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Embryo Transfer (ET) Embryo collection Donor cow Repeat Superovulation AI Fresh embryo Frozen embryo Recipient Estrus synchronization Embryo transfer Calf Advantage of ET Advantage of ET Treatment for repeat breeding cow Efficiency of ET on repeat breeding cow No of ET No of Preg Preg Rate (%) Normal cow 28 23 82 Repeat breeding cow 23 16 70 Efficiency of ET of IVF embryos after AI on repeat breeding cow Heifer Cow No of ET No of Preg Preg Rate (%) AI+ET 61 30 49.2 ET 61 18 29.5 AI+ET 273 114 41.5 ET 137 28 20.4 Superovulation of the cow Progesterone Day Estrus AI Luteal phase 10 11 12 13 14 15 Estrus/ovulation FSH with decreasing manner (2 times / day, or days) PGF2α Principle Induction of growth and ovulation of multiple follicles FSH treatment (decreasing manner) Stimulate multiple follicle growth Timing of FSH treatment Stimulate 2nd follicular wave PGF2α treatment Control of CL regression and induction of estrus/ovulation FSH treatment (decreasing manner) Half life of FSH: hours Twice / day, to days, 28-50 IU (total amount ) PGF2α treatment Inject at days after 1st FSH injection Estrus will occur at 36-48 hours after PGF2α injection Ovulation will occur at 24-36 hours after estrus ＊ eCG (equine chronic gonadotropin, PMSG) Similar to FSH and LH Half life: 40 hours Large molecular weigh: induce antibody and reduce response Follicular wave occurrence 2-wave cycle: 2nd wave occur at 9-10 days after estrus 3-wave cycle: 2nd wave occur at 8-9 days after estrus, 3rd wave occur at 15-16 days after estrus Follicle selection FSH level decrease at 2-3 days after wave occurrence Follicular growth depend on LH after follicle selection Endogenous FSH FSH peak appear at one day before wave occurrence Treatment with exogenous FSH Optimum timing: from one day before to days after wave occurrence Improvement of response to hormonal treatment Aspiration of large follicle or estradiol treatment Induction of new follicular wave by removing suppressive effects of large follicle on the growth of another follicles Aspiration of large follicle New follicular wave will occur within days Induction of atresia of large follicle by estrogen injection New follicular wave will occur at 3-4 days after injection Progeaterone releasing device: CIDR, PRID Luteal phase could be made artificially Growth of bovine embryo Oocyte Ovary fertilization 2-cell fimbriae 4-cell isthmus ampulla oviduct Days after fertilizaion 8-cell 16-cell Morula Blastcyst Uterotubal junction Uterus horn Haching Embryo collection Uterus flushing at days after insemination （ embryos enter to uterus at 4-5 days after ovulation) Flushing medium Balloon catheter Filter unit Uterus flushing using balloon catheter Anesthesia to stop rectal contraction Remove mucus of luteal phase Balloon catheter Injection of air or saline into balloon Location of balloon Location of balloon Flushing medium PBS Glucose （ 1g/L ） Sodium pyruvate （ 0.036g/L ） Bovine serum albumin （ 3g/L ） Antibiotics 3-way flushing line Filter unit for embryo collection Pore size: 70µm Embryo diameter: 120-140 µm Microscope Searching embryo How to recover flushing medium Searching embryos Development of bovine embryo Immature oocyte Unfertilized oocyte 2-cell embryo 4-cell embryo 8-cell embryo 16-cell embryo Early blastcyst Blastcyst Morula embryo Expanded blastcyst Hatching blastcyst Hatched blastcyst Evaluation of the embryos Regularity of shape (embryo and zona) Compactness of blastmeres Variation in cell size Color and texture of cytoplasm Overall diameter Abnormal Presence of extruded cells Irregular shape Presence of vesicles Presence of vesicles Normal Regular shape Presence of extruded cells Dark color Presence of degenerated cells Clear color A rank: Degenerated area 50% Evaluation of embryo quality Pregnancy rate Synchronization of estrus cycle between donor and recipient Difference in time of estrus cycle Synchronization of estrus cycle between donor and recipient Synchronization of estrus cycle Twice injection of PGF2α with 12 days apart Hormonal treatment same as timed-AI Ovsynch, PreSynch/Ovsynch CIDR-Synch PRID Follicular growth Oulation Ovulation Progesterone FSH 10 15 Days after estrus 20 ・ or wave of follicular gorwth ・ FSH stimulate small follicle growth (recruitment) Hypothalamus Hypothalamus Pituitary Pituitary （−） LH surge （＋） Pituitary FSH FSH （＋） LHRH LHRH LHRH Hypothalamus Ovary Small follicle growth by FSH （−） Inhibin Inhibition of follicle growth by dominant follicle （＋） Signal for ovulation from mature follicle Estradiol [...]... oocyte 2-cell embryo 4-cell embryo 8-cell embryo 16-cell embryo Early blastcyst Blastcyst Morula embryo Expanded blastcyst Hatching blastcyst Hatched blastcyst Evaluation of the embryos Regularity of shape (embryo and zona) Compactness of blastmeres Variation in cell size Color and texture of cytoplasm Overall diameter Abnormal Presence of extruded cells Irregular shape Presence of vesicles Presence of vesicles