Chromosomal characteristics of Tho-Tho cattle (Bos indicus) in Nagaland, India

Tho-Tho cattle is a semi-wild indigenous cattle available in Nagaland, India. Tho-Tho cattle has been associated with tribal culture of 16 tribes of Nagaland besides its use for meat, milk and draught purposes. A cytogenetic analysis was conducted in a total of 10 numbers of Tho-Tho cattle (5 males and 5 females) from 3 districts of Nagaland hills situated at mean sea level (msl) ranging from 1000 to 10000 ft (4 nos. from one district and 3 nos. each from other 2 districts). The statistical design included 5 males and 5 females Tho-Tho cattle for gathering data on cytogenetic parameters viz. (i) karyotype, (ii) relative length, centromeric index and arm ratio of chromosome and (iii) construction of ideograms. Statistical tools used for analysis of variation of relative length of each autosome, centromeric index; arm ratio of the ‘X’ chromosome between male and female are done by using the Student’s t-test. Duncan's multiple range test (DMRT) was used to isolate means if the effect was found significant. The study revealed that(i) Tho-Tho cattle has 29 pairs of autosomes (ii) out of 29 pairs, 23 pairs of autosomes did not differ significantly (P > 0.05) between male and female Tho-Tho cattle in terms of relative length (iii) autosomes are acrocentric, (iv) relative length of 4th, 5th, 6th, 7th and 9th autosomes are significantly (P < 0.05) higher in male; however, 19th autosome of female is significantly (P < 0.05) higher than male (v) X-chromosome in both the sexes is clearly distinguishable and is submetacentric (vi) Y-chromosome (to the total haploid genome) of male Tho-Tho cattle is 1.77 ± 0.10% and it is acrocentric (vii) centromeric index of “X” chromosome in male is 33.75% and in female 32.76% and (viii) Arm ratio of “X” chromosome is found to be 0.66 in male and 0.67 in female.


INTRODUCTION
Tho-Tho cattle is a local name of a semi wild indigenous cattle, which is found in Sub Himalayan region of North Eastern India, particularly in Nagaland hills at an altitude of 1000 to 10000 ft.msl.No literature is available on Tho-Tho cattle as no research has been done yet on this animal.Tho-Tho cattle are popular by different names for different tribes within their own dialects.This cattle is medium in structure (average length in male is 108.2 ± 1.5 cm and in female 106.8 ± 1.7 cm and average height in male is 120.4 ± 1.6 cm and in female 109.8±1.5 cm) and closely associated with tribal rituals, religious and socio economic activities of Naga tribes.This animal is Email: hchoudhury2002@yahoo.co.inAuthor(s)

CI (%) AR
Figure 1.Variation (mean±SE) in centromeric index (CI, %) and arm ratio (AR) of X chromosome in male and female Tho-Tho cattle.
mainly used for meat purpose and also for dairy and tilling and draught purposes.Currently, meagre scientific information is available on the qualitative and quantitative traits of this animal.The animal did not have attract due attention from the appropriate authority due to its remote habitat and semi wild behaviour.Therefore, this study was designed with an objective of investigating chromosomal characteristics to explore its potential as domestic cattle.Investigation on chromosomal profile in livestock provides a useful tool to identify and judge the fertility status of the animal even at an early age (Basumatary, 2003).The knowledge of chromosomal characteristics of Tho-Tho cattle and their effects on economic traits will also be helpful in planning animal breeding strategies.

METHODOLOGY
A cytogenetic analysis was conducted in a total of 10 Tho-Tho cattle (5 males and 5 females) from 3 districts of Nagaland hills situated at mean sea level (msl) ranging from 1000 to 10000 ft (4 from one district and 3 each from other 2 districts).Based on the secondary data on habitat of Tho-Tho cattle collected from Department of Animal Husbandry and Veterinary, Forest and environment, National Research Centre on Mithun and village heads; Kohima, Phek and Zunheboto districts of Nagaland were selected for the survey.Sample size was purposefully fixed at 5 males and 5 females.Cytogenetic parameters studied were (i) karyotype, (ii) relative length, centromeric index and arm ratio and (iii) construction of ideogram.For karyotype study a total of 100 karyotypes (50 from 5 males 50 from 5 females) were prepared to study the morphometry of chromosomes following the protocol described by Moses (1974).

Karyotype preparation
The chromosomes were counted and numbered on the printed copy of the image.Length of each chromosome was determined using the image analysis software of the Nikon microscope (Eclipse 80i) and digital copy of the image.The homologous pairs of chromosomes were identified by matching their absolute length.The chromosome pairs were then arranged in a decreasing order of length and a digital image of the full set was prepared using the Adobe photoshop software.

Estimation of relative length, centromeric index and arm ratio of the chromosomes
Length of chromosomes was measured from centromere to telomere in case of acrocentric chromosomes and tip of short arm to the tip of long arm in case of submetacentric chromosomes.
Length of the homologous chromosomes; X-chromosome in female and in case of males, lengths of "X" and "Y" were measured separately and calculated using the formula (Mean±SE) described by Choudhury et al. (1997).The centromeric index of the submetacentric chromosomes was calculated using the formula (Choudhury et al., 1997).

Construction of idiograms
A total of 30 karyotypes from 5 males and 5 females were taken for preparation of idiograms (Figures 2 and 3) as described by Rowley (1974).Statistical tools used were for analysis of variation of relative length of each autosome, centromeric index; arm ratio of the 'X' chromosome between male and female are done by using the Student's t-test.Duncan's multiple range test (DMRT) was used to isolate means if the effect was found significant.

RESULTS
Ecological and environmental conditions are presented in Table 1.The results of the cytogenetic characters are presented in Table 2.The cytogenetic analysis of Tho-Tho cattle revealed a diploid number of 60 XX in female and 60 XY in male.The morphometric measurements obtained from each pair of chromosomes and sex chromosomes were expressed in terms of relative length   (The metaphase plate from male and female Tho-Tho cattle are presented in Plates 1 and 2).The significant results are; (i) autosomes in both sexes of Tho-Tho cattle are acrocentric, (ii) relative length (mean ± SE) of the autosomes ranges from 1.93 ± 0.05 to 5.19 ± 0.12% in case of male and 2.10 ± 0.04 to 5.38 ± 0.16% in case of female, (iii) karyotype of the female Tho-Tho cattle has two large submetacentric 'X' chromosome, whereas the male Tho-Tho cattle has one large submetacentric 'X' chromosome and a small acrocentric 'Y' chromosome, (iv) 'X' chromosome contributes 5.70 ± 0.10% in male and 5.45 ± 0.09% in female, which are larger than the first pair of autosomes, (v) 'Y' chromosome contributes 1.77 ± 0.10% towards the total haploid genome in male and it was the smallest complement of male karyotype, (vi) length of first autosome pair is 5.19 ± 0.12% in male and 5.38 ± 0.16% in female, whereas the twenty-ninth smallest autosomes contributes 1.93 ± 0.05% in male and 1.88 ± 0.10% in female respectively and (vii) the centrometric index of 'X' chromosome (Figure 1) was 33.75% in male, but in case of female it was 32.76% (Viii) while the arm ratio (Figure 1) of "X" chromosome was 0.66 in case of male, it was 0.67 in case of female Tho-Tho cattle.Statistical analysis showed Tho-Tho cattle population amounts to number 175897 which is 39% of the total indigenous cattle of the state of Nagaland.The study area which constitutes 3 districts has a population of Tho-Tho cattle of 37000 which is 5.33% of the total cattle population of the state.Analysis of cytogenetic characters revealed that the relative length of 23 pairs of autosomes did not differ significantly (P < 0.05) between male and female Tho-Tho cattle.The relative length of 4 th , 5 th , 7 th , and 9 th number of autosomes were significantly (P < 0.05) higher in male (4.53 ± 0.04%, 4.42 ± 0.06%, 4.14 ± 0.02% and 3.85 ± 0.03%) than that in female (4.34 ± 0.04%, 4.22 ± 0.05%, 3.97 ± 0.05% and 3.75 ± 0.02%).
But the relative length of 19 th number autosome was found to be significantly (P < 0.05) higher in female (2.81 ± 0.04%) than that in male (2.67 ± 0.03%).The relative length of X-chromosomes were 5.70 ± 0.10% and 5.45 ± 0.09% in male and female Tho-Tho cattle respectively and the difference was non-significant (P < 0.05).The centromeric index and the arm ratio were also non- significant between male and female Tho-Tho cattle.

