W171: Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[05/20/2003] [10/12/2003] [03/05/2004]

Date of Annual Report: 05/20/2003

Report Information

Annual Meeting Dates: 01/11/2002 - 01/11/2002
Period the Report Covers: 01/01/2001 - 12/01/2001

Participants

Rorie, Rick - Arkansas; Rosenkrans, Charlie - Arkansas; Anderson, Gary - California; Seidel, George - Colorado; Wheeler, Matt - Illinois; Youngs, Curt - Iowa; Godke, Bob - Louisiana; Adams, Gregg - Saskatoon; Mapletoft, Reuben - Saskatoon.

Brief Summary of Minutes

The meeting was called to order at 7:00 p.m. by Chairman Charlie Rosenkrans. Members were welcomed, and Charlie expressed regrets to the group from Jerry Yang (Connecticut), Tom Bunch (Utah), and Fred Menino (Oregon) who were unable to attend. Charlie Rosenkrans appointed Curt Youngs as acting secretary due to Jerry Yang‘s absence at the meeting. Jerry will resume his duties as secretary next year. Bob Godke moved and Rick Rorie seconded to have Curt Youngs assume the duties of president next year. Motion passed unanimously.

Bob Godke gave a brief overview of the history and purposes of the W-171 USDA Regional Research Project for the benefit of our new members. Neither Kelvin Koong (administrative advisor) nor Mark Mirando (CSREES) was able to attend, so the group did not receive comments from the administrative advisor or CSREES representative. Charlie Rosenkrans volunteered to contact both Dr. Koong and Dr. Mirando to request that any pertinent information be shared with the W-171 members via either e-mail or snail mail.

Bob Godke moved and Rick Rorie seconded to remove from the table the motion to add the Nevada station to the W-171 group. Motion passed. After prolonged discussion, a consensus was reached that insufficient information was available on which to base a decision concerning the "fit" of the proposed Nevada members with the objectives of the W-171 project. It was decided that Charlie Rosenkrans should contact the Nevada station to request that they attend the next W-171 meeting in order to answer questions for the group.

Options for the 2003 W-171 meeting were discussed. If the next meeting is held near the time of the International Embryo Transfer Society (IETS) annual meeting, it was agreed that it should be held before IETS in order to minimize conflicts with spring semester teaching schedules. Three options were proposed: Los Angeles (Ed Fonda -host?), San Francisco (Gary Anderson - host?), or Denver (George Seidel - host?). Bob Godke will contact Ed Fonda to see if he would be willing to host the group if we meet in Los Angeles. If the meeting were to be held at IETS in Auckland, New Zealand, Robin Tervit of the local organizing committee is the contact person. Members not in attendance at this year‘s meeting should be contacted about their input, and a decision should be made by September 1, 2002. Curt Youngs shall be in charge of final arrangements.

Charlie Rosenkrans reminded everyone to send to him, in electronic form, their accomplishments under each objective and their publication list no later than February 15, 2002. Please be sure to include graduate student theses and dissertations in the publication list.

No new applications for membership were submitted to the W-171 group during the past year, so no action on membership was necessary. Discussion was held concerning persons that potentially could be recruited to become W-171 members. Two suggestions were put forth: Pete Hansen, University of Florida (to be contacted by Curt Youngs), and Allan King, University of Guelph (to be contacted by Rick Rorie).

Discussions were held concerning the joint research projects. The first project centered on the efficiency of the Heatwatch system for detection of estrus. Four stations (Louisiana, Arkansas, Utah, and Iowa) indicated they had used the system with cattle. One station (Saskatchewan) used the system in wapiti, one station in sheep (Arkansas), and one station with goats (Louisiana). The system doesn‘t work well in goats, as there are too many false mounts (ovulations or lack thereof verified via laparotomy). A discussion was held concerning the efficiency of the system in dairy cattle versus beef cattle, as well as the influence of body condition score. An idea was proposed to enhance multi-species use by mounting a transmitter on a male and a receiver on a female. Bob Godke will send Gregg Adams a draft protocol for a group project. John Riesen also expressed interest in the protocol.

The second project discussion centered on the IFAS grant proposal that had been submitted to examine the effect of oocytes age and semen quality on the efficiency of in vitro fertilization. It was suggested that costs should be updated: 70 cents/oocyte and $28/straw of semen. It was agreed that we should try to gather preliminary data in the upcoming year. Charlie Rosenkrans agreed to e-mail the experimental design to all stations.

No substantial progress had been made on markers of oocytes quality. The microchannel device is still viewed as highly desirable, but due to lack of availability this project will need to be deferred. Once available, though, the device could be used across stations to compare factors such as time of maturation. Reuben Mapletoft asked if Matt Wheeler would be willing to organize a pre-conference (IETS) or post-conference symposium on oocyte quality. The W-171 group could then refine the experimental design for projects in the coming year. It was agreed that a specific action plan should be developed. The plan should name the person responsible and a deadline should be indicated.

Individual station reports were then given for the stations in attendance. Interesting discussions ensued.

Reuben Mapletoft moved and Bob Godke seconded to adjourn the meeting. The motion carried, and the meeting was adjourned at 9:40 p.m.

Respectfully submitted,

Curt Youngs

Acting Secretary

Accomplishments

Objective 1. Understanding the developmental biology and underlying mechanisms of fertilization and embryonic development. A. Managing the Female LA studied benefits of having a vasectomized bull or androgenic cow to aid in getting females to cycle and detecting estrus. There was no advantage of these animals in AI pregnancy rates, possibly because cattle were well managed. AR studied 3 commercially available methods of electronic monitoring of estrous behavior in cattle; all three were effective, and there were differences in cost and convenience. Sask studied timing of AI, 23-33 h after E2 injection and CIDR removal. No effects were noted on pregnancy rates or sex ratios of calves over this time interval. IA developed software to assist producers in choosing from a plethora of estrus synchronization procedures for cattle with inputs of costs, expected success rates, etc. B. Studies Focusing on Sperm CA studied the DRKD protein on porcine sperm, which first appears in the epididymis. Several isoforms were found, and amounts varied seasonally. LA studied deterioration of stallion epididymal sperm when castrated testes-epididymides were stored at 50C post-castration for up to 4 days. Sperm remained reasonably viable for 3 days. WA studied cooling and storing stallion sperm at 50C; it was found that egg yolk-based extenders resulted in much less chromatin damage than milk-based extenders. CA studied evaluation of bull and stallion sperm via flow cytometry, concentrating on the JC-1 stain. Interpretation of data using this stain remains problematic. IA studied use of centrifugation of sperm in Bovipure to sex bovine sperm; no effect on sex ratio was found, but cleavage rates improved slightly. LA injected sperm directly into follicles of mares, but results were not promising. C. The Oocyte, Embryo, Fetus, and Offspring LA studied potential benefits of flushing post-partum beef cows with high energy feed and/or injection of bovine growth hormone for transvaginal recovery of oocytes; all cows also received an FSH injection. Oocyte recovery and blastocyst development rates after IVF were lower with the flushing treatment than no treatment; growth hormone was beneficial alone but not with energy supplementation. A similar experiment was done with mares and equine growth hormone, and there was no benefit from growth hormone. Recovered equine oocytes were fertilized via sperm injection, with some pregnancies and a foal resulting after embryo transfer. Sask showed that echo-texture of bovine follicles is correlated with oocyte quality and subsequent blastocyst production. CO showed that roscovitine could reversibly arrest nuclear maturation of equine oocytes. Embryo quality was improved by transient arrest followed by sperm injection. WI attempted to slow bovine maturation rates by culture at 350C. Blastocyst production declined at lower temperatures and longer maturation times. WI also showed that maturation of bovine oocytes with LH was superior to FSH in terms of embryo production. IL developed microfluidic devices to mature bovine oocytes and remove cumulus cells, and they showed that development of mouse embryos was enhanced by these procedures. OR characterized metalloproteinases and their inhibitors in pregnant and non-pregnant ovine uteri and day 9-15 embryos. All genes studied were transcribed in pregnant and non-pregnant uteri, but some were missing in embryos. OR also used day-7 bovine embryos to study endodermal cell migration on various extracellular matrix proteins in vitro. CO studied calves resulting from IVF with sexed and control sperm; embryos were cultured with and without insulin. There was no effect of sexing, but insulin may have led to more problems with neonatal calves. Objective 2. Development of genetically modified animals to enhanced desirable traits. A.. Cloning by Nuclear Transplantation Utah studied potential benefits of activating oocytes with mitogens. This approach turned out not to be promising. IL studied various methods of activating porcine oocytes parthenogenetically. Some approaches were quite successful. CT studied additional permutations and combinations of approaches to activation of bovine oocytes. Chemical plus electrical combinations resulted in up to 40% parthenogenetic blastocysts. UT studied Ca++ release due to the arginine-glycine-aspartic acid tripeptide. This approach turned out to release IP3 sensitive Ca++ stores. CT studied a novel approach to removal of chromosomes from oocytes, hypertonic sucrose. This greatly facilitated the process, as chromosomes migrated to surface blebs. LA showed that bovine cumulus cells were superior nuclear donors to fibroblasts. They also studied using bovine cytoplasts to process nuclei from equine fibroblasts, with reasonable rates of early development of embryos; they surmised that this approach could be used for screening procedures. TX studied microarrays of gene expression of cloned vs IVF embryos. Most genes were expressed similarly, but a number of genes from the donor cells continued to be expressed inappropriately in the first few cleavage divisions. Utah studied telomerase expression, which was not different in IVF vs cloned bovine embryos. CO studied d-75 cloned pregnancies. There was more IGF-2 mRNA in livers of clones, but less IGF-1 and -2 in cotyledons of cloned placentae; umbilical cords were thicker in clones. No differences could be found in amounts of mRNA for angiogenic factors between clones and controls. LA found that there was less variation in blood hormone levels within bovine clonal sets than within other animals, but that variation in growth rates was not reduced. B. Transgenesis and Stem Cells CA studied porcine embryonic germ cell incorporation into chimeras by comparing blastocoele injection vs addition to the perivitelline space pre-compaction. Both procedures resulted in success at low rates. UT showed that the lytic peptide shiva-1 could be expressed in milk of transgenic mice at moderate rates using a bovine casein promoter. LA made 8 transgenic kids by cloning from a line of cells derived from a transgenic fetus sired by a transgenic buck. IL made 2 lines of transgenic pigs, one for bovine alpha lactalbumin and one for IGF-1. Transgenic mothers resulted in faster growth of suckling piglets when transgenic for alpha lactalbumin but not IGF-1. Piglets suckling sows transgenic for IGF-1 had higher jejunal villi than controls. Future Plans AR will investigate the effect of bovine somatotropin on embryo quality, embryo transfer pregnancy rates, and follicular wave dynamics. CA will characterize sperm and oocyte molecules involved with fertilization and utility of embryonic germ cells for transgenesis. CO will study bovine and equine in vitro oocyte maturation and culture, as well as gene expression in placentae of cloned versus control pregnancies. CT will investigate methods for oocyte activation and enucleation, as well as methods for oocyte cryopreservation. IL will continue research with transgenic swine and embryo culture in microfluidic devices. IA will compare the efficacy of dinoprost versus cloprostenol for induction of estrus in postpartum dairy cows. LA will investigate various in vitro embryo culture systems and will continue research with nuclear transfer and transgenesis in farm animals. OR will study transcription factor expression and cellular differentiation during bovine embryonic development. TX will continue studies with differential gene expresssion in cloned versus control embryos. UT will evaluate protein supplment and gorwth factors for use in in vitro embryo culture. WA will examine the relationship between sperm DNA quality and fertility rates. WI will investigate methods for bovine oocyte cryopreservation.

