1. www.nias.go.kr Embryonic development of oocytes fertilized with frozen-thawed sperm derived from epididymis in young Hanwoo bull ABSTRACT : In general, it has been believed that semen collection should be carried out from 15 months of age in bulls. However, if we can use more young bulls’ semen to in vitro production of embryos, fertilizing ability and embryonic developmental competence can be predicted. Thus, we examined the embryonic developmental competence of oocytes fertilized with frozen-thawed sperm derived from epididymis in Hanwoo bull at 13 months of age (puberty). Blastocyst development rate of oocytes fertilized with frozen-thawed sperm in epididymis was similar to that of commercial frozen-thawed sperm (29.6 vs. 32.0 %, respectively). In conclusion, frozen-thawed sperm from epididymis in Hanwoo bull at 13 months of age has fertilizing ability and embryonic developmental competence. Key Words) Bull, Embryonic development, Epididymis, Hanwoo, Sperm In vitro production of embryos · Collection of oocytes : follicles (2-8 mm) of ovaries derived from slaughterhouse · IVM : TCM-199, 10% FBS, Na-pyruvate, FSH, LH, EGF, 10-15 COCs/ 45 ul droplet, 5% CO 2, 20% O 2, 38°C, 22 h · IVF : IVF-TALP, 18 µg heparin, 3mg/ml BSA, 2 x 10 6 cells/ml, 10-15 COCs/ 50 ul droplet, 5% CO2, 5% O2, 90% N 2, 38°C, 18 h · IVC : mSOFai, 3 mg/ml BSA, 15-25 COCs/ 50 ul droplet 5% CO 2, 5% O 2, 90% N 2, 38°C, 170 h Evaluation of in vitro development of embryos fertilized with epididymal sperm · Cleavage rate (Day 2) · Blastocyst rate (Day 7-8) ※ Day 0 = start of IVF Generally, epididymal sperm of young bull has been showed low fertilizability. Not elucidated the epididymal sperm fertilizability and embryonic developmental competence in young bull (Hanwoo) at 13 months of age. Therefore, we examined sperm ability when epididymial sperm in young bull was used to in vitro fertilization. 1.OWINY, O. D., BARRY, D. M., AGABA, M. & GODKE, R. A. 2009. In vitro production of cattlexbuffalo hybrid embryos using cattle oocytes and African buffalo (Syncerus caffer caffer) epididymal sperm. Theriogenology, 71, 884-94. 2.VIEIRA, L. A., GADEA, J., GARCIA-VAZQUEZ, F. A., AVILES-LOPEZ, K. & MATAS, C. 2013. Equine spermatozoa stored in the epididymis for up to 96h at 4 degrees C can be successfully cryopreserved and maintain their fertilization capacity. Anim Reprod Sci, 136, 280-8. Origin of sperm (Replicate) No. of oocytes % of blastocyst a % of cleavage Epididymis (4)7329.6 ± 7.5 NS 79.1 ± 13.7 NS Control b (3) 9232.0 ± 9.984.5 ± 3.6 Table 1. Blastocyst development after fertilization in vitro with frozen-thawed sperm derived from epididymis in Hanwoo Bull at 13 months. Values are expressed as Mean ± SD. a blastocyst development after in vitro fertilization (day = 0). b Commercial frozen-thawed sperm was collected from adult bull at15 months after birth. NS: Values in a row with superscript NS are not significant difference (P>0.05) 2. Oocyte-penetrating capacity of cauda epididymal sperm · 2 pronuclei (2PN) · Penetrated with more than 2 sperm (polyspermy) · Enlarged sperm head with anaphase II or telophase II (ESH; Enlarged sperm head) Surgical castration of testis Collection of epididymal sperm in Hanwoo · Extraction of epididymal sperm in Hanwoo bull at 13months of age · Surgical castration (testicles) Separation of testis and epididymis Preparation of epididymis Isolation of cauda epididymis Minced cauda epididymis Collection of sperm pronucleus polar body pronucleus polar body Fig.1. Penetration rate of the oocytes fertilized with epididymal sperm in Hanwoo bull at 13 months. Head; caput Body; corpus Tail; cauda 1. In vitro production of embryo using cauda epididymal sperm