Buffalo Bulletin Vol.29 No.4 - International Buffalo Information Center

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International Buffalo Information Centre

BUFFALO BULLETIN ISSN : 0125-6726

(IBIC) Aims IBIC is a specialized information center on water buffalo. Established in 1981 by Kasetsart University (Thailand) with an initial financial support from the International Development Research Center (IDRC) of Canada. IBIC aims at being the buffalo information center of buffalo research community through out the world.

Buffalo Bulletin is published quarterly in March, June, September and December. Contributions on any aspect of research or development, progress reports of projects and news on buffalo will be considered for publication in the bulletin. Manuscripts must be written in English and follow the instruction for authors which describe at inside of the back cover.

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Main Objectives

S. Sophon

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International Buffalo Information Centre, Office of University Library, Kasetsart University

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Original Article

Buffalo Bulletin (December 2010) Vol.29 No.4

SUPEROVULATORY RESPONSE TO FSH AND EMBRYO RECOVERY RATE IN PANDHARPURI BUFFALOES (Bubalus bubalis)

D.V. Patel*, S.P. Singh, H.R. Shukla, C.P. Devanand and R. Kasiraj

ABSTRACT The superovulatory responses using either 400 mg or 600 mg of FSH (Folltropin-V) in eight Pandharpuri buffaloes used repeatedly were studied. All buffaloes except one responded (minimum 2 CL / follicle) for superovulation in all seasons. Overall response (mean±SE) to superovulation was 61 ovulations (3.39±0.46), 21 follicles/cysts (1.40±0.41), 33 total embryos (1.94±0.39) and 24 viable embryo recoveries (1.41±0.32). The response was non-significantly higher in buffaloes injected with 400 mg (4.13± 0.52-total ovulations) than 600 mg (2.80±0.64). Use of different bull semen had no significant difference in viable embryo recovery.

ability, producing a calf every 13 months. Under average management conditions and hot -dry climate these buffaloes yield 6 to 7 liters of milk per day and under good management they have been reported to yield up to 15 liters of milk per day (Ambardekar, 2000). Farmers are interested in more milk production with higher fat percentage. This has resulted in increase in inseminations of Pandharpuri buffaloes by Murrah semen leading to reduction in the number of pure-bred Pandharpuri buffaloes. Considering the need of conserving Pandharpuri buffalo germplasm in the form of embryos and semen, the present study was undertaken to assess the superovulatory response to FSH and embryo recovery.

Keywords: buffalo, Pandharpuri, superovulation, embryo recovery

MATERIALS AND METHODS Selection of animals and general management A total of eight pure bred, disease free, multiparous, non-pregnant and lactating, Pandharpuri buffaloes were selected from different parts of Pandharpur region of Maharashtra. The buffaloes were examined per-rectally before purchase to ascertain the normalcy of the reproductive organs. All the buffaloes were bred by using frozen semen of four elite Pandharpuri

INTRODUCTION Pandharpuri buffaloes, mainly found in Solapur, Kolhapur and Sangli regions of Maharashtra state, India, and typically have long sword-like horns, black body coats and comparatively medium-sized bodies. These buffaloes are famous for their high reproductive

Sabarmati Ashram Gaushala, Bidaj Farm, PO- Lali, Dist, Kheda, Gujarat-387120, India *E-mail: [email protected] 244

Buffalo Bulletin (December 2010) Vol.29 No.4

buffalo bulls having more than 50 percent post thaw motility. All the animals were maintained under uniform stall fed condition throughout the study. Animals were fed with 3 kg maintenance ration and extra ration (0.4 kg per kg milk production) based on their production. The animals were regularly dewormed (pre- and post- monsoon using Fenbendazole, orally) and were vaccinated before onset of monsoon using Triovac (Indian Immunologicals).

procedure (Misra et al., 1990) using 18 G Rusch catheter (Minitub, Germany) and DPBS media (IMV, France) added with 0.1% bovine serum albumin (BSA, Fraction V, Sigma). All the recovered embryos were evaluated as per standards given by International Embryo Transfer Society Manual (IETS) and viable embryos were frozen in 1.4 M glycerol (as described by Misra et al., 1992) using Ni-cool MS 21 biofreezer (Air Liquide, Bussy Saint Georges, France). Data analysis The data collected were analysed statistically and the difference in superovulatory response was tested using the student’s t-test. The data were also analysed for the effect of batch of flushing (I, II and III), dose rate of FSH (400 mg and 600 mg), SOV (superovulatory) CL (big, medium and small), day of starting of SOV (9th, 10th and 11th day of oestrous

Superovulation, embryo recovery and freezing of embryos The donor buffaloes were induced for oestrus with either single or double i/m injection of prostaglandin (PG) Dinoprost - 0.75 mg (Iliren, Hoechst). The animals were examined per-rectally at 72 h after PG injection for presence of ovarian follicle, uterine tone and vaginal discharge. The animals found to be in oestrus were further selected for superovulatory treatment. Repeated flushings were done in different months, i.e. March (Batch I), April to July (Batch II) and October (Batch III). The selected animals were checked for the quality and number of corpus lutea (CL) before superovualtion. The animals were superovulated with either 400 mg (n=8) or 600 mg (n=10) of FSH (Folltorpin-V, Veterepharm, Canada). FSH injection was initiated between day 9 and 11 after oestrus continuously for 5 days in tapering doses (details shown in Table 1). PG was administered with the 7th and 8th FSH dose. All the animals were inseminated four times using frozen Pandharpuri semen at 36, 48, 60, and 72 h after the first PG injection. The number of corpora lutea was counted per-rectally before flushing the buffaloes. At 5.5 to 6 day, flushing was carried as per the standard

cycle) and bull (bulls 1, 2, 3 and 4) on the superouvlatory response.

RESULTS The overall response for superovulation in different batches and embryo recovery are presented in Table 2. The overall response for superovulation was 94.44 percent (17/18). All the animals that responded for superovulation were flushed. The total number of embryos recovered was 33 with 24 of them being viable and frozen subsequently. Effects of batch of flushing, dose rate of FSH, SOV CL, day of starting of SOV and bull on the superovulatory response are presented in Table 3. The overall mean (±SE) number of ovulations, anovulatory follicles, embryo recovery 245

Buffalo Bulletin (December 2010) Vol.29 No.4

Table 1. Superovulation treatment in Pandharpuri buffaloes. Day 0 (Heat) 10th day 11th day 12th day 13th day 14th day 15th day 16th day 21th day

Time Any time Morning Evening Morning Evening Morning Evening Morning Evening Morning Evening Morning Evening Morning Evening Morning

400 mg group Check for oestrus Check CL FSH- 60mg FSH- 60mg FSH- 50 mg FSH- 50 mg FSH- 40 mg FSH- 40 mg FSH- 30 mg + PG FSH- 30 mg + PG FSH- 20 mg FSH- 20 mg + AI AI AI AI Flush

600 mg group Check for oestrus Check CL FSH-100 mg FSH-100 mg FSH- 80 mg FSH- 80 mg FSH- 60 mg FSH- 60 mg FSH- 40 mg + PG FSH- 40 mg + PG FSH- 20 mg FSH- 20 mg + AI AI AI AI Flush

Table 2. Flushing details in Pandharpuri buffaloes.

Particulars No. of animals Programmed No. of animals responded No. of animals flushed Total no. of CL Total anovulatory follicles Total Embryo Recovery Viable Embryo Recovery UFO / Zona Degenerated Embryos Total Embryos Frozen

Batch 1 (March-06)

(April to July-06)

Batch 3 (October-06)

Cumulative

7

5

6

18

7 7 29 5 16 12 2 2 12

4 5 15 5 8 4 1 3 4

6 5 17 11 9 8 1 0 8

17 17 61 21 33 24 4 5 24

Batch 2

246

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Table 3. Effect of batch of flushing, dose rate of FSH, SOV CL, day of starting of SOV and bull on the superovulatory response

Particulars Overall Dose 400 mg 600 mg Batch Batch 1 Batch 2 Batch 3 SOV day 9 10 11 SOV CL Big Medium Small Bull PB-201 PB-203 PB-209 PB-210

Total Animal (Flushed) 18 (17)

Ovulations

Anovulatory follicles

Embryo recovery

Viable embryo

3.39±0.46

1.40±0.41

1.94±0.39

1.41±0.32

8 (8) 10 (9)

4.13±0.52 2.80±0.64

0.88±0.44 1.90±0.67

2.38±0.42 1.56±0.63

1.75±0.41 1.11±0.48

7 (7) 5 (5) 6 (5)

4.14±0.59 3.00±1.18 2.83±0.70

0.71±0.47 1.00±0.45 2.67±0.92

2.29±0.47 1.60±1.03 1.80±0.66

1.71±0.47 0.80±0.49 1.60±0.75

5 (5) 10 (9) 3 (3)

4.40±0.81 2.80±0.66 3.67±0.67

0.00±0.00a 2.10±0.64b 1.70±0.33

2.80±0.49 1.33±0.58 2.33±0.88

2.00±0.63 0.78±0.32 2.33±0.88

5 (5) 5 (5) 8 (7)

