In the present study, both in vitro and in vivo experiments were performed to investigate modes of action of selected EDCs including mercury chloride (HgCl2), bisphenol A (BPA), vinclozolin (VZ) and Di-(2-ethylhexyl)-phthalate (DEHP) on male reproductive physiology in fish. Results obtained from in vitro studies showed that EDCs decrease sperm motility via disruption in energetics and damage to spermatozoa; however the effective doses are higher than reported in the environmentally relevant concentrations. In contrast, in vivo studies showed adverse effects of BPA, VZ, and DEHP on sperm quality at environmentally relevant concentrations. Decrease in sperm quality is associated with disruption in testicular steroidogenesis, particularly inhibition of androgens. Only BPA induced vitellogenin production, associated with increase in transcripts of estrogen receptor, androgen receptor and cytochrome P450 mRNA at the high dose approaching environmental level. Inhibition of androgen production is due to disruption in transfer of cholesterol into the steroidogenesis pathway, because transcript of steroidogenic acute regulatory protein mRNA was decreased. In addition, BPA acts through sex steroid-mediated mechanism while DEHP mode of action is sex-steroid independent mechanism.

The aim of the work was to evaluate the quality of boar ejaculate in terms of sperm morphology and the influence on fertility of sows. The data came from 58 sperm collections from 8 boars of one line. The boars were housed in the semen collection centre under the same conditions and were in age from 11 to 21 months. In the ejaculate analysis, the evaluation of the pathologically changed sperms was performed by determining the frequency of the finding of individual morphological changes. To evaluate the effect of semen on the litter size, data from 123 successful inseminations and subsequent births were processed. The results showed that boars in most cases produced ejaculate with an average volume with a lower sperm concentration. The incidence of morphologically abnormal sperms was normal for almost all boars. The most common sperm abnormalities were immature sperms and defects of sperm flagella. It was confirmed that more piglets were born after using insemination doses made from sperm ejaculate with the higher sperm count. Furthermore, when using ejaculate with a morphologically abnormal sperm count below 15%, more piglets were born than when the sperm count was 1525%. In the case of immature sperms, a negative correlation with the number of born piglets has been proven.

Devido às mudanças climáticas, a adaptação das raças de touro às condições ambientais torna-se um aspecto importante para a expressão do seu potencial genético. O objetivo deste trabalho foi avaliar a performance reprodutiva de touros da raça Braford, através do Índice de Temperatura e Umidade (ITU) e as suas relações com as características seminais de touros mantidos em diferentes climas do Brasil. Foram utilizados setenta e seis touros da raça Braford (5/8 Hereford x 3/8 Nelore), de seis propriedades rurais localizadas nos municípios de Rondonópolis (MT), Porto Nacional (TO) e Rio Verde (GO); Uruguaiana (RS), São Gabriel (RS) e Lages (SC). A avaliação seminal foi realizada 4 vezes em cada local. Os dados climáticos de temperatura e umidade foram coletados a cada hora de estações automáticas do Instituto Nacional de Meteorologia de cada localidade. O ITU foi analisado no período de 30, 18 (espermiogênese) e 12 dias (trânsito epididimário) antes da coleta seminal. Os maiores ITU foram observados no verão na localidade de Uruguaiana (83,69) e durante a estação chuvosa em Rondonópolis (80,15) Os menores valores de ITU foram observados durante o outono e inverno de Lages (51,66 e 50,03) e na estação chuvosa de Rio Verde (72,05). Na qualidade seminal, turbilhonamento (T), motilidade (M) e vigor (V) são menores no inverno de São Gabriel (2,87 ± 0,61; 60,60 ± 10,80 e 2,87 ± 0,51). Os defeitos espermáticos totais foram superiores em Uruguaiana (21,42 ± 1,26). Em Rio Verde, T, M e V foram menores (2,69 ± 0,17; 63,79 ± 4,01 e 2,69 ± 0,17) e maiores percentuais de defeitos maiores e menores (29,01 ± 3,24 e 16,02 ± 1,80). A motilidade mostrou correlação negativa com o ITU de 30 e 18 dias (-0,121 e -0,163; P<0,01), equanto