title: Roscovitine treatment improves synchronization of donor cell cycle in G0/G1 stage and in vitro development of handmade cloned buffalo (Bubalus bubalis) embryos.
abstract: This study investigated the effects of serum-starvation, total confluence, and roscovitine treatment on cell-cycle synchronization of buffalo ear skin fibroblasts to the G0/G1 stage and on the developmental competence of cloned embryos. Serum starvation of total confluence cultures for 24 h had a higher (p<0.05) proportion of cells at G0/G1 stage (94.4%) compared with serum starved cyclic and nonstarved confluent cultures (76.8 and 86.0%, respectively), whereas differences between cyclic cells with or without serum starvation were not significant. The proportion of cells at G0/G1 was higher (p<0.05) with 20 and 30 μM roscovitine treatment than that with 10 μM (94.4, 96.4, and 86.6%, respectively), which was similar to that for total confluence (86.0%). MTT assay showed that cell viability decreased as dose of roscovitine increased. The blastocyst rate was significantly higher (p<0.05) when nuclear transfer embryos were reconstructed using donors cells from total confluence, confluence serum starved, and roscovitine-treated (20 and 30 μM) groups (48.8, 48.9, 57.9, and 62.9%, respectively) compared to nontreated cyclic cells (20.2%). However, the cleavage rate and total cell number of cloned embryos were similar for all the groups. The number of ICM cells was improved by 30 μM roscovitine treatment (45.25 ± 2.34). The cryosurvival rate of blastocysts derived from cells synchronized with 20 or 30 μM roscovitine was higher compared to that for total confluence group (33.6, 37.8 vs. 23.8%). In conclusion, treatment with 30 μM roscovitine is optimal for harvesting G0/G1 stage cells for producing high quality cloned buffalo embryos, and that it is better than serum-starvation or total confluence for cell synchronization.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
authors.forename: Naresh L
authors.initials: NL
authors.lastname: Selokar
authors.forename: Monika
authors.initials: M
authors.lastname: Saini
authors.forename: Mushariffa
authors.initials: M
authors.lastname: Muzaffer
authors.forename: G
authors.initials: G
authors.lastname: Krishnakanth
authors.forename: Ambika P
authors.initials: AP
authors.lastname: Saha
authors.forename: Manmohan S
authors.initials: MS
authors.lastname: Chauhan
authors.forename: Radheysham
authors.initials: R
authors.lastname: Manik
authors.forename: Prabhat
authors.initials: P
authors.lastname: Palta
authors.forename: Pavneesh
authors.initials: P
authors.lastname: Madan
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
mesh_terms: D000818:Animals; D002020:Buffaloes / Q000196:embryology / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D019976:Cloning, Organism / Q000379:methods; D042522:DNA Replication Timing / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D053652:Nuclear Transfer Techniques; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000502:physiology; D000077546:Roscovitine; D013997:Time Factors
chemical_list: D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine