Dogslife is the first large-scale, longitudinal cohort study of canine lifestyle, morphology and health. The project involves recruiting the owners of UK-based, Kennel Club registered Labrador Retrievers and asking them to submit data about their dogs via an online questionnaire repeatedly as the dogs age. In this thesis, I have analysed Dogslife data regarding the lifestyle, morphology and health of Labrador Retrievers up to four years of age. A validation study was initially undertaken in order to understand the quality of the Dogslife data because this would underlie all future investigations. Owners were visited and veterinary records scrutinised. It was determined that Dogslife illness reports were subject to recall decay and that minor changes would improve the usability of the questionnaire. Accelerometers were subsequently sent to a subset of the cohort and aspects of the Dogslife exercise questionnaire were found to be correlated to accelerometer readings indicative of sedentary, light and moderate to vigorous exercise. Overall, Dogslife dogs were exercised for over two hours each day with the time spent being dominated by time off lead and on other activities. Dogs in England spent less time exercising than those in Scotland and Wales and dogs in family households spent less time exercising than those in single adult households or households comprising more than one adult. Despite being pedigree animals, the males in the cohort were 2-3cm taller than the breed standard. On average, the females met the breed standard but there was wide variation for both sexes. Working dogs in the cohort were over 2kg lighter than household pets and chocolate coloured dogs were 1.4kg heavier than their black and yellow counterparts. Dogs in multi-dog households were 0.5kg lighter than those in households with no other dog. Heavier dogs spent less time fetching, chasing and retrieving and on other exercise. Over 6,000 signs of illness were reported to Dogslife in the first three and half years and approximately half of them did not involve a veterinary visit. Reported signs were dominated by vomiting and diarrhoea, both of which peaked when the dogs were between 3-6 months of age. For the first time, rates of diarrhoea were shown to be positively associated with human population density in the UK. Limber tail was found to be associated with swimming in the cohort and working dogs were more likely to develop the condition than pets. Genetic analyses identified regions of interest that might predispose the dogs to limber tail on chromosomes 6 and 30. Data from the Dogslife project provide a unique resource for investigating the epidemiology of Labrador Retrievers. This thesis creates a platform for all such future investigations.

The colony-stimulating factor-1 receptor (CSF-1R) is expressed by the mononuclear phagocytic lineage, and is important for the development of these cells from their progenitors and also for promoting their survival and activation after maturation. The receptor has two ligands, CSF-1 and IL-34, which induce the formation of a stable dimer between two receptor monomers. This leads to intracellular autophosphorylation of tyrosine residues and subsequent signalling cascades, leading to rapid protein expression, cytoskeleton remodelling and cellular motility. Although CSF-1R signalling is crucial for normal embryogenic development and other physiological functions mediated by the phagocytic lineage, it has also been found to promote the pathogenic progression of cancer. Tumour-associated macrophages (TAMs) can comprise a large proportion of the cellular population in several solid tumours. These cells promote several hallmarks of cancer malignancy, such as increased neovascularization, tissue invasion, induction of metastases and immunosuppression. In this work, it was confirmed that CSF-1 had a prominent role in inducing cancer-promoting cellular phenotypes. Both canine cancer cells and macrophages respond to this cytokine, respectively increasing cancer cell proliferation and reducing inflammatory activation. Given the importance of CSF-1R signalling in the tumour microenvironment, antibodies were generated with the objective of blocking receptor function. Mice were immunized with either the extracellular region or the dimerization domain of the CSF-1R. Hybridomas were produced using the primed splenocytes, and monoclonal antibody (mAb) candidates were selected based on their performance in immunostaining and on their capacity to inhibit CSF-1R+ cells. The best antibodies were subjected to speciation. Chimeric antibodies maintained the ability of the parental mAbs to inhibit macrophage proliferation following CSF-1R stimulation. However, the mAbs possessed moderate affinity and specificity for their target, failing to stain monocytes and presenting a degree of cross-reactivity. The binding properties of one of such mAbs were altered by PCR-induced mutations, generating semi-synthetic antibody libraries. These were screened by phage display, yielding novel clones that show reduced cross-reactivity with unrelated proteins and retain the property of inhibiting macrophage survival. These results are a step in the development of therapeutic monoclonal antibodies for cancer treatment in dogs.

