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An integrated system combining a magnetically-driven micromotor and a synthetized protein-based hyaluronic acid (HA) microflake is presented for the in situ selection and transport of multiple motile sperm cells (ca. 50). The system appeals for targeted sperm delivery in the reproductive system to assist fertilization or to deliver drugs. The binding mechanism between the HA microflake and sperm relies on the interactions between HA and the corresponding sperm HA receptors. Once sperm are captured within the HA microflake, the assembly is trapped and transported by a magnetically-driven helical microcarrier. The trapping of the sperm-microflake occurs by a local vortex induced by the microcarrier during rotation-translation under a rotating magnetic field. After transport, the microflake is enzymatically hydrolyzed by local proteases, allowing sperm to escape and finally reach the target location. This cargo-delivery system represents a new concept to transport not only multiple motile sperm but also other actively moving biological cargoes.

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

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För att bedöma mäns spermiekvalitet och därmed pars chanser till en graviditet har standardparametrar tidigare använts som fertilitetsindikator, men har visat sig vara en mindre tillförlitlig metod. Därför har en ny metod som bygger på flödescytometri och kallas Sperm Chromatin Structure Assay (SCSA) utvecklats som fokuserar på spermiernas arvsmassa, och som då har visat sig vara en mycket mer tillförlitlig metod. Syftet med denna studie var att jämföra DNA Fragmentations Index (DFI) -värde mellan svenska och danska män och se om en signifikant skillnad finns. För att uppfylla detta späddes och färgades spermaproven in och analyserades med SCSA, som därefter gav hur stor andel av spermiernas DNA som är defekt, vilket presenterades som ett procentuellt DFI-värde. Resultatet visade en signifikant skillnad av medianvärdet mellan svenska och danska män med avseende på DFI, där svenska män hade högre medianvärde än de danska männen. Detta i kombination med tidigare forskning som gjordes mellan svenska och danska män och som visade att svenska män hade bättre kvalitet än danska män med avseende på standardparametrarna, stödjer tidigare forskning som visar på att det ej finns ett samband mellan standardparametrar och DFI. Dock hade ytterligare forskning av skillnaderna mellan svenska och danska män behövts, för att validera fynden i denna studie. / To assess men's sperm quality and thus couples' chances of pregnancy, standard parameters have previously been used as a fertility indicator, but have proven to be a less reliable method. Therefore, a new method based on flow cytometry called Sperm Chromatin Structure Assay (SCSA) has been developed which focuses on the sperm's genetic mass, and which has been shown to be a much more reliable method. The purpose of this study was to compare the DNA Fragmentation Index (DFI) value between Swedish and Danish men and see if a significant difference exists. To fulfill this, the sperm samples were diluted and stained and analyzed with SCSA, which then gave the ratio of the sperm's DNA that is defective, which was presented as a percentage DFI value. The result showed a significant difference in the median value between Swedish and Danish men with regard to DFI, where Swedish men had a higher median value than the Danish men. This, in combination with previous research that has been done between Swedish and Danish men which showed that Swedish men had better quality than Danish men in regards to the standard parameters, supports previous research that shows that there is no connection between standard parameters and DFI. However, further research into the differences between Swedish and Danish men is needed to validate the findings in this study.

