A novel approach for in vitro production of bovine embryos: use of the Oxoid atmosphere generating system.
The importance of the incubator type is often overlooked when protocols for in vitro production of embryos are evaluated. In this study the ability of a standard CO2 Heraeus incubator and the Oxoid CO2Gen atmosphere-generating system to support bovine in vitro oocyte maturation, fertilization and embryo development is described for the first time. The Oxoid CO2Gen gas generating system, originally designed for the growth of bacteria, is based on the chemical reaction of ascorbic acid and air. When the sachet with ascorbic acid is placed in the confined volume of the airtight AnaeroJar, an atmosphere of 6% CO2 in 15% O2 is created, which is comparable to the 5% CO2 and 20% O2 used for standard in vitro production of bovine embryos. In the first set of experiments oocyte in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) were allocated to one or the other of the culture systems. In the second set of experiments IVM and IVF took place in the Heraeus incubator, while IVC was allocated either to the Heraeus or to the AnaeroJar. During experiments the AnaeroJar was placed in the Heraeus incubator to ensure identical incubation temperatures of 38.8 degrees C. A standard protocol was used for production of embryos: 23 h of IVM in TCM-199, 20 h of IVF with frozen-thawed washed spermatozoa in TALP medium and 7 days of IVC (8 days after insemination) in B2 medium with bovine oviduct epithelial cells. In the first set of experiments, based on a total of 766 inseminated oocytes, the Day 8 blastocyst rates were the same in the Heraeus incubator and the AnaeroJar: 30% vs. 30% with oviduct cell coculture, and 21% vs. 18% without coculture. In the second set of experiments, based on 1963 inseminated oocytes, the average blastocyst rates were 27% vs. 32% from the Heraeus incubator and the AnaeroJar. In 2 of 6 replicates blastocyst rates were lower in the Heraeus incubator than in the jar; in the remaining replicates they were alike. No differences were noted in blastocyst kinetics or morphology. In conclusion, the Oxoid gas generating system seems to be a cheap, convenient and stable alternative to expensive CO2 incubators, not only for the growth of bacteria, but also for in vitro production of bovine embryos.
Avery
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Department of Clinical Studies, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark. bma@kvl.dk
Melsted
J K
JK
Greve
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T
D000818:Animals; D002245:Carbon Dioxide / Q000502:physiology; D002417:Cattle / Q000196:embryology* / Q000502:physiology; D005314:Embryonic and Fetal Development; D005260:Female; D005307:Fertilization in Vitro / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D007185:Incubators / Q000662:veterinary; D008297:Male; D009865:Oocytes / Q000502:physiology; D010100:Oxygen / Q000502:physiology; D011897:Random Allocation; D012098:Reproduction / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology
D002245:Carbon Dioxide; D010100:Oxygen