In total, four experiments were conducted to determine the therapeutic and safety effects of the nutraceutical, turmeric and its active ingredient curcumin on canine and equine. Two studies were conducted on client-owned, moderately arthritic canines, studying the therapeutic and safety effect of curcumin’s anti-inflammatory properties. In Exp. 1, two different dosages, 500 mg, SID of 95% curcumin and 250 mg, BID of 95% liposomal-curcumin, were evaluated in ten moderately arthritic dogs over five months. The dogs in the 95% curcumin group had an overall greater significance in pain reduction by Day 60. Exp. 2, was a follow-up experiment to Exp. 1. In Exp. 2, two different dosages, 500 mg, SID or 100 mg, SID of 95% curcumin, were evaluated in ten moderately arthritic dogs over five months. Findings showed that dogs in the 500 mg, SID group had an overall greater significance in pain reduction by Day 60. Experiment 3 and 4 were a two-part project looking at the anti-microbial and anti-inflammatory properties of turmeric, curcumin, and liposomal-curcumin in cecally-cannulated equine. Exp. 3, was a two-part in vitro study, the first part looked at the anti-microbial effects of turmeric, curcumin, and liposomal-curcumin in reducing opportunistic bacteria found in the equine hindgut, including Streptococcus bovis/equinus complex (SBEC) (P = 0.0056), E. coli K-12 (P = 0.5114), Escherichia coli general (P = 0.1083), Clostridium difficile (P < 0.001), and Clostridium perfringens (P = 0.2439). Treatment D, 95% liposomal-curcumin, numerically reduced the concentration of all five opportunistic strains, and was therefore selected for use in the follow-up in vitro experiment. The second in vitro studied the effects of four different dosages, 15 g, 20 g, 25g, and 30 g of 500 mg/g of 95% liposomal-curcumin at reducing the concentration of SBEC (P < 0.0001), E. coli K-12 (P = 0.0124), E.coli general (P = 0.032), C. difficile (P = 0.5608), and C. perfringens (P = 0.4214). In Exp. 4, 500 mg/g of 95% liposomal-curcumin at 15 g, 25 g, and 35 g, were tested in vivo for anti-inflammatory and anti-microbial therapeutic effects. In total, four experiments were conducted to determine the therapeutic and safety effects of the nutraceutical, turmeric, and its active ingredient curcumin on canines and equines. Two studies were conducted on client-owned, moderately arthritic canines, studying the therapeutic and safety effect of curcumin’s anti-inflammatory properties. In Exp. 1, two different dosages, 500 mg, SID of 95% curcumin and 250 mg, BID of 95% liposomal-curcumin, were evaluated in ten moderately arthritic dogs over five months. The dogs in the 95% curcumin group, overall, had a greater reduction in pain by Day 60. Exp. 2, was a follow-up experiment to Exp. 1. In Exp. 2, two different dosages, 500 mg, SID or 100 mg, SID of 95% curcumin, were evaluated in ten moderately arthritic dogs over five months. We observed that dogs in the 500 mg, SID group had an overall greater significance in pain reduction by Day 60. Experiment 3 and 4 were conducted as a two-part project looking at the antimicrobial and anti-inflammatory properties of turmeric, curcumin, and liposomal-curcumin. The purpose of these studies were to investigate both form and dose of turmeric and its active ingredient, curcumin, on reducing opportunistic bacteria found in the equine hindgut. The bacterial strains of interest included Streptococcus bovis/equinus complex (SBEC), Escherichia coli K-12, Escherichia coli general, Clostridium difficile, and Clostridium perfringens. Exp. 3, was a two-part in vitro study; the first part looked at the antimicrobial effects of turmeric, curcumin, and liposomal-curcumin (LIPC) on reducing opportunistic bacteria found in the equine hindgut, including SBEC (P = 0.006), E. coli K-12 (P = 0.50), E. coli general (P = 0.11), C. difficile (P < 0.0001), and C. perfringens (P = 0.24). The follow-up in vitro 24 h batch culture examined four different dosages (15 g, 20 g, 25 g, and 30 