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Title: ウシの体外受精における精子処理法と体外受精由来の初期胚の体外培養法に関する研究
Other Titles: Studies on sperm treatments and culture systems of early stage bovine embryos in relation to in vitro fertilization.
Authors: 青柳, 敬人 Browse this author
Issue Date: 25-Dec-1991
Abstract: The effects of semen treatments and semen from different bulls on in vitro fertilization results and the effect of culture systems of early stage embryos after in vitro fertilization wereexamined in order to establish a stable procedure to produce bovine blastocysts derived from in vitro maturation and in vitro fertilization. ***Effects of two treatments on semen from different bulls on in vitro fertilization results of bovine oocytes*** The spermatozoa from six different bulls were treated with caffeine or caffeine plus Ca-ionophore A23187 and their effects on in vitro fertilization, cleavage and development into morulae of in vitro matured bovine oocytes were investigated. ln vitro fertilization results(formation of both pronuclei, cleavage and development to ≧ four-cell stage) were significantly(P<0.01)higher using caffeine plus Ca-ionophore A23187 than those using only caffeine. The rates of fertilization and first cleavage were only slightly variable among the bulls. However, the present data showed significant variability in the formation of both pronuclei(36 to 75%) of fertilized embryos and development to the ≧ four-cell stage(39 to 71%) by using semen from different bulls. Development into morulae of embryos recovered from the rabbit oviduct did not show any significant differences in relation to sperm treatments or individual bulls. ***Effects of culture systems on the development of in vitro fertilized bovine embryos into blastocysts*** The effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development into blastocysts of bovine eight-cell embryos derived from in vitro maturation and fertilization were examined. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Fertilized embryos resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 plus 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within the rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly(P<0.05)increased developmentinto blastocysts(39.0 to 50.7%) when compared to co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen-thawed embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were subsequently born. The results have demonstrated that co-culture of embryos with oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells is a useful method for enhancing development of in vitro fertilized bovine eight cell stage embryos into blastocysts and for producing normal calves using frozen-thawed embryos derived from in vitro maturation and fertilization. It is obvious that this study is useful to stabilize production of blastocyst stage embryo by bovine in vitro fertilization and contribute to the practical application of bovine embryo transfer.
Conffering University: 北海道大学
Degree Report Number: 乙第4008号
Degree Level: 博士
Degree Discipline: 獣医学
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)

Submitter: 青柳 敬人

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