26 SPERM REPRODUCTIVE POTENTIAL OF FETAL FIBROBLAST CELLS ON A NUCLEAR TRANSFER–CLONED BULL AND ON AN ORIGINAL PATERNAL BULL: A COMPARATIVE ANALYSIS

Abstract

Recently, cloning technology has produced newborn calves from fetal fibroblast cells and somatic cells. These cloned calves appear to be normal and healthy in terms of reproductive ability. Therefore, we undertook this study to compare the reproductive ability of cloned sperm from the clones themselves and sperm obtained from the frozen semen of the paternal bull (KPN-178). Two cloned bulls from fetal fibroblast cell nuclear transfers (Korean HanWoo Clone-38, 34 months old, and Clone-39, 30 months old) were selected. Sperm from the cloned groups (C-38 and C-39), cloned-38 fresh sperm, and sperm from the paternal bull (KPN-178) were analyzed for the following sperm reproductive characteristics: sperm motility, by computer-assisted sperm analysis (CASA); sperm mitochondrial activity, by flow cytometer after mitotracker staining; and sperm viability, by flow cytometer after staining with live/dead sperm viability kits, both PI and SYBR. Sperm motility was evaluated with Percoll-treated or non-Percoll-treated frozen-thawed spermatozoa. In the Percoll-treated group, total motility of the paternal bull sperm (KPN-178: 75.14%) was significantly lower than that of cloned sperm (C-38: 90.42%; C-39: 87.62%: C-38 fresh: 93.97%; P < 0.05). The non-Percoll-treated group showed different results compared to the Percoll results (P < 0.05), i.e. the total motility of C-38 (92.47%) and C-38 fresh (92.47%) sperm was significantly higher than that of C-39 (79.52%) and KPN-178 (78.41%) sperm. The mitochondrial activity staining rate of KPN-178 (58.22%) sperm was low compared to that of C-38 (88.65%), C-39 (89.45%), C-38 fresh (88.66%) sperm. The sperm viability tests showed similar results (P < 0.05) for all sperm groups: C-38 (PI: 42.53% and SYBR: 46.28%), C-39 (PI: 39.03% and SYBR: 66.18%), C-38 fresh (PI: 34.32% and SYBR: 57.10%), and KPN-178 (PI: 14.43% and SYBR: 47.86%). However, the PI staining of KPN-178 was lower than that of the other sperm groups. Next, in vitro fertilization (IVF) was investigated to compare the reproductive ability of C-38, C-39, KPN-178, and Control (normal bull) sperm. The results, development to the 2-cell stage (157/228, 68.90%; 85/116, 73.28%; 78/114, 68.42%; 61/79, 77.21%, respectively) and blastocyst rates (41/228, 18.0%; 35/116, 30.17%; 26/114, 22.81%; 20/79, 25.32%, respectively) were evaluated (P < 0.05). The IVF rates were not different among cloned bull, paternal bull, and normal bull sperm. A cloned female bovine was successfully impregnated by cloned bull sperm (C-38) using artificial insemination. The calf resulting from this prenancy showed no signs of phenotypical abnormalities. These results suggest that the physiology of cloned bulls and the quality of semen from cloned bulls show no evidence of any deleterious effects.