TY - JOUR
T1 - Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank
AU - Nsabimana, E.
AU - Kišidayová, S.
AU - Macheboeuf, D
AU - Newbold, C. J.
AU - Jouany, J. P.
N1 - Nsabimana, E., Kišidayová, S., Macheboeuf, D., Newbold, C. J., Jouany, J. P. (2003). Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank. Applied and Environmental Microbiology, 69 (7), 3826-3832.
PY - 2003/7
Y1 - 2003/7
N2 - The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25degreesC and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayova, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30degreesC, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2degreesC/min and 2.5degreesC/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.
AB - The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25degreesC and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayova, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30degreesC, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2degreesC/min and 2.5degreesC/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.
KW - OLIGOTRICH PROTOZOA
KW - DIGESTION
KW - IN-VITRO
KW - ENTODINIUM-CAUDATUM
KW - GROWTH
KW - DIPLOPLASTRON-AFFINE INVITRO
KW - CRYOBIOLOGY
KW - ENTODINIOMORPHID PROTOZOA
KW - CULTIVATION
KW - SHEEP
UR - http://hdl.handle.net/2160/9405
U2 - 10.1128/AEM.69.7.3826-3832.2003
DO - 10.1128/AEM.69.7.3826-3832.2003
M3 - Article
SN - 0099-2240
VL - 69
SP - 3826
EP - 3832
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 7
ER -