3.60±0.80 4.40±1.20 2.60±0.50

0.80±0.37 1.20±0.58 1.13±0.82

1.80±0.92 2.40±0.93 1.71±0.38

1.80±0.92 1.40±0.51 1.14±0.32

4 (4) 2 (2) 5 (4) 7 (7)

4.50±0.96 3.50±0.50 2.40±0.98 3.43±0.89

1.50±0.29 1.50±1.50 0.80±0.58 1.86±0.94

3.00±0.91 1.50±0.50 1.00±0.58 2.00±0.65

2.25±0.63 1.00±0.00 0.50±0.29 1.57±0.61

Means in the same column within categories with different superscript differ significantly (P0.05, data not shown). The literature suggests higher serum urea concentrations are associated with lower fertility in buffaloes (Qureshi et al., 1999). To sum up, administration of insulin during the post-AI mid-luteal phase failed to alter

(Thatcher et al., 1995). Comparing different days in the treatment group, plasma insulin was highest on day 12 (p0.05) from corresponding controls. This could be due to the fact that blood samples on day 12 were collected about 24 h after insulin administration (day 11). Although in present trial, the preparation of insulin used was long-acting, it has been suggested that the rise in plasma insulin after insulin treatment is maintained for 12 h (Kirkwood et al., 1991). 252

C 6.82±1.50 8.60±1.51 9.46±1.21 9.72±1.88 8.02±1.75 8.50±1.70 9.60±2.04

I 4.95±1.21A 8.62±1.44AB 8.39±1.47AB 13.49±1.41B 11.31±1.62B 9.44±1.97AB 9.88±2.46AB

Plasma insulin (μIU/ml) C 59.02±1.31 55.53±1.59 59.42±2.24 61.37±2.27 59.08±1.92 66.40±2.94 60.67±1.55

I 62.90±2.91 56.21±2.87 64.50±1.88 60.82±2.13 62.19±1.91 66.40±2.16 65.20±1.89

Blood glucose (mg/dl) C 71.52±5.29 68.04±5.22 68.44±4.22 67.24±4.53 63.16±3.04 59.47±2.55 60.34±4.08

I 71.42±2.47 67.38±2.73 67.43±2.50 65.38±2.47 68.83±2.63 67.47±2.88 65.52±3.22

Plasma cholesterol (mg/ dl)

Means bearing different superscript (A, B) in a column differ significantly (P0.01) than 2-D, 4-D, 8-D and denuded oocytes without support of a cumulus cell monolayer (negative control) but showed equal significance with (positive control) oocytes surrounded with compact cumulus cells. Oocytes do not have receptors for gonadotrophins and oestradiol while receptors for both types of hormones are present only in adjacent follicular cells (Lawrence et al., 1980). Therefore signals induced by oestrogen or gonadotrophins in follicular cell are transduced into the oocyte via gap junctions (Downs et al., 1988). Niwa and Chang (1975) and Thibault (1977) have reported that certain regulatory signals are specifically required for oocyte maturation. These signals operate most actively during 6-8 h after the initiation of maturation. A low or almost negligible maturation rate of bovine oocytes was observed when cumulus cells were removed before in vitro culture (Leibfried and First 1979; Fukui and Sukuma, 1980; Dahlhousen et al., 1981). Nagai (1994) reported that after collection of oocytes, selection of cumulus-oocytes complex is extremely important for successful maturation and fertilization. However, poor recovery of immature oocytes is one of the problems for in vitro maturation, and fertilization. To overcome the poor recovery problem, in the present experiment, the denuded oocytes with normal cytoplasm were matured on the

spermatozoa/ml. Oocytes and spermatozoa were co-incubated for 20 h at 39oC under 5% CO2 in air. To study of the fertilization process some of the oocytes after 20 h of sperm-oocyte co-incubation were fixed in acetic acid and ethanol (1:3) stain with aceto-orcein, and observed for presence of two pronucles. Statistical analysis The effect of cumulus cells monolayer on in vitro maturation and fertilization rate of denuded oocytes were analyzed by using a 2x2 fractional design of chi square test (Snedecor and Cochran, 1967).

RESULTS AND DISCUSSION At the time of recovery of oocytes from follicles there is great destruction of surrounding cumulus cells. Hence large numbers of oocytes were observed without cumulus cells (denuded) having normal cytoplasm. It has been reported earlier that cumulus cells play an important role in providing nutritional support to developing oocytes. (Buccione et al., 1990; Haekwon and Schueltz, 1991). Another possible role of cumulus cells is to provide metabolic support. In vitro observation demonstrates that these cells produce significant amounts of protein in the medium (Rabahi et al., 1993). In the present study, the denuded oocytes having normal cytoplasm were matured on different days of monolayers and the maturation and fertilization rate of these oocytes were studied. The percent maturation and normal fertilization rate are summarized in Table 1. 258

Buffalo Bulletin (December 2010) Vol.29 No.4

different days of cumulus cell monolayer. It was observed that denuded buffalo oocytes, during IVM culture, showed 25.60% maturation rate compared with 78.72% in oocytes enclosed with cumulus cells. The denuded oocytes were then cultured for 24 h on 2-D, 4-D, 6 and 8-D old cumulus cell monolayers and the result indicated that 6-D old cumulus cell monolayer can support a higher maturation rate 60.81% than 2-D, 4-D and

17.30%, 32.80% and 18.30%, in 2-D, 4-D, 6-D and 8-D, respectively. The percent normal fertilization rate in the positive control group was significantly higher (P>0.01) than those of the other groups. Also the percent normal fertilization was significantly higher (P>0.01) in 6-D as compared to 2-D, 4-D and 8-D and denuded oocytes without support of a cumulus cells monolayer. The completion of nuclear maturation does

8-D cumulus cell monolayer. These results indicate that 6- D old cumulus cell monolayers reestablish coupling between oocytes and cumulus cells and that coupling facilitate the entry of essential products and the communication of instructive signals to the oocytes. The percent normal rates of fertilization of compact cumulus enclosed oocytes (positive control group) and denuded oocytes (negative control group) without support of a cumulus cells monolayer were 81.20% and 7.69%, respectively. The percent normal fertilization rates were 7.40%,

not necessarily identify normal cytoplasmic maturation. There are several essential components required for completion of both nuclear and cytoplasmic maturation, the most important being the presence of cumulus cells (Trounson, 1992). The percent normal fertilization rate of compact cumulus enclosed was significantly higher as compared to denuded oocytes matured without cumulus cell monolayer and denuded oocytes matured on different days of monolayer. The denuded oocytes matured on 6-D monolayers showed significantly higher maturation as compared

Table 1. In vitro maturation and fertilization of denuded buffalo oocytes with cumulus cells monolayer.

Oocytes in M-II/Total oocytes % M-II Oocytes in 2 PN/Total Oocytes % 2 PN

Days of cumulus cell monolayer

Control +ve

Control -ve

2-D

4-D

6-D

8-D

74/94

21/82

22/61

25/72

45/74

24/80

(78.72)a

(25.60)b

(36.06)b

(34.72)b

(60.81)c

(30.00)b

69/85

11/143

12/162

26/150

24/73

23/126

(81.20)a

(7.69)b

(7.40)b

(17.3)bc

(32.8)bcd

(18.3)b

Control +ve, control -ve: Denuded oocytes without monolayer a, b, c, d, means different superscript signs differ significantly (P0.05), demonstrating that the null hypothesis of mutationdrift equilibrium is fulfilled in this population. A second approach, a qualitative geographical method, detected no mode shift in the frequency distribution of alleles showing a normal L-shaped curve, where the alleles with the lowest frequencies (0.01-0.1) were observed to be most abundant (Figure 2). A normal L-shaped distribution of a plot of allelic frequency class versus proportion of alleles reinforces the result that the Mehsana buffalo population has not experienced any recent bottleneck. In conclusion the study thus presents valuable insight into the existing genetic variability in Mehsana buffalo. The high degree of variability demonstrated implies that this population is a rich reservoir of genetic diversity. This fact together with its demonstrated functional superiority as high milk production consolidate the significance of its conservation as a valuable pure breed and its utilization in agricultural exploitation as a source for indigenous buffalo improvement to achieve better production.