g) of 500 mg/g of LIPC, at reducing the concentration of opportunistic bacteria. These results were utilized to determine the dosing rate in vivo. Exp. 3, in vitro, evaluated the efficacy of antimicrobial and anti-inflammatory properties of LIPC dosed at 15, 20, 25, and 35 g. These results were utilized to determine the dosing rate in vivo. Exp. 4, in vivo, evaluated the efficacy of antimicrobial and anti-inflammatory properties of LIPC dosed at 15, 25, and 35 g compared to a control. In vivo, LIPC’s antimicrobial properties, at 15 g, significantly decreased (P = 0.02) SBEC compared to other treatments. In addition, C. perfringens tended (P = 0.12) to decrease as LIPC dose increased. Non-significant results in digestion, blood parameters, and range of motion suggest there were no adverse side effects from oral dosing increasing doses of curcumin. Valerate decreased (P = 0.005) linearly as LIPC dose increased. As LIPC dose increased, butyrate and iso-valerate decreased (P ≤ 0.03) linearly. However, acetate tended (P = 0.10) to increase linearly as the dose of LIPC increased. Treatment did not affect (P ≥ 0.19) any of the other individual VFAs measured, but increasing doses of LIPC tended (P = 0.10) to increase total VFA concentrations. Additionally, LIPC tended (P = 0.11) to increase total VFA concentrations when compared to control. In the future, further work should be conducted examining liposomal-curcumin’s antimicrobial properties in canine and anti-inflammatory properties in equine over a longer period of time

Made available in DSpace on 2014-06-11T19:32:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-03Bitstream added on 2014-06-13T18:44:21Z : No. of bitstreams: 1 disantis_gw_dr_botfmvz_prot.pdf: 4486819 bytes, checksum: 22bfb6868fb6b4112ac603f50cd0b4e4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A neoplasia intra-epitelial prostática (HGPIN) e a atrofia inflamatória proliferativa (PIA) são lesões potencialmente pré-malignas encontradas na próstata humana (De Marzo et aI., 1999; Bostwick e Qian, 2004). HGPIN tem sido relatada na próstata canina com características semelhantes às observadas em humanos (Waters et aI., 1997), porém a PIA ainda não foi caracterizada nesta espécie. O presente trabalho objetivou a avaliação imunoistoquímica do índice proliferativo (PCNA e Ki67), da expressão de proteínas pró-apoptose (caspase-3), genes supressores de tumores (p-53), genes inibidores de apoptose (bcl-2) e de moléculas de adesão (E-caderina), além da avaliação histoquímica do estado proliferativo (AgNOR) e da determinação de mensurações nucleares por análise quantitativa computadorizada de imagens (AQCI) em focos HGPIN, PIA e em ácinos normais da próstata canina. Considerando os resultados de ácinos normais, constatou-se que as lesões estudadas apresentam alto índice proliferativo; capacidade proliferativa no compartimento epitelial secretor; ausência de expressão de p-53; perda de expressão de E-caderina; padrão de AgNOR semelhante ao de ácinos normais; e núcleos celulares maiores e com fator de circunferência maior. Focos de PIA exibem ainda índice apoptótico semelhante ao de ácinos normais e predomínio do linfócitos T, quando considerado o infiltrado Iinfocítico. Tais achados aproximam estas duas lesões entre si e sugerem que possam estar envolvidas no processo de transformação neoplásica da próstata canina. / Prostatic intraepithelial neoplasia (HGPIN) and proliferative inflammatory atrophy (PIA) are potentially premalignant lesions, found in human prostate (De Marzo et aI., 1999; Bostwick e Qian, 2004). HGPIN have been reported in canine prostate (Waters et aI., 1997), and share the same aspects with the humans, and PIA hadn't been described in the dogs. The aim of this work was to evaluate, by immunohistochemistry proliferative index (PCNA and KI-67), proapoptotic proteins expression (caspase 3), tumour suppressor gene (p-53), adhesion molecules (Ecadherin), histochemical proliferative status (AgNOR) and nuclear measurement by image computer