The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

The studies presented in this thesis were designed to elucidate whether the abundance of ovulation-inducing factor/nerve growth factor (OIF/NGF) in alpaca semen can be used as a biomarker to predict male fertility. The neurotrophin, OIF/NGF has been identified in camelid, cattle and human semen. It is only in camelids, however, that an ovulation-inducing role for OIF/NGF has been described. The information gathered from several studies clearly demonstrate that this protein is the stimulus responsible for initiating the ovulatory cascade in camelids. In addition, intramuscular administration of OIF/NGF resulted in a dose-dependent response in terms of ovulation rate, corpus luteum (CL) lifespan, luteinizing hormone (LH) and progesterone secretion. I hypothesized that the quantity of OIF/NGF differs among male alpacas and this abundance arbitrates ovulation and pregnancy rates as well as CL formation and function. To substantiate this hypothesis, the following questions were answered: 1) can OIF/NGF in alpaca semen be quantified using a radioimmunoassay; 2) does the concentration and total abundance of OIF/NGF in alpaca semen vary within and among male ejaculates; 3) what is the glandular source of OIF/NGF that contributes to the male ejaculate; 4) is OIF/NGF concentration or abundance related to parameters associated with male fertility; 5) can OIF/NGF concentration or total abundance in the ejaculate discriminate fertile and subfertile males using both retrospective and prospective approaches; and 6) can power Doppler ultrasonography be used to assess the luteotrophic effect of OIF/NGF in tissue vasculature of the developing CL? I discovered that the source and the amount of OIF/NGF varies among species. In llamas, OIF/NGF is produced by both the corpus and disseminate portions of the prostate gland. In rats, OIF/NGF was detected in testis interstitial cells and in the lumen of the coagulating gland (anterior prostate). Ovulation-inducing factor/NGF secretion by the ampullae and vesicular glands contributed to its presence in bull (cattle and bison) ejaculates. In elk and white tail deer, OIF/NGF was detected in the ampullae and prostate glands, respectively. To gain an understanding of the abundance of OIF/NGF in ejaculates and changes in its concentration within and among males, OIF/NGF levels in semen were quantified using the radioimmunoassay. The assay developed exhibited parallel displacement curves among recombinant NGF, OIF/NGF purified from llama seminal plasma, llama and bull (cattle) seminal plasma. Ovulation-inducing factor/NGF comprised a greater percentage of the total protein found in camelid ejaculates than in cattle. Ovulation-inducing factor/NGF concentration correlated positively with sperm concentration and negatively with pH and semen volume, while total abundance of OIF/NGF was related to total prostate area and OIF/NGF concentration. Although a correlation was found between sperm concentration, neither OIF/NGF concentration nor total abundance was associated with higher ovulation, pregnancy or live birth rates. A clear association of the quantity of OIF/NGF in the male ejaculate at breeding and CL form and function was not evident. The measurement of CL vasculature by power Doppler ultrasonography, however, was able to determine nonpregnancy in alpacas earlier than the assessment of changes in CL diameter. In summary, my results did not support the hypothesis that the measurement of OIF/NGF concentration or total abundance in alpaca semen can be used to predict fertility in male alpacas.

