Sperm phenotype is an essential indicator of the male ejaculate quality and may have a significant impact on male reproductive success. Sperm phenotypes are considerably variable across species but variation is also found among males within species. This thesis examines (1) variation in sperm phenotypes among males in barn swallows (Hirundo rustica), (2) changes in male ejaculate quality with age, (3) relationships between sperm morphology and motility, (4) effects of sperm phenotypes (morphology and motility) on male fertilization success, using a large dataset of 174 observation for 130 males, and (5) the influence of female reproductive environment on sperm motility. From the tested variables, only midpiece length correlated with male age. Older males had shorter midpiece but no relationship between male age and reproductive success was found. Sperm length negatively affected sperm motility and, simultaneosly, relative midpiece length posively correlated with sperm motility. No correlation was found between the male reproductive success and sperm motility, presence of abnormalities, length of outermost tail feathers or age. Males with shorter relative midpiece were more successful in within-pair paternity, and males with shorter sperms but longer relative midpiece were more successful in...

Made available in DSpace on 2014-06-11T19:33:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-18Bitstream added on 2014-06-13T20:25:12Z : No. of bitstreams: 1 silva_mr_dr_jabo.pdf: 414659 bytes, checksum: 6652a4427059f17af722c81fb586a4e3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Diante do grande impacto da fertilidade dos touros sobre as eficiências reprodutiva e produtiva em rebanhos comerciais, o presente trabalho foi realizado visando avaliar as características testiculares e seminais como critérios de seleção de touros jovens da raça Nelore. As correlações fenotípicas foram, de modo geral, baixas ou nulas (P<0,01) entre as características testiculares, seminais e de crescimento. Todas as características testiculares ou seminais deverão ser contempladas no exame andrológico, visto a independência existente entre as mesmas e a importância destas na comprovação da fertilidade de touros. Os componentes de (co)variância e os valores genéticos foram estimados pelo método da amostragem de Gibbs pelo programa GIBBS2F90, sob modelo animal, o qual incluiu como efeitos fixos, os grupos de contemporâneos e a idade do animal e como aleatório o efeito genético aditivo direto. As estimativas de herdabilidade para perímetro escrotal aos 18 meses, perímetro escrotal no andrológico, volume testicular, formato testicular, aptidão andrológica, defeitos espermáticos maiores, menores e totais foram: 0,42; 0,41; 0,23; 0,22; 0,10; 0,16; 0,04 e 0,15. As correlações genéticas entre perímetro escrotal aos 18 meses com as demais características testiculares e aptidão andrológica foram baixas a altas, de 0,20 a 0,84 e, baixas com as seminais, de -0,16 a -0,24. A circunferência escrotal como critério de seleção deverá promover ganho genético rápido por seleção individual e, por resposta correlacionada, aumento do volume testicular, mudança para formas mais esféricas dos testículos, melhoria da qualidade seminal e aumento do número de animais aptos à reprodução nas condições extensivas de criação no Brasil. / Considering the great impact of fertility of bulls on reproductive and productive efficiency in commercial herds, the objective of this study was to evaluate the testicular and seminal traits as selection criteria of Nellore young bulls. The residual correlations between the traits were analyzed and, in general, the phenotypic associations were small (P<0.01) or null between the testicular, seminal and growth traits. The testicular or seminal traits should be considered in breeding soundness examination, due to their independence of each other and the importance to fertility of bulls. The genetic (co)variance components and genetic values were estimated by the Gibbs sampling method, using the program GIBBS2F90, under an animal model, which included, as fixed effects, the contemporary groups and animal age and, as random effect, the additive genetic effect. The heritability estimates for scrotal circumference at 18 months, scrotal circumference, testicular volume, testicular format, breeding soundness classification, major sperm defects, minor and total sperm defects were: 0.42, 0.41, 0.23, 0.22, 0.10., 0.16, 0.04 and 0.15. Genetic correlations between scrotal circumference at 18 months and others testicular traits and breeding soundness classification were low to high (0.20 to 0.84) and, with seminal traits was low (-0.16 to - 0.24). The scrotal circumference as a selection criterion should result in rapid genetic gain by individual selection, and as correlated response, should result in testicular volume, changes to more spherical shapes of the testicles, improved sperm quality and increased number of satisfactory bulls for breeding under extensive conditions in Brazil.

