Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T20:08:58Z No. of bitstreams: 1 127110020.pdf: 531553 bytes, checksum: 0170d654c8a7662821baf128927374e8 (MD5) / Made available in DSpace on 2015-03-08T20:08:58Z (GMT). No. of bitstreams: 1 127110020.pdf: 531553 bytes, checksum: 0170d654c8a7662821baf128927374e8 (MD5) Previous issue date: 2014-08-12 / A técnica de seleção espermática para fecundação in vitro (FIV) deve proporcionar além, da recuperação eficiente de espermatozoides, a manutenção da integridade celular. Diversos métodos vêm sendo propostos para a seleção de espermatozoides bovinos destinados a FIV, sendo o método de centrifugação em gradientes de densidade descontínuos de Percoll® o mais utilizado. Em bovinos esta técnica de seleção vem sendo amplamente modificada no que se refere ao volume, número de gradientes, tempo e força de centrifugação, visando aumentar a recuperação espermática, proporcionando um melhor rendimento da dose. Contudo, a etapa de centrifugação, presente nestes protocolos, pode causar danos irreversíveis aos espermatozoides e assim influenciar nas taxas de fecundação. O presente estudo teve como objetivo determinar a influência da força e do tempo de centrifugação, assim como a associação do meio Cushion Fluid® na segunda centrifugação na seleção espermática por gradientes de Mini Percoll® modificado, através da avaliação de características morfofuncionais, formação de espécies reativas ao oxigênio (EROs), defesas antioxidantes, taxa de fecundação in vitro e desenvolvimento embrionário em três experimentos. No experimento I, um pool contendo sêmen de dois touros Bos taurus taurus com fertilidade conhecida foi submetido ao método de separação por Mini Percoll®, sendo as amostras divididas em quatro grupos e submetidos a duas forças (2200 ou 9000 X g) e dois tempos de centrifugação (1 e 3 minutos). No experimento II e III, um pool de sêmen de dois touros Bos taurus taurus com fertilidade conhecida foram processados pelo método de Mini Percoll® modificado, sendo adicionado uma quantidade de 150μL de Cushion Fluid® (Minitübe, Tiefenbach, Germany) durante a segunda centrifugação. Foram realizadas avaliações morfofuncionais e bioquímicas das amostras do sêmen destinado a FIV, assim como a capacidade fecundante e o desenvolvimento embrionário dos oócitos fecundados com os espermatozoides selecionados. No primeiro experimento foi observada uma melhora no vigor dos espermatozoides após o gradiente de Mini Percoll®, para todos os tratamentos. No segundo experimento, a taxa de recuperação espermática não diferiu entre os tratamentos, sendo 42,0% ± 4,9 para T1 e 38,6% ± 3,6 para T2 (grupo com adição do Cushion Fluid® na segunda centrifugação):. Na morfologia espermática o T2 (5,1 ± 1,1) apresentou menor taxa de espermatozoides com defeitos maiores do que os tratamentos T0 (Pré-Percoll) e T1 (8,8 ± 0,8 e 5,8 ± 1,0). Neste mesmo experimento, os níveis de EROs apresentaram-se aumentados (P < 0,05) no meio com Cushion Fluid® (T2), no entanto, a avaliação das defesas antioxidantes não diferiram entre os tratamentos. No experimento III, não foram observadas diferenças na taxa de fecundação. Este trabalho demonstrou que o uso de uma menor força e tempo (2200 X g/1 min.) de centrifugação pode ser utilizado sem interferir nas taxas de recuperação espermática. O uso do meio Cushion Fluid® durante a centrifugação de espermatozoides bovinos não influenciou a taxa de fecundação e desenvolvimento embrionário até 48 horas. / The technique of sperm selection for in vitro fertilization (IVF) should provide addition, the sperm efficient recovery, the maintenance of cellular integrity. Several methods have been proposed for bovine sperm selection for IVF, and the discontinuous density gradients Percoll® ( Sigma Aldrich – St. Louis MO, USA) centrifugation method on the most used. In cattle this selection technique has been extensively modified with respect to the volume, number of gradients, time, and centrifugal force, to increase the sperm recovery, providing a better yield of the dose. However, the centrifugation step present in these protocols, may cause irreversible damage to sperm and thus influence fertilization rate. The present study aimed to determine the influence of centrifugation force and time, as well as the association of the Cushion Fluid® centrifugation on sperm selection by gradients Mini Percoll® modified by evaluating morphological and functional characteristics, ROS formation, antioxidant defenses, rate of in vitro fertilization and embryo development. Three experiments carry out. In experiment I, a pool containing two semen Bos taurus taurus bulls was subjected Mini Percoll® separation method, samples being divided into four groups, with two centrifugal forces (2200 or 9000 X g) and two times (1 and 3 min). In