Orientador: João Carlos Pinheiro Ferreira / Banca: Eunice Oba / Banca: Fabiana Ferreira Souza / Banca: Anthony Cesar Castilho / Banca: Anibal Ballarotti Nascimento / Resumo: O objetivo desse estudo foi descrever as características do corpo lúteo (CL) durante a luteólise parcial, em diferentes dias do ciclo estral, após desafio com sub-dose de cloprostenol sódico. Para tanto foram realizados dois experimentos que foram descritos em dois artigos. No artigo I, 32 vacas foram submetidas à sincronização da ovulação (Ovsynch + P4), sendo dessas, 28 ovularam entre 24 e 32 horas (h) após último GnRH (Dia 0) do protocolo. No D6 as vacas foram divididas aleatoriamente em três tratamentos (momento 0h): Controle (2 mL, salina, i.m.; n=10), 2XPGF (duas doses de 500 μg de cloprostenol sódico com 2 horas de intervalo, i.m.; n=8) ou 1/6PGF (83,3 μg de clorprostenol sódico, i.m.; n=10). Amostras de sangue e volume luteal, foram coletadas antes dos tratamentos e a cada 8 h durante às 48 h de observações. Também foram coletadas duas biopsias de CL nos momentos 24 e 40 h pós-tratamento. Valores com P < 0,05 foram considerados diferentes estatisticamente. Nos animais do tratamento 2XPGF, a diminuição da concentração plasmática de progesterona (P4) foi observada a partir de 16 h permaneceu diminuindo até 48 h pós-tratamento. No tratamento 1/6PGF foi observada a luteólise parcial, caracterizada pelas menores concentrações da P4 no momento 16 h seguido do aumento de nos momentos seguintes. As marcações de COX-2 e PGDH foram intensas nos momentos 24 e 40 h, porem a marcação para StAR foi intensa somente no momento 40 h. Ainda no tratamento 1/6PGF o volume luteal também diminuiu com 24 h e esse momento foi caracterizado pela menor área das células luteais grandes (LLC), comparada com o Controle, também algumas células positivas para a marcação de FAS e FAS-L. Nos momentos seguintes o volume luteal aumentou assim como a área das células tiveram tendência em aumentar. No artigo II, 39 vacas foram divididas em dois grupos e submetidas a sincronização da ovulação (Ovsynch + P4), sendo dessas ... / Abstract: We aimed describe the CL characteristics during partial luteolysis, in different days of estrous cycle, after cloprostenol sodium low-dose challenge. Therefore, two projects were conduct and describe in two articles. In the first article, 32 cows were submitted to ovulation synchronization (Ovsunch +P4), 28 cows ovulated between 24 and 32 hours (h) after last GnRH (Day 0) of ovulation synchronization protocol, and were used in the project. At D6, cows were randomly divided in three treatments (moment 0): Control (2 mL of saline 0.9%; IM, n=10), 2XPGF (two doses of 500 μg of cloprostenol sodium with 2 hours interval, n=8) and 1/6PGF (83.33 μg of cloprostenol sodium, IM, n=10). Blood samples and luteal volume were collect before treatments and every 8 h, during 48 h of observations. We also collect two CL biopsies at 24 and 40 h post-treatments. The data was considered statically different when P < 0.05. Animals from 2XPGF had progesterone concentration (P4) decreased 16 h after treatment and it remain low until 48 h after treatment. In 1/6PGF, we observe a partial luteolysis, with decrease on circulating P4 and luteal volume 16 h, after that, we observe an increase in the next moments. The COX-2 and PGDH staining were more intense at 24 and 40 h, however the staining for StAR was intense only on 40 h moment. Still in 1/6PGF treatment, the luteal volume also decrease after 24 h and this moment was characterized by large luteal cells (LLC) minor area than Control, also some positive cell for FAS and FAS-L staining. In the next moments, luteal volume increase as well as cell area tend increase. In the second article, 39 cows were submitted to ovulation protocol (last GnRH = D0), 34 ovulated between 24 and 32 h after GnRH of protocol. The cows were synchronized in a way that data collect were at the same day. Each estrous cycle day were submitted in the same 3 treatments described in Article I: D7 (n=5/2XPGF, n=5/1/6PGF e n=6/control) and ... / Doutor

