Livestock Research for Rural Development 22 (5) 2010 Notes to Authors LRRD Newsletter

Citation of this paper

Performance of growing kids on rations with Lablab (Lablab purpureus) grains as protein source

Sultan Singh, S S Kundu, A S Negi* and V C Pachouri

Plant Animal Relationship Division, Indian Grassland and Fodder Research Institute,
Jhansi-284003 (India)
singh.sultan@rediffmail.com
* Central Institute for Research on Medicinal and Aromatic Plants, Lucknow, India

Abstract

A 105 days growth experiment was conducted to evaluate the milled dry lablab grains (LLG) as alternate protein source to ground nut cake (GNC) in ration of kids. For this, 15 kids of local goat breed (Bundelkhandi) were divided into 3 groups (LL0, LL50 and LL100) of 5 animals each and were offered a dry grass based diet (mixed grasses; Cenchrus ciliaris, Sehima nervosum and Dicanthium annulatum) ad lib and concentrates to meet their nutritional requirements. In LL0 (control) concentrate mixture GNC was the sole protein source, whereas in LL50 and LL100, 50 and 100 % of the GNC was replaced by LLG on N basis, respectively. After 60 days feeding, a digestion -cum metabolism trial (7d) was conducted to determine feed intake, nutrients digestibility, nutritive value, N balance along with rumen fermentation and growth rate. Rumen liquor samples were collected at the end of trial, while body weights were recorded at 15 days interval.

 

Crude protein (CP) and ether extract (EE) contents were lower in LLG (27.50 and 1.60) than GNC (33.7 and 4.50 %). LLG supplementation did not influence (P>0.05) the intake, while roughage intake (%) was (P>0.05) higher in LL100 (1.91) than LL0 (1.6). Concentrate intake (%) in 30 minutes of feeding was higher (P<0.05) in LL50 (88.2) and LL100 (96.8) than LL0 (80.1). However the meal size (g/2 h) was comparable amongst the dietary groups. Digestibility (%) of DM, OM, NDF, ADF and cellulose were comparable (P>0.05), while LLG supplementation increased (P<0.05) CP digestibility in LL50 (73.5) and LL100 (73.4) compared to GNC in LL0 (67.4%). In sacco DMD of LLG (95.3) was higher (P<0.05) than GNC (74.8%). Digestible crude protein (DCP) and total digestible nutrients (TDN) contents were comparable among dietary groups. Digestible energy (DE) contents were 2.9, 2.8 and 3.2 K cal/g DM in LL0, LL50 and LL100, respectively. N balance tended to increase (P>0.05) in LLG supplemented group. N balance (% of absorbed) was significantly (P<0.05) higher in LL50 (61.50) and LL100 (54.3) than LL0 (36.9). Inclusion of LLG in the ration of LL50 and LL100 increased (P>0.05) growth rate of kids and it varied from 33.4 to 39.7 g/d across groups. Total-N and total volatile fatty acids (TVFA) production was higher (P>0.05) in LL50 (112 and 164) and LL100 (110 and 163) than LL0 (96.2 mg/100 ml and 147 mmol/l.).

 

LLG can substitute GNC as protein source in the concentrate mixture of kids with positive associative effect on roughage intake, nutrient utilization, rumen fermentation and body growth with better N utilization.

Key words: eating pattern, goat kids, growth, lablab grains, protein supplement


Introduction

Resource poor small farmers and landless laborers in rural and suburb parts of India rear small ruminants, particularly the goats, as a part of their livelihood. Goats are raised on common grazing lands, roadsides and crop residues supplemented with local tree leaves. These grasses and crop residues are deficient in energy, protein and minerals and are unable to meet the nutritional requirements of producing animals unless these are supplemented with energy and protein sources.  Occasionally, goats are supplemented with kitchen wastes and cereal grains, while traditional protein sources available are mainly used for lactating animals, as protein is the most expensive feed component.  This has resulted in the low productivity of small ruminants due to nutritional imbalance, particularly of protein. To meet the protein need of small ruminants, attention was given to exploit the available alternate protein sources. Legumes (soybean, mung beans, peanuts, pigeon peas, cowpea, chick pea and lablab) are primarily grown to produce grain for human consumption or for processing, but the grain, or the byproduct their off, is also a valuable source of feed for livestock. Alike other legume lablab purpureus grains also contain anti-nutritional factors like tannins, phytate, trypsin inhibitors and polyphenols (Deka and Sarkar 1990; Ramakrishana et al 2006). These compounds due to their astringency and bitterness affect intake and palatability of diet and also interfere with the availability of dietary nutrients specially minerals and protein.  The activity of these compounds can be reduced by certain processing methods like seed coat removal, soaking, cooking, roasting and boiling (Deka and Sarkar 1990; Ramakrishana et al 2006).

 

In the recent, interest for the use of legume grains in the ration of livestock seems to increase throughout the globe (Dixon and Hosking 1992; Lanza et al 2003; Christodoulou et al 2005; Singh et al 2006; Liponi et al 2007).  Even when most of the legume grains may be originally intended for human consumption, grains that fail to meet the required quality standards for the market could make an important source of protein and high energy in the feedlot rations.

 

Lablab is a field crop mostly confined to the peninsular region and cultivated to a large extent in Karnataka and adjoining districts of Tamil Nadu, Andhra Pradesh and Maharashtra and one of the major sources of protein in the diets of these southern states of India. Karnataka contributes nearly 90 % both in terms of area and production in the country. Karnataka state records production of about 18,000 tonnes from an area of 85,000 hectares. Dolichos lablab (Lablab purpureus) is a high yielding legume shrub (5-6 t/h) even with the minimum rainfall and management (Tacheba and Moyo 1988). Lablab grains contain 20-28% protein with high contents of vitamins A, B and C. Lablab has lower proteinase activity (2.4-3.2 units/mg seed) compared to most of the other legume grains (Deka and Sarkar 1990). The objective of this work was to evaluate the lablab grains (LLG) as an alternate of a traditionally used protein source, groundnut cake (GNC), in the ration of kid goats.

 

Materials and methods 

Animal and feeding groups

 

Fifteen healthy growing local goats (Bundelkhandi) below six months of age, having average body weight of 10.0 kg were randomly divided into three dietary groups (LL0, LL50 and LL100) of 5 animals each in a completely randomized design. Animals were offered chaffed dry mixed grass ad lib and isonitrogenous concentrate mixture (consisting of maize grain, wheat bran, groundnut cake -GNC and/or lablab grains-LLG, common salt and mineral mixture) to meet the nutritional requirements (ICAR 1985). Experimental diets were as follows:

LL0: grass adlib + concentrate mixture-CM1 (GNC as 100 % protein source)

LL50: grass adlib + concentrate mixture-CM2 (LLG and GNC each 50 % as protein source)

LL100: grass adlib + concentrate mixture-CM3 (LLG as 100 % protein source)

Kids were housed and individually tied with small mesh chains. Animals were offered concentrate mixture daily between 9-10 a.m. and dry mixed grass was offered ad lib at 12 a.m. and 14 p.m. as basal roughage. Quantity of concentrate offered to each kid was revised at fortnight interval as per the change in animal body weight. Animals were provided clean drinking water daily twice at 11 a.m. and 16 p.m. hours after 1 to 2 hours feeding of concentrate and roughage (grass).

 

Feeds

                                                                                                                                               

Mixed grasses comprising mainly Cenchrus ciliaris, Sehima nervosum with small proportion of Chryosopogon fulvus and Dicanthium annulatum naturally grown in pasture area at Central Research Farm of Indian Grassland and fodder Research Institute (IGFRI), Jhansi were harvested at mature stage and stored as hay. Kids were offered ad lib chaffed grass along with concentrate mixture consisting of crushed barley, wheat bran, mineral mixture (containing 72% Ca, 9.0% P, 0.06% Cu, 0.01% Co, 0.09% Mn, 0.02% I, 0.15% Zn and 40% Fe), common salt and groundnut cake/ or milled dry lablab grains (Table1). LL0: 100 % GNC, LL50: 50:50 GNC: LLG and LL100: 100% LLG, respectively to make the concentrates iso-nitrogenous (19.0%).

Table 1.  Ingredient composition of concentrate mixtures fed to animals of different groups

Ingredients

 LL0

LL50

LL100

Groundnut cake

 40

20

-

Wheat bran

 17

22

25

Maize

 40

30

22

Milled lablab grain

 -

25

50

Common salt

 2

2

2

Mineral mixture

 1

1

1

CP of diet

 19.28

19.28

19.28

Growth and metabolism trial

 

In 120 days experimental period, initial 15 days were not included in the growth period and it was treated as adjustment period. The meteorological records collected from the observatory of the Institute during experimental period are given in Table (2).

