Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Livestock producers, farmers, research scientists, and reproduction companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? We have trained numerous undergraduate students and visiting scientists in this technology. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Work will be continued under a W-3171 Multistate Project. We plan to expand our studies with transgenic swine and cattle. We plan to continue our work analyzing embryo culture and manipulation in microfluidic channels in collaboration with the University of Wisconsin and BORN Animal Biotechnology, Inc. Finally, we plan to continue to expand our efforts in the area of stem cell biology, differentiation and regenerative biology. We plan to continue to optimize novel microfluidic devices for oocyte maturation and embryo culture.
Impacts
What was accomplished under these goals?
Objective 1 We have developed a method to culture single porcine and bovine oocytes to maturation, fertilize them in vitro using conventional methods and then culture them singly to the blastocysts stage. Objective 2 We have demonstrated the ability of MAC-T cells to b used as a screening method to test transgene constructs in cattle. It has been previously demonstrated (IETS 2011) that Panama is applying the biotechnology of in vitro embryo production (IVP) into their bovine reproduction management systems. This present work demonstrates the ability to apply the IVP technology across two distant country borders. We here demonstrate that a country (Dominican Republic, DR) that does not have a bovine IVP lab can take advantage of fresh bovine IVP embryos for transfer using distant IVP facilities in another country (Panama , ~1,500km away). The objective of this study was to demonstrate that a model system for large-scale commercial in vitro bovine embryo production for beef and dairy producers, that do not have IVP technology in their home country, could be developed producing comparable results. Since the same laboratory provides IVP services toboth countries, a special sanitary protocol was developed in order to legalize the exchange of biological materials (oocytes/embryos). The data obtained in the Dominican Republic was compared to Panamanian client data because identical conditions were utilized for IVP. Cattle production systems were similar as Brahman (a Zebu type of cattle) is the most popular breed in both countries. Oocytes were collected from 10 different herds in Panama and 4 different herds in the DR. The oocytes were transported in a oocyte transporter in both instances. However, oocytes from the DR were transported in InVitro Brasil™ maturation medium from 12-18 hr and in Panama from 6-12 hr before they were placed in a standard CO2 incubator. In both cases the oocytes were matured for 24 hr before fertilization with conventionally frozen Brahman semen in InVitro Brasil™ fertilization medium followed by culture up to 7 days in InVitro Brasil™ embryo culture medium. The embryos were transferred on day 7, either in Panama or the DR. They were transported by car in Panama and via airplane back to the DR. A comparison of oocyte number and quality, cleavage, embryo production and pregnancy rate was made using the same in vitro production system for Brahman Donors from September 2012 until May 2013. The difference between sites in the relative number of viable oocytes, relative number of cleaved oocytes among viable oocytes, relative number of embryos produced among cleaved oocytes and relative number of embryos produced among viable oocytes was tested using Fisher’s exact test. Pregnancy rate was analyzed with X2 We realize that these results represent field data, however we believe the present work is a significant step in demonstrating the potential for wide commercial-scale dissemination of the IVP technology between distant countries. The number of embryos produced in Panama was slightly but significantly higher than those produced in the DR but this is likely due to the larger number of donors and oocytes from the Panama herds. However, the pregnancy rate was higher in the DR likely due to the health status of the DR recipients. These data illustrate that in vitro embryo production using Brahman donors could be used as a tool to improve and spread superior genetics. Furthermore, this technique can serve as a model for other Central American and Caribbean countries under similar management systems.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Nagele, A., Gom�s Jr., E., Ruiz, A., Nasser, L.F., Feli�, S., Rodr�guez, E., Mojica, K., Basso, A.C., Pontes, J.H.F., Rabel, R.A.C., Rodriguez-Zas, S.L. and Wheeler, M.B. Comparison of commercial in vitro embryo production and pregnancy rates of Brahman donors in Panama vs. Dominican Republic. Reproduction, Fertility and Development 26:185.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience was scientists, students and livestock producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? We sent a student to Panama and the Dominican Republic to learn and assist in this project with colleagues in those countries. How have the results been disseminated to communities of interest? These results have been published in the Journal of Reproduction, Fertility and Development and will be presented at the International Embryo Transfer meetings in January 2014 in Reno, Nevada. What do you plan to do during the next reporting period to accomplish the goals? We plan to continue our work in improving bovine in vitro embryo production as well as expand our efforts in embryo culture and production of genetically modified cattle.