Observed and expected heterozygosity ranged from 0.27 (CSSM43) to 0.84 (CSSM22), and from 0.62 (ILSTS29) to 0.83 (CSSM61, CSSM43, ILSTS61) respectively. PIC for each locus ranged from 0.58 at (ILSTS29) to 0.81 at (CSSM61), with an average of 0.73. The allele numbers and heterozygosity levels observed across the loci indicate presence of reasonably high level of genetic variability in Mehsana buffalo. The high PIC values observed for most of the markers are indicative of the usefulness of microsatellites for biodiversity evaluation in this breed. Table 2 also shows FIS and P value observed for Hardy-Weinberg equilibrium at each microsatellite locus in this population. In both cases eight microsatellites showed deviation from Hardy-Weinberg equilibrium. This might be attributed to selection in the population, presence of null alleles, sample relatedness, linkage with loci under selection, population heterogeneity. Heterozygosity deficiency (FIS >0) was also observed for eight loci in this study, and that might be due to presence of null alleles. Overall FIS value was observed to be 0.13. The higher value indicates within population inbreeding directly or indirectly. Observed heterozygosity was significantly lower than expected for locus CSSM43, and this may be attributed to presence of null allele. The marker did not amplify occasionally or only one allele was amplified in multiplex reaction. The analysis also showed absence of any bottleneck in Mehsana buffalo in the recent past. The first approach based on heterozygosity excess works on the principle that in a recently bottlenecked population, the observed gene diversity is higher than the expected equilibrium gene diversity (Heq), which is computed from the observed number of alleles (k), under the assumption of a constant-size (equilibrium) population. None of the calculated P

ACKNOWLEDGEMENT Authors are thankful to Dudhsagar Research and Development Association (DURDA), Mehsana, for providing blood samples and financial assistance for our research work.

REFERENCES Barker, J.S.F., S.S. Moore, D.J.S. Hetzel, D. Evans, S.G. Tan and K. Byrne. 1997. Genomic diversity of Asian water buffalo (Bubalus 268

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genomic DNA from leukocytes. Nucl. Acid. Res., 19: 408. Moore, S.S. and D. Vankan. 1994. The application of DNA fingerprinting to parentage verification in cattle. Australian Biotechnology, 4(2): 107-108. Navani, N., P.K. Jain, S. Gupta, B.S. Sisodia and S. Kumar. 2002. A set of cattle microsatellite DNA markers for genome analysis of riverine buffalo (Bubalus bubalis). Anim. Genet., 33: 149-154. Oliver, A. 1983. A brief survey of some of the important breeds of cattle, ICAR, New Delhi, Misc. Bulletin, 17: 45. Pundir, R.K., G. Sahana, N.K. Navani, P.K. Jain, D.V. Singh, Satish Kumar and A.S. Dave. 2000. Characterization of Mehsana Buffaloes in India. Animal Genetic Resources Information, 28: 53-62. Singh, D.V. 1992. Breed characterization of Mehsana buffaloes and strategies for their genetic improvement. Ph. D. Thesis, NDRI Deemed University, Karnal, Haryana, India. Yeh, C.G., J.K. Kogi, M.T. Holder, T.M. Guerra, S.K. Davis and J.F. Taylor. 1997. Caprine

bubalis): microsatellite variation and comparison with protein coding loci. Anim. Genet., 28: 103-115. Botstein, D., R.L. White, M. Skolnick and R.W. Davis. 1980. Construction of a genetic linkage map in man using restriction fregment length polymorphism. Amer. J. Hum. Genet., 32: 324-331. Cornuet, J.M. and G. Luikart. 1996. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics, 144: 2001-2014. Di Berardino, D. and L. Iannuzzi. 1984. Detailed description of RBA-banded chromosomes of river buffalo (Bubalus bubalis L.). Genet. Sel. Evol., 16: 249-260. Goudet, J. 2001. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Available from http://www. unil.ch/izea/softwares/fstat.html. Updated from Goudet (1995). Gupta P.R. 1997. Dairy India, 5th ed. Statistics. p. 153-195. John, S.W., G. Weitzner, R. Rozen and C.R. Scriver. 1991. A rapid procedure for extracting Continued from p. 261

Trouson, A. 1992. The production of ruminant embryos of in vitro of bovine oocytes in vitro. Anim. Reprod. Sci., 28: 125-137. Younis, A.I., K.A. Zuelke, K.M. Harper, M.A.L. Olivecra and B.G. Bracketi. 1991. In vitro fertilization of goat oocytes. Biol. Reprod., 44: 1177-1182.

Reprod. Fertil., 51: 1-5. Totey, S.M., G. Singh, M. Taneja, C.H. Pawshe and G.P. Talwar. 1992. In vitro maturation fertilization and development of follicular oocytes from buffalo (Bubalus bubalis). J. Reprod. Fertil., 95: 597-607.

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Original Article

Buffalo Bulletin (December 2010) Vol.29 No.4

STUDIES ON CERTAIN PHYSICAL AND BIOCHEMICAL PARAMETERS OF SYNOVIAL FLUID FROM THE TIBIO-TARSAL JOINT OF BUFFALOES (Bubalis bubalis) Dharmendra Sonowal, U.K. Garg, G.P. Jatav, Nidhi Shrivastava, S.P. Jagtap and S.C. Khanna

ABSTRACT

INTRODUCTION

A total number of 50 apparently healthy, adult buffaloes of both the sexes were examined for different physical and biochemical parameters of synovial fluid from the tibio-tarsal joints. The physical and biochemical parameters which was studied during present investigation were as follows: clarity , colour, specific gravity, pH, viscosity, spontaneous clot formation and total volume (ml), ALT (IU/L), glucose (mg/dl), total protein(g/dl) and bilirubin (mg/dl). Out of the 50 samples, 30% of the samples were colorless clear whereas 70% of the samples found were slightly yellow in colour and 90% of the samples had clear transparency whereas 10% were showed slight turbidity. The material was collected from buffaloes brought in the Cantonment Board slaughter house, Mhow (M.P.).

Synovial fluid is found in the diarthroidal joints filling the joint cavity which lubricates the joints, nourishes the articular cartilage and is produced by synovial membrane. Normally synovial fluid is composed of hyaluronic acid, lubricin, proteinases and phagocytic cells. (Wikipedia, 2009) Synovial fluid is a clear, transparent strawcolored fluid but during any disease condition of any etiological origin affecting joints may bring changes in the cellular, chemical content and physical characteristic. Synovial fluid can provide valuable information regarding various arthritic conditions which may be infectious, rheumatic, autoimmune, neoplastic etc. and can be a valuable diagnostic aid. Although, synovial fluid examination will not always provide a specific diagnosis, it does give an indication of the degree of synovitis and metabolic derangement within the joints. (Tyagi and Krishnamurthy, 1974; Jani et al., 1994; Pal et al., 1994; Barvalia et al., 1995). As the literature on the quality and quantity of synovial fluid of buffalo is scanty, there is a need to undertake a study related to synovial fluid. Keeping this in view, the present study was conducted to analyze the physical properties, cytological contents and biochemical parameters of the synovial fluid of apparently healthy buffaloes and is being presented for publication through this

Keywords: synovial fluid, tibio-tarsal joint, buffalo, ALT, bilirubin, buffalo, clarity, colour, glucose, pH, specific gravity, spontaneous clot formation, total protein, total volume, viscosity

Department of Veterinary Pathology, College of Veterinary Science, Mhow, M.P., India 270

Buffalo Bulletin (December 2010) Vol.29 No.4

using an Auto analyzer, Chem-5 V 2 plus with kits supplied by Merck.

article.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Synovial fluid was collected from 50 apparently healthy adult animals, which showed no signs of any arthritic condition immediately before slaughter in Mhow Cantonment Board slaughter house for meat purposes from the lateral aspect of the tibio-tarsal joint by arthrocentesis using a sterile 20 ml syringe having an 18 gauge needle. The collected fluid was immediately transferred to a dry, sterile vial without any anticoagulant. The gross appearance, physical properties and total volume were recorded at the time of collection. All the tests except for glucose were conducted within 12 h of collection of samples while the glucose estimation was done within one hour of collection. The pH of synovial fluid and the specific gravity were estimated by using pH paper and a urinometer, respectively. The viscosity was evaluated by the method employed by Baniadam and Razi (2005) in which string formation of 2.5-5 cm was considered to be an indication of good viscosity. Total leucocyte count was carried out on uncentrifuged synovial fluid samples in an Improved Brightline Neubauer counting chamber with the help of WBC diluting fluid by the method similar to that used during hematology (Jain, 1986). As to biochemical parameters, glucose was estimated by method based on GOD-POD, total protein was estimated by the biuret method, end point, bilirubin was estimated by the method of Jendrassik and Grof, and ALT was estimated by IFCC method. These parameters were estimated by

The synovial fluid of buffalo was clear colorless to slightly yellow. Out of the 50 samples, 30% of the samples were colorless clear whereas 70% of the samples were slightly yellow and 90% of the samples had clear transparency whereas 10% had slight turbidity. The average total volume, pH and specific gravity of synovial fluid was found to be 17.65±1.01ml, 7.3 and 1.013, respectively, which was in accordance with the findings of Soliman et al. (1975). The total volume of synovial fluid collected depends on the size of the joint, because the volume collected from older animal having larger joints was greater than that from the younger animals having smaller joints. Exercise and inactivity also influence the amount of fluid (Jubb et al., 1985). Viscosity in 100% of the samples was found to be good as evaluated by the length of the string produced. A significant decrease in relative viscosity was observed following induced arthritis in buffalo calves (Jani et al., 1994). Viscosity varies with species and joint, e.g., fluid from larger joints is usually less viscous and cellular than from that from smaller joints. Synovial fluid viscosity also varies inversely with temperature. Synovial fluid viscosity depends almost entirely on hyaluronic acid concentration, which decreases considerably if it is hydrolyzed enzymatically or if its polymerization is destroyed by enzymes or by physical processes. (Swenson, 2000) Absence of clot formation in any of the samples indicates that normal fluid does not contain fibrinogen and other macromolecules of plasma, but these proteins may accumulate if a 271

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joint is injured as it may increase the permeability of synovial membrane and they diffuse through it. However, in the present study no such finding was observed in any of the samples. The total number of leucocytes counted on uncentrifuged samples ranged from 300-1250/ cu.mm with an average of 425/cu.mm, which was in accordance with the findings of Soliman et al., 1975. The count may be considered as a normal count or a count during non-inflammatory condition. During pathological processes, cellular content is altered. The total leucocyte count will be helpful in distinguishing inflammatory from noninflammatory conditions. There was no significant difference in the measured physical parameters in respect of age, sex and side of the joints. The values of different physical and biochemical parameters of synovial fluid are presented in the Tables 1 and 2.