que os defeitos totais estão correlacionados positivamente com todos os períodos de ITU (0,234; 0,207 e 0,198; P<0,01). Apesar dos altos índices de ITU, a qualidade seminal não mudou significamente para reprovar algum touro ao exame andrológico durante todas as estações, mostrando que os touros Braford são capazes de trabalhar a campo sob diferentes climas quente, demonstrado pelo ITU. / Due to climate change, the adaptation of bull breeds to environmental conditions becomes an important aspect for the expression of genetic potential. The objective of this study was to evaluate the reproductive performance of Braford bulls, through the Temperature and Humidity Index (THI) and its relationships with the seminal characteristics of bulls sustained in different climates of Brazil. Seventy-six Braford bulls (5/8 Hereford x 3/8 Nellore) were used, of six farms located in the cities of Rondonópolis (MT), Porto Nacional (TO) and Rio Verde (GO); Uruguaiana (RS), São Gabriel (RS) and Lages (SC). The seminal evaluation was performed four times at each location. Temperature and air humidity data were collected every hour from the automatic weather station at the National Institute of Meteorology. The THI was analyzed in the period of 30, 18 (spermiogenesis) and 12 days (epididymal transit) before seminal collection. The highest THI were observed in the summer of Uruguaiana (83.69) and in the rainy season of Rondonópolis (80.15) The lowest values of THI were observed in the autumn and winter of Lages (51.66 and 50.03), and in the rainy season of Rio Verde (72.05). In sperm quality, mass motion (MM), motility (M) and vigor (V) are lower in the winter of São Gabriel (2.87 ± 0.61; 60.60 ± 10.80 and 2.87 ± 0.51). The total defects were higher in Uruguaiana (21.42 ± 1.26). In Rio Verde, MM, M and V were lower (2.69 ± 0.17; 63.79 ± 4.01 and 2.69 ± 0.17) and higher percentages of major and minor defects (29.01 ± 3.24 and 16.02 ± 1.80). Motility showed a negative correlation with THI of 30 and 18 days (-0.121 e -0.163; P<0.01), while the total defects are positively correlated with all periods of THI (0.234; 0.207 and 0.198; P<0.01). Despite the high THI indexes, the sperm quality has not changed significantly to reject a bull during BBSE exam during all seasons, showing that Braford bulls are able of field service under different hot climates, as demonstrated by the THI.

At least 30 million men are infertile around the world, identifying male factor infertility as a global health issue. In the past 70 years, evidence of a significant general decline in sperm quality has been reported, prompting concerns about the implications for reproductive health. Over the same period, there have been substantial changes in human lifestyles. New technologies, such as mobile phones and wi-fi, have been proposed to have a negative impact on a range of health outcomes, from an increased risk of cancer to a decrease in fertility. However, these links remain controversial. Over the last 30 years, the introduction of assisted reproductive technologies (ART) has offered infertile patients, particularly men with severe male factor infertility, a successful treatment option. However, miscarriage rates associated with fertility treatment can be as high as 30% and how this risk had changed over time was unclear. In addition, there are natural fluctuations in human health, including seasonal changes to birth rates. However, the clinical implications of these fluctuations need to be established. In this thesis, using an integrated approach that combined epidemiological research with laboratory investigations, I show that sperm quality is negatively affected by exposure to RF-EMR from mobile phones and wi-fi. I also identified a seasonal summer increase in sperm motility and morphology that followed patterns of seasonality in birth rates and in the success of assisted conception cycles. I showed that although the number of successful conceptions from ART has increased over time, there has been an equal increase in miscarriage rates. Male reproductive health continues to be under-researched when compared with the female, this inequality needs to be addressed in order to understand the causes of the decline in male fertility and the relationship this has with subsequent reproductive success.

