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Citation of this paper

Disassociate bound protein in tree leaves by trypsin hydrolysis and its effect on feed utilization, microbial protein and weight gain in sheep – an experimental study

B Srinivas, N Swain and N P Singh 

Division of Animal Nutrition, Central Sheep and Wool Research Institute,
Avikanagar-304501, Via. Jaipur, Rajasthan, India

bandla_srinivas@rediffmail.com

Abstract 

Twenty indigenous Malpura breed of sheep of average age 8.6+0.4 mo and body weight (BW) 15.9+0.7 kg were randomly divided into 2 equal groups. Animals were fed Alianthus excelsa tree leaves (200 g) as roughage at 08:00 h. Uneaten leaves removed at 14:00 h and experimental group (EG) fed feed mix consisted trypsin treated tree leaves (200g) and concentrate supplement (CS) (300g) at 14:00 h. Control group (CG) fed feed mix without trypsin treatment.  BW changes recorded at 5 d intervals for 45 d. After 25 d of preliminary period of feeding, a metabolism trial for 7 d was conducted.

 

Non protein nitrogen (NPN) fraction in feed mix increased (P< 0.05) after trypsin treatment. Dry matter intake (P<0.01) and digestibility were higher in EG than CG, yet there was 50% reduction in weight gain. CP digestibility reduced (P<0.05) after trypsin treatment and contrary was true for hemicelluloses (P<0.01) digestibility.  Fecal excretion of nitrogen was higher in EG than CG but vice versa was true in urinary excretion. Excretion of purine derivatives in urine was also 39 % lesser (P<0.05) in EG than CG. MBP absorbed (P<0.05) in CG and EG was 8.42 and 4.51 mM, d-1, respectively. Residual trypsin appears to be still active in rumen environment and might have damaged the MBP production. Microbial N, g kg-1 digestible organic matter intake (DOMI) was 21.48 and 10.36, respectively in CG and EG (P<0.05). Lower efficiency of MBP (g/kg DOMI) in EG was indicative of imbalance in protein and energy compared to CG. N retention was lesser besides inefficiency in the metabolizable N utlization in EG than CG (P< 0.05). Weight gain also affected because of lesser metabolizable N availability per MJ of ME and higher heat increment caused energy inefficiency. 

 

It is concluded that trypsin was effective in improving the solubility of bound protein fraction in tree leaves however, it had adverse affect on rumen microbial synthesis, protein digestibility and induced protein-energy imbalance. Its adverse impact on weight gains were observed after 30 d of feeding and hence, not suitable for its use in ruminants unlike in non-ruminants. Alternative source of proteolytic enzyme to disassociate bound protein in tree leaves without any adverse affect on rumen may need to be explored.

Keywords: derivatives, digestibility, enzyme, feed, microbial N, proteolysis, purine rumen, sheep, weight gain


Introduction

Tree-fodder is important feed resource in semi-arid regions to feed sheep round the year (Singh and Patnayak 1977, Kumawat and Chaudhary 2004). Although protein content of some of the tree leaves is higher than grasses, their degradation is not complete due to binding with cell wall polysaccharides particularly with maturity (Singh and Srinivas 1998, Karabulut et al 2006). Disassociating these bound proteins is necessary to improve protein availability from scanty feed resources in semi-arid regions. Trypsin is commonly used protease for site-specific hydrolysis of peptide fragments in metabolomics to derive bioactive peptides (Daniel 2003, Mota et al 2006). It is a very potent proteolytic enzyme catalyzing the breakdown of peptide bonds of protein involving lysine and/or arginine into smaller oligopeptides in the small intestine of either non-ruminants or ruminants (Alpers 1986). Proteolytic enzymes, used in the diets of non-ruminant animals to improve feed utilization efficiency, have been seldom tried in ruminant diet owing to high proteolytic activity in rumen (Bedford 1993, Kung 1996). Improving the hydrolysis of feed protein by trypsin to polypeptides or oligopeptides or free amino acids can optimize the growth of bacteria (Fadda et al 1999, Russell et al 1992), enhance nutrient uptake, digestibility and bioactivity in animal system (Wu 1998, Pintado et al 1999, Foregeding et al 2002). There are hardly any attempts in the past to disassociate cell wall bound protein by proteolytic enzymes in the feedstuffs of ruminants even though it is one of the alternatives to improve protein intake of sheep in arid and semi-arid regions in general and, particularly during lean periods of feed availability.