Impacts
What was accomplished under these goals?
It has been previously demonstrated (IETS 2011) that Panama is applying the biotechnology of in vitro embryo production (IVP) into their bovine reproduction management systems. This present work demonstrates the ability to apply the IVP technology across two distant country borders. We here demonstrate that a country (Dominican Republic, DR) that does not have a bovine IVP lab can take advantage of fresh bovine IVP embryos for transfer using distant IVP facilities in another country (Panama , ~1,500km away). The objective of this study was to demonstrate that a model system for large-scale commercial in vitro bovine embryo production for beef and dairy producers, that do not have IVP technology in their home country, could be developed producing comparable results. Since the same laboratory provides IVP services to the both countries, a special sanitary protocol was developed in order to legalize the exchange of biological materials (oocytes/embryos). The data obtained in the Dominican Republic was compared to Panamanian client data because identical conditions were utilized for IVP. Cattle production systems were similar as Brahman (a Zebu type of cattle) is the most popular breed in both countries. Oocytes were collected from 10 different herds in Panama and 4 different herds in the DR. The oocytes were transported in a oocyte transporter in both instances. However, oocytes from the DR were transported in InVitro Brasil™ maturation medium from 12-18 hr and in Panama from 6-12 hr before they were placed in a standard CO2 incubator. In both cases the oocytes were matured for 24 hr before fertilization with conventionally frozen Brahman semen in InVitro Brasil™ fertilization medium followed by culture up to 7 days in InVitro Brasil™ embryo culture medium. The embryos were transferred on day 7, either in Panama or the DR. They were transported by car in Panama and via airplane back to the DR. A comparison of oocyte number and quality, cleavage, embryo production and pregnancy rate, was made using the same in vitro production system for Brahman Donors from September 2012 until May 2013. The difference between sites in the relative number of viable oocytes, relative number of cleaved oocytes among viable oocytes, relative number of embryos produced among cleaved oocytes and relative number of embryos produced among viable oocytes was tested using Fisher’s exact test. Pregnancy rate was analyzed with X2. We realize that these results represent field data, however we believe the present work is a significant step in demonstrating the potential for wide commercial-scale dissemination of the IVP technology between distant countries. The number of embryos produced in Panama was slightly but significantly higher than those produced in the DR but this is likely due to the larger number of donors and oocytes from the Panama herds. However, the pregnancy rate was higher in the DR likely due to the health status of the DR recipients. These data illustrate that in vitro embryo production using Brahman donors could be used as a tool to improve and spread superior genetics. Furthermore, this technique can serve as a model for other Central American and Caribbean countries under similar management systems. We showed that movement of bovine oocytes between two countries was feasible to produce in vitro fertilized cattle embryos.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yuan, Y., Wheeler, M.B. and Krisher, R.L. Disrupted redox homeostasis and aberrant redox gene expression in porcine oocytes contribute to decreased developmental competence. Biol. Reprod. (2012) 87(4):78, 110,doi:10.1095/biolreprod. 112.099952.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The outputs from this project have been published in peer-reviewed journals and presented at international scientific meetings. Results form this work have also been incorporated into my teaching materials for my classes at the University of Illinois. Results from this project have been presented at the International Embryo Transfer Society Annual Meeting. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Advancements in micro and nanotechnology have allowed scientists a powerful platform to study biological systems. Micro-fluidics is one area of advancement with great promise. Micro-fluidics deals with the behavior, specific control, and manipulation of microliter and nanoliter volumes of fluid. The small-scale design of these microfluidic devices permits laminar flow, characterized as parallel streams flowing without disruption between currents. With the introduction of micro-technology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and they provide a more in vivo like environment for the cells to grow and hence can serve as a better way of culturing the cells. In the current study we examine the influence of microfluidic devices on the development of stem cells. Adipose derived stem cells were isolated from pigs and were seeded in a microfluidic device to differentiate towards adipogenic, osteogenic and chondrogenic lineages using specific differentiation promoting mediums. Five thousand cells were seeded per channel at a density of 5 million cells per ml. The microchannel dimensions were 5mm long, 1mm wide and 200 micrometers deep. Cells were maintained for 14 days and then stained with respective staining dyes: Oil Red O for adipogenesis, Alizarin Red for osteogenesis and Toluidine Blue for chondrogenesis. Cells differentiated towards adipogenic lineage contained small lipid droplets, which stained red with Oil Red O stain, during osteogenic differentiation the cells formed large nodules and stained positive for the presence of calcium and the chondriogenic differentiating cells showed the presence of proteoglycans (blue in color) when stained with toluidine blue. We seeded ADSCs in five channels for each differentiation lineage and all the channels gave positive staining results. We conclude that microfluidic channels support proliferation and differentiation of adipose derived stem cells. This system uses small amounts of culture medium, experiments with different culture compositions can be efficiently performed, and culture manipulations can be automated using fluid handling robotics. Because microfluidics can deal with small number of cells, the characteristics of cellular structure and function and the microenvironment of the stem cells can be understood in a more precise manner. The miniaturization of cell culture platforms allows the observation of cellular behaviour at the scale found in living systems.
Publications
- Wilson, S.M., Goldwasser, M.S., Clark, S.G., Monaco, E., Bionaz, M., Hurley, W.L., Rodriguez-Zas, S., Feng, L., Dymon, Z. and Wheeler, M.B. 2012. Adipose-derived mesenchymal stem cells enhance healing of mandibular defects in the ramus of swine. J. Oral Maxillofac. Surg. 70:e193.
- Monaco, E., Bionaz, M. Rodriguez-Zas, S., Hurley, W.L. and Wheeler, M.B. 2012. Transcriptomics comparison between porcine adipose and bone marrow mesenchymal stem sells during in vitro osteogenic and adipogenic differentiation. PLoS ONE 7(3): e32481. doi:10.1371/journal.pone.0032481.
- Chen, K., Hawken, R., Flickinger, G.H., Rodriguez-Zas, S.L., Rund, L.A., Wheeler, M.B., Abrahamsen, M., Rutherford, M.S., Beever J.E. and Schook, L.B. 2012. Association of the porcine transforming growth factor beta type I receptor (TGFBR1) gene with growth and carcass traits. Animal Biotechnology, 23: 43 , 2012, doi.org/10.1080/10495398.2011.630897.
- Yuan, Y., Wheeler, M.B. and Krisher, R.L. 2012. Disrupted redox homeostasis and aberrant redox gene expression in porcine oocytes contribute to decreased developmental competence. Biol. Reprod. 87(4):78, 1, doi:10.1095/biolreprod.112.099952.
- Bionaz, M., Mkrtschjan, M., Kyrouac, D., Hollister, S.J. and Wheeler, M.B. 2012. In vitro migration of adipose-derived stem cells from GFP pigs into polycaprolactone scaffolds treated with FGF or BMP2. Reproduction, Fertility and Development 24:219
- Hollister, S.J., Wheeler, M.B., Feinberg, S.E. and Murphy, W.L. 2012. The importance of large animal models for translational research in bone tissue engineering. Reproduction, Fertility and Development, 24:287.
- Feltrin, C., Mohamad-Fauzi, N., Gaudencio Neto, S., Martins, L.T., Almeida, J.L., Salviano, M.B., Freire, A.K., Carneiro, I.S., Rios, D.B., Freire, R.R., Wheeler, M.B., Murray, J.D., Maga, E.A., Bertolini, L.R. and Bertolini, M. 2012. Effect of the cytoplast source and karyoplast type on the development of handmade cloned embryos in goats. Reproduction, Fertility and Development, 24:127.
