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Alternative tests to assess viability of bovine embryos
THERIOGENOLOGY WTIVE
TESTS 'I0ASSESS VIABILITYOF I30VINEEMBRYOS
J.-P. Fienard,Y. Menem,* and Y. Heyman 1.N.fi.A. Station de Physiologicanimale, 78350 Jouy-en-Jonas,and *IX&A., Iaboratoirede Biolcgie 406, 69621 Villeurbanne cedex, France In order to assess the viability of embryos more accurately, we have experimented with several techniques other than subjective observation of gross morphology. Amount of fluorescenceliberatedby fluoresceindiacetate (FDA) solutions in live blastomeres after only 3 min of incubationat room temperaturewere used to score 84 day-7 to -8 embryos: Hatched blastocysts Pregnancy rate Fluores after transfer Score cence No. embryos (%) after 48 hr culture 35147 (74.4%) 1 bright 55 (65.4%) 6/8 (75%) 2 partial 15 (17.8%) 216 (33%) 4/9 (44.4%) none developed 3 none 14 (16.6%) none developed Accuracy of scoring, however, was no greater than with the simple gross morphologicalassessmentat the time of recovery with the exceptionof 3 embryos (3.5%)classifiedas good that scored 3 on the FDA test. Diamino-phenyl-indol (DAPI), a non-toxic compound that binds specifically to the DNA of the cells, was less effective than morphologicalobservations. All the embryos classifiedmorpholcgically as degenerate fluoresced brightly after 3 min of incubation, but9118 brightly fluorescingembryos, which had been classifiedmorphologically as good before incubation, developed in hatched blastocysts after culture for 48 br, and three developed in vivo after transfer. After 20 hr in vitro, lactate dehydrogenasealways appeared in the medium when embryos were markedly degenerate,but was never found with the apparentlynormal embryos, which developed in vivo when transferred to recipients after culture. G6PDH, GPDH or succinodehydrogenase were released by day-7 or -8 blastocysts developing apparently normally within 20hr of culture, but these enzymes could not be considered as markers of viability as they were also released by degenerated blastocysts. The same situation apparently occurs with lysosomal enzymes such as beta hexosaminidase,which was releasedby day-8 embryos hatching in vitro or degeneratingafter 48 hr of culture. When day-7 to -8 normal or degenerateembryos were cultured 48 hr in a chemically defined medium, no glucose uptake was detectable. When cultured for 20 hr, 25/48 (52%) day-10 to -11 blastocysts collected just before their elongation phase took up a significant amount of glucose (more than 50 ug). There were no morphological differences between the blastocyststhat did or did not take up glucose. Following nonsurgical transfer, 9113 (69.2%) of blastocysts that did take up glucose and 2114 (14.2%)of those that did not, developed into fetuses. For older, elongated day-12 blastocysts, a marked glucose uptake was detectable after only 5 hr of culture; 15/43 (35%) of the developed embryos took up glucose (60.7+ 3.0~); however, in contrast to day-10 to -llembryos, this uptake was correlated to the size of the embryos (r=0.92)but there was no direct relation to viability after transfer (9/14 (64%) embryos with glucose uptake developed vs. 4/6 (67%) with no glucose uptake). Although hatching blastocysts take up more free amino acids in culture than other day-7 to -10 stages, this technique is too difficult to be used rountinelyto assess the viabilityof embryos. JANUARY
1982 VOL. 17 NO. 1
TESTS 'I0ASSESS VIABILITYOF I30VINEEMBRYOS
J.-P. Fienard,Y. Menem,* and Y. Heyman 1.N.fi.A. Station de Physiologicanimale, 78350 Jouy-en-Jonas,and *IX&A., Iaboratoirede Biolcgie 406, 69621 Villeurbanne cedex, France In order to assess the viability of embryos more accurately, we have experimented with several techniques other than subjective observation of gross morphology. Amount of fluorescenceliberatedby fluoresceindiacetate (FDA) solutions in live blastomeres after only 3 min of incubationat room temperaturewere used to score 84 day-7 to -8 embryos: Hatched blastocysts Pregnancy rate Fluores after transfer Score cence No. embryos (%) after 48 hr culture 35147 (74.4%) 1 bright 55 (65.4%) 6/8 (75%) 2 partial 15 (17.8%) 216 (33%) 4/9 (44.4%) none developed 3 none 14 (16.6%) none developed Accuracy of scoring, however, was no greater than with the simple gross morphologicalassessmentat the time of recovery with the exceptionof 3 embryos (3.5%)classifiedas good that scored 3 on the FDA test. Diamino-phenyl-indol (DAPI), a non-toxic compound that binds specifically to the DNA of the cells, was less effective than morphologicalobservations. All the embryos classifiedmorpholcgically as degenerate fluoresced brightly after 3 min of incubation, but9118 brightly fluorescingembryos, which had been classifiedmorphologically as good before incubation, developed in hatched blastocysts after culture for 48 br, and three developed in vivo after transfer. After 20 hr in vitro, lactate dehydrogenasealways appeared in the medium when embryos were markedly degenerate,but was never found with the apparentlynormal embryos, which developed in vivo when transferred to recipients after culture. G6PDH, GPDH or succinodehydrogenase were released by day-7 or -8 blastocysts developing apparently normally within 20hr of culture, but these enzymes could not be considered as markers of viability as they were also released by degenerated blastocysts. The same situation apparently occurs with lysosomal enzymes such as beta hexosaminidase,which was releasedby day-8 embryos hatching in vitro or degeneratingafter 48 hr of culture. When day-7 to -8 normal or degenerateembryos were cultured 48 hr in a chemically defined medium, no glucose uptake was detectable. When cultured for 20 hr, 25/48 (52%) day-10 to -11 blastocysts collected just before their elongation phase took up a significant amount of glucose (more than 50 ug). There were no morphological differences between the blastocyststhat did or did not take up glucose. Following nonsurgical transfer, 9113 (69.2%) of blastocysts that did take up glucose and 2114 (14.2%)of those that did not, developed into fetuses. For older, elongated day-12 blastocysts, a marked glucose uptake was detectable after only 5 hr of culture; 15/43 (35%) of the developed embryos took up glucose (60.7+ 3.0~); however, in contrast to day-10 to -llembryos, this uptake was correlated to the size of the embryos (r=0.92)but there was no direct relation to viability after transfer (9/14 (64%) embryos with glucose uptake developed vs. 4/6 (67%) with no glucose uptake). Although hatching blastocysts take up more free amino acids in culture than other day-7 to -10 stages, this technique is too difficult to be used rountinelyto assess the viabilityof embryos. JANUARY
1982 VOL. 17 NO. 1