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Effect of partial lipid removal from in vitro produced bovine zygotes on further development in vitro and on the freezing tolerance of blastocysts
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Theriogenology EFFECT OF PARTIAL LIPID REMOVAL FROM IN VITRO PRODUCED BOVINE ZYGOTES ON FURTHER DEVELOPMENT IN VITRO AND ON THE FREEZING TOLERANCE OF BLASTOCY STS C. Diez, D. Le Bourhis, Y. Heyman and J.P. Renard. Biologie du Developpement. INRA. 78352, Jouy-en-Josas (France).
In vitro-produced (IVP) bovine embryos are more sensitive to standard cryopreservation methods than in vivo ones. Intracellular lipids may have an important influence on this greater sensitivity to freezing (Leibo et al, Theriogenology 43:265,1995). The present work was designed to examine the effects of lipid removal from IVP bovine zygotes, with respect to further development in vitro and in vivo (Exp. I), and to their freezing tolerance at the blastocyst stage (Exp. 2). IVP bovine zygotes were centrifuged at 158OOgfor 7 min in the presence of Cytochalasin B ($&ml) to polarize cytoplasmic lipid droplets within the cells. The resultant lipid layer was then removed by micromanipulation. After lipid removal, “lipid free” and control embryos were cocultured for 7 days on VERO cell monolayers in B2 medium+lO% fetal calf serum (FCS) at 39°C to evaluate their in vitro survival and development to hatched blastocysts. In Experiment 1, a total of 970 embryos were delipated (13 replicates) and subsequent development in vitro was compared to that of 583 control IVP zygotes (13 replicates). The rates of cleavage and development to blastocysts were respectively 96.6% and 45.7% for “lipid free” and 91.5% and 46.2% for control zygotes. No significant differences were found in the rates of development of “lipid free” and controls zygotes (x2 test: p>O.O5). The developmental competence of “lipid free” blastocysts was further confirmed by transferring twelve embryos to synchronous recipient heifers (I embryo/recipient). Eight recipients established pregnancy as assessed by positive plasma progesterone test on Day 2 I, and six (50%) were confirmed pregnant on Day 90 by ultrasonography. In Experiment 2, “lipid free” and control IVP blastocysts on Day 7 of culture, were equilibrated in PBS containing 1.36M glycerol and 0.2SM sucrose, loaded into 0.25 ml straws and cooled from 20 to -6°C at S”C/min The straws were seed, cooled at 0 3Wmin to -30°C and then plunged into liquid nitrogen. Straws were thawed by agitation in 20°C water bath for 30 set and the content was expelled into a solution of 0.25M sucrose in PBS. Embryos were then washed in PBS+20% FCS and cocultured for 3 days to assess further development and hatching. TABLE 1, In vitro survival of “lipid free” or control bovine blastocysts after freezing/thawing Number Developing In Vitro (%) Number 24 h 48 h 72 h (hatching) Treatment thawed 37 (50.7) 41 (56.2) a 33 (45.2) a “Lipid free” 73 IS (22.4) h Control 67 21 (31.3) 22 (39.8) h x2: values with different superscripts within column differ significantly, n*h:pcO.02. The survival rate of “lipid free” blastocysts after freezing/thawing was significantly higher than that of control IVP blastocysts (Table I), and resulted in a higher hatching rate after 3 days of culture. These results suggest that the removal of approximately 90% of intracellular lipids from zygotes has no detrimental effect on in vitro and in vivo survival of bovine embryos, and that this treatment improves freezing tolerance of IVP embryos at the blastocyst stage
Theriogenology EFFECT OF PARTIAL LIPID REMOVAL FROM IN VITRO PRODUCED BOVINE ZYGOTES ON FURTHER DEVELOPMENT IN VITRO AND ON THE FREEZING TOLERANCE OF BLASTOCY STS C. Diez, D. Le Bourhis, Y. Heyman and J.P. Renard. Biologie du Developpement. INRA. 78352, Jouy-en-Josas (France).
In vitro-produced (IVP) bovine embryos are more sensitive to standard cryopreservation methods than in vivo ones. Intracellular lipids may have an important influence on this greater sensitivity to freezing (Leibo et al, Theriogenology 43:265,1995). The present work was designed to examine the effects of lipid removal from IVP bovine zygotes, with respect to further development in vitro and in vivo (Exp. I), and to their freezing tolerance at the blastocyst stage (Exp. 2). IVP bovine zygotes were centrifuged at 158OOgfor 7 min in the presence of Cytochalasin B ($&ml) to polarize cytoplasmic lipid droplets within the cells. The resultant lipid layer was then removed by micromanipulation. After lipid removal, “lipid free” and control embryos were cocultured for 7 days on VERO cell monolayers in B2 medium+lO% fetal calf serum (FCS) at 39°C to evaluate their in vitro survival and development to hatched blastocysts. In Experiment 1, a total of 970 embryos were delipated (13 replicates) and subsequent development in vitro was compared to that of 583 control IVP zygotes (13 replicates). The rates of cleavage and development to blastocysts were respectively 96.6% and 45.7% for “lipid free” and 91.5% and 46.2% for control zygotes. No significant differences were found in the rates of development of “lipid free” and controls zygotes (x2 test: p>O.O5). The developmental competence of “lipid free” blastocysts was further confirmed by transferring twelve embryos to synchronous recipient heifers (I embryo/recipient). Eight recipients established pregnancy as assessed by positive plasma progesterone test on Day 2 I, and six (50%) were confirmed pregnant on Day 90 by ultrasonography. In Experiment 2, “lipid free” and control IVP blastocysts on Day 7 of culture, were equilibrated in PBS containing 1.36M glycerol and 0.2SM sucrose, loaded into 0.25 ml straws and cooled from 20 to -6°C at S”C/min The straws were seed, cooled at 0 3Wmin to -30°C and then plunged into liquid nitrogen. Straws were thawed by agitation in 20°C water bath for 30 set and the content was expelled into a solution of 0.25M sucrose in PBS. Embryos were then washed in PBS+20% FCS and cocultured for 3 days to assess further development and hatching. TABLE 1, In vitro survival of “lipid free” or control bovine blastocysts after freezing/thawing Number Developing In Vitro (%) Number 24 h 48 h 72 h (hatching) Treatment thawed 37 (50.7) 41 (56.2) a 33 (45.2) a “Lipid free” 73 IS (22.4) h Control 67 21 (31.3) 22 (39.8) h x2: values with different superscripts within column differ significantly, n*h:pcO.02. The survival rate of “lipid free” blastocysts after freezing/thawing was significantly higher than that of control IVP blastocysts (Table I), and resulted in a higher hatching rate after 3 days of culture. These results suggest that the removal of approximately 90% of intracellular lipids from zygotes has no detrimental effect on in vitro and in vivo survival of bovine embryos, and that this treatment improves freezing tolerance of IVP embryos at the blastocyst stage