Orientador: Frederico Ozanam Papa / Resumo: O processo de criopreservação acarreta estresse oxidativo à célula espermática e a adição de antioxidantes aos meios de refrigeração e congelação de sêmen pode auxiliar na proteção dos espermatozoides contra o dano induzido pelas espécies reativas de oxigênio (ERO), incluindo perda da motilidade de forma irreversível, peroxidação lipídica e fragmentação do DNA, interferindo na capacidade de fertilização do espermatozoide. Dentre os diferentes antioxidantes, o Butil-hidroxitolueno (BHT) é um análogo sintético da vitamina E, cujo efeito protetor é atribuído a dois mecanismos: o aumento da fluidez da membrana plasmática após a incorporação do antioxidante, e o segundo por conter a cascata de peroxidação lipídica pela conversão de peroxil em hidroperóxido lipídico. O BHT foi testado em diversas espécies demonstrando melhora na motilidade, integridade de membrana plasmática e viabilidade espermática; porém, na espécie equina, a inclusão de BHT em diluentes para criopreservação de sêmen necessita ser melhor avaliada. Deste modo, o objetivo deste trabalho foi avaliar a influência do BHT nas células espermáticas de equinos submetidas à criopreservação e sua capacidade de conter a peroxidação lipídica e produção de espécies reativas de oxigênio quando os espermatozoides foram submetidos a estresse. / Abstract: The process of cryopreservation brings out oxidative stress to spermatic cell and the addition antioxidant of into cooling and freezing semen extenders could assist in protection of spermatozoa against the damage induced by reactive oxygen species (ROS), including irreversible loss in motility, lipid peroxidation and DNA fragmentation, interfering with the fertilizing capacity of sperm. Among different antioxidants, the butylated hydroxytoluene (BHT) is a synthetic analogue of vitamin E, whose protective effect is attributed to two mechanisms: the increase in sperm membrane fluidity after the addition of antioxidant, and the lipid peroxidation cascade for converting peroxy radicals into lipid hydroperoxides. BHT was tested in different species and the results include improvement in motility, spermatic membrane integrity and sperm viability. However, the addition of BHT in horse semen extenders for cryopreservation purposes demands further research. Thereby, the aims of this study were (1) to evaluate the influence of BHT on equine spermatic cells submitted to cryopreservation and (2) the capacity of BHT to contain the lipid peroxidation and the production of reactive species of oxygen when sperm cells were submitted to stress. / Mestre

As espécies reativas de oxigênio (ROS) podem ser responsáveis por causar danos às membranas dos espermatozoides, fragmentação de DNA, entre outros fatores, influenciando assim na fertilidade principalmente no processo de criopreservação do sêmen. A melatonina (MEL) e o ácido ferúlico (AF) são potentes agentes antioxidantes que poderiam atuar no controle da produção de ROS no sêmen equino. Este estudo teve como objetivo avaliar o efeito dos antioxidantes AF e MEL na criopreservação do sêmen equino. Foram utilizados 5 ejaculados de 4 garanhões. Dentre os tratamentos aplicados, foram utilizadas duas concentrações de cada antioxidante (AF 0,5mM, AF 1,2mM, MEL 2mM e MEL 1µM) além do controle (diluidor de congelação convencional BotuCrio®), totalizando 5 tratamentos. As variáveis analisadas foram cinética espermática pelo sistema CASA (programa SCA - Sperm Class Analyser), morfologia, integridade de membranas plasmática, acrossomal e potencial de membrana mitocondrial, com o uso das sondas fluorescentes PI, Hoescht 33342, FITC-PSA e JC-1 além da produção de (ROS) pelo espermatozoide com a sonda fluorescente CellRox Deep Red®. Comparações entre os tratamentos foram realizadas pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3) e as diferenças entre eles foram localizadas através do teste de Duncan. A probabilidade de P0,05 foi considerada como diferença significativa. Os resultados para características da motilidade tiveram diferença significativa em alguns aspectos, porém nenhum tratamento foi superior ao controle. Houve diminuição no percentual de defeitos maiores nas amostras tratadas com AF 1,2mM, MEL 2mM e MEL 1µM comparadas ao grupo controle. No que diz respeito à integridade de membranas, o tratamento MEL 1µM apresentou porcentagens significativamente melhores nas células com membrana plasmática intacta, acrossomo intacto e alto potencial de membrana mitocondrial, quando comparadas ao grupo controle. As células em estresse oxidativo não se diferenciaram entre os tratamentos. O uso da sonda fluorescente CellRox Deep Red® foi validado para espermatozoides de equinos. Foram utilizados 4 ejaculados de 4 garanhões aos quais eram submetidos aos tratamentos T0 (fração do ejaculado não submetida à indução do estresse oxidativo), T50 (50% da amostra não induzida e 50% induzida ao estresse oxidativo) e T100 (amostra induzida ao estresse oxidativo). Os dados de porcentagem de células positivas (com estresse oxidativo) foram submetidos à análise de regressão polinomial pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3). O valor do coeficiente de determinação (R2) foi igual a 0,88 e a probabilidade de P0,05 foi considerada significativa. Pode-se concluir que o tratamento MEL 1µM contribui para a preservação da integridade de membranas espermáticas durante o processo de criopreservação do sêmen equino e que a sonda fluorescente CellRox Deep Red® é eficiente na detecção de espécies reativas de oxigênio no espermatozoide de garanhões. / Reactive oxygen species (ROS) can be