Devido às mudanças climáticas, a adaptação das raças de touro às condições ambientais torna-se um aspecto importante para a expressão do seu potencial genético. O objetivo deste trabalho foi avaliar a performance reprodutiva de touros da raça Braford, através do Índice de Temperatura e Umidade (ITU) e as suas relações com as características seminais de touros mantidos em diferentes climas do Brasil. Foram utilizados setenta e seis touros da raça Braford (5/8 Hereford x 3/8 Nelore), de seis propriedades rurais localizadas nos municípios de Rondonópolis (MT), Porto Nacional (TO) e Rio Verde (GO); Uruguaiana (RS), São Gabriel (RS) e Lages (SC). A avaliação seminal foi realizada 4 vezes em cada local. Os dados climáticos de temperatura e umidade foram coletados a cada hora de estações automáticas do Instituto Nacional de Meteorologia de cada localidade. O ITU foi analisado no período de 30, 18 (espermiogênese) e 12 dias (trânsito epididimário) antes da coleta seminal. Os maiores ITU foram observados no verão na localidade de Uruguaiana (83,69) e durante a estação chuvosa em Rondonópolis (80,15) Os menores valores de ITU foram observados durante o outono e inverno de Lages (51,66 e 50,03) e na estação chuvosa de Rio Verde (72,05). Na qualidade seminal, turbilhonamento (T), motilidade (M) e vigor (V) são menores no inverno de São Gabriel (2,87 ± 0,61; 60,60 ± 10,80 e 2,87 ± 0,51). Os defeitos espermáticos totais foram superiores em Uruguaiana (21,42 ± 1,26). Em Rio Verde, T, M e V foram menores (2,69 ± 0,17; 63,79 ± 4,01 e 2,69 ± 0,17) e maiores percentuais de defeitos maiores e menores (29,01 ± 3,24 e 16,02 ± 1,80). A motilidade mostrou correlação negativa com o ITU de 30 e 18 dias (-0,121 e -0,163; P<0,01), equanto que os defeitos totais estão correlacionados positivamente com todos os períodos de ITU (0,234; 0,207 e 0,198; P<0,01). Apesar dos altos índices de ITU, a qualidade seminal não mudou significamente para reprovar algum touro ao exame andrológico durante todas as estações, mostrando que os touros Braford são capazes de trabalhar a campo sob diferentes climas quente, demonstrado pelo ITU. / Due to climate change, the adaptation of bull breeds to environmental conditions becomes an important aspect for the expression of genetic potential. The objective of this study was to evaluate the reproductive performance of Braford bulls, through the Temperature and Humidity Index (THI) and its relationships with the seminal characteristics of bulls sustained in different climates of Brazil. Seventy-six Braford bulls (5/8 Hereford x 3/8 Nellore) were used, of six farms located in the cities of Rondonópolis (MT), Porto Nacional (TO) and Rio Verde (GO); Uruguaiana (RS), São Gabriel (RS) and Lages (SC). The seminal evaluation was performed four times at each location. Temperature and air humidity data were collected every hour from the automatic weather station at the National Institute of Meteorology. The THI was analyzed in the period of 30, 18 (spermiogenesis) and 12 days (epididymal transit) before seminal collection. The highest THI were observed in the summer of Uruguaiana (83.69) and in the rainy season of Rondonópolis (80.15) The lowest values of THI were observed in the autumn and winter of Lages (51.66 and 50.03), and in the rainy season of Rio Verde (72.05). In sperm quality, mass motion (MM), motility (M) and vigor (V) are lower in the winter of São Gabriel (2.87 ± 0.61; 60.60 ± 10.80 and 2.87 ± 0.51). The total defects were higher in Uruguaiana (21.42 ± 1.26). In Rio Verde, MM, M and V were lower (2.69 ± 0.17; 63.79 ± 4.01 and 2.69 ± 0.17) and higher percentages of major and minor defects (29.01 ± 3.24 and 16.02 ± 1.80). Motility showed a negative correlation with THI of 30 and 18 days (-0.121 e -0.163; P<0.01), while the total defects are positively correlated with all periods of THI (0.234; 0.207 and 0.198; P<0.01). Despite the high THI indexes, the sperm quality has not changed significantly to reject a bull during BBSE exam during all seasons, showing that Braford bulls are able of field service under different hot climates, as demonstrated by the THI.