Publications

REFEREED JOURNAL ARTICLES: ARKANSAS Brown, A H, D W Kellogg, Z B Johnson, R W Rorie, W K Coblentz, B A Sandelin, K E Lesmeister and W R Jackson. 2001. Growth, luteal activity and pregnancy rates of three breed types of dairy heifers in a forage based development program. Prof. Anim. Sci. 17:20-26 Burke, J.M., R.W. Rorie and G.A. Perry. 2002. Changes in ovarian function in mature beef cows grazing endophyte-infected tall fescue. Theriogenology (in press). Burke, J M, R W Rorie, E L Piper and W G Jackson. 2001. Reproductive responses to grazing endophyte-infected tall fescue in postpartum beef cows. Theriogenology 56:357-369. Kreider, D L, R Rorie, D Brown, F Miller and S Wright. 2001. Enhancement of ovulation rate and litter size in gilts. J. Anim. Sci. 79:1691-1696. Melnychuk, V.L., M.A. Cooper, R.W. Rorie, J.D. Kirby and N.B. Anthony. 2001. Characterization of ovary status using ultrasound techniques. J. Poultry Sci. (in press) Rorie, R.W., T.R. Bilby, and T.D. Lester. 2002. Application of electronic estrus detection technology to reproductive management of cattle. Theriogenology (in press). CALIFORNIA Berger, T. and Dally, M. 2001. Do sire-dam interactions contribute significantly to fertility comparisons in heterospermic insemination trials? Theriogenology 56:535-543. Bertolini, M., J.B. Mason, S.E. Beam, G.F. Carneiro, M.L. Sween, D.J. Kominek, A.L. Moyer, T.R. Famula, R.D. Sainz and G.B. Anderson. 2002. Morphology and morphometry of in vitro-and in vitro-produced concepti from early pregnancy to term and association with high birth weights. Theriogenology (in press). Carneiro, G., P. Lorenzo, C. Pimental, L. Pagoraro, M. Bertolini, B. Ball, G. Anderson and I. Liu. 2001. The influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol and fetal calf serum and in vitro maturation and parthenogenic development in equine oocytes. Biology of Reproduction 65:899-905. Gravance, C.G., Garner, D.L., Miller, M.G. and Berger, T. 2001. Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function. Reprod. Tox. 15:5-10. Jelks, K., Berger, T., Horner, C., and Miller, M.G. 2001. Chlorohydrin induced changes in sperm fertilizing ability in the rat: association with diminished sperm ATP levels and motility. Reprod. Tox. 15:11-20. COLORADO Choi, Y.H., E.M. Carnevale, G.E. Seidel, Jr. and E.L. Squires. 2001. Effects of gonadotropins on bovine oocytes matured in TCM-199. Theriogenology 56:661-670. Choi, Y.H., Y.G. Chung, G.E. Seidel, Jr. and E.L. Squires. 2001. Developmental capacity of equine oocytes matured and cultured in equine trophoblast- conditioned media. Theriogenology 56:329-339. Landim-Alvarenga, F.C., M.A. Alvarenga, G.E. Seidel, Jr., E.L. Squires and J.K. Graham. 2001. Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187. Theriogenology 56:937-953. Lane, M., M.K. O‘Donovan, E.L. Squires, G.E. Seidel, Jr. and D.K. Gardner. 2001. Assessment of metabolism of equine morula and blastocysts. Molec. Reprod. Develop. 59:33-37. Oberstein, N., M.K. O‘Donovan, J.E. Bruemmer, G.E. Seidel, Jr., E.M. Carnevale and E.L. Squires. 2001. Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methods. Theriogenology 55:607-613. Seidel, G.E., Jr. 2001. Cloning, transgenesis, and genetic variance in animals. Cloning and Stem Cells 4:251-256. Coutinho da Silva, M.A., E.M. Carnevale, L.J. Maclellan, G.E. Seidel, Jr. and E.L. Squires. 2002. Effect of time of oocyte collection and site of insemination on oocyte transfer in mares. J. Anim. Sci. (in press). Li, G.-P., G.E. Seidel, Jr. and E.L. Squires. 2002. Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa. Theriogenology (in press). Li, G.-P., G.E. Seidel, Jr. and E.L. Squires. 2002. Improved cleavage of bovine ICSI ova cultured in heparin-containing medium. Theriogenology (in press). CONNECTICUT Deng, M.Q. and X. Yang. 2001. Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection. Molec. Reprod. Devel. 58:1-6. Dinnyes, A., L. Liu, Y. Dai, M. Barber, J. Xu, P. Zhou, and X. Yang. 2001. Development of cloned embryos from adult rabbit fibroblast cells. Biol. Reprod. 64:257-263. Jeong, B.S. and X. Yang. 2001. Cysteine, glutathione, and percoll treatments improve porcine oocyte maturation and fertilization in vitro. Molec. Reprod. Devel. 59:330-335. Tian, XC, C. Kubota, and X. Yang. 2001. Cloning of aged animals - a medical model for tissue and organ regeneration. Trends in Cardiovascular Medicine 11:313-317. Xu, J. and X. Yang. 2001. Telomerase activity in early bovine embryos derived from pathenogenetic activation and nuclear transfer. Biol. Reprod. 64:770-774. Yang, X. and J. Xu. 2001. Developmental changes of telomerase in oocytes and embryos. Advances in Reproduction 6:63-68. Enright, B., M. Taneja, J. Riesen, X.C. Tian, J. Fortune, and X. Yang. 2002. Normal onset of puberty in cloned heifers of an aged Holstein cow. Biol. Reprod (in press). Govoni, K.E., X.C. Tian, G.M. Kazmer, M. Taneja, B.P. Enright, A.L. Rivard, X. Yang, and S.A. Zinn. 2002. Age-related changes of the somatotropic axis in cloned Holstein calves. Biol. Reprod. (in press). Liu, J.L., L.-Y. Sung, M. Barber, and X. Yang. 2002. Hypertonic medium treatment for localization of nuclear material in bovine metaphase II oocytes. Biol. Reprod. (in press). Tian, X.C., P. Lonergan, A.E.O. Evans, and X. Yang. 2002. Dynamics of MPF and MAPK in oocytes matured in vitro for 20 vs. 40 hours. Molec. Reprod. Devel. (in press). ILLINOIS Boston, W.S., G.T. Bleck, M.B. Wheeler, D.J. Miller. 2001. The effects of increased expression of alpha-lactalbumin in transgenic mice on milk yield and pup growth. J. Dairy Sci. 84:620-622. Donovan, S. M. , M. H. Monaco, G. T. Bleck, J. B. Cook, M. S. Noble, W.L. Hurley and M.B. Wheeler. 2001. Transgenic over-expression of bovine alpha-lactalbumin and human insulin-like growth factor-I in porcine mammary gland. J. Dairy Sci. 84 (E. Suppl.): E216-E222. (http://www.adsa.org/jds/papers/2001/jds_es216.pdf) Glasgow, I.K., Zeringue, H.C., Beebe, D.J., Choi, S.-J., Lyman, J., Chan, N.G., Wheeler, M.B. 2001. Handling individual mammalian embryos using microfluidics. IEEE Transactions on Biomedical Engineer 48:570-577. Wheeler, M.B. And Walters, E.M., 2001. Transgenic technology and applications in swine. Theriogenology 56:1345-1370. Zeringue, H., M. B. Wheeler and D. J. Beebe, 2001. Removal of cumulus from mammalian oocytes using microfluidic techniques. Biomedical Microdevices 3:219-224. IOWA Youngs, C.R. 2001. Factors influencing the success of embryo transfer in the pig. Theriogenology 56:1311-1320. LOUISIANA Han, Y., M. Meintjes, K. Graff, R. Denniston, L. Zhang, C. Ziomek and R.A. Godke. 2001. Production of fresh and frozen-thawed transplant offspring from latter stage IVF-derived caprine embryos. Vet. Rec. 149:714-716. Meintjes, M., B. Eilts, K. Graff, R. Denniston, D. Paccamonti and R. Godke. 2001. Transvaginal intrafollicular sperm cell injection in the cyclic mare. Internat‘l. Symp. Equine Embryo Transf. Monograph 3:39-40. Murakami, M., A. Boediono, O. Perez, E. Ferguson, R. S. Denniston, E. Behboodi and R. A. Godke. 2001. The use of adult bovine skin fibroblasts cultured at different intervals prior to nuclear transfer. Arch. Anim. Breeding 44:175-177. Poleo, G.A., R. S. Denniston, B. Reggio, R. A. Godke and T. R. Tiersch. 2001. Fertilization of eggs of Zebrafish (Danio rerio) by intracytoplasmic sperm injection. Biol. Reprod. 65:321-326. Reggio, B.C., A.N. James, H. L. Green, W. G. Gavin, E. Behboodi, Y. Echelard and R. A. Godke. 2001. Cloned transgenic offsring in goats resulting from somatic cell nuclear transfer in goats: Oocytes derived from FSH-stimulated and nonstimulated ovaries. Biol Reprod. 65:1528-1533. Reggio, B.C., M. Sansinena, R.A. Cochran, A. Guitreau, J.A. Carter, R.S. Denniston and R.A. Godke. 2001. Nuclear transfer embryos in the mare. Internat‘l. Symp. Equine Embryo Transf. Monograph 3:45-46. Boediono, A., T. Suzuki and R.A. Godke. The development of in vitro-derived hybrid and straightbred beef cattle embryos during in vitro culture. Anim. Reprod. Sci. (in press). Carter, J.A., S. Bellow, M. meintjes, O. Perez, E. Ferguson and R. A. Godke. Transvaginal ultrasound-guided oocyte aspiration for production of embryos in vitro. Arch. Anim. Breeding (in press). Cochran, R., M. Meintjes, D. Hylan, M. Sansinena, R. S. Denniston D. Paccamonti and R.A. Godke. Follicle growth in mares for oocyte production. In: From Epididymis to Embryo. Havemeyer Foundation. (in press) Han, Y., Y. Han, R.S. Denniston, A. Boediono, C. Ziomek and Godke. Development of IVF-derived caprine embryos with four different somatic cell co-cultures. Small Ruminant Res. (in press). Murakami, M., S. Ideguchi, M. Fahrudin and R.A. Godke. Bovine nuclear transplant using fluorescent embryos after microinjection with the EGFP gene. Anim. Reprod. Develop. (in press). Pinto, C.R.F., D.L. Paccamonti, B.E. Eilts, C.R. Short and R.A. Godke. Effect of nitric oxide synthetase inhibitors on ovulation in hCG-stimulated mares. Theriogenology (in press). Sansinena, M., Paccamonti, D., C. Cochran, M. Meintjes, B. Reggio, R. S. Denniston, C. Pinto, B. Eilts and R.A.Godke. Developing the intracytoplasmic sperm injection (ICSI) procedure for the horse. In: From Epididymis to Embryo. Havemeyer Foundation. (in press). Songasen, N., I. Yu, S. Murton, D. Paccamonti, B.E.Eilts, R. A. Godke and S.P. Leibo. Osmotic sensitivity of Canine Spermatozoa. Cryobiology (in press). Yu, I.J., N. Songsasen, R.A. Godke and S.P. Leibo. Differences among male dogs in response of their spermatozoa cryopreservced at various coolling and warnimg rates. Cryobiology (In press). OREGON Al-Aghbari, A.M. and A.R. Menino, Jr. 2002. Survival of oocytes recovered from vitrified sheep ovarian tissues. Anim. Reprod. Sci.(in press) Schilperoort-Haun, K.R. and A.R. Menino, Jr. 2002. Evaluation of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 on bovine inner cell mass outgrowth in vitro. In Vitro Cell. Dev. Biol.-Animal (in press). UTAH Kitiyanant, Y., J. Saikhun, B. Chrisalee, K.L. White and K. Pavasuthipaisit. 2001. Somatic Cell Cloning in Buffalo (Bubalus bubalis): Effects of interspecies cytoplasmic recipients and activation procedures. Cloning and Stem Cells 3:97-104. Thonabulsombat, C., W.A. Reed, J.D. Morrey, K. Bates, R. Smart and K.L. White. 2001. Transgenic mice carrying a gene for green fluorescent protein fused to a lytic peptide, Shiva 1, under control of the bovine 8-Casein regulatory region. Transgenics 3:183-197. Viets, L.N., K.D. Campbell and K.L. White. 2001. Pathways involved in RGD-mediated calcium transients in mature bovine oocytes. Cloning and Stem Cells 3:105-113. Wang, S., W.C. Foote, D.L. Sutton, A. Maciulis, J.M. Miller, R.C. Evans, G.R. Holyoak, J.W. Call, T.D. Bunch, W.D. Taylor and M.R. Marshall. 2001. Preventing experimental vertical transmission of scrapie by embryo transfer. Theriogenology. 56:315-327. Wang, S., K.E. Panter, R.C. Evans, and T.D. Bunch. 2001. The effects of pokeweed mitogen (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation and embryo development in vitro. Anim. Reprod. Sci. 67:215-220. WASHINGTON Bosch, P., J.M. deAvila, J.E. Ellington, and R.W. Wright, Jr. 2001. Heparin and Ca2+-free medium can enhance release of bull sperm attached to oviductal epithelial cell monolayers. Theriogenology 56:247-260. WISCONSIN Rutledge, J.J. 2001. Greek temples, tropical kine and recombination load. Livestock Production Science 68:171-179. Rutledge, J.J. 2001. Use of embryo transfer and IVF to bypass effects of heat stress. Theriogenology 55:105-111. Al-Katanani, Y.M., M. Drost, R.L. Monson, J.J. Rutledge, C.E. Krininger III, C.J. Block, W.W. Thatcher, and P.J. Hansen. 2002. Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions. Theriogenology (in press). Men, H., R.L. Monson, and J.J. Rutledge. 2002. Effect of maturation process and maturation protocols on bovine oocyte freezing tolerance. Theriogenology (in press). BOOKS, CHAPTERS IN BOOKS, PROCEEDINGS, AND THESES: CALIFORNIA Berger, T. and Dally, M. 2001. Is there a sire-dam interaction in sperm fertilizing potential? California Agriculture 55:25. COLORADO Choi, Y.H., G.E. Seidel, Jr., S. Boyazoglu and E.L. Squires. 2001. Polyvinylalcohol is superior to bovine serum albumin in equine IVF medium. Proc. 5th Int. Symp Equine Embryo Transfer, T. Katila and J.F. Wade (Eds), Havemeyer Foundation Monograph Series No. 3, pp. 28-29. Garner, D., J. Schenk and G. Seidel, Jr. 2001. Chromatin stability in sex-sorted sperm. Proc. VII Int. Congr. Androl., pp. 3-7. Landim-Alvarenga, F.C., S.E.A. Boyazoglu, G.E. Seidel, Jr. and E.L. Squires. 2001. Effect of fetuin on zona hardening and the distribution of cortical granules in equine oocytes matured in vitro. Proc. 5th Int. Symp Equine Embryo Transfer, T. Katila and J.F. Wade (Eds), Havemeyer Foundation Monograph Series No. 3, pp. 26-27. Oberstein, N., M.K. O‘Donovan, J.E. Bruemmer, M. Lane, G.E. Seidel, Jr., E.M. Carnevale and E.L. Squires. 2001. Cryopreservation of equine embryos by open pulled straw, cryoloop or conventional slow cooling methods. Proc. 5th Int. Symp Equine Embryo Transfer, T. Katila and J.F. Wade (Eds), Havemeyer Foundation Monograph Series No. 3, pp. 54-56. O‘Donovan, M.K., N. Oberstein, J.E. Bruemmer, M. Lane, G.E. Seidel, Jr., D.K. Gardner and E.L. Squires. 2001. Metabolic activity of equine blastocysts cryopreserved in ethylene glycol and galactose. Proc. 5th Int. Symp Equine Embryo Transfer, T. Katila and J.F. Wade (Eds), Havemeyer Foundation Monograph Series No. 3, pp. 57-59. Seidel, G.E., Jr. 2001. Cloning (Genetics). McGraw-Hill Yearbook of Science and Technology, pp. 98-100. Seidel, G.E., Jr. 2001. Sexing bovine sperm. Proc. 34th Ann. Mtg. Am. Assoc. Bovine Practitioners, pp. 106-108 and Reprinted in substantially similar form in Ann. Proc. Korean Societies of Animal Reproduction (2001), pp. 6-10. Seidel, G.E., Jr. 2001. Procedures for sexing bovine sperm. In: Advances in Beef Cattle Science, K.A. Beauchemin and D.H. Crews, Jr. (Eds). Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, pp. 235-239. Seidel, G.E., Jr. and J.F. Hasler. 2001. El sexado de semen bovino. Proc. 4b Simposio Internacional de Reproduccion Animal, pp. 25-30. CONNECTICUT Dinnyes, A., X.C. Tian, and X. Yang. 2002. Cloning of Rabbits. In (J.B. Cibelli, R.P. Lanza, K.H.S. Campbell, and M.D. West, Eds.) Principles of Cloning, Academic Press, San Diego, CA (in press). Liu, L., M. Deng, X.C. Tian, and X. Yang. 2002. Principles and practice of oocyte activation in mammals. In (Daulat Talsiani, Ed.) Introduction to Mammalian Reproduction (in press). ILLINOIS Hickman, Debra. 2001. Efficacy of In Vitro Culture of Murine Preimplanation Embryos in Static And Dynamic Media Environments. M.S. Thesis, University of Illinois at Urbana-Champaign, 76 pgs. Malusky, Samantha. 2001. Cloning and Sequence Analysis of Porcine Sucrase-Isomaltase Promoter and Transmembrane Region. M.S. Thesis, University of Illinois at Urbana-Champaign, 64 pgs. Miller, D.J. and Wheeler, M.B. 2001. Cloning dairy cattle: How is it done and what is the future? Illinois Dairy Report: Focus on the Future, Illinois Agricultural Experiment Station pgs 28-30. Noble, M.S., Wheeler, M.B., Hurley, W.L. 2001. Association of total milk solids intake and litter growth: Data from a-lactalbumin transgenic sows. Illinois Swine Report: Illinois Agricultural Experiment Station, pp. 131-133. Raty, Stephannie. 2001. Design and Testing of Microfluidic Devices for Static Culture of Preimplantation Mammalian Embryos. M.S. Thesis, University of Illinois at Urbana-Champaign, 70 pgs. IOWA Sieren, K.R. 2001. The Use of BoviPure for the Separation of X and Y Chromosome-Bearing Bovine Sperm Cells. M.S. Thesis, Iowa State University, 61 pgs. Sieren, K.R. and C.R. Youngs. 2001. Assessment of BoviPure for the in vitro production of bovine embryos. Beef Research Report, Iowa State University, A.S. Leaflet R1768, pp. 142-144. Youngs, C.R. 2001. Cattle breeding in the 21st century. Proc. of the 30th Annual Cornbelt Cow-Calf Conference, February 24, Ottumwa, IA, pp. 1-9. Youngs, C.R. 2001. Sheep and goat reproduction. Proc. of the Student American Veterinary Medical Association Symposium, March 8-10, Ames, IA, pp. 122-128. Youngs, C.R., Ed. 2001. Proc. of the International Conference Commemorating the 50th Anniversary of the First Successful Embryo Transfer in the Pig. Theriogenology 56(8):1285-1369. LOUISIANA Bellow, M.S., B.A. Didon, D.L. Davis, C. Murphy, C.E. Ferguson, R.A. Godke and B.N. Day. 2001. A procedure for nonsurgical transvaginal ultrasound-guided oocyte aspiration in the sow. Cook Veterinary Symposium on Transvaginal Oocyte Aspiration/In Vitro Fertilization in the Pig (Omaha, Nebraska). pp. 28-29. Carter, J.A., M.S. Bellows, M. Meintjes, O. Perez, E. Ferguson and R.A. Godke. 2001. Trans-vaginal ultrasound-guided oocyte aspiration in farm animals for in vitro embryo production. Cook Veterinary Symposium on Transvaginal Oocyte Aspiration/In Vitro Fertilization in the Pig (Omaha, Nebraska). pp. 17-27. Ferguson, C.E., O. Perez, C. Airhart and R. A. Godke. 2001. Estrous synchronization using MGA or altrenogest in crossbred beef heifers. LA. Beef Cattle Res. Rep. 31:31-33. Fernandez, J.M., J.E. Miller, and R.A. Godke (2001) Conservation plan for the American Gulf Coast Native sheep. In: Proc. Vth Congreso Iberoamericano de Razas Autoctonas y Criollas, Havana, Cuba (November 28 - December 1, 2000). pp. 42-43. Gentry, G.T., R.P. Del Vechio, and R.A. Godke. 2001. Estrual behavioral patterns in prostaglandin-induced and naturally cycling recipient females from a commercial embryo transfer facility. LA. Beef Cattle Res. Rep. 31:5-8. Gentry, G.T., Jr., R.P. Del Vecchio D. Faber and R.A. Godke. 2001.The effect of prostaglandin F2 (Lutalyse.) on the behavioral estrus patterns in beef cattle. Louisiana Cattleman (June Issue). 2 pages. Godke, R.A. New assisted reproductive technologies: Their potential for use in large animals. Proc. Zool. Biotechnol Prod. Conf. (Portugal) (In Press). Perez, O., R. Richard III, H.L. Green and R.A. Godke. 2001. Ultrasound-guided transvaginal oocyte recovery from postpartum beef cows. LA. Beef Cattle Res. Rep. 31:9-11. OREGON Paul, K. B. 2001. Membrane-type Matrix Metalloproteinase and Inhibitor Expression in Sheep Embryos and Uterus. M.Sc. Thesis, Oregon State University, Corvallis, OR. UTAH Wang, S., K.E. Panter, R.C. Evans and T.D. Bunch. 2001. Bovine embryo development in vitro in medium containing pokeweed mitogen or phytohemagglutinin. Proceedings Western Section Amer. Soc. Anim. Sci. 52: 436-439. Wang, S., K.E. Panter, L.F. James, D.R. Gardner, R.C. Evans and T.D. Bunch. 2001. In vitro development of bovine embryos treated with isocupressic acid (ICA), a pine needle abortifacient toxin. Proceedings Western Section Amer. Soc. Anim. Sci. 52: 440-443. White, K. L., T.D. Bunch, S. Mitalipov and W.A. Reed. 2001. Application of ARTs and nuclear transfer in exotic and endangered species. In (D.P. Wolf and M. Zelinski-Wooten, Eds.) Comparative Endocrinology: Assisted Fertilization and Nuclear Transfer in Mammals. Humana Press Inc., Totowa, NJ, pp. 229-251. WISCONSIN Rutledge, J.J. 2001. Biotechnology enables matching global genetic resources with local environmental niches in the Tribe Bovini (true callte). Proc. Symp. Int‘l. Conf. on Agricultural Science and Technology, November 7-9, Beijing, China, pp. ABSTRACTS: ARKANSAS Bilby, T.R. and R.W. Rorie. 2001. Effectiveness of maturational treatments to block porcine oocyte germinal vesicle breakdown without reducing subsequent nuclear maturation and cleavage. Theriogenology 55:463. Bilby, T.R. and R.W. Rorie. 2001. Effects of sequence and timing of gonadotropin supplementation on porcine oocyte maturation and subsequent cleavage in vitro. J Anim Sci 79(Suppl 2):20. Burke, J.M., R.W. Rorie, G.A. Perry, and D.H. Keisler. 2001. Changes in ovarian function in mature beef cows grazing endophyte-infected (E+) tall fescue. Biol. Reprod. 64(Suppl. 1):344. CALIFORNIA Ducummon, C. and T. Berger. 2001. Cytotoxic necrotizing factor, a bacterial activator of the monomeric G?protein Rho?A, enhances spontaneous acrosome reactions in porcine sperm. Biol. Reprod. 64(Suppl 1):300. Sartini, B.L. and T. Berger. 2001. Localization of DRKD sperm protein on boar sperm. Biol. Reprod. 64(Suppl 1):277. Williams, J., B.L. Sartini, and T. Berger. 2001. Flow cytometric analysis of boar sperm mitochondrial function with JC-1. Biol. Reprod. 64(Suppl 1):301-302. COLORADO DeLille, A.J.A.E., R.V. Anthony and G.E. Seidel, Jr. 2001. Characteristics of placental and fetal tissues from Day 75 nuclear cloned bovine pregnancies. Theriogenology 55:263. Lu, K.H., T.K. Suh and G.E. Seidel, Jr. 2001. In vitro fertilization of bovine oocytes with flow-cytometrically sorted and unsorted sperm from different bulls. Theriogenology 55:431. Seidel, G.E., Jr. and L.J. Maclellan. 2001. Effect of peptide Fert Plus on bovine in vitro fertilization and development. Theriogenology 55:437. CONNECTICUT Deng, M. and X. Yang. 2001. Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection. Theriogenology 54:502. Enright, B.P., M. Taneja, D. Schreiber, J. Riesen, X.C. Tian, and X. Yang. 2001. Puberty and early follicular dynamics in adult somatic cell derived cloned heifers. Theriogenology 54:267. Lee, J.-W., B.-S. Jeong, J. Riesen, T. Hoagland, and X. Yang. 2001. Fertilization and embryonic development following injection of frozen-thawed spermatozoon into in vitro matured porcine oocytes. Theriogenology 54:506. Taneja, M., R. French, H. Levine, D. Tauro-Miller, and X. Yang. 2001. Clinical and pathological status of cloned calves born pre-term. Theriogenology 54:293. ILLINOIS Chan, N.G., S. Raty , H.C. Zeringue, D.J. Beebe, M.B. Wheeler. 2001. Development of microfabricated devices for embryo production: Embryo biocompatibility. Theriogenology 55:332. Clark, S.G., J. Davis, D. J. Beebe, M.B. Wheeler. 2001. Biocompatibility of porcine sperm cells in polydimethylsiloxane (PDMS). Theriogenology 55:421. Hassun, P., Silva, I.G., Wheeler, M.B., Baracat, E.C., Freitas, V., Verreschi, I.T.N. 2001. Analysis of polymorphisms in the QM gene in women with premature ovarian dysfunction. Brazilian Human Genome Meeting. Monaco, M.H., Cook, J.B., Tappenden, K., Wheeler, M.B., Donovan, S. M. 2001. Intestinal lactase activity is increased in piglets suckling IGF-transgenic sows. The FASEB Journal: A644. Raty , S., J. Davis , M.B. Wheeler, D.J. Beebe. 2001. Culture in microchannels enhances in vitro culture of preimplantation mouse embryos. Theriogenology 55:241. Walters, E.M., D.J. Beebe, M.B. Wheeler. 2001. In vitro maturation of pig oocytes in PDMS and silicon microfludic devices. Theriogenology 55:497. IOWA Dikeman, M.A., D.R. Strohbehn, and C.R. Youngs. 2001. Development of a web-based scheduling program for synchronization of estrus in cattle. J. Anim. Sci. 79(Suppl.2):15. Funk, D.J. and C.R. Youngs. 2001. Embryo transfer, in vitro fertilization, cloning and transgenic production in farm animals: A review. Proc. of the Student American Veterinary Medical Association Symposium, March 8-10, Ames, IA, p. 89 Pugh, M.L., M. Pence, J.N. Caamaqo, S. Robbe, L.L. Timms, J.U. Thomson, and C.R. Youngs. 2001. Use of a vaginal mucus electrical resistance probe to alter insemination time and calf gender distribution in beef heifers. J. Anim. Sci. 79(Suppl. 2):66-67. Pugh, M.L., K.R. Sieren, L.L. Timms, and C.R. Youngs. 2001. Use of a vaginal mucus electrical resistance probe to alter insemination time and calf gender distribution in dairy heifers. J. Dairy Sci. 84(Suppl. 1):64. Sieren, K.R. and C.R. Youngs. 2001. Evaluation of BoviPure for the in vitro production of bovine embryos. Theriogenology 55:438. LOUISIANA Bartels, P., Y. Friedmann, K. Lubbe, D. Mortimer, L.A. Rasmussen and R.A. Godke. 2001. The live birth of an eland (Taurotragus oryx) calf following estrous synchronization and artificial insemination using frozen-thawed epididymal sperm. Theriogenology 55:381. Bellow, M.S., B.A. Didon, D.L. Davis, C. Murphy, C.E. Ferguson, R.A. Godke and B.N. Day. 2001. A procedure for nonsurgical transvaginal ultrasound-guided oocyte aspiration in the sow. Theriogenology 55:528. Chen, L.H., E. Behboodi, B.C. Reggio, H. Liem, M.M. Destrempes, H.L. Green, C.A. Ziomek, R.S. Denniston, Y. Echelard, R.A. Godke and H.M. Meade. 2001. Production of transgenic goats from a transfected fibroblast cell line. Theriogenology 55:259. Cochran, R., M. Meintjes, D. Hylan, M. Sansinena, R. S. Denniston D. Paccamonti and R.A. Godke. 2001. Follicle growth in mares for oocyte production. In: From Epididymis to Embryo. Havemeyer Foundation Workshop. (October 18-21, 2001) New Orleans. p. 21.(Abstract) Gentry, G.T., R.P. Del Vecchio and R.A. Godke. 2001. Effect of the presence of the male on characteristics of estrus of synchronized and naturally cycling crossbred heifers. J. Anim. Sci., Southern Section (Abstr.). Paccamonti, D., M. Sansinena, C. Cochran, M. Meintjes, B. Reggio, R. S. Denniston, C. Pinto, B. Eilts and R.A.Godke. 2001. Developing the intracytoplasmic sperm injection (ICSI) procedure for the horse. In: From Epididymis to Embryo. Havemeyer Foundation Workshop. (October 18-21, 2001) New Orleans. p. 26. (Abstract). Perez, O., A. Boediono, E. Ferguson, C. Airhart, R. Richard III and R.A. Godke. 2001. Oocyte and embryo production from FSH-treated postpartum beef cows shortly after calving. Theriogenology 55:501. Poleo, G. A., R. S. Denniston, B. Reggio, R. A. Godke and T.R. Tiersch. 2001. Intracytoplasmic sperm injection (ICSI) in Zebrafish eggs. Proc World Aquaclture Soc. (Orlando, FL). Songsasen, N., I. Yu, D. Paccamonti, B.E. Eilts, S. Mourton, R.A. Godke and S.P. Leibo. 2001. Osmotic sensitivity of canine spermatozoa. Proc. 37th Annual Mtg. Society of Cryobiology (Boston). p. 350, No. 60. Songsasen, N., I. Yu, D. Paccamonti, B.E. Eilts, S. Mourton, R.A. Godke and S.P. Leibo. 2001. Survival and binding ability of canine spermatozoa stored at 4*C. Proc. 37th Annual Mtg. Society of Cryobiology (Boston). P. 361,No. 61. Taylor, P.J., S. Taylor, A.N. James, R.S. Denniston and R.A. Godke. 2001. Alpaca offspring born after cross species embryo transfer to llama recipients. Theriogenology 55:401. UTAH Panter, K.E., S.Wang, L.F. James, W. Garfield, R.C. Evans and T.D. Bunch. 2001. Effects of Cyclopamine and synthetic analog on bovine oocyte maturation in vitro. Theriogenology 55:491. Wang, S., K.E. Panter, L.F. James, W. Garfield, R.C. Evans and T.D. Bunch. 2001. The effects of steroidal alkaloids from Veratrum californium on bovine embryo development in vitro. Theriogenology 55:345. WISCONSIN Al-Katanani, Y.M., M. Drost, R.L. Monson, J.J. Rutledge, C.E. Krininger III, C.J. Block, and P.J. Hansen. 2001. Pregnancy rates in lactating dairy cattle following timed embryo transfer under heat stress condition. J. Anim. Sci. (Suppl): (in press). MISCELLANEOUS PUBLICATIONS: COLORADO Seidel, G.E., Jr. 2001. What about sexed semen? Hoard‘s Dairyman 146:322. Seidel, G.E., Jr. 2001. Sexing bovine sperm. Handout for National Association of Animal Breeders Mexican Seminar Series. CONNECTICUT Tian, X.C. 2001. How safe is milk from cloned cows? University of Connecticut College of Agriculture Newsletter. Yang, X. and X.C. Tian. 2001. Don‘t ban therapeutic cloning. Commentary in Hartford Currant, December 2, 2001. IOWA Youngs, C.R. 2001. A new millennium for the Jersey breed. Iowa Jersey Today 7(2):5. Youngs, C.R. 2001. The Magnificent Seven... hormones controlling reproduction in the cow (Part I). Iowa Jersey Today 7(3):6. Youngs, C.R. 2001. Use of a vaginal mucus electrical resistance probe as a breeding tool for dairy cattle. Swiss Valley Dairyman, November issue, pp. 6-7.