The effect of gamete storage on sperm quality has received considerable attention in recent years. Previous studies have shown that spermatozoa stored in vivo or in vitro for a long time can lost their motility and fertilization capacity. Moreover, it have been concluded that male fertilization potential is highly dependent, not only on spermatozoa motility parameters, but also on organic components including protein composition of seminal plasma. On the other hand, spermatozoa motility and protein profiles of seminal plasma are highly dependent on conditions of storage. Therefore, additional data about effects of in vivo and in vitro storage on quality sperm parameters and protein composition of seminal plasma are essential for development of fish artificial reproduction methods. In the current work the influence of in vitro and in vivo storage on parameters of sperm motility, DNA integrity, antioxidant defensive system and seminal plasma protein composition were studied. As a conclusion, the results of this study provide new data on sperm quality and quantity parameters of chondrostean and teleost fish species with respect to in vivo and in vitro storage capacities, which should be beneficial for the development of aquaculture of these species. The data confirmed that protein patterns in seminal plasma varied during in vivo storage, depending on time of sperm collection. Furthermore, the altered proteins are probably involved in enzymatic pathways that regulate spermatozoa movement. In practice, the results presented in this thesis should help to improve management and optimize the development of protocols for artificial reproduction.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-04-26T12:49:23Z No. of bitstreams: 1 Maiana Silva Chaves.pdf: 890339 bytes, checksum: 482b52d7baa7cd8512faba3b39aaca01 (MD5) / Made available in DSpace on 2017-04-26T12:49:23Z (GMT). No. of bitstreams: 1 Maiana Silva Chaves.pdf: 890339 bytes, checksum: 482b52d7baa7cd8512faba3b39aaca01 (MD5) Previous issue date: 2015-05-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of semen from 5/8 Girolando bulls is a way to turn aviable genetic material from a specialized breed in the harshest environments. In this sense, the objective of this study was to analyze in vitro the semen from this breed, concomitant to the comparison of semen changes the same sample subjected to cooling (24 and 48 hours) and freezing, the seminal analysis of bulls exposed to the same conditions and checking correlations between them and mainly obtain standard values for l in vitro seminal assays. Analyzes of total motility, progressive motility, curvilinear speed and beat cross frequency showed statistical difference between the cooling and freezing treatments, with the bull factor influencing the results of plasma membrane integrity, acrosome membrane potential and chromatin condensation. Among the variables, the integrity of the plasma membrane was the variable that presented the highest correlation with the other studied. The in vitro seminal analyzes of 5/8 Girolando bulls subjected to low temperatures will contribute to the knowledge of standard values for the analyzed variables. / A utilização do sêmen de touros 5/8 Girolando é uma maneira de disponibilizar material genético de uma raça especializada em ambientes mais hostis. Nesse sentido, o objetivo desse trabalho foi analisar in vitro o sêmen de reprodutores dessa raça, concomitante a comparação das alterações do sêmen da mesma partida submetido a refrigeração (24 e 48 horas) e congelação, às análises seminais de touros expostos às mesmas condições verificando correlações e principalmente, obtenção de valores padrões de análises seminais in vitro. Análises de motilidade total, motilidade progressiva, velocidade curvilínea e batimento flagelar cruzado mostraram diferença estatística entre os tratamentos de refrigeração e congelação, com o fator touro influenciando nos resultados de integridade de membrana plasmática, potencial de membrana acrossomal e condensação da cromatina. Dentre as variáveis, a integridade da membrana plasmática foi a variável de maior correlação com as demais estudadas. As análises seminais in vitro de touros 5/8 Girolando submetidos a baixas temperaturas contribuirá para o conhecimento dos valores padrões para as variáveis analisadas.

O presente estudo visou investigar o efeito da refrigeração do sêmen da jaguatirica sobre o Índice de Motilidade Espermática [IME=(%M+MPx5)/2; %M = proporção de espermatozóides móveis; MP = motilidade progressiva], integridade acrossomal (IA) e capacitação espermática; assim como avaliar a eficácia da técnica FITC-PNA/IP na avaliação simultânea da viabilidade espermática (VE) e IA. Sete jaguatiricas foram eletroejaculadas, sendo utilizados apenas ejaculados (n=16) apresentando %M>=60% e MP>=3. Avaliou-se a IA por meio da Coloração Simples. Os ejaculados foram diluídos 1:1 na Variante do Diluente de PLatz e submetidos aos Protocolos de Transporte: Temperatura Ambiente e Refrigeração, - 0,23ºC/min, (Experimento 1); ou apenas Temperatura Ambiente (Experimentos 2 e 3). Após 2h, as alíquotas foram reaquecidas, reavaliando-se os parâmetros observados antes do transporte. Os espermatozóides foram lavados por centrifugação em meio F10 de Ham, ressuspensos nesse meio e processados conforme o experimento: (1) após pré-incubação (38ºC; 5%CO2) durante 0, 1, 2 e 4 horas, foram retiradas alíquotas a cada intervalo para serem incubadas (30 min) na ausência e na presença do cálcio ionóforo A23187 (Ca2+Ion) (1mM), avaliando-se IA e IME; (2) após pré-incubação por 0, 1 e 2h, foram incubadas alíquotas na ausência e presença de 1 e 2mM de Ca2+Ion, avaliado-se IA e IME; (3) pré-incubados por 9h, sendo retiradas alíquotas a cada hora, para as avaliações da IA e VE, (a) separadamente através da Coloração Simples e do IME, ou (b) simultaneamente através da técnica FITC-PNA/IP. A refrigeração causou declínio (p<0,02) da IA (71,0%) e IME (67,1), em comparação aos valores observados antes do transporte (88,5%; 85,4), enquanto a manutenção das amostras à temperatura ambiente não afetou (p>0,1) essas variáveis (84,8%; 76,4). Dentre as amostras refrigeradas, aquelas expostas ao Ca2+Ion sofreram redução (p<0,01) na IA (52,4%) frente ao controle (55,56%). Já nas amostras transportadas à temperatura ambiente, não foi observada diferença (p>0,1) entre os grupos com e sem ionóforo (64,41% vs. 63,87%). Quando analisados os tempos separadamente, o único tratamento em que houve efeito (p<0,05) do Ca2+Ion sobre a IA foi aquele refrigerado e pré-incubado por 2h. Foi verificada redução (p<0,05) nos valores de IME e IA devida à simples incubação, mesmo na ausência do Ca2+Ion. A concentração de 2µM dessa substância foi mais efetiva na indução da reação acrossômica que 1µM. Apesar dos fluorocromos FITC-PNA/IP terem se ligado aos espermatozóides, nas regiões esperadas, a proporção de células marcadas variou aleatoriamente durante pré-incubação, sem correlação (p>0,1) com IME. A IA avaliada pela Coloração Simples apresentou correlação positiva (r=0,77; p<0,0001) com IME, decrescendo (p<0,0001) durante pré-incubação. A refrigeração mostrou-se desvantajosa frente à manutenção do sêmen à temperatura ambiente, pois foi deletéria à função e às membranas dos espermatozóides. A refrigeração tornou-os capazes de responder ao estímulo do Ca2+Ion, característica observada nos espermatozóides capacitados. O ensaio de reação acrossômica induzida pelo Ca2+Ion deve ser aperfeiçoado para permitir avaliação acurada da capacitação espermática na jaguatirica. A Coloração Simples associada à avaliação do IME foi mais eficiente e menos laboriosa, frente á técnica FITC-PNA/IP, na avaliação da IA e VE. / This study aimed to investigate the effect of ocelot semen refrigeration on Sperm Motility Index [SMI=(%M+PMx5)/2; %M = proportion of motile spermatozoa ; PM = Progressive Motility], acrossomal integrity (AI) and sperm capacitation. Another objective was to evaluate the FITC-PNA/IP technique efficacy on evaluating simultaneously sperm viability (SV) and AI. Five ocelots, were electroejaculated, the semen was evaluated and only ejaculates (n=16) presenting %M>=60% and PM>=3 were used. Sperm AI was evaluated using Fast Green / Rose Bengal staining (FGRB). The ejaculates were diluted 1:1 in Platz Diluent Variant and subjected to the transportation protocols: Room Temperature and Cooling, -0.23ºC/min, (experiment 1); or only Room Temperature (experiments 2 and 3). After 2 hours, the aliquots were rewarmed and samples were taken to re-evaluate the parameters observed before the transport. The spermatozoa were washed in Ham’s F10 medium, ressuspended in fresh medium and processed differently, according the experiment: (1) after pre-incubation (38ºC; 5%CO2) during 0, 1, 2 and 4 hours, samples were taken at each time point to be incubated in the absence and presence of 1mM calcium ionophore A23187 (Ca2+Ion), SMI and AI were evaluated; (2) after pre-incubation during 0, 1 and 2h, aliquots were incubated in the absence and presence of 1 and 2 mM Ca2+Ion; SMI and AI were evaluated; (3) after pre-incubation during 9h, aliquots were taken every hour to compare the evaluation of SV and AI (a) separately by the FGRB staining and SMI or (b) simultaneously by the FITC-PNA / IP technique. Cooling caused decline (p<0.02) on AI (71.0%) and SMI (67.1), when compared to values observed before transportation (88.5%; 85.4). Maintenance at room temperature didn’t affect (p>0.1) these variables (84.8%; 76.4). Among cooled samples, spermatozoa exposed to Ca2+Ion showed smaller (P<0.01) AI value (52.4%) compared to the group incubated without that substance (55.56%). For samples transported at room temperature, it wasn’t observed difference (P>0.05) between the groups with and without ionophore (64.41% vs. 63.87%). When time intervals were analysed separately, the only treatment in which there was effect (p<0,05) of Ca2+Ion on AI was the group refrigerated and pre-incubated for 2h. There was a reduction (p<0,05) on SMI and AI due simply to incubation, even in the absence of Ca2+Ion. The 2µM concentration of this substance was more effective to induce acrosome reaction than 1µM. FITC-PNA and IP fluorocromes bound spermatozoa at the expected sites. However, proportion of marked cells varied randomly during pre-incubation, and didn’t correlate (p>0,1) with SMI. IA evaluated by FGRB staining showed positive correlation (r=0,77; p<0,0001) with SMI, decreasing (p<0,0001) during incubation. Cooling was disadvantageous compared to maintaining semen at room temperature, since it was deleterious to spermatozoa membranes and function, and made those cells capable to answer the Ca2+Ion challenge, a characteristic observed in capacitated spermatozoa. Ca2+Ion induced acrosome reaction assay must be improved to allow accurate evaluation of sperm capacitation on ocelots. FGRB staining associated to SMI evaluation was more efficient and easier to perform, than FITC-PNA/IP technique, for AI and SV investigation.

