67 ORGAN WEIGHT VARIATION IN CLONED PIGS

Previously, we compared adult clones and naturally bred control females using a series of physiological and genetic parameters and found considerable variation in certain traits in clones. In order to more fully understand this clonal variation we examined the organ weight differences among a group of clones from a single litter. Briefly, in vitro-matured oocytes were stripped of their cumulus cells at 46–48 h postmaturation by vortexing in TCM 199 containing 5% FCS with 0.1% hyaluronidase, and placed in Ca-free NCSU-23 with 4 mg mL-1 BSA. Oocytes were stained in Ca-free NCSU-23 containing 4 mg mL-1 BSA and 5 µg mL-1 Hoechst 3334 for 10 min, and enucleated in Ca-free, salt buffered, NCSU-23 containing 5% FCS and 7.5 µg mL-1 of cytochalasin B, Puromycin-resistant fetal fibroblasts grown for 35 days in D-MEM/F-12 containing 15% FCS and 1.65 µg mL-1 puromycin were synchromized by contact inhibition for 1-2 days. Cells were trypsinized, resuspended in salt buffered NCSU-23 with 10% FCS and placed into the perivitelline space. Couplets wer fused by 2 V AC for 2 s followed by 2 pulses DC of 1.4 kv cm-1 for 50 µs. Following fusion, the oocytes were placed in NCSU-23 containing 4 mg mL-1 BSA for 1.5–2 h. The oocytes were then electroactivated by 2 pulses of 1.2 kg cm-1 for 50 µs, allowed to recuperate for 10–15 min, and transferred into the oviduct of a naturally synchronized recipient. A total of 286 doublets were transferred into 4 recipients one of which became pregnant and gave birth to 9 clones (9/81). One clone died soon after birth. The remaining 8 clones were weaned at three weeks and kept as a group until Day 120 at which time the animals were euthanized for organ recovery. Weights were determined for major organs, and coefficients of variation (CV) calculated for both absolute weights, and corrected total weight. Corrected weights were calculated as weight of organ divided by weight of clone. CV for absolute and corrected weights, respectively, were: uterus, 15.1% and 5%; left kidney, 13.2% and 6.9%; right kidney, 11% and 6.8%; spleen, 29.7% and 27.1%; liver, 20.6% and 19.7%; lung, 37.5% and 32%; heart, 20.2% and 15.5%; and brain, 5.6% and 9.5%. Both absolute and corrected weights indicated that there is a high degree of variation in some of the organs among a group of identical clones. To determine whether the variation we observed is typical of non-cloned swine, we compared the variations in the clones to those observed in a group of 3 age-matched, sex-matched, breed-matched controls that had been kept with the clones for approximately 90 days. Relative coefficients of variation of corrected organ weights between clones and non-cloned animals were: uterus, 0.18; left kidney, 0.34; right kidney, 0.34; spleen, 2.58; liver, 1.08; lung, 1.06; heart, 0.59; and brain, 0.7. In summary, these results indicate that some organs in cloned animals have a high degree of weight variability, most likely due to epigenetic aberrations of overriding environmental effects.

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