In the present study ALT, total protein and glucose were found to be 17.82±0.71 IU/L, 0.94±0.06 g/dl, 62.89±3.69 mg/dl, respectively. The finding of ALT is slightly higher as compared to the value reported by Baniadam and Razi, 2005 i.e.15.08±2.75. Total protein of the synovial fluid of buffaloes was reported 882.10-900.20 mg% and 0.96±0.08 g/dl, respectively (Soliman et al., 1975; Kumar et al., 2007). The low total protein in the synovial may be due to reduced filtration of protein molecules through synovial membrane because of its higher molecular size (Tulamo et al., 1989). Glucose was reported 59.25±3.39 mg/dl and 49.1±5.72 from the synovial fluid of buffaloes. (Baniadam and Razi, 2005; Kumar et al., 2007). There was no significant difference between groups according to age, sex and side of the joint for the measured biochemical parameters, except ALT and LDH according to sex (Baniadam and Razi Jalali, 2005).

Table 1. Physical characteristics of tibio-tarsal synovial fluid. Parameters

Observations

Clarity Colour Specific gravity pH Viscosity Spontaneous clot formation Total volume (ml)

Clear Colorless -slightly yellow 1.013 (range :1.010-1.015) 7.3 (range:7.28-7.32) Good None 17.65±1.01 ml

Table 2. Chemical characteristic of tibio-tarsal synovial fluid. Parameters ALT (IU/L) Glucose (mg/dl) Total protein (g/dl) Bilirubin (mg/dl) ± standard error

Results 17.82±2.01 62.89±3.69 0.94±0.06 0.11±0.02

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Jain, N.C. 1986. Schalms Veterinary Haematology, 4th ed. Vol. 12. Lea and Febiger, Philadelphia.

Bilirubin was also found in samples having slightly yellowish tinge and was not reported by any of the earlier workers. Bilirubin-0.11±0.02 mg/dl (range-0.00-0.41mg/dl).The bilirubin along with ALT and AST might be useful in assessing the condition of the liver and bone health of the affected animals.

p. 121-123. Jani, B.M., B.M. Nigam and A.K. Nisal. 1994. Synovial fluid transfusion with and without corticosteroid in induced arthritic buffalo calves: changes in synovial fluid. Indian J. Anim. Sci., 64(1): 18-21. Jubb, K.V.F., P.C. Kennedy and N. Palmer. 1985. Pathology of Domestic Animals, 3rd ed. Vol. 1. Academic Press (London). 93. Krishnamurthy and R.P.S. Tyagi. 1973. Studies on stifle synovial fluid of bovines. II. Certain biochemical parameters of the fluid in normal buffalo calves. Indian Vet. J., 50(1): 79-82. Kumar, S., V.K. Sharma and N.S. Jadon. 2007. Biochemical parameters of serum and synovial fluid in buffaloes. Indian Vet. J., 84(11): 106-107. Pal, M., H.P. Singh and N.S. Jadon. 1994. Synovial alteration in experimental arthritis in buffaloes. Indian Vet. J., 71(7): 698-702. Soliman, M.K., S. Elamrosi and L.B. Youssef. 1975. Studies on the normal synovial fluid from the tibio-tarsal joint of buffaloes. Indian Vet. J., 52(1): 19-23.

A general trend of increase in all the biochemical parameters had been observed as the colour of the synovial fluid progresses from colorless to slightly yellowish. Synovial fluid can be considered as a dialysate of blood plasma except for hyaluronic acid content, which is almost entirely responsible for its viscosity. In comparison to blood plasma of buffalo, total protein was significantly low in synovial fluid, which may be due to lesser permeability of synovial membrane and capillaries to protein, but it increases significantly following induced arthritis in buffalo calves. (Jani et al., 1994) The value of glucose is in and around blood plasma, ALT is a bit higher than blood plasma and bilirubin is absent in colorless samples and in samples where it is found either around blood plasma or lower.

REFERENCES

Swenson Melvin, J. 2000. Dukes Physiology of Domestic Animals, 9th ed. p. 434-437. Tulamo, R.M., L.R. Bramlage and A.A. Gabel. 1989. Sequential clinical and synovial fluid changes associated with acute infectious arthritis in the horse. Equine Vet. J., 21: 325331. Tyagi, R.P.S. and D. Krishnamurthy. 1974. The pattern of total protein and protein fractions in synovial fluid and serum of normal and affected (subluxation of patella) bovines. Indian Vet. J., 51(2): 156-158.

Baniadam, A. and M. Razi Jalali. 2005. Studies on the normal synovial fluid from the carpal joints of Iranian water buffaloes: physical and biochemical parameters. Indian J. Vet. Res., 6(1): 17-20. Barvalia, D.R., B.M. Jani, P.H. Tank and R.R. Parsania. 1995. Synovial fluid changes in Escherichia coli lipopolysaccharide induced arthritis and evaluation of intra-articular dexamethasone therapy in buffalo calves. Indian J. Vet. Surg., 16(2): 101-106. en.wikipedia.org/wiki/Synovial fluid (2009) 273

Original Article

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GENETIC POLYMORPHISM IN THE ALDO-KETO REDUCTASE FAMILY 1 MEMBER B1 (AKR1B1) GENE OF MURRAH BUFFALO BULLS (Bubalus bubalis) V.B. Ahir1, M.T. Panchal2, A.K. Tripathi1, P.G. Koringa1 and C.G. Joshi1

ABSTRACT

is the basic material for improving livestock through selection. Embryo implantation is an important step in the establishment of pregnancy and is marked by distinctive biological processes that occur during the preimplantation and early post implantation periods. Preimplantation development directs the formation of an implantation-, or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and foetal development can be sustained (Watson, 1992; Watson and Barcroft, 2001). Preimplantation development encompasses the period from fertilization to implantation, which occurs on 1734 days for cow (Ko, 2004). Bovine preimplantation embryo development is under constant control of genes activated from either the maternal or the embryonic genome. Large-scale association studies by genotyping many single nucleotide polymorphisms (SNPs), in individuals with wellcharacterized phenotypes are considered as promising methods for identifying the cause of many complex traits. Ideally, such studies should be free of biological hypotheses and be done at the whole genome level to maximize the likelihood of success (Tsuchihashi and Dracopoli, 2002). AKR1B1 is an enzyme in carbohydrate metabolism that converts glucose to its sugar alcohol form, sorbitol, using NADPH as the reducing agent (Chung and LaMendola, 1989).

The present study was conducted to investigate the existence of polymorphism at the AKR1B1 locus by the PCR-RFLP method. The genotypes were confirmed by checking specific, clearly distinguishable DNA band patterns resulting from digestion with the restriction enzyme Nde I. Among the 41 animals studied, 18 samples produced two bands of 463 bp and 333 bp referred as AA genotype, whereas 23 animals produced three bands of 796 bp, 463 bp and 333 bp referred as AG genotype. None of the animals revealed GG genotype. The allelic frequencies of ‘A’ and ‘G’ alleles were found to be 0.725 and 0.275, respectively. Association analysis revealed that none of the genotypes were associated with any semen quality traits studied. The current study is the first report of DNA based genotyping of AKR1B1 gene of this indigenous buffalo breed of India. Keywords: AKR1B1 gene, Murrah buffalo, PCRRFLP, genotype

INTRODUCTION The variation in DNA sequence, as it causes the variation in the performance of animals

Department of Animal Biotechnology, 2Department of Animal Reproduction, Gynaecology and Obstetrics, Veterinary College, Anand Agricultural University, Anand - 388001, Gujarat, India

