| Title |
Investigators | Department | Objectives | Approach Keywords | Progress Reports | Impact Statements | Publications | |
Project * COL00293A(See Project History for COL00293) | |
| Title | Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock |
| Investigator(s) | Seidel, Jr., G |
| Department | Biomedical Sciences |
| Objectives | 1) Understand the biology and underlying mechanisms of gamete development, fertilization and embryogenesis. 2) Refine methods for production of genetically modified animals to improve livestock production efficiency. |
| Approach | We will study bovine oocytes and their associated cumulus cells from 1 to 2 mm and 3 to 4 mm follicles from abattoir ovaries along with in vivo-derived controls from greater than or equal to 6-mm follicles. We will study differences in mRNA content for specific genes by making corresponding labeled cDNAs and hybridizing them to cDNA microarrays for 591 human genes. Genes identified in this screen then will be examined for differences among groups more rigorously using reverse transcriptase plus real time PCR. Resulting information will be used to design improved methods of oocyte maturation. |
| Keywords | bovine, embryo, in vitro culture, oocyte maturation, microarrays, mRNA |
| Progress Reports | |
| 2005 | Our efforts toward this Regional Project center on two areas: Genes regulating oocyte maturation in cattle and cryopreservation of embryos via vitrification. We are making good progress in the former area by validating the various techniques needed to evaluate RNA in oocytes, but have not generated much data to date. Therefore, this report will concentrate on vitrification. We have focused on two aspects of cryopreservation of bovine embryos via vitrification: simplifying procedures to make them practical and developing procedures that do not include animal products, such as proteins from blood. The latter circumvents possible problems with virus contamination. We have made considerable progress on both fronts. We have developed a simple procedure to vitrify embryos so that the containers (thin plastic straws) can be thawed on the farm and the thawed embryos transferred to the uterus directly without even seeing the embryos at this step. Our preliminary results are very promising in terms of embryo survival and pregnancy rates. Dispensing with animal products in media also has been successful with no apparent decrease in efficacy. One area in which we failed was to simplify the vitrification procedure to a single step of adding embryos to cryoprotectant followed by plunging the container into liquid nitrogen. While most embryos survived this process, nearly half did not, so we had to be content with a 2-step procedure of adding cryoprotectant, which still is fairly simple. |
| 2006 | Vitrification is an effective approach to cryopreserving preimplantation bovine and equine embryos. Dozens of protocols have been published that differ in types and concentrations of cryoprotectants, timing of procedures, and the nature of the vitrification container. However , only a few studies have involved transferring the hundreds of embryos per treatment needed for thorough evaluation of pregnancy rates. We have designed a vitrification procedure that is practical under field conditions. Cryoprotectant is added to embryos in two steps, and embryos are vitrified in 0.25-ml straws. Diluent containing 1 M galactose is aspirated into 0 .25-ml straws, then air, and then vitrification solution and embryos followed by air and more diluent. These columns are mixed post-warming so that embryos can be directly transferred nonsurgically into recipients. Both vitrification solutions and diluent have been added to a medium that contains no animal products. Promising pregnancy rates have been obtained with both Bos taurus and Bos indicus embryos. |
| Impact | |
| 2005 | The simplified method of cryopreserving embryos has already been commercialized for equine embryos, and is expected to result in a commercial product for bovine embryos next year. While vitrification does not fit all commercial situations for technical reasons, we expect this technology to become widely applied in the bovine embryo transfer industry. |
| 2006 | The simplified method of cryopreserving embryos already has been commercialized for equine embryos, and is expected to result in a commercial product for bovine embryos next year. While vitrification does not fit all commercial situations for technical reasons, we expect this technology to become widely applied in the bovine embryo transfer industry. |
| Publications | |
| 2005 |
Campos-Chillon, L., T. Suh, E. Carnevale and G. Seidel, Jr. 2005. Effect of meiotic arrest by roscovitine and subsequent IVM time on developmental competence of immature bovine oocytes. Reprod. Fertil. Dev. 17:271 (abstr. #241). Coutinho da Silva, M.A. 2005. Effects of Milk Proteins on Intracellular Ca2+ Concentration and Binding of Stallion Sperm to Zonae Pellucidae. Ph.D. Dissertation, Colorado State University, Fort Collins, CO. Coutinho da Silva, M.A., G.E. Seidel, Jr., J.K. Graham, E.L. Squires and E.M. Carnevale. 2005. Effects of milk proteins and extracellular calcium concentration on the intracellular calcium concentration of stallion sperm. Theriogenology 64:802. Eldridge-Panuska, W.D., V. Caracciolo di Brienza, G.E. Seidel, Jr., E.L. Squires and E.M. Carnevale. 2005. Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos. Theriogenology 63:1308-1319. Horvath, G., L. Solti and G. Seidel. 2005. Vitrification of bovine oocytes treated with cholesterol-loaded methyl-beta-cyclodextrin. Reprod. Fertil. Dev. 17:193-194 (abstr. #87). Kutvolgyi, G., T. Suh, E. Carnevale and G. Seidel, Jr. 2005. Use of pentoxifylline and hyaluronic acid for stallion sperm separation. Reprod. Fertil. Dev. 17:310 (abstr. #319). Kutvolgyi, G., T. Suh, E. Carnevale and G.E. Seidel, Jr. 2005. Morphologic evaluations after using pentoxifylline and hyaluronic acid for stallion sperm separation. Reprod. Dom. Anim. 40:407. Lindbloom, S. 2005. Roles of EGF-Like Growth Factors and Phosphodiesterases in Initiation of Oocyte Maturation in the Equine Follicle. M.S. Thesis, Colorado State University, Fort Collins , CO. Preis, K., G. Seidel, Jr. and D. Gardner. 2005. Markers of oocyte competence during in vitro maturation. Reproduction Abstr. Series 32:48. Seidel, G.E., Jr. 2005. Mapping and sequencing the bovine genome - implications for the beef industry. Proc. 2005 Colorado Nutrition Round Table, pp. 1-9. Seidel, G.E., Jr., E.M. Carnevale, E.L. Squires, J.F. Hasler and R.J. Mapletoft. 2005. New approaches to cryopreservation: Vitrification of bovine and equine embryos. Proc. VI Simposio Internacional Reproduccion Animal, Cordoba, Argentina, pp. 327-342. Suh, T.K. and G.E. Seidel, Jr. 2005. Activation of protein kinase C and subsequent inhibition of protein phosphorylation improve embryonic development of bovine oocytes after ICSI. Biol. Reprod. (Special Issue), pp. 156-157. Suh, T.K., G.E. Seidel, Jr. and S. Purcell. 2005. Effect of protein kinase C activation followed by kinase inhibition on embryonic development of in vivo-derived equine oocytes after ICSI. Proc. Havemeyer Symposium, p. 19. Walker, D. and G. Seidel. 2005. Vitrification of bovine embryos without animal-derived products. Reprod. Fertil. Dev. 17:153 (abstr. #6). Walker, D.J. 2005. A Simple Efficacious Method for Vitrifying Bovine Embryos. M.S. Thesis, Colorado State University, Fort Collins, CO. Zhang, M., K.H. Lu and G.E. Seidel, Jr. 2005. Blastocyst development of male and female bovine embryos produced by IVF with flow cytometrically-sorted sperm. Reprod. Fertil. Dev. 17:306-307 (a |
| 2006 |
Campos-Chillon, L.F., D.J. Walker, J.F. De La Torre-Sanchez and G.E. Seidel, Jr. 2006. In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 65:1200-1214. De La Torre-Sanchez, J.F., D.K. Gardner, K. Preis, J. Gibbons and G.E. Seidel, Jr. 2006. Metabolic regulation of in vitro-produced bovine embryos II. Effects of phenazine ethosulfate, sodium azide, and 2,4-dinitrophenol during post-compaction development on glucose metabolism and lipid accumulation. Reprod. Fertil. Develop. 18:597-607. De La Torre-Sanchez, J.F., K. Preis and G.E. Seidel, Jr. 2006. Metabolic regulators of in vitro-produced bovine embryos I. Effects of metabolic regulators at different glucose concentrations with embryos produced by semen from different bulls. Reprod. Fertil. Develop. 18:585-596. Horvath, G. and G.E. Seidel, Jr. 2006. Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-β-cyclodextrin. Theriogenology 66:1026-1033. Schenk, J.L., T.K. Suh and G.E. Seidel, Jr. 2006. Embryo production from superovulated cattle following insemination of sexed sperm. Theriogenology 65:299-307. Seidel, G.E., Jr. 2006. Modifying oocytes and embryos to improve their cryopreservation. Theriogenology 65:228-235. Seidel, G.E., Jr. and D.J. Walker. 2006. Pregnancy rates with embryos vitrified in 0.25-ml straws. J. Reprod. Develop. 52 (Suppl):S71-S76. Walker, D.J., L.F. Campos-Chillon and G.E. Seidel. 2006. Vitrification of in vitro-produced bovine embryos by addition of ethylene glycol in one step. Reprod. Domest. Anim. 41:467-471. |