21 CLONING OF ANATOLIAN GREY BULL

Abstract

One of the application fields of nuclear transfer (NT) is to increase the population of endangered mammals. In the present study, cloning of native Anatolian Grey Cattle living semi-wildly especially in the Marmara Region was aimed. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 50 μg mL^–1 of sodium pyruvate, 1% v/v penicillin-streptomycin (10.000 U mL^–1 of penicillin G, 10 000 mg mL^–1 of streptomycin), 50 ng mL^–1 of epidermal growth factor, 0.5 μg mL^–1 of FSH, 5 μg mL^–1 of LH (Arat et al. 2004 Reprod. Fertil. Dev. 16, 135). After removing the cumulus cells of matured oocytes (MII), meiotic spindles and first polar bodies were removed. As nuclear material source, cartilage and fibroblast cells obtained from the ear tissue of 3-year-old Anatolian Grey Bull were used. This animal is kept under the National Conservation Program of the Turkish Government. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 supplemented with 10% FBS (Arat et al. 2002 Biol. Reprod. 66, 1768–1774). After transfer of single cells to enucleated oocytes, oocyte-cell couples were fused by a DC pulse of 2.66 kV cm^–1 for 30 μs in the Zimmerman medium. After fusion, fused NT units were activated using a combination of calcium ionophore (5 μm for 5 min), cytochalasin D (2.5 μg mL^–1) and cycloheximide (CHX, 10 μg mL^–1) for 1 h and CHX alone for 4 h. In the first experiment, after activation, NT units were cultured in Sage cleavage medium (Cooke et al. 2002 Fertil. Steril. 78, 1254–1260; Tang et al. 2006 Hum. Reprod. 21, 1179–1183) supplemented with 8 mg mL^–1 of BSA for 72 h and then developing embryos were divided into 4 groups. After 72 h of culture, all cleaved embryos from cartilage cells in groups I, II, III, and IV were respectively cultured in Sage blastocyst media supplemented with 8 mg mL^–1 of BSA, 8 mg mL^–1 of BSA + 5% FCS, 4 mg mL^–1 of BSA + 5% FCS, and 4 mg mL^–1 of BSA + 100 ng mL^–1 of insulin-like growth factor for additional 4 days. Differences among groups were analyzed by one-way ANOVA after arcsin square transformation (P = 0.05). In group III, development rate to blastocyst (27.3%) was significantly higher than the other groups (18.2, 21.2, and 15.0% for groups I, II, and IV, respectively). In the second experiment, development rates of embryos from cartilage or fibroblast cells, cultured in the group III medium treatment above, were compared. Development rate to blastocyst of 1-cell embryos from cartilage cells (30.8%) was significantly higher than the rate of embryos from fibroblast cells (20.8%). Forty-two good quality embryos from cartilage cells and 11 embryos from fibroblast cells were transferred into 23 Holstein dairy recipient cows (2–5 blastocysts/cow). Day 35 pregnancies were diagnosed in 10 cows from cartilage cells (53% 10/19; one of them was over to 100 days) and in 1 cow from fibroblast cells (50% 1/2). Two recipient cows carrying embryos from fibroblast cells were not yet examined for pregnancy. This cloning study is the first on the Anatolian Grey Cow and is still going on.

This study was supported by grants from TUBITAK, Turkey (TOVAG-104O360 and KAMAG-106G005).