Made available in DSpace on 2016-01-26T18:55:34Z (GMT). No. of bitstreams: 1 Aline Silva.pdf: 343244 bytes, checksum: 747b2ff671732dd29f9e774a854fb28c (MD5) Previous issue date: 2012-09-20 / The Bulls have a great influence on the genetic composition of livestock so it is necessary to know the evidence of modern evaluation and apply them, thus obtaining, breeding for reproductive traits through the use of semen quality. The spermiogram conjunction with techniques such as scrotal thermogram and electrophoresis, to detect possible proteins in seminal plasma markers of fertility may contribute significantly to the selection of superior breeding. This study will use 10 Nelore bulls aged 30 months raised on a farm in the municipality of Presidente Prudente-SP, which will be evaluated during 6 months of the year (spring and summer), will be held 12 semen samples with an interval of 15 days. Be done spermiogram electrophoresis on polyacrylamide gel (SDS-PAGE) and the measured surface temperature for half of the scrotum infrared thermometer for obtaining the temperature gradient scrotal animals. The environment will be monitored daily for the minimum and maximum temperatures and relative humidity. The goal from study to study the influence of climatic factors on the surface temperature of the scrotum, spermiogram and protein profile (SDS-PAGE) of seminal plasma in Nellore bulls in the spring and summer seasons. / Os touros têm uma grande influência sobre a composição genética dos rebanhos por isso é necessário conhecer as provas modernas de avaliação e aplicá-las, obtendo, assim, melhoramento genético para as características reprodutivas, por meio do uso de sêmen com qualidade. O espermiograma aliado às técnicas como termograma escrotal e a eletroforese, para detecção de possíveis proteínas do plasma seminal marcadoras de fertilidade poderão contribuir significativamente na escolha de reprodutores superiores. Neste estudo foram utilizados 10 touros da raça Nelore com idade de 24 meses criados em uma propriedade rural no município de Presidente Prudente SP, que foram avaliados durante os meses de agosto a novembro, quando foram realizadas 12 colheitas de sêmen com intervalo de 15 dias. Foram realizados o espermiograma, a eletroforese do plasma seminal em gel de poliacrilamida (SDS-PAGE), aferida a temperatura da superfície do escroto, por meio de termometria de infravermelho para a obtenção do gradiente de temperatura escrotal destes animais e dosagem sérica de testosterona e cortisol O ambiente foi monitorado diariamente, para as temperaturas mínima e máxima, umidade relativa do ar e índices pluviométricos. O objetivo desde estudo foi estudar a influência dos fatores climáticos na temperatura da superfície do escroto, espermiograma e perfil protéico (SDS-PAGE) do plasma seminal em touros Nelore, nas estações primavera e verão.