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-28T17:39:10Z No. of bitstreams: 1 Daniele Missio.pdf: 779272 bytes, checksum: 6b39f5d8e9f05222e1d9034c1daa6e30 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-28T17:39:27Z (GMT) No. of bitstreams: 1 Daniele Missio.pdf: 779272 bytes, checksum: 6b39f5d8e9f05222e1d9034c1daa6e30 (MD5) / Made available in DSpace on 2018-09-28T17:39:27Z (GMT). No. of bitstreams: 1 Daniele Missio.pdf: 779272 bytes, checksum: 6b39f5d8e9f05222e1d9034c1daa6e30 (MD5) Previous issue date: 2017 / Na produção in vitro de embriões (PIV) bovinos são utilizadas diferentes técnicas de seleção espermática, a fim de recuperar o maior número de espermatozoides com boa qualidade e remover componentes do sêmen e diluente. Dentre essas técnicas, a seleção por gradientes descontínuos de Percoll, é a mais utilizada atualmente em laboratórios de PIV de bovinos. Porém essa técnica vem sendo modificada principalmente com o aumento da utilização do sêmen sexado. O sêmen sexado possui características distintas do sêmen convencional, como baixo número de células por dose e baixa motilidade pós-descongelamento. Assim, o desenvolvimento de um método eficiente para a seleção espermática, que aumente a taxa de recuperação sem prejudicar a qualidade dos espermatozoides é essencial. O objetivo do presente estudo foi avaliar os efeitos do volume de Percoll na recuperação espermática, qualidade dos espermatozoides, estresse oxidativo e cinética do desenvolvimento embrionário de embriões bovinos PIV. Palhetas de sêmen convencional ou sexado foram descongeladas a 35º C por 20 s e distribuídas em alíquotas iguais em três diferentes volumes de Percoll: 300 μL de cada gradiente de Percoll (90, 60 e 30%), Controle; 100 μL de cada gradiente de Percoll, P100; e 200 μL de cada gradiente de viii Percoll, P200. Qualidade espermática, taxa de fecundação e cinética do desenvolvimento embrionário até 48 horas pós-inseminação foram avaliadas. Para o sêmen convencional, a motilidade, o vigor e a taxa de recuperação foram maiores nos tratamentos P100 e P200 comparado ao Controle (P < 0,05), enquanto os níveis de espécies reativas de oxigênio (ROS), peroxidação lipídica e atividade da enzima superóxido dismutase (SOD) não foram influenciados pelos tratamentos. Para o sêmen sexado, P100 aumentou a velocidade curvilínea, velocidade média da trajetória e amplitude de deslocamento lateral da cabeça (P < 0,05). A taxa de recuperação foi maior em P100 do que no Controle e P200 (P < 0,05). A formação de ROS foi menor em P100 do que em P200 e Controle, e a atividade SOD foi menor em P100 do que no Controle. As taxas de fecundação e clivagem, momento da primeira clivagem e número de células foram similares entre P100 e Controle (P > 0,05). Portanto, o volume de Percoll de 100 μL aumentou a taxa de recuperação espermática sem prejudicar a qualidade dos espermatozoides e desenvolvimento embrionário, permitindo assim, aumentar o número de gotas inseminadas/ dose e diminuir custos. / For in vitro production (IVP) of bovine embryos, different sperm selection techniques are used to recover the highest number of sperm with good quality and to remove components of the semen and diluent. Among these techniques, the selection by discontinuous Percoll gradients is currently the most used in bovine IVP laboratories. However, this technique has been modified mainly with the increase of the use of sex-sorted semen. The sex-sorted semen has distinct characteristics of conventional semen, such as low number of cells per dose and low post-thaw motility. Thus, the development of an efficient method for sperm selection, which increases the rate of recovery without impairing the quality of sperm is essential. The aim of the present study was to evaluate the effects of Percoll volume on recovery rate, sperm quality, oxidative stress and embryo development kinetics in bovine embryos IVP. Conventional or sex-sorted semen straws were thawed at 35°C for 20 s and distributed in equal aliquots into three different Percoll volume: 300 μL of each Percoll gradient (90, 60, and 30%), Control; 100 μL of each Percoll gradient, P100; and 200 μL of each Percoll gradient, P200. Sperm quality, fertilization rate, and embryonic development kinetics up to 48 h post-insemination were evaluated. For conventional semen, motility, vigor, and recovery x rate improved in the P100 and P200 treatments compared to the Control (P < 0.05), whereas reactive oxygen species (ROS) levels, lipid peroxidation, and superoxide dismutase enzyme (SOD) activity were not influenced by treatments. For sex-sorted semen, P100 increased sperm curvilinear velocity, average path velocity, and amplitude of lateral head displacement (P < 0.05). Recovery rate was higher in P100 than in the Control and P200 (P < 0.05), formation of ROS was lower in P100 than in the Control and P200, and SOD activity was lower in P100 than in the Control. Fertilization and cleavage rates, moment of first cleavage, and cell number were similar between P100 and the Control (P > 0.05). Therefore, a Percoll volume of 100 μL increased sperm recovery rate without damage to sperm quality or early embryonic development, allowing to increase the number of inseminated drops / dose and to reduce costs.