Impact Statements

Methods have been developed that enable the harvest of oocytes from cattle, horses, goats, and pigs using transvaginal ultrasound-guided procedures. This development allows oocytes to be harvested from cyclic, postpartum, and early pregnant females for subsequent embryo production using standard in vitro fertilization protocols, increasing the efficiency of animal production per female.
Methods for enhanced in vitro oocyte maturation, in vitro fertilization, and in vitro culture of embryos have been developed. This breakthrough enables embryos to be produced in vitro but transferred to recipient females using standard non-surgical embryo transfer techniques, leading to increased improvement in livestock production.
Improved methods for oocyte activation and enucleation have been developed. These enhancements to existing procedures will significantly aid in the more efficient production of nuclear transfer offspring.
Offspring produced via in vitro fertilization with sexed versus unsexed bovine sperm were compared and were found to be normal and not different from one another.
An innovative microchannel culture device shows great promise for individual culture of oocytes and embryos. This development will enable researchers to more easily identify genes of importance to embryonic development that would be more difficult to perform with culture of groups of oocytes or embryos.

Date of Annual Report: 10/12/2003

Report Information

Annual Meeting Dates: 08/14/2003 - 08/15/2003
Period the Report Covers: 01/01/2002 - 12/01/2002

Participants

Rorie, Rick - Arkansas; Rosenkrans, Charlie - Arkansas; Berger, Trish -California; Seidel, George - Colorado; Yang, Jerry - Connecticut; Tian, Cindy - Connecticut; Wheeler, Matt - Illinois; Youngs, Curt - Iowa; Godke, Bob - Louisiana; Menino, Fred - Oregon; Bunch, Tom - Utah; Rutledge, Jack - Wisconsin; Beattie, Craig - Nevada (guest); Hu, Ching Yuan - Oregon State University Agricultural Experiment Station; Mirando, Mark - CSREES.

Brief Summary of Minutes

The annual meeting was called to order by Chairman Curt Youngs at 8:00 AM. Members in attendance were Rick Rorie and Charles Rosenkrans (Arkansas), Trish Berger (California), George Seidel (Colorado), Jerry Yang and Cindy Tian (Connecticut), Matt Wheeler (Illinois), Curt Youngs (Iowa), Bob Godke (Louisiana), Fred Menino (Oregon), Tom Bunch (Utah) and Jack Rutledge (Wisconsin). Craig Beattie (Nevada) attended as a potential new member. Also present were project administrators Ching Yuan Hu (Oregon State Agri. Exp. Station) and Mark Mirando (CSREES).

After review, Jack Rutledge made a motion to approve the minutes of the previous meeting. The motion was seconded by Tom Bunch, and approved unanimously by the members.

Dr. Hu reported that although the W171 project has a very good history, a review by the Regional Implementation Coordination Committee (RCIC) found that the project is missing at least some of the required annual reports. Dr. Hu indicated that currently the 2000 and 2001 (FY 1999 and 2000 activities) annual reports were missing, and that annual reports must be submitted via NIMSS web site (www.lgu.umd.edu) within 60 days of the annual meeting. Discussion followed that members have prepared and submitted an annual report each year, but members were never informed of the requirement to post the report via the NIMSS web site. After discussion, a plan of action was adopted that members from each station should email to Chairman Youngs (by Sept 15th) the following information (from their Jan. 2000 and 2001 station reports): 1) list of publications, 2) summary of accomplishments for each objective 3) bulleted list of impacts, and 4) description of future research plans. Chairman Youngs will prepare and submit the necessary reports to get reporting requirements up-to-date.

Dr. Hu indicated that the project must be revised and submitted by January 15, 2004, or the project will be terminated. He also reported that projects have changed from a regional to a national concept and discussed the project proposal evaluation process (Appendix H: Multistate Research Committee Evaluation Form). It was suggested that the project revision emphasize the measurable impacts, application of the information and how the project fits into national goals.

Also, extramural funding resulting from participation and cooperation within the project should be included in the final project report and project revision. Project membership was briefly discussed and Dr. Hu reported that anyone could become a member of the project with approval of his or her experiment station.

Dr. Mirando presented information on personnel changes at CSREES, as well as current and projected budgets. Electronic submission of NRI competitive grants is scheduled to begin in FY2005. The trend in grant funding is for larger, longer-term awards. New NRI programs for FY2003 include functional genomics, agricultural security, obesity and human nutrition, and air quality. The NRI request for applications for FY 2004 should be available in September.

Craig Beattie gave a presentation on his research into identification of: 1) genes that account for phenotypic variation of pig conceptus growth and survival, and placental vascularity, and 2) genes that control the differentiation process into adult stem cells. Following the presentation, Charles Rosenkrans made a motion to remove from table, the appendix previously submitted to the committee by Dr. Beattie for consideration. The motion was seconded by Jerry Yang and approved unanimously by the members. Jack Rutledge moved and Tom Bunch seconded a motion for Craig Beattie to join the W171 regional project. The motion was approved unanimously.

Bob Godke was nominated by Jerry Yang to become secretary of W171 for the coming year. The nomination was seconded by Matt Wheeler and unanimously approved by members. Rick Rorie will become chairman for the coming year.

Possible dates were discussed for our next meeting and for any final work on the project revision. January 9 and 10, 2004 were discussed, in conjunction with the IETS meeting in Portland. Curt Youngs agreed to contact Jennifer Gavel at IETS for possible meeting room arrangements.

The objectives, job assignments and time lines for the project revision were discussed. It was agreed that objective 1 of the revised project will be changed to: Understand the biology and underlying mechanisms of gamete development, fertilization and embryogenesis. Objective 2 will remain unchanged as: Refine methods for production of genetically modified animals to improve livestock production efficiency.

Bob Godke nominated Matt Wheeler to serve as coordinator the project revision. The motion was seconded by Jerry Yang and approved unanimously by the committee members. It was agreed that October 3 would be the deadline for stations to submit to Matt Wheeler their literature review and references, and their proposed methods with references for each objective. It was decided that all references are to be submitted in Journal of Animal Science format with inclusive page numbers. Matt agreed to prepare a rough draft of the revision and send it to the stations for review by November 7. Dr. Hu suggested each station do a CRIS search to avoid duplication of efforts in the revised project.

Matt Wheeler nominated Bob Godke to coordinate the critical review and summary of the current project. The motion was seconded by Jerry Yang and approved unanimously. A deadline of October 3 was set for stations to submit their station project summaries and publications to Bob Godke.

The meeting was adjourned at approximately 11:50 AM for lunch. The meeting reconvened after lunch at 1:05 PM.

Matt Wheeler discussed his research using microfluidic chambers for in vitro embryo production. Discussions were initiated for designing cooperative projects incorporating the microfluidic chambers into oocyte maturation, fertilization and blastocyst development studies. Two species groups were designated to employ the chambers in these studies: a swine group led by Trish Berger and a cattle group led by Jack Rutledge. Monday, Sept. 15, 2003, was set as the deadline for submission of research proposals employing the chambers by interested PIs to the respective species leaders. The website for literature on the technology associated with the microfluidic chambers is: www.minitubeofamerica.com.

Discussions moved to genetic analysis of embryos and using microarrays for analysis of gene expression. Craig Beattie provided input with respect to the design of experiments utilizing this technology. Matt Wheeler suggested incorporating the technology for designing or identifying measures of embryo viability.

The effects of in vitro conditions on the sex ratio of IVF-derived embryos was also discussed. Utilizing the Heatwatch system was also mentioned but was regarded as peripheral as a collaborative research focus considering the overall interest of the membership.

Chairman Youngs solicited comments from the remaining members regarding station activities. Genes on the Y-chromosome that affect male fertility was brought up by George Seidel as a potential collaborative research topic.

Following remarks from the individual stations, Dr. Hu reminded the membership to include theses in the publication list.

Jerry Yang distributed announcements for the First New England Symposium on Regenerative Biology and Medicine to be held on Sept. 15-16, 2003 at the University of Connecticut.

Solicitation of new members was briefly discussed. The meeting concluded at 2:35 PM.

Respectfully submitted,

Rick Rorie

W171 Secretary

Accomplishments

A greater understanding of preservation techniques for porcine spermatozoa was gained, which will enable refinement of techniques for porcine in vitro embryo production. A porcine oocyte plasma membrane interaction protein was identified and characterized. Chemically-defined media for in vitro maturation of bovine oocytes were optimized. A practical method of vitrifying bovine embryos was developed. A novel method to improve development of cloned embryos by pretreatment of donor cells was identified. A culture system was devised which can be used to improve efficiency of cryopreservation of in vitro produced embryos. An alternative system for in vitro embryo manipulation in livestock was developed and tested. Milk production and piglet growth can be significantly increased by the introduction of a single gene that is involved in milk synthesis. The intralabial route of administration of prostaglandin F2a for synchronization of postpartum dairy cows was found to be comparable to the intramuscular route of administration. The simple and inexpensive treatment of embryo transfer recipient females with aspirin may be a viable method to enhance pregnancy rate. Improvements were made in the in vitro culture system for bovine IVF-derived embryos. Documented that epididymal sperm stored in the testes for 24 h at 4:C can be used for the in vitro production of bovine embryos capable of establishing good pregnancy rates. The expression pattern of three members of the ETS family of transcription factors (Ets-1, Ets-2, and Elf-1), in bovine and ovine oocytes and embryos from the zygote stage to Day 15 of pregnancy was documented. Wortmannin and genistein were shown to be inhibitors of in vitro bovine embryonic development.

Publications

REFEREEED JOURNAL ARTICLES ARKANSAS Burke, J.M. and R.W. Rorie. 2002. Changes in ovarian function in mature beef cows grazing endophyte-infected tall fescue. Theriogenology 57:1733-1742. Looper, ML, CF Rosenkrans, Jr., ZB Johnson, AH Brown, Jr., JA Hornsby, and JL Perkins. 2002. Relationships among growth characteristics of replacement Angus heifers. The Prof Anim Scientist 18:120-126. Melnychuk, V.L., M.A. Cooper, J.D. Kirby, R.W. Rorie, and N.B. Anthony. 2002. Use of ultrasonography to characterize of ovary status in chicken. J. Poultry Sci. 81:892-895. Rorie, R.W., T.R. Bilby and T.D. Lester. 2002. Application of electronic estrus detection technology to reproductive management of cattle. Theriogenology 57:137-148. CALIFORNIA Bertolini, M., Mason, J.B., Beam, S.E., Carneiro, G.F., Sween, M.L., Kominek, D.J. Moyer, A.L., Famula, T.R., Sainz, R.D. and Anderson, G.B. 2002. Morphology and morphometry of in vitro-and in vitro-produced concepti from early pregnancy to term and association with high birth weights. Theriogenology 58:973-994. Bertolini, M., Beam, S.E., Shim, H., Bertolini, L.R., Moyer, A.L., Famula, T.R. and Anderson, G.B. 2002. Growth, development and gene expression by in vitro-and in vitro-produced day-7 and day-16 bovine embryos. Molecular Reproduction and Development 63:318-328. Carmona, E., Weerachatyanukul, W., Soboloff, T., Fluharty, A.L., White, D., Promdee, L., Ekker, M., Berger, T., Buhr, M., and Tanphaichitr, N. 2002. Arylsulfatase A is present on the pig sperm surface and is involved in sperm-zona pellucida binding. Dev. Biol. 247:182-196. Fieni, F., Rowe, J., VanHoosear, K., Burucoa, C., Oppenheim, S., Anderson, G., Murray, J. and BonDurant, R. 2002. Presence of caprine arthritis-encephalitis virus (CAE) infected cells in flushing media following oviductal-stage embryo collection. Theriogenology 57:931-940. COLORADO Cibelli, J.B., K.H. Campbell, G.E. Seidel, M.D. West and R.P. Lanza. 2002. The health profile of cloned animals. Nature Biotechnology 20:13-14. Coutinho da Silva, M.A., E.M. Carnevale, L.J. Maclellan, G.E. Seidel, Jr. and E.L. Squires. 2002. Effect of time of oocyte collection and site of insemination on oocyte transfer in mares. J. Anim. Sci. 80:1275-1279. Landim-Alvarenga, F.C., S.E.A. Boyazoglu, L.R. Carvalho, Y.H. Choi, E.L. Squires and G.E. Seidel, Jr. 2002. Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle. Anim. Reprod. Sci. 71:181-191. Li, G.-P., D.-Y. Chen, L. Liau, Z.-M. Han, Z.-Y. Zhu and G.E. Seidel, Jr. 2002. Rabbit cloning: Improved fusion rates using cytochalasin B in the fusion buffer. Molec. Reprod. Develop. 61:187-191. Seidel, G.E., Jr. and D.L. Garner. 2002. Current status of sexing mammalian spermatozoa. Reproduction 124:733-743. CONNECTICUT Du, F., L.-Y. Sung, X.C. Tian and X.Yang. 2002. 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Chung, D.D. Varner, M.E. Westhusin, R.C. Burghardt and K. Hinrichs . 2002. Use of piezo-driven direct nuclear injection and activation with stallion sperm extract to produce horse nuclear transfer embryos. Theriogenology. 58:771-774. Hanna C.B., C.R. Long, M.E. Westhusin and D.C. Kraemer. 2002. Arresting canine oocytes in the germinal vesicle phase of meiosis I. Theriogenology. 57:722. Williams B., T. Shin, L. Liu, G. Flores-Foxworth, J. Romano, J. Rugila, D. Dean, R. Burghardt, M. Westhusin and D. Kraemer. 2002. Interspecies nuclear transfer of desert bighorn sheep (ovis canadensis mexicana). Theriogenology 57:457. Willingham-Rocky L.A,, C.B.M. Hanna, M.E. Westhusin and D.C. Kraemer. 2002. Effects of porcine somatotropin and epidermal growth factor on the resumption of meiosis of canine oocytes matured in vitro. Theriogenology. 57:742. UTAH Bunch, T.D. , S. Wang,, N.E. Cockett, T.L. Shay, J. M. Miller, A. Maciulis, R.C. Evans, G.R. Holyoak, W.C. Foote and J.W. Call. 2002. The PRPN gene polymorphic distribution in embryo transfer flocks used for studying scrapie transmission. Plant, Animal & Microbe Genomes X. P134, p 111. Li, G.P., K.L. White, K.I. Aston and T.D. Bunch. 2002. Effect of culture conditions on the development and chromosomal composition of nuclear transfer (NT) bovine embryos. Mole. Biol. Cell 13:645. Panter, K.E., S. Wang, L.F. James, W. Gaffield, R.J. Molyneux, R.C. Evans and T.D. Bunch. 2002. The effects of steroidal glycolakaloids from potato tubers (Solanum tuberosum) on bovine oocytes maturation and subsequent embryo development in vitro. Theriogenology 57:734. Pate, B.J., K.D. Campbell, L.D. Meerdo, K.L. White. 2002. Inhibition of bovine oocyte development after fertilization using the kinase inhibitors wortmannin and genistein B. Mole. Biol. Cell 13:623. Wang, S., K.E. Panter L.F. Jame, D.R. Gardner, W. Gaffield, R.J. Molyneux, B.L. Stegelmeier, R.C.Evans and T.D. Bunch. 2002. Effects of isocupressic acid (ICA), a pine needle abortifacient toxin, on the viability of bovine preimplantation embryos. Theriogenology. 57:529. White, K.L., M. Passipieri, T.D. Bunch, K.D. Campbell, L.D. Meerdo and B.J. Pate. 2002. Effects of arginine-glycine-aspartic acid (RGD) -containing snake venom peptides on parthenogenetic development and in vitro fertilization of bovine oocytes. Mole. Biol. Cell 13:622. WISCONSIN Fischer-Brown, A.E., R.L. Monson, J.R. Fredrickson and J.J. Rutledge. 2002. Antioxidant use with FCS and BSA supplementation in SOF medium: effect on development and cell number. Theriogenology 47:518. Men, H., R.L. Monson and J.J. Rutledge, 2002. Detection of DNA damage in bovine metaphase II oocytes caused by cryopreservation. Theriogenology 57:471. Westberg, S.A., R.L. Monson and J.J. Rutledge. 2002. Higher hatching rates obtained with BSA supplementation compared with FCS. Theriogeneology 57:530. MISCELLANEOUS PUBLICATIONS ARKANSAS Golden, C, C Rosenkrans, Jr., and Z Johnson. 2002. Effects of ascorbic acid and alpha-tocopherol on cryopreserved boar sperm. Arkansas Anim Sci Report 499:120-123. Post, N.M., D.L. Kreider, R. Rorie, T. Lester and K. Cole. 2002. Estrus synchronization and timed artificial insemination with melengesterol acetate plus GnRH in beef females. Arkansas Animal Science, Research Series 499:126-129 R.W. Rorie, T.R. Bilby and T.D. Lester. 2002. Comparison of Electronic Mount Monitors for Detection of Estrus in Cattle. Arkansas Animal Science, Research Series 499:124-125. R.W. Rorie and T.D. Lester. 2002. Comparison of Lutalyse and Estrumate for Inducing Estrus in Beef Cows and Heifers. Arkansas Animal Science, Research Series 499:130-131. IOWA Youngs, C. 2002. 2002 IETS foundation student research competition. Embryo Transfer Newsletter 20(1):4. Youngs, C.R. 2002. The magnificent seven hormones controlling reproduction in the cow (Part II). Iowa Jersey Today 8(1):5-6. Youngs, C.R. 2002. Synchronization of estrus in dairy cattle using prostaglandin F2a (Part I). Iowa Jersey Today 8(2):10. Youngs, C.R. 2002. Synchronization of estrus in dairy cattle using prostaglandin F2a (Part II). Iowa Jersey Today 8(3):9-10. LOUISIANA Ferguson, C.F., O. Perez, K. Hebert , J. Cartmill and R.A. Godke. 2002. Using altrenogest to synchronize estrus in beef heifers. Louisiana Beef Cattle Research Report (In press). Gentry, G.T.Jr., R.P. Del Vecchio and R.A. Godke. 2002. The effect of different types of spotter animals on estrus and pregnancy rates after artificial insemination in beef cattle. Louisiana Cattleman. pp.11-13. Hebert, K., D. Hylan, R.S. Denniston and R.A Godke. 2002. Administering prostaglandin F2a twice daily for 5 days to nursing beef cows starting 5 days after calving. Louisiana Beef Cattle Research Report (In press).

Impact Statements

The procedures developed for vitrifying bovine embryos are simple, rapid, and do not require expensive equipment and may replace current procedures for freezing.
Being able to produce pregnancies from epididymal sperm from testes after 24 hours of storage at 4: would allow cattle producers to save germplasm from a deceased bull.
The improved methods for in vitro production of bovine embryos in defined media will enable designing better experiments, as well as lead to improved methods for commercial use, perhaps decreasing the incidence of abnormal calves.
Aspirin may be an effective blocker of prostaglandin F2a synthesis that could enhance pregnancy rate following embryo transfer.
Increasing the useful life of porcine spermatozoa will allow scientists and producers to use valuable male genetic material over a longer period of time.

Date of Annual Report: 03/05/2004

Report Information

Annual Meeting Dates: 01/09/2004 - 01/10/2004
Period the Report Covers: 01/01/2003 - 12/01/2003

Participants

Hu, Ching Yuan (chingyuan.hu@oregonstate.edu) - Oregon State University; Mirando, Mark (mmirando@csrees.usda.gov) CSREES; Rorie, Rick (rrorie@uark.edu) - University of Arkansas; Rosenkrans, Charles (crosenkr@uark.edu) - University of Arkansas; Seidel, George (gseidel@colostate.edu) - Colorado State University; Godke, Robert (rgodke@agctr.lsu.edu) - Louisiana State University; Leibo, Stanley (sleibo@acres.org) - Audubon Center for Research of Endangered Species; Wheeler, Matt (mbwheele@uiuc.edu) - University of Illinois; Youngs, Curt (cryoungs@iastate.edu) - Iowa State University; Menino, Fred (alfred.r.menino@oregonstate.edu) - Oregon State University; Mapletoft, Reuben (reuben.mapletoft@usask.ca) - University of Saskatchewan; White, Kenneth (kwhite@cc.usu.edu) - Utah State University; Keefer, Carol (ckeefer@umd.edu) - University of Maryland; Krisher, Rebecca (rkrisher@purdue.edu) - Purdue University; Schrick, Neal (fschrick@utk.edu) - University of Tennessee; Edwards, Lannett (jedwards@utk.edu) - University of Tennessee;

Brief Summary of Minutes

Minutes from the W-171 Meeting

Portland, Oregon

January 10, 2004

The meeting was opened at 6:05 pm by Bob Godke, the W-171 secretary, since Rick Rorie, the chairperson, was held up by weather conditions.

New Business: Dr. Mark Miranda the CSREES Administrator made opening comments and review the status of the USDA budget.

W-171 Administrative Advisor, Dr. C. Y. Hu from Oregon State University, then gave opening comments relating to the procedure to submit the revised project proposal. Dr. Hu indicated that the project review will occur in May of 2004, if the group can get the revised project into the NIMSS system as soon as possible. Dr. Hu then reviewed the steps that needed to be completed to get the revised W-171 proposal sent out for outside review. He indicated that once the revised proposal was submitted to the NIMSS system, each station was to submit Appendix E from their experiment station activating that station scientists to become members of the new Mullti-State Research Project.

New members, Rebecca Krisher from Perdue, Neal Schrick from the University of Tennessee and Lannett Edwards from the University of Tennessee were welcomed to the group.

Old Business: Matt Wheeler reported on the project rewrite and thanked everyone who worked with him to get the revised proposal completed by this meeting. Bob Godke report that he had finished the Critical Review section and thanked the members from each station for contributing the information requested over the last two and a half months. He indicated that there were over 70 pages of publication alone reported by members of the contributing stations. Curt Youngs indicated that the revised project proposal prepared by Matt Wheeler and the critical review prepared by Bob Godke should be e-mailed to him, so that he could get into the NIMSS system the following week.

Dr. Hu indicated that we needed to suggest names of respected scientists in the field as potential reviewers of the revised project. A list of 10 reproductive physiologists was suggested by the members present. Bob Godke was to contact members from the list to identify four scientists that would agree to review the proposal. Godke was asked to contact four or five scientists from the list to conduct the outside review and then send their names and addresses to the administrative advisor.

Station project reports were made in an abbreviated format. It was suggested that we contact IFIS to obtain funds to support a seminar program. Baton Rouge, Louisiana (LSU) was selected for next Technical Committee meeting of new W-1171 in February of 2005. Bob Godke was elected as the new W-171 Chairperson and Rebecca Krisher was elected as the new Secretary by a unanimous vote.

A motion was made and seconded to adjourn the meeting at 10:10 pm.

Respectfully submitted,

Robert A. Godke

W-171 Secretary

Accomplishments

It was demonstrated that phosphatase 1 is an important regulator of expression of tPA, PAI-1 and b-actin in bovine oocyte-cumulus cell complexes matured in vitro. Oocyte plasma membrane proteins were investigated that are potentially involved in sperm-oocyte interaction. Two potential boimarkers were identified for evaluation of oocyte maturation. An alternative culture system for in vitro embryo manipulation in livestock was fabricated and tested. Improvements were made in systems of in vitro production of bovine embryos are resulting in embryos fairly similar to those produced in vivo. The generation of artificial gametes from somatic cells was studied and improvement was made in the rate of meiosis-like divisions. The levels of epigenetic markers of donor cells at different passages, tissue type and cell cycle stage for nuclear transfer was investigated. It was shown that milk production and piglet growth can be significantly increased by the introduction of single genes, which are involved in milk synthesis and intestinal maturation, respectively. Nuclear transfer studies, using colcemid treated oocytes as recipient cytoplasts resulted in an increase in the number of blastocysts and hatched blastocysts. The degree of differentiation effects reprogramming capacity within a single lineage of donor cells used in somatic cell NT. Appropriate expression of DNA methyltransferases was found to be important for normal mammalian development to proceed in both cloned embryos and others produced by natural mating or in vitro. To demonstrate how germplasm from deceased bulls can be saved, epididymal sperm stored in the testes for 24 h at 4ºC was used in a standard in vitro fertilization procedure, producing good quality embryos and subsequent pregnancies in recipient beef females. A very sensitive test for fertility in cattle was developed, using competitive fertilization procedures. Bovine somatotropin was shown to adversely affect fertility, when given the second week of gestation. A simple, inexpensive procedure was developed for cryopreservation of bovine embryos. Calves resulting from sexed sperm were shown to be normal except being about 90% of the selected sex, rather than 50:50. Investigated the potential usefulness of the intralabial route of administration of prostaglandin F2alpha for synchronization of postpartum dairy cows for use as recipients.

Publications

Refereed Journal Articles/Refereed Book Chapters ARKANSAS Baublits, R.T., A.H. Brown, Jr., Z.B. Johnson, C.F. Rosenkerans, Jr., R.W. Rorie and B.A. Sandelin. 2003. Variation in the percentage of female births over time in five breeds of registered cattle. Prof. Anim. Sci. 19:204-210. Steelman, C.D., R.W. McNew, R.B. Simpson, R.W. Rorie, J.M. Phillips and C.F. Rosenkrans, Jr. 2003. Evaluation fo alternative tactics for management of insecticide-resistant horn flies (diptera muscidae). J. Econ. Entomol. 96:892-901. CALIFORNIA Bertolini, M., J.B. Mason, S.E. Beam, G.F. Carneiro, M.L. Sween, D.J. Kominek, A.L. Moyer, T.R. Famula, R.D. Sainz and G.B. Anderson. 2002. Morphology and morphometry of in vitro-and in vitro-produced concepti from early pregnancy to term and association with high birth weights. Theriogenology 58:973-994. Bertolini, M., S.E. Beam, H. Shim, L.R. Bertolini, A.L. Moyer, T.R. Famula and G.B. Anderson. 2002. Growth, development and gene expression by in vitro-and in vitro-produced day-7 and day-16 bovine embryos. Molecular Reproduction and Development 63:318-328. Carmona, E., W. Weerachatyanukul, T. Soboloff, A. L. Fluharty, D. White, L. Promdee, M. Ekker, T. Berger, M. Buhr, and N. Tanphaichitr. 2002. Arylsulfatase A is present on the pig sperm surface and is involved in sperm-zona pellucida binding. Dev. Biol. 247:182. Sartini, B.L. and T. Berger. 2003. Changes in oocyte plasma membrane binding sites on boar spermatozoa with capacitation and acrosome reactions. Reproduction 125:865. Rui, R., H. Shim, A.L. Moyer, D. Anderson, M.C. Penedo, J.D. Rowe, R.H. BonDurant and G.B. Anderson. Attempts to enhance production of chimeras from porcine embryonic germ cells and preimplantation porcine embryos. Theriogenology (In press). Maga, E.A., G. Sargent, H. Zeng, S. Pati, D. Zarling, S. M. Oppenheim, N.M.B. Collette, A.L. Moyer, J.S. Conrad-Brink, J. D. Rowe, R.H. BonDurant, G.B. Anderson, and J.D. Murray. Increased efficiency of transgenic livestock production. Transgenic Reseach (In press). CANADA Bo, G.A. and Mapletoft, R.J., 2003. Superovulacion en Bovinos. En: Reproduccion en los Animales Domisticos. Tomo II. R. Ungerfeld (Editor). Melibea Ediciones, pages 515-538. Bo, G.A., Palasz, A.T. and Mapletoft, R.J., 2003. Criopreservacion de Embriones. En: Reproduccion en los Animales Domisticos. Tomo II. R. Ungerfeld (Editor). Melibea Ediciones, pages 551-566. Vassena, Rita, Mapletoft, Reuben J., Allodi, Stefano, Singh, Jaswant, Adams, Gregg P., 2003. Morphology and Developmental Competence of Bovine Oocytes relative to Follicular Status. Theriogenology, 60, 923-932. Colazo, Marcos German, Kastelic, John Patrick, Mapletoft, Reuben John., 2003. Effects of Estradiol Cypionate (ECP) on Ovarian Follicular Dynamics, Synchrony of Ovulation and Fertility in CIDR-based, Fixed-time AI Programs in Beef Heifers. Theriogenology, 60, 855-865. Martinez, M.F., Mapletoft, R.J., Kastelic, J.P. and Carruthers, T.D., 2003. The Effects of Three Gonadorelin Products on Luteinizing Hormone Release, Ovulation, and Follicular Wave Emergence in Cattle. Canadian Veterinary Journal, 44:125-131. Vassena, R., Adams, G.P., Mapletoft, R.J., Pierson, R.A. and Singh, J, 2003. Ultrasound Image Characteristics of Ovarian Follicles in Relation to Oocyte Competence and Follicular Status in Cattle. Animal Reproduction Science, 76, 25-41. COLORADO Bass, L. D., D. J. Denniston, L. J. Maclellan, P. M. McCue, G. E. Seidel, Jr., and E. L. Squires. 2003. Methanol as a cryoprotectant for equine embryos. Theriogenology. Submitted. Choi, Y. H., F. C. Landim-Alvarenga, G. E. Seidel, Jr., and E. L. Squires. 2003. Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes. J. Anim. Sci. 81:2080-2087. Coutinho da Silva, M. A., E. M. Carnevale, L. J. Maclellan, K. A. Preis, G. E. Seidel, Jr., and E. L. Squires. 2003. Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled and frozen stallion semen. Theriogenology. In Press. Flint, A. F., P. L. Chapman, and G. E. Seidel, Jr. 2003. Heterospermic insemination of cattle using flow-sorted sperm. J. Anim. Sci. 81:1814-1822. Franz, L. C., Y.-H. Choi, E. L. Squires, G. E. Seidel, Jr., and K. Hinrichs. 2003. Effects of roscovitine on maintenance of the germinal vesicle in horse oocytes, subsequent nuclear maturation, and cleavage rates after intracytoplasmic sperm injection. Reproduction 125:693-700. Garner, D. L., and G. E. Seidel, Jr. 2003. Past, present and future perspectives on sexing sperm. Can. J. Anim. Sci. 83:375-384. Glover, M. D., and G. E. Seidel, Jr. 2003. Increased messenger RNA for allograft inflammatory factor-1, LERK-5, and a novel gene in 17.5-day relative to 15.5-day bovine embryos. Biol. Reprod. 69:1002-1012. Li, G.-P., G. E. Seidel, Jr., and E. L. Squires. 2003. Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa. Theriogenology 59:1143-1155. Li, G.-P., G. E. Seidel, Jr., and E. L. Squires. 2003. Improved cleavage of bovine ICSI ova cultured in heparin-containing medium. Theriogenology. In Press. Lindsey, A. C., D. D. Varner, G. E. Seidel, Jr., J. E. Bruemmer, and E. L. Squires. 2003. Hysteroscopic or rectally guided, deep-uterine insemination of mares with spermatozoa stored at 18 h at either 5ºC or 15ºC prior to flow-cytometric sorting. Anim. Reprod. Sci. Submitted. Mendes, J. O. B., Jr., P. D. Burns, J. F. De La Torre-Sanchez, and G. E. Seidel, Jr. 2003. Effect of heparin cleavage rates and embryo production with four bovine sperm preparation protocols. Theriogenology 60:331-340. Seidel, G. E., Jr., 2003. Economics of selecting for sex: The most important genetic trait. Theriogenology 59:585-598. Seidel, G. E., Jr. 2003. Sexing mammalian sperm ? Intertwining of commerce, technology, and biology. Anim Reprod Sci. In Press. Zhang, M., K. H. Lu, and G. E. Seidel, Jr. 2003. Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically-sorted, stained, and unsorted sperm from different bulls. Theriogenology 60:1657-1663. CONNECTICUT Enright, B. P., B. S. Jeong, X. Yang, and X. C. Tian. 2003a. Epigenetic characteristics of bovine donor cells for nuclear transfer: Levels of histone acetylation. Biol Reprod 69: 1525-1530. Enright, B. P., C. Kubota, X. Yang, and X. C. Tian. 2003b. Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin a or 5-aza-2‘‘‘‘-deoxycytidine. Biol Reprod 69: 896-901. Lee, J. W., X. C. Tian, and X. Yang. 2003a. Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection. Biol Reprod 68: 1341-1347. Lee, J. W. et al. 2003b. Production of cloned pigs by whole-cell intracytoplasmic microinjection. Biol Reprod 69: 995-1001. Savage, A. F. et al. 2003. Behavioral observations of adolescent holstein heifers cloned from adult somatic cells. Theriogenology 60: 1097-1110. Suzuki, H., Y. Saito, N. Kagawa, and X. Yang. 2003. In vitro fertilization and polyspermy in the pig: Factors affecting fertilization rates and cytoskeletal reorganization of the oocyte. Microsc Res Tech 61: 327-334. Tian, X. C., C. Kubota, B. Enright, and X. Yang. 2003. Cloning animals by somatic cell nuclear transfer - biological factors. Reprod Biol Endocrinol 1: 98. Xu, J., and X. Yang. 2003. Will cloned animals suffer premature aging - the story at the end of clones‘‘‘‘ chromosomes. Reprod Biol Endocrinol 1: 105. Yang, X. 2003. Toward therapeutic cloning and regenerative biology research forum: To clone or not to clone. Reprod Biol Endocrinol 1: 97. ILLINOIS Wheeler, M.B., Walters, E.M., Clark, S.G. (2003). Transgenic animals in biomedicine and agriculture:outlook for the future. Animal Reproduction Science 79:265-289. Wheeler, M.B., Malusky, S.A. (2003). Transgenesis in mammals: status and perspectives. Acta Scientiae Veterinariae 31:119-136. IOWA Godke, R.A. and C.R. Youngs. 2003. Biotechnology: Embryo Transfer Technology. In (W. Pond, Ed.) Encyclopedia of Animal Science. Marcel Dekker, New York, NY (in press). LOUISIANNA Boediono, A., T. Suzuki and R.A. Godke. 2003. Comparison of hybrid and prurebred in vitro-derived cattle embryos during in vitro culture. Anim. Reprod. Sci. 78:1-11. Cochran, R., M. Meintjes, D. Hylan, M. Sansinena, R. S. Denniston D. Paccamonti and R.A. Godke. 2003. Follicle growth in mares for oocyte production. In: From Epididymus to Embryo. Havemeyer Fdn. Monograph 6:51-54. Devireddy,R.V., B. Fahrig, R.A. Godke and S.P. Leibo. The effect of extracellular ice and glycerol on boar sperm water transport parameters: Convergence of theoretical and empirical rates of optimal freezing. Molec. Reprod. Develop. (In press). Godke, R.A., K. Hebert, A. James, R.S. Denniston and D. Paccamonti. 2003. The potential use of epididymal sperm from bulls and stallions for assisted fertilization. Havemeyer Fdn. Monograph (In press). Godke, R.A. and C. R. Youngs. Biotechnology: Embryo technology in cattle. In: Encyclopedia of Animal Science. W.G. Pond & A.W. Bell (eds.). Marcel Dekker:New York (In press). (Book Chapter) Gomez, M., C.E. Pope, A. Giraldo, L. Lyons, R. Harris, A. King, A. Cole, R. Godke and B. Dresser. 2003. Cloned African Wildcats born from a domestic cat. Cloning & Stem Cells. (Submitted). Han, Y., Y. Han, R.S. Denniston, A. Boediono, C. Ziomek and Godke. Development of IVF-derived caprine embryos with four different somatic cell co-cultures. Small Ruminant Res. (Accepted). Klump, A, A. Aljarrah, M., Sansinena M., D. Hylan, C. Pinto, B. Eilts, R. Godke and D. Paccamonti. 2003. Follicular aspiration during transition to advance the onset of cyclicity. Pferdeheilkunde Equine Vet. J. 19(6):609-610. Landry, A.M., D.J. Landry, L.R. Gentry, H.L. Green, K.L. Koonce, Y. Echlard and R.A. Godke. Endocrine profiles and growth patterns of goats produced by nuclear transfer. Cloning & Stem Cells (In Final Prepartion). Leibo, S.P. and N. Lane. The early history of gamete cryobiology. In: Life in the Frozen State. E. Benson, B. Fuller & N. Lane (eds.). Taylor & Francis:London. (In press). (Book Chapter) Luster, S.M., M. Murakami, S.P. Leibo, R.S. Denniston Y. Echelard, and R.A. Godke. Pregnancies resulting from nuclear transfer embryos derived from vitrified bovine oocytes. Cryobiology (In press). Massip A, Leibo SP, Blesbois E. Cryobiology of gametes and the breeding of domestic animals. In: Life in the Frozen State. E. Benson, B. Fuller & N. Lane (eds.) Taylor & Francis:London (In press). (Book Chapter) Murakami, M., T.R. Davidson, C.E. Ferguson, Y. Echelard and R. A. Godke. Use of the amniotic cavity of the developing chick embryo for culturing bovine 8-cell embryos derived from somatic cell nuclear transfer. Molec. Reprod. Develop. (In press). Murakami, M., S. Ideguchi, M. Fahrudin, T. Otoi, R.A. Godke and T. Suzuki. 2003. Influence of the DNA amount per injection on the development and EGFP expression in bovine embryos. Arch. Anim. Breeding 1:25-30. Murakami, M., O. Perez, C.E. Ferguson, E. Behboodi, R.S. Denniston and R.A. Godke. 2003. Using in vivo-recovered oocytes and adult somatic cells from the same donor for nuclear transfer in cattle. Vet Rec. 153: (In press). Pinto, C.R.F., D.L. Paccamonti, B.E. Eilts, C.S. Venugopalan, C.R. Short, L.R. Gentry, D.L. Thompson Jr. and R.A. Godke. 2003. Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin. Theriogenology 60:819-827. Pinto, C.R.F., D.L. Paccamonti, B.E. Eilts, C.R. Short , L. Gentry, D.L. Thompson and R.A. Godke. Evidence of nitric oxide-mediated modulation of equine granulosa cell steroidogenesis. Theriogenology (Submitted). Sansinena, M., D. Hylan, K. Hebert, R.S. Denniston and R.A. Godke. 2003. Benteng (Bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer. Theriogenology (Under review). Sansinena, M., Paccamonti, D., C. Cochran, M. Meintjes, B. Reggio, R. S. Denniston, C. Pinto, B. Eilts and R.A. Godke. 2003. Developing the intracytoplasmic sperm injection (ICSI) procedure for the horse. In: From Epididymus to Embryo. Havemeyer Fdn. Monograph 6:69-74. Sansinena, M., S.A. Taylor, P.J. Taylor, R.S. Denniston and R.A. Godke. 