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Sperm competition is a widely-recognized and powerful selective force. Male accessory glands are organs found across animal taxa that can influence sperm performance, and thus may be selected for in competitive contexts. In fishes, these organs are in fact rare, but display great diversity in form and function across species. Although the accessory gland is known to play a role in mate attraction, parental care, fertilization, or post-copulatory competition in a few select species, the role of this organ remains a mystery in most species. Many fishes with accessory glands also exhibit alternative reproductive tactics (ARTs), which add an extra layer of complexity to how species respond to sperm competition. Because males of different ARTs typically experience different levels of sperm competition risk, it’s possible they may differentially invest in accessory glands to overcome this competition. In this thesis, I used the plainfin midshipman fish (Porichthys notatus), a species with both ARTs and an accessory gland, to experimentally investigate the role of the accessory gland in sperm competition and uncover how this organ may differ between ARTs. Over a two-year period, I studied tactic-specific investment in the accessory gland in fish from the beaches of British Columbia. I also examined the effects of seminal fluid, produced in part by the accessory gland, on sperm performance and morphology. I found that males adopting the ‘guarder’ male tactic invested more in one region (the lobules) of this organ, while males adopting the smaller ‘sneaker’ male tactic invested more the other region of the gland (the nodes). Using data collected over five years, I found that guarder males also invested more in their whole accessory glands. Additionally, I report that sperm swam faster in the presence of seminal fluid, and seminal fluid increased sperm head size in both male tactics and increased midpiece size in guarder males. These results suggest that the plainfin midshipman accessory gland may have dual functions, one of which may be to aid sperm competitive ability through enhancements in swimming speed and potentially more successful sperm morphology. Taken together, the results of my thesis improve our knowledge of the role of non-sperm components like seminal fluid and the accessory gland in sperm competition, and demonstrate how species with ARTs can have varying physiological responses to such competition. Only a handful of studies have considered the effects of seminal fluid on sperm performance. By examining sperm competition in a more biologically relevant way and incorporating the effects of a little-studied organ that impacts sperm competition, we should be able to more generally and accurately appreciate the dynamics of post-copulatory competition and fertilization. / Thesis / Master of Science (MSc)

Background: Infertility has become a growing concern across the world as cases continue to increase each year. Research has now shifted to identifying novel biomarkers to predict male fertility. While mtDNAcn has recently been found to show promising results as potential biomarker, its regulation remains unclear. Method: Triplex probe-based PCR was used to quantify mtDNA levels, while 850K Array was used to measure methylation levels. A-clustering algorithm followed by generalized estimating equations (GEE) lead to clustering of individual CpG sites, containing a minimum of 2 CpGs within 1000 base pairs of each other. These clusters were used for analysis of the association between mtDNAcn and DNA methylation within sperm. Metascape1 was used to annotate gene ontology terms. Result: Generalized estimating equation model analysis produced 6,038 FDR significant (q Conclusion: Thus, we show that sperm mtDNAcn is strongly associated with sperm DNA methylation and the associated implicates mtDNAcn as an influence on infertility.

