638 research outputs found
Much alike, yet different: Digital innovation labs in family/non-family business
No full textSchleef M, Steinlechner J, Stummer C. Much alike, yet different: Digital innovation labs in family/non-family business. In: Bitran I, Conn S, Gernreich C, Huizingh E, Torkkeli M, Yang J, eds. Proceedings of the XXXII ISPIM Innovation Conference. LUT Scientific and Expertise Publications; 2021: 1-13
Investigation on the chemical and thermal behavior of recycling agglomerates from EAF by-products
No full textIn addition to the blast furnace converter route, electric steel production in the electric arc furnace (EAF) is one of the two main production routes for crude steel. In 2019, the global share of crude steel produced via the electric steel route was 28%, which in numbers is 517 million metric tons of crude steel. The production and processing of steel leads to the output of a variety of by-products, such as dusts, fines, sludges and scales. At the moment, 10–67% of these by-products are landfilled and not recycled. These by-products contain metal oxides and minerals including iron oxide, zinc oxide, magnesia or alumina. Apart from the wasted valuable materials, the restriction of landfill space and stricter environmental laws are additional motivations to avoid landfill. The aim of the Fines2EAF project, funded by the European Research Fund for Coal and Steel, is to develop a low-cost and flexible solution for the recycling of fines, dusts, slags and scales from electric steel production. During this project, an easy, on-site solution for the agglomeration of fine by-products from steel production has to be developed from lab scale to pilot production for industrial tests in steel plants. The solution is based on the stamp press as the central element of the agglomeration process. The stamp press provides the benefit of being easily adapted to different raw materials and different pressing parameters, such as pressing-force and-speed, or mold geometry. Further benefits are that the stamp press process requires less binding material than the pelletizing process, and that no drying process is required as is the case with the pelletizing process. Before advancing the agglomeration of by-products via stamp press to an industrial scale, different material recipes are produced in lab-scale experiments and the finished agglomerates are tested for their use as secondary raw materials in the EAF. Therefore, the tests focus on the chemical and thermal behavior of the agglomerates. Chemical behavior, volatilization and reduction behavior of the agglomerates were investigated by differential thermogravimetric analysis combined with mass spectroscopy (TGA-MS). In addition, two melts with different agglomerates are carried out in a technical-scale electric arc furnace to increase the sample size
Mapping the optical absorption of a substrate-transferred crystalline AlGaAs coating at 1.5 µm
No full textThe sensitivity of 2nd and 3rd generations of interferometric gravitational wave detectors will be limited by thermal noise of the test-mass mirrors and highly reflective coatings. Recently developed crystalline coatings show a promising thermal noise reduction compared to presently used amorphous coatings. However, stringent requirements apply to the optical properties of the coatings as well. We have mapped the optical absorption of a crystalline AlGaAs coating which is optimized for high reflectivity for a wavelength of 1064nm. The absorption was measured at 1550nm where the coating stack transmits approximately 70% of the laser light. The measured absorption was lower than (30.2 +/- 11.1)ppm which is equivalent to (3.6 +/- 1.3)ppm for a coating stack that is highly reflective at 1530nm. While this is a very promising low absorption result for alternative low--loss coating materials, further work will be necessary to reach the requirements of <1ppm for future gravitational wave detectors.