responsible for causing damage to the membranes of sperm, DNA fragmentation, among other factors influencing fertility especially in cryopreservation. Melatonin (MEL) and ferulic acid (FA) are potent antioxidants that could act in the control of ROS production in equine semen. This study aimed to evaluate the effect of antioxidants AF and MEL in cryopreservation of equine semen. Five ejaculates from four stallions were used. Among the treatments, we used two concentrations of each antioxidant (AF 0.5mM, AF 1.2mM, MEL 2 mM and MEL 1µM) beyond the control (conventional freezing extender BotuCrio®), totaling five treatments. The parameters analyzed were sperm kinetics with the CASA system (SCA program - Sperm Class Analyzer), morphology, plasma and acrossomal membrane integrity mitochondrial membrane potential, using fluorescent probes PI, Hoechst 33342, FITC-PSA and JC- 1 over production ROS by the sperm with the fluorescent probe CellRox Deep Red®. Comparisons between treatments were performed by generalized linear model (PROC GLM) of SAS (version 9.3) and the differences between them were located with the Duncan test. The probability of P0.05 was considered significant. The results for the motility characteristics were significant differences in some aspects, but no treatment was superior to the control. There was a decrease in the percentage of major defects in the samples treated with AF 1.2mM, MEL 2 mM and MEL 1µM compared to the control group. Regarding to membrane integrity, treatment MEL 1µM showed significantly better in percentages of cells with intact plasma membrane, intact acrosome and high mitochondrial membrane potential compared to the control group. Cells with oxidative stress not differ between treatments. The fluorescent probe CellRox Deep Red® was validated for equine sperm previously. Was used ejaculates of 4 stallion which were subjected to the treatments T0 (fraction of the ejaculate not subjected to induction of oxidative stress), T50 (50% sample uninduced and 50% induced to oxidative stress) and T100 (sample induced to oxidative stress). The data of percentage of positive cells (with oxidative stress) were submitted to polynomial regression analysis based on generalized linear model (GLM PROC) of SAS (version 9.3). The value of the coefficient of determination (R2) was 0.88 and a probability of P0.05 was considered significant. It can be conclude that the treatment MEL 1µM contributes to the preservation of the integrity of sperm membranes during the equine sperm cryopreservation process and the fluorescent probe CellRox Deep Red® is efficient in the detection of reactive oxygen species in stallions sperm.

Foi estudado a influência das estações não reprodutiva e reprodutiva, dos crioprotetores glicerol e etilenoglicol e do processo de congelação e descongelação sobre a motilidade, o vigor, a morfologia e o complexo DNA-proteína de espermatozóides em garanhões, comparando o sêmen fresco, o exposto a congelação e descongelação sem crioprotetores, o exposto aos crioprotetores sem congelação e o exposto aos crioprotetores com congelação e descongelação. Foram utilizados seis garanhões da raça Mangalarga Paulista, colhendo 12 ejaculados de cada animal na estação não reprodutiva (1) e 12 ejaculados na estação reprodutiva (2), analisando a motilidade, o vigor, a concentração, a morfologia e a patologia do complexo DNA-Proteína. A patologia do complexo DNA-Proteína foi avaliada em sêmen fixado com etanol-ácido-acético glacial 3:1 (v/v), tratado com HCL 4N a 25oC e corado com azul de toluidina a 0,025% em tampão Mcllvaine, empregando microscopia óptica com aumento de 1000x. Os resultados mostraram que a motilidade, o vigor, a morfologia e a anomalia do complexo DNA-Proteína dos espermatozóides diferem entre os grupos congelado e não congelado (P<0,05). Para o grupo fresco, a taxa de patologia do complexo DNA-Proteína apresentou diferença significativa (P<0,05) entre as estações 1 e 2. O processo de congelação e descongelação exerce influência negativa sobre o complexo DNA-Proteína de espermatozóides em garanhões. / To study the effects of freezing and thawing on DNA-protein complexes in spermatozoa, 12 ejaculate were collected from each of 6 stallions (Mangalarga Paulista breed) in each of two consecutive breeding seasons. Motility, vigor, concentration, morphology and incidence of the DNA-protein complex pathologies of spermatozoa were evaluated and compared among fresh semen, semen expose to freezing and thawing without cryoprotectants and semen exposed to the cryoprotectors glycerol and ethylene glycol left at room temperature or frozen. Incidence of the DNA-protein complex pathologies was evaluated in semen smear fixed in na ethanol-acetic-acid mixture (3:1, v/v) for 1 minute, washed in 70% ethanol for 3 minutes and air dried. To induce nuclear metachromasy, smears were treated with 4N HCL at 25ot for 20 minutes followed by rinsing in distilled water. Preparation were stained with a 0.025% toluidine blue solution in Mailvaine buffer for 15 minutes. Frequency of spermatozoa exhibiting nuclear metachromasy was determined in 1000 cells/animal by light microscopy at 1000X magnification. Motility, vigor, morphology and DNA-protein complex pathologies of spermatozoa were different for semen frozen compared to not frozen (P<0.05). For fresh semen, there were effects of seasons on the incidence of DNA-protein complex pathologies (P<0.05) For semen frozen without cryoprotectants there were significant effects seasons (P<0.05) The process of cryopreservation and thawing influences negatively the DNA-protein complexes in stallion spermatozoa.

