title: Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: efficiency and in vitro developmental competence.
abstract: The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry-mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea.
authors.forename: Hyun-Tae
authors.initials: HT
authors.lastname: Jo
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea.
authors.forename: Jae-Il
authors.initials: JI
authors.lastname: Bang
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea.
authors.forename: Seong-Su
authors.initials: SS
authors.lastname: Kim
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea.
authors.forename: Byung-Hyun
authors.initials: BH
authors.lastname: Choi
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea.
authors.forename: Jong-In
authors.initials: JI
authors.lastname: Jin
authors.affiliation: Dairy Cattle Improvement Center, National Agricultural Cooperation Federation, Goyang-si, Gyeonggi-do, Republic of Korea.
authors.forename: Heyng-Lyool
authors.initials: HL
authors.lastname: Kim
authors.affiliation: Korea Sexing Biotech Inc., Daegu, Gyeongsangbuk-do, Republic of Korea.
authors.forename: In-Suk
authors.initials: IS
authors.lastname: Jung
authors.affiliation: Korea Sexing Biotech Inc., Daegu, Gyeongsangbuk-do, Republic of Korea.
authors.forename: Tae-Kwang
authors.initials: TK
authors.lastname: Suh
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea; Department of Animal Production, Faculty of Agriculture Cairo University, Giza, Egypt.
authors.forename: Nasser
authors.initials: N
authors.lastname: Ghanem
authors.affiliation: Department of Animal, Dairy, and Veterinary Sciences, School of Veterinary Medicine, Center for Integrated BioSystems, Utah State University, Logan, UT, USA.
authors.forename: Zhongde
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea. Electronic address: ikong7900@gmail.com.
authors.forename: Il-Keun
authors.initials: IK
authors.lastname: Kong
mesh_terms: D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D002417:Cattle / Q000196:embryology*; D002452:Cell Count; D002469:Cell Separation / Q000379:methods / Q000662:veterinary; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005434:Flow Cytometry / Q000662:veterinary; D008297:Male; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000145:classification / Q000166:cytology
keywords: Bovine; Embryo development rate; Intracytoplasmic sperm injection; In vitro fertilization; Karyotyping; Sex-sorted sperm