Rolled up microtubes for the capture, guidance and release of single spermatozoa

Hybride Mikroschwimmer, die einen biologischen Antrieb und eine künstlich hergestellte Mikrostruktur enthalten sind ein attraktiver Ansatz um kontrollierte Bewegung auf kleinstem Maßstab zu erreichen. In dieser Dissertation wird ein neuer hybrider Mikroschwimmer vorgestellt, der aus ferromagnetischen Nanomembranen besteht, die sich zu Mikroröhrchen aufrollen und in der Lage sind, einzelne Spermien einzufangen. Dieser Mikrobioroboter nutzt die starke Antriebskraft der Spermazelle um das magnetische Mikroröhrchen fortzubewegen. Die vorliegende Arbeit beschreibt, wie dieser Mikroschwimmer seine Bewegung vollzieht und wie verschiedene Faktoren wie Temperatur, Radius der Mikroröhrchen, Eindringtiefe der Spermien in das Röhrchen und Länge der Röhrchen einen Einfluss auf sein Verhalten haben. Richtungskontrolle wird durch externe magnetische Felder realisiert und es wird dargestellt, wie dies zur Trennung der Mikrobioroboter aus einer Mischung von Spermien und Mikroröhrchen genutzt werden kann. Weiterhin werden zwei Oberflächenmodifizierungsmethoden angewandt um die Kupplungseffizienz zwischen Mikroröhrchen und Spermien zu erhöhen. In diesen Methoden wird das extrazelluläre Protein Fibronektin auf die innere Röhrchenoberfläche aufgebracht und dient als Bindungsstoff für Spermien. Schließlich wird durch den Einbau temperatursensitiver Material in die Mikroröhrchen ein ferngesteuerter Freisetzungsmechanismus für die Spermazelle vorgestellt. Dabei falten sich die Mikroröhrchen bei kleinen Temperaturerhöhungen auf und setzen die Zelle frei. Diese Arbeit diskutiert letztendlich das Potential solch eines hybriden Mikroschwimmers für die Anwendung in assistierter Reproduktion.:TABLE OF CONTENTS SELBSTSTÄNDIGKEITSERKLÄRUNG 0 ABSTRACT 1 TABLE OF CONTENTS 3 1 MOTIVATION AND GOALS 5 1.1 MINIATURIZATION: “THERE IS PLENTY OF ROOM AT THE BOTTOM…” 5 1.2 SPERMBOTS: POTENTIAL IMPACT 7 2 BACKGROUND AND STATE-OF-THE-ART 11 2.1 MICROBIOROBOTICS 11 2.2 SPERM MORPHOLOGY AND THEIR JOURNEY TO THE EGG 15 2.3 INFERTILITY AND ASSISTED REPRODUCTION TECHNIQUES 19 2.4 SINGLE CELL RELEASE 22 2.5 STIMULI-RESPONSIVE MATERIALS 25 3 MATERIAL AND METHODS 29 3.1 ROLLED UP TECHNOLOGY 29 3.2 TREATMENT OF BOVINE SPERMATOZOA 32 3.2.1 Preparation of Spermbots 32 3.2.2 Speed Measurements 33 3.2.3 Separation On Chip 33 3.3 SURFACE MODIFICATION OF MICROTUBES 34 3.3.1 Surface Chemistry 35 3.3.2 Microcontact printing 39 3.4 POLYMER TUBE FABRICATION 44 3.4.1 Synthesis of photosensitive monomer 4-Acryloylbenzophenone 44 3.4.2 Synthesis of poly (N-isopropylacrylamide-co-Acryloylbenzophenone) 46 3.4.3 Photolithography of polymeric films 48 3.5 VIABILITY TESTS 51 4 RESULTS AND DISCUSSION 53 4.1 CHARACTERIZATION OF SPERMBOTS 55 4.2 TEMPERATURE INFLUENCE 60 4.3 MAGNETIC CONTROL 62 4.4 SEPARATION ON CHIP 68 4.5 EFFECT OF DECREASED MICROTUBE LENGTH 72 4.6 COUPLING EFFICIENCY 74 4.7 THERMORESPONSIVE POLYMERIC MICROTUBES FOR CELL RELEASE 80 4.8 SPERM VIABILITY TESTS 94 5 SUMMARY AND CONCLUSIONS 97 6 OUTLOOK 101 7 LIST OF FIGURES 107 8 LIST OF TABLES 113 9 ABBREVIATIONS 115 10 CURRICULUM VITAE 117 11 LIST OF PUBLICATIONS 119 JOURNAL ARTICLES 119 CONTRIBUTIONS TO