Contribution to and reconsideration of knowledge in small animal neonatology and gonadectomy in the dog

Part 1: Physiology of the neonate and indicators for its well-being Veterinary neonatology is a rather young field of research. Important differences between the physiology of the neonate compared to the adult have to be taken into consideration in its clinical evaluation. The first topic of the present PhD thesis describes the current knowledge of neonatal physiology and different methods for evaluation of the newborns’ viability. Methods such as the Apgar scoring system and blood gas analysis are successfully used in veterinary medicine. Birth weight and neonatal growth have been evaluated as parameters to estimate a puppy’s risk for neonatal mortality. Although such a correlation has been proven, the populations used for this prove are composed of a high number of different breeds. To evaluate the usefulness of such an approach a comparison with a breed-specific evaluation has been made (Appendix 1). This comparison has shown important differences and the importance of breed-specific investigations of birth weight and growth of the neonate. Part 2: Current knowledge on beneficial and noxious effects of gonadectomy Elective gonadectomy is an important part of the daily veterinary practice. Although beneficial effects of gonadectomy on the dog have been proven, over the last two decades the number of studies reporting an increased risk for neoplastic and other disorders in the castrated animal increased. These reports, although predominantly retrospective, statistical investigations have caused concern and doubt within practitioners and owners. Easy access to information granted by the internet increased this doubt in the group of owners. Results of these investigations have been cited and described in scientific papers, as well as on non-scientific web-sites. In both, although most frequently in the latter, results of studies have been mentioned without describing or considering the study design, the population and the context in which the results have been presented. The lack of this information lead to premature conclusions which have an important impact on many stakeholders. It is therefore that the second part of the present PhD thesis presents a literature review of beneficial and noxious effects of gonadectomy. Prostatic neoplasia in the dog was used as an example of problematic differences in study design, population composition and interpretation of the results (Appendix 2). This part further presents the motivation for the experimental design of Part 3. 5 Part 3 and 4: Immunohistochemical evaluation of steroid hormone receptor expression in extra-genital tissue in the bitch motivated by reports on negative long-term health effects of gonadectomy Hormone receptor expression has proven to be of importance not only in the research of a pathological pathway, but gave also indications on possible treatment options (e.g. human breast cancer). In veterinary medicine the research on hormone receptor expression is not yet as commonly used as in human medicine. The presence of hormone receptors in a tissue may be considered the basis for a direct effect of a hormone on a tissue. Nevertheless, their absence does not exclude the possibility that a certain hormone may not have an impact following a secondary pathway. Reports on effects of gonadectomy on extra-genital tissue gave reason to investigate the expression of Estrogen and Progesterone receptors. Immunohistochemistry is a viable method to evaluate hormone receptor expression. Samples of 14 bitches between the age of 2.1 to 16.7 years were taken within a maximum of 4 hours postmortem. Ten bitches resulted gonadectomized at the moment of death. Collected tissues include the right cardiac ventricle, the splenic hilum and the popliteal and retromandibular lymph nodes. Samples were formalin- fixed, paraffin-embedded and stained with hematoxylin and eosin for histological evaluation. Expression of progesterone receptor (PR) and estrogen receptor alpha (ERa) was evaluated with anti-human antibodies: monoclonal rabbit PR (Clone 1E2) and monoclonal mouse ERa (Clone EP1) using an automatic immunostainer (Ventana BenchMark GX, Roche Diagnostic). Reactivity with canine tissue was confirmed using dog uterus and ovaries showing strong positive nuclear staining for both ERa and PR. Tissues of 11 dogs were considered physiologic or mildly compromised, whereas 3 dogs presented neoplasia in heart (n=2) and spleen (n=1). IHC showed negative immunostaining within investigated tissues for neither ERa not PR. Prior to drawing final conclusions, additional tests have to be performed to confirm the absence of ERa and PR expression. Despite the preliminary results in our study, we consider further research on hormone receptor expression necessary to understand the possible impact of gonadectomy on extra-genital tissue in the dog.