DISCUSSION
The karyotype analysis of Tho-Tho cattle revealed a diploid (2n) chromosome number in all the complete metaphase plates.All the 29 pairs of autosomes in both sexes of Tho-Tho cattle were found to be acrocentric.The larger chromosomes were distinguishable from the smaller ones but the decrease in size was so gradual that further sub-classification was not possible (Plates 1 and 2).Similar results are also reported by Yadav (1981) in Sahiwal, Tharparkar, brown Swiss, Holstein Friesian and Jersey, Sarkhel (1988) in Gir, Jersey and Holstein Friesian, Nivsarkar et al. (1992) in Tharparkar, Kumar et al. (1995) in Hariana, Vijh et al. (1996) in Rathi, Vij et al. (1997) in Nagori, Choudhury et al. (1997) in Assam local cattle and Dayananda (2000) in Nimari breeds of cattle.
The morphology of sex chromosomes is a distinguishing factor differentiating Bos indicus from other species.The cattle chromosome can easily be distinguished from that of goat as the later has an acrocentric 'X' and metacentric 'Y' chromosome.This can further be characterized by small acrocentric 'Y' chromosome which is not found in Bos taurus, Bos banteng (Bali cattle), Bos grunniens (Yak), and Bos mutus (Wild yak).Sarkhel (1988) reported that the crossbred males from exotic sire have a small submetacentric 'Y' chromosome.Absence of the small submetacentric chromosome revealed the purity of Tho-Tho cattle as an indigenous breed of the country.The results of the present study in respect of relative length are in close agreement with Sarkhel (1988), Vij et al. (1997) and Choudhury et al. (1997) in Gir, Hariana, Nagori and Assam local cattle respectively.Although, it was difficult to identify the exact position of 'Y' chromosome in the karyotype without banding, the smallest acrocentric chromosome of the chromosomal complement was considered as 'Y' chromosome, based on the reports of earlier studies by Gupta et al. (1974), Yadav (1981) and Sarkhel (1988) in different breeds of Bos indicus cattle and the absence of smaller meta/submetacentric chromosome.However, Vij et al. (1997) reported that the 'Y' chromosome should be placed between 27 th and 28 th autosomes o f the karyotype.
The values of centromeric index are also in close agreement with that of Yadav (1981), Sarkhel (1988), Vijh et al. (1996) and Dayananda (2000) in different indigenous breeds of cattle.
The Tho-Tho cattle belongs to an indigenous breed (Bos indicus) which can be characterized by the chromosome number, morphology of autosomes and sex chromosome.The unique relative length of chromosome determines its similarity with other indigenous breeds of India.However, complete characterization is possible only after comparison with many other indigenous breeds and different types of bandings (G-banding, C-banding and High resolution banding), which may reveal the exact structure of chromosome specific for the breed.

Conclusion
The experimental findings reveals that the cytogenetic characteristics of Tho-Tho cattle are at par with other high yielding indigenous breeds of cattle.There are also some positive traits in Tho-Tho cattle such as (i) resistance to disease, (ii) tolerance to extreme weather conditions, (ii) draught power, (iv) good carcass quality, and (v) efficient conversion of low quality feeds.Tho-Tho cattle play a crucial role in the livelihood of the small farmers.Therefore, the extension and institutional systems should take necessary steps to conserve, propagate and upgrade this valuable indigenous germplasm.Suitable breeding strategies of Tho-Tho cattle and farmers' awareness programme are also required for development of cultural, socio economic, dairy, draught and agricultural sector of Naga people.
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Karyotype of a male Tho-Tho cattle.
Karyotype of a female Tho-Tho cattle.

Table 1 .
Month wise variation in average temperature, dew point, relative humidity and rainfall in different districts.

Table 2 .
Relative length (mean ±SE) of the chromosomes (haploid set) of Tho-Tho cattle.
a,b Indicates values within row differ significantly (P<0.05).