Jessica Steinlechner, Iain W Martin, Angus Bell, Garrett Cole, Jim Hough, Steven Penn, Sheila Rowan, Sebastian Steinlechne
Somatostatin agonist pasireotide inhibits exercise stimulated growth in the male Siberian hamster (Phodopus sungorus)
No full textR.Dumbell was supported by a University of Aberdeen PhD studentship and a research visit grant awarded by the British Society of Neuroendocrinology. Further support was provided by the Scottish Government Rural and Environment Science and Analytical Services Division (Barrett and the German Research Foundation (DFG; STE 331/8-1; Steinlechner lab). We are grateful for technical assistance from Dana Wilson at RINH and Siegried Hiliken at UVMH, and thank Dr Claus-Dieter Mayer of Biomathematics & Statistics Scotland for valuable advice on statistical analysis.Peer reviewe
Characterization and process development for the selective removal of Sn, Sb, and As from anode slime obtained from electrolytic copper refining
No full textThe aim of this work was to develop a process for the removal of Sn, Sb and As from anode slime out of copper refinery to disburden a subsequent pyrometallurgical processing for precious metals refinement. For this reason, a detailed literature survey was conducted, followed by a characterization to find the present compounds/alloys and their morphology. A newly developed process concept for the separate extraction of the afore mentioned three target metals was developed and verified by leaching experiments, combined with thermodynamic calculations on their behavior under varying conditions. In this context, the influence of leaching temperature, alkalinity of leaching solution, and solid-liquid ration were evaluated on the extraction yields of Sn, As, and Sb, as well as how to exploit these findings to obtain separate streams enriched in the respective metals
Developing a new process to agglomerate secondary raw material fines for recycling in the electric arc furnace - The fines2EAF project
No full textRecent years have seen a worldwide change in the environmental policy towards circular economy approaches. It is estimated that steel-making activities in Europe produce about 80 million tonnes annually of by-products and waste, equivalent to half of the European steel production, of which more than 10 million tonnes is waste for disposal. This waste of resources and land area is not sustainable and has to be decreased in the future. The Fines2EAF project aims to increase the value of steelmaking residues by internal recycling and (re)use in the form of agglomerates. The benefit of this strategy is threefold: improved utilization of residues, internal recovery of valuable materials and reduction of the amount of dumped materials. The approach followed is the development of an innovative process to produce cement-free agglomerates based on primary and secondary raw material fines, alternative binder systems and a hydraulic stamp press. In addition, a new pre-treatment process for fines based on microwave heating is investigated. The first results of the lab-scale investigation of the fines pre-treatment to reduce the amount of zinc, volatiles and alkalis are presented. Six materials from two steel plants have been tested in a laboratory microwave furnace. Also presented are first results of the agglomeration of fines using a laboratory press
Developing a new process to agglomerate secondary raw material fines for recycling in the electric arc furnace - the Fines2EAF project
No full textRecent years have seen a worldwide change in the environmental policy towards circular economy approaches. It is estimated that steel-making activities in Europe produce about 80 million tonnes annually of by-products and waste, equivalent to half of the European steel production, of which more than 10 million tonnes is waste for disposal. This waste of resources and land area is not sustainable and has to be decreased in the future. The Fines2EAF project aims to increase the value of steelmaking residues by internal recycling and (re)use in the form of agglomerates. The benefit of this strategy is threefold: improved utilization of residues, internal recovery of valuable materials and reduction of the amount of dumped materials. The approach followed is the development of an innovative process to produce cement -free agglomerates based on primary and secondary raw material fines, alternative binder systems and a hydraulic stamp press. In addition, a new pre-treatment process for fines based on microwave heating is investigated. The first results of the lab-scale investigation of the fines pre-treatment to reduce the amount of zinc, volatiles and alkalis are presented. Six materials from two steel plants have been tested in a laboratory microwave furnace. Also presented are first results of the agglomeration of fines using a laboratory press
Suitability of Self-Reducing and Slag-Forming Briquettes for Electric Arc Furnace Use Based on Laboratory Tests