Table 2.  Meteorological observations recorded during feeding period

Month

Temp, 0C

RH, %

Rainfall, mm

Max.          Min.

I/ 8.30 am    II/2.00 pm

 March

35.2        12.4

74.0              25.0

0.0

 April

42.0        20.0

45.0              19.0

0.0

 May

41.0        25.3

60.0             26.0

22.4

 June

39.2       27.2

67.0             40.0

19.4

RH: relative humidity

Animals were weighed at 15 days interval to record the growth rate, and the requirements of animals were revised fortnightly as per their body weight change.  After 60 days of feeding a digestion-cum metabolism trial of 7 days duration was conducted. Representative samples of feeds offered, feces, urine voided and refusals were collected daily and pooled on individual basis for further chemical analysis. Pooled samples of feed offered, refusals and feces were dried at 60 0C and ground to pass through 1mm sieve using Willey mill for further chemical analysis. At the end of metabolism trial rumen liquor samples were collected before feeding (0 h) for 2 consecutive days through perforated stomach tube and analyzed separately for individual animal for volatile fatty acids (VFA) and nitrogen metabolites.  

 

Eating pattern

 

To observe the consumption/eating rate of concentrate and grass, animals were initially offered weighed amount of concentrate in feeding troughs at 9 a.m. and intake was estimated at 30 minute and 1h of feeding. This followed offering roughage (grass) to animals at 10 a. m. Subsequent determination of grass consumption was recorded at 2 (12 a.m.), 4 (14 p.m.) and 24 h of feeding. This procedure was repeated for 3 consecutive days. The total intake (24 h) was determined by weighing the refusal next day (9 a.m.). For meal size (g/2h) estimation amount of concentrate and grass consumed by individual animal was pooled.   

 

Analytical techniques

 

Dry matter in feeds, faeces and fodder was estimated by drying in hot air oven at 80 0C for 24 hours. Fresh feces and urine samples collected during metabolism trial were preserved in diluted and concentrated sulfuric acid, respectively, and were analyzed for N by micro-kjeldhal method. Ground feeds and fecal samples were analyzed for CP, EE and total ash contents (AOAC 1997). Cell wall contents (NDF, ADF, cellulose and lignin) were estimated as per the method of Van Soest et al (1991).  In sacco degradability of concentrate mixtures was analyzed by method of Mehrez and Orskov (1977). The energy contents in feeds, feces and refusals were estimated by the method of O’ Shea and Maguire (1962).  The rumen liquor samples were analyzed for pH (digital pH meter of Systronic make), TVFA (Briggs et al 1957), total-N (Mckenzie and Wallace 1954) and ammonia -N (Conway 1957).

 

Statistical analysis

 

The database was subjected to analysis of variance for completely randomized design (Snedecor and Cochran 1967) using the following model:

Yij=m  +Di + eij (general linear model)

Where:

m is the general mean,
Di the effect of ith diet (1-3) and
eij is the random error

 

Results and discussion 

Chemical composition

 

Crude protein contents of LLG (27.5) were lower than the GNC (33.7%, Table 3).

Table 3.  Chemical composition (%) and gross energy (GE); kcal/g DM) of dry grass, concentrates mixture and feed ingredients

Ingredients

OM

CP

NDF

ADF

Cellulose

Hemi-cellulose

Lignin

EE

GE

Milled lablab grain

94.7

27.5

33.5

17.1

14.9

16.4

1.42

1.50

5.01

GNC

92.1

33.7

38.1

27.1

15.0

11.0

6.71

4.50

4.42

Maize

95.7

8.5

20.3

8.70

5.72

11.6

2.50

4.70

4.73

Wheat bran

94.1

14.2

35.2

11.3

7.91

23.9

1.87

3.42

4.12

CM1

93.2

20.4

38.5

9.8

7.2

28.7

2.21

4.56

4.61

CM2

93.5

20.2

37.6

8.7

6.5

28.9

1.90

3.25

4.46

CM3

93.7

20.2

33.2

8.1

6.1

25.1

1.47

1.82

4.64

Dry grass

93.2

3.94

70.0

47.9

39.0

32.1

6.59

0.7

4.45

CM1: concentrate mixture for LL0 group; CM2: concentrate mixture for LL50 group; and CM3: concentrate mixture for LL100 group, respectively