A criopreservação de sêmen humano tem sido amplamente utilizada como método de preservação da fertilidade. A fim de reduzir os danos causados pelo processo de congelamento, diversos estudos utilizaram substâncias antioxidantes e estimulantes, porém, sua eficácia, bem como seu papel no controle funcional dos espermatozoides, não está bem estabelecida na literatura. Assim, o objetivo do estudo foi avaliar o efeito da suplementação de melatonina e cafeína nas características estruturais e funcionais de espermatozoides pré-criopreservação e pós-descongelamento. Para isso, este estudo prospectivo utilizou 30 amostras seminais de pacientes entre 19 e 45 anos de idade da rotina do Laboratório Androscience entre maio de 2013 e maio de 2017. Todas as amostras foram classificadas como normozoospérmicas, segundo análise seminal inicial de acordo com os critérios da Organização Mundial da Saúde (OMS). Em seguida, as amostras foram criopreservadas com Human Tubal Fluid modificado sem suplemento ou com 2mM de melatonina. Após o descongelamento, as amostras foram analisadas ou suplementadas com 2 mM de cafeína, incubadas por 15 minutos. Ao final dos experimentos, obtivemos 4 grupos: Amostras pós-descongelamento sem suplementação (CONT), amostras suplementadas com cafeína (CAF), melatonina (MEL) e cafeína + melatonina (CM). Parâmetros seminais de contagem, motilidade e cinética, analisados pelos critérios da OMS e pelo software SCA®, a atividade mitocondrial, pela coloração por diaminobenzidina, avaliação da taxa de fragmentação de DNA, pelo método SCSA® e a dosagem dos níveis de radicais livres de oxigênio (ROS), pelo método de luminescência, foram realizados pré-criopreservação e pós-descongelamento com ou sem suplementação. Os dados foram analisados pelo teste T de Student pareado e pela análise de variâncias de uma via com medidas repetidas, seguida pelo pós-teste de Holm-Sidak, e adotado um alfa de 5%. Observamos redução significativa concentração espermática (p < 0,001), motilidade total (p < 0,001) e progressiva (p < 0,001), parâmetros cinéticos (p < 0,009) e atividade mitocondrial (p < 0,001) e aumento das taxas de fragmentação de DNA (p=0,046) e ROS (p=0,052) nas amostras CONT quando comparadas com as amostras a fresco. Quando suplementadas com cafeína e melatonina, observamos aumento da motilidade progressiva nos grupos CAF (p=0,005) e CM (p=0,048) e aumento da atividade mitocondrial no grupo CM (p < 0,05). Podemos concluir que a associação da suplementação com cafeína e melatonina nas amostras pré-criopreservação e pós-descongelamento demonstrou ser uma ferramenta importante para ser aplicada em amostras criopreservadas para atuarem como substâncias estimuladoras de motilidade espermática / Human semen cryopreservation has been widely used as a method of preserving fertility. In order to reduce the damages caused by the freezing process, several studies used antioxidant and stimulant substances, however, their efficiency as well as their role in the functional control of spermatozoa have not been well established in literature. Thus, the goal of this study was to investigate the effect of melatonin and caffeine supplementation on the structural and functional characteristics of pre-cryopreservation and post-thaw spermatozoa. For this, this prospective study used 30 seminal samples from patients aged from 19 to 45 years in the routine of the Androscience Laboratory between May 2013 and May 2017. All samples were classified as normozoospermic, according to the initial seminal analysis following criteria of the World Health Organization (WHO). Then, the samples were cryopreserved with modified Human Tubal Fluid without supplement or with 2 mM of melatonin. After thawing, the samples were analyzed or supplemented with 2 mM of caffeine, and incubated for 15 minutes. At the end of the experiments, we obtained 4 groups: Post-thaw samples without supplementation (CONT), samples supplemented with caffeine (CAF), melatonin (MEL) and caffeine + melatonin (CM). Seminal parameters for count, motility and kinetics, analyzed by WHO criteria and by the SCA® software, mitochondrial activity, by diaminobenzidine staining, evaluation of DNA fragmentation rate, by the SCSA® method, and dosage of radical oxygen species (ROS) levels, by the luminescence method, pre-cryopreservation and post-thaw were carried out with or without supplementation. The data were analyzed by the paired Student\'s t-test and by one-way analysis of variance with repeated measurements, followed by the Holm-Sidak post-test, adopting an alpha of 5%. We observed a significant reduction in sperm concentration (p < 0.001), total (p < 0.001) and progressive (p < 0.001) motility, kinetic parameters (p < 0.009) and mitochondrial activity (p < 0.001), and increased rates of DNA fragmentation (p=0.046) and ROS (p=0.052) production in the CONT samples when compared with fresh sample. When supplemented with caffeine and melatonin, we observed an increase in progressive motility in the CAF (p=0.005) and CM (p=0.048) groups, and an increase in mitochondrial activity in CM group (p < 0.05). We can conclude that that the combination of caffeine and melatonin supplementation in pre-cryopreservation and post-thaw samples proved to be an important tool to be applied in cryopreserved samples to act as substances that stimulate sperm motility