Many chemicals present in the aquatic environment can interfere with physiological functions in fish. Exposure to chemicals can be revealed by the use of biomarkers. Induction of 7-ethoxyresorufin O-deethylase (EROD) activity is a commonly used biomarker for exposure to CYP1A inducers such as dioxins and polyaromatic hyrdrocarbons. Vitellogenin is a frequently used biomarker for estrogenic compounds in various fish species whereas a biomarker for androgens, spiggin, is only found in sticklebacks. The main objectives of this thesis were to evaluate gill EROD activity as a biomarker and the three-spined stickleback as a model species in ecotoxicological studies. EROD activities were measured in gill, liver and kidney in rainbow trout (Oncorhynchus mykiss) caged in urban areas in Sweden. EROD induction was most pronounced in the gill. Also in fish caged at reference sites, with an expected low level of known CYP1A inducers, a marked gill EROD induction was found. One suggested inducer in rural waters is humic substances (HS). To evaluate the EROD-inducing capacity of HS, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to HS of natural or synthetic origin. Both kinds of HS caused significant EROD induction. Gill EROD activities were also induced in sticklebacks exposed to ethynylestradiol (EE2) and β-naphthoflavone (βNF), alone and in combinations. Production of vitellogenin was induced in sticklebacks exposed to ≥50 ng EE2/l and a significant decrease in spiggin production was observed in individuals exposed to 170 ng EE2/l. Results from this thesis further strengthen the contention that gill EROD activity is a very sensitive biomarker for CYP1A inducers and that the stickleback is a suitable biomonitoring species, especially for exposure to CYP1A inducers. The finding that not only classical CYP1A inducers but also HS and high EE2 concentrations stimulate gill EROD activity is of significance for the interpretation of biomonitoring data.

Introduction. Sperm parameter analysis is the standard method of male fecundity testing; however, minimal evidence supports associations between individual sperm parameters and reproductive outcomes. Our previous work shows strong associations between sperm mitochondrial DNA copy number (mtDNAcn) and time-to-pregnancy (TTP) in general populations, and between mtDNAcn and fertilization outcomes in clinical populations. Thus it is possible for sperm mtDNA biomarkers to act as summary measures of semen quality. In this study, we developed a sperm quality index (SQI) from semen parameters and compared its ability to measure fecundity to sperm mtDNAcn. Methods. We received 384 semen samples from the Longitudinal Investigation of Fertility in the Environment Study. Sperm mtDNAcn and mtDNA deletions (mtDNAdel) were quantified using a triplex probe-based qPCR method. The SQI was developed by ranking and summing select sperm parameters within the study population, including sperm concentration, sperm count, normal morphology, high DNA stainability, and DNA fragmentation to create a cumulative index. Discrete-time proportional hazards models were used to determine fecundability odds ratios (FOR), indicating associations between mtDNAcn, SQI, and TTP. Receiver operating characteristic (ROC) analyses determined the validity of the SQI and mtDNAcn as predictors of pregnancy within 12 months. Results. The SQI was highly associated with mtDNAcn, both continuously (Spearman Rho: -0.487; p-value: <0.001) and in deciles (ANOVA p-value: <0.001). The SQI (FOR: 1.25; 95% confidence interval (CI): 1.09, 1.43) and mtDNAcn (FOR: 0.754; 95% CI: 0.657, 0.866) performed similarly in discrete-time survival models and indicated a significant decrease and increase in TTP, respectively. MtDNAcn more effectively predicted pregnancy within 12 months (AUC: 0.703; 95% CI: 0.617, 0.789) than the SQI (AUC: 0.642; 95% CI: 0.531, 0.753). With multiple predictors, mtDNAcn outperformed summary models, with addition of the SQI and percent normal morphology minimally increasing model efficacy (AUC: 0.718, 95% CI: 0.617, 0.819). Conclusion. The association between the SQI and mtDNAcn suggest that mtDNAcn may serve as a summary biomarker for overall sperm quality. Neither individual nor summed sperm parameters are useful indicators of couple fecundity and reproductive outcomes compared to mtDNAcn. These results suggest that mtDNAcn has potential for use as a biomarker of fecundity.