Similarly the cell wall contents (NDF, ADF, cellulose and lignin) were lower in LLG than GNC. CP contents of GNC were lower and fiber fractions were higher than earlier findings (Mahanta et al 1999) but similar to findings of Singh et al (2006). The variation in GNC chemical constituents may be attributed to the method of processing and genetic differences as well as hull contents. CP contents of concentrate mixtures were comparable while, cell wall contents were lower in LLG-supplemented groups (LL50 and LL100) than GNC based concentrate mixture (LL0). Lower fiber fraction in LL50 and LL100 groups may be due less cell wall contents fraction in LLG. The EE content was significantly (P<0.05) lower in LLG (1.6%) compared to GNC (4.50%). Concentration of NDF, ADF, cellulose and lignin was 38.5, 9.8, 7.2, 2.21 and 33.2, 8.1, 6.1 and 1.47% in LL0 and LL100 group, respectively. CP, EE and fiber fractions in lablab seeds reported by Ismartoyo et al (1993) are consistent with the present findings. Rain fed legumes CP contents varies from 260-310 g/ kg (Hadjipanayiotou et al 1985).

 

Dry matter intake and nutrients digestibility

 

Supplementation of LLG did not influence (P>0.05) the total DMI (g/ day) in animals of LL50 (341) and LL100 (398) compared to LL0 (395 Table 4).

Table 4.  Dietary intake and nutrients digestibility in kids maintained on different dietary regimens

Attributes

LL0

LL50

LL100

Pooled SEM

Dry matter intake

 

 

 

 

Total DMI ,g/d

395

341

398

33.4

% body wt 

3.01

3.28

3.44

0.21

g kg/ W0.75

58.8

58.7

63.1

2.01

Roughage intake, % body wt

1.5

1.8

1.9

0.12

Concentrate,  % body wt

1.4

1.4

1.5

0.13

Roughage concentrate ratio

52:48

54:46

56:44

 

Water intake,  l/day

1.64

1.31

1.56

0.13

Nutrients digestibility, %

 

 

 

 

DM

64.7

63.1

64.6

2.39

OM

67.8

65.2

67.6

3.02

EE

86.0b

83.0b

72.0a

2.14

CP

67.4a

73.5b

73.4b

2.12

NDF

59.6

55.0

59.5

2.64

ADF

53.4

50.0

54.1

2.45

Cellulose

66.5

60.0

66.2

2.49

ab means in the same row for each parameter with different superscripts are significantly different (p <0.05)

However the kids exhibited higher (P>0.05) roughage intake in LLG supplemented groups (LL50 1.8 and LL100 1.9) than GNC supplemented group (LL0 1.5%). This shows that LLG had positive associative effect on roughage intake (dry grass). Roughage to concentrate ratio varied from 52:48 in LL0 to 56:44 in LL100. This further indicates increased roughage intake due to LLG supplementation. Supplementation with 6g/kg live weight of lablab purpureus seeds increased the roughage and total diet intake in goats (Ismartoyo et al 1993), which supports the present findings. However these workers further reported that lablab supplemented at higher levels (12-g/kg live weight) decreased roughage intake in goats. Lower proportion of LLG in the present study (25 and 50 % in concentrate mixture of LL50 and LL100) compared to sole grain feeding might be responsible for enhanced microbial growth and hence higher rate of passage resulting in higher intakes. Earlier workers had reported increased (Cheva-Isarakul et al 1991; Cheva-Isarakul 1992; Singh et al 2006) and decreased (Baumlin 1986; Devendra 1978) DMI on use of legume grains in ration of ruminants. Water intake (l/d) was comparable between GNC (LL0 1.64) and LLG supplemented groups (LL50 1.31 and LL100 1.56).