1

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using the Proteinase K method as described by Aravindakshan et al. (1998) and dissolved in 0.3X TE (pH 8.0) buffer. The primer sequences (AKR1B1 F: 5’- ACCAGGGCTTACCTGGAAGT -3’ and AKR1B1 R: 5’- GGTCAATGGGCCTTAGGATT -3’) for amplification of the AKR1B1 gene (Kia, 2007) were used. The PCR was carried out in a final volume of 25 μl reaction mixtures in 0.2 ml thin wall PCR tubes. Each PCR tube contained 12.5 μl of 2X master mix (MBI, Fermentas) containing 0.05 U/μl Taq DNA polymerase (recombinant), MgCl2 (4 mM) and dNTPS (0.4 mM of dATP, dCTP, dGTP, dTTP of each), 3 μl (30 ng/ μl) genomic DNA, 1 μl of 10 picomole of each forward and reverse primer and 7.5 μl of DNaseRNase free water. The protocol for PCR amplification for AKR1B1 consisted of an initial denaturation at 94oC for 5 minutes, followed by 35

This reaction, in particular the sorbitol produced, is important for the function of various organs in the body. Sorbitol is subsequently metabolized to fructose by sorbitol dehydrogenase. Fructose produced from sorbitol is used by sperm cells; also, fructose can be used as an energy source for glycolysis and glyconeogenesis. AKR1B1 participates in glucose metabolism and osmoregulation and is supposed to play a protective role against toxic aldehydes derived from lipid peroxidation and steroidogenesis that could affect cell growth or differentiation when accumulated (Lefrancois-Martinez et al., 2004). High-level glucose concentration which triggers apoptosis during preimplantation in murine embryos (Riley et al., 2004) has led to the over expression of the AKR1B1 gene (Mohan et al., 2002). The AKR1B1, gene known for its 20α-hydroxysteriod dehydrogenase activity, was found to be upregulated in both biopsies derived from blastocysts resulting in no pregnancy and resorption. It may determine the fate of the embryo through the involvement in apoptic pathway (El-Sayed et al., 2006).

cycles of denaturation at 94oC for 30 seconds, annealing at 58oC for 30 seconds. and extension at 72oC for 60 seconds. with final extension at 72oC for 5 minutes. The amplification was carried out in Minicycler (MJ Research) PCR machine. The amplified PCR products of the AKR1B1 gene were resolved on 2% agarose gel electrophoresis along with MassRular Law range DNA Ladder (Range, 80-1031 bp). Restriction digestion of the PCR products was carried out to confirm the identity of the PCR products. The PCR product of AKR1B1 gene was digested with 5.0 U of Nde I restriction enzyme by incubating in a water bath for 14-16 h at 37oC in a 200 μl capacity PCR tube. After restriction digestion, the PCR products were electrophoresed on 2% agarose gel (according to the expected size of fragments) along with 100 bp molecular weight marker and undigested PCR product as a positive control.

MATERIALS AND METHODS The experimental material for the present study comprised of 41 semen ejaculates collected from 41 Murrah buffalo bulls using AV method and evaluated for various semen quality traits, viz., ejaculate volume (ml), individual motility (%), sperm concentration (106/ml), live and dead sperm count (%) and post-thaw motility (%) at Amul Research and Development Association (ARDA), Ode centre, Anand. Genomic DNA was extracted

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Figure 1. Polymorphic pattern of AKR1B1 gene 796 bp PCR fragment digested with Nde I. PC: Psotive control, L: Molecular marker. Lane 1, 2, 3, 4, 5, 6, 7, 810, 12, 14, 15, 17 Homozygous AA type. Lane 9, 11, 13, 16, 18 Heterozygous AG type. Table 1. Genotypes, genotype frequencies and allelic frequencies in Murrah buffalo bulls. Breed

Sample No.

Murrah

41

Genotype Frequencies AA AG GG 0.44 0.56 0.00 (18) (23) (0)

Allele Frequencies A G 0.725 0.275

Table 2. Mean (±SEM) of different semen quality traits for the AKR1B1 loci.

Genotype

AA Mean ± SEM AG Mean ± SEM * (p< 0.05)

Motility (%)

Motility after Thawing (%)

Live and Dead Sperm Count (%)

3.81±0.48a

73.06±0.92a

53.89±1.036a

84.67±0.77a

2.99±0.28a

73.91±1.75a

54.78±0.86a

86.04±0.57a

Sperm Concentration (106/ml)

Volume (ml)

1494.83±119.99a 1523.78±89.38a

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RESULTS AND DISCUSSION

Association analysis of AKR1B1 gene with various semen quality traits To study the association the AKR1B1 gene with semen quality traits, genotypes were compared with various semen quality parameters using SPSS software version 12 for calculating means and standard errors of mean (SEM) and the F-test was used at the 5% level of significance. The means for various semen quality parameters, viz., volume (ml), concentration (106/ml), motility (%), motility after thawing (%) and live and dead count (%) were found to be 3.35±0.27, 1511.07±112.25, 73.54±1.05, 54.39±0.66 and 85.44±0.47, respectively. The results were found to be non- significant (P>0.05) (Table 2) and hence it was concluded that there was no association of genotypes GG and GH with any of the semen quality traits studied.

The PCR amplification generated a 796 bp segment from the buffalo AKR1B1 gene homologous to the bovine AKR1B1 gene of similar length (Figure 1). The target sequence which includes part of intron 7 of the bovine AKR1B1 gene has one polymorphic Nde I site due to a A to G transition mutation (Kia, 2007). Allele A of bovine AKR1B1 having one internal site for Nde I was represented by two fragments of 463 and 333 bp, while allele G is comprised of an intact fragment of 796 bp with no internal site for Nde I restriction enzyme. Genotype AA results in a two fragments of 463 and 333 bp, AB in three fragments of 796, 463 and 333 bp and AG in one fragment of 796 bp on 2.0% agarose gel electrophoresis (Figure 1). In the present study, the amplified product of 796 bp when digested with Nde I enzyme revealed two distinct genotypes, viz. AA and AG, in 18 and 23 Murrah buffaloes, respectively, while the GG genotype was absent in Murrah buffaloes. For unambiguous typing, some of the samples were digested with excess unit of enzymes (20 U) and for extended duration of digestion to confirm that there was no partial digestion. The allele and genotype frequencies for the AKR1B1 locus were calculated using POP GENE32 software (Yeh et al.,1999) and are given in Table 1. Because of the paucity of the literature on the AKR1B1 gene in the buffalo, it is difficult to compare with other observations. However, Kia (2007) reported the allele frequencies were 0.82 and 0.18, respectively, for the ‘A’ and ‘G’ alleles in bos taurus. The genotype GG was not detected. These frequencies are not much different from our findings and indicate ‘A’ as predominant over the ‘G’ allele.

ACKNOWLEDGEMENTS Authors are thankful to Amul Research and Development Association (ARDA), Ode, Anand for providing necessary help, facility and co-operation during collection of the semen sample for the study.

REFERENCES Aravindakshan, T.V., A.M. Nainar and K.A. Nachimuthu. 1998. Simple and efficient method for isolating high molecular weight DNA from bovine sperm. Indian Vet. J., 75: 314-317. Chung, S. and J. LaMendola. 1989. Cloning and sequence determination of human placental aldose reductase gene. J. Biol. Chem., 264: 14775-14777. 277

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El-Sayed, A., M. Hoelker, F. Rings, D. Salilew, D. Jennen, E. Tholen, M.A. Sirard, K. Schellander and D. Tesfaye. 2006. Largescale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients. Physiol. Genomics, 28: 84-96. Kia, H.D. 2007. Identification and SNP detection for preimplantation active genes and their association with embryo development and male fertility in cattle. Dissertation, Bonn University. Ko, M.S.H. 2004. Embryogenomics of pre-

retinoid X receptors, retinaldehyde dehydrogenase, and peroxisome proliferator activated receptor gamma in bovine preattachment embryos. Biol. Reprod., 66: 692-700. Riley, J.K., J.M. Heeley, A.H. Wyman, E.L. Schlichting and K.H. Moley. 2004. Trail and killer are expressed and induce apoptosis in the murine preimplantation embryo. Biol. Reprod., 70: 1365-1373. Tsuchihashi, Z. and N.C. Dracopoli. 2002. Progress in high throughput SNP genotyping methods. The Pharmacogenomics Journal, 2: 103-

implantation mammalian development: current status. Reprod. Fertil. Dev., 16(1-2): 79-85. Lefrancois-Martinez, A.M., J. Bertherat, P. Val, C. Tournaire, N. Gallo-Payet, D. Hyndman, G. Veyssiere, X. Bertagna, C. Jean and A. Martinez. 2004. Decreased expression of cyclic adenosine monophosphate-regulated aldose reductase (AKR1B1) is associated with malignancy in human sporadic adrenocortical tumors. J. Clin. Endocrinol. Metab., 89: 3010-3019. Mohan, M., J.R. Malayer, R.D. Geisert and G.L. Morgan. 2002. Expression patterns of

110. Watson, A.J. 1992. The cell biology of blastocyst development. Mol. Reprod. Dev., 33: 492504. Watson, A.J. and L.C. Barcroft. 2001. Regulation of blastocyst formation. Front. Biosci., 6: D708-730. Yeh Francis, C., R.C. Yang, B.J. Boyle Timothy, Z.H. Ye and J.X. Mao. 1999. POPGENE version 1.32, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada. Available: http://www.ualberta.ca/fyeh/.