experiment II and III, the pool has been processed by the Mini Percoll® modified method, being added to an amount of 150μLix of Cushion Fluid® (Minitübe, Tiefenbach, Germany) in the second centrifugation in the treated group. Morphofunctional and biochemical evaluation of semen samples for IVF, as well as the fertilizing capacity and embryo development of oocytes fertilized with sperms were selected performed. In the first experiment an improvement was observed in the vigor of sperm after gradient Mini Percoll® for all treatments. In the second experiment, the recovery rate sperm did not differ between treatments (T1: 42.0 ± 4.9 and T2 (with adding Cushion Fluid® the second centrifugation): 38.6 ± 3.6). In sperm morphology T2 (5.1 ± 1.1) showed a lower sperm rate with major defects than T0 (Pre-Percoll) and T1 (8.8 ± 0.8 and 5.8 ± 1.0 treatments ). In this same experiment, ROS levels were presented increased (P <0.05) in the medium with Cushion Fluid® (T2), however, the evaluation of antioxidant defenses did not differ between treatments. In experiment III, no differences in fertilization rate were observed. This study demonstrated that the use of a smaller force and time (2200 X g / 1 min.) to centrifugation can be used without interfering with the sperm recovery rate. The use of the medium Cushion Fluid® during centrifugation of bovine sperm did not influence the fertilization rate and embryo development to 48 hours.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The incidence of male infertility is increasing, with up to 50% of infertile males having “unexplained” (idiopathic) infertility. Newly developed molecular techniques have great value in detecting subtle causes of male infertility, as compared to idiopathic infertility which may be explained by standardizing and optimizing sperm functional and structural tests in non-human primate (NHP) sperm. The aim of the study was to evaluate sperm functionality utilizing the sperm of two NHP species, i.e.1) the rhesus monkey (Macaca mulatta) and 2) the vervet monkey (Chlorocebus aethiops), and further evaluate the effect of physiological media (including commonly used, and newly formulated sperm wash and sperm capacitating media) on NHP sperm functionality. Sperm functionality was evaluated by investigating the following sperm functions i.e.: sperm motility, vitality, acrosome reaction (AR), hyperactivation, and mitochondrial membrane potential (MMP). Sperm functional tests included computer-aided semen analysis (CASA), motility analysis, BrightVit staining for sperm vitality, flourescenin isothiocyanate (FITC)- conjugated peanut agglutinin (PNA) staining for sperm acrosome integrity, induction of hyperactivation by stimulants (sperm preparation media containing capacitating ingredients), and mitochondrial inhibitor (Oligomycin-A) for testing MMP. All functional and structural tests were investigated in both species, except for acrosome integrity, mitochondrial inhibition and functional tests compared over time that could not be successfully completed and investigated in the rhesus species. Motility analysis tests proved that within the vervet species, the use of different physiological media results in statistically significant differences in motility and kinematic parameters over a 1 hour time period. Hyperactivation tests proved that capacitating physiological media produced significantly higher percentages hyperactivation when compared to sperm wash media within the vervet species over a 1 hour time period. Furthermore, within both NHP species, sperm structural analysis (vitality and acrosome integrity) results showed that no significant differences are present when making use of different physiological media over a period of 1 hour incubation. The incubation of vervet sperm with different concentrations of mitochondrial inhibitor, Oligomycin-A (0 μM, 5 μM, and 25 μM), resulted in motility inhibition over a 1 hour incubation period. By the evaluation of these tests it was found that the use of different sperm wash [Human tubal fluid (HTF), Ham‟s F-10® and HD Sperm Wash Plus (HDSWP)] and sperm capacitation media [Human tubal fluid with added caffeine (HTFC) and HD Sperm Capacitating Plus (HDSCP)] resulted in significantly different results within sperm functional tests as compared to sperm structural tests. The study indicates that the composition of media, varying from simple to more complex, used for semen preparation plays an important role in determining NHP sperm functionality. Based on these findings further investigation in larger NHP sample groups and human sperm are required to evaluate the role of certain ingredients in the development of more cost-effective media producing satisfactory results in terms of sperm functionality for artificial reproductive technologies (ART).