Hyperthyroidism may lead to an increase in oxidative stress (OS) in testis, may cause male reproductive disorders, among them, the loss in the sperm quality. The effect of resveratrol (RSV) on sperm motility and on OS parameters in testis of euthyroid and hyperthyroid rats was investigated. Hyperthyroidism was induced by daily intraperitoneal injection (i.p.) of triiodothyronine (T3) (100 μg/kg, i.p.) for six weeks. After two weeks, concomitantly to T3 treatment, animals received daily injections of RSV at dose of 1 mg/kg e 10 mg/kg (i.p.) during four weeks, totaling six experimental weeks. At the end of experimental period animals were euthanized for removal of organs. Sperm of cauda epididymal was collected for sperm motility and morphology analysis Testis were homogenized for determination of lipoperoxidation by lipid hydroperoxides and thiobarbituric reactive substances (TBARS) levels; activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST); and non-enzimatic antioxidant glutathione (GSH). Hyperthyroid rats presented lower sperm motility, higher lipid hydroperoxides and TBARS levels, lower CAT and GPx activities and higher GST activity in testis than animals of control groups. RSV treatment at doses of 1 mg/kg and 10 mg/kg was able to prevent the loss on sperm motility induced by hyperthyroidism. In addition, RSV decreased lipid hydroperoxides and TBARS levels; reversed the decrease in CAT and GPx activities; and also prevented the increase in GST activity caused by hyperthyroidism in adult rat testis. Together these data show the protective effect of RSV in the testis, preserving sperm motility, and protecting testis against oxidative damage caused by hyperthyroidism, which suggests the RSV as a possible target of studies in the search for therapeutic strategies in order to preserve testicular function. / O hipertireoidismo pode levar ao aumento do estresse oxidativo (EO) nos testículos, podendo causar desordens na função reprodutiva masculina, entre elas, a perda na qualidade espermática. Este estudo avaliou o efeito do resveratrol (RSV) na qualidade espermática e em parâmetros de EO no testículo de ratos eutireóideos e hipertireóideos. O hipertireoidismo foi induzido pela injeção intraperitoneal (i.p) diária de triiodotironina (T3) (100 μg/kg) durante seis semanas. Após duas semanas do início do experimento, concomitante ao tratamento com T3, os animais começaram a receber injeções diárias de RSV nas doses de 1 mg/kg ou 10 mg/kg (i.p.) por quatro semanas, totalizando as seis semanas de tratamento. No final do período experimental, os animais foram eutanasiados para retirada dos órgãos. Os espermatozóides da cauda do epidídimo foram coletados para análise de motilidade e morfologia. Os testículos foram homogeneizados para determinação da lipoperoxidação através dos níveis de hidroperóxidos lipídicos e de substâncias que reagem ao ácido tiobarbitúrico (TBARS); da atividade das enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx) e glutationa-S-transferase (GST); e dos níveis do antioxidante não-enzimático glutationa (GSH). Os ratos hipertireóideos apresentaram menor motilidade espermática, maiores níveis de hidroperóxidos lipídicos e de TBARS, menor atividade da CAT e da GPx e maior atividade da GST do que os animais dos grupos controle. O tratamento com RSV, nas doses de 1 mg/kg e 10 mg/kg, foi capaz de prevenir a perda de motilidade espermática induzida no hipertireoidismo. Em adição, o RSV também diminuiu níveis de hidroperóxidos lipídicos e de TBARS; reverteu a diminuição na atividade da CAT e da GPx; além de prevenir o aumento na atividade da GST causado pelo modelo de hipertireoidismo em testículos de ratos adultos. Em conjunto estes dados mostram o efeito protetor do RSV no testículo, preservando a motilidade espermática e protegendo contra danos oxidativos causados pelo hipertireoidismo, o que sugere o RSV como um possível alvo de estudos na busca de estratégias terapêuticas com o objetivo de preservar a função testicular.

Philosophiae Doctor - PhD / Numerous studies on mammalian spermatozoa have reported large variations in the dimensions of the main sperm structural components, namely the head, midpiece and flagellum. These variations in sperm architecture are believed to be adaptations for functioning of spermatozoa in complex environments outside the male reproductive system. The midpiece of the mammalian permatozoon contains a varied number of mitochondria, but the reason for the marked difference in the size and structure of this sperm component is not clear. This study confirmed the variations in the sperm morphometry of seven selected mammalian species and revealed unique features of the sperm midpiece and sperm mitochondria of these seven species. Evaluation of several sperm kinematic parameters revealed the unique swimming characteristics of the different spermatozoa. The importance of using standardized motility parameters was highlighted as well as the assessment of different subpopulations of spermatozoa in order to produce more reliable and comparable data. Investigating the role of sperm mitochondria in human sperm metabolism indicated that these organelles are related to sperm function in terms of sperm motility. Furthermore, it was suggested that glycolysis and mitochondrial respiration are linked processes and that both are important for the maintenance of human sperm motility. By optimizing and employing standardized experimental procedures and analysis techniques, this study was able to confirm the species specificity of almost all the sperm parameters evaluated, while also elucidating the phylogenetic relatedness of the non-human primate species. In conclusion, the present study has confirmed that the various midpiece morphometry parameters are related to the remaining sperm morphometry parameters as well as to the sperm kinematic parameters. These proposed associations between the various sperm parameters were used to explain the sperm velocity of two hypothetical and morphologically different sperm structures. Therefore, the results of the current study support the idea of co-evolution between sperm components in mammalian spermatozoa and propose that the midpiece morphometry parameters that are selected for in these spermatozoa are midpiece volume, total number of mitochondrial gyres, thickness of the mitochondrial sheath and mitochondrial height. / South Africa

Sexual selection plays an important role in the evolution of animals. Today we already know that it takes place not only before copulation (precopulatory sexual selection), but also after copulation. This type of sexual selection is called postcopulatory sexual selection, and occurs mainly in promiscuous species, where females mate with multiple males. In this case, sperm competition occurs in the female reproductive tract. To increase the likelihood of their reproductive success, males began to develop surprisingly diverse sperm adaptations at the morphological, physiological, or behavioural levels. These adaptations often affect sperm velocity (motility), which is a key factor for successful egg fertilization. However, the result of reproductive success can also be influenced by females, who may prefer sperm with a certain phenotype in the process of cryptic female choice, and thus, for example, obtain better genes for offspring. In some species, females even can have the ability to sort and store sperm in specialized organs in which the sperms are nourished for some time, and then used to fertilize the egg. Even in this case, the storage of sperm is often affected by sperm morphology. Due to these mechanisms of postcopulatory sexual selection, sperm are under strong selection pressure, which can...