COLLECTED EDITIONS/PROCEEDINGS 121 12 ACKNOWLEDGEMENTS 123 13 REFERENCES 125The search for autonomously moving, highly functional and controllable microdevices is a purpose of current micro/nanobiotechnology research, especially in the area of biomedical applications, for which reason, biocompatible solutions are in demand. In this thesis, a novel type of hybrid microswimmer is fabricated by the combination of rolled up thin nanomembranes with bovine spermatozoa. The microbiorobot presented here uses the powerful motion of the sperm flagella as a propulsion source for the magnetic microtube. This work demonstrates how the microswimmer performs its motion and how several factors such as temperature, radius of the microtube, penetration of the cell inside the microtube and length of the tube have influence on its performance. Directional control mechanisms are offered by external magnetic fields and are presented to be useful for the on-chip separation of the microbiorobots from a mixture of cells and microtubes. Two surface modification methods are presented as means to improve the coupling efficiency between the microtubes and the sperm cells. By these surface functionalizations, the extracellular matrix protein fibronectin is attached on the inner microtube walls and serves as binding agent for the spermatozoa. Finally, a remote release mechanism for the sperm cells is demonstrated by the incorporation of thermoresponsive material into the microtubes, which makes them fold and unfold upon small temperature changes. This work discusses the potential of such microswimmers for the application in assisted reproduction techniques and gives an outlook on future perspectives.:TABLE OF CONTENTS SELBSTSTÄNDIGKEITSERKLÄRUNG 0 ABSTRACT 1 TABLE OF CONTENTS 3 1 MOTIVATION AND GOALS 5 1.1 MINIATURIZATION: “THERE IS PLENTY OF ROOM AT THE BOTTOM…” 5 1.2 SPERMBOTS: POTENTIAL IMPACT 7 2 BACKGROUND AND STATE-OF-THE-ART 11 2.1 MICROBIOROBOTICS 11 2.2 SPERM MORPHOLOGY AND THEIR JOURNEY TO THE EGG 15 2.3 INFERTILITY AND ASSISTED REPRODUCTION TECHNIQUES 19 2.4 SINGLE CELL RELEASE 22 2.5 STIMULI-RESPONSIVE MATERIALS 25 3 MATERIAL AND METHODS 29 3.1 ROLLED UP TECHNOLOGY 29 3.2 TREATMENT OF BOVINE SPERMATOZOA 32 3.2.1 Preparation of Spermbots 32 3.2.2 Speed Measurements 33 3.2.3 Separation On Chip 33 3.3 SURFACE MODIFICATION OF MICROTUBES 34 3.3.1 Surface Chemistry 35 3.3.2 Microcontact printing 39 3.4 POLYMER TUBE FABRICATION 44 3.4.1 Synthesis of photosensitive monomer 4-Acryloylbenzophenone 44 3.4.2 Synthesis of poly (N-isopropylacrylamide-co-Acryloylbenzophenone) 46 3.4.3 Photolithography of polymeric films 48 3.5 VIABILITY TESTS 51 4 RESULTS AND DISCUSSION 53 4.1 CHARACTERIZATION OF SPERMBOTS 55 4.2 TEMPERATURE INFLUENCE 60 4.3 MAGNETIC CONTROL 62 4.4 SEPARATION ON CHIP 68 4.5 EFFECT OF DECREASED MICROTUBE LENGTH 72 4.6 COUPLING EFFICIENCY 74 4.7 THERMORESPONSIVE POLYMERIC MICROTUBES FOR CELL RELEASE 80 4.8 SPERM VIABILITY TESTS 94 5 SUMMARY AND CONCLUSIONS 97 6 OUTLOOK 101 7 LIST OF FIGURES 107 8 LIST OF TABLES 113 9 ABBREVIATIONS 115 10 CURRICULUM VITAE 117 11 LIST OF PUBLICATIONS 119 JOURNAL ARTICLES 119 CONTRIBUTIONS TO COLLECTED EDITIONS/PROCEEDINGS 121 12 ACKNOWLEDGEMENTS 123 13 REFERENCES 12

Microfluidic Reduction of Osmotic Stress in Oocyte and Zygote Vitrification.

Microfluidic cryoprotectant exchange enables vitrification of murine zygotes with superior morphology as indicated by a smoother cell surface and higher developmental competence compared to conventional methods. Bovine oocyte vitrification also benefit as evidenced by higher lipid retention. Experimental observations and mathematical analysis demonstrate that the microfluidic advantage arise predominantly from eliminating high shrinkage rates associated with abrupt and uneven exposure to vitrification solutions that readily occur in current manual protocols. The microfluidic cryoprotectant exchange method described has immediate applications for improving animal and human oocyte, zygote, and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sub-lethal damage should be broadly useful for cryobiology.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107056/1/davlai_1.pd

Mechanisms of seasonal reproduction in cattle

This study was conducted to evaluate the effect of season on reproductive parameters in cattle in the temperate climate of the UK. In the first study, reproductive fertility data were collected from a local dairy herd. The results revealed that cows born in autumn were inseminated at an earlier age (P15°C and a temperature humidity index (THI) range from 40 to 59 compared with >60 units (10 days before and 21 days after insemination). In the second study, ovarian tissues were collected from a local abattoir to investigate the effect of season on follicular populations, corpus luteum (CL) development and incidence of multiple ovulations. There were no effects of season on antral follicle count. However, individual and total CL weight was heavier in the autumn. Additionally, season influenced multiple ovulations with a higher incidence (P7mm in cows with multiple CLs was higher (P<0.05) than cows with a single CL. The individual CL weight was heavier in single ovulation cows. However, the total luteal tissue weight and total progesterone (P4) content of luteal tissue was higher in cows with multiple ovulations. The third study investigated the effects of culturing granulosa cells (GCs) under low (5%; physiological oxygen (O2)) conditions rather than traditional culture (20%; atmospheric O2) systems. Granulosa cells from antral follicles were cultured in fibronectin coated plates in M199 for up to 144 hour (h) under physiological (5%) and atmospheric (20%) O2 tension. Melatonin was added at one of four concentrations (0, 20, 200, 2000 pg/ml). The number of viable GCs was greater (P<0.05) under 5% O2 than 20% O2. Reactive oxygen species (ROS) generation was similar under both physiological and atmospheric O2, but was reduced (P<0.05) by treatment with melatonin. Oestrogen concentration (P<0.001) and aromatase activity (P<0.014) were also influenced by O2 tension in a time dependent manner. Both oestradiol (E2) production and aromatase enzyme activity were maintained for up to 144 h of culture under 5% O2 conditions. Progesterone production was increased under 20% O2 compared with 5% O2 (P<0.05). Additionally, the expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) mRNA increased (P<0.05) with time under 20% O2, but remained unchanged at 5% O2. Haemoglobin subunit alpha 1 (HBA) transcript was increased (P<0.05) under 5% O2 levels. The final study quantified the effect of temperature and melatonin on GC function. Cells were cultured for up to 144 h under 5% O2 tension. Treatments commenced after 48 h of culture and consisted of two incubation temperatures (37.5 vs 40.0°C) and four melatonin treatments. Melatonin increased cell number at high temperature (40.0°C). However, BCL2-associated X protein (BAX) mRNA expression was greater (P<0.05) in GCs cultured at 40.0°C than at 37.5°C by 144 h. Culture temperature did not affect ROS, but melatonin reduced (P<0.001) generation of ROS. Oestradiol production increased with time (P<0.001) and was not affected by temperature. In contrast, high temperature reduced P4 production (P<0.001) at 144 h of culture. Similarly, the effect of melatonin treatment depended on temperature; melatonin increased P4 production at 37.5C, while reducing P4 at 40.0C. Temperature increased acetylserotonin O-methyltransferase (ASMT) mRNA expression (P<0.05) though there was no significant effect of temperature and melatonin on tumour protein p53 (P53), HSD3B1, superoxide dismutase (SOD1 and SOD2), HBA and heat shock protein family A (Hsp70) member 1A (HSPA1A) gene expression. The results of this thesis contribute to our understanding of the effects of season on ovarian function and seasonal variation in cattle fertility particular in temperate climate regions where season influenced puberty, conception rate, incidence of multiple ovulations and CL development. In in vitro studies, low O2 (5%) enhance cell proliferation, reduced luteinisation and altered steroidogenesis as well as increasing the expression of HBA mRNA. Culture at higher temperature reduced P4 production and increased apoptotic mRNA while addition of melatonin reduced ROS generation and influenced P4 production. This new approach to culture could offer a valuable system for future investigation of the physiological function of cells in vitro

Advanced Knowledge Application in Practice

The integration and interdependency of the world economy leads towards the creation of a global market that offers more opportunities, but is also more complex and competitive than ever before. Therefore widespread research activity is necessary if one is to remain successful on the market. This book is the result of research and development activities from a number of researchers worldwide, covering concrete fields of research

Evaluation of the ingestive behaviour of the dairy cow under two systems of rotation with slope

