34 EFFECT OF GENOTYPE AND CELL LINE ON THE EFFICIENCY OF LIVE CALF PRODUCTION BY SOMATIC CELL NUCLEAR TRANSFER

The efficiency of production of live calves using somatic cell nuclear transfer was compared among 52 different cell lines representing 43 different genotypes. Cell lines were not genetically modified. Nuclear transfer was performed according to methods described by Cibelli et al., 1998 Science 280, 1256–1258, with modifications. All cells were derived from either explant cultures or enzyme digests of skin biopsies and were cyropreserved and thawed at least 48 hours prior to nuclear transfer. Cells were harvested using either pronase or trypsin at 70 to 90% confluence. Oocytes were either activated prior to fusion or immediately after fusion using ionomycin. The couplets were then cultured in cycloheximide and cytochalsin B for 6 hours. In 36 cases (84%), at least one healthy calf was produced from the initial trial which included transfer to 10 to 20 recipients for each cell line. For 4 of the 7 cases where the initial cell line failed to produce a live calf, a new cell line was derived and the process repeated. In one case where the data are available from the second cell line, 5 live calves were produced from 20 recipients receiving embryos (25%). Results from the other repeated cell lines are pending. For 5 of the different genotypes, nuclear transfer was done at about the same time using two different cell lines, and 4 of these have produced healthy calves from both cell lines. In one case, one cell line produced live calves, and no calves were produced from the other cell line. In total, 167 calves were born, of which 107 are alive and healthy as of this writing (64%), and range in age from 1 to 25 months. There are 86 calves older than 6 months of age and no losses have occurred as calves have aged into early adulthood. Forty-four (26%) of the calves were stillborn, failed to convert to neonatal circulation or were euthanized within 48 hours of birth. The most frequent reason for euthanasia was severe contracture of the limbs (arthrogryposis). This defect occurred even within cell lines that also gave rise to healthy calves, although it was more prevalent with certain cell lines. Other complications among the normal calves born were those of an abnormally large umbilicus or umbilical vessels. In addition, 16 calves were lost after the first 48 hours (13%). Two of these losses were due to accidents and 9 of them were due to complications from umbilical infections. The other 5 calf loses resulted from complications common to young calves such as clostridial infection and ruptured abomasum. Recent improvements in cell line derivation and embryo culture techniques, as well as a higher incidence of natural birth and improved neonatal management, have resulted in healthy calf production efficiencies (from embryos transferred) greater than 30% for 5 independent genotypes. The number of healthy calves produced per embryo transferred was 11 of 20 (55%), 5 of 10 (50%), 5 of 10 (50%), 4 of 11 (36%), and 3 of 10 (30%), for each of these genotypes, respectively. There was no correlation between the efficiency of blastocyst production and pregnancy outcome for the cell lines evaluated in this study. In conclusion, the efficiency of live healthy calf production using somatic cell nuclear transfer remains variable, depending on both the cell line and the genotype. However, efficiencies approaching those obtained using conventional embryo transfer is possible.

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