quantitative analysis (AQCI) in HGPIN focus, PIA e normal acinus in canine prostate. Comparing the normal acinus results, the lesions showed higher proliferative index, secretory epithelial cells capable of proliferation, absence of p-53 expression, loss of E-cadherin expression, AgNOR patterns similar to normal acinus, cellular nucleus bigger and with higher nuclear round factor. PIA had the same apoptotic index as the normal acinus and mainly T Iymphocytes in the inflammatory infiltrate. Our findings allow us to consider these two lesions close to each other, and they may be involved in the process of neoplastic transformation of canine prostate.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-21Bitstream added on 2014-06-13T21:07:28Z : No. of bitstreams: 1 barreto_cs_dr_botfmvz.pdf: 336891 bytes, checksum: 2ec6600a65ab0775c0d9c612d2474e34 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O feto prematuro não sobrevive à vida extra-uterina devido a imaturidade de órgãos vitais como os pulmões e rins. O objetivo desse trabalho foi avaliar a maturidade renal através da identificação de proteínas de baixa massa molecular; pulmonar através da citologia com azul de Nilo (0,1%), teste de Clements modificado e densidade óptica dos fluidos; alfa-fetoproteína através da eletroforese em SDS-PAGE (12%) e concentração de cortisol nos líquidos fetais por radioimunoensaio. Foram obtidas 40 amostras de líquido amniótico e alantoideano de 16 cadelas gestantes pela cesariana. Ajustes no teste de Clements favorecem a análise de fluidos caninos promovendo resultados confiáveis (p<0,05) para as diluições. Células morfologicamente diferentes e com porcentagem de células orangeofílicas e cianofílicas refletem a maturidade pulmonar sugerindo feto em estágio de pós-maturidade. Na densidade óptica (DO) dos líquidos fetais encontramos 220l0,190 para os líquidos amniótico e 0,250l0,02 para o alantoideano sendo indicativo de maturidade (= 0,150). A concentração proteica nos fluidos estudados reflete a fisiologia fetal (p<0,05). A dosagem de cortisol indica a exposição do feto no ambiente uterino (p<0,05). A IOD da banda protéica de 66kDa foi de 15,94 para o líquido amniótico e 13,3 para o alantoideano. Cinco bandas proteicas presentes nos dois fluidos podem espelhar a maturidade renal (32; 30; 28; 20 e 12). Com base nestes dados conclui-se que a obtenção de informações básicas sobre a maturidade e viabilidade do feto canino podem ser avaliada com os testes propostos. / The premature fetus is unable to survive in extra-uterine environment due to immaturity of its vital organs as the lungs and kidneys. The objective of this work was to evaluate the renal maturity through the identification of low molecular mass proteins; pulmonary maturity through the cytology with Nile blue (0,1%), modified Clements test and optic density of fluids; moreover, the alpha-fetoprotein was evaluated through electrophoresis using SDS-PAGE (12%); and the cortisol concentration from fetal liquids was determinated by radioimunoassay. Forty amniotic and allantoic liquid samples from 16 pregnant bitches were colleted by punction during cesarean section. The Clements test was adapted to canine fluid analysis to promote true results (p<0,05) for the diluitions. Cells morphologically and with percentage of different orangeo and cyanophilia cells reflect the maturity. The optic density (DO) of the fetal fluids liquids were 220l0,190 and 0,250l0,02 to the amniotic and allantoic liquid, respectively, indicating maturity (= 0,150). The protein concentractions reflect the fetal physiology (p<0,05) in evaluated fluids. The cortisol concentration indicates the fetal exposition in the uterine environment (p<0,05). The IOD of 66 kDa protein band was 15,94 to amniotic and 13,30 to allantoic liquid. Five protein band in two fluids can reflect the renal maturity (32; 30; 28; 20 e 12). Based in these results we conclude that the tests considered in the study can evaluate the maturity and viability of canine fetus.

