The present study was carried out to evalute the effect of glucose and lactate on in vitro produced bovine embryos using m-SOF medium. In the first and second experiments, cumulus oocyte complexes (COCs) were matured (22 h), fertilized (5 h) and cultured (9 days) in medium containing 0, 3.3, 5, 10, 15 mM lactate or 0, 1.5, 3, 5 mM glucose in m-SOF for in vitro production of embryos. In the third experiment, 1.5 mM of glucose was suspended in m-SOF at 0, 48, 72, 96 and 120 h after IVF. The cleavage rates of embryos were not significantly different among the concentrations of lactate, however the blastocyst production rate was significantly higher (p<0.05) with a concentration of 3.3 mM (34.8%) than those of 10 and 15 mM (18.9 and 15.7%, respectively). The cleavage rate of embryos was significantly higher (p<0.02) with a concentration of 0 mM of glucose (66.9%) than those of 1.5, 3, and 5 mM (41.2, 46.5 and 40.3%, respectively). The blastocyst production rate was significantly higher (p<0.02) with concentration of 0 mM (35.6%) than those of 3 and 5 mM (16.7 and 19.6%, respectively). When the embryos were cultured in m-SOF suspended with 1.5 mM of glucose at 48, 72, 96 and 120 h after IVF, the blastocyst production rates were significantly higher (p<0.05) at 96 and 120 h (51.6 and 50.8%) than those of 48 and 72 h (32.0 and 33.3%). These results indicate that 3.3 mM of lactate was more effective than that of other concentrations for in vitro embryo production, and that glucose inhibited embryonic development in early stage embryos, but sustained their post-morula stage.

View full abstract

In this paper, we show a protocol of a refined and useful application of rapid fluorescence in situ hybridization (FISH) for sexing bovine blastocysts. About ten blastomeres per bovine blastocyst are fixed in an equal volume of PBS(-) and fixative freshly made of acetic acid and methanol (1:3, v/v), and mounted onto a clean glass slide, on which the fixative is dropped. The PCR primer pair is designed from a bovine male- specific DNA sequence termed as BC1.2. The following sequences of the oligonucleotide primers are used: upstream 5'-ATCAGTGCAGGGACCGAGATG-3' and downstream 5'AAGCAGCCGATAAACACTCCTT-3'. A Dig-labeled probe produced by PCR is added to the hybridization mixture (as a final concentration, 50% formamide, 10% dextran sulfate, 30 ng/μl BSA and 2 × SSC, pH 7.0). The hybridization mixture adjusted is dropped on each glass slide, and denatured at 72°C for 8 min on an aluminum block. Immediately after denaturation, they are hybridized at 38.5°C for 5 min, washed in 0.5 × SSPE at 72°C for 5 min and followed by washing in PN buffer (0.1% sodium phosphate, pH 8.0, 0.1% NP-40) for 2 min. The Dig is detected by incubation with anti-Dig-fluorescein (1.0 μg/ml) in PN buffer plus 1% Block Ace at 38.5°C for 5 min. After washing with PN buffer, the blasomeres are counterstained with propidium iodide (0.3 μg/ml) in an anti-fade solution. The observation is carried out under an epifluorescence microscope. The whole process of FISH can be carried out within almost 1 hr. This protocol with a male-specific DNA probe is a powerful tool for sexing bovine embryos.

View full abstract