Orientador: Frederico Ozanam Papa / Resumo: Os garanhões possuem um conjunto completo de glândulas sexuais acessórias, compostas pelas bulbouretrais, a próstata, vesículas seminais e ampolas dos ductos deferentes. Dentre as afecções que acometem essas estruturas, a vesiculite seminal é a de maior ocorrência, consistindo na colonização de uma ou ambas as vesículas por bactérias, sendo a Pseudomonas aeruginosa a mais frequente. No ejaculado é observada uma grande percentagem de neutrófilos e hemácias podem estar presentes, caracterizando os quadros de piospermia e hemospermia, respectivamente. O diagnóstico definitivo é realizado por meio da endoscopia das vesículas, onde é possível visualizar o conteúdo purulento, associado à cultura bacteriana do lavado da glândula. O tratamento é desafiador, pois a antibioticoterapia apresenta baixa eficácia. Visto a importância desta enfermidade e a dificuldade do tratamento, este estudo teve como objetivo abordar os principais aspectos e avaliar a eficácia do ácido metacresolsulfônico e formaldeído para o tratamento da vesiculite seminal em garanhões, considerando os parâmetros seminais e características histopatológicas das vesículas seminais. Três garanhões adultos (n=3), com aspectos reprodutivos normais, tiveram a vesiculite seminal induzida por Pseudomonas aeruginosa através da endoscopia. Após 8 semanas de infecção, foi realizado tratamento local utilizando solução a 30% do ácido metacresolsulfônico e formaldeído (Lotagen®). O experimento consistiu em avaliações semanais, ante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Stallions have a complete set of accessory sex glands, composed of the bulbourethral, prostate gland, seminal vesicles and ampullae. Among the diseases that affect these structures, seminal vesiculitis is the most frequent, consisting the colonization of one or both vesicles by bacteria, whose Pseudomonas aeruginosa is the most frequent. In ejaculate is observed a large percentage of neutrophils and erythrocytes may be present, characterizing piospermia and hemospermia, respectively. The definitive diagnosis is made through the endoscopical evaluation of the seminal vesicles, where it is possible to visualize the purulent contents, associated to gland lavage and bacterial culture. Treatment is challenging because antibiotic therapy is ineffective. Considering the importance of this disease and the difficulty of treatment, this study aims to address the most important aspects and evaluate the efficacy of metacresolsulfonic acid and formaldehyde for the treatment of vesiculitis in stallions, considering seminal parameters and histopathological characteristics. Three adult stallions (n = 3), with normal reproductive aspects, had seminal vesiculitis induced by Pseudomonas aeruginosa through endoscopy. After 8 weeks of infection, local treatment was performed using 30% solution of metacresolsulfonic acid and formaldehyde (Lotagen®). The experiment consisted of weekly evaluations, before and after experimental infection and after treatment. The characteristics of semen and seminal ... (Complete abstract click electronic access below) / Mestre

As espécies reativas de oxigênio (ROS) podem ser responsáveis por causar danos às membranas dos espermatozoides, fragmentação de DNA, entre outros fatores, influenciando assim na fertilidade principalmente no processo de criopreservação do sêmen. A melatonina (MEL) e o ácido ferúlico (AF) são potentes agentes antioxidantes que poderiam atuar no controle da produção de ROS no sêmen equino. Este estudo teve como objetivo avaliar o efeito dos antioxidantes AF e MEL na criopreservação do sêmen equino. Foram utilizados 5 ejaculados de 4 garanhões. Dentre os tratamentos aplicados, foram utilizadas duas concentrações de cada antioxidante (AF 0,5mM, AF 1,2mM, MEL 2mM e MEL 1µM) além do controle (diluidor de congelação convencional BotuCrio&reg;), totalizando 5 tratamentos. As variáveis analisadas foram cinética espermática pelo sistema CASA (programa SCA - Sperm Class Analyser), morfologia, integridade de membranas plasmática, acrossomal e potencial de membrana mitocondrial, com o uso das sondas fluorescentes PI, Hoescht 33342, FITC-PSA e JC-1 além da produção de (ROS) pelo espermatozoide com a sonda fluorescente CellRox Deep Red&reg;. Comparações entre os tratamentos foram realizadas pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3) e as diferenças entre eles foram localizadas através