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

Darbo tikslas: įvertinti skirtingais racionais šertų veislinių bulių spermos kokybę. Darbo uždaviniai: 1. Išnagrinėti ir įvertinti skirtingus veislinių bulių racionus; 2. Nustatyti tiriamųjų bulių spermos kokybę; 3. Įvertinti skirtingų racionų įtaką veislinių bulių spermos kokybiniams rodikliams. Išvados: 1. B fermos buliai reproduktoriai gavo 16,4 MJ AE, 52,35 žaliųjų baltymų, 20,66 g fosforo, 40,9 mg vario, 859,2 mg cinko, 28290 TV vitamino D bei 122700 TV vitamino A daugiau nei A fermos buliai, kur šios medžiagos yra ypač svarbios reprodukcinei sistemai ir spermos kokybei. 2. A fermoje laikomų bulių išskirtas spermos tūris buvo 5,60 (±0,11) ml, koncentracija 1287,61 (±38,04) mln/cm3, gyvybingų spermatozoidų 81,22 (±0,57) proc., tiesiai judančių spermatozoidų 65,73 (±0,76) proc. B fermoje laikomų bulių vidutinis išskiriamas spermos tūris buvo 8,50 (±0,25) ml, koncentracija 1371,54 (±67,07) mln/cm3, gyvybingų spermatozoidų 80,13 (±0,37) proc, tiesiai judančių spermatozoidų 67,81 (±0,47) proc. 3. B fermoje laikomų bulių išskirtas spermos tūris buvo 2,90 ml (P<0,001), koncentracija – 84,92 mln/cm3 (P>0,05), tiesiai judančių spermatozoidų – 2,07 proc. didesnis (P>0,05) nei A fermoje laikomų bulių. Vienintelis priešingai rodęs rodiklis, t.y. gyvybingi spermatozoidai buvo 1,09 proc. (P<0,05) mažesnis už A fermos bulių. 4. Bulių davusių iki 4 ml spermos kiekį, koncentracija vidutiniškai nuo 1351,11(P<0,001) (A buliai) ir 2218,25 (P<0,001) (B buliai) sumažėjo iki 1097,43 (P<0,001)... [toliau žr. visą tekstą] / The aim of this research is evaluate the quality of bull semen, that are fed different diets. Research goal of the paper: 1. Analyze and evaluate different breeding bull rations; 2. Analyze bull’s semen quality; 3. Evaluate the influence of different diets on breeding bull semen quality (semen volume, concentration, percent viable sperm, directly moving spermatozoa percent). The results of different bull’s diet show that bulls of farm B got more mineral and vitamin needed for reproductive system than bulls from farm A. The breeding bulls of farm B were given 16.4 MJ 52.35 crude protein, 20.66 g of phosphorus, 40.9 mg of copper, 859.2 mg of zinc, 28,290 IU of vitamin D and 122 700 IU of vitamin A more than bulls from farm A. This material is particularly important for the reproductive system and sperm quality. Breeding bull’s sperm quality indicators showed that the semen characteristics (volume, concentration, and right-moving sperm) were on average superior to those bulls whose diets were additionally given mineral - vitamin supplements. The semen quality of bulls is changing dependent on different bull semen volume released. It showed that increasing volume of the sperm determine decreasing in sperm concentration mln/cm3.