The objective of two studies was to manipulate the essential fatty acid content of turkey semen by enhancing the dietary levels of either n-3 polyunsaturated fatty acids (PUFA) or n-6 PUFA and determine the effect on fertility. In 1999 (Trial 1), and again in 2000 (Trial 2), Large White tom turkeys, 37 weeks of age, were fed one of three diets substituted with chicken fat, soybean oil, or menhaden fish oil. Chicken fat provided the industry's standard ratio of n-6 to n-3, soybean oil provided a greater ratio of n-6 to n-3, and fish oil provided a lower ratio of n-6 to n-3. Contemporary hens were inseminated weekly with semen collected from each group of toms. The effects of dietary lipids on tom body weights, fertility, motility, perivitelline layer sperm penetration percent, and live vs. dead sperm were analyzed. Whereas body weight increased linearly from 31 to 56 weeks of age (WOA), there was no effect of dietary treatment. As measured by the Accudenz® procedure, there were differences in sperm motility due to dietary treatment during 48 and 51 WOA during Trial 1. During Trial 2, sperm motility differences were observed at 53 WOA with the soybean oil-treated toms having the largest absorbance reading and the chicken fat-treated toms having the largest absorbance reading during 56 WOA. The live vs. dead sperm cells during Trial 1 revealed differences among the toms prior to treatment and post treatment. No dietary effects on percent live vs. dead sperm cells were observed during Trial 2. Once per mo, eggs were collected for a one-week period to analyze for sperm penetration of the perivitelline layer. In Trial 1, sperm from toms fed chicken fat produced more penetrations (holes) during 36, 48, and 52 WOA. In Trial 2, sperm penetration values were lower for toms fed fish oil during 42, 47, and 51 WOA. Whereas there were significant differences in fertility, hatch of total eggs, and hatch of fertile eggs among treatments in Trial 1, a bacterial contamination on the farm during weeks seven through fourteen may have contributed to these findings. No significant differences due to treatment were found in these parameters during the second study. The fatty acid analysis of spermatozoa collected at the conclusion of Trial 2 revealed significant differences in total n-3 and total n-6 content, leading to significant differences in the ratio of total n-6 to total n-3. The mixed results indicated the fertilizing ability of domesticated turkey spermatozoa may not be affected by the n-6 to n-3 ratio in the diet of the tom. / Master of Science

No / The Comet assay is one of the most versatile tools in toxicology today and can be used to measure responses in both diploid (peripheral blood lymphocytes) and haploid (sperm) primary cells in humans. This chapter will discuss how these cells are employed to determine if they have differential responses to chemical and physical agents in healthy and disease-affected individuals and how such information can be of use to man.

<p>The chromatin integrity in sperm cells is vital for successful pregnancy. In this</p><p>study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization</p><p>(IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were</p><p>purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test</p><p>Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation</p><p>Index (DFI) of 30 % and the results correlated with fertilization, cleavage and</p><p>pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower</p><p>cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro</p><p>may be independent of the degree of DNA-damage, the embryonic development could be</p><p>seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a</p><p>DFI threshold of 30 %.</p>

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2013-09-29 23:45:35.395

In externally fertilizing fishes, multiple factors of the spawning environment may affect the sperm viability, and thus the fertilization rate. In this thesis, the sperm activation effect of osmolality of non-electrolytes and electrolytes activation media, pH and ion channel inhibitors on Nile tilapia, Oreochromis niloticus, and the effect of environmentally relevant pollutants (cadmium, malathion and rotenone) on sperm fitness (motility and morphology) were investigated. Seminal fluid samples collected from male fishes (200-250g) were subjected to activation treatments, then analyzed for sperm motility using motility score, and motility variables using Hobson sperm tracker for straight line velocity (VSL), beat cross frequency (BCF) and percentage of motile cells (MOT). For the ion channel inhibitors and pollutants, the effect on sperm motility variables of VSL, VCL (curvilinear velocity) and LIN (linearity) were determined. Multivariate analysis was also carried out to determine the effects of ion channel inhibitors and pollutants on sperm subpopulations. The effects of pollutants on sperm morphology were observed using microscopy techniques, namely, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Sperm motility was initiated when the sperm were exposed to hypoosmotic electrolytes and non-electrolytes solution. We also found that sperm show optimal activity at pH range of 6-8 which depicts that the effect of pH on sperm motility is negligible. Lanthanum (calcium channel blocker) and flunarizine (sodium-calcium exchanger pump blocker) were found to inhibit sperm motility at 25 and 5 µM, respectively, suggesting that both ion channels play a significant role in sperm activation in O. niloticus. In contrast amiloride, ouabain and quinine showed no effects on activation, indicating that epithelial sodium channels, sodium-potassium ATPase and voltage gated potassium channels respectively are unlikely to have major roles in sperm activation or motility. The spermatozoa of Oreochromis niloticus were uniflagellate with clearly differentiated oval-shaped head, midpiece and flagellum. Sperm exposed to hypoosmotic shock showed swelling of the midpiece and sleeve structure. The pollutants showed dose- and time-dependent effect on sperm motility of the fast linear sperm subpopulation. Sperm morphology was not affected. Sperm motility was inhibited at 0.44, 0.03 and 0.063 µM, cadmium, malathion and rotenone respectively. Both cadmium and malathion exerted effects very quickly after exposure. The effect of cadmium, which can exert toxicity by calcium antagonism, is consistent with the effects of calcium channel blockes and further supports an important role for calcium in sperm activation and motility. Malathion had effects at relatively low, environmentally relevant concentrations, suggesting the presence of functionally important acetylcholinesterase activity in sperm, and also the presence of activation cytochrome P450 activity. Rotenone, a well known mitochondrial poison, affected motility only after 15 min of pretreatment. The alteration of sperm trajectories in fast linear spermatozoa subpopulation by pollutants at submicromolar concentrations as demonstrated in our study implies potentially serious consequences for fish populations in polluted environments. Furthermore the results indicate that fish sperm motility as assessed by CASA could be an ecologically relevant, sensitive, and ethically acceptable method for toxicity testing in environmental risk assessment.

Estudos recentes avaliaram o papel do Magnetic Activated cell Sorting (MACS, separação celular por ativação magnética) para reduzir a percentagem de espermatozoides apoptóticos e melhorar a qualidade seminal. No entanto, a eficiência do uso do MACS isoladamente, antes ou depois do processamento seminal pelos métodos clássicos, como o centrifugação em gradiente de densidade (DGC), ainda não foi estabelecida, de modo que o protocolo de uso do método não foi adequadamente estabelecido. Desta forma, o objetivo do presente estudo foi avaliar se o MACS sozinho, antes (MACS-DGC) ou depois do processamento seminal pelo DGC (DGC-MACS) melhora a seleção espermática quando comparado ao processamento pelo DGC. Realizou-se um estudo prospectivo experimental avaliando amostras de sêmen de 15 homens saudáveis. A mesma amostra foi dividida em 4 alíquotas e processada por: DGC, DGC-MACS, MACS-DGC e MACS. Após o processamento, foram analisadas a integridade do DNA espermático pelo método TUNEL - TdT-mediated dUTP Nick End Labeling (marcação de quebras no DNA por dUTP e deoxinucleotidil terminal transferase) assim como a concentração de espermatozoides, a motilidade progressiva e a morfologia, segundo os últimos critérios adotados pela Organização Mundial da Saúde. A percentagem de dano ao DNA foi significativamente menor no grupo MACSDGC, quando comparado com DGC e MACS, e semelhante a do grupo DGC-MACS. A concentração de espermatozoides recuperados nos grupos de DGC e MACS foi similar e significativamente maior do que MACS-DGC e DGC-MACS, que foram semelhantes entre si. A motilidade progressiva dos espermatozoides recuperados foi semelhante nos grupos MACS-DGC e DGC e significativamente maior do que nos grupos DGC-MACS e MACS. O percentual de espermatozoides com morfologia normal foi significativamente maior no grupo MACS-DGC quando comparado ao DGC-MACS e MACS, e semelhante quando comparado com DGC. Desta forma, evidenciou-se que ambos os métodos combinados promovem a recuperação de espermatozoides com menor percentagem de dano ao DNA espermático. O MACS isoladamente ou aplicado após o DGC promove redução significativa dos espermatozoides progressivos recuperados, assim como da percentagem de espermatozoides com morfologia normal. Todavia, o MACS aplicado antes do DGC promove a recuperação de amostras com elevada percentagem de espermatozoides com motilidade progressiva e morfologia normal, aliada a baixa percentagem de DNA fragmentado, sugerindo ser o melhor protocolo de uso desta metodologia, cuja importância clínica precisa ser avaliada em estudos clínicos bem delineados. / Recent studies evaluated the role of Magnetic Activated Cell Sorting (MACS) in order to reduce the percentage of apoptotic sperm and improve seminal quality. However, the effectiveness of using MACS alone, before or after seminal processing by classical methods, such as the density gradient centrifugation (DGC), has not yet been established. Thus, the role of the present study was evaluate whether: MACS alone, before (MACS-DGC) or after seminal processing by DGC (DGC-MACS) improves sperm selection when compared to DGC. A prospective experimental study was conducted evaluating semen samples from 15 healthy men. The same sample was divided into 4 aliquots and processed by: DGC, DGC-MACS, MACS-DGC and MACS. After processing, the integrity of the sperm DNA was analyzed by TUNEL - TdT - mediated dUTP method Nick End Labeling (marking DNA breaks by dUTP and deoxynucleotidyl terminal transferase), sperm concentration, progressive motility and morphology according to the last criteria adopted by the World Health Organization. The percentage of DNA damage was significantly lower in the MACS-DGC group, when compared to DGC and MACS, and similar to the DGC-MACS group. The concentration of spermatozoa recovered in the DGC and MACS groups was similar and significantly higher than MACS-DGC and DGC-MACS, which were similar to each other. The progressive motility was similar in the MACS-DGC and DGC groups and significantly higher than in the DGC-MACS and MACS groups. The percentage of spermatozoa with normal morphology was significantly higher in the MACS-DGC group when compared to DGC-MACS and MACS, and similar when compared to DGC. Thus, it was evidenced that both methods combined promote the recovery of spermatozoa with a lower percentage of DNA damage. MACS alone or applied after the DGC promotes a significant reduction of the progressive sperm retrieved, as well as the percentage of spermatozoa with normal morphology. However, the MACS applied before the DGC promotes the recovery of samples with a higher percentage of spermatozoa with progressive motility and normal morphology, combined with low percentage of fragmented DNA, suggesting to be the best protocol, whose clinical importance needs to be evaluated in well-delineated clinical studies.