No full textThe in-plant recycling routes of side streams produced in electric arc furnace (EAF) steelmaking remain underexplored. Briquetting is an attractive technique to enable their recycle. Briquettes introduced into EAF must possess certain mechanical and chemical properties. However, no standard is available to determine the suitability of briquettes used in the EAF process. Herein, eight side streams are characterized, and used to produce seven different briquettes to be used in EAF. Briquettes tested consist of four self-reducing briquettes and three slag-forming briquettes produced using different recipes. The briquettes are subjected to several mechanical and thermal tests which reflect their intended use in EAF. The mechanical tests include compression and drop tests, and the thermal tests include optical dilatometry, thermogravimetric analysis (TGA)–derivative thermogravimetry (DTG)–mass spectrometry (MS), and full-scale briquette reduction tests. Moreover, melting trials are performed to assess the melting behavior of selected briquettes and their interaction with slag. Suitability of briquettes characteristics is assessed based on values from the literature and against reference ferroalloys and lime stones used in one of the steel plants. Two briquettes are deemed suitable for EAF use, while three briquettes are deemed unsuitable, and two briquettes are considered of limited use
Characterization of the interaction of porcine spermatozoa with uterine epithelial cells and the coinciding gene expression with regard to modulation of a successful artificial insemination procedure
No full textDie künstliche Besamung des Schweines hat in den letzten 20 Jahren weltweit zunehmend an Bedeutung gewonnen. Durch Optimierung des Besamungsmanagements gelingt eine hohe Fertilitätsrate von nahezu 90%. Allerdings werden hierfür Besamungsportionen mit mindestens 1 x 109 Spermien benötigt, wodurch der effiziente Einsatz von Besamungsebern eingeschränkt wird. Dieser Umstand limitiert auch die Etablierung neuer biotechnischer Verfahren in der Schweinereproduktion, hervorzuheben ist die geschlechtsspezifische Sortierung von Spermien anhand der Geschlechtschromosomen mittels Durchflusszytometrie. Um die benötigte Spermienzahl pro Besamungsportion senken zu können, muss auf aufwendige Techniken wie tief intrauterine Besamung und laparoskopische Eileiterbesamung zurückgegriffen werden, da bei diesen Verfahren die durch die Uteruspassage bedingte Verluste vermieden werden können. Damit die künstliche Besamung mit einer reduzierten Spermienkonzentration innerhalb der konventionellen Besamungstechniken erfolgreich durchgeführt werden kann, müssen daher grundlegende uterine Mechanismen der Spermienselektion aufgezeigt werden.
Ziel dieser Arbeit war es, anhand eines besseren Verständnisses sowohl der uterinen Spermienretentionsmechanismen als auch der immunologischen Reaktionen im porcinen Uterus neue Möglichkeiten für die Optimierung des Besamungsmanagements beim Schwein aufzuzeigen. Demzufolge wurden die nachstehend genannten Hypothesen aufgestellt:
1. Die Retention von Spermien im Uterus wird durch die Bindung von Spermien an das uterine Epithelzellgewebe bedingt.
2. Die Spermien-Epithelzellbindung im Uterus wird durch Seminalplasma moduliert.
3. Die Bindung von Spermien an uterine Epithelzellen beeinflusst die Genexpression im Uterus und hat dadurch Auswirkungen auf spätere Ereignisse wie Implantation und Embryonalentwicklung.
In einem ersten in-vivo-Versuch wurden präovulatorischen Jungsauen eine von 5 verschiedene Inseminatvarianten intrauterin appliziert: PBS allein (PBS); Seminalplasma allein (SP); Nebenhodenschwanzsperma in PBS (NHS); Spermien in PBS (PBSSp); Spermien in Seminalplasma (SPSp). Das Inseminat hatte dabei jeweils ein Volumen von 80 ml und enthielt gegebenenfalls 3 x109 Spermien. Eine unbesamte Negativkontrolle (NC) vervollständigte die Untersuchungsgruppen. Zwei Stunden nach Behandlung wurden die Tiere geschlachtet und der Reproduktionstrakt entfernt. Auf die Spülung eines der beiden Uterushörner mit 20 ml einer PBS-Lösung folgte die Aufbereitung von Gewebeproben zur Herstellung histologischer Schnitte und einer anschließenden Präparation des uterinen Epithelzellgewebes zur Gewinnung von RNA für Genexpressionsstudien. Das zweite Uterushorn diente einer Gramfärbung zum Ausschluss symptomloser Infektionen. Die aus dem Uterus gewonnene Spülfüssigkeit wurde hinsichtlich der Quantität rückgespülter Spermien und Leukozyten untersucht. Die geringsten Mengen der in das Uteruslumen eingewanderter Leukozyten sowie die größte Anzahl an rückgespülten Spermien konnten in der Untersuchungsgruppen SPSp gezählt werden. Die RNA-Proben wurden mittels eines speziell gefertigten Mikroarray-Chips auf die Expression von 349 immunrelevanten Genen untersucht. Der Gruppenvergleich NC versus SPSp ergaben mit 15 die meisten der differentiell regulierten Gene. Die in dieser Gruppe regulierten Gene CD163 und PPAR-alpha wurden nachfolgend mittels immunhistologischen Untersuchungen näher untersucht. Der Nachweis von CD163-Protein gelang, jedoch wurden keine signifikanten Unterschiede hinsichtlich der detektierten Menge an CD163 im uterinen Epithelzellgewebe festgestellt. Für PPAR-alpha konnte keine Expression im uterinen Epithelzellgewebe ermittelt werden. Die Validierung der Mikroarray Ergebnisse mittels Real-time PCR zeigte weitgehende Übereinstimmungen.