 

Looking into the paucity of literature on the adversity of trypsin in ruminants, microbial protein production (MBP) was taken as an appropriate indicator to evaluate its overall effect on the rumen environment. MBP production in rumen has not only quantitative significance to provide quality protein to the animal down the gut but also, an ultimate marker for optimum conditions of nutrient fermentation in rumen. This investigation was conducted with the objectives; 1. To increase the solubility of bound protein in the tree leaves using trypsin as proteolytic enzyme and, 2.  To evaluate exogenous trypsin affect on the MBP production and nutrient utilization in sheep.

 

Materials and methods 

Substrate and trypsin hydrolysis

 

Sun dried Alianthus excelsa leaves were taken as substrate for trypsin hydrolysis. Trypsin from bovine pancreas (1:3000, bovine pancreas, MW 23, 281, M/s Merck India Ltd.) was used for hydrolysis. A stock solution of 50 g of trypsin prepared in 250 ml of 10-4 N HCl (pH 4) and stored in refrigerator at 4oC till further use.  Enzymatic hydrolysis of substrate was performed in 20 L capacity polypropylene buckets in an open shed at 40oC of ambient air temperature. A 50 ml of trypsin stock solution was diluted to 2 L with tap water to make the final concentration of enzyme to 500 mg/100ml.    This was added to 2 Kg of Alianthus excelsa leaves  and kept for 12 h. Contents in the bucket were thoroughly mixed at every 2-3 h intervals.  After 12 h of soaking, 3 kg of concentrate supplement (CS; consisted groundnut cake 25, maize 30, wheat bran 40, mineral mixture 3 and common salt 2 parts) was mixed thoroughly with the tree leaves to adsorb any hydrolyzed protein that was leached into the solution from the leaves. This complete feed mix was again hydrated with another 1 L of water and kept overnight to complete the reaction of any residual enzyme in the substrate. Trypsin treated Feed mix (TFM) was exposed to air for 8 h to reduce moisture content as sheep prefers dry feeds over mash. Simultaneously, a blank diet was also prepared without adding trypsin to the solution.  TFM was prepared every day for 45 d using the trypsin stock solution.

 

Animals and feeding

 

Twenty Malpura breed of indigenous male sheep of average age 8.6 + 0.4 mo and body weight (BW) of 15.9 + 0.7 kg were randomly divided into two equal groups. Animals were dewormed prior to experimentation. Animals in both the groups were fed 200 g of Alianthus excelsa leaves at 08:00 h. Uneaten tree leaves were removed at 14:00 h, followed by feeding either 500 g of TFM (experimental group; EG) or a similar quantity of blank diet  (control group; CG) that is without trypsin treatment. Drinking water was provided to the sheep 4 times in a day in 4-6 h interval. Weight change in sheep was recorded at 5 d intervals for a period of 45 d.

 

Metabolism trial

 

After 25 d of preliminary feeding, a metabolism trial was conducted for 7d. Animals were accommodated in metabolic cages 2 d before the sampling for acclimatization. Urine collection bucket was acidified by adding 10% H2SO4 (v/v) solution. Faecal and urine output was measured at 08:30 h every day. Faecal samples were sub-sampled to 1/50th part in 2 quantities. One part was oven dried at 100 + 5 0C for 24 h. The other part was acidified with 25% H2SO4 (v/v) in a glass bottle and kept for nitrogen (N) estimation. Urine quantity was measured and diluted uniformly to 2 L with tap water. Diluted urine was mixed, filtered through glass wool and 20 ml was stored in 50 ml capacity polypropylene bottles on each day. Urine samples stored at –20 0C to estimate purine derivatives. Another sub-sample of 20 ml of urine was pooled separately for 7 d to estimate Nitrogen (N). Feed offered and orts samples were collected every day, oven dried and pooled for 7 d. At the end of metabolism trial sheep were shifted to individual stalls and, feeding continued for another fortnight.