A vitamina D é uma molécula versátil que possui ação genômica e não genômica em múltiplas reações dos seus órgãos que possuem a expressão de seu receptor (VDR), inclusive do trato reprodutivo de ambos os sexos, além dos seus clássicos efeitos no metabolismo ósseo, e na homeostase do cálcio e fosfato. Novas evidências a partir de estudos experimentais e com humanos sugerem que a vitamina D está envolvida nas funções dos órgãos reprodutivos masculinos, influenciando na espermatogênese. O objetivo do presente estudo foi avaliar a influência dos níveis séricos de 25(OH)D com os parâmetros de qualidade seminal em homens normozoospérmicos e com alterações nos parâmetros seminais. Nesse estudo retrospectivo, dos 508 pacientes atendidos no período de 2009 até 2017 com todas as dosagens séricas e análise seminal, selecionamos os dados de 260 pacientes que atenderam aos critérios de inclusão, de uma clínica médica de Andrologia da cidade de São Paulo. Eles foram divididos em dois grupos: Grupo normozoospermicos (NZG; N=124) e Grupo com parâmetros Seminais Alterados (SAG; N=136). Foram considerados as dosagens séricas de 25(OH)D, e as variáveis de confusão ambientais, como uso de álcool, tabaco, sedentarismo, índice de massa corporal (IMC), e presença de varicocele. As analises seminais foram realizadas e classificadas de acordo com o manual da OMS 2010, e foram consideradas para o estudo os parâmetros de pH, volume, motilidade total e progressiva, morfologia por OMS e Kruger. Além disso, foram consideradas os exames de Cariótipo e Micro deleção no Cromossomo Y. As análises estatísticas foram realizadas com o SPSS versão 19.0. Correlação de Spearman, Mann-Whitney e um modelo de regressão multivariada foram aplicadas. Significância considerada foi de P <= 0.05. A distribuição das médias dos níveis séricos de 25(OH)D foi significativamente menor nos pacientes do grupo com parâmetros seminais alterados (P =0.016), e foi encontrado uma correlação positiva entre os níveis séricos de 25(OH)D e todos os parâmetros de qualidade seminal analisados, excetos de pH e volume. Foi descrita correlação forte entre 25(OH)D e motilidade total (r=0,225; P =0,001). Os resultados obtidos nesse estudo mostram que os níveis séricos de 25(OH)D possuem uma correlação positiva com os parâmetros de motilidade, concentração e morfologia do espermatozoide, de forma independente. Esses achados indicam que a adequação dos níveis de vitamina D podem ser um importante coadjuvante no tratamento da infertilidade masculina / Vitamin D is a versatile signaling molecule, that targets also male reproductive organs, in addition to the classic effects on bone, calcium and phosphate homeostasis. Accumulating evidence from animal and human studies suggests that it is involved in reproduction functions in both genders. This study aimed to evaluate the vitamin D correlation with semen quality in male with seminal parameters alteration and normozoospermic diagnosis. We selected 260 men (aged 18 to 60 y.o.) from a private andrology reference medical clinic for this observatory study. They were divided in two groups: Normal seminal parameters (NZG N=124) and Abnormal seminal parameters (SAG N=136). 25(OH) vitamin D serum concentration were collected such as lifestyle data available. Semen was analyzed according to WHO 2010 guidelines, ph, volume, motility, concentration, morphology, strict criteria and sperm functional tests were performed (ROS, CK, beads). In addition, karyotype, frequency of varicocele, smoking, alcohol ingestion, and body composition were considered. Statistical analysis was performed by SPSS program version 19.0 (SPSS Inc., Chicago, IL). Spearman correlation, Mann-Whitney test and regression model were applied. Statistical significance was considered with P value < 0. 05. The 25(OH)D average distribution concentration were significant lower in Abnormal seminal parameters group (P =0.016), and all parameters had a positive correlation with 25(OH)D serum levels. The highest coefficient value was observed in the association of total motility with Vitamin D (P =0.001). Our results demonstrated that 25(OH)D levels has a positive influence on spermatogenesis and semen quality, suggesting that vitamin D replacement should highly be concerned on male fertility treatment