In einem zweiten Besamungsversuch wurde die Zeitkinetik der uterinen Immunantwort untersucht. Nach Instillation von entweder PBS allein oder Spermien in Seminalplasma in präpuberale Jungsauen wurde der Reproduktionstrakt zu verschiedenen Zeitpunkten (15 min, 2h und 6h nach Besamung, sowie zum Zeitpunkt der Ovulation) chirurgisch entfernt. Die weiteren Untersuchungen wurden gemäß dem ersten In-vivo-Versuch durchgeführt. Wie im ersten Versuchsabschnitt fanden sich hierbei im Gruppenvergleich NC versus SPSp mit 17 die meisten differentiell regulierten Gene, allerdings zeitverschoben zu 6 Stunden nach der Besamung. Ausserdem zeigte es sich, dass zum Zeitpunkt der Ovulation jegliche immunologische Reaktion des Uterus abgeschlossen war.
Zusammenfassend lässt sich aus den Ergebnissen der hier vorliegenden Arbeit schließen:
Die Besamungsportionen, welche sowohl Spermien als auch Seminalplasma enthielten, also den Komponenten des Eberejakulates im Natursprung entsprachen, wiesen den geringsten Anteil ins Uteruslumen eingewanderter Leukozyten auf, bei gleichzeitig vergleichsweise hoher Rate rückgespülter Spermien. Darüber hinaus konnte in beiden Versuchsabschnitten gezeigt werden, dass es bei Kontakt des Uterusepithels mit Spermien und Seminalplasma in Kombination zu einer signifikanten Änderung im Genexpressionsmusters im Vergleich mit der Negativkontrolle kommt.
Dieses verdeutlicht und unterstreicht die These, dass die Kombination aus Spermien und Seminalplasma einen nicht unerheblichen Einfluss auf die Modulation der porcinen, uterinen Immunantwort hat. Es ist zu vermuten, dass die Einflussnahme auf die uterine Genexpression durch Seminalplasma-vermittelte Bindung der Spermien an das Uterusepithel erfolgt, auch wenn der eigentliche Beweis für diese Bindung noch aussteht. Diese Bindung könnte auch einen Teil der Verantwortung für die hohen uterinen Spermienverluste tragen.
Ein weiterer interessanter Befund dieser Studie ist, dass jegliche Änderungen in der Genexpression zum Zeitpunkt der Ovulation nicht mehr nachweisbar sind. Derzeit laufende in-vitro Untersuchungen haben zum Ziel die Spermien-Epithelzellbindung direkt nachzuweisen und den Bindungsmechanismus zu entschlüsseln.During the past two decades artificial insemination in pigs has become more a more important worldwide. Optimized insemination management has led to high fertilisation rates of up to 90 %. However, at least 1 x 109 spermatozoa per AI portion are needed to succeed. This limits the efficient use of boars and ejaculates. Furthermore the application of innovative biotechnological methods in pig reproduction such as sex differentiation of spermatozoa by flow-cytometry, are margined. In order to reduce the number of spermatozoa for successful insemination, complex techniques are needed such as deep intra-uterine and laparoscopic-oviductal insemination, as bypassing the uterus avoids sperm selection. To enable conventional artificial insemination but with reduced numbers of spermatozoa, a better understanding of essential uterine mechanisms of sperm selection is needed.
The aim of this thesis is to identify, by an improved understanding of sperm retention mechanisms as well as the immunological reaction of the porcine uterus, new methods to optimize artificial insemination management in the pig. Thus the following hypotheses were aligned:
1. The retention of porcine spermatozoa in the uterus is mediated by binding of the spermatozoa to the uterine epithelial cell tissue.