Recently, several glycerophosphocholine biomarkers for multiple sclerosis were discovered in serum, plasma, and cerebrospinal fluid; little is known, however, about brain glycerophosphocholine metabolism during multiple sclerosis despite evidence that lysophosphocholines can elicit demyelination experimentally. Using a lipidomics approach, glycerophosphocholine metabolites in the hippocampus and temporal-parietal-entorhinal cortex of female C57BL/6J mice subjected to experimental autoimmune encephalomyelitis (a mouse model of multiple sclerosis) were quantified and compared to metabolite levels in healthy mice. To control for potential hormonal regulation, glycerophosphocholine metabolites from these same regions were quantified across the estrous cycle in healthy female N5 C57BL/6J x C3h/HeJ mice. I found that several critical glycerophosphocholine metabolites were significantly decreased over the course of experimental autoimmune encephalomyelitis in both brain regions, although the hippocampus was more affected compared to the temporal-parietal-entorhinal cortex. Similarly, hippocampal glycerophosphocholine metabolism was more responsive to fluctuations in female sex hormones than the cortex. Overall, these results suggest that glycerophosphocholine metabolism differs not only between brain regions, but also between conditions, namely experimental autoimmune encephalomyelitis and the estrous cycle.

At the author’s request, the abstract has been removed due to the confidential nature of the thesis. It will be added once the embargo period has passed.

There is a very few papers to provide an overview of the characteristics of the estrous cycle, the relationship of the estrous cycle to physiological manifestations such as the pH of the vaginal environment, as well as the dynamics of the vaginal microbiota in wild mice. The aim of this thesis is to contribute to the understanding of the dynamic relationship between external influences and the physiology of the female reproductive system, to develop a reliable methodology for measuring the pH of the vaginal microenvironment in mice as well as to quantify the overall abundance of some bacterial taxons by comparing sequencing and qPCR methods. The results suggest that the physical presence of the male in the cage has the most significant effect on the prolongation of the estrus phase, in contrast to non-significant olfactory stimulation of the urine. Fluctuation in the pH of the vaginal environment have also been shown to be cyclic, and the qPCR method shows that the composition of the vaginal microbiota, during the estrus phase, differs significantly from other phases of the estrous cycle, as we confirmed by 16S rRNA sequencing. Thus, these results provide a comprehensive view of the variability of the estrous cycle with an emphasis on the variability of the vaginal microbiota and the change in the...

The objective was to evaluate the insulin-leptin interactions in grazing mares and their impact on aspects of reproductive function using a series of four studies. Fourteen mares were first examined, 10 grazing and 4 confined to stalls in four 22-h studies in April, August, October (2005) and January (2006). There was a positive relationship between insulin and leptin concentrations (r = 0.50; P &lt; 0.001). The second study used 24 mares maintained at pasture and fed supplements rich in either FF, or SS, or pasture forage. Nonstructural carbohydrate (NSC) content of forage was correlated to insulin concentration (r = 0.55; P &lt; 0.01). Insulin was associated with leptin (r = 0.55; P &lt; 0.001) and progesterone (r = 0.48; P &lt; 0.001). In the third study nine mares adapted to FF, SS or forage only underwent two FSIGT tests; once during the luteal phase and again during the follicular phase of the estrous cycle. Minimal model analysis was used to describe insulin sensitivity (SI), glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and the disposition index (DI). SI was lower (P &lt; 0.001) in the luteal phase (3.1 &plusmn; 0.6) compared to the follicular phase (5.0 &plusmn; 0.6). In the fourth study 15 mares had their follicular fluid collected during the luteal and follicular phase of the estrous cycle. Insulin concentration (ln (x+1)) was 52% higher (P &lt; 0.01) in large (&gt; 25 mm) follicles (1.4 ± 0.1 mIU/L) than either medium (16 to 25 mm) or small (le; 15 mm) follicles (0.9 &plusmn; 0.1; 0.9 &plusmn; 0.1 mIU/L, respectively) irrespective of estrous cycle phase. A correlation was observed between follicular fluid (FFL) leptin and plasma leptin (r = 0.30; P &lt; 0.001). A similar relationship was observed between FFL insulin and plasma insulin (r = 0.25; P &lt; 0.001). Plasma insulin and leptin were positively associated (r = 0.45, P &lt; 0.0001), along with FFL insulin and FFL leptin (r = 0.46, P &lt; 0.0001). Both leptin and insulin were affected by season and this observation contradicts the use of single sample analysis for determining detrimental concentrations of these hormones. / Ph. D.