A vitamina D é uma molécula versátil que possui ação genômica e não genômica em múltiplas reações dos seus órgãos que possuem a expressão de seu receptor (VDR), inclusive do trato reprodutivo de ambos os sexos, além dos seus clássicos efeitos no metabolismo ósseo, e na homeostase do cálcio e fosfato. Novas evidências a partir de estudos experimentais e com humanos sugerem que a vitamina D está envolvida nas funções dos órgãos reprodutivos masculinos, influenciando na espermatogênese. O objetivo do presente estudo foi avaliar a influência dos níveis séricos de 25(OH)D com os parâmetros de qualidade seminal em homens normozoospérmicos e com alterações nos parâmetros seminais. Nesse estudo retrospectivo, dos 508 pacientes atendidos no período de 2009 até 2017 com todas as dosagens séricas e análise seminal, selecionamos os dados de 260 pacientes que atenderam aos critérios de inclusão, de uma clínica médica de Andrologia da cidade de São Paulo. Eles foram divididos em dois grupos: Grupo normozoospermicos (NZG; N=124) e Grupo com parâmetros Seminais Alterados (SAG; N=136). Foram considerados as dosagens séricas de 25(OH)D, e as variáveis de confusão ambientais, como uso de álcool, tabaco, sedentarismo, índice de massa corporal (IMC), e presença de varicocele. As analises seminais foram realizadas e classificadas de acordo com o manual da OMS 2010, e foram consideradas para o estudo os parâmetros de pH, volume, motilidade total e progressiva, morfologia por OMS e Kruger. Além disso, foram consideradas os exames de Cariótipo e Micro deleção no Cromossomo Y. As análises estatísticas foram realizadas com o SPSS versão 19.0. Correlação de Spearman, Mann-Whitney e um modelo de regressão multivariada foram aplicadas. Significância considerada foi de P <= 0.05. A distribuição das médias dos níveis séricos de 25(OH)D foi significativamente menor nos pacientes do grupo com parâmetros seminais alterados (P =0.016), e foi encontrado uma correlação positiva entre os níveis séricos de 25(OH)D e todos os parâmetros de qualidade seminal analisados, excetos de pH e volume. Foi descrita correlação forte entre 25(OH)D e motilidade total (r=0,225; P =0,001). Os resultados obtidos nesse estudo mostram que os níveis séricos de 25(OH)D possuem uma correlação positiva com os parâmetros de motilidade, concentração e morfologia do espermatozoide, de forma independente. Esses achados indicam que a adequação dos níveis de vitamina D podem ser um importante coadjuvante no tratamento da infertilidade masculina / Vitamin D is a versatile signaling molecule, that targets also male reproductive organs, in addition to the classic effects on bone, calcium and phosphate homeostasis. Accumulating evidence from animal and human studies suggests that it is involved in reproduction functions in both genders. This study aimed to evaluate the vitamin D correlation with semen quality in male with seminal parameters alteration and normozoospermic diagnosis. We selected 260 men (aged 18 to 60 y.o.) from a private andrology reference medical clinic for this observatory study. They were divided in two groups: Normal seminal parameters (NZG N=124) and Abnormal seminal parameters (SAG N=136). 25(OH) vitamin D serum concentration were collected such as lifestyle data available. Semen was analyzed according to WHO 2010 guidelines, ph, volume, motility, concentration, morphology, strict criteria and sperm functional tests were performed (ROS, CK, beads). In addition, karyotype, frequency of varicocele, smoking, alcohol ingestion, and body composition were considered. Statistical analysis was performed by SPSS program version 19.0 (SPSS Inc., Chicago, IL). Spearman correlation, Mann-Whitney test and regression model were applied. Statistical significance was considered with P value < 0. 05. The 25(OH)D average distribution concentration were significant lower in Abnormal seminal parameters group (P =0.016), and all parameters had a positive correlation with 25(OH)D serum levels. The highest coefficient value was observed in the association of total motility with Vitamin D (P =0.001). Our results demonstrated that 25(OH)D levels has a positive influence on spermatogenesis and semen quality, suggesting that vitamin D replacement should highly be concerned on male fertility treatment