Sperm function is pivotal to successful sexual reproduction. The phenotype of sperm is defined by the male’s genotype and by the environment sperm encounter during their travel to the oocyte. During their functional lifespan, sperm encounter a variety of environments: After manufacture in the testis, they are stored in males before they are ejaculated along with seminal fluids and transferred to and stored in females for hours up to years before getting a chance to fertilise an egg. The sperm environment in male and female reproductive tract will be determined by male and female genotype, but also by environmental factors that affect sperm directly or indirectly by altering male and female condition. Like somatic cells, sperm age and decline in function over time due to the accumulation of cellular damage. Reactive oxygen species (ROS) that emerge as a by-product of aerobic metabolism and environmental stress are believed to be the main cause of cell senescence. Spermatozoa are in particular susceptible to ROSinduced damage because they have only limited defence and repair mechanism. As it contains many polyunsaturated fatty acids, the sperm membrane is especially prone to peroxidation by ROS and can consequently become leaky. The condition of the sperm membrane can hence be used to assess sperm age. Sperm quality has frequently been measured as sperm viability even though this approach has several biological and technical pitfalls. I developed an osmotic sperm stress test to assess sperm quality and predict future sperm performance that circumvents several of these pitfalls. Further, using osmotic stress to challenge the sperm membrane and observing sperm viability in a longitudinal approach is probably more meaningful in predicting future sperm performance than sperm viability per se. An essential abiotic factor that affects sperm directly and indirectly during storage in males and females is temperature. Ectotherms that inhabit different climates like D. melanogaster can be expected to be locally adapted to temperatures, particularly in fitness-relevant reproductive traits. I assessed the joint and isolated effects of thermal adaptation, of rearing and of ambient temperature on sperm quality by measuring sperm osmotic stress resistance, ejaculate effects on the induction of egg-laying in females, male fertility as well as female fertility and sperm storage in two D. melanogaster strains from Zambia (warm-adapted) and two from Sweden (cold-adapted). I found complex G x E interactions on male and female reproductive traits. Sperm quality was generally higher in the cold-adapted strains and showed negative carry-over effects of hot-rearing, demonstrating the important role of male genotype and developmental temperature on sperm quality. In contrast, there were positive carry-over effects of hot-rearing on male fecundity and male and female fertilisation rate in the hot-adapted strains, supporting local adaptation to heat stress. To investigate direct effects of the female reproductive tract environment on stored sperm, I genetically manipulated female D. melanogaster with a spermathecalspecific GAL4 driver line and hoped to test proposed candidate genes associated with female sperm storage. My results suggested sperm storage defects in the driver line that may either be an unexpected side-effect of the insertion of the GAL4 driver into a spermathecal serine endopeptidase or of the genetic background of the driver line.:Chapter 1 General introduction 1 Sperm phenotype 1 Basic sperm morphology 2 Sperm ageing 3 Environmental effects on sperm 5 Male sperm storage 6 Female sperm storage (FSS) 8 Phase 1: Recruitment of sperm into storage 10 Phase 2: Sperm maintenance 11 Phase 3: Release of sperm from storage 16 References 18 Chapter 2 More pitfalls with sperm viability staining and a viability-based stress test to characterize sperm quality 29 Abstract 29 Author contributions 30 Introduction 31 Methods 33 Methods of sperm viability staining in ecology and evolution 33 Empirical study 33 Statistical analysis 38 Results 38 Methods of sperm viability staining in ecology and evolution 38 Sperm survival examined with cross-sectional vs. longitudinal sampling 43 Sperm viability in the bedbug 43 Sperm viability in the fruitfly 45 Discussion 47 SV heterogeneity 48 Protocol