2. The interactions between spermatozoa and the epithelium are mediated by seminal plasma components.
3. The binding of spermatozoa to uterine epithelial cells influences the gene expression in the uterus and has effects on later events such as nidation and embryonic development.
In the first of two in vivo trials, pre-ovulatory gilts were artificially inseminated intra-uterine with one of five different inseminate treatments: PBS only (PBS); seminal plasma only (SP); epididymal sperm in PBS (NHS); spermatozoa in PBS (PBSSp); spermatozoa in seminal plasma (SPSp). Volumes of 80 ml were used containing 3 x 109 spermatozoa respectively. A not inseminated negative control (NC) completed the treatment groups. Two hours after treatment animals were slaughtered and the reproductive tract was extracted. One uterine horn was flushed with 20 ml PBS and then used to gain samples for histological slices as well as salvaging uterine epithelium tissue for RNA extraction for later gene expression studies. The other horn served for a gram stain to detect possible latent infections. In the gathered fluid from the uterine flush the number of spermatozoa and leukocytes was quantified. The lowest number of luminal leukocytes and coinciding with it, the highest number of retrieved spermatozoa were found in the treatment group SPSp. The RNA samples were analysed for the expression of 349 immunorelevant genes with a custom made micro-array chip. The groups NC versus SPSp showed 15 differentially regulated genes, which was the highest number in all groups compared. The expression of two genes, CD136 and PPAR-alpha, were additionally analysed on protein level using immunohistological methods. CD136 could be detected, but no significant change in the amount of CD163 protein was detected in the uterine tissue. PPAR-alpha could not be shown to be expressed in the epithelial tissue. The micro-array results were validated by quantitative PCR.
In the second insemination trial the time associated kinetics of the uterine immune response were examined. After instillation of either PBS only or spermatozoa in seminal plasma to pre-puberal gilts, the reproductive tract was extracted surgically at different times post AI (15min, 2h, and 6h post “insemination” as well as time of ovulation). The succeeding investigations were undertaken in the same ways as for the first trial. Repeating the results from the first trial, the NC and the treatment group SPSp showed 17 differentially regulated genes, but shifted in time, namely after 6 hours post insemination. Also, it was observed that at time of ovulation all immunological responses in the uterus were completed.
From the findings in these studies it can be summarized that:
Insemination portions that contained spermatozoa as well as seminal plasma, which therefore correspond to compounds in boar ejaculates in natural service, showed the lowest number of leukocytes migrated into the uterine lumen and at the same time high numbers of retained spermatozoa. Moreover, in both trials it could be shown that contact of the uterine epithelium with spermatozoa in presence of seminal plasma lead to a significant regulation of the gene expression of the endometrium compared to the negative control. This emphasises the hypothesis that the combination of spermatozoa and seminal plasma has an significant effect on the modulation of the porcine maternal immune response after insemination. We propose that this effect on the uterine gene expression is caused by seminal plasma mediated binding of spermatozoa to the uterine epithelium even though this binding is still to be substantiated. This binding may also account for the high sperm losses. A further interesting finding of this study is that by the time of ovulation no changes in gene expression could be detected comparing inseminated and not inseminated animals. Currently on going experiments are aimed at verifying and identifying this sperm-epithelial cell binding
Experimental analysis of epigenetic modulation in prepuberal and adult bovine oocytes