2003. Production of nuclear transfer llama (Lama glama ) embryos from in vitro fertilized llama oocytes. Cloning & Stem Cells 5(3):191-198. Sansinena, M.J., B. Reggio, D. Hylan, A. Klumpp, S.K. Lyle, R.S. Denniston, D. Paccamonti and R.A. Godke. 2003. Effect of different pre-maturation treatments on in vitro maturation of equine oocytes and subsequent competence for nuclear transfer. Havemeyer Fdn. Monograph (In press). Songsasen, N., I. Yu, M. Gomez and S.P. Leibo. 2003. Effects of meiosis-inhibiting-agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes. Molec. Reprod. Develop. 65:435-445. VandeVoort, C.A., S.P. Leibo and A.F.Tarantal. 2003. Improved collection and developmental competence of immature macaque oocytes. Theriogenology 59:699-707. Yu, I., N. Songsasen, Y-J. Kim, B.L. Dresser and S.P. Leibo. 2003. Development in culture of canine ovarian and testicular fibroblasts after cryopreservation. Cryo-Letters (In press). Wirtu, G., C.E. Pope, J. Vaccaro, E. Saart, A. Cole, R.A. Godke and B.L. Dresser. Dominance hierachy in a small herd of female eland (Taurotragus oryx) in captivity. Zoo Biol. (In press). Wirtu, G., C.E. Pope, P. Damiani, F. Miller, B.L. Dresser, R.A. Godke and B.D. Bavister. Development of in vitro-derived bovine embryos in protein-free media: Effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure. Reprod. Fertil. Develop. (Submitted). TEXAS Westhusin M., K. Hinrichs, Y.H. Choi, T. Shin, L. Liu and D. Kraemer. 2003. Cloning Companion Animals (Horse, Cat, Dog). Cloning and Stem cells. (in press). Golding, M.C. and M.E. Westhusin. 2003. Analysis of DNA (cytosine-5) methyltransferase mRNA sequence and expression in preimplantation embryos, fetal and adult tissues. Gene Expression Patterns 3: 551-558. Willingham-Rocky L.A., K. Hinrichs, M.E. Westhusin and D.C. Kraemer. 2003. Effects of stage of cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro. Reproduction (in press). Choi Y.H., L.B. Love, M.E. Westhusin and K. Hinrichs. 2003. Remodling of equine somatic cell nuclei after transfer to equine or bovine cytoplasts, and effect of timing of activation treatment on in vitro development of horse nuclear transfer embryos. Biol. Reprod. (in press). Long CR, S.C. Walker, R.T. Tang and M.E. Westhusin. 2003. New commercial opportunities for advanced reproductive technologies in horses, wildlife, and companion animals. Therio. 59:139-149. Choi Y.H., Y.G. Chung, S. Walker, M.E. Westhusin and K Hinrichs. 2003. In vitro development of equine nuclear transfer embryos: Effects of oocyte maturation and amino acid composition during embryo culture. Zygote 11:77-86. UTAH Woods, G.L., K.L. White, D.K. Vanderwall, Guang-Peng Li, K.I. Aston, T.D. Bunch, L.N. Meerdo, and B.J. Pate. 2003. A mule clone from fetal cells by nuclear transfer. Science. 301: 1063. WASHINGTON Jiang, Zhihua, M. Zhang, V. Wasem, J. Michal, H. Zhang, R.W. Wright. 2003. Census of Genes Expressed in Porcine Embryos and Reproductive Tissues by Mining an Expressed Sequence Tag Data Base Based on Human Genes. Biol. of Reprod. 69:1177-1182. WISCONSIN Block, J., M. Drost, R.L. Monson, J.J. Rutledge, R. M. Rivera, F.F. Paula-Lopes, O.M. Ocon, C.E. Krininger III, J. Liu and P.J. Hansen. 2003. Use of insulin-like growth factor-1 during embryo culture and treatment of redipients with GnRH to increase pregnancy rates following the transfer of in vitro produced embryos to heat-stress, lactating cows. J. Anim. Sci. 81: 1590-1602. Fischer-Brown, Amy and Jack Rutledge. 2003. Practical and research applications of elongation stage bovine embryos. Embryo Transfer Newsletter 21: 11-15. Men H, Monson RL, Parrish JJ, Rutledge JJ. 2003. Degeneration of cryopreserved bovine metaphase II oocytes via apoptosis during subsequent culture. Cryobiology 47:73-81. Men H, Monson RL, Parrish JJ. Rutledge JJ. 2003. Detection of direct DNA damage in bovine metaphase II oocytes caused by cryopreservation. Mol Reprod Dev 64:245-250. Books, chapters (non-refereed) in books, proceedings, and theses CANADA Mapletoft, R.J., Colazo, M.G., Martinez, M.F. and Kastelic, K.P., 2003. Esteres de Estrogeno para la Sincronizacion de la Emergencia de la Onda Follicular y la Ovulacion en Animales Tratados con Dispositivos con Progesterona. V Simposio Internacional de Reproduccion Animal. Heuta Grande, Cordoba, Argentina, pages 55-70. Kastelic, K.P. and Mapletoft, R.J., 2003. Causas no Infecciosas de Muerte Embrionaria en Ganado Bovino. V Simposio Internacional de Reproduccion Animal. Heuta Grande, Cordoba, Argentina, pages 149-160. Barth, A.D., Thundathil, J.C. and Mapletoft, R.J., 2003. Importancia de la Calidad Seminal y el Uso de FIV para el Estudio de Defectos Espermaticos. V Simposio Internacional de Reproduccion Animal. Heuta Grande, Cordoba, Argentina, pages 205-222S. Mapletoft, R.J., Martinez, M.F., Colazo, M.G. and Kastelic, J.P., 2003. The Use of Controlled Internal Drug Release Devices for the Regulation of Bovine Reproduction. Journal of Animal Science, 81(E. Suppl. 2):E28?E36. COLORADO Checura, C. 2003. Cryopreservation of Bovine Oocytes: Effects of Macromolecules in Vitrification Solutions. M.S. Thesis, Colorado State University, Fort Collins, CO. DeLille, A. 2003. Physical and Molecular Characteristics of Day 75 Nuclear Transfer Cloned Bovine Conceptuses. Ph.D. Dissertation, Colorado State University, Fort Collins, CO. Garner, D. L., and G. E. Seidel, Jr. 2003. Applications of flow cytometry in animal reproduction. In Press in Flow Cytometry in Biotechnology, L.A. Sklar (ed). Oxford University Press (USA). Seidel, G. E., Jr. 2003. Cloning I. Scientific background. In Press in Encyclopedia of Bioethics. S.G. Post (ed). MacMillan, New York. Seidel, G. E., Jr. 2003. Metabolism of bovine embryos. Pages 3-8 in Proc. Ann. Conv. Am. Embryo Transfer Assoc. Seidel, G. E., Jr. 2003. Vitrification of bovine embryos. Pages 9-12 in Proc. Ann. Conv. Am. Embryo Transfer Assoc. Seidel, G. E., Jr. 2003. Transgenic vertebrates ? The long road to practical application. Pages 1-2 in Proc. Transgenic Animal Research Conference IV, Tahoe City, CA. Seidel, G. E., Jr., R. P. Elsden, and J. F. Hasler. 2003. Embryo Transfer in Dairy Cattle. 3rd Edition. Hoard?s Dairyman Books, Fort Atkinson, WI. 96 pages. Tubman, L. 2003. Characteristics of Calves Produced with Sperm Sexed by Flow Cytometry/Cell Sorting. M.S. Thesis, Colorado State University, Fort Collins, CO. CONNECTICUT Lee, J.W. 2003. Production of pig embryos following microinjection of somatic cells and sperm. Ph.D. thesis. University of Connecticut. Enright, B. 2003. Development and subsequent reproductive performance of cloned cattle derived from adult somatic cells. Ph. D. thesis. University of Connecticut. Kubota, C. 2003. Studies on the development of bovine somatic cell nuclear transfer and its application for the production of super sire bull. Ph. D. thesis. University of Kagoshima. He, J. 2003. Quantification of extracellular matrix gene expression in single bovine oocytes and preimplantation embryos derived from IVF using real time RT-PCR. MS thesis. University of Connecticut. ILLINOIS Clark, Sherrie G., Ph.D., December 2003, dissertation title, "In vitro maturation, fertilization and embryo culture of porcine gametes in a microfluidic environment", University of Illinois at Urbana-Champaign, 106 pgs. Etherton, T.D., Bauman, D.E., Beattie, C.M., Bremel ,R.D., Cromwell, G.L, Kapur, V., Varner, G., Wheeler, M.B., Wiedmann, M. (2003). Biotechnology in Animal Agriculture: An Overview. Council for Agricultural Science and Technology Issue Paper, 12 pgs. Jackson, Kevin S., Ph.D. May 2003, dissertation title, "Trophinin: Expression and regulation in the porcine uterus and embryo during the time of embryo implantation", University of Illinois at Urbana-Champaign, 124 pgs. Roseman, Hayley M., M.S., October 2003, thesis title. "Porcine oocyte maturation and embryo culture within polydimethylsiloxane-borosilicate microchannels" , University of Illinois at Urbana-Champaign, 92 pgs. LOUISIANA Fahrig, B. 2003. Cryopreservation by Pellet Freezing of Epididymal and Ejaculated Spermatozoa from Male Dogs. M.S. Thesis. Louisiana State University. Baton Rouge. James, A.N. 2003. Preservation of Sperm Harvested from the Rat, Caprine, Equine and Bovine Epididymis. Ph.D. Dissertation. Louisiana State University. Baton Rouge. Leibo, S.P. 2003. Basic cryobiology of embryos. In: Andrology and Embryology Review Course Manual. American Association of Bioanalysts. St. Louis, MO. 14:1-15. Nel-Themaat, L., T. Harding, J.E. Chandler, J.F. Chenevert, P. Damiani, M.J. Fernadez and R.A. Godke. 2003. Freezability of sperm ejaculates collected multiple times weekly from endangered Louisiana Native rams. Proc Amer. Zoo Assoc. (In press). Nel-Themaat, L., T. Harding, J.E. Chandler, J.F. Chenevert, P. Damiani, M.J. Fernadez and R.A. Godke. 2003. Freezability of endangered Louisiana Native ram sperm. Proc. Environ-mental State of State Conf. (ESOS) VIII. (In press). Perez. O. 2003. Oocyte Production in the Postpartum Beef Cattle. Ph.D. Dissertation. Louisiana State University. Baton Rouge. Sansinena, M. 2003. Somatic Cell Interspecies Nuclear Transfer. Ph.D. Dissertation. Department of Animal Sciences. Louisiana State University. Baton Rouge. OREGON Peoples, M. 2003. The effects of the phosphatase inhibitor, okadaic acid, on plasminogen activator activity in bovine oocyte-cumulus cell complexes matured in vitro. Senior Thesis, Oregon State University, Corvallis, OR. WISCONSIN Jensen, Nancy L. Bovine In Vitro Embryo Production using Avian White Yolk. M.S. Thesis, Department of Animal Sciences, University of Wisconsin. Abstracts ARKANSAS Rorie, R.W., C.F. Rosenkrans and A.J. Aishman. 2004. Effects of bovine somatotropin treatment on the AI pregnancy rate in dairy heifers. Reprod. Fert. Dev. CALIFORNIA Batchelder, C.A., K.A. Hoffert, M. Bertolini, A.L. Moyer and G.B. Anderson. 2004. Development of bovine nuclear transfer embryos cloned from follicular donor cells in sequential stages of differentiation. Annual Meeting, International Embryo Transfer Society, January 12-14, 2004, Portland, OR. Hoffert, K.A., C.A. Batchelder, M. Bertolini, A.L. Moyer and G.B. Anderson. 2004. Angiogenesis in cloned and IVF-derived bovine pregnancies at day 30 of gestation. Annual Meeting, International Embryo Transfer Society, January 12-14, 2004, Portland, OR. McCarthy, M., E. E. At-Taras, B. J. Nitta-Oda, C. Ducummon, C. Pearl, C., J. F. Roser, and T. Berger. 2003. Reduction of endogenous estrogens reduces epididymal boar sperm fertility. Biol. Reprod. 68 (Suppl 1):301. Khalil, M.B., K. Chakrabandhu, E. Carmona, H. Xu, N. Vuong, P. Kumarathasan, S. da Silva, D. Carrier, P. Wong, M. Buhr, T. Berger, and N. Tanphaichitr. 2003. Pig sperm rafts exhibit zona-pellucida binding ability and contain mammalian male germ cell specific sulfogalactosylglycerolipid and arylsulfatase A. Biol. Reprod. 68 (Suppl 1): 167. Khalil, M.B., K. Chakrabandhu, P. Wong, M. Buhr, N. Vuong, J. Mayne, E. Carmona, T. Berger, P. Kumarathasan, H. Xu, S. Wong, M. Na, D. Carrier, M. Mbikay, and N. Tanphaichitr. 2003. Sperm rafts bind to the egg zona pellucida and contain sulfogalactosylglycerolipid. Mol. Biol. Cell 14 (suppl). Mason, J.B., J. Najarian, E.A. Maga, J. Conrad-Brink, J.D. Murray and G.B. Anderson. 2003. The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice. Third Biennial Conference on Transgenic Animal in Agriculture, August 12-15, 2003, Lake Tahoe, CA. CANADA Colazo, M.G., Whittaker, P.R., Kastelic, J.P. and Mapletoft, R.J. 2003. CIDR-based protocols for synchronizing bovine embryo transfer recipients without estrus detection. Journal of Animal Science, 81 (Suppl. 1)/Journal of Dairy Science, 86 (Suppl. 1), 183 (Abstract). Colazo, M.G., Gavaga, Q.A., Kastelic, J.P. and Mapletoft, R.J., 2003. The Effects of Estradiol Cypionate (ECP) at CIDR Removal and Interval to Fixed-time AI on Pregnancy Rates in Beef Cattle. Theriogenology, 59, 215. (Abstract). Kastelic, J.P., Colazo, M.G., Martinez, M.F. and Mapletoft, R.J., 2003. Effects of a CIDR or MGA, Reproductive Status and Estradiol, GnRH or pLH Treatment on Pregnancy Rate to Fixed-time AI in Beef Heifers. Theriogenology, 59, 223. (Abstract). Martínez, M.F., Colazo, M.G., Kastelic, J.P. and Mapletoft, R.J., 2003. Effects of Estradiol and Progesterone on Plasma Steroid and Gonadotrohin Concentrations in CIDR-treated Ovariectomized Cows. Theriogenology, 59, 224. (Abstract). Nasser, L.F., Bo, G.A., Reis, E.L., Menegati, J.A., Marques, M.O., Mapletoft, R.J. and Baruselli, P.S., 2003. Superovulatory Response during the First Follicular Wave In Nelore (Bos Indicus) Donors. Theriogenology, 59, 530. (Abstract). COLORADO Bass, L .D., D. J. Denniston, L. J. Maclellan, P. M. McCue, G. E. Seidel, Jr., and E. L. Squires. 2003. Effect of methanol as a cryoprotectant for equine embryos. Proc. 19th Equine Nutr. Physiol. Soc. Symp. Campos-Chillon, L. F., J. F. De La Torre-Sanchez, and G. E. Seidel, Jr. 2003. Two-step vitrification and in-straw dilution of in vitro produced bovine embryos. J. Anim. Sci. 81 (Suppl. 1):143 (abstr. #565). Checura, C. M., and G. E. Seidel, Jr. 2003. Effect of macromolecules for bovine oocyte vitrification. Theriogenology 59:207. Coutinho da Silva, M. A., E. M. Carnevale, G. E. Seidel, Jr., and E. L. Squires. 2003. Effect of semen extender and centrifugation through Percoll on binding of stallion sperm to bovine zonae pellucidae. Proc. Soc. Theriogenology. In Press. De La Torre, J. F., and G. E. Seidel, Jr. 2003. Timing of dinitrophenol treatment during in vitro culture of bovine embryos. J. Anim. Sci. 81 (Suppl. 1):142 (abstr. #564). De La Torre-Sanchez, J. F., and G. E. Seidel, Jr. 2003. Effects of glucose concentration and presence of EGF and hormones on bovine oocyte maturation. J. Anim. Sci. 81 (Suppl. 1):186 (abstr. #M32). Flint, A., Z. Brink, and G. E. Seidel, Jr. 2003. Use of heterospermic insemination and genotyping embryos to compare fertility of flow-sorted sperm from individual bulls. Theriogenology 59:507. Schenk, J. L., W. B. Henderson, and G. E. Seidel, Jr. 2003. Timed insemination of superovulated heifers with sexed sperm. J. Anim. Sci. 81 (Suppl. 1):118 (abstr. #470). Seidel, G. E., Jr., Z. Brink, and J. L. Schenk. 2003. Use of heterospermic insemination with fetal sex as the genetic marker to study fertility of sexed sperm. Theriogenology 59:515. Tubman, L. M., Z. Brink, T. K. Suh, and G. E. Seidel, Jr. 2003. Normality of calves resulting from sexed sperm. Theriogenology 59:517. Walker, D. J., J. F. De La Torre-Sanchez, and G. E. Seidel, Jr. 2003. Effects of glucose concentration and presence of EGF and hormones on bovine oocyte maturation. J. Anim. Sci. 81 (Suppl. 1):33 (abstr. #132). CONNECTICUT Enright, B.P., X. C. Tian. 2003. Epigenetic Characteristics of Donor Cells for Nuclear Transfer: Levels of Histone Acetylation. Theriogenology 58:251. Bormann, C. L., B. P. Enright, X. Yang. 2003. Increased parthenogenetic development of bovine oocytes activated with a combinations of phosphorylation and protein synthesis inhibitors. Theriogenology 58:471. Jeong, B.-S., F. Xue, X. C. Tian. 2003. 5?-aza-2?-deoxycytidine induces expression of PLP1, an X-linked gene normally silent in bovine adult fibroblasts Theriogenology 58:422. Lee, J.-W., A. Dinnyes, X. Yang. 2003. Development of transgenic embryos following injection of freeze-dried sperm in pigs. Theriogenology 58:305. Liu, J.-L., L.-Y. Sung, F. Du, M. Julian, S. Jiang, M. Barber, J. Xu, X. C. Tian, X. Yang. 2003. Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer. Theriogenology 58:271. Nedambale, T. L., S. A. Chaubal, F. Du, W. Groen, D. C. Faber, A. Dinnyes, X. Yang. 2003. Vitrification methods for cryopreservation of in vitro produced cattle embryos. Theriogenology 58: 307. Wang L., B.-S. Jeong, E. Duan, X. C. Tian. 2003. Effect of donor cell age on number of population doubling of feline fibroblasts cultured in vitro Theriogenology 58:535. ILLINOIS Bonde, S., Varzavand, A., Zou, G.-M., Field, E., Walters, E.M., Wheeler, M.B., Zavazava, N. (2003) Porcine-Derived Embryonic Stem Cells are Immune-Privileged and Resistant to Natural Killer Cell Lysis. American Journal of Transplantation (Suppl. 5), S546. Clark, S.G. Walters, E.M. Beebe, D.J., Wheeler, M.B. (2003). A Novel Integrated In Vitro Maturation and In Vitro Fertilization System In Swine. Theriogenology 59:441. Clark, S.G., Walters, E.M., Beebe, D. J., M.B. Wheeler. (2003). Determination of the BSA content needed for culture of porcine embryos fertilized in micro channels. Biol. Reprod. 68 (Suppl. 1) 336. Donovan ,S.M, Hartke, J.L., Monaco, M.H., Wheeler, M.B. (2003) Over-expression of IGF-I effects on piglet intestinal growth. J. Dairy Sci 86 (Suppl. 1): 134. Gronlund D.E., Hurley, W.L, Monaco, M.H., Wheeler, M.B., Donovan, S.M. (2003) Over-expression of insulin-like growth factor-I in lactating porcine mammary tissue has a differential effect on amino acid transport systems. J. Dairy Sci 86 (Suppl. 1): 190. Hartke J.L., Monaco, M.H., Wheeler, M.B., Donovan, S.M. (2003) Effect of a short-term fast on intestinal disaccharidase activity and villus morphology in piglets suckling insulin-like growth factor-I (IGF-I) transgenic sows. J. Dairy Sci 86 (Suppl. 1): 134. Hartke J.L., Monaco, M.H., Wheeler, M.B., Donovan, S.M. (2003) Intestinal growth and development in piglets suckling insulin like growth factor-I (IGF-I) transgenic sows. J. Dairy Sci.86 (Suppl. 1): 311. Marshall, K.M., Shanks, R.D., Wheeler, M.B. (2003) Increased nursing intensity stimulated greater lactational response in sows expressing a bovine alpha-lactalbumin transgene. Biol. Reprod. 68 (Suppl. 1) 202. Monaco, M.H., Hurley, W.L, Wheeler, M.B., Donovan, S.M. (2003) Insulin-like growth factor-I (IGF-I) modulates the process of mammary apoptosis after weaning in IGF-I transgenic pigs. J. Dairy Sci 86 (Suppl. 1): 301. Walters, E.M., Clark, S.G, Roseman, H., Beebe, D.J., Wheeler, M.B. 2003. Production of Live Piglets Following In Vitro Embryo Culture in Microfluidic Environment. Theriogenology 59:353. LOUISIANA Barry, D.M., G. Herholdt, A. Visser, B.B. Hzimhanovich and R.A. Godke. 2003. Development of mammalian embryos and stem cells at micro-gravity conditions at the international space station. Theriogenology 59:337. Damiani, P., G. Wirtu, F. Miller, C.E. Pope, A. Cole, R.A. Godke and B.L. Dresser. 2003. Development of Giant eland antelope (Taurotragus derbianus) embryos following nuclear transfer with Common eland (Taurotragus oryx) and bovine (Bos taurus) oocytes. Theriogenology 59:390. Ferguson, C.E., D.J. Kesler, T.R. Davidson, B. Lenard and R.A. Godke. Saving pregnancies in beef cattle after a luteolytic dose of prostaglandin. Theriogenology (In press). Godke, R.A., Sansinena,M.J., B. Reggio, D. Hylan, A. Klumpp, S.K. Lyle, R.S. Denniston, and D. Paccamonti. 2003. Effect of different pre-maturation treatments on in vitro maturation of equine oocytes and subsequent competence for nuclear transfer. Proc. Havemeyer Workshop. p.13. Godke,R.A., K. Hebert, A. James, R.S. Denniston, D. Paccamonti. 2003. The potential use of epididymal sperm from bulls and stallions for assisted fertilization. Proc. Havemeyer Workshop. p. 21. Gómez, M.C., C.E. Pope, A.M. Giraldo, L. Lyons, R.F. Harris, A. King, A. Cole, R.A. Godke and B.L. Dresser. Birth of African Wildcat cloned kittens. Theriogenology (In press). Klump, A, Aljarrah A, Hylan D, Pinto C, Sansinena M, Eilts B, Godke RA and Paccamonti D. 2003. Follicular aspiration during transition to advance the onset of cyclicity. Proc. Leipzig Expert Workshop on Equine Reproductive Medicine (Abstract). Klumpp, A. M., R.S. Denniston D. Paccamonti, S.P. Leibo, and R.A. Godke. Embryonic development after holding bovine oocytes in undiluted follicular fluid for a 6-hour prematuration period. Theriogenology (In press). Landry, A.M., Y. Echelard, M. Murakami, M.G. Collins, R.S. Denniston and R.A. Godke. 2003 Effect of electrofusion method and time of cleavage on bovine embryo development after blastomere fusion at the 2-cell stage. Theriogenology 59:355. Moisan, A.E., J.W. Lynn, S.P. Leibo, R.S. Denniston and R.A. Godke. Volumetric changes of bovine oocytes resulting from dehydration in disaccharides solution. Theriogenology (In press). Nel, L., R.A. Godke, B.L. Dresser and P. Damiani. Isolation, culture and cryopreservation of somatic cells obtained from fresh and cooled semen, and fresh milk of gulf coast native sheep. Theriogenology (In press). Sansinena, M.J. Hylan, D., Klump, A., Reggio, B, Denniston, R.S. and Godke, R.A. 2003. Effect of roscovitine prematuration on in vitro maturation of equine oocytes from small ovarian follicles and subsequent nuclear transfer efficiency. Proc World Equine Vet. Med. Assoc. (Abstract). Sansinena ,M., P. Taylor, S. Taylor, R.S. Denniston and R.A. Godke. 2003. Production of interspecies nuclear transfer embryos using male and female llama (Lama glama) cell lines. Theriogenology 59:287. Wirtu, G., P. Damiani, B. Bavister, F. Miller, B. Dresser, R. Godke and E. Pope. 2003. Effects of eland and bovine serum on the development of in vitro-derived bovine embryos. Theriogenology 59:354. Wirtu, W., C. E. Pope, P. Damiani, B.L. Dresser, R.A. Godke and B.D. Bavister. Insulin improves frequency of cleavage and subsequent development of bovine embryos in vitro. Theriogenology (In press). WISCONSIN Fischer-Brown A., D. Northey, J. Parrish, and J. Rutledge 2003. Post-transfer growth of in vitro-produced bovine embryos. Biol Reprod 68 (Supp 1):252. Fischer-Brown A., R. Monson, D. Northey, J. Rutledge, K. Albrecht, and D. Schaefer 2003. Reduced dietary protein improves pregnancy rates following transfer of in vitro produced bovine embryos. Theriogenology 59(1):364. d. Miscellaneous publications CANADA Martinez, M.F., Adams, G.P., Kastelic, J.P. and Mapletoft, R.J., 2001. Artificial Insemination without Heat Detection in Beef Heifers. Large Animal Veterinary Rounds, 1, 2. Mapletoft, R.J., Schmutz, S.M. and Searcy, G.P., 2000. Observations on the Inheritance of a Bleeding Disorder in Simmental Cattle. Simmental Country, 21, 18. Mapletoft, R.J., Barth, A.D., Kastelic, J.P. and Coulter, G.H., 1999. Fertility and Puberty in Young Beef Bulls. Simmental Country, February 1999, 80-81. LOUISIANA Ferguson, C.F., O. Perez, K. Hebert , J. Cartmill and R.A. Godke. 2003. Using altrenogest to synchronize estrus in beef heifers. LSU AgCenter Beef Cattle Research Report 32:55-56. Hebert, K., D. Hylan, R.S. Denniston and R.A Godke. 2003. Administering prostaglandin F2a twice daily for 5 days to nursing beef cows starting 5 days after calving. LSU AgCenter Beef Cattle Research Report 32:58-59. Godke, R.A. and R.S. Denniston. 2003. Biotechnology and Cattle Production in the Future. OvaGenix Information Day Report 1:47-53. Godke, R.A., R.S. Denniston and B. Reggio. 2003. Animal biotechnology and the future. Louisiana Agric. pp. 10-14. IOWA Youngs, C.R. 2003. Use of gonadotropin releasing hormone for controlled breeding of dairy cattle. Iowa Jersey Today 9(1):9-10.

Impact Statements

Information from this multistate project will improve techniques employed for cloning and genetic engineering in livestock, impacting on animal agriculture, animal and human health.
Development of efficient oocyte and embryo culture systems allow for improvement of livestock via associated technologies.
Procedures developed for vitrifying bovine embryos will decrease costs and increase flexibility of cryopreserving embryos.
Demonstration that calves from sexed semen are normal allow farmers can use these procedures with minimal concerns.
Beta-lactalbumin and IGF transgenic swine may allow for improvement of piglet growth and health in swine production systems.
Impact 6; Understanding of the role of bST in reproduction may lead to development of procedures to avoid adverse effects on fertility; Impact 7; The competitive fertilization model developed enables accurate comparisons of treatments for fertility at lower cost;Impact 8; Cloning studies identified cell types best-suited for use in cloning and oocyte treatment to increase the number of NT morulae and blastocysts for transfer.