Made available in DSpace on 2016-07-18T17:53:10Z (GMT). No. of bitstreams: 1 Aline Silva.pdf: 343244 bytes, checksum: 747b2ff671732dd29f9e774a854fb28c (MD5) Previous issue date: 2012-09-20 / The Bulls have a great influence on the genetic composition of livestock so it is necessary to know the evidence of modern evaluation and apply them, thus obtaining, breeding for reproductive traits through the use of semen quality. The spermiogram conjunction with techniques such as scrotal thermogram and electrophoresis, to detect possible proteins in seminal plasma markers of fertility may contribute significantly to the selection of superior breeding. This study will use 10 Nelore bulls aged 30 months raised on a farm in the municipality of Presidente Prudente-SP, which will be evaluated during 6 months of the year (spring and summer), will be held 12 semen samples with an interval of 15 days. Be done spermiogram electrophoresis on polyacrylamide gel (SDS-PAGE) and the measured surface temperature for half of the scrotum infrared thermometer for obtaining the temperature gradient scrotal animals. The environment will be monitored daily for the minimum and maximum temperatures and relative humidity. The goal from study to study the influence of climatic factors on the surface temperature of the scrotum, spermiogram and protein profile (SDS-PAGE) of seminal plasma in Nellore bulls in the spring and summer seasons. / Os touros têm uma grande influência sobre a composição genética dos rebanhos por isso é necessário conhecer as provas modernas de avaliação e aplicá-las, obtendo, assim, melhoramento genético para as características reprodutivas, por meio do uso de sêmen com qualidade. O espermiograma aliado às técnicas como termograma escrotal e a eletroforese, para detecção de possíveis proteínas do plasma seminal marcadoras de fertilidade poderão contribuir significativamente na escolha de reprodutores superiores. Neste estudo foram utilizados 10 touros da raça Nelore com idade de 24 meses criados em uma propriedade rural no município de Presidente Prudente SP, que foram avaliados durante os meses de agosto a novembro, quando foram realizadas 12 colheitas de sêmen com intervalo de 15 dias. Foram realizados o espermiograma, a eletroforese do plasma seminal em gel de poliacrilamida (SDS-PAGE), aferida a temperatura da superfície do escroto, por meio de termometria de infravermelho para a obtenção do gradiente de temperatura escrotal destes animais e dosagem sérica de testosterona e cortisol O ambiente foi monitorado diariamente, para as temperaturas mínima e máxima, umidade relativa do ar e índices pluviométricos. O objetivo desde estudo foi estudar a influência dos fatores climáticos na temperatura da superfície do escroto, espermiograma e perfil protéico (SDS-PAGE) do plasma seminal em touros Nelore, nas estações primavera e verão.

Seminal fluid proteins (SFPs) have been shown to affect the physiology,behaviour and immune responses of mated females in some species. Thisopen window for manipulation of female’s fitness allows the possibility forcomplex evolutionary dynamics between the SFPs and proteins of femalesthat would counter the effects of the former, the female reproductive proteins (FRPs). Also, the bean beetles of the Bruchinae subfamily are pests to pre-ferred species of plant hosts. The hosts have a great variety of secondary defensive metabolites between them and to detoxify those compounds, each beetle species is expected to have a well adapted arsenal of digestive proteinsfor a specific host. I carried out a comparative study with four species of bean beetles with the aim to identify patterns of selection in the proteins mentioned. Expression data for one of those species, Callosobruchus maculatus, has allowed to identify its SFPs, FRPs and digestive proteins and with orthology inference I identified their orthologues in the other three species. Then I estimated theratio of non-synonymous to synonymous substitution rates (ω) for each protein by using codeML of the PAML package and used them as a proxy for estimating selection. FRPs had about the same ω values as conserved genes found across the Arthropod phylum, while the SFPs and digestive proteins hadhigher ω values, indicating more relaxed purifying selection. I also performed tests of positive selection and have identified 92 digestive proteins, 9 FRPs and 26 SFPs as potential targets for future functional work. Finally, I examined the scenario of co-evolution between SFPs and FRPs because of direct interaction. By correlating branch-specific ω values for each possible pairs of proteins I found that SFPs are associated on average more with FRPs than with digestive or conserved genes, as expected. The same was true for the FRPs. Also I examined the possibility of factors contributing to the association such as expression levels, sex-biased expression and protein function. Using linear regression models I found that expression levels and proteinfunction do predict in some degree the ω estimates and could thus also affectthe correlations examined. High gene expression levels reduce the overall ωvalues of genes, also known as E-R anticorrelation. Sex-biased expression does not affect the overall ω values, but does affect the intensity of the E-R anticorrelation, with it being less prominent in male-biased genes and more prominent towards female-biased genes.

MSCAGR (Animal Science) / Department of Animal Science / The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on goat buck semen. Six ejaculates were collected from six indigenous bucks by means of electro-ejaculator method, and semen was pooled, and replicated 10 times. Raw semen were randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. All six groups were analysed for spermatozoa motility rates using computer-aided sperm analysis (CASA). The spermatozoa viability for all groups were assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Both the Triladyl® and Bioxcell® washed semen groups were diluted (1:4) with Phosphate Buffered Saline (PBS) then centrifuged at 1500 x g for ten min and seminal plasma was aspirated using 1 mL sterile plastic pipette. Semen samples were diluted (1:4) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5 ºC for 2 hours. Following equilibration, semen parameters were analysed. Thereafter, the semen samples were loaded into straws and placed 5 cm above a liquid nitrogen vapour for 10 min, and then stored at -196 ºC until use. Following one month of storage, frozen semen straws per treatment group were thawed at 37 ºC for 30 seconds, then semen parameters were analysed again. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. Total Spermatozoa motility rate of Bioxcell® (92.5±4.6), (68.2±13.5) and Triladyl® (94.9±5.5), (63.1±15.1) were significantly reduced (P < 0.05) following equilibration and freeze-thawing process, respectively on washed semen groups. Live and normal spermatozoa percentages were drastically reduced in Bioxcell® (5.2±4.9) and Triladyl® (6.9±8.6) washed semen groups, following freeze-thawing. There was a significantly lower number of spermatozoa with high mitochondrial membrane potential in non-washed semen extended with Triladyl® (68.7±26.8) compared to non-washed semen extended with Bioxcell® (49.8±20.1) following the freeze-thawing process. In conclusion, the freezing-thawing process did reduce the indigenous buck semen parameters irrespective of removal or non-removal of seminal plasma. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of buck semen.