Made available in DSpace on 2017-07-10T18:13:17Z (GMT). No. of bitstreams: 1 Mauricio Spagnolo Adames.pdf: 2594661 bytes, checksum: 4bbc4710428addfb51d141108a994f72 (MD5) Previous issue date: 2014-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the effects of fructose as a modulator of sperm movement on the optimization of the sperm: oocyte in artificial reproduction procedures with the use of cryopreserved semen in Rhamdia quelen. Through the Computer Assisted Sperm Analysis (CASA) were evaluated in four replicas the motility rates, speed and time of sperm activation of the cryopreserved semen after thawing (10 seconds after activation) into six activating solutions containing fructose, concentrated at 0.0; 0.9; 1.8; 2.7; 3.6 and 4.5%. For the testing of fertilization an factorial experimental design (5 x 6) composed of five sperm:oocyte ratio (1x104, 3x104, 5x104, 7x104 e 9x104), six activating solutions containing fructose (0.0; 0.9; 1.8; 2.7; 3.6 and 4.5%) and three replicas or experimental blocks ("pools" of oocytes from three female groups). The effects were evaluated over variable rates of fertilization, hatching and larval normality. In the evaluation of sperm variables a regression analysis showed effect (p<0,05) of the solutions in motility, speed and duration of sperm activation. It was also verified effect (p<0,05) in the index obtained from clustering of such variables called index of sperm movement, with results of theoretical peak performance when 2.85% of fructose in solutions was employed. In the fertilization assay the analysis of response surface showed interactive effect (p<0.05) between sperm:oocyte ratio and fructose concentrations in activating solutions, on the fertilization and hatching rates and of the clustering index of two variables called index of reproductive success. According to the statistical model, sperm:oocyte ratio can be reduced keeping up the rates of fertilization and hatching. The inclusion of fructose in activating solutions has promoted similar theoretical maximum levels for fertilization and hatching when compared to fertilization only in distilled water, however with a saving of 17.77% of mobile spermatozoa: oocyte. The larval normality showed effect (p<0.05) just for the block. It is concluded that the sperm:oocyte ratio can be reduced through the employment of fructose-based activating solutions, without affecting the fertilization, hatching rates and larval normality. / O objetivo deste estudo foi avaliar os efeitos da frutose como modulador do movimento espermático sobre a otimização da relação espermatozoide:ovócito em procedimentos de reprodução artificial com o uso do sêmen do Rhamdia quelen criopreservado. Através do Computer Assisted Sperm Analysis (CASA) foram avaliadas em quatro réplicas a motilidade, a velocidade e o tempo de duração da ativação espermática do sêmen criopreservado após o descongelamento (10 segundos após a ativação) em seis soluções ativadoras contendo frutose na concentração de 0,0; 0,9; 1,8; 2,7; 3,6 e 4,5%. Para o ensaio de fertilização foi aplicado um delineamento experimental em esquema fatorial (5 x 6) composto de cinco relações espermatozoides:ovocito (1x104, 3x104, 5x104, 7x104 e 9x104), seis soluções ativadoras contendo frutose (0; 0,9; 1,8; 2,7; 3,6 e 4,5%) e três replicas ou blocos experimentais ( pools de ovócitos provenientes de três grupos fêmeas). Os efeitos foram avaliados sobre as taxas de fertilização, eclosão e normalidade larval. Na avaliação das variáveis espermáticas, a análise de regressão mostrou efeito (p<0,05) das soluções na motilidade, velocidade e tempo de duração da ativação espermática. Também foi verificado efeito (p<0,05) no índice obtido pelo agrupamento destas variáveis, denominado de índice de movimento espermático, com resultados de máximo desempenho teórico quando empregado 2,85% de frutose na solução ativadora. No ensaio de fertilização a analise de superfície de resposta mostrou efeito interativo (p<0,05) entre a relação espermatozoides:ovócito e as concentrações de frutose das soluções ativadoras, sobre as taxas de fertilização e de eclosão e do índice de agrupamento das duas variáveis denominado índice de sucesso reprodutivo. De acordo com o modelo estatístico a relação espermatozoides:ovócito foi reduzida mantendo-se as taxas de fertilização e eclosão. A inclusão de frutose nas soluções ativadoras promoveu níveis máximos teóricos semelhantes para fertilização e eclosão quando comparada à fertilização apenas em água destilada, porém com uma economia de 17,77% de espermatozoides móveis. A normalidade larval apresentou efeito (p<0,05) apenas do bloco. Conclui-se que a relação espermatozóides:ovócito pode ser reduzida através do emprego de soluções ativadoras a base de frutose, sem causar prejuízos ás taxas de fertilização e eclosão e normalidade larval.