 

Laboratory analysis

 

DM content was determined by drying samples in a forced draught oven at 100 oC for 24 h (AOAC 1995). Oven dried samples were weighed and ground in a lab mill (M/s Jaico laboratory, India) using 1 mm sieve. Organic matter (OM) content was obtained by difference after ashing the dried sample in a muffle furnace at 600 oC (AOAC 1995).  N in feces and urine was determined by Kjeldahl procedure (CP = 6.25 X N) and ether extract (EE) by petroleum ether (AOAC 1995). Solubility of protein was analysed as suggested by Licitra et al (1996). Non Protein Nitrogen (NPN) was determined as difference between total N and true Protein N (TP; estimated by precipitating protein by trichloroacetic acid, AOAC 1995).  Buffer insoluble protein (PBIP) was analyzed by soaking 5 g of sample in 500 ml of borate-phosphate buffer (pH 6.7) for 4 h at room temperature, filtered and N content in the residue was estimated by standard Kjeldahl method. PBIP values were subtracted from TP to derive true soluble protein (Licitra et al 1996). Cell wall bound N fractions such as NDF insoluble (NDIP) and ADF insoluble (ADIP) protein were analyzed from NDF and ADF residues respectively, by Kjeldahl method (AOAC 1995). NDIP was subtracted from PBIP to derive NDS soluble protein (fraction B2). Difference between NDIP and ADIP was taken as protein insoluble in NDS but soluble in ADS. ADIP was considered as indigestible (Licitra et al 1996, Chalupa and Sniffen 1996). Neutral detergent fiber (NDF) analysis did not use amylase and expressed inclusive of residual ash (Van Soest et al 1991). Acid detergent fiber without adjustment for residual ash (ADF), cellulose and, lignin were analyzed according to methods of AOAC (1995). Hemicelluloses were expressed as difference between NDF and ADF (Van Soest et al 1991). Gross (GE) and digestible energy (DE) was estimated by adiabatic bomb calorimeter (M/s Gallenkamp Bomb Calorimeter, UK). Metabolizable energy (ME), heat increment (HI) and energy efficiency were determined based on the empirical models recommended by ARC (1990).

 

Microbial protein analysis

 

MBP was estimated from the purine derivatives excreted in the urine (Fujihara et al 1987, Chen et al 1995).  Urine samples were stored in refrigerator at 40 C. These samples were then thawed to 250 C and diluted further 10 times to maintain the concentration of purine derivatives within the range of 5-50 mg l-1.  Diluted urine samples were pooled into triplicate for each animal. Allantoin standards were prepared for 10 to 60 mg l-1 with 10 mg increment (nr. A-7878, Sigma, USA). Allantoin concentration was determined by colorimetric method at 522 nm using UV-visible spectrometer (Cintra 10e Double beam, M/s GBC Scientific Equipments, Australia). Xanthine and hypoxanthine were estimated by degrading to uric acid using Xanthine Oxidase (nr. X-1875, Sigma, USA). Standards of uric acid (nr.U-0881, Sigma, USA) were prepared in the concentration 20 to 100 mg/l with 20 mg increment and absorbance read at 293 nm. Uric acid was determined by uricase (M/s Sigma Chemicals, USA) method and standard working concentrations were prepared 5, 10, 20, 30 and 40 mg/l. Allantoin concentration in urine was calculated using linear regression analysis. Uric acid estimations derived from the natural logarithmic function of Ln (X) and Ln (Y). Purines absorption (X) was calculated using following equation on the basis of total purine excreted (Y) in urine. 
 

Y = 0.84X + (0.150 W0.75 e-0.25X)


Calculation of X from Y was performed by means of the Newton-Raphson iteration process as below:

where,

ƒ (x) = 0.84X + 0.150 W0.75 e-0.25X  - Y  and,

the derivative of ƒ’(Xn) = 0.84-0.038 W0.75 e-0.25X .

 

The supply of microbial nitrogen (MN) to the duodenum was calculated as:

MN (g/day) = X(70 x 0.83 x 0.116x1000) = 0.727 X

Where,

the digestibility of microbial purine was 0.83,

N concentration in purine (mg/mmol) was 70 and,

0.116 was the ratio of purine-N : total-N in mixed rumen microbes.

