Abstract
Anatolian grey cattle (endangered native Anatolian cattle) as 1 male (clone 1) and 4 females (clones 2–5) were produced from cells of 1 male and 1 female cattle by somatic cell nuclear transfer (SCNT) in a previous study. In this study, we examined the reproductive potential of these cloned animals, which are now 4 and 5 years old. The parameters evaluated by phase contrast microscopy for motility, TUNEL for DNA fragmentation, eosin staining for viability, Hoechst 33258 staining and hypo-osmotic swelling test (HOST) for membrane integrity, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) for acrosome integrity of frozen-thawed spermatozoa, as well as birth and survival of calves following insemination with frozen-thawed semen of cloned and nuclear donor bull and normal bull. Six ejaculates and 3 samples per ejaculate from each bull were tested, and the Mann-Whitney U test was used to analyse the data. The spermatological parameters of cloned bull semen – volume, concentration, and motility of fresh – were within accepted limits for artificial insemination (4.60 ± 0.47 mL, 1.55 ± 0.21 × 109 spermatozoa mL–1, 80.00 ± 1.07%, respectively). Frozen-thawed sperm motility and viability rate were higher in the cloned bull (56.6%, 56.7%) than in its nuclear donor (47%, 43%; P < 0.05). Intact membrane and DNA fragmentation rate of cloned bull and its nuclear donor bull sperm were similar (P > 0.05) but the intact acrosome rate of cloned bull was higher than that of its nuclear donor (P < 0.05). Low rates in frozen-thawed sperm of nuclear donor can be related to storage time of sperm which were frozen 5 years before. One (clone 4) of the cloned grey heifers was artificially inseminated with frozen semen from nuclear donor bull and the other (clone 5) was naturally mated with a Holstein bull. Two healthy calves were delivered naturally. When same cloned cows (clones 4–5) and 2 other cloned heifers (clones 2–3) were artificially inseminated with frozen semen of the cloned grey bull, clones 2 and 4 gave birth to 2 healthy female calves. One cloned cow (clone 3) aborted in the third month of gestation and other one (clone 5) is currently 8 months pregnant. Two calves of clone 4 and 5 are 17 months old and 2 other calves of clone 2 and 4 are now 6 and 1 months old. Except for clone 3, our results show that cloned Anatolian grey bull and cows produced from frozen cells in gene bank have normal fertility.
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