In CBA/NJ mice, splenic natural killer (NK) cell activity varies with stages of estrous. Susceptibility of ICR mice to intravaginal inoculation of herpes simplex virus type 2 (HSV-2) decreases with age. Susceptibility of female ICR and CBA/NJ mice to HSV-2 inoculated intravaginally and intraperitoneally was examined during the estrous cycle. In cycling ICR mice, greatest susceptibility to intravaginal inoculation was observed during diestrous and the least during metestrous. CBA/NJ mice were most susceptible to intravaginal inoculation of HSV-2 during diestrous. ICR mice were ovariectomized to mimic diestrous and found to be highly susceptible to intravaginal inoculation at all virus doses. No difference in susceptibility among phases of the estrous cycle was seen following intraperitoneal inoculation.

O trabalho teve por objetivo estudar o ciclo ovariano de fêmeas de gato-mourisco (Herpailurus yaguaroundi), mantidas em cativeiro, utilizando técnicas de extração e quantificação de metabólitos fecais de hormônios esteróides sexuais e corticosterona. A mensuração dos metabólitos fecais de estradiol, progesterona e cortisol foi efetuada por meio da técnica de radioimunoensaio (RIE). Foram utilizadas oito exemplares fêmeas, mantidas na Fundação Parque Zoológico de São Paulo, São Paulo. As colheitas foram realizadas cinco vezes por semana durante o período de nove meses. As amostras foram acondicionadas individualmente em sacos plásticos e mantidas em freezer a - 20°C. A mediana da duração do ciclo estral (n=33) de gato-mourisco, definido por dois picos consecutivos de metabólitos fecais de estradiol, encontrada no presente estudo, foi de 24 dias; e a duração média±EPM do estro (n=41) foi de 8,65±0,38 dias. O valor da mediana dos valores basais para os metabólitos de estradiol foi de 4,67ng/g de fezes, e dos valores de pico 87,32ng/g de fezes. Para as progestinas os valores encontrados para mediana foram de 0,75ug/g de fezes e 7,49ug/g de fezes para valores basais e de pico respectivamente. As variações no perfil de progestinas sugeriram não terem ocorrido ovulações espontâneas. Não foi encontrada correlação entre progestinas e corticosterona durante os períodos de gestação, lactação e após a retirada dos filhotes. / The aim of this study was analyze the ovarian cycle of captive jaguarundi (Herpailurus yagouaroundi) trough fecal steroid. Radioimmunoassay (RIA) was used for the measurement of estradiol, progesterone and cortisol metabolites in feces. Fecal samples were collected five days per week from eight females kept at Fundação Parque Zoológico de São Paulo during nine months. These samples were placed into zip-lock bags and stored in freezer at - 20°C. The median of estrous cycle length, defined by two consecutive peaks of fecal estradiol metabolites, found in this study was 24 days (n=33 cycles) and the mean (±SEM) of estrous duration was 8,65±0,38 days (n= 41 cycles). The median concentrations of fecal estradiol metabolites were 4,67ng/g of feces and 87,32ng/g of feces for basal and peak values respectively. Progestins median found was 0,75ug/g of feces for basal and 7,49ug/g of feces for peak values. The fluctuations in progestins profiles suggested that spontaneous ovulation did not occur. No correlations were found between progestins and fecal corticosterone during pregnancy, lactation and kitten removal.

Cabras leiteiras das raças Saanen e Toggenburg (n=10) mantidas num rebanho particular no Estado de São Paulo tiveram seus níveis séricos de progesterona e estradiol comparados às concentrações fecais, durante o período correspondente a três ciclos estrais. O perfil hormonal foi confrontado com as manifestações comportamentais de estro visando uma contribuição ao conhecimento do ciclo das cabras destas raças em condições brasileiras, que leve a um manejo reprodutivo mais eficiente. As correlações entre níveis séricos e fecais foram calculadas, sugerindo a validade do modelo de dosagem hormonal nas fezes em cervídeos. Foi verificada correlação extremamente significante entre as concentrações séricas de progesterona e as de progestinas fecais, com r=0,83 e p<0,0001. Entre as concentrações séricas de estradiol e as de seus metabólitos nas fezes não foi verificada correlação significante, com r=-0,16 e p<0,3916. A partir da observação dos resultados e sua análise estatística, pode-se concluir que, dentro das condições deste trabalho, as concentrações de progestinas fecais apresentam variação correspondente à da progesterona sérica ao longo do ciclo estral das cabras; por outro lado, diferentemente das observações de outros autores, o mesmo não ocorre com os metabólitos fecais do estradiol em relação ao estradiol sérico. / Saanen e Toggenburg dairy goats (n=10) kept in a private flock in São Paulo State had their serum levels of progesterone and estradiol compared to faecal concentrations of the respective metabolites, through an interval corresponding to 3 estrous cycles. Hormonal profile was checked with behavioural manifestations of estrous searching for a better knowledge of the cycle of these breeds in brazilian environment, leading to a more efficient reproductive management. Correlations between serum and faecal levels were calculated, suggesting that the model of faecal hormone measurements could be valuable in cervidae. It was found an extremely significant correlation between progesterone serum concentration and progestin faecal concentration, with r=0,83 and p<0,0001. Between estradiol serum concentrations and its faecal metabolites? concentrations no significant correlation was found, with r=-0,16 and p<0,3916. Based on results observation and statistical analysis, we can assume that, in the circumstances of this trial, progestin faecal concentrations show a variation profile similar to that one of serum progesterone, through estrous cycle of goats; however, this fact could not be observed for estradiol metabolites, compared to serum estradiol concentrations.

Comparações da dinâmica ovariana entre mais de duas raças equinas, sob condições padronizadas, não são encontradas na literatura. Objetivando comparar a dinâmica folicular e luteal, foram realizados exames diários de ultrassonografia durante um intervalo interovulatório contemporâneo, em éguas de pequeno porte (grupo Mini Pony – MP; n=10), médio porte (grupo Large Pony – LP; n=9) e grande porte (grupo Brasileiro de Hipismo – BH; n=12). Concluiu-se que os três grupos diferiram quanto ao máximo diâmetro do folículo pré-ovulatório (FPO) (mm) (MP=36,15; LP=40,95; BH=46,66), diâmetro do FPO um dia antes da ovulação (mm) (MP=35,8; LP=40,55; BH=46,48) e crescimento diário médio do FPO (mm/dia) (MP=2,6; LP=3,05; BH=3,51). O grupo MP diferiu dos demais quanto ao número de folículos por onda ovulatória (MP=4,8; LP=10,11; BH=9,75), número de folículos por dia (MP=4,19; LP=10,27; BH=10,63), número de folículos maiores ou iguais a 10mm (MP=2,98; LP=5,88; BH=5,98), diâmetro do FPO à divergência (mm) (MP=22,62; LP=24,81; BH=25,58), diâmetro do segundo maior folículo à divergência (SMF) (mm) (MP=15,56; LP=21,25; BH=21,83), diferença de diâmetro entre FPO e SMF à divergência (mm) (MP=7,25; LP=3,56; BH=3,75) e área do corpo lúteo (CL) (mm2) (MP=436,1; LP=674,4; BH=720,4). Não houve diferença entre os grupos quanto à duração do ciclo (dias) (MP=21,9; LP=20,22; BH=20,58), ocorrência de divergência (MP=80%; LP=88,89%; BH=100%), dias decorridos entre a emergência do FPO e SMF (MP=0,8; LP=0,89; BH=0,92) e diferença de diâmetro entre estes na emergência (mm) (MP=0,4; LP=0,44; BH=0,5) e na divergência (mm) (MP=7,25; LP=3,56; BH=3,75); dias entre emergência e divergência (MP=5,12; LP=5,5; BH=5,5) e divergência e ovulação (MP=7,12; LP=6,62; BH=6,8), número de ondas menores (MP=0,3; LP=0,33; BH=0,42) e duração do CL (dias) (MP=12,4; LP=14,67; BH=13,92). / Comparisons of ovarian dynamics between more than two equine breeds, under standardized conditions, are not found in the literature. The objective of this study was to compare follicular and luteal dynamics during one contemporary intervulatory interval by daily ultrasonography examinations in small size mares (Mini Pony group - MP; n=10), medium size (Large Pony group - LP; n=9) and large size (Brazilian Warmblood group - BH; n=12). It was concluded that all three groups differed regarding maximum diameter of the preovulatory follicle (POF) (mm) (MP=36.15; LP=40.95; BH=46.66), maximum diameter of POF one day before ovulation (mm) (MP=35.8; LP=40.55; BH=46.48) and the mean daily growth of POF (mm / day) (MP=2.6; LP=3.05; BH=3.51). The MP group differed from LP and BH groups regarding number of follicles per ovulatory wave (MP=4.8; LP=10.11; BH=9.75), number of follicles per day (MP=4.19; LP=10, 27; BH=10.63), number of follicles equal or greater than 10mm (MP=2.98; LP=5.88; BH=5.98), diameter of POF at deviation (mm) (MP=22.62; LP=24.81; BH=25.58), diameter of second largest follicle (SLF) at deviation (MP=15.56; LP=21.25; BH=21.83), diameter difference between FPO and SLF at deviation (mm) (MP=7.25; LP=3.56; BH=3.75), corpus luteum (CL) area (mm2) (MP=436.1; LP=674.4; BH=720.4). There was no difference between groups regarding cycle length (MP=21.9; LP=20.22; BH=20.58), occurrence of deviation (MP=80%; LP=88.89%; BH=100%), days between emergence of POF and SLF (MP=0.8, LP = 0.89, BH = 0.92), and diameter difference between POF and SLF at emergence (mm) (MP=0.4; LP=0.44; BH=0.5) and at deviation (mm) (MP=7.25; LP=3.56; BH=3.75); days between emergence and deviation (MP=5.12; LP=5.5; BH=5.5), deviation and ovulation (MP=5.12; LP=6.62; BH=6.8), number of minor waves (MP = 0.3; LP = 0.33; BH = 0.42) and CL lifespan (days) (MP=12.4; LP=14.67; BH=13.92).