 

Therefore, efficiency of microbial N synthesis used here was expressed as grams of microbial N per kilogram of digestible organic matter (DOM) apparently digested in the rumen (DOMR). DOMR derived by multiplying DOM with a factor of 0.65.

 

Statistical analysis

 

Body weight changes were subjected to repeated measure analysis by taking 9 levels (weeks) for within group. Intake, digestibility, N and energy and ruminal microbial protein synthesis data were analysed by paired sample student t- test that included the standard error between two means. Variations among means with P < 0.05 to 0.001 were accepted as representing tendencies to differences among the groups. These analyses used the standard procedures of statistical package for social sciences (SPSS), V 14.0 for Windows.

 

Results 

Chemical composition of CS, Alianthus excelsa leaves and feed mix is presented in table 1.


Table 1.  Chemical composition of diet (% on DM basis)

Parameter

Concentrate

Tree Leaves

Feed Mix

Control

Experimental

DM

94.6+0.79

97.0+0.14

85.5+0.64

86.0+0.42

OM

91.5+0.28

89.0+0.05

87.9+0.21

87.3+0.19

CP**

17.0+0.11

14.8+0.88

15.6+0.12a

16.9+0.11b

EE

4.1+0.03

1.8+0.04

3.3+0.18

3.1+0.27

TA

8.5+0.03

11.0+0.06

12.1+0.21

12.7+0.19

NDS

71.8+0.95

59.7+5.51

69.7+0.65

70.4+0.21

NDF

28.2+1.39

40.4+5.51

30.3+0.66

29.6+0.21

ADF

19.4+0.06

28.9+4.61

19.6+0.31

20.2+0.50

Hemicelluloses

8.7+0.41

14. 8+3.21

10.7+0.80

9.4+0.46

Cellulose

4.7+0.40

11.5+0.90

8.8+0.12

9.4+0.20

Lignin

4.9+0.06

9.3+0.75

6.9+0.12

7.1+0.07

a bmeans in the same row for each parameter with different superscripts are significantly different **(P< 0.01)


No significant variation was observed in the chemical composition of feed mix (P>0.05) between CG and EG except CP (P<0.01) content. NPN was high (P<0.05) in treated mix than in blank diet (table 2). 


Table 2.  Soluble and insoluble nitrogen fractions of feed (g/kg DM)

Parameter

Group

SEM

P-Value

Control

Experimental

Total Protein**

156a

169b

1.10

0.002

Non Protein Nitrogen*

45.0a

50.2b

1.19

0.048

True Soluble Protein

69.5

70.3

2.35

0.743

Protein Insoluble in NDS

16.1

16.2

0.84

0.972

Protein Insoluble in ADS

14.0

12.7

2.86

0.675

Indigestible Protein

20.5

16.5

3.05

0.283

a bmeans in the same row for each parameter with different superscripts are significantly different   *(P< 0.05), **(P<0.01) ; NDS = Neutral detergent solution, ADS = Acid detergent solution.


Other than NPN, no change in any other soluble and indigestible protein (P> 0.05) was observed. However, both NPN and true soluble protein together were increased while cell wall bound and insoluble protein fractions were decreased after trypsin treatment of feed with probability of significance was 0.13 (P < 0.13) and 0.20 (P < 0.20), respectively.

 

BW changes were not significantly different between both the groups till 30d of feeding (Figure 1).


Figure 1.  Body weight gain pattern of sheep fed on blank diet (CG) or trypsin treated feed mix (EG)


Thereafter, weight gain in EG was significantly (P< 0.05) lower than CG. Sheep in CG gained 1.54 kg higher BW during 45 d compared to EG (table 3).


