19 Success of handmade cloning in a commercial setting

The purpose of this field trial was to determine whether handmade cloning could be used in a commercial setting to produce, transport, and implant embryos into recipients and to determine blastocyst and pregnancy rates. Donor animals and recipients were housed on 2 commercial farms, Farm A and Farm B. Ear notches were collected, grown in DMEM 10% FCS and 1% penicillin-streptomycin, and incubated at 38°C. Ovaries from a local abattoir were processed to collect cumulus-oocyte complexes for cloning. The cloning process included the following: (1) addition of demecolcine to maturation media at 22h, (2) bisection at 24 to 26h, (3) fusion at 25 to 27h, (4) activation at 30h, and (5) culture at 36h. After maturation, the cumulus cells were removed from the oocytes by incubating in 0.1% (wt/vol) hyaluronidase in HEPES-buffered TCM-199 with 2% (vol/vol) steer serum (T2) for 5min, followed by vortexing for 3min. The resulting cumulus-free oocytes were incubated in maturation media containing 0.5µg mL−1 of demecolcine for 2h. Next, the zona pellucida was removed with 0.2% (wt/vol) pronase in T2. An ultrasharp cutting blade was used to bisect the oocytes under a stereomicroscope, producing karyoplasts containing extrusion cones and cytoplasts. Fusion of 2 cytoplasts with a fibroblast was performed on a BTX fusion slide (San Diego, CA, USA) using a single direct current pulse of 100V for 9 µs. After fusion, the reconstructed embryos (REC) were incubated in SOFaaci for 3h until activation. The REC were activated with 10µM calcium ionophore for 5min in T2, followed by incubation in SOFaaci containing 2mM DMAP for 6h. Activated REC were individually cultured in well-of-the-wells (Vajta et al. 2000Mol. Reprod. Dev. 55, 256-264) containing SOFaaci without serum in 6% CO2, 5% O2, and 89% N2 for 7 days. For transport, 2-mL transfer tubes were filled with 400 uL of SOFaaci; overlayed with oil; gassed with 6% CO2, 5% O2, and 89% N2; loaded with 1 embryo per tube; and placed into a 39°C portable incubator. On Farm A, 34 REC were produced, with 13 developing to blastocyst stage (38% blastocyst rate). After a 1.5-h transport, 7 grade 1 expanded blastocysts were implanted into 7 synchronized recipients. At the 90-day pregnancy check, 3/7 (42%) were pregnant. On Farm B, 35 REC were produced, with 14 grade 1 morulas or early blastocysts developing (40% blastocyst rate). After a 6-h transport, 9 morulas or early blastocysts were implanted into 9 synchronized recipients. At the 90-day pregnancy check, 2/9 (22%) were pregnant. Overall, 5/16 (31%) of recipients remained pregnant by month 8 of gestation. In conclusion, handmade cloning is a practicable method to produce, transport, and implant embryos into recipients in a commercial setting.