standardization and recommendations 49 Sperm viability vs. sperm quality 50 Sperm stratification 51 Conclusion 52 References 52 Chapter 3 Effects of temperature and thermal adaptation on sperm stored in male and female Drosophila melanogaster 56 Abstract 56 Introduction 57 Material and Methods 61 Fly populations and culture 61 Temperature treatments 62 Wing length 63 Quality of sperm stored in males 63 Male effects on female fertility 64 Female effects on sperm 64 Statistical analysis 66 Results 67 Wing length at 19° and 29°C 67 Sperm viability under osmotic stress 68 Male effects on female fertility 70 Female effects on stored sperm 73 Fertilisation rate 75 Sperm storage 77 Does the decrease in sperm predict female fecundity? 80 Discussion 82 Phenotypic plasticity vs. local adaptation 82 Sperm effects, seminal fluid effects and female effects on fertility 83 Supernumerary spermathecae 87 Conclusion 87 References 89 Chapter 4 Effect of spermathecal proteins on female fertility and sperm storage in Drosophila melanogaster 94 Abstract 94 Introduction 95 Material and Methods 99 Fly Stocks 99 UAS/GAL4 crosses and controls 101 General experimental procedure 103 Experimental fly strains 103 Parameters used to assess sperm storage capability 103 Mating procedure 104 Oviposition and progeny development 104 Part I. RNAi screen (Experiments 1 and 2) 109 Material and methods 109 Experiment 1: Preliminary RNAi screen 109 Experiment 2: RNAi screen 109 Results 110 Experiment 1: Preliminary RNAi screen 110 Experiment 2: RNAi screen 114 Part II: Spermathecal secretory function (Experiments 3 to 5) 121 Material and Methods 121 Experiment 3: Survey of fertility effects of the spermathecal secretory function 121 Experiment 4: Verifying experiment 1 122 Experiment 5: Verifying experiment 2 122 Results 122 Experiment 3: Survey of fertility effects of the spermathecal secretory function 122 Experiment 4: Verifying experiment 1 127 Experiment 5: Verifying experiment 2 128 Part III. Trpa1 and temperature effects 130 Material and Methods 130 Results 131 Sperm number and fertilisation rate 140 Discussion 151 Effect of knock-down of genes with putative role in sperm storage in SSC on fertility 151 Effect of impaired spermathecal secretory function on fertility 153 Effects of enhanced secretory function of the SSC on fertility and sperm storage 154 Wildtype variation in fertility (and sperm storage) with temperature 155 Conclusion 157 References 157 Chapter 5 General discussion 164 Measuring sperm quality in male and female storage organs 164 Environmental effects on sperm stored in males and females 166 Effect of the direct environment on sperm stored in females 168 Conclusion 169 References 170 Acknowledgements 175 Supplementary Material 176 Composition of corn/yeast food 176 Composition of yeast food 176 Chapter 2 176 Chapter 3 177 Full models 177 Survival and mating rate 179 Interaction plots males 181 Fertility of focal males 184 Sex ratio of the progeny of focal males 185 Female fertility 185 Chill coma recovery assay 187 Results 187 Chapter 4 189 Experiment 3 189 Experiment 6 189

Theory predicts that in species with a greater risk of sperm competition, males will invest more in ejaculate traits. In species with alternative mating tactics (AMTs), males of different phenotypes will be under different sperm competition risk. Because minors sneak inside other males\' territories to mate they should always face sperm competition. Major males, on the other hand, defend territories and have more chance of mating exclusively with females. For majors, the risk of sperm competition is theoretically lower. The main prediction from game theory models for species with AMTs is that majors invest less in ejaculate traits than minors. However, when the proportion of minors in the population increases, majors should invest more in ejaculate traits, reaching a similar level of ejaculate expenditure to minors. In this study, we tested these predictions with a meta-regression analysis of 29 fish species with AMTs. As a proxy for the risk of sperm competition, we ranked each species according to a sperm competition rank with five levels, from 1 (low risk of sperm competition) to 5 (high risk of sperm competition). Overall, we found that minors invest more in ejaculate traits than majors. We also