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Dec 14, 2010 - Sanjeev Singh • Suneet Sharma • J. S. Arora •. B. C. Sarkhel. Received: 7 January 2009 / Accepted: 18 October 2010 / Published online: 14 ...

Biochem Genet (2011) 49:242–250 DOI 10.1007/s10528-010-9402-8

Molecular and Biochemical Evaluation of Indian Draft Breeds of Cattle (Bos indicus) Sanjeev Singh • Suneet Sharma • J. S. Arora B. C. Sarkhel



Received: 7 January 2009 / Accepted: 18 October 2010 / Published online: 14 December 2010 Ó Springer Science+Business Media, LLC 2010

Abstract Glutathione peroxidase-1 (GPX-1) enzyme detoxifies peroxides by reacting with the GSH (reduced glutathione) responsible for the maintenance of the integrity of essential biomolecules. This study was conducted on 100 animals of two Indian draft breeds of cattle (Bos indicus), Nimari, and Malvi. Genomic DNA was isolated from blood samples, and four fragments (80, 71, 78, and 442 bp) of the GPX-1 gene were amplified by polymerase chain reaction. PCR-SSCP analysis in 12% PAGE with silver staining revealed polymorphism in three of the fragments (80, 71, and 442 bp) in these two cattle breeds. Breed differences for the blood biochemical parameters (serum creatine kinase and lactic acid level) and overall draft ability were studied. The genetic polymorphism identified for the GPX-1 gene in this investigation would help in the identification of alleles related to draft capacity in these animals for future genetic improvement. Keywords Bos indicus  Draft ability  GPX-1  Genetic polymorphism  PCR-SSCP

Introduction India is primarily an agriculture-based country. With few exceptions, a majority of Indian farmers possess small land holdings and largely depend on animals or manual power for agricultural operations. Two-thirds of rural transportation and 70% of the energy input in farm operations is contributed by draft animal power. Animal power is a renewable natural resource, which can assist not only in production but also in land and water management and conservation. Compared with various national and state research programs conducted to improve milk S. Singh (&)  S. Sharma  J. S. Arora  B. C. Sarkhel Biotechnology Centre, Jawaharlal Nehru Krishi Vishwavidyalaya, Jabalpur, MP 482004, India e-mail: [email protected]; [email protected]

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production, the programs for improving draft animals have been meager, resulting in an enormous waste of this resource. Therefore, there is a need to generate a simple and reproducible method for the assessment of draft capacity of animals that can be utilized for their genetic improvement. Strenuous aerobic exercise depletes antioxidants from the skeletal muscles, and sometimes also from other organs. Glutathione is an essential cofactor for the antioxidant enzyme glutathione peroxidase-1 (GPX-1), which detoxifies peroxides by reacting with the reduced form of GSH, responsible for the maintenance of the integrity of essential biomolecules. The bullocks of the Malvi and Nimari breeds are excellent draft animals, well adapted to the tropical conditions of their respective breeding tracts. There is no scientific information available on molecular markers, draft ability, and biochemical parameters related to exercise in Malvi and Nimari breeds. Reports available in the literature for variability in the GPX-1 gene pertain to Homo sapiens (Hamanishi et al. 2004). No report is available on genetic polymorphism of the GPX-1 gene in any breed of cattle in the world. This gene is essentially involved in the antioxidant stress mechanism. Therefore, polymorphism in the GPX-1 gene was investigated by the PCR single-stranded conformation polymorphism (SSCP) technique, and its association with biochemical parameters (serum creatine kinase and lactic acid) and overall draft ability was analyzed in the Malvi and Nimari breeds of cattle.

Materials and Methods Genomic DNA Extraction The investigation was carried out on 100 unrelated animals of the Nimari and Malvi breeds (50 animals of each breed) of cattle. Blood samples were collected from two government cattle breeding farms of Madhya Pradesh state of India. Genomic DNA was isolated by the phenol–chloroform extraction method (Sambrook and Russell 2001). Quantification and quality were checked with a spectrophotometer. PCR Amplification and Genotyping of the Samples PCR amplification was carried out using four primer pairs to identify the genetic polymorphisms (Table 1). PCR was carried out using 100 ng of genomic DNA in a 25 ll reaction mixture. The reaction mixture consisted of 2.5 mM of each dNTP, 1.5 mM MgCl2, 0.6 U Taq DNA polymerase, and 5 pmol each of forward and reverse primers. The PCR program was optimized for 35 cycles at 53/55°C annealing temperature. It consisted of initial denaturation at 95°C for 5 min, denaturation at 94°C for 45 s, annealing at 53/55°C for 45 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min. The samples were checked for amplification by electrophoresis in 3.5% horizontal agarose gel at 60 V for 1 h and 30 min, along with a 100 bp ladder. PCRSSCP was performed for all the samples. A 12% polyacrylamide gel was made for SSCP study. A 4 ll volume of PCR product was mixed with 9 ll denaturing

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Table 1 PCR primers used for amplification of GPX-1 Location

Primer Sequence (50 –30 )

Exon 2

F1-ATGACCGACCCTAAGTTCATCAC

Amplicon size (bp)

Annealing temperature (°C)

80

53

71

53

78

55

442

53

R1-CCCACCAGGAACTTCTCGAA Exon 2

F-CCCGTGCGCAGGTACAG R-GGACAGCAGGGTTTCAATGTC

Exon 1

F2-GCTGCTCATTGAGAACGTAGCA R2-CGCCGCTGCAGGTCAT

Exon 2 and 30 UTR

For-GAAAAGTGCGAGGTGAATGG Rev-GCTGTGGTCTGGGAAAGG

formamide dye in 0.5 ml PCR tubes. This solution was denatured at 95°C for 5 min followed by snap chilling in ice for 15 min. The denatured PCR products were then run in 12% native PAGE at a constant 250 V for 3 h in vertical gel electrophoresis. This was followed by silver staining for the identification of the SSCP patterns in individual samples (Bassam et al. 1991). Nucleotide Sequencing of the GPX-1 Gene Fragment A 442 bp fragment of GPX-1 was amplified by PCR. This fragment was subjected to PCR-SSCP analysis followed by direct sequencing to identify the genetic variation at the nucleotide level, and alleles were identified. Estimation of Overall Draft Ability The draft capacity of the bullocks was estimated as overall draft ability by the method described by Thomas (2000). Six bullocks of each breed were analyzed for carting and plowing for the assessment, using the formula Overall Draft Ability = [100 - (A ? B)]/2, where A is ability with respect to carting, or (increase in time for final km/time taken for initial km) 9 100, and B is ability with respect to plowing, or (increase in time to cover one lap in closing stages/time taken to cover one lap in initial stages) 9 100. For carting (A), a single pneumatic animal cart of specified design was employed. The animal pulled a total load (including cart) of 200% of its body weight over a distance of 10 km. The study was done on a labeled road. For plowing ability (B), a 200-meter labeled elliptical track having approximately 50% sand and 50% fine clay was used for plowing continuously for 2 h. The time taken for a complete lap during the initial stage and toward the end of 2 h was noted. The measurements were repeated six times and an average was taken. Estimation of Biochemical Parameters For biochemical analysis, the serum samples of the same six bullocks of each breed taken for the study of overall draft ability were analyzed for pre- and post-carting

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and plowing using standard kits. The serum creatine kinase and lactic acid levels were estimated using standard kits supplied by Trinity, Biotech (UK). Statistical Analysis A paired t-test was performed to test for significance of difference between pre- and post-carting and plowing within the Malvi and Nimari breeds. To compare the differences between breeds in these parameters, an analysis of variance (ANOVA) was performed using factorial range design. Mean values were compared using Duncan’s multiple range test (Steel and Torrie 1960). The genotypic frequency of SSCP patterns was calculated by the gene counting method (Falconer and Mackay 1996).

Results PCR-SSCP Analysis of GPX-1 Gene Fragments Analysis of an 80 bp fragment of the Nimari breed revealed three SSCP patterns (Fig. 1); two patterns were also observed in the Malvi breed, making two genotypes for Malvi and three for Nimari. This investigation revealed that this gene fragment is polymorphic in both breeds. The genotype frequencies of the two SSCP patterns for the Malvi breed were 0.74 and 0.26, respectively. The three Nimari patterns had genotypic frequencies of 0.78, 0.12, and 0.10. Similarly, a 71 bp fragment revealed two SSCP patterns in both breeds (Fig. 2). The Malvi breed had genotype frequencies of 0.87 and 0.13, whereas the corresponding values for the Nimari breed were 0.93 and 0.07. The 78 bp fragment, however, revealed a single monomorphic pattern in both breeds (Fig. 2). The 442 bp fragment was found to be polymorphic in the Malvi breed, having three SSCP patterns (Fig. 3), but monomorphic in the Nimari breed. The frequencies of the Malvi patterns were 0.53, 0.42, and 0.03. Nucleotide Sequencing of the 442 bp GPX-1 Gene Fragment The larger 442 bp gene fragment was subjected to direct sequencing. The sequencing of the 442 bp PCR products revealed a C (A allele) to T (B allele) transition at nucleotide position 189. The nucleotide sequences were submitted to the NCBI gene bank with accession numbers FJ467558 and FJ467559. The 80, 71, and 78 bp fragments amplified in the study could not be sequenced because of their smaller size and limitation of cloning facilities during the study. Overall Draft Ability The measurement of overall draft ability was based on two subparameters, carting and plowing. The average observed for Malvi bullocks was 34.8 ± 0.5; for Nimari bullocks, the corresponding value was 33.6 ± 0.6. Although the t-test for

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246 Fig. 1 Three SSCP patterns in 12% PAGE for an 80 bp fragment of the GPX-1 gene (exon 2) in the Nimari breed of cattle

Fig. 2 SSCP patterns in 12% PAGE for (left) a 71 bp fragment of the GPX-1 gene (exon 2) in the Nimari breed of cattle and for (right) a 78 bp fragment (exon 1) in the Malvi breed

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Fig. 3 SSCP patterns in 12% PAGE for a 442 bp fragment of the GPX-1 gene in the Malvi breed of cattle

comparison of the two breeds revealed a nonsignificant difference (P [ 0.05), there was a general trend to higher values in Malvi bullocks. Estimation of Biochemical Parameters The mean levels of serum creatine kinase in the Malvi bullocks were 64.73 ± 3.41 U/l for pre-carting and 108.97 ± 6.35 for post-carting, and 67.57 ± 4.76 for pre-plowing and 106.17 ± 9.36 for post-plowing. In the Nimari bullocks, the corresponding values were 97.83 ± 9.18 pre-carting and 194.75 ± 7.99 post-carting, and 101.75 ± 25.81 pre-plowing and 280.13 ± 38.37 post-plowing. Creatine kinase levels increased significantly (P \ 0.01) immediately after the study in both genetic groups, as revealed by a t-test. Duncan’s multiple range test in the two breeds showed that the values for creatine kinase increased significantly (P \ 0.05) in Nimari bullocks from pre- to post-carting and postplowing. The serum lactic acid levels recorded in the Malvi bullocks were 98.3 ± 4.77 mg/dl pre-carting and 125.9 ± 7.33 post-carting, and 98.97 ± 7.04 pre-plowing and 117.63 ± 8.65 post-plowing. The corresponding values in the Nimari bullocks were 270.54 ± 18.85 pre-carting and 362.04 ± 20.75 post-carting, and 289.88 ± 10.55 pre-plowing and 357.50 ± 17.38 post-plowing. Duncan’s test revealed a significant difference (P \ 0.05) for the mean values between the two breeds for both carting and plowing.