No full textOozyten von präpuberalen Rindern weisen eine deutlich geringere Blastozystenrate im Vergleich zu adulten Rindern auf. Ziel dieser Arbeit war es, weitere Erkenntnisse zur Entwicklungskompetenz präpuberaler und adulter boviner Oozyten durch Untersuchung der mRNA-Expression von entwicklungsrelevanten nichtgeprägten Genen (GDF9, SLC2A1, PRDX1 und ZAR1) sowie deren genspezifischer Methylierung zu erlangen. Die Analyse der Genexpression erfolgte mittels Real-time PCR. Für die genspezifische Methylierungsanalyse wurde die "Limiting Dilution Methode" eingesetzt. Zusätzlich wurde der Methylierungsgrad von zwei Satellitensequenzen (Repeatsequenzen) untersucht, um einen Einblick in das globale Methylierungsmuster zu bekommen. Für die Untersuchungen wurden bovine Oozyten aus vier präpuberalen und zwei adulten Tiergruppen gewonnen. Diese wurden nach morphologischer Beurteilung in die Klasse 1-2 (mit gutem Entwicklungspotential), und Klasse 3 (mit eingeschränktem Entwicklungspotential) eingeteilt. Für die einzelnen Untersuchungen wurden ungereifte und in vitro gereifte Oozyten der verschiedenen Untersuchungsgruppen eingesetzt. Drei der vier Kälberversuchsgruppen erhielten 48h vor jeder OPU-Sitzung eine FSH und zwei dieser Untersuchungsgruppen eine zusätzliche intraovarielle Injektion mit IGF-1 oder IGF K (0,01 M Essigsäure) (Injektionskontrolle). Auch ein Teil der Kühe erhielt 48h vor der Punktion eine hormonelle Follikelstimulierung. Für die Arbeit wurden präpuberale Kälber ab dem 6. Lebensmonat zweimal wöchentlich über einen Zeitraum von drei Wochen punktiert. Die Punktion wurde nach einer dreiwöchigen Pause bis zum Erreichen des 9. Lebensmonats wiederholt. Zur Kontrolle wurden adulte Kühe mit mindestens zwei Laktationen genutzt. Folgende Ergebnisse konnten erzielt werden: 1. Die Gesamtzahl der punktierten Follikel je Tier war in der Gruppe "Kuh" (38±19) signifikant höher als in der Gruppe "Kuh FSH" (36±16). Weitere signifikante Unterschiede zwischen den übrigen Untersuchungsgruppen konnten nicht beobachtet werden. 2. Bei Tieren der Gruppe "Kuh" (8±2) und "Kalb FSH K" (8±2) konnte gegenüber Tieren der Gruppen "Kalb" (5±2) und "Kalb FSH" (6±2) eine signifikant höhere Anzahl Follikel je Tier und OPU-Sitzung festgestellt werden. 3. Eine signifikant höhere Anzahl Oozyten wurde zwischen den Gruppen "Kalb FSH K" (26) und "Kalb FSH" (15) sowie zwischen "Kalb FSH K" (26) und "Kuh FSH" (19) beobachtet. 4. Für die Gene GDF9, SLC2A1, PRDX1 und ZAR1 konnten in ungereiften und gereiften Oozyten zwischen den einzelnen Untersuchungsgruppen keine signifikanten Unterschiede dargestellt werden. Beim Vergleich innerhalb der Untersuchungsgruppen wurde nach Maturation für die Gene GDF9 und ZAR1 ein signifikanter Rückgang der mRNA-Menge in allen Gruppen beobachtet. Für SLC2A1 wurde, mit Ausnahme der i.m. und i.o stimulierten Kälber, ein signifikanter Rückgang in den Gruppen "Kuh, Kalb, Kuh FSH und Kalb FSH" festgestellt. Für das Gen PRDX1 wurde in den Gruppen "Kuh, Kalb, Kuh FSH, Kalb FSH und Kalb FSH 1" ein signifikanter Rückgang nachgewiesen. 5. Das Methylierungsmuster für die Bovine testis satellite I Sequenz wurde in ungereiften und gereiften Oozyten der morphologischen Klasse 1-3 bestimmt. Für ungereifte Oozyten der Klassen 1-3 wurden signifikante Unterschiede zwischen den Gruppen "Kalb" (75,6%), "Kalb FSH1" (56,1%) und "Kuh" (53,8%) beobachtet. Nach der morphologischen Differenzierung wurden für ungereifte Oozyten der Klassen 1-2 signifikante Unterschiede zwischen den Gruppen "Kuh" (49,6%) und "Kalb" (74,6%) sowie zwischen "Kuh" (49,6%) und "Kuh FSH" (69,8) festgestellt. Für gereifte Oozyten der Klassen 1-2 bestand ein signifikanter Unterschied zwischen der Gruppe "Kalb FSH1" (71,7%) und "Kalb" (53,3%). Bei ungereiften Oozyten der Klasse 3 wurde eine signifikant höhere Methylierung der Gruppe "Kalb" (77%) gegenüber "Kuh" (56,6%) und "Kalb FSH1" (52,9%) ermittelt. Oozyten aus der Gruppe "Kalb FSH" (68,2%) wiesen eine signifikant höhere Methylierung gegenüber "Kalb FSH1" auf, aber