do teste de Duncan. A probabilidade de P0,05 foi considerada como diferença significativa. Os resultados para características da motilidade tiveram diferença significativa em alguns aspectos, porém nenhum tratamento foi superior ao controle. Houve diminuição no percentual de defeitos maiores nas amostras tratadas com AF 1,2mM, MEL 2mM e MEL 1&micro;M comparadas ao grupo controle. No que diz respeito à integridade de membranas, o tratamento MEL 1&micro;M apresentou porcentagens significativamente melhores nas células com membrana plasmática intacta, acrossomo intacto e alto potencial de membrana mitocondrial, quando comparadas ao grupo controle. As células em estresse oxidativo não se diferenciaram entre os tratamentos. O uso da sonda fluorescente CellRox Deep Red&reg; foi validado para espermatozoides de equinos. Foram utilizados 4 ejaculados de 4 garanhões aos quais eram submetidos aos tratamentos T0 (fração do ejaculado não submetida à indução do estresse oxidativo), T50 (50% da amostra não induzida e 50% induzida ao estresse oxidativo) e T100 (amostra induzida ao estresse oxidativo). Os dados de porcentagem de células positivas (com estresse oxidativo) foram submetidos à análise de regressão polinomial pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3). O valor do coeficiente de determinação (R2) foi igual a 0,88 e a probabilidade de P0,05 foi considerada significativa. Pode-se concluir que o tratamento MEL 1&micro;M contribui para a preservação da integridade de membranas espermáticas durante o processo de criopreservação do sêmen equino e que a sonda fluorescente CellRox Deep Red&reg; é eficiente na detecção de espécies reativas de oxigênio no espermatozoide de garanhões. / Reactive oxygen species (ROS) can be responsible for causing damage to the membranes of sperm, DNA fragmentation, among other factors influencing fertility especially in cryopreservation. Melatonin (MEL) and ferulic acid (FA) are potent antioxidants that could act in the control of ROS production in equine semen. This study aimed to evaluate the effect of antioxidants AF and MEL in cryopreservation of equine semen. Five ejaculates from four stallions were used. Among the treatments, we used two concentrations of each antioxidant (AF 0.5mM, AF 1.2mM, MEL 2 mM and MEL 1&micro;M) beyond the control (conventional freezing extender BotuCrio&reg;), totaling five treatments. The parameters analyzed were sperm kinetics with the CASA system (SCA program - Sperm Class Analyzer), morphology, plasma and acrossomal membrane integrity mitochondrial membrane potential, using fluorescent probes PI, Hoechst 33342, FITC-PSA and JC- 1 over production ROS by the sperm with the fluorescent probe CellRox Deep Red&reg;. Comparisons between treatments were performed by generalized linear model (PROC GLM) of SAS (version 9.3) and the differences between them were located with the Duncan test. The probability of P0.05 was considered significant. The results for the motility characteristics were significant differences in some aspects, but no treatment was superior to the control. There was a decrease in the percentage of major defects in the samples treated with AF 1.2mM, MEL 2 mM and MEL 1&micro;M compared to the control group. Regarding to membrane integrity, treatment MEL 1&micro;M showed significantly better in percentages of cells with intact plasma membrane, intact acrosome and high mitochondrial membrane potential compared to the control group. Cells with oxidative stress not differ between treatments. The fluorescent probe CellRox Deep Red&reg; was validated for equine sperm previously. Was used ejaculates of 4 stallion which were subjected to the treatments T0 (fraction of the ejaculate not subjected to induction of oxidative stress), T50 (50% sample uninduced and 50% induced to oxidative stress) and T100 (sample induced to oxidative stress). The data of percentage of positive cells (with oxidative stress) were submitted to polynomial regression analysis based on generalized linear model (GLM PROC) of SAS (version 9.3). The value of the coefficient of determination (R2) was 0.88 and a probability of P0.05 was considered significant. It can be conclude that the treatment MEL 1&micro;M contributes to the preservation of the integrity of sperm membranes during the equine sperm cryopreservation process and the fluorescent probe CellRox Deep Red&reg; is efficient in the detection of reactive oxygen species in stallions sperm.

Foi estudado a influência das estações não reprodutiva e reprodutiva, dos crioprotetores glicerol e etilenoglicol e do processo de congelação e descongelação sobre a motilidade, o vigor, a morfologia e o complexo DNA-proteína de espermatozóides em garanhões, comparando o sêmen fresco, o exposto a congelação e descongelação sem crioprotetores, o exposto aos crioprotetores sem congelação e o exposto aos crioprotetores com congelação e descongelação. Foram utilizados seis garanhões da raça Mangalarga Paulista, colhendo 12 ejaculados de cada animal na estação não reprodutiva (1) e 12 ejaculados na estação reprodutiva (2), analisando a motilidade, o vigor, a concentração, a morfologia e a patologia do complexo DNA-Proteína. A patologia do complexo DNA-Proteína foi avaliada em sêmen fixado com etanol-ácido-acético glacial 3:1 (v/v), tratado com HCL 4N a 25oC e corado com azul de toluidina a 0,025% em tampão Mcllvaine, empregando microscopia óptica com aumento de 1000x. Os resultados mostraram que a motilidade, o vigor, a morfologia e a anomalia do complexo DNA-Proteína dos espermatozóides diferem entre os grupos congelado e não congelado (P<0,05). Para o grupo fresco, a taxa de patologia do complexo DNA-Proteína apresentou diferença significativa (P<0,05) entre as estações 1 e 2. O processo de congelação e descongelação exerce influência negativa sobre o complexo DNA-Proteína de espermatozóides em garanhões. / To study the effects of freezing and thawing on DNA-protein complexes in spermatozoa, 12 ejaculate were collected from each of 6 stallions (Mangalarga Paulista breed) in each of two consecutive breeding seasons. Motility, vigor, concentration, morphology and incidence of the DNA-protein complex pathologies of spermatozoa were evaluated and compared among fresh semen, semen expose to freezing and thawing without cryoprotectants and semen exposed to the cryoprotectors glycerol and ethylene glycol left at room temperature or frozen. Incidence of the DNA-protein complex pathologies was evaluated in semen smear fixed in na ethanol-acetic-acid mixture (3:1, v/v) for 1 minute, washed in 70% ethanol for 3 minutes and air dried. To induce nuclear metachromasy, smears were treated with 4N HCL at 25ot for 20 minutes followed by rinsing in distilled water. Preparation were stained with a 0.025% toluidine blue solution in Mailvaine buffer for 15 minutes. Frequency of spermatozoa exhibiting nuclear metachromasy was determined in 1000 cells/animal by light microscopy at 1000X magnification. Motility, vigor, morphology and DNA-protein complex pathologies of spermatozoa were different for semen frozen compared to not frozen (P<0.05). For fresh semen, there were effects of seasons on the incidence of DNA-protein complex pathologies (P<0.05) For semen frozen without cryoprotectants there were significant effects seasons (P<0.05) The process of cryopreservation and thawing influences negatively the DNA-protein complexes in stallion spermatozoa.

As biotécnicas da reprodução na espécie equina avançaram na última década e os criadores de equinos começaram a questionar as possibilidades de interferência na determinação do sexo dos potros. A determinação do sexo é importante, pois o sexo do potro possui grande influência no valor comercial do mesmo. O objetivo do presente estudo foi avaliar a influência do momento da cobertura em relação ao momento da ovulação no sexo dos potros e outros fatores como: idade da égua e garanhão; indutor da ovulação; ovário em que ocorreu a ovulação e o diâmetro do folículo pré-ovulatório também foram avaliados. O estudo foi realizado em Haras na região de Bagé e Aceguá (RS) – Brasil (31°24'06.1''S 54°07'47.5''W) e (31°30'16.0''S 54°07'45.5''W) nas estações de monta de 2011 a 2015. Utilizou-se 259 ciclos reprodutivos de 160 éguas e 22 garanhões da raça Puro Sangue de Corrida. Informações como agente indutor de ovulação utilizado (Deslorelina; n = 187 ou hCG; n = 72); data da cobertura (n = 259); momento da ovulação em relação a cobertura (+ 24 horas; n = 69 e - 24 horas; n = 190); idade da égua (G1: até 8 anos; n = 123, G2: 9 até 14 anos n = 110 e G3: >14 anos; n = 26); idade do garanhão (Até 14 anos; n = 11 e >15 anos; n = 11) ; ovário em que a ovulação ocorreu (Direito; n = 122 e Esquerdo; n = 137); foram registrados e avaliados No total de potros nascidos, 136 foram machos (52,51%) e 123 foram fêmeas (47,49%). O tempo decorrido após a cobertura não influenciou o sexo dos produtos, nas éguas que ovularam com – 24h após a indução da ovulação: 104 potros (54,74%) eram machos e 86 (45,26%) eram fêmeas, enquanto que nas éguas que ovularam com + 24h, 32 potros (46,38%) eram machos e 37 (53,62%) eram fêmeas. A porcentagem de fêmeas nascidas em relação a idade da égua foi de 46,34% (n=57), 47,27% (n=52) e 46,15% (n=12) nos grupos G1, G2 e G3 respectivamente. Garanhões com idade até 15 anos tiveram 44,14% (n=49) de fêmeas e com mais de 15 anos 49,66% (n=73) eram fêmeas. Não houve diferença no sexo dos produtos quanto ao agente indutor da ovulação (Deslorelina x hCG) e ovário em que ocorreu a ovulação. O presente estudo concluiu que nenhum dos fatores estudados alterou a proporção macho:fêmea dos potros nascidos. / The biotechnologies of reproduction in equine species have been improved in the last decade and the horse breeders started to inquire about the possibility of intervention regarding to the sex determination in foals. Sex determination is important because the sex of the foal has a great influence