O objetivo deste trabalho foi avaliar de maneira detalhada e sistemática os perfis hormonais sanguíneos, fecais, urinários e salivares das progestinas e estrógenos durante o ciclo estral induzido de ovinos. Foram colhidas amostras diárias durante um período de 60 dias de sete fêmeas adultas (n=8) saudáveis e sexualmente maduras. Antes do início da fase de colheita das amostras, todos os animais foram submetidos ao protocolo de tratamento hormonal para indução e sincronização do cio durante dozes dias. O primeiro ciclo ovariano de cada animal desse experimento, detectado logo após a indução do cio foi descartado e seus valores não foram utilizados nas análises hormonais, pois poderiam estar sob o efeito dos hormônios exógenos. A concentração dos progestágenos foi determinada pelas técnicas analíticas de Radioimunoensaio (RIE) e Enzimaimunoensaio (EIE) e os estrógenos por RIE. Houve correlações entre as concentrações de progesterona medidas nas matrizes sérica e fecal, sérica e salivar, fecal e salivar (r=0,90, p<0,0001; r=0,90, p<0,0001; r=0,92, p<0,0001, respectivamente) durante os ciclos estrais observados (n=15). Obtivemos correlação (r=0,74, p<0,0001) entre as concentrações dos estrógenos quantificados nas matrizes sérica e fecal, mas não entre estas concentrações e aquelas medidas na matriz salivar. Não obtivemos nenhuma correlação entre as concentrações medidas na matriz urinária com as quantificadas nas outras matrizes para nenhum dos hormônios estudados. Obtivemos correlação entre as concentrações de progesterona medidas na matriz fecal pelos métodos de RIE e EIE (r=0,78, p<0,0001) e também na matriz salivar pelos dois métodos empregados (r=0,81, p<0,0001). Os resultados do presente experimento indicam que os imunoensaios utilizados podem ser utilizados para a avaliação das concentrações de progestágenos nas matrizes fecal e salivar durante o ciclo estral em ovinos. / The aim of the present work was evaluate the hormonal profiles of progestins and estrogens in blood, feces, urine and saliva during the induced estral cycle in ovine. Samples were collected daily a 60-day period from eight adult (n=8) cycling ewes. The animals were previously submitted to a protocol of estrus induction and synchronization for twelve days. In order to avoid the effect of exogenous hormones, the first cycle immediately after the synchronization was not considered for hormonal analysis. Progestagen concentrations were quantified by two analytical techniques, radioimmunoassay (RIA) and enzyme immunoassay (EIA). Estrogen concentrations were assessed by radioimmunoassay. Correlations in progesterone concentrations were found to be significant for serum and feces, serum and saliva and feces and saliva (r=0.90, p<0.0001; r=0.90, p<0.0001; r=0.92, p<0.0001, respectively) during the estrous cycles (n=15). Estrogen concentrations in the serum and feces were also positively correlated (r=0.74, p<0.0001). Salivary concentrations of estrogens were not correlated with fecal or serum concentrations of the same hormone. No correlation was found between urinary concentrations and concentrations found in other matrixes for both progestagens and estrogens. Concentrations of progestagens obtained using RIA and EIA were correlated on feces (r=0.78, p<0.0001) and saliva (r=0.81, p<0.0001). Results indicate that both immunoassays used in the present experiment can be used to evaluate progestagen concentrations on fecal and salivary matrixes during the estrous cycle of sheep.