 

Results presented in Table 4 revealed that nutrients digestibility was comparable among dietary groups except CP and EE. Ether extract digestibility was higher (P<0.05) in LL0 (86.0) than LL100 (72.0), while CP digestibility was significantly (P<0.05) lower in former (LL50 67.4) than later group (LL100 73.4%). Higher CP digestibility in LL100 may be due to more solubility of LLG than GNC in respective concentrate mixtures as it is evident from the higher in sacco degradability of LLG than GNC recorded in the present study. Similar to our findings Ismartoyo et al (1993) observed higher  CP and similar NDF and ADF digestibility in kids fed LLG at different levels (3-12g/kg live weight) of LLG. Decreased EE digestibility with supplementation of cowpea and pigeon pea grains in sheep has also reported (Cheva-Isarkul 1992; Singh et al 2006). Feed intake, nutrient utilization and feed to gain ratio of kids were similar when soyabean meal replaced partly/completely by common vetch or broad bean grains (Koumas and Economides 1987). In sacco degradability of LLG was significantly (P<0.05) higher than GNC at all the tested hours (0-48) of incubation. It varies from 17.9 to 74.8 in GNC and 30.6 to 95.3 % in LLG across the incubation hours (Figure 1).


Figure 1.  In sacco degredebility of GNC and Lablab grains at different hours of incubation

This shows that LLG are more soluble than GNC. Winged bean had higher CP solubility (98.2) than soybean (96.9%) reported by Mutia and Uchida (1993) confirms that legume grains are more degradable than conventional oil cake protein sources. Singh et al (2006) also reported higher degradability of cowpea grain based concentrate mixture. 

 

Nutritive value and nitrogen balance

 

Protein source and its nature did not influence the nutritive value as TDN, DCP and DE contents of dietary groups were comparable (P>0.05; Table 5).

Table 5.  Nutritive value and nitrogen balance in kids fed different diets                                           

Attributes

LL0

LL50

LL100

Pooled SEM

Nutritive value

 

 

 

 

DCP%

10.6

10.3

8.60

0.91

TDN%

65.5

63.2

66.2

3.08

DE kcal g/DM

2.90

2.80

3.2

0.14

Nitrogen balance, g/d

 

 

 

 

Intake

7.6

7.3

7.2

0.31

Feacal

2.5

1.9

2.1

0.27

Absorbed

5.1

5.4

5.1

 

Urinary

3.1

2.0

2.3

0.29

Balance

2.0a

3.4b

2.8b

0.32

% of total intake

21.1

45.3

38.8

4.75

% of absorbed

36.9a

61.5b

54.3b

5.92

ab means in the same row for each parameter with different superscripts are significantly different (p <0.05)

Kids were in positive nitrogen balance (g/ day) and differed (P>0.05) amongst the groups. Nitrogen intake was similar in dietary groups, however, fecal and urinary nitrogen execration was (P>0.05) higher in LL0 (2.5, and 3.1) than LL50 (1.9 and 2.0) and LL100 (2.1 and 2.3). Nitrogen balance (g/day) was significantly (P<0.05) higher in LL50 (3.4) and LL100 (2.8) than LL0 (2.0). This indicates higher utilization and metabolism of N in animals from LLG than GNC. Further the balance of absorbed nitrogen (%) was higher (P<0.05) in LL50 (61.5) and LL100 (54.3) than LL0 (36.9). Increased DE, TDN and DCP with nitrogen balance in lablab grain supplemented groups may be associated to higher solubility of protein and higher contents of soluble carbohydrates in LLG than GNC protein source.  Increased DE and nitrogen balance in sheep fed increasing level of pigeon pea seeds and cowpea grains reported by Cheva-Isarakul (1992) and Singh et al (2006) supports the present finding. Barajas et al (1999) recorded increased (P=0.04) DE content of the diet 2.4 % (3.93 vs 3.47 M cal/kg) with chickpea seeds incorporation. DE and TDN value of pigeon pea seeds (3.2 K cal/g DM and 71.1 %) has been reported earlier (Cheva-Isarakul 1992). Significantly higher N-balance in LL100 indicates that LLG contain enough protein and digestible nutrient to promote protein retention for growth and production. These findings are on the line of Egan (1986), who concluded that supplemental protein source, its chemical composition can alter nitrogen conditions in the reticulo-rumen affecting microbial protein synthesis and or/or rate of fiber digestion but also can provide additional amino acids in the small intestine.

 

Growth performance

 

Average daily gain (g/ day) tended to increase (P>0.05) in LL50 (35.5) and LL100 (39.7) with regard to LL0 (33.4) ( Table 6).

