KARYOLOGICAL STUDIES ON EMBRYO TRANSFER AND ARTIFICIAL INSEMINATION BORN ONGOLES*

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1 KARYOLOGICAL STUDIES ON EMBRYO TRANSFER AND ARTIFICIAL INSEMINATION BORN ONGOLES* S. Sai Reddy 1, B. Ramesh Gupta 2, R. Vinoo 3, K. Sudhakar 4 and M. Mahender 5 College of Veterinary Science, Rajendranagar Hyderabad ABSTRACT A total of 450 metaphase spreads, consisting of ET born and AI born Ongole cows were examined for studying the chromosome profiles. The diploid chromosome number in both the groups was 60, XX. All the Autosomes were acrocentric, while the X-chromosome was submetacentric in appearance. Relative length of the autosomes of ET born and AI born cows varied from 1.95 to 4.97 and 1.95 to 4.99 per cent, respectively. The contribution of the X-chromosome to the total genome was the highest in ET as well as AI born cows. No differences either in chromosome number or in morphology were detected between the ET born and AI born cows. Key words: Ongole cattle, ET and AI born, Karyotype, Relative chromosome lengths INTRODUCTION The Ongole is a dual-purpose and pride breed of Andhra Pradesh, famous for its hardiness, ability to thrive on coarse roughages, adaptability to high humid temperature and disease resistance. The capability to produce number of progeny per sire increased tremendously with the introduction of Artificial Insemination (A.I) and with the advent of Embryo Transfer Technology it is also become possible to have more number of progeny from a single elite cow. Such a Technology was employed on Ongoles also and large number of calves were produced. But chromosomal studies on AI and ET born Ongoles were not undertaken previously. Hence, the present study was undertaken to study the chromosome profiles of ET and AI born cows. MATERIALS AND METHODS A total of ten Ongole cows consisting of five born by the Embryo Transfer Technology (ETT) and five born by Artificial Insemination (AI) technique born at the Cattle Project, Livestock Forms part of the Ph.D. Thesis submitted by the first author to SVVU, Tirupati 1 Asst. Prof., Dept. of Animal Genetics and Breeding, Veterinary College, Korutla 2 Professor and Head, Dept. of Animal Genetics and Breeding 3 Sr. Scientist, Cattle Project, LRS, Lam farm, Guntur 4 Prof. & Head, Dept. of Livestock Production and Management 5 Associate Prof., Dept. of Livestock Production and Management 39

2 Research Station, Lam farm, Guntur during the years 2002 to 2004 were utilized to study the variations in chromosome profiles, if any, between the two groups. Blood samples (3 to 5 ml) were collected aseptically by puncture of external jugular vein into sterile heparinated vacutainer tubes and transported to the laboratory as quickly as possible by maintaining the cold chain. The cultures were set up within 12 hours of blood collection. The short term lymphocyte culture technique as described by Moorehead et al. (1960) was employed with slight modifications. For each culture, RPMI 1640 (Hi Media) was used as the cell culture medium (8 ml), PHA-M (0.20 ml) as mitotic agent, foetal calf serum (2.5 ml) as nutrient supplier, antibiotic and antimycotic solution (0.20 ml) to prevent contamination, heparin (0.1 ml) to avoid cell agglutination and whole blood sample (0.5 ml) were added to the culture medium. The tubes were then incubated at 370C for 72 hours in CO2 incubator. Colchicine (0.2 ml) of was added to each culture tube, 45 minutes prior to the harvest of cultures. After 72 hours, the culture vials were taken out of the incubator, contents mixed gently and transferred to 15 ml capacity centrifuge tubes and centrifuged at 1500 rpm for ten minutes. The supernatant was discarded, leaving about 0.5 ml of the medium above the cell button. The contents were then subjected to the hypotonic treatment by adding 8 ml M potassium chloride solution to each centrifuge tube. The tubes were kept in the water bath at 37o C for 20 minutes. This was followed by the centrifugation at 1500 rpm for 10 minutes and again the supernatant was removed leaving a cell button of about 0.5 ml at the bottom of the tube. Then the cells were fixed in freshly prepared and pre-cooled Cornoy s fluid (methanol and glacial acetic acid in 3:1 ratio) and stored at 40 C overnight. The cell fixations were repeated 2 to 3 times the next day. About 20 micro litres of the cel1 suspension was forcefully dropped onto the cool and wet slide Karyological studies on... held at 45o angle and scanned under a phase contrast microscope to determine the quality of the metaphase spreads. Then the slide was stained with the 4% Giemsa stain for 10 minutes. The chromosomal spreads were photographed by inbuilt Olympus Pro-ges camera with 100 magnification using photographing software directly on computer. The length of a chromosome was measured with a dial type Vernier Calliper (Mitutoyo, Japan). The chromosomes were paired in descending order of their lengths for karyotyping. The percent relative length of individual chromosome was calculated by a formula given below. Length of individual chromosomes Relative length = 100 Total length of genome including X-chromosomes RESULTS AND DISCUSSION The metaphase spreads of the ET born and AI born cows are presented in Figures 1 and 2 and the respective karyotypes prepared are shown in Figures 3 and 4. The means for relative length of the autosomes and X-chromosome of both groups of cows are given in Table 1 and the same are presented as idiograms in Figures 5 and 6. A total of 450 Giemsa stained metaphases were examined under the microscope and found that all the metaphases contained a diploid chromosome number of 60, XX consisting of 58 autosomes and 2 X-chromosomes in both ET and AI born cows. None of the metaphases contained any numeric chromosome anomalies. Gupta et al. (1974) and Balaji et al. (2006) also observed the diploid chromosome number of 60XX in Zebu cows. 40

