title: Regeneration of cryoresistance of in vitro rumen ciliate cultures.
abstract: The purpose of this study was to investigate factors affecting mechanical- and cryo-resistance of the rumen ciliates Entodinium caudatum (E.c.), Entodinium furca monolobum (E.f.m.), Entodinium simplex (E.s.), Diplodinium denticulatum (two clones, D.d.01 and D.d.02), Diploplastron affine (D.a.) and Epidinium ecaudatum forma caudatum (E.e.c.) after long-term in vitro cultivation. Following prolonged in vitro cultivation (more than six months), the ciliates were very sensitive to both centrifugation and 5% (v/v) dimethylsulphoxide, with motility decreased to: 39 and 23% for E.c., 66 and 32% for E.f.m., 46 and 27% for D.d. 01, 64 and 41% for D.a., and 44 and 28% for E.e.c., respectively. Thus, cryopreservation was unsuccessful. The effect of supplementing the ciliate growth medium with rumen fluid, glycine-betaine, proline, myo-inositol, linoleic acid, Sel-Plex or insulin, together with the effect of the source of rumen fluid on ciliate resistance to centrifugation, dimethylsulphoxide and freezing was also tested. The omission of rumen fluid from the growth medium resulted in the loss of cryoresistance after one-month cultivation. Supplementing the growth environment with a combination of glycine-betaine, proline, linoleic acid, Sel-Plex, insulin plus improved quality rumen fluid significantly enhanced survival of the ciliates after the freezing-thawing procedure (from 1 to 33% survival in un-supplemented vs. supplemented for E.c., P<0.01; 4-40% E.f.m., P<0.01; 0-17% D.d., P<0.05; 5-7% D.a. and 4-36% E.e.c., P<0.01).
authors.affiliation: Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovak Republic. kisiday@saske.sk
authors.forename: Svetlana
authors.initials: S
authors.lastname: Kisidayová
authors.forename: Zora
authors.initials: Z
authors.lastname: Váradyová
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Michałowski
authors.forename: C Jamie
authors.initials: CJ
authors.lastname: Newbold
mesh_terms: D000818:Animals; D001622:Betaine / Q000737:chemistry / Q000494:pharmacology; D002465:Cell Movement; D049109:Cell Proliferation; D016798:Ciliophora / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000494:pharmacology; D005615:Freezing; D066298:In Vitro Techniques; D007294:Inositol / Q000737:chemistry; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D019787:Linoleic Acid / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D012417:Rumen / Q000378:metabolism; D012756:Sheep / Q000469:parasitology; D016800:Trichostomatida / Q000378:metabolism*
chemical_list: D003451:Cryoprotective Agents; D007328:Insulin; D001622:Betaine; D007294:Inositol; D011392:Proline; D019787:Linoleic Acid