PhD / An important component of fish health is an optimally functioning reproductive system. The Luvuvhu River Catchment in the Limpopo Province, South Africa, is a tropical, high-risk malaria area where 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), an endocrine disrupting chemical (EDC), has been used annually since 1945 as a malaria vector control. DDT is known to affect testes morphology and motility of fish sperm. As such, testicular histology and sperm motility (kinematic) parameters were studied as reproductive indicators of the reproductive capacity for two wild, indigenous fish species (Oreochromis mossambicus and Clarias gariepinus) from the currently DDT sprayed area. Three field studies were carried out over two years (2007 – 2008), including two high flow (HF) periods and one low flow (LF) period [HF 1 (March 07), LF (October 07), HF 2 (February 08)]. Both species were sampled from three sites on the Luvuvhu River for testicular histology and computer assisted sperm analysis (CASA), during all three field studies. The sites included a reference site outside the DDT sprayed area, Albasini Dam (AD), and two exposed sites within the DDT sprayed area, Xikundu Weir (XW) and Nandoni Dam (ND). CASA, based on open-source software, was used for the first time in South Africa to assess sperm kinematic parameters of indigenous fish species in field conditions. These included percent motile sperm (% MOT), curvilinear velocity (VCL μm s-1), velocity of an average path (VAP μm s-1), straight line velocity (VSL μm s-1), linearity (LIN %), progression (PROG μm), and average efficiency (AVE. EFF.). Water and sediment samples were collected during all field studies from the three sites for metal and EDC analysis. Controlled laboratory studies were also carried out on the sperm of both species, externally sourced from aquaculture farms equipped to breed and raise fish in toxicant free water. The laboratory studies involved in vitro exposure of spermatozoa to two different, but environmentally relevant, concentrations of both DDT (DDT 1: 0.27 μg L-1; DDT 2: 0.5 μg L-1) and 1,1-dihloro-2,2-bis(p-chlorophenyl)ethylene (DDE) (DDE 1: 0.11 μg L-1; DDE 2: 1.0 μg L-1) with the aim to provide data to support the possible outcomes found in the field studies using CASA. Furthermore, peroxidation of sperm lipids was assayed by production of malondialdehyde (MDA) after in vitro exposure of spermatozoa to DDT and DDE. DDT and its metabolites were found in varying concentrations in the water from all three sites (0.1 μg L-1 – 1.2 μg L-1). Levels of dieldrin (3.5 μg L-1) and lindane (9.4 μg L-1) residues were also found at XW in HF 2. The histological results revealed alterations to testis tissue of both species at all three sites. The testes were assessed through the identification of alterations and an organ index was calculated: Testes Index (IT). The index is indicative of the histological response in the respective tissue type. O. mossambicus at XW had the highest mean IT value during LF (7.45 ± 5.73) and for all field studies combined (5.47 ± 4.63), primarily due to the occurrence of testicular oocytes (intersex), where the frequency of prevalence was 72.73% and 58.82% respectively. These results were statistically higher than the laboratory control (C) group. The CASA results showed statistical differences primarily for O. mossambicus, where motility parameters were lower at XW when compared to AD. Laboratory exposures found a decrease in sperm motility (% MOT) between the control (C) group and the DDT 1, DDE 1 and DDE 2 exposed groups for C. gariepinus. No significant differences were seen for lipid peroxidation (MDA). On the other hand, no significant differences were seen in CASA parameters between the control and exposed laboratory groups for O. mossambicus, but there was an increase in MDA production from the control to the DDT 1 exposure group.