Este trabalho teve como objetivo principal avaliar o sêmen da jararaca ilhoa, Bothrops insularis, quanto ao volume, motilidade, vigor e concentração, além de verificar se estes paramentos estão relacionados ao comprimento ou massa dos animais ou se variam ao longo das estações, uma vez que esta espécie possui ciclo reprodutivo sazonal. Para tanto, foram avaliadas amostras de sêmen de 18 machos, com comprimento rostro-cloacal (CRC) variando de 43,5 a 73,70 cm, pertencentes ao plantel do Laboratório de Ecologia e Evolução do Instituto Butantan, entre os meses de agosto de 2012 e maio de 2013. O sêmen de Bothrops insularis apresentou coloração variando de esbranquiçada a amarelada e consistência cremosa e espessa. Foi possível observar amostras de sêmen viáveis em todos os 18 animais avaliados, indicando que animais com CRC igual ou acima de 43,5 cm já são sexualmente maduros. O espermatozoide de Bothrops insularis é filiforme, possui cabeça alongada, fina e com formato pontiagudo, seguindo o padrão morfológico descrito para outros Squamata. Não foi observada nenhuma relação entre o comprimento, massa e cada um dos parâmetros seminais avaliados. Foi observada variação significativa da motilidade e da concentração do sêmen de Bothrops insularis entre as estações, porém o volume e o vigor do sêmen não variaram. Foi no outono, época de cópula da espécie, que o sêmen apresentou as maiores médias para os parâmetros avaliados. / This study aimed to evaluate sperm parameters of the golden lancehead, Bothrops insularis, including appearance, sperm motility, vigor, volume and concentration, determine if these parameters varied with body size or weight and to verify whether these vestments vary throughout seasons, since this species has a seasonal reproductive cycle. Samples of semen of 18 males with snout-vent length (SVL) ranging from 43.5 to 73.70 cm, belonging to the squad of the Ecology and Evolution Laboratory at Instituto Butantan, were collected between August 2012 and May 2013. Sperm color from Bothrops insularis ranged from whitish to yellowish, and a creamy and thick consistency. Viable sperm was observed in samples from all 18 males evaluated, indicating that animals with SVL equal to or above 43.5 cm are sexually mature. Sperm of Bothrops insularis is thready, has elongated, thin and pointed shaped head, following the morphological pattern described for other Squamata. No relationship between length, mass and each of seminal parameters evaluated was observed. Significant variation of concentration and motility of semen from Bothrops insularis between seasons was observed, but volume and vigor did not change. It was during Fall, mating season of the species, that sperm had the highest means for all parameters evaluated.