title: Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos.
abstract: BACKGROUND
  The in vitro culture of presumed zygotes derived from single cow ovum pick-up (OPU) is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality.
  
  
  METHODS
  Cumulus oocyte complexes (COCs) were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured in vitro, fertilized in vitro with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1) control OPU (controlOPU) with single cow OPU-derived presumed zygotes (2~8); (2) agar chip-embedded slaughterhouse helper embryo coculture (agarOPU) with ten presumed zygotes including all presumed zygotes from a cow (2~8) and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2); and (3) slaughterhouse in vitro embryo production (sIVP) with ten slaughterhouse ovary-derived presumed zygotes, each in 50 μL droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR.
  
  
  RESULTS
  The coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P < 0.01). Genes predicted to be involved in implantation failure and/or embryo resorption were down-regulated (P < 0.05) in control OPU zygotes (CD9, 0.4-fold; AKRAB1, 0.3-fold) and in cocultured zygotes (CD9, 0.3-fold; AKRAB1, 0.3-fold) compared to sIVP blastocysts (1.0-fold). Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P < 0.05 to P < 0.01) in control OPU zygotes (PGSH2, 5.0-fold; TXN, 4.3-fold; PLAU, 1.7-fold) and cocultured zygotes (PGSH2, 14.5-fold; TXN, 3.2-fold; PLAU, 6.8-fold) compared to sIVP (1.0-fold) blastocysts. However, the expression of PLAC8, TGF-β1, ODC1, ATP5A1 and CASP3 did not differ between the three culture groups.
  
  
  CONCLUSIONS
  Results show that the agar chip-embedded helper embryo coculture system enhances developmental competence and embryo quality in cultures of limited numbers of high pedigree single cow OPU presumed zygotes.
authors.affiliation: Division of Applied Life Science (BK21 program), Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Gautam Kumar
authors.initials: GK
authors.lastname: Deb
authors.forename: Jong In
authors.initials: JI
authors.lastname: Jin
authors.forename: Tae Hyun
authors.initials: TH
authors.lastname: Kwon
authors.forename: Byung Hyun
authors.initials: BH
authors.lastname: Choi
authors.forename: Jae Il
authors.initials: JI
authors.lastname: Bang
authors.forename: Shukla Rani
authors.initials: SR
authors.lastname: Dey
authors.forename: In Rae
authors.initials: IR
authors.lastname: Cho
authors.forename: Il Keun
authors.initials: IK
authors.lastname: Kong
mesh_terms: D000003:Abattoirs; D000818:Animals; D017849:Animals, Inbred Strains; D017209:Apoptosis; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D001947:Breeding / Q000379:methods; D002417:Cattle; D054885:Cumulus Cells / Q000502:physiology; D004477:Ectogenesis; D046149:Embryo Culture Techniques / Q000662:veterinary*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D018507:Gene Expression Regulation, Developmental; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D007202:Indicators and Reagents / Q000737:chemistry; D054315:Oocyte Retrieval / Q000662:veterinary*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012685:Sepharose / Q000737:chemistry; D015053:Zygote / Q000166:cytology / Q000502:physiology*
chemical_list: D007202:Indicators and Reagents; D012333:RNA, Messenger; D012685:Sepharose