Table 6.  Average body weight gain of kids maintained on different experimental rations

Attributes

LL0

LL50

LL100

Pooled SEM

Initial weight, kg

9.85

10.45

10.7

1.59

Final weight, kg

13.3

14.2

14.8

1.60

Total gain, kg

3.52

3.75

4.07

 

Growth rate,  g/d

33.4

35.5

39.7

6.26

Relatively more DE value and higher (P<0.05) N balance in LLG supplemented groups might have lead to increased microbial protein synthesis for relatively higher growth. Feeding Lablab purpureus at the level of 3 or 6 g/ kg live weight has no effect on growth rate, however Lablab purpureus at 12 g/ kg live weight level reduced the growth rate of sheep (Ismartoyo et al 1993). Cheva-Isarakul (1992) observed higher body weight gain in pigeon pea seeds supplemented diets than soyabean meal based dietary group. Growth rate of kids in the present study is lower than expected; it may be due to adverse climatic conditions (High temperature) as evident from the meteorological observations (Table 2). Kids on field bean and common vetch or broad supplemented diets performed (growth rate) equally well with those on soyabean meal (Koumas and Economides 1987). Anderson et al (1989) and Kung et al (1991) observed no significant difference (P>0.05) in growth parameters of cattle and sheep, respectively fed grains of lupine or soybean oil cake as protein supplement. Contrary to it Tracy et al (1988) observed low N retention and low weight gain in animals receiving diet supplemented solely with lupines than with soybean cake only.

 

Rumen fermentation and eating pattern

 

Total volatile fatty acid and total-N concentration was higher (P>0.05) in LL50 (164 and 112) and LL100 (163 and 110) than LL0 (147 mmol/l and 96.2 mg/100 ml. Table 7).

Table 7.  Rumen fermentation pattern of animals on different dietary regimens

Attributes

LL0

LL50

LL100

Pooled SEM

pH

5.86

5.98

5.95

0.10

TVFA, mmol/l

147

164

163

9.87

Total-N, mg/100 ml

96.2

112

110

4.36

Ammonia-N, mg/100 ml

21.7

21.9

22.7

1.91

Higher total-N in LLG-supplemented groups may be associated to higher solubility of LLG than GNC. Higher TVFA production in LLG supplemented groups may also be related with higher roughage intake in these groups.  Increased concentration of TVFA, total-N and NH3-N in sheep due to cowpea seeds supplementation has been reported (Singh et al 2006). Further the NH3-N concentration in the rumen liquor of all dietary groups’ animals was within the levels reported for maximum microbial growth efficiency (15-20 mg/ 100 ml; Boniface et al 1986).

 

Concentrate consumption (%) in initial 30 min of feeding was significantly (P<0.05) higher in LL100 (96.8) and LL50 (88.2) than LL0 (80.1% Table 8). However at 1 h of feeding concentrate consumption was comparable amongst the groups.

Table 8.  Eating pattern of animals offered concentrate mixture of different protein sources

Attributes

LL0

LL50

LL100

Pooled SEM

Concentrate intake % in 30 m

80.1a

88.2b

96.8b

5.17

Concentrate intake % in 1h

95.5

95.1

100

2.65

Meal size (g) in first 2 h

318

310

323

15.0

% meal consumption/2 h

71.5

67.5

72.9

3.92

Total intake 24 h

446

432

447

19.1

ab means in the same row for each parameter with different superscripts are significantly different (p <0.05)

The higher intake of concentrate in LL50 and LL100 group may be associated to easy grinding and mastication of LLG during eating than GNC. Similar results with cowpea grains in sheep have been reported (Singh et al 2006). Meal consumption (%/2h) was 71.5, 67.5 and 72.9 in LL0, LL50 and LL100, respectively. Total intake of diet in 24 h were 446, 432 and 447 g in LL0, LL50 and LL100, respectively. Studies on eating pattern of goats with LLG as protein supplement are very sporadic, however inconsistent dietary intake due to different legume seeds in various species has been reported earlier (Cheva-Isarakul 1991; Baumlin 1986; Singh et al 2006). 

 

Conclusions

 

Acknowledgements 

Authors are thankful to Director, Indian Grassland and Fodder Research Institute (IGFRI), Jhansi for providing necessary facilities to carry this work. Technical assistance of Sh Ram Kishan and Rashid Ahmed is duly acknowledged.

 

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Received 22 November 2009; Accepted 21 February 2010; Published 1 May 2010

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