The ingestive behaviour of grazing animals is modulated by the vegetation characteristics, topography and the type of stocking method. This research was carried out in 2019, at the Rumipamba CADER-UCE. It aimed to evaluate the impact of two contrasting stocking methods of dairy cows grazing a pasture with an average of slope >8.5%. Four dairy cows were set to graze a 0.4 ha paddock for 5 days for continuous stocking methods, while for the electric fence methods the dairy cows were restricted to 0.2 ha and the fence was moved uphill every 3 hours, repeating this process four times a day. Cow were equipped with activity sensors for 12 h per day. The whole procedure was repeated 2 times after realizing an equalization cuts and both paddocks, a rest time of 30 days and a random reassignment of paddocks to one of the treatments. The cows showed a difference in terms of the percentage of grazing P=0.0072, being higher with the electric fence (55% of the measurement time). From rising-plate-meter estimates of available biomass along the grazing periods, we calculated despite similar forage allowances (electric fence = 48.06 kg DM/cow/d and continuous = 48.21 DM/cow/d) a higher forage intake was obtained in the electric fence treatment (17.5 kg DM/cow/d) compared the continuous stocking (15.7 kg DM/cow/d) (P=0.006). In terms of milk production animals grazing under the differences electrical fence stocking method tended (P=0.0985) to produce more milk (17.39 kg/d) than those grazing in the continuous system (15.16 kg/d) due to the influence of the slope (P=0.05), while for milk quality the protein content was higher for the electric fence (33.7 g/l) than the continuous method (30.5 g/l) (P=0.039). None of the other milk properties differed between methods (P>0.05)

The use of high sugar grasses as a strategy to improve nitrogen utilization efficiency:A meta-analysis

publishersversio

12th Man in Space Symposium: The Future of Humans in Space. Abstract Volume

The National Aeronautics and Space Administration (NASA) is pleased to host the 12th IAA Man in Space Symposium. A truly international forum, this symposium brings together scientists, engineers, and managers interested in all aspects of human space flight to share the most recent research results and space agency planning related to the future of humans in space. As we look out at the universe from our own uniquely human perspective, we see a world that we affect at the same time that it affects us. Our tomorrows are highlighted by the possibilities generated by our knowledge, our drive, and our dreams. This symposium will examine our future in space from the springboard of our achievements

XIII International Congress in Animal Hygiene, June 17-21, 2007, Tartu, Estonia "Animal health, animal welfare and biosecurity" : proceedings. Volume 1

KonverentsikogumikOn behalf of both the Organising Committee and the Scientific Committee, I am pleased to welcome you in Tartu, Estonia, to participate at the XIII International Congress of the International Society for Animal Hygiene (ISAH). The ISAH (www.isah-soc.org) was founded in 1970 and has today members from 48 countries throughout the world. ISAH can be considered as a group of scientists contributing to efficient, sustainable animal farming with healthy animals, providing wholesome food in a sound environment. Veterinarians and non-veterinary academic scientists (animal science, agricultural economics, engineers, microbiologists, public health professionals, epidemiologists etc., etc) and respective professionals in animal husbandry, who work and/or do research and education in the field of animal hygiene, can apply for a membership of ISAH, and are most welcome to attend ISAH congresses. The first ISAH congress was held in Budapest in 1973. The last ISAH main congress took place in Warsaw, Poland in 2005 and the last in-between symposium in Saint-Malo, France in 2004. Starting from Warsaw congress in 2005, the ISAH, considering the need for a more flexible and frequent exchange of scientific and practical knowledge, organizes its congresses every second year. The present, XIII ISAH congress in Tartu, Estonia, in June 17–21, 2007 is organised under the device "Animal health, animal welfare and biosecurity”. The scientific programme, trying to follow the scope of the ISAH and receive the feedback from modern animal husbandry and food production, concentrates with more profoundness on the following subjects: interaction between the environment and health and welfare of individual animal and herds; managing animal health in large dairy units; ensuring animal welfare during transportation and slaughter; economical implications considering animals’ health; possibilities of precision livestock farming in maintaining good health and welfare of animals; measures for prevention the development and spread of diseases and pathogens in animals including those posing risk to human health (zoonoses); food safety relevant infections and contaminations such as residues in food derived from animals; influence of the animal production on the environment and public health. The Proceedings from the XIII ISAH Congress are herewith presented. The papers on lectures from invited speakers, oral and poster presentations from 11 parallel sessions are included in this excellent compilation. In general, the printed contribution to the ISAH-2007 congress illustrates clearly the broad scientific field of the ISAH and related to it activities. I hereby would like to express my most sincere gratitude in the address of ISAH-2007 organising and scientific committees. Special thanks go to Frens Conference Services for their excellent organizational and technical contribution and to AS Triip for their outstanding printing job of these proceedings. We also appreciate different companies and organisations for their considerable financial support which gave us the opportunity to keep the registration fees affordable. Finally, we thank all participants, contributors, chairpersons, organisational and technical assistants for your considerable efforts – you made the ISAH-2007 in Tartu real success. We wish you all interesting and pleasant congress and enjoyable stay in Tartu. A. Aland Editor Chairman of the ISAH-2007 Organising Committe