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Discussion GPX-1 Gene Polymorphism The PCR-SSCP technique was successfully used to explore the genetic polymorphism in the exon 1 and 2 regions of the GPX-1 gene. The polymorphism of the 442 bp fragment was further confirmed by direct sequencing. No report on genetic polymorphism of the GPX-1 gene is available for any breed of cattle. Thus, this is the first such report on this gene in cattle. Similar findings for GPX-1 gene polymorphism in humans have been reported by various workers (Lee et al. 2006; Ichimura et al. 2004). In this investigation, the exon 2 region of the GPX-1 gene in Bos indicus was found to be polymorphic. A single nucleotide polymorphism was observed in the exon 2 region. A silent mutation was observed at position 189 of the 442 bp fragment. Ratnasinghe et al. (2000) observed that in Homo sapiens in the exon 2 region of the GPX-1 gene, a variation of proline and leucine at position 198 is associated with differences in the antioxidant stresses, and leads to an increase in the chances of cancer in humans. Glutathione peroxidase-1 plays an important role in the antioxidant defense of the vascular wall. Functional variants in the GPX-1 gene are linked to atherosclerosis because of antioxidant stresses (Hamanishi et al. 2004). The silent mutation at position 189 of the 442 bp fragment may affect splicing or control transcription activity of the gene and subsequently the structure and activity of the GPX-1 protein (enzyme). Hence, this genetic variant may be used in association studies to compare differences in the oxidative stress in draft animals during exercise. Studies in humans and animals indicate that GSH (reduced glutathione) is depleted by exercise. Exercise increases the body’s oxidative burden by calling on the tissues to generate more energy. Thus, it would be interesting to study the association of GPX-1 gene polymorphism with blood glutathione levels in bulls of these two breeds. Estimation of Draft Ability and Biochemical Parameters Carting and plowing are the two major types of study performed by draft animals in agriculture. Any program for genetic improvement of draft ability should be aimed at improving the capacity for both types of work (Thomas 2000). The present experiments were based on carting and plowing. The slightly higher average value observed for Malvi bullocks (34.81 ± 0.52 vs. 33.60 ± 0.59 for Nimari bullocks) was barely significant, indicating increased pulling power for the breed. This trend needs to be confirmed by testing a larger population for each breed. There was a significant increase in the level of serum creatine kinase in Nimari bullocks (P \ 0.01) for both pre- to post-carting and pre- to post-plowing. An elevated level is associated with strenuous exercise. The increase in Nimari bullocks may be due to an increase in muscle enzyme activity. This may cause increased membrane permeability or cellular damage, resulting in its leakage and accumulation in plasma. Thus, the lower value in the Malvi breed indicates its superiority for draft capacity over Nimari. A significant increase in blood serum creatine kinase

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level was also observed by Veigh and Tarrant (1983) in stressed young bulls. These findings also confirm those of Balogh et al. (2001) and Art et al. (1990) in horses, in which a significant increase in levels after exercise was observed. There is also a significant increase (P \ 0.05) in serum lactic acid levels immediately after work in Nimari bullocks, compared with Malvi, for both pre- to post-carting and pre- to post-plowing. During intense muscular activity, the circulatory system fails to bring enough oxygen and glucose to the muscle. This results in utilization of muscle glycogen as a reserve fuel and its breakdown to form lactate. Thus, due to a scarcity of oxygen, lactate cannot be metabolized further in muscle, causing increased blood levels. A lower level of serum lactic acid in Malvi is indicative of less oxidative stress, making Malvi bullocks superior to Nimari in this respect. Increased blood lactic acid levels in Kangayam and crossbred bullocks were reported by other workers (Sreekumar and Thomas 2000; Yawalikar et al. 2005). A post-exercise elevation in plasma lactate level has also been reported in horses (White et al. 1995; Art et al. 1990; Harris and Snow 1988). These findings are in consonance with the present findings for the two cattle breeds. It is interesting to note that the basal serum creatine kinase and lactic acid levels in Nimari bullocks were substantially higher (50.85 and 184.10% more) than in the Malvi. The ability to alter gene expression is a key factor in the adaptive repertoire of animals with wide temperature tolerances (Willmer et al. 2005). Higher creatine kinase and lactic acid levels suggest more stressful conditions for the animal. The Nimari breed has been adapted to the adverse tropical climatic conditions of the Nimar tract of Madhya Pradesh, where the peak environmental temperature during the summer season may reach 45–46°C. Moreover, the breed has been adapted to the strong and hard soil texture prevailing in the fields characteristic of this region. This investigation revealed genetic polymorphism in the exon 2 region of the GPX-1 gene in Bos indicus. Overall draft ability and estimation of the biochemical parameters of serum creatine kinase and lactic acid revealed breed differences during oxidative stress. A lower value for the two biochemical parameters in Malvi animals, along with higher draft ability, is indicative of the breed’s better draft capacity. The results, however, need to be tested in a larger sample before drawing a firm conclusion in this context. The variations observed in this study at the molecular and biochemical level can be used to evaluate and compare oxidative stresses in animals of draft breeds. Acknowledgments The authors thank the Director of the Biotechnology Centre and Director of Research Services, JNVV, Jabalpur, for providing the necessary facilities, and Incharges of the Malvi and Nimari cattle breeding farms, MP, India, for supplying the biological materials.

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