on the commercial value of the foal. The aim of the present study is was to evaluate the influence of the time of the breeding in relation to time of ovulation in the sex of the foals, besides analyzing other factors such as: the age of the mare and the stallion; ovulation inductor, ovary and the diameter of the preovulatory follicle. The study was accomplished in studs located Bagé/RS and Aceguá/RS – Brazil (31°24'06.1''S 54°07'47.5''W) and (31°30'16.0''S 54°07'45.5''W) during the breeding seasons from 2011 to 2015, using 259 reproductive cycles of 160 mares and 22 stallions of Thoroughbred breed. Information like the induction of ovulation agent that was used (Deslorelin; n = 187 or hCG; n = 72); date of breeding (n = 259); time of ovulation in relation to the breeding (+24 hours; n = 69 and -24 hours; n = 190); age of the mare (G1: up to the age of 8; n = 123 G2: from the age of 9 to the age of 14; n = 110 and G3: >14 years old; n = 26); age of the stallion (up to the age of 14; n = 11 and >15 years old; n = 11); ovary in which the ovulation occurred (Right; n = 122 and Left; n = 137) were catalogued and evaluated As result, 136 (52,51%) were born colts and 123 (47,49%) were born fillies. The elapsed time from breeding to ovulation did not influence on the sex of the product, on mares that ovulated in less than 24 hours after the ovulation induction: 104 foals (54,74%) were male and 86 (45,26%) were female, whereas in the mares that ovulated in more than 24 hours, 32 foals (46,38%) were male and 37 (53,62%) were female. The percentage of born females regarding to the age of the mare was 46,34% (n = 57), 47,27% (n = 52) and 46,15% (n = 12) in the groups G1, G2 and G3, respectively. From stallions up to the age of 15 years, 44,14% (n = 49) were females and from those which were older than 15 years old, 49,66% (n = 73) were females. There was no difference regarding the sex of the product in relation to the ovulation inducer agent (Deslorelin x hCG) and ovary in which the ovulation occurred. None of the factors studied modified the male:female proportion of the born foals.

Em todas as espécies de mamíferos estudadas, o gene PLCζ é responsável pelo aumento de cálcio no momento da fecundação do oócito. Recentemente, o gene WBP2NL, também esta sendo estudado por sua possível capacidade de iniciar a ativação de oócitos. O gene TNF-α é uma citocina pró-inflamatória que pode ser encontrada no plasma seminal, mas seu efeito na viabilidade espermática ainda não está claro. O objetivo desse trabalho foi investigar a expressão dos genes PLCζ, WPB2NL e TNF-α no espermatozoide e suas relações com a qualidade e fertilidade em equinos. Foram utilizados ejaculados de 40 garanhões da raça Crioula, de criatórios localizados próximos a Porto Alegre. A concentração espermática foi avaliada em câmara de Neubauer. As demais análises microscópicas foram realizadas através do Computer Assisted Sperm Analysis AndroVision. A análise da integridade física da membrana foi realizada utilizando-se sondas fluorescentes A integridade funcional da membrana plasmática foi avaliada por meio do teste hiposmótico. Para análise estatística foi utilizada correlação de Pearson, com nível de significância de P<0,05. Foi encontrada correlação positiva do gene PCLζ com integridade funcional da membrana plasmática (0,449), integridade física da membrana plasmática (0,438), taxa de prenhez (0,454), motilidade total (0,486), motilidade progressiva (0,413); correlação negativa com motilidade lenta (-0,427) e espermatozoides imóveis (-0,405). Não foram encontradas correlações dos genes WBP2NL e TNF-α. Concluí-se que a expressão do gene PCLζ no Espermatozoide equino pode ser utilizada com um marcador para a qualidade seminal e fertilidade do garanhão. / In all species of mammals studied, the PLCζ gene is a factor of calcium increase at the time of oocyte fertilization. Recently, the WBP2NL gene is also being studied for its potential ability to initiate oocyte activation during fertilization. The TNF-α is a pro-inflammatory cytokine found in seminal plasma but it effect on motility and viability sperm still unclear. The aim of this study was to verify the expression of PLCζ, WPB2NL and TNF-α genes in spermatozoa and their relationship with quality and fertility in horses. We used the ejaculates of 40 Crioulo stallions, were used from horse farms located near Porto Alegre. After semen collection, the ejaculate was diluted in a commercially available extender. Microscopic analysis of total and progressive motility was performed using a computer assisted sperm analysis system AndroVision. Membrane physical integrity was assessed by fluorescent probes and membrane functionality was analyzed by the hypo-osmotic test (HOST) Statistical analysis was performed using the Pearson correlation, with a significance level of P<0.05. A moderate positive correlation of the PCLζ gene was found with functional plasma membrane function (0.449), physical organization of the plasma membrane (0.438), conception rate (0.454), total motility (0.486), progressive motility (0.413) and negative correlation with slow motility (-0.427) and spermatozoa properties (-0.405). No correlation on WBP2NL and TNF-α genes were found, showing that those genes expression should not be used as a marker of seminal quality and fertility in stallions. It can be concluded that expression of the PCLζ gene on equine spermatozoa can be used as a marker for seminal quality and fertility in the stallion.

