and total protein levels were lower during PB than during LB (p < 0,01). The highest number of bears in the study area of the year happened in LB (p < 0,01) in response to increased appetite during the hyperphagia phase because the study area is also the place used for food administration. Blood glucose concentration was positively correlated to body weight (p < 0,02; r = 0,40) and was the lowest during LB (p < 0,01). Finally, in the third and last experience the influence of several factors on the seminal quality of the more concentrated fraction (rich fraction) of the ejaculate were studied: seasonality (as main environmental factor) and age, testicular volume, testosterone levels, and social status (as main individual male factors). Factors dependent on the male were evaluated only during PB to minimize seasonal influences. As of the second fortnight of April (PB) the rich fraction of the ejaculate did not present changes with respect to LB in the parameters pH, osmolality, volume, spermatozoa concentration and production, MI, MP, VAP, VSL, LIN, ALH, iACRO (viable spermatozoa with intact acrosomes), VIAB (viable spermatozoa), and MIT (spermatozoa with high mitochondrial membrane potential). During EB, seminal samples showed lower volume, VAP, VSL, LIN and iACRO compared to LB (p < 0,05), but they still can be considered a good quality sample for cryopreservation. The male social status did not have any influence in seminal characteristics. However, significant differences were found in some seminal characteristics depending on the testicular volume and age of the male, but such differences do not imply clear improvements in seminal quality for any of the groups. Bears from the age group between 10 and 20 years old had lower spermatozoa concentration (p < 0,05), which could be related to higher ejaculation frequencies at that age group. Serum testosterone levels below 1 ng/mL were associated with lower percentages of motile spermatozoa and decreases in VAP and VSL (p < 0,05). Therefore, it can be asserted that, although sexually mature male brown bears present a marked social hierarchy and important variations associated with reproductive seasonality in ethological, morphological and physiological aspects, their seminal quality is appropriate for cryopreservation into the genetic resource bank during all the periods comprising the reproductive season and regardless of all the individual factors studied in this doctoral dissertation.