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Original Article

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MOLECULAR CHARACTERIZATION OF THE GROWTH HORMONE GENE IN RIVERINE BUFFALOES

Aruna Pal1 and P.N. Chatterjee2

ABSTRACT

growth hormone genotypes monomorphic haplotype.

Growth hormone, a protein hormone, has a remarkable role in growth, reproduction and milk production. The present investigation was aimed at studying the genetic variation in growth hormone gene locus 4th exon, 4th intron and 5th exon in

due

to

their

Keywords: growth hormone gene, PCR-RFLP, genetic polymorphism, phylogenetic tree, haplotype

Murrah buffalo bulls by PCR-RFLP. The growth hormone gene was amplified by PCR using oligonucleotide primers standardized for Bos taurus species. A 428 bp fragment of growth hormone gene spanning over 4th exon, 4th intron and 5th exon was amplified and digested with Alu I restriction enzyme using a cattle specific primer. The sizes of the amplification products were similar in cattle and buffalo. Growth hormone gene in buffalo reveals monomorphism since no variation was found. The result indicates strong conservation of DNA sequence between cattle and buffalo. Nucleotide sequence variations and amino acid variation were observed at growth hormone gene between Bubalus bubalis and Bos taurus species reveals 97.1% nucleotide identity 12 nucleotide substitutions, but a single amino acid change. A phylogenetic tree constructed for buffalo with other species revealed buffalo were closer to cattle and yak compared to other species. The growth, milk production, seminal and sexual behavioral parameters could not be associated with buffalo

INTRODUCTION Growth hormone is a protein hormone consisting of 191 amino acids. Reports suggest that growth hormone has remarkable role in growth, reproduction and milk production. Growth hormone helps in body growth and metabolism through protein synthesis, protein deposition in tissues and organs (Gluckman et al., 1987), increased nitrogen retention (Hart and Johnson, 1986), gluconeogenesis and cell division (Neathery et al., 1991), intestinal calcium absorption, thereby enhancing overall bone growth and stimulating chondrocyte proliferation, (Boyd and Baumann, 1989). It has been found that growth hormone increases the metabolic priority of the mammary gland (Elvinger et al., 1988; Binelli et al., 1995). Growth hormone augments testosterone action in the maintenance of spermatogenesis (Boccabella et al., 1963) and acts on sertoli cells releasing IGF-I, which then acts on the leydig cells of the testis and

Additional Block Animal Health Centre, Jashpur, Birbhum, E-mail: [email protected] 2 Additional Block Animal Health Centre, Chatra, Birbhum, West Bengal, India

1

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Bengal,

India,

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Murrah buffalo bulls maintained at Artificial Breeding Complex of the National Dairy Research Institute, Karnal, Haryana, India, and Frozen Semen bull station, Salboni, Paschim Medinipur, West Bengal and other bull breeding stations in West Bengal, India.

releases testosterone in mice (Ritzen, 1983; Waites et al., 1985). In the case of AI bulls, a positive relationship between GH and IGF-I concentrations and semen quality has been observed, with a higher (3.2 times) level of GH in seminal plasma than in blood plasma (Davis et al., 1985). The growth hormone gene has been assigned to the 19q17 position of bovine chromosome (Hediger et al., 1990). Bovine growth hormone gene consists of five exons and four introns (Gordon et al., 1983). Genetic polymorphism at growth hormone gene mainly arises from several point mutations at the gene. Polymorphism studies of bovine growth hormone gene have been conducted on the coding (Lechniak et al., 1999), intron (Hoj et al., 1993) and promoter regions (Rodrigues et al., 1999) of the gene. The buffalo contributes about 54 percent of the total milk produced in India. Although the economic importance of buffaloes has always been known, yet very little work has been carried out to exploit the genetic potential of this animal. Though studies have been carried out on characterization in cattle, similar studies in buffaloes are scarce. The reports regarding polymorphism studies in buffaloes are very scanty, so the present investigation was carried to find the polymorphism of growth hormone gene in buffalo and to associate

Sample and data About 10 ml venous blood was collected from the jugular vein of each animal in a sterile 50 ml polypropylene vial containing 0.5 M EDTA as anticoagulant. Data on birth weight, three-month body weight, six month body weight, expected predicted difference (EPD) and superiority (percent) over the herd average of the bulls were collected from the records maintained. The data on behavioral traits such as libido score, reaction time, Flehmen’s response, requirement of mounting stimulus and various seminal traits like semen volume, sperm mass activity, seminal consistency and individual fresh sperm motility, post thaw sperm motility and number of semen doses per collection were collected during daily semen collection and evaluation. The ejaculates were collected twice a week. For all practical purposes the freezing of semen was done with the mass activity more than 3.5 and the lower standard for sperm concentration remain 20x106 motile sperm per semen dose. The

such polymorphic pattern with growth traits, milk production traits, seminal and sexual behavioral traits, needed during semen collection and evaluation in artificial breeding programme for genetic improvement of buffalo.

bulls exhibiting Flehmen’s response were coded as 1.0 and 2.0 when no response was obtained. Likewise when external stimulus was needed for mounting, the code was 1.0 and when no stimulus was needed, the code was 2.0. Libido was scored on a 10-point scale (Chenoweth, 1976). Reaction time was measured as the time taken by a bull from its introduction to female (a dummy in the present study) until first ejaculation, and it is an important

MATERIALS AND METHODS Animals The present study was conducted with 130 280

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(PTC-200, MJ Research, USA) in two stages. In the first stage, initial denaturation at 94oC for 5 minutes, denaturation at 94oC for 1 minute, annealing at 46oC for 45 seconds, extension at 72oC for 50 seconds were carried out for 10 cycles. In the next stage, denaturation at 94oC for 1 minute, and annealing at 48oC for 45 seconds were carried out, followed by final extension at 72oC for 50 seconds for the next 25 cycles.

indication of male sexual behaviour. The bulls exhibiting Flehmen’s response were coded as 1.0 and 2.0 when no response was obtained. Likewise when external stimulus (as whistling sound) was needed for mounting, the code was 1.0 and when no stimulus was needed the code was 2.0. DNA preparation Genomic DNA was isolated from blood samples following the phenol- chloroform extraction method described by Sambrook and Russel (2001). DNA was dissolved in TE buffer and was kept in a water bath at 60oC for 2 h to dissolve pellet properly in buffer. The quality of DNA was checked through spectrophotometry. DNA samples with O.D. ratio between 1.7 and 1.9 were considered as good and used for further study. The samples beyond this range were re-extracted by the phenol-chloroform extraction method. DNA quality was also checked by running the sample in 0.8 percent agarose gel electrophoresis. The DNA samples devoid of smear were used for further study.

RFLP and polyacrylamide gel electrophoresis The 428 bp amplicon was digested with Alu I enzyme to identify polymorphism of growth hormone gene. Amplified product was digested with 10 U Alu I restriction enzyme at 37oC for overnight and finally, reaction was stopped after adding 0.5 m EDTA. The digested product was separated through 8% non-denaturing polyacrylamide gel run at 100V for 5 h. The gel was stained with silver nitrate, as described by Bassam et al. (1991), with slight modifications where 10% glacial acetic acid was used for fixing the DNA bands for 30-45 minutes, before staining with 0.1% silver nitrate for 30 minutes. The developer used was 3% sodium carbonate containing 300 μl of formalin. After staining, the fragments were visualized and documented in gel doc system.

DNA amplification A 428 bp fragment of the growth hormone gene spanning over 4th exon, 4th intron and 5th exon was amplified with forward (5′CCGTGTCTATGAGAAGC3′) and reverse (5′GTTCTTGAGCAGCGCGT3′) pr imer sequences. PCR was carried out in a final volume of 25 μl reaction mixture containing 80-100 ng DNA, 2.5 μl 10X PCR assay buffer, 200 mM of each dNTP, Taq DNA polymerase, 20 pmM of each primer and 2 mM MgCl2. To check contamination, a negative control, labelled ‘C’ with master mix devoid of template DNA was made. PCR-reactions were carried out in a thermocycler

Nucleotide sequencing The PCR products were concentrated to 50 ng/μl by pooling several tubes to precipitate by the isopropanol procedure. In order to obtain clean fragment for sequencing, the PCR products were separated by electrophoresis in a TAE agarose gel containing ethidium bromide using standard protocols. The desired PCR product band was excised using a clean, sterile razor blade or scalpel (band was visualized in a medium or long 281

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wavelength (e.g., ≥300 nm) UV light, and excised quickly to minimize exposure of the DNA to UV light). The minimum agarose slice was transferred to a 1.5 ml microcentrifuge or screw cap tube and then purified by using commercially available gel extraction kits (Qiagen). Quantification was done by loading one μl of eluted sample in 1% agarose gel and comparing with standard molecular marker (Phi X 174 DNA ladder or 100 bp DNA ladder). Only samples with good concentration (>50 ng/μl) were selected and subjected to sequencing.