Beurteilung von nativen und aufgetauten Spermatozoen fertiler und subfertiler Hengste mit Hilfe der Phasenkontrast- und Transmissionselektronenmikroskopie. Institut für Veterinär-Pathologie der Veterinärmedizinischen Fakultät, Universität Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In dieser Arbeit wurde die Ultrastruktur von nativen und tiefgefrorenen Spermien mittels Phasenkontrast- und Transmissionselektronenmikroskopie (TEM) untersucht. Für die Beurteilung der Spermienmotilität und der Morphologie von in Formolzitrat fixierten Spermien standen jeweils drei Ejakulate von 50 Hannoveraner Hengsten des Niedersächsischen Landgestüts Celle zur Verfügung. Aus dieser Gruppe wurden drei fertile, drei subfertile Hengste und 6 Hengste mittlerer Fertilität ausgewählt, von denen sowohl die Nativ-Proben als auch eine Tiefgefrierprobe (TG-Probe) für die TEM im Institut für Veterinär-Pathologie der Universität Leipzig gemäß des Standardprotokolls des Institutes aufbereitet wurden. Die Spermien wurden gewaschen und das Seminalplasma der nativen Proben oder der Verdünner der TG-Proben abpipettiert und durch eine 5%-ige Glutaraldehydlösung in einem 0,1 M Kakodylatpuffer (pH 7,2) ersetzt. Die Fixierungslösung wurde anschließend entfernt und das Pellet gewaschen und danach mit Gelatine gemischt. Die spermienreichen Stellen wurden aus der Gelatine herausgeschnitten und in Glutaraldehyd aufbewahrt. Nach einer Nachfixierung in OsO4 und einer Entwässerung in Ethanollösungen erfolgte eine Einbettung in einer Eponmischung. Nach einer Polymerisation von 5 Tagen wurden die eingebetteten Eponblöckchen angetrimmt und die Semi- und Ultradünnschnitte angefertigt. Die Ultradünnschnitte wurden auf ein Kupfergrid gelegt, mit Uranylazetat und Bleizitrat kontrastiert und mit dem Transmissionselektronenmikroskop (Zeiss EM 900, Oberkochem) bei 80 kV analysiert. In den nativen Proben wurden insgesamt 360 Spermien pro Hengst beurteilt, in den TG-Proben 120 Spermien pro Hengst. Die Qualität der elektronenmikroskopischen Aufnahmen war sehr gut, doch die Plasmamembran zeigte fixierungsbedingte Artefakte. Nach dem Auftauen waren die Bilder heller und der Kontrast etwas geringer. Es gab eine Zunahme an Akrosomdefekten, akrosomreagierten Spermien und Beschädigungen der Plasmamembran, der Mitochondrien, sowie der Mantel- und Ringfasern. Durch die Membranbeschädigungen trat auch eine Verringerung der Anzahl proximaler und distaler Zytoplasmatropfen auf. Sowohl geschwollene Akrosome mit einer niedrigeren Dichte der akrosomalen Matrix als auch Mitochondrien mit einer zu hellen mitochondrialen Matrix waren typische Befunde in den TG-Proben. Die Studie der Ultrastruktur und die wahrgenommenen Defekte führten zur Erstellung eines Standardprotokolls für die transmissionselektronenmikroskopische Beurteilung von Hengstspermien. Die Beurteilung mittels TEM sollte aber nicht zu einer quantitativen, sondern zu einer qualitativen Aussage führen. Sie ermöglicht die Diagnose von Kern- (Kerndeformationen und Taschenbildung im Kern) und Akrosomabweichungen (deformierte Akrosome mit oder ohne Vakuolenbildung, abgehobene Akrosome und akrosomreagierte Spermien), Anomalien der Mitochondrien (Unterbrechung der Mitochondrienscheide, zu viele Mitochondrien, anormale Dichte der mitochondrialen Matrix), Defekten des Axonemas (Ordnung oder Anzahl der Mikrotubuli, Mantel- und Ringfasern) und der Anwesenheit immaturer Spermienvorstufen. Diese Methode eignet sich für die Diagnostik subfertiler Hengste mit normalen Spermienparametern bei der routinemäßige Spermienbeurteilung und kann sowohl in nativen als auch in TG-Proben angewendet werden. Im Vergleich zur Phasenkontrastmikroskopie waren die elektronenmikroskopischen Bilder wegen ihrer stärkeren Vergrößerung und der Darstellung innerer Spermienstrukturen viel aussagekräftiger. Für die Beurteilung von Halsansatzdefekten, abweichende Geißelformen und Mehrfachmißbildungen ist die Phasenkontrastmikroskopie die am besten geeignete Methode. / Evaluation of fresh and frozen-thawed semen samples of fertile and subfertile stallions by light microscopy and transmission electron microscopy. Institut of Pathology of the Faculty of Veterinary Medicine, University of Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In this study the ultrastructure of fresh and frozen-thawed semen samples of 50 stallions from the National Stud of Lower Saxony (Celle, Germany) were evaluated by light microscopy and transmission electron microscopy (TEM). Three ejaculates of each stallion were available for the motility analysis and the morphological analysis by lightmicroscopy after fixation in formol citrate. Based on the fertility data, the ejaculates of 12 stallions (3 fertile stallions, 3 subfertile stallions and 6 stallions of average fertility) were selected for the morphological analysis by TEM. The native samples and one frozen-thawed sample from these stallions were prepared for the TEM at the Institute of Pathology of the Faculty of Veterinary Medicine, Uni-versity of Leipzig. The sperm cells were washed and the seminal plasma from the native samples and the diluents of the frozen-thawed samples were replaced by a 5%-glutaraldehyde solution in a 0,1 M cacodylate buffer pH 7,2. The fixative was removed, the pellet was washed again and mixed with gelatin. The sperm rich fraction in the gelatin mass was excised and stored in glutaraldehyde. A second fixation in OsO4 was followed by a dehydratation in ethanol and a polymerization phase in epon. After 5 days of polymerization the starred samples were used for semi- and ultratight cuts. The latter were placed on a copper grid, contrasted with uranyl acetate and lead citrate and analyzed with the transmission electron micro-scope (EM 900) by 80 kV. In the fresh samples, 360 sperm cells were examined per stallion, whereas in the frozen-thawed samples only 120 sperm cells per stallion were evaluated. The microscopic pictures were of a high quality. However, the sperm plasma membrane showed some fixation artifacts. In the thawed samples a lower contrast was noticed than in the fresh samples. The sperm cells in the frozen-thawed samples showed an increase in acrosome defects, acrosome reactions, damage of the cell plasma membrane, mitochondria, fibrous sheet and outer dense fibers. The latter defect was associated with a decrease in proximal and distal cytoplasmatic droplets. Swollen acrosomes with a lower matrix density and a bright mitochondrial matrix were typically present in the cryopreserved samples. The ultrastructural defects in these samples, examined by TEM, have led to the development of a standard evaluation protocol with the most common sperm defects in stallion semen. TEM is an expensive and time consuming technique, which cannot be used to obtain quantitative results, but is considered as an accurate method for the qualitative examination of semen samples in cases of unexplained subfertility. TEM can especially be recommended for the diagnosis of nuclear (nuclear malformations and pouches) and acrosomal defects (acrosome deformations, acrosome vacuoles, detached acrosomes and acrosome reactions), mitochondrial (mitochondrial sheet defects, mitochondrial proliferation, decrease in mitochondrial matrix density) and axonema malformations (anormal position or quantity of microtubules and fibrous sheet or outer dense fibers defects) and the detection of immature sperm cells in ejaculates. The results of this study state that TEM can be useful for the evaluation of both fresh and frozen-thawed semen samples. Compared to the light microscopic evaluation of stallion sperm, the TEM images give more precise information because of their higher magnification rate and the ability to reveal internal sperm structures. However, light microscopy remains the best method to detect sperm neck defects, deformed tailes and sperm cells with multiple heads or tails.

Anti-Müllerian hormone (AMH) is a homodimeric glycoprotein that is best known for its role in regression of the Müllerian duct in the male fetus. Accumulating evidence indicates that AMH also has an important role during different physiological processes after birth. In contrast to other species, relatively little is known about AMH in the horse. In chapter one, developmental and seasonal changes in serum AMH concentrations in male horses were determined, and the use of AMH for determination of retained cryptorchid testes was established. In chapter two, the interrelationship between plasma AMH concentrations, antral follicle counts (AFC), and age in mares was evaluated. Molecular and hormonal changes in the equine follicle with regard to variations in antral follicle count and follicular development were examined in chapter three. In chapter four, the effect of AFC on age-related changes in follicular parameters in light-type horse mares was examined. Peripheral AMH concentrations were significantly higher in prepubertal colts than in postpubertal stallions and varied with season in mature stallions with higher concentrations during the physiological breeding season. Furthermore, serum AMH concentrations were significantly higher in cryptorchid stallions compared to intact stallions or geldings. Circulating AMH concentrations varied widely amongst mares of the same age while the repeatability of AMH was high within and between estrous cycles. More importantly, AMH concentrations were positively associated with AFC, and this relationship increased with mare age. In addition, variations in AMH concentrations or AFC were associated with molecular differences in granulosa cells of growing follicles, and the expression of AMH and genes co-expressed with AMH in the equine follicle as well as intrafollicular AMH concentrations decreased during follicular development. Finally, the inter-ovulatory interval and length of the follicular phase is increased in aged mares with low AFC. In conclusion, AMH is a useful biomarker for cryptorchidism in stallions and ovarian reserve in mares. Furthermore, follicular function was interrelated to AFC or AMH based upon molecular differences in growing follicles, while age-related changes in follicular parameters are linked to differences in AFC.