3 Fig.1 Metaphase spread of ET born cow (L 703) Sai Reddy et al., Fig.2 Metaphase spread of AI born cow (L 700) Fig.3 Karyotype of ET born cow ( L 703) shwoing diploid chromosome number 2n = 60, XX Fig.4 Karyotype of AI born cow ( L 700) shwoing diploid chromosome number 2n = 60, XX 41

4 Karyological studies on... Table 1. Mean relative length (%) of chromosomes of ET born and AI born cows Chromosome ET born (n 1 = 5, n 2 = 305) AI born (n 1 = 5, n 2 = 145) Mean SE Mean SE a b X Means followed by the same superscript in a rows do not differ significantly (P? 0.05) n 1 = number of cows n 2 = Number of metaphases examined 42

5 Sai Reddy et al., Fig.5 Idiogram showing the relativr length of Et born cows All the autosomes were found to be acrocentric in both the groups, which was in agreement with the findings of Gupta et al. (1974) in Sahiwal and Red Sindhi cows and Kumarasamy et al. (2006) in Ongole. In the present study, X- chromosome was the first longest and submetacentric in appearance in both the groups, indicating its highest contribution to the total genome. This supported the findings of Chaudhuri et al. (1996) in Assamese local cattle, Balaji et al. (2006) in Deoni cattle and Kumarasamy et al. (2008) in Umblachery cattle. However, Kumarasamy et al. (2006), in Ongole cattle reported that X-chromosome was the second longest in the karyotype. The mean relative length of the autosomes varied from 1.95 to 4.97 and 1.95 to 4.99 percent in ET and AI born cows, respectively, which are comparable with the reports made by Balaji et al. (2006) in Deoni cattle. The relative length of chromosome 20 of ET born cows was significantly longer (2.86%) than that of the AI born cows (2.77%). Apart from this, no other differences either in chromosome number or morphology could be detected between the ET born and AI born cows, suggesting that ET born cows were as good as the AI born cows with respect to their chromosome profiles. However, more detailed studies by including more number of animals and chromosome bandings may be undertaken to draw more valid conclusions. REFERENCES Balaji, R., Ramesh Gupta, B., Narsimha Rao, G. and Narsa reddy, G.V. (2006). Cytogenetic characterization of Deoni cattle. Indian Journal of Animal Research, 40(1): Chaudhuri, A.D., Goswami, R.N. and Das, B. (1996). Studies on karyotypes of Assamese local cattle. Indian Veterinary Journal, 73: Gupta, P., Singh and Ray Chaudhuri L. (1974). Chromosomes of Indian breeds of cattle. The Nucleus, 17: Kumarasamy, P., Sivaselvam, S.N., Thara, S., Thanga Raju, P. and Mahalinga Nainar, A. (2006). Karyological studies on Ongole cattle. Indian Veterinary Journal, 83:

6 Kumarasamy, P., Sivaselvam, S.N., Rajendran, R., Thanga Raju, P. and Mahalinga Nainar, A. (2008). Chromosomal characterization of Umblachery breed of cattle (Bos indicus) a famous South Indian breed of Tamilnadu, India. Indian Journal of Science and Technology, 1 (6): 1-3. Karyological studies on... Moorehead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M. and Hungerford, D.A. (1960). Chromosome preparation of leucocytes cultured from human peripheral blood. Experimental Cell Research, 20: Nagpure, N.S., Pawde, A.M. and Koul, G. L. (2001). Karyological studies in Hariana, Holstein Friesian and their cross breds. Indian Journal of Animal Sciences, 72: