title: Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos.
abstract: Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 × 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 ± 0.03) was the highest in three observation areas (P < 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 × 10(6) group (10 × 10(6) sperm/mL) were higher than in the 1 × 10(6) and 5 × 10(6) groups (P < 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P < 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% ± 2.61%) than the modified standard IVF technique (24.55% ± 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation.
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Jingchun
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Sibing
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Xianjing
authors.initials: X
authors.lastname: He
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Sun
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Qianyu
authors.initials: Q
authors.lastname: He
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
authors.forename: Shengjun
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Tsushima-Naga, Okayama, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Funahashi
authors.affiliation: Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China. Electronic address: elj863@163.com.
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology*; D005260:Female; D005306:Fertilization / Q000502:physiology; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000662:veterinary*; D008928:Mitochondria / Q000502:physiology; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000502:physiology*
keywords: Dairy cattle; IVF; Microfluidic sperm sorter; Sex-sorted embryo; Sex-sorted sperm