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Developmental Characteristics of Interspecific Hybrid Embryos

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Developmental Characteristics of Interspecific Hybrid Embryos DEVELOPMENTAL CHARACTERISTICS OF INTERSPECIFIC HYBRID EMBRYOS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph bv DAWN A. KELK In partial fulfilLment of requkements for the degree of Doctor of Philosophy Jdy, 1997 Bibbaièque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services senrices bibliographiques 395 Wellington Street 395. rue Wemgtcm ût&waON KlAW Otlawa ON KIA ON4 Canada canada The author bas granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive pamettant à la National L&rary of Canada to Bibliothèque nationale du Canada de reproduce, loan, disîriiuîe or sell reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. ia forme de microfiche/fï?m, de reproduction sur papier ou sur fonnat électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neitha the droit d'auteur qui protège cette thèse. thesis nor substantial extracts hmit Ni la thèse ni des extraits substantiels may be printed or otherwise de cebci ne doivent être imwés reproduced without the author's ou autrement reproduits sans son permission. autorisation, DEVnOPMENTAL CHARACTERISTICS OF INTERSPECIFIC HYBRID EMBRYOS Dawn A. Kelk Advisor: University of Guelph, 1997 Dr. W. A. King Establishment of an embryo capable of development to term involves precisely regdated nudear and cytoplasmic events. Interspeefic hybrids provide unique emb ryo models with morpho logical, b iochemical and temporal markers of development which enable investigation of these interactions. This study explores the feasibility of producing and utilizing interspecific hybrid embryos of cattle, sheep and goats as models to examine the respective roles of the maternd and patemal contributions to the embryo and the interactions of the nucleus and cytoplasm. Interspeafic hybrid embryos were created under in vivo and/or in uitro conditions. Ram spermatozoa penetrated intact bovine oocytes under in oitro conditions, but the resulting embryos arrested at the 4-8-ceil stage. This developmental =est was exhibited even when bovine X ovine hybrid zygotes were transferred to sheep oviducts for in oiuo culture, illustrating that the arrest was not an effect of the in oitro culture. Construction of nuclear transfer hybrids between ovine karyoplasts and bovine cytoplasts resulted in the same pattern of developmentd arrest as in the bovine X ovine hybrid embryos. This indicates that the developmental =est exhibited by the hybrid embryos is not an intragenomic incompatibfity between the bovine and ovine componentç of the nudeus but an incompatibility between nudeus and cytoplasm. Autoradiogaphic detection of 3~-ur~dineincorporation provided evidence for Iow levels of genomic activiv at the 2-ceIl stage of all types of embryos. ki ail but bovine X ovine hybrids, transcriptional activity inaeased through the 4-ceII stage and culminated in a burst of transcriptional activity by the laie &cd stage. Bovine X ovine hybrid embryos exhibited generous transcriptional activïty at the 4ceU stage but no burst of activity as the ernbryos arrested at the 48-cd stage. Ovine X caprine hybrid embryos develop to the blastocyst stage, hatch in vitro and establish pregnancies when transferred to sheep or goats. The pregnancies however, have retarded fetal devdopment, incompletely formed placentornes and ultimately fail during the second month of gestation. In summary, most interspecific hybrid embryos can be produced readily under in oioo or in vitro conditions. This study confirms the potential of producing and utilizing interspecific hybrid embryos from domestic ruminants as models to study embryonic devdopment. ACKNOWLEDGEMENTS I wish to thank the following people: Dr. Wh. King for his support, guidance and willingness to help at any time. Man made the duration of this project an enjoyable and rewarding experience through his encouragement and direction. Dr. C.J. Gartley for enduring di of the fnistations and successes of the in vivo aspects of the projed Thanks for maintaining a postitive atmosphere during the times when nothhg seemed to be working and for appreciating my nuv enthusiasm over a few pregnant sheep when things worked wd. The members of my advisory cornmittee; Dr. A. Hahnd and Dr. K. Goodrowe for their advice and assistance throughout the course of this researdi and during preparation of this manuscript. The staff at the Ponsonby Sheep and Eramosa Research Stations, the Veterinary Teadiing Hospital and the Animal Saence Meat Laboratory for their cooperative assistance. Cathy Britton and Pam Jordan for being partidarly accommodating to my requests. My fellow graduate students from the WAK lab and the department of Biomedical Sciences and all the faculty and staff for help in the laboratory as well as comments and constructive criticisms. Elizabeth St. John for help preparing culture media. United Breeders Inc. for supplying frozen bu11 semen. NSERC, OMAFRA and OGS for their suppoa of the research. FinaUy, 1 wiçh to thank my parents and family for th& constant support. DECLARATION OF WORK PERFORMED 1 declare that with the exception of the items Ïndicated below, ail the work reported in this thesis was perfonned by me. AU laparoscopic and surgical ernbryo transfers and surgical embryo flushes of ewes and does were performed by Cathy Gartley with my assistance. Ultrasound diagnosis of ewe and doe pregnancies was occassionally assisted and confirmed by Cathy Gartley. Bovine ovarïes were collected from the abattoir by Lawrence Afflu. AU media used were made by either myself or Elizabeth St. John. Progesterone assays were performed by Claudia Jiminez. Histological preparations of placentomes were performed by Mary John. Nudear transfer micromanipulations were performed by Marie-Cede Lavoir with my assistance. Photographs were printed by a commercial photo laboratory. Methods ....................................................................................................... In uivo production of ovine, caprine and hybrid ernb ryoç ........ Experimental animals................................................................... Synchronization and superovulation of sheep and goats .... Breedïng ........................................................................................... Confirmation of breeding ............................................................ Embryo coIleaion .......................................................................... Surgical embryo fi ush ................................................................ Slaughter flush ............................................................................ In uitro production of embryos ........................................................ In vitro oocyte maturation .......................................................... Collection of ovaries ................ .................................................. Isolation of cumulus-oocyte complexes................................. Culture of cumulus-oocyte complexes................................... Preparation of oocytes for fërtilization................................... collection ............................................................................ Sperm preparation ......................................................................... In vitro fertilization ...................................................................... oviduct explant preparation ....................................................... Buffalo rat liver cdpreparation ................................................ Culture of embryos ........................................................................ Fertiliza tion assay .......................................................................... Cytogenetic analysis of embryos ....................................................... Embryo transfer of in oifro produced bovine X ovine hybrid embryos ......................................................................................... Experimental animals................................................................... Synchroniza tion of sheep reapients ......................................... Transfer of embryos....................................................................... Recovery of embryos ........................................................................... Results .......................................................................................................... Isolation of cumulussocyte complexes.......................................... In vivo production of ovine, cap~eand hybrid embryos ........ Penehation and development of in uïtro produced embryos ... Bovine and ovine crosses ...................................*........................ Caprine and ovine crosses ........................................................... Discussion.................................................................................................... Isolation of cumulus-oocyte complexes.......................................... 64 Caprine and ovine crosses ................................................................ 65 Bovine and ovine crosses .................................................................. 67 CHAPTER 2: INTERSPECIFIC HYBRID EMBRYO TRANSFERS AND PREGNANCIES........................................................................... Introduction...............................................................................................
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