In der Schweinezucht werden konventionell 1-3 x109 Spermien als Frischsamen in einem Volumen von 80-100 ml intrauterin versamt. Im Vergleich zur Rinderbesamung, bei der mitunter 2 x106 Spermien zu Trächtigkeiten führen, sind Ejakulate von Ebern ineffizient. Die einzige Möglichkeit geringe Spermienzahlen erfolgreich zu versamen besteht darin die Spermienablage nahe dem Ort der Befruchtung im distalen Isthmus des Eileiters durchzuführen. Die spezies-spezifische Bindung von Säugerspermien an diverse Oberflächenzellen des weiblichen Traktes, die der Kapazitation und Hyperaktivierung vorangehen, setzt die Erkennung von Kohlenhydraten durch lektin-ähnliche Proteine der Spermienplasmamembran voraus Daher wurde vermutet, dass eine mutmaßliche Bindung porziner Spermien an das Uterusepithel ebenfalls Protein-Kohlenhydrat vermittelt ist. Ziel der vorliegenden Arbeit war die Etablierung eines Zellkulturmodells aus primären Uterusepithelien der Sau (Sus scrofa), um mögliche Gründe für die hohen Spermienzahlen in der Schweinbesamung zu untersuchen und zu identifizieren. Porzine uterine Epithelien wurden mittels Verdau mit Trypsin/EDTA (1x) gewonnen. Zellen wurden auf Collagen (rat tail, type I) auf Kollagen-beschichteten Deckgläschen aus Glas in 6-Well Kulturschalen ausgesät. Die Anheftung der Zellen an die Kollagen-Matrix wurde nach 12-36 Stunden beobachtet. Erste Kolonien bildeten sich nach fünf bis sieben Tagen und Konfluenz wurde nach zehn bis fünfzehn Tagen erreicht. Der Nachweis der Zellart erfolgte mittels Immunfluoreszenz-Färbung mit einem Epithelien-spezifischen Antikörper (anti-Cytokeratin-19). Eberspermien banden innerhalb weniger Minuten an die UEC und verblieben währenddessen motil. Eine verringerte Spermienbindung wurde sowohl auf Aortenendothelien als auch fötalen Fibroblasten des Schweins beobachtet. Dies weist auf das Vorhandensein spermienspezifischer Liganden auf den UEC hin. Spermien, wie auch UEC wiesen hohe Bindungsintensitäten für Lektine die an Glc-NAc, (WGA) sowie Gal-NAc (sWGA) binden. Die Sättigung von Sialinsäure führte zu einer stark verminderten Spermienbindung auf den UEC. Bei einer Sättigung von Glc-NAC wurde kein Unterschied beobachtet. Dies kennzeichnet Sialinsäure als den vermutlich wichtigsten Kohlenhydratrest in der Spermien-Epithelzellbindung.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility. / AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid, beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer rondom die ware potensiaal van hierdie parameter weens die gebrek aan standardisering van verskillende kleuringstegnieke wat gebruik word vir die evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue® (SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp. Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate, word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer (SCA®). Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het. Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel. Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge, word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer. Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie sperme met goeie beweeglikheid.

The European earwig exhibits a remarkable male-dimorphism in forceps morphology that is associated with alternative reproductive tactics under the control of a conditional evolutionarily stable strategy. Populations on the small, rocky islands of the Farnes off the Northumberland coast are known to sustain populations with dramatically higher morph ratios than observed on the UK mainland. A survey conducted of island and mainland sites around the UK showed that the dimorphic populations of the Farnes are similar to other islands and that mainland populations generally exhibit low morph ratios. Additionally, a correlation between morph ratio and population density was found lending support to the hypothesis that the ESS thresholds that define the morph ratios have diverged through local adaptation. A set of seven microsatellite markers are presented that were developed from a Farne island population of F. auricularia with one additional, previously published locus. These eight markers exhibit genetic variability within and between populations and as such can potentially be applied at a range of scales, from broad-scale phylogeography to within population parentage studies. A phylogeographic study of the UK populations using these markers suggests a single postglacial colonisation from mainland Europe and give further support to the local adaptation hypothesis of ESS threshold evolution. A study of ejaculate size in F. auricularia showed that the males transfer free sperm at a steady rate and that the morphs do not differ in the number of sperm per ejaculate. Measurements of change in body-mass were found to be ineffective measures of ejaculate size, but that macrolabic males lost more weight during copula than brachylabic males. This may be the result of differential investment in accessory ejaculate components between the morphs, as a result of the differing risk of sperm competition.

Rodents exhibit diverse biology, mating systems and diverse sperm morphology. Different levels of sperm competition have therefore been found in this group. Generally, it is accepted that sperm competition in animals causes the prolongation of sperm tails. However, what other sperm morphological adaptations in rodents are affected by sperm competition? Species-specific sexual selection likely optimizes sperm morphology. Sperm physiology, morphology and competition may be influenced, ultimately, by environmental factors. This Ph.D. thesis focuses on sperm competition in murine rodents. The thesis contains three research articles (including one as a submitted manuscript). In addition, another published article, partialy related to the topic of this thesis, is represented in the Appendix. The sperm morphology in rodents plays an important role in sperm competition. Longer tails and apical hooks are usually found in the species with relatively larger testes and with higher percentage of multiple paternity (i.e. rate of promiscuity), that is in species with the higher risk of sperm competition. The goal of the first study was to investigate the relationships between relative testis weight and sperm traits (apical hook and tail lengths and variance in this traits). The apical hook length was positively...

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