RESULTS AND DISCUSSION Identification of Genotypes The PCR amplification generated a 428 bp segment from growth hormone gene which is homologous to the cattle growth hormone gene of similar length (Figure 1), thus it indicates strong conservation of DNA sequences in both species. A single restriction pattern was observed in all the samples of Murrah buffalo bulls producing four bands consisting of 265 bp, 96 bp, 51 bp and 16 bp (Figure 2) and was assigned as the LL genotype corresponding to leucine homozygote for cattle. Thus, all the buffalo bulls were found to be monomorphic, which was in agreement with the report of Biswas et al. (2003) , who observed 70 Murrah, 32 Bhadawari, 30 Jaffarabadi, 30 Surti and 30 Nagpuri and found only the LL genotype with AluI PCR-RFLP. The present study is also in agreement with the studies conducted by Mitra et al. (1995). Aravindakshan et al. (1997) also reported monomorphism of growth hormone gene at 3rd

Sequence data analysis Sequence data were analyzed mostly by DNASTAR software. Database search The database search of sequences for a possible match to the DNA sequence of growth hormone gene was conducted using the BLAST algorithm available at the National Center for Biotechnology Information (NCBI, Bethesda, MD). Translated protein sequences of different growth hormone genes were also subjected to the BLAST algorithm.

intron of growth hormone gene using MspI as restriction enzyme. Therefore, this monomorphism of the buffalo may be a species specific characteristic of buffalo. Thus, the gene and genotypic frequencies were found to be 1.00.

Statistical analysis The frequencies of gene and genotypes were estimated for the identified locus as per the method suggested by Falconer and Mackay (1996). Association study with the growth, milk production, seminal and sexual behavioral parameters with the genotype of growth hormone gene could not be achieved due to monomorphism of the alleles of growth hormone gene.

Nucleotide sequencing Monomorphism of the genotype of growth hormone gene was confirmed by nucleotide sequencing. The nucleotide sequence as well as the derived amino acid sequence of growth hormone gene of riverine buffalo (Gene Bank Accesion number GU223914 ) have been depicted in Figure 3. Nucleotide sequence variations and amino acid variation were observed at the growth hormone gene between Bubalus bubalis and Bos taurus 282

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Table 1. Mean±S.E. of growth traits of Murrah bulls. Birth Weight (kg) 33.83±0.95

3 M Body Weight (kg) 62.53±2.24

6 M Body Weight (kg) 100.88±3.64

Table 2. Mean±S.E. of behavioural characteristics Murrah bulls. Libido Score (Score)

Reaction Time (Second)

5.07±0.46

43.13±11.36

Flehmen’s Response (Score) 1.33±0.09

Mounting Stimulus (Score) 1.25±0.10

Table 3. Percent of behavioural and seminal characteristics shown Murrah bulls. Flehmen’s Response (%)

Mounting Stimulus (%)

Seminal Consistency (%)

72.22

50.00a 40.00b 10.00c –d

65.00

a = Creamy; b = Lemon; c = Milky; d = Watery. Table 4. Mean±S.E. of seminal characteristics of Murrah bulls. Semen Volume (ml)

Mass Activity (0-5)

Individual Motility (%)

Individual Motility (Score)

2.70±0.48 2.10±0.13 58.40±4.15

3.18±0.23

PostPostSemen Thaw Thaw Doses per Motility Motility Collection (%) (Score) (No.) 44.98±1.63 2.80±0.10 133.59±9.98

Table 5. Mean±S.E. of production traits of Murrah. EPD (kg) 242.52±26.14

Superiority (%) 11.03±1.20

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Figure 1. PCR product of buffalo and cattle growth hormone gene in agarose gel electrophoresis. Lane C represents control. Lane 1, 2 represents PCR product obtained from cattle bulls. Lane 3, 4 represents PCR product obtained from Murrah buffalo bulls.

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Figure 2. PCR-RFLP of 428 bp fragment of growth hormone gene in buffalo bulls. Lane P : 428 bp PCR product. Lane M: 100bp marker. Lane 1-6 : LL genotype. * indicates the presence of non-specific bands.

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Nucleotide sequence: CTGAAGGACCTGGAGGAAGGCATCCTGGCCCTGATGCGGGTGGGGATGGCGTTGTGGGTCCC TTCCATGCTGGGGGCCAT GCCCGCCCTCTCCTGGCTTAGCCAGGAGAACGCACGTGGGCTGGGGGAGAGAGATCCCTGCT CTCTCACTCTTCCTAGCA GTCCAGCCTTGACCCACCCCAAACCTTTTCCCCTTTTGAAACCTCCTTCCTCGCCCTTCTCCAA GCCTGTAAGGGAGGGT GGAACATGGAGCGGGCAGGAGGGAGCTGCTCCTGAGGGCCCTTCGGCTCTCTGTCTCTCCCTC CCTTGGCAGGAGCTGGA AGATGGCACCCCCCGGGCTGGGCAGATCCTCAAGCAGACCTATGACAAATTTGACACAAACAT GCGCAGTGACGACGCGC TGCTCAAGAAC Amino acid sequence: LKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKN

Figure 3. Sequence report :Amplified 411 bp fragment of growth hormone gene exon 4, intron4 and exon5 in Bubalus bubalis breeds ( Gene bank acc no. GU223914).

286

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6.1

2

64.0

94.6

21.6

100.0

5.1

1

14

15

16

17

18

94.7

3

3

94.8

13.6

84.2

13.0

96.7

25.2

5.5

9.7

70.9

83.9

10.6

87.1

28.5

91.5

8.5

***

47.3

47.5

4

4

96.6

15.7

82.3

12.9

100.0

25.2

8.0

11.7

73.6

83.5

4.7

87.5

28.4

91.1

***

92.0

47.3

47.5

5

5

5.0

99.5

21.6

93.3

63.4

100.0

92.8

80.6

44.3

30.1

93.3

8.2

100.0

***

47.7

47.7

97.1

99.5

6

6

100.0

31.2

100.0

31.1

100.0

43.3

28.4

28.4

94.4

100.0

28.7

100.0

***

40.5

76.4

76.4

40.3

40.2

7

7

8.8

94.4

19.8

88.8

65.4

100.0

88.3

76.9

48.2

29.9

87.5

***

42.1

92.3

48.7

48.9

91.7

92.3

8

8

94.4

17.1

86.8

14.9

100.0

26.7

8.5

12.9

77.3

91.0

***

48.7

76.2

47.2

95.5

90.2

46.8

46.9

9

9

31.7

88.6

29.4

91.0

65.7

95.1

80.7

80.5

44.9

***

47.8

75.8

43.9

75.8

49.9

49.9

75.2

76.3

10

10

48.2

79.6

49.8

80.5

66.8

90.9

68.8

70.5

***

67.0

51.8

65.2

46.3

67.1

53.1

54.2

66.2

67.4

11

11

81.8

17.4

83.1

15.1

91.7

29.4

9.7

***

71.1

75.6

22.6

91.5

17.6

95.1

23.1

23.1

93.9

95.1

12

12

93.9

11.8

84.2

12.4

95.0

24.8

***

44.7

55.0

50.9

92.0

48.7

76.4

47.5

92.5

94.7

47.0

47.2

13

13

100.0

31.6

100.0

30.8

87.7

***

79.2

34.2

47.6

47.0

77.9

43.7

67.4

43.7

78.9

78.9

43.3

43.4

14

14

66.5

100.0

65.6

100.0

***

48.4

46.4

70.4

56.6

57.6

44.6

56.8

43.0

57.9

44.6

46.1

57.2

57.6

15

15

99.0

3.6

94.1

***

44.3

75.4

88.7

39.2

50.8

47.8

86.7

48.4

74.7

47.2

88.2

88.2

46.8

46.9

Figure 4. Genetic similarity of buffalo with other species with respect to growth hormone gene.

100.0

22.6

95.1

64.8

100.0

94.2

100.0

12

82.1

45.9

8.8

13

81.6

11

30.7

29.3

43.8

9

94.6

8

10

95.1

8.2

7

3.0

100.0

0.5

92.9

100.0

4

***

93.3

6

92.4

3

2

97.1

5

3.0

92.8

2

***

1

1

Percent Similarity in upper triangle. Percent Divergence in lower triangle.

Pair Distances of ALL SPP CORRECTED.meg J. Hein (Weighted).