eine niedrigere gegenüber "Kalb FSH K" (81,9%). Die Oozyten der Gruppe "FSH K" wiesen eine signifikant höhere Methylierung gegenüber "Kalb FSH1" auf. Beim Vergleich der Methylierung nach in vitro Reifung der Klassen 1-2 innerhalb jeder Untersuchungsgruppe wiesen Oozyten der Gruppen "Kuh" (49,6% vs. 64,9) und "Kalb FSH1" (60,6%vs.71,7%) eine signifikante höhere, die Gruppe "Kalb" (74,6% vs. 53,3%) eine signifikant niedrigere Methylierung auf. 6. Für die Methylierung der Bos taurus a-Satellite I Sequenz konnten zwischen den einzelnen Versuchsgruppen und morphologischen Klassen keine signifikanten Unterschiede beobachtet werden. Für die Gruppen "Kuh FSH" und "Kalb FSH" wurde nach der Reifung der Oozyten mit guten Entwicklungspotential eine signifikante Abnahme der Methylierung (76,5 vs. 52,5% und 72,8 vs. 57,8%) ermittelt. 7. Für die entwicklungsrelevanten, nichtgeprägten Gene SLC2A1, PRDX1 und ZAR1 wurde in ungereiften und gereiften Oozyten aller Untersuchungsgruppen eine genspezifische Hypomethylierung beobachtet. Für GDF9 konnte aufgrund fehlender CpGs keine Aussage zur Methylierung getroffen werden. Zusammenfassend ist festzustellen, dass in der vorliegenden Arbeit erstmals an ungereiften und gereiften bovinen Oozyten aus präpuberalen und adulten Tieren die genspezifische Methylierung von entwicklungsrelevanten, nicht geprägten Genen untersucht wurde. Hierbei wurden Alter und hormonelle Behandlung der Tiere berücksichtigt. Innerhalb des Forschungsprojektes wurde das Methylierungsmuster von zwei Repeatsequenzen zwischen verschiedenen Versuchsgruppen und Entwicklungsstadien der Oozyten bestimmt. Die Resultate der Repeatsequenzierung von einzelnen Untersuchungsgruppen können als Referenzwert für zukünftige Untersuchungen eingesetzt werden. Die hier gewonnenen Ergebnisse geben erste Hinweise auf das genspezifische Methylierungsgeschehen in ungereiften und gereiften Oozyten bei präpuberalen und adulten Rindern. Dieses sollte durch weitere Studien und Analyseverfahren bestätigt bzw. überprüft werden.Oocytes derived form prepuberal cattle develop to the blastocyst stage at a significantly reduced rate compared to oocytes from adult cattle. The aim of this thesis was to gain new insights into the developmental competence of prepuberal and adult bovine oocytes. To achieve this, we investigated the mRNA expression profiles of developmentally important non-imprinted genes (GDF9, SLC1A2, PRDX1 and ZAR1) as well as their gene specific methylation status. The gene expression profiles were analyzed by Real Time PCR. For the gene specific methylation analysis the “Limiting Dilution” method was employed. Additionally, we investigated the methylation level of two satellite sequences (repeat sequences), in order to gain insight into the global methylation pattern. The bovine oocytes for these assays were retrieved from four prepuberal and two adult animal groups. The oocytes were classified regarding their morphological properties into class 1-2 (good developmental potential) and class 3 (limited developmental potential). Immature and in vitro matured oocytes of the different groups were used. Three groups of prepuberal animals were administered FSH 48h prior to each OPU session. Additionally, two of the FSH treated groups also received intraovarian injections of IGF-1 and IGF K (0.01 M acetic acid/ injection control), respectively. A subgroup of adult animals also received hormonal follicle stimulation 48h before puncture. For this thesis, we used prepuberal calves with an age of 6 months. These calves were subjected to OPU twice weekly over a period of three weeks. Puncture was repeated every three weeks until the animals were 9 months old. As control, adult cows with at least two lactations were used, with a mean of three lactations
The following findings resulted from this work:
1. The total number of punctured follicles per animal was significantly higher in the group “Cow” (38±19) than in the group “Cow FSH” (36±16). No other significant differences were observed between the remaining groups.
2. Animals from the group “Cow” (8±2) and group “Calf FSH+IGF K” (8±2) showed a significantly higher number of follicles per animal and OPU session compared to the groups “Calf” (5±2) and “Calf FSH” (6±2).
3. A significantly higher number of oocytes was observed between the groups “Calf FSH+IGF K” (26) and “Calf FSH” (15) as well as between “Calf FSH+IGF K” (26) and “Cow FSH” (19).
4. For the transcripts of the genes GDF9, SLC1A2, PRDX1 and ZAR1 no differences could be shown between the respective groups for immature and matured oocytes. However, within the same group, a significant reduction in mRNA levels was observed for GDF9 and ZAR1 for all groups. For SLC2A1 a significant reduction, with the exception of the intramuscular and intraovarian stimulated calves, could be shown for the groups “Cow”, “Calf”, “Cow FSH” and “Calf FSH”. The transcript for PRDX1 was significantly reduced in the groups “Cow”, “Calf”, “Cow FSH”, “Calf FSH”, “Calf FSH+IGF 1”.
5. The methylation pattern for the Bovine testis satellite I sequence was examined in immature and matured oocytes of the morphological classes 1-3. For immature oocytes of the quality 1-3 significant differences were found between the groups “Calf” (75.6%), “Calf FSH+IGF-1” (56.1%) and “Cow” (53.8%). After morphological differentiation significant differences were observed between the groups “Cow” (49.6%) and “Calf” (74.6%) as well as for the groups “Cow” (49.6%) and “Cow FSH” (69.8%) for immature oocytes of the quality 1-2. For matured oocytes of high quality (class1-2) a significant difference was shown between “Calf FSH+IGF-1” (71.7%) and “Calf” (53.3%). For immature oocytes of reduced quality (class 3) a significantly enhanced methylation was observed for the group “Calf” (77%) when compared to “Cow” (56.6%) and “Calf FSH+IGF-1” (52.9%). Oocytes from the group “Calf FSH” (68.2%) showed a significantly higher methylation compared to “Calf FSH+IGF-1”, although a reduced methylation level compared to “Calf FSH+IGF K” (81.9%).
When the methylation levels before and after in vitro maturation of oocytes class 1-2 within each group are compared, oocytes of the groups “Cow” (49.6% vs. 64.9 %) and “Calf FSH+IGF-1” (60.6% vs. 71.7%) showed a significantly higher methylation level, the group “Calf” (74.6% vs. 53.3%) a significantly lower methylation level after maturation.
6. For the methylation status of the Bos Taurus α-Satellite I sequence were no significant differences were found between the individual groups and morphological qualities.For the groups “Cow FSH” and “Calf FSH” a significant reduction of methylation was observed for oocytes with good developmental potential after maturation (76.5 vs. 52.5% and 72.8 vs. 57.8%).
7. A gene specific hypomethylation was shown for the developmentally important non- imprinted genes SLC2A1, PRDX1 and ZAR1 in immature and matured oocytes of all groups. For GDF9 no information regarding methylation could be gathered due to the lack of CpGs.
In conclusion, this is to our knowledge the first report on gene specific methylation of developmentally important non-imprinted genes in immature and in vitro matured bovine oocytes from prepuberal and adult animals. For these experiments age and hormonal status of the animals were taken into consideration. In this project the methylation patterns of two repeated sequences of oocytes from different treatments and age groups and developmental status were determined. The results from the analysis of the repeat sequencing for the different groups have the potential to be used as a point of reference in advanced studies.
The results from this investigation give first cognitions regarding gene specific methylation processes in immature and matured oocytes from prepuberal and adult cattle that need to be validated in further studies
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