Die Brachyura umfassen ca. 7000 Arten. Die Variationsbreite ihrer morphologischen Merkmale spiegelt sich in den männlichen und weiblichen Reproduktionssystemen wider und machen sie zu einem herausfordernden Untersuchungsgegenstand. Obwohl sich zahlreiche Studien mit der Phylogenie der Brachyura befasst haben, fehlen eindeutige Ergebnisse. Manche Studien stützen sich weiterhin auf deren Teilung in die Podotremata, die Heterotremata und die Thoracotremata (die beiden letzteren bilden die Eubrachyura), die auf der Position der Gonoporen basiert. In dieser Arbeit wurden die männlichen Kopulations- und weiblichen Reproduktionssysteme von vier eubrachyuren Arten untersucht. Zur Analyse ihrer inneren Morphologie, wurden die Gonopoden in der ersten und zweiten Studie μCT-gescannt und 3D-rekonstruiert. Zusätzlich wurden in allen Studien rasterelektronenmikroskopische Untersuchungen angewendet, um Informationen über die Oberflächenstrukturen der Gonopoden zu erhalten. Alle Untersuchungen des weiblichen Systems wurden mit bewährten histologischen Methoden und Lichtmikroskopie durchgeführt. In der ersten und zweiten Studie wurde diese detaillierte Strukturanalyse durch 3D-Rekonstruktion ergänzt. Die Ergebnisse dieser Studien wurden in Zusammenhang mit der vorhandenen Literatur interpretiert, um Merkmale des männlichen und weiblichen Reproduktionssystems zu definieren und deren Potenzial für phylogenetische Untersuchungen zu diskutieren. Außerdem wird ein evolutives Scenario bezüglich der Transformation der hier vorgeschlagenen Merkmalszustände des weiblichen Reproduktionssystems diskutiert. Die Gonopoden sind wertvoll, um Artenzugehörigkeiten zu Brachyurengruppen zu identifizieren, sind aber für Untersuchungen großskaliger Brachyurenphylogenie ungeeignet. Einige der weiblichen Merkmale können ausschließlich heterotremen oder thoracotremen Weibchen zugeordnet werden. Die vorgeschlagenen Szenarien deuten darauf hin, dass einige dieser Charaktere mehrfach entstanden sind. / The Brachyura comprise approximately 7000 species. The variability of their morphological traits is reflected in the male copulatory and the female reproductive systems that make them a challenging object of investigation. Numerous studies addressed the brachyuran phylogeny but unambiguous results have yet to be presented. Some studies still rely on the division of Brachyura into the Podotremata, the Heterotremata and the Thoracotremata (the latter two forming the Eubrachyura) that is based on the position of the male and female gonopores. In this work, the male copulatory and female reproductive systems of four eubrachyuran species were investigated. In the first and the second study, the gonopods were µCT-scanned and 3D-reconstructed to analyse their internal morphology. Additionally, in all studies scanning electron microscopy was used in order to obtain information about the surface structures of the gonopods. All investigations of the female system were conducted using approved histological methods and light microscopy. In the first and second study, this detailed structural analysis was complemented by 3D-reconstruction. The results of these studies are evaluated in comparison with the existing literature in order to define characters of the male copulatory and female reproductive system and discuss their potential for phylogenetic investigations. Additionally, an evolutionary scenario of the transformations of the herein proposed character states of the female reproductive system is discussed. Without additional information from the female reproductive system, the gonopod morphology is valuable to identify species affiliations to certain groups but remains inconclusive for large-scale brachyuran phylogeny. Some of the female characters found in these studies can explicitly be assigned to heterotreme or thoracotreme females. The proposed scenarios suggest, that some, if not all of these characters probably have evolved multiple times.