- Monaco, E. ,Bionaz, M., Lima, A., Hurley, W.L. and Wheeler, M.B. 2012. Transcriptomic comparison between porcine adipose and bone marrow mesenchymal stem cells during in vitro osteogenic and adipogenic differentiation. Reproduction, Fertility and Development 24:219.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The outputs form this project have been published in peer-reviewed journals and presented at national and international scientific meetings. Results from this work have also been incorporated into my teaching materials for my classes at the University of Illinois. Results from this project have been presented at the International Embryo Transfer Society Annual Meeting and the Society for the Study of Reproduction. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Production and tracking of individual embryos in vitro would be a useful technique for many purposes. In vitro embryo production includes multiple steps, including oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC). The objective of this study was to evaluate the effect of a novel microfluidic system in both a dynamic and static environment for individual oocyte IVM, on oocyte nuclear maturation and developmental competence after standard IVF/IVC. These microfluidic well inserts can mature a large number of oocytes individually, and each oocyte can be tracked easily. Each insert is fabricated of polydimethylsiloxane and is composed of microwells. Each microwell is connected to adjacent microwells via microchannels underneath the surface of inserts, allowing communication to occur between individual cumulus oocyte complexes. To create a dynamic culture environment, the culture dish was placed on a rocking platform inside the incubator with a speed of 10 rpm during maturation. Oocytes were matured in either the microfluidic well system or control conditions, in dynamic or static culture environments. For fertilization, gametes were co-incubated and then cultured for 6 days, when cleavage, blastocyst development and blastocyst cell number were determined. Data were analyzed by ANOVA for balanced data with culture system (microfluidic well or control) and environment (dynamic or static) as fixed factors, and differences determined by Bonferroni (All-Pairwise) Multiple Comparison Test. The percentage data was arcsin transformed. There were no significant differences in the percentage of nuclear maturation, percentage of embryonic cleavage or blastocyst total cell number between treatments. Blastocyst development was significantly decreased when oocytes were matured in this dynamic environment compared to static environment. Importantly the blastocyst development of oocytes matured in the microfluidic well system was equal to that of oocytes matured in standard control conditions. In conclusion, a dynamic environment during oocyte maturation in either standard or microfluidic well systems was detrimental to blastocyst formation. The microfluidic well system can successfully mature oocytes individually without compromising blastocyst formation after IVF/IVC. These results demonstrate that this microfluidic system provides an efficient way to successfully mature oocytes individually. Further work in optimizing this microfluidic system for individual egg fertilization and embryo culture will provide the opportunity to fully integrate each step of individual in vitro embryo production.
Publications
- Monaco, E., Bionaz, M., Lima, A., Hurley, W.L. Wheeler, M.B. 2011. ADSC and BMSC present large similarities in transcriptome prior to and during adipogenic and osteogenic differentiation. Reproduction, Fertility and Development 23:253-254.
- Bionaz M., Jensen T., Monaco E., Dymon Z., Maki A.J., Hurley W.L. and Wheeler M.B. 2011. Unsorted, freshly isolated porcine adipose-derived stem cells are more efficacious in bone healing compared to purified CD34+ adipose-derived stem cells. Reproduction, Fertility and Development 23:253-253.
- Feltrin, C., Machado, M.,. Queiroz, L.M.V., Peixer, M.A.S., Malard, P.F., Santana, G.M. Bertolini, M., Wheeler, M.B. and Rodrigues , J.L. 2011. Effectiveness of microwell-based in vitro culture systems for bovine zona-free cloned embryos. Reproduction, Fertility and Development, Vol. 23:124-124.
- Monaco, E., Bionaz, M., Hollister, S. and Wheeler, M.B. 2011. Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells. Theriogenology 75:1381-1399.
- Polak, S.J., Lan Levengood, S.K., Maki, A.J., Clark, S.G., Wheeler, M.B. and Wagoner Johnson, A.J. 2011. Analysis of the roles of microporosity and BMP-2 on multiple measures of bone regeneration and healing in calcium phosphate scaffolds. Acta Biomaterialia (In Press).
- Wheeler, M.B., 2011. Transgenic animals in agriculture. Nature Education. (Accepted).
- Sears, K.E., Bormet, A.K., Rockwell, A., Powers, L.E., Noelle Cooper, L. and Wheeler, M.B. 2011. Developmental basis of mammalian digit reduction: A case study in pigs. Evolution and Development. 13:533-541.
- Wilson, S.M., Goldwasser, M.S., Clark, S.G., Monaco, E., Bionaz, M., Hurley, W.L., Rodriguez-Zas, S., Feng, L., Dymon, Z,. and Wheeler, M.B. 2011. Adipose-derived mesenchymal stem cells enhance healing of mandibular defects in the ramus of swine. J. Oral/Maxillofacial Surgery. (In Press).
- Gottlieb, S. and Wheeler, M.B. 2011, Genetically engineered animals and public health: Compelling benefits for health care, nutrition, the environment, and animal welfare. BIO (Biotechnology Industry Organization), 37 pgs., 2nd ed.
- Chen, J.R.S., Nasser, L.F., Penteado, L., Mendizabal, M., Basso, A.C., Pontes, J.H.F., Bionaz, M. and Wheeler, M.B. 2011. Comparison of commercial in vitro embryo production of Brahman donors under Brazilian versus Panamanian management. Reproduction, Fertility and Development 23:210-210.