El penegal, Helicolenus dactylopterus dactylopterus (Pisces: Scorpaeniformes), és una espècie que habita profunditats d'entre els 200 i 1000 m i presenta una clara distribució batimètrica en funció de la seva talla. Presenta fecundació interna i a l'interior de l'ovari conté estructures d'emmagatzematge que permeten emmagatzemar l'esperma durant períodes de temps considerablement llargs. Les cèl·lules sexuals masculines es mantenen viables gràcies a diverses substàncies nutritives que obtenen de la bossa citoplasmàtica i de l'epiteli criptal que delimita les estructures d'emmagatzematge. Aquest epiteli és també responsable de la seva protecció. Un cop els ous han assolit la maduresa, els espermatozoides són alliberats al lumen ovàric i es dóna la fertilització. És una espècie zigòpara: allibera òvuls fecundats que han estat retinguts al tracte reproductiu femení durant un curt període de temps i, per tant, els embrions són alliberats en estadis molt primerencs de desenvolupament. Així, el penegal presenta una estratègia reproductiva evidentment eficaç. / The bluemouth, Helicolenus dactylopterus dactylopterus (Pisces: Scorpaeniformes), is a species that inhabits the seabed at depths between 200 and 1000 m and shows a clear size-dependent bathymetric distribution.It has internal fertilization and, inside the ovary, there are storage structures that allow it to store sperm for considerably long periods of time. These male sexual cells remain viable thanks to several nutritious substances they obtain from the cytoplasmic bag and from the cryptal epithelium that delimits the storage structures. This epithelium is also responsible for their protection. Once the eggs have reached maturity, the spermatozoa are freed in the ovarian lumen and fertilization occurs. This species is zygoparous: liberation of fertilized ova, which have been retained in the female reproductive tract during a short period of time and, therefore, the embryos are freed in very early stages of development. Thus, the bluemouth exhibits an obviously effective reproductive strategy.

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.

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.

Over three decades ago, W.H.O. declared infertility as a public health issue; due to its impact on millions of people worldwide. While cases of infertility could be multifactorial (affecting both male and female), 50% of cases are due to male factor infertility and this is mostly characterised by reduced sperm motility (asthenozoospermia). Assisted Reproduction Technology (ART) is the only treatment option available for this condition. Over 20 years ago, non-selective phosphodiesterase inhibitors (PDEi), such as pentoxifylline, were shown to enhance motility of human spermatozoa; however, contradictory results and stimulation of premature acrosome reaction has precluded their clinical use. Advancement in our knowledge have now made it clear that human sperm express several different PDEs and these are compartmentalised at different regions of the cells. By using type-specific phosphodiesterase inhibitors, differential modulation of sperm motility can be achieved without affecting other sperm function such as acrosome reaction. Additionally, by enhancing sperm function through PDE inhibition, there is a possibility of increasing IVF rates. The objective of this thesis is to: (1) examine the effect of phosphodiesterase inhibitors on spermatozoa in order to identify compounds that have clinically relevant enhancement of human sperm motility; (2) identify the signalling pathway(s) involved in the motility enhancing effects of identified compounds by targeting the modulator and mediator of cyclic nucleotides; (3) develop an animal IVF model to assess effects of Ibudilast on fertilization; and (4) optimise high performance liquid chromatography (HPLC) techniques for routine detection of cyclic nucleotides in sperm cells. A two phase drug screening approach was used to systematically and comprehensively screen series of compounds in order to identify those that have clinically relevant enhancement of human sperm motility. In phase 1, 6 compounds (out of 43 compounds) were found to have strong effects on poor motility samples, with magnitude of response ≥60% increase in percentage total motility. Additionally, these compounds significantly enhanced sperm penetration into cervical mucus substitute (p≤0.05), and they did not affect sperm acrosomal integrity nor cause externalisation of phosphatidylserine (p=0.6 respectively). 63% of IVF samples treated with compounds #26, #37 and #38 had significant increase in percentage total motility. For ICSI samples, compounds #26, #37 and #38 were the most effective. In respect to total motility, 88%, 81% and 79% of samples treated with these compounds showed significant increases in total motility, and 94%, 93% and 81% of samples showed significant increases in percentage of progressive cells, respectively. Analysis of the signalling pathways, using PKA, sGC and PKG inhibitors, showed that chosen PDE inhibitors were working predominantly through PKA signalling pathways. Additionally, this study revealed that this pathway is needed for the maintenance of basal progressive motility and hyperactivation in human sperm. Animal IVF studies showed that addition of Ibudilast (compound #26) during sperm-oocyte incubation leads to higher IVF rates. Lastly, this study used an HPLC system to detect cAMP in boar sperm. This was done to explore if HPLC system can be used for high throughput detection of cyclic nucleotides in mammalian sperm.

Sperm function has been studied in multiple research fields as it is essential to male fertility. In previous studies a variety of sperm traits have been examined as an assessment of sperm function. Among those traits, sperm viability, sperm motility and sperm metabolism are often commonly examined. However, sperm function can be influenced by both environmental and genetic factors. Specifically, nuclear genome has been demonstrated to play a role in sperm function, especially in sperm competitive capacity. There are increasing evidence for effects of mitochondrial genome on sperm function. Mitochondrial genetic variance has been suggested to affect sperm length and sperm viability in seed beetle and sperm metabolism in rodent. Given the coordinated collaborations between nuclear and mitochondrial genomes in OXPHOS, replication and transcription of mitochondrial genome as well as intergenomic signalling, potential mitonuclear effects on sperm function are expected even though empirical evidence so far remains less. A recent review summarised all the previous work on environmental effects on sperm and found that various factors affects sperm function but largely neglected in ecology and evolution. In the study, we used D. melanogaster as a model to disentangle both genetic and environmental components of sperm function at sperm cell, ejaculate and offspring levels. We found environmental effects on sperm function in D. melanogaster. Specifically, sperm incubation buffers affect sperm viability in chapter 2 and dietary PUFAs influence sperm volume and metabolism in chapter 4. Nuclear effects were found on sperm viability, sperm quality and male fertility in chapter 3. Mitochondrial genome was found to have an effect on sperm function, i.e. sperm viability and sperm quality differed among mitochondrial haplotypes examined. In addition, sperm function was further modified by the interaction of nuclear and mitochondrial genomes in ageing male. Sperm quality and fertilization success were suggested to be dependent on age-related mitonuclear interaction in chapter 3. Moreover, we examined the mitonuclear coadaptation hypothesis in the function of D. melanogaster sperm. No evidence for mitonuclear coadaptation hypothesis was found for sperm function in D. melanogaster as there were no difference between coadapted and non-coadapted lines in sperm traits examined. Lastly, we found that sperm viability, sperm quality and sperm metabolic rate cannot predict male fertility in D. melanogaster as correlation analysis revealed no relationship between them. Our experiment explored and disentangled the genetic and environmental components of sperm function at multiple levels in D.melanogaster systematically. Our results suggested that both mitochondrial and nuclear genome as well as the interaction between them play a role in sperm function in D. melanogaster. In addition to genetic components, our findings revealed environmental components of Drosophila sperm and suggested that it was phenotypic plastic.