16

16

23.0

95.6

***

46.9

57.2

44.3

49.7

79.3

64.3

76.0

49.0

82.7

41.7

81.5

50.3

49.7

80.7

81.5

17

17

100.0

***

46.4

96.5

44.3

74.9

89.2

22.1

51.0

48.3

85.0

46.8

74.7

45.6

86.0

87.7

45.2

45.3

18

18

***

44.8

80.5

45.8

56.5

43.2

47.2

97.1

65.2

74.7

46.9

91.8

40.2

95.2

46.4

46.9

94.2

95.2

SHEEP

RAT

PIG

MOUSE

MONKEY

HUMAN

HORSE

GOAT

ELEPHANT

DONKEY

DOG

DEER

CHICKEN

CATTLE

CAT

CAMEL

BUFFALO

YAK

Buffalo Bulletin (December 2010) Vol.29 No.4

Buffalo Bulletin (December 2010) Vol.29 No.4

dog elephan cat camel Pig donkey horse mouse rat Cattle yak Buffalo Goat Sheep deer human monkey Chick

18.1 18

16

14

12 10 8 6 Nucleotide Substitutions (x100)

4

2

0

Figure 5. Phylogenetic analysis of buffalo with other species with respect to growth hormone gene.

and yak compared to other species (Figure 5), thus bovines were grouped together. Buffalo were genetically closer to goat and sheep. This pattern was similar to the phylogenetic tree constructed based on the nucleotide sequence of CD14 gene from different species (Pal and Chatterjee, 2009). Deer was also clustered with the above mentioned species, indicating ruminants were grouped together. Buffalo was found to be genetically most distant to chicken, thus depicting completely different lineage. The average value of the phenotypic parameters including growth traits (Table 1), reproductive traits (Tables 2, 3, 4) and production traits (Table 5) have been depicted. The growth, milk production, seminal and sexual behavioral parameters could not be associated with buffalo growth hormone genotypes due to their monomorphic haplotype.

species reveals 97.1% nucleotide identity. Comparison of the growth hormone gene sequence of cattle and buffalo, pertaining to 4th exon, 4th intron and 5th exon revealed 12 nucleotide substitutions, but a single amino acid change (Figure 4). The remaining three codons were synonymous. The nucleotide sequence organization is similar to other species, as goat, sheep, mouse, human, monkey, camel, cat, chicken, deer, dog, donkey, elephant, horse, yak, pig and rat (www. ncbi.nlm. nic.in) Phylogenetic study of buffalo with other species with respect to growth hormone gene The phylogenetic tree for buffalo with other species has been depicted in Figure 5. The phylogenetic tree constructed for buffalo with other species revealed the buffalo was closer to cattle

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CONCLUSION

Anal. Biochem., 196: 80-83 Binelli, M., W.K. Vanderkooi and L.T. Chapin. 1995. Comparison of growth hormone releasing factor and somatotropin: body growth and lactation of primiparous cows. J. Dairy Sci., 78: 2129-2139 Biswas, T.K., T.K. Bhattacharya, A.D. Narayan, S. Badola, P. Kumar and A. Sharma. 2003. Growth hormone gene polymorphism and its effect on birth weight in cattle and buffalo. Asian Australus. J. Anim. Sci., 16: 494-497. Boccabella, A.V. 1963. Re-initiation and restoration of spermatogenesis with testosterone propionate and other hormone after a long term post-hypophysectorny regression period. Endocrinology, 72: 787-798. Boyd, R.D. and D.E. Baumann. 1989. Mechanism of action of somatotropin in growth, p. 257293. In Campion, D.R., G.J. Haeesman and R.J. Martin (eds.) Current Concepts of Animal Growth Regulation. Plenum Press, New York, USA. Chenoweth, P.J. 1976. Behavioural considerations of the natural breeding bull. Proc. Soc. Theriogenology. 19. Davis, S.L., P.L. Senger, D.L. Ohlson and K.L. Hossner. 1985. Temporal patterns of growth hormone in blood and seminal plasma of mature dairy bulls. J. Anim. Sci., 60: 35-39. Elvinger, F., H.H. Head and C.J. Wilcox. 1988. Effect of bovine somatotropin on milk yield and composition. J. Dairy Sci., 71(6): 15151525. Falconer, D.S. and T.F.C. MacKay. 1996. Introduction to Quantitative Genetics, 4th ed. Longman Group Ltd., Essex. England. Gluckman, P.D., B.H. Breier and S.R. Davis. 1987. Physiology of the somatotropic axis with particular reference to the ruminant. J. Dairy

Bovine growth hormone gene specific primers amplified the buffalo growth hormone and PCR amplification yielded a 428 bp fragment spanning from 4th exon, 4th intron and 5th exon from buffalo DNA homologous to that of cattle. AluI restriction enzyme yields 265 bp, 96 bp, 51 bp and 16 bp fragments. Based on available information in cattle, all the buffalo DNA samples were genotyped as LL. Thus the monomorphic pattern of growth hormone gene in buffaloes may be a species specific characteristics of buffalo.

ACKNOWLEDGEMENTS The facilities and support of the National Dairy Research Institute, Karnal, Haryana,India and the help provided during sample collection from the Artificial Breeding Complex, Joint Director, Composite State Animal Husbandry Farm, Salboni and Laboratory facilities provided by Indian Veterinary Research Institute, Izatnagar, UP, India, are acknowledged.

REFERENCES Aravindakshan, T.V., A.M. Nainar, P. Ramadass and K. Nachimuthu. 1997. Genetic polymorphism within the growth hormone gene of cattle and buffalo (Bubalus bubalis) detected by polymerase chain reaction and endonuclease digestion. Int. J. Anim. Sci., 12: 5-8. Bassam, B.J., G. Gaetano-Anolles and P.M. Gresshoff. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. 289

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and buffalo. J. Anim. Breed. Genet., 112: 7174. Neathery. M.W., C.T. Crowe, G.F. Hartnell, J.J. Veensuizen, J.O. Reagan and D.M. Blackmon. 1991. Effects of sometribove on performance, carcass composition and chemical blood characteristics of dairy calves. J. Dairy Sci., 74(11): 3933-3939. Pal, A. and P.N. Chatterjee. 2009. Molecular cloning and characterization of CD14 gene in goat. Small Ruminant Res., 82(2-3): 8487. Ritzen, E.M. 1983. Chemical messengers between Sertoli cells and neighbouring cells. J. Steroid Biochem., 19: 499. Rodrigues, C.V., E. Das Neto, Lopez and L.E. Pinheiro. 1999. A variation of simple repeat sequence in the promoter region of the bovine growth hormone (bGH) gene in beef cattle is similar to water buffalo. J. Anim. Breed. Genet., 116: 15-19. Sambrook, J. and D.W. Russel. 2001. Molecular Cloning - A Laboratory Manual, 3nd ed. Vol. 3. Cold Spring Harbour Laboratory Press, New York, USA. Waites, G.M.H., A.C. Speight and N. Jenkins. 1985. The functional maturation of the Sertoli cell and Leydig cell in the mammalian testis. J. Reprod. Fertil., 75: 317.

Sci., 70(2): 442-466. Gordon, D.F., D.P. Quick, C.R. Erwin, J.E. Donelson and R.A. Maurer. 1983. Nucleotide sequence of the bovine growth hormone chromosomal gene. Mol. Cell. Endocrinol., 33: 81-95. Hart, I.C. and I.D. Johnson. 1986. Manipulation of milk yield with growth hormone, p. 105-124. In Haresign, W. and D.J.A. Cole (eds.) Recent Advances in Animal Nutrition. Butterworths, London, UK. Hediger, R., S.E. Johnson, W. Barendse, R.E. Drinkwater, S.S. Moore and J. Hetzel. 1990. Assignment of the GH gene locus to 19q26qter in cattle and to 11q25qter in sheep by in situ hybridization. Genomics, 8: 171174. Hoj, S., M. Fredholm, N.J. Larsen and V.H. Nielson. 1993. Growth hormone gene polymorphism associated with selection for milk fat production in lines of cattle. Anim. Genet., 24: 91-96. Lechniak, D., G. Machnik, M. Szydlowski and M. Switonski. 1999. Growth hormone gene polymorphism and reproductive performance of AI bulls. Theriogenology, 52: 1145-1152. Mitra, A., P. Schlee, C.R. Balakrishnan and F. Pirchner. 1995. Polymorphisms at growth hormone and prolactin loci in Indian cattle

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Original Article

Buffalo Bulletin (December 2010) Vol.29 No.4

COMPARATIVE STUDIES OF CERTAIN MACRO MINERALS DURING VARIOUS REPRODUCTIVE STATES IN BUFFALOES R. Chaurasia1, H.S. Kushwaha1, D. Chaurasia2, M.K. Gendley2 and A.K. Santra2

ABSTRACT

Murrah buffaloes. Differences in levels of bound forms of serum calcium and phosphorus were nonsignificant among the various groups.

Thirty healthy Murrah buffaloes of 5-9 years of age in their second to fifth lactation were randomly chosen from well-organized dairies located at Jabalpur and screened as per the approved technical programme to elucidate alteration in concentration of some macro elements (Na, K, Ca, P and Mg) in serum of normal cyclic, repeat breeder and anoestrus Murrah buffaloes. Gynecological examinations were employed for the diagnosis of reproductive states of animals. The selected 30 Murrah buffaloes were divided into three groups; each group comprised of 10 animals for the generation of experimental date. Blood samples were collected from animals of different groups on the day of estrus from normal cyclic, repeat breeder and anoestrus buffaloes on the same day as gynaecoclinical examination. The serum was prepared following routine procedure. The concentrations of total, free and bound forms of serum sodium, potassium and magnesium were non-significant among the three groups. The total and free forms of serum calcium were significantly (P