- Maki, A.J., Clark, S.G., Woodard, J.R., Goldwasser, M. and Wheeler, M.B. 2011. A critical-size craniofacial bone defect model in the Yorkshire pig. Reproduction, Fertility and Development 23:159-159.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The outputs from this project have been published in peer-reviewed journals and presented at national and international scientific meetings. Results from this work have also been incorporated into my teaching materials for classes at the University of Illinois. Results from this project have been presented at the International Embryo Transfer Society Annual Meeting, the International Society on Stem Cell Research, the Society for the Study of Reproduction Meetings and the Korean National Academy of Science Biotechnology Conference. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Mammalian embryo development is still relatively inefficient in vitro. Much research has been conducted on the chemical environment, or culture medium, surrounding the embryo, but little attention has been given to the actual physical culture environment, which has changed very little over the years. The application of microfluidics to embryo production in vitro is a tantalising approach that may alleviate some of the limits that traditional microdrop culture places on embryo development and research into gamete and embryo physiology. These devices may lead to enhanced in vitro embryo development and quality by more closely mimicking the in vivo environment. Initial work in this area is promising and gives us proof-of-principle that these unique microfluidic systems may indeed be applicable to in vitro culture of gametes and embryos. The present paper reviews the advantages of microfluidics for in vitro embryo production: how the platforms are manufactured, the current uses of microfluidics in assisted reproduction, static v. dynamic culture environments, individual gamete and embryo culture and the future directions of microfluidic application to in vitro embryo production and manipulation. Finally, preliminary data from our laboratory using a new microfluidic well insert for porcine, bovine and murine embryo culture were obtained.
Publications
- Kim, D., Monaco, M.A., Lima, A.S., Kong, H.-Y., Hurley, W.L. and Wheeler, M.B. 2010. Morphologic and transcriptomic comparison of adipose and bone marrow derived porcine stem cells in alginate hydrogel. Cell and Tissue Research 341:359-370.
- Krisher, R.L. and Wheeler, M.B. 2010. Towards use of microfluidics for individual embryo culture. Reproduction, Fertility and Development 22:32-39.
- Lan Levengood, S.K., Polak, S.J., Wheeler, M.B., Maki, A.J., Clark, S.G., Jamison, R.D. and Wagoner Johnson, A.J. 2010. Multiscale osteointegration as a new paradigm for the design of calcium phosphate scaffolds for bone regeneration. Biomaterials 31, 3552-3563.
- Lan Levengood, S.K., Polak, S.J., Wheeler, M.B., Maki, A.J., Clark, S.G., Jamison, R.D. and Wagoner Johnson, A.J. 2010. The effect of BMP-2 on micro and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity. Acta Biomaterialia 6(8):3283-3291.
- Monaco, E., Bionaz, M., Lima, A.S., Hurley, W.L. and Wheeler, M.B. 2010. Internal controls for quantitative polymerase chain reaction of porcine adult mesenchymal stem cells. Stem Cell Research and Therapy, 1:7 doi:10.1186/scrt7
- Bionaz, M., Monaco, E., Tanaka, T. and Wheeler, M.B. 2010. Current and emerging biotechnologies for livestock production. The 37th National Academy of Sciences (Korea) International Symposium, Biotechnology in Agriculture and Fisheries, October 22, 2010, pgs 121-182, ISSN 1225-30X.
- Fahrenkrug, S.C., Blake, A., Carlson, D.F., Doran, T., Van Eenennaam, A., Faber, D., Galli, C., Hackett, P.B., Li, N., Maga, E.A., Murray, J.D., Stotish, R., Sullivan, E., Taylor, J.F., Walton, M., Wheeler, M.B., Whitelaw, B. and Glenn, B.P. 2010. Precision genetics for complex objectives in animal agriculture. J. Anim. Sci. 88:2530-2539.
- Paczkowski, M. and Krisher R.L. 2010. Aberrant protein expression is associated with decreased developmental potential in porcine oocytes. Molecular Reproduction and Development 77:51-58. Published Online: 2 Sep 2009; DOI 10.1002/mrd.21102.
- Wheeler, M.B., Monaco, E., Bionaz, M. and Tanaka, T. 2010. The Role of existing and emerging biotechnologies for livestock production: Toward holism. Acta Scientiae Veterinariae. 38(Supl 2): s463-s484.
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