Canine myxomatous mitral valve disease (MMVD) is the most common cardiac disease in dogs affecting all breeds, and it shares many similarities with the equivalent human disease. From the only transcriptomic report for canine MMVD published in 2006, serotonin signalling was identified as a contributing factor and has been widely studied since. Two transcriptomic profiling studies in human MMVD have also identified oxidative stress response and bone morphogenic protein signalling contributing to disease pathology. All studies at the transcriptional level have identified a variety of biological functions in MMVD suggesting dynamic extracellular matrix (ECM) remodelling processes are on-going. Moreover, cellular changes found in MMVD are somewhat reminiscent of the events seen in early heart valve, suggesting possible re-activation of signalling pathways of which those driving development and endothelial-to-mesenchymal transition (EndoMT) are particularly interesting. EndoMT, in which endothelial cells change their identity to mesenchymal phenotype and migrate into the cardiac jelly underneath the endothelium, is a crucial mechanism in valvulogenesis. Whether or not gene regulation of EndoMT and valve development also plays a role in MMVD is unknown. In this study, the MMVD cellular changes in the Cavalier King Charles Spaniel (CKCS), a breed with the highest prevalence, earliest onset, and rapid progression of the disease, was investigated. Secondly, transcriptional profiling was conducted using the latest canine microarray chips, a single affected breed (CKCSs), stringent sample quality control and statistical thresholds, with quantitative polymerase chain reaction (Q-PCR) for data validation. After transcriptional mapping, multi-platform in silico analysis was conducted to identify relationship between differentially expressed genes and their relevant biological functions. Next, a comparison study using immunohistochemistry was performed on different severities of myxomatous valves to localize the proteins of interest. Finally, to model the transcriptional factors and their downstream targets, mitral valve endothelial cell (MVEC) clones were derived from the canine normal mitral valves for future in vitro studies. Cellular changes of MMVD between CKCS and non-CKCS populations showed no difference in their distribution, number and phenotypic markers. Global genomic expression analysis identified similar (inflammation, up-regulation of serotonin receptor and bone morphogenic protein) and novel biological functions (epithelial-to-mesenchymal transition) compared to the previous study in 2006. Key transcriptional factors and genes associated with EndoMT including SNAI1, TAGLN, ACTA2, ACTG2, HAS2, and CTNNB1 were found up-regulated, and NID1, LAMA2, CDH5 were down-regulated in the MMVD group. In myxomatous mitral valves, increased expression of HAS2 in myofibroblasts, SNAI1 expression in endothelial cells, and co-expression of CDH5 and α-smooth muscle actin (α-SMA) also suggested the presence of EndoMT compared to normal valves. Nevertheless, there is also evidence of EndoMT in normal valves (α-SMA positive endothelial cells) which might suggest contribution to life-long valve re-modelling. In addition, there was a decreased expression of microRNAs associated with modulation of extracellular matrix transcripts, including miR-23, miR-29, and miR-218, indicating epigenetic regulation in MMVD. Based on the cellular changes, MMVD in CKCS appears to be representative of MMVD in all breeds and the early-onset of MMVD in that breed does not lead to different end-stage pathology. Novel biological functions such as EndoMT, were identified by transcriptional profiling, and by using powerful bioinformatic tools providing insight into understanding gene regulation in MMVD. Furthermore, a relationship between developmental biology processes and MMVD pathogenesis was established, with a likely important role for epigenetics in disease pathogenesis.

Made available in DSpace on 2014-06-11T19:24:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-03Bitstream added on 2014-06-13T20:27:07Z : No. of bitstreams: 1 mangia_sh_me_botfmvz.pdf: 1746429 bytes, checksum: 82e2d527d1969123d65920e8659de0fc (MD5) / Este estudo teve por objetivos testar a eficácia da ribavirina em cães acometidos de cinomose na fase neurológica; avaliar os efeitos colaterais da mesma através do monitoramento hematológico e provas bioquímicas; avaliar a resposta imunológica no sistema nervoso central dos cães através do exame de líquor; adaptar uma dose e via de administração adequada da ribavirina em cães e testar a eficácia do DMSO como permeante de membranas biológicas atuando como vetor da ribavirina. Foram utilizados 20 cães com sinais clínicos neurológica, divididos em dois grupos de tratamento, sendo que um grupo recebeu a ribavirina e outro a associação da ribavirina e o DMSO, sem distinção de sexo e raça, com idades até seis anos e tempo de evolução máximo de 10 dias. Todos os animais foram avaliados clinicamente e realizado o teste de imunofluorescência direta de sangue para inclusão no estudo. Os exames complementares foram realizados de forma rotineira, o líquor foi colhido antes e após o tratamento, as drogas foram administradas durante 15 dias consecutivos e realizada a avaliação clínica diária dos animais. Pelos resultados observamos que a ribavirina demonstrou atividade efetiva contra o vírus da cinomose, exercendo leves efeitos colaterais na medula óssea, sistema imune e sistema gastro-intestinal. No líquor observamos diminuição da produção de anticorpos e verificamos que o DMSO tornou a ação da ribavirina mais eficaz. / This study aimed to test the ribavirin efficacy in dogs infected with canine distemper virus in neurological stage; to evaluate this collateral effects by hematological and biochemical evaluation; to evaluate the immunological response on dogs central nervous system (CNS) by cerebrospinal fluid (CSF) analysis; to adapt a dose and an adequate administration route of ribavirin in dogs and to test the efficacy of dimethylsulphoxide (DMSO) in increasing the permeability of biological membranes acting as ribavirin vector. Twenty dogs with neurological signs, divided in two treatment groups, without sex and breed distinction, till six years old and a maximum evolution time of 10 days were used. All animals were selected based on clinical sings and the blood direct fluorescent test (DFA). The hemogram, biochemical and urinalsis test were carried out. At the complementary tests, CSF was collected before and after the treatment, and ribavirin and its association with DMSO were administered during 15 days, consecutive, and the animal clinical evaluation was carried out daily. Based on the results, ribavirin demonstrated effective activity against canine distemper virus, besides its light collateral effects in bone marrow, immune and gastrointestinal systems. In CSF, the lowering of antibody production, and the positive effects of DMSO on ribavirin efficacy were observed.

Orientador: Nereu Carlos Prestes / Banca: Maria Denise Lopes / Banca: Fabiana Ferreira de Souza / Banca: Luiz Henrique de Araújo Machado / Banca: Maria Isabel de Mello Martins / Resumo: O feto prematuro não sobrevive à vida extra-uterina devido a imaturidade de órgãos vitais como os pulmões e rins. O objetivo desse trabalho foi avaliar a maturidade renal através da identificação de proteínas de baixa massa molecular; pulmonar através da citologia com azul de Nilo (0,1%), teste de Clements modificado e densidade óptica dos fluidos; alfa-fetoproteína através da eletroforese em SDS-PAGE (12%) e concentração de cortisol nos líquidos fetais por radioimunoensaio. Foram obtidas 40 amostras de líquido amniótico e alantoideano de 16 cadelas gestantes pela cesariana. Ajustes no teste de Clements favorecem a análise de fluidos caninos promovendo resultados confiáveis (p<0,05) para as diluições. Células morfologicamente diferentes e com porcentagem de células orangeofílicas e cianofílicas refletem a maturidade pulmonar sugerindo feto em estágio de pós-maturidade. Na densidade óptica (DO) dos líquidos fetais encontramos 220l0,190 para os líquidos amniótico e 0,250l0,02 para o alantoideano sendo indicativo de maturidade (= 0,150). A concentração proteica nos fluidos estudados reflete a fisiologia fetal (p<0,05). A dosagem de cortisol indica a exposição do feto no ambiente uterino (p<0,05). A IOD da banda protéica de 66kDa foi de 15,94 para o líquido amniótico e 13,3 para o alantoideano. Cinco bandas proteicas presentes nos dois fluidos podem espelhar a maturidade renal (32; 30; 28; 20 e 12). Com base nestes dados conclui-se que a obtenção de informações básicas sobre a maturidade e viabilidade do feto canino podem ser avaliada com os testes propostos. / Abstract: The premature fetus is unable to survive in extra-uterine environment due to immaturity of its vital organs as the lungs and kidneys. The objective of this work was to evaluate the renal maturity through the identification of low molecular mass proteins; pulmonary maturity through the cytology with Nile blue (0,1%), modified Clements test and optic density of fluids; moreover, the alpha-fetoprotein was evaluated through electrophoresis using SDS-PAGE (12%); and the cortisol concentration from fetal liquids was determinated by radioimunoassay. Forty amniotic and allantoic liquid samples from 16 pregnant bitches were colleted by punction during cesarean section. The Clements test was adapted to canine fluid analysis to promote true results (p<0,05) for the diluitions. Cells morphologically and with percentage of different orangeo and cyanophilia cells reflect the maturity. The optic density (DO) of the fetal fluids liquids were 220l0,190 and 0,250l0,02 to the amniotic and allantoic liquid, respectively, indicating maturity (= 0,150). The protein concentractions reflect the fetal physiology (p<0,05) in evaluated fluids. The cortisol concentration indicates the fetal exposition in the uterine environment (p<0,05). The IOD of 66 kDa protein band was 15,94 to amniotic and 13,30 to allantoic liquid. Five protein band in two fluids can reflect the renal maturity (32; 30; 28; 20 e 12). Based in these results we conclude that the tests considered in the study can evaluate the maturity and viability of canine fetus. / Doutor

Orientador: Wilter Ricardo Russiano Vicente / Banca: Maria Denise Lopes / Banca: Camila Infantosi Vannucchi / Resumo: O objetivo desta pesquisa foi avaliar os efeitos da suplementação de cisteína e cisteamina no desenvolvimento meiótico de oócitos caninos durante o processo de maturação ín vítro. Os oócitos foram coletados de sete cadelas hígidas em fase pré-ovulatória imediata, submetidas à ovario-histerectomia. Os COC's selecionados foram cultivados por um período de 72 horas em quatro meios diferentes: A (controle) - TCM199 suplementado com BSA (3 mg/mL) + FSH (5 J.Lg/mL) + LH (10 f.Lg/mL) + progesterona (2 f.Lg/mL) + estradiol (2 f.Lglml); 8 - controle + 0,1mM de cisteína; C - controle + 100J.1M de cisteamina; D - controle + 0,1 mM de cisteína + 100J.1M de cisteamina. Os resultados demonstraram que não houve diferença significativa entre os tratamentos (p<0,05), ou seja, a suplementação de compostos antioxidantes no meio de maturação não favoreceu a competência meiótica. Além disso, neste estudo pode-se inferir que para cada fase do ciclo estral, talvez seja necessário um período de maturação diferenciado. / Abstract: The aim of this research was to evaluate the effects of the cysteine and cysteamine supplementation on meiotic deveropment of canine oocytes dunng the process of in vitro maturation. The oocytes were collected atter ovanohysterectomy from seven healthy bitches in immediate preovulatory stage. The selected COC's were cultured by a period of 72 hours in four different media: A (control) - TCM199 supplemented with BSA (3 mg/mL) + FSH (5 J-Lg/mL) + LH (10 J-Lg/mL) + progesterone (2 f.Lg/mL) + estradiol (2 f.LglmL); 8 - control + 0,1mM of cysteine; C - control + 100JlM of cysteamine; O - control + 0,1 mM of cysteine + 100J,lM of cysteamine. The present study demonstrated that there was not significant difference among the treatments (p<0,05), in other words, the supplementation of antioxidant in the medium of maturation didn't favor the meiotic competence. Besides, in this study it can be inferred that for each stage of the oestrus cycle, perhaps it is necessary a different maturation penod. / Mestre

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