categorized the ejaculate expenditure of males, according to the original variables quantified in the studies that were included in our analysis and found that minors invest more energy in the production of gonads than majors. Additionally, minors and majors have a similar investment in sperm number and sperm quality, but majors allocate more sperm to females. Overall, the sperm competition rank did not influence the magnitude of the difference in investment of majors and minors. The differential investment in gonad mass between majors and minors should represent an increase in sperm numbers, but our data showed that majors and minors are not producing different amount of sperm. Therefore, the higher investment in gonad mass can be related to minors mating more frequently than majors. minors are not able to produce sperm in greater quantities than majors, but they probably can replenish sperm faster than majors. Against theoretical predictions, sperm quality does not respond to differences of sperm competition, probably because sperm quality is not under such strong selection as gonad mass. Our findings suggest that, in fishes with alternative mating tactics, both majors and minors are under strong selection from sperm competition, even when the risk of polyandry is low / A teoria prevê que em espécies sob maior risco de competição espermática, os machos irão investir mais em características do ejaculado. Em espécies com táticas alternativas de acasalamento (AMTs), machos de fenótipos diferentes estão sob diferentes riscos de competição espermática. Uma vez que machos minors (i.e., machos furtivos) se esgueiram para dentro do território de outros machos para acasalar, eles provavelmente sempre enfrentam competição espermática. Machos major, por outro lado, defendem territórios e possuem uma chance maior de acasalar exclusivamente com fêmeas. Para os majors, o risco de competição espermática é teoricamente menor. A principal previsão de modelos de teoria dos jogos para espécies com AMTs é que majors investem menos em características de ejaculado do que minors. Entretanto, quando a proporção de minors em uma população aumenta, os majors devem investir mais em características do ejaculado, alcançando um nível similar de investimento em ejaculado que os minors. Neste estudo, nós testamos essas previsões com uma meta-regressão de 29 espécies de peixes com AMTs. Como proxy para o risco de competição espermática, nós classificamos cada espécie de acordo com um ranking de competição espermática. Esse ranking utiliza características de história de vida e demografia de cada espécie, e possui cinco níveis, de 1 (baixo risco de competição espermática) até 5 (alto risco de competição espermática). De maneira geral, nós encontramos que minors investem mais em características de ejaculado do que majors. Nós também categorizamos o investimento em ejaculado dos machos de acordo com as variáveis originais quantificadas nos estudos que foram incluídos na nossa análise e encontramos o resultado de que minors investem mais na produção de gônadas para seu próprio tamanho do que majors. Além disso, minors e majors apresentam investimento similar em número de espermatozoides e qualidade espermática, mas majors alocam mais esperma para as fêmeas. Em geral, o ranking de competição espermática não influenciou a magnitude da diferença de investimento entre majors e minors. O investimento diferencial em massa gonadal entre majors e minors deveria representar um aumento no número de espermatozoides, porém nossos dados mostraram que majors e minors não estão produzindo quantidades diferentes de esperma. Assim, um investimento maior em massa gonadal pode estar relacionado aos minors acasalarem mais frequentemente que os majors. minors não conseguem produzir esperma em maiores quantidades que os majors, mas eles provavelmente conseguem repor seu estoque de esperma mais rápido que os majors. Contrário às previsões teóricas, a qualidade espermática não responde às variações de competição espermática, provavelmente porque a qualidade espermática não está sob forte seleção como a massa gonadal. Nossos resultados sugerem que, em peixes com táticas alternativas de acasalamento, tanto os majors como os minors estão sob forte seleção da competição espermática, mesmo quando o risco de poliandria é baixo