Table 3.  Dry matter intake and nutrient digestibility

Parameter

Group

SEM

P-Value

Control

Experimental

Initial body weight, kg

16.0

15.8

0.46

0.732

Final body weight, kg*

18.7a

17.2b

0.46

0.020

Average weight gain, g d-1*

60.2a

29.6b

13.1

0.020

Total DM intake, g/d**

526a

556b

6.53

0.006

Total DM intake, g, Kg W0.75

34.4a

39.2b

1.96

0.065

Digestibility of nutrients, %

Dry matter

60.6

62.7

2.34

0.407

Organic matter

65.6

66.1

1.93

0.782

Crude protein*

75.7 a

50.3b

6.85

0.014

Ether extract

54.3

45.9

7.52

0.313

Total ash

24.1

36.0

3.06

0.098

Neutral detergent fiber

51.1

59.4

4.19

0.098

Acid detergent fiber

41.9

45.4

8.11

0.649

Hemicelluloses**

38.5a

43.3b

1.09

0.007

Cellulose

45.1

56.7

4.99

0.068

Energy

63.4

66.9

4.07

0.435

a bmeans in the same row for each parameter with different superscripts are significantly different   *(P< 0.05), **(P<0.01).


Contrary to 2-fold lower growth rate, dry matter intake (DMI) of sheep in EG was significantly higher (P< 0.01) than CG. Digestibility of CP (P< 0.05) and hemicelluloses (P< 0.01) were significantly different between two groups. Digestible CP intake was higher in CG than EG (P< 0.05) but contrary was true for GE intake (P< 0.05).

 

Allantoin, xanthine and hypoxanthine and, uric acid in urine were 41 (P<0.05), 15 (P> 0.05) and 36 per cent (P< 0.01) lesser in EG compared to CG (table 4).


Table 4.  Nutritive value of ration and microbial protein production and nitrogen partition

Parameter

Group

SEM

P-Value

Control

Experimental

Allantoin, mM/d*

4.91a

2.55b

0.71

0.021

Xanthine + Hypoxanthine, mM/d

0.95

0.78

0.06

0.129

Uric Acid, mM/d**

1.38a

0.87b

0.12

0.008

Total purine derivatives (TPD) excreted, mM/d**

7.24 a

4.20b

0.71

0.008

Allantoin, % of TPD)

66.2

60.9

4.7

0.309

Xanthine + Hypoxanthine, % of TPD

13.4a

18.3b

1.6

0.026

Uric Acid, % of TPD

20.4

20.8

3.8

0.923

MBP Absorbed, mM/d*

8.42 a

4.51b

0.96

0.023

Microbial Nitrogen, g/d*

6.36 a

3.28 b

0.87

0.023

Microbial Nitrogen, g/Kg DOMI*

21.5 a

10.4 b

2.75

0.013

Microbial Nitrogen, g/Kg DOMR*

31.9 a

15.9 b

3.24

0.013

a bmeans in the same row for each parameter with different superscripts are significantly different   *(P< 0.05), **(P<0.01), ***(P< 0.001)


Total purine derivatives excreted in the urine of EG were 39 per cent lesser than CG. Contrary to expectation, TFM had negative affect on the MBP absorbed (P< 0.05) or microbial N flow to intestine (P<0.05). Efficiency of microbial N synthesis in terms of DOMI or DOMR (P<v0.05) also decreased due to trypsin treatment of feed mix.

 

N absorbed from the gut was 2.51+0.91 g/d higher in CG than EG (P<0.05). On contrary, urinary N excretion was 0.65+0.29 g/d (P<0.05) lesser in EG compared to CG (Table 5).


Table 5.  Nitrogen and Energy partitioning

Parameter

Group

SEM

P-Value

Reference

Experimental

Nitrogen (N) Partition

Total  N Intake, g/day ***

13.0 a

14.7 b

1.48

0.001

Faecal Output, g/day**

3.16 a

7.33b

1.03

0.010

Digested N, g/day*

9.88a

7.37b

0.91

0.040

Urine Output, g/day*

4.19a

3.53b

0.29

0.046

Metabolizable N, g/day*

5.70a

3.84b

0.82

0.042

Energy (E) Partition

Gross Energy, MJ/day*

8.10a

8.48b

0.10

0.012

Digestible Energy, MJ/day

5.13

5.65

0.28

0.125

Faecal & Fermentative heat losses, MJ

2.97

2.83

0.36

0.719

Metabolizable Energy, MJ/day

4.21

4.68

0.25

0.116

Metb-N : Metb-E ratio*

1.35a

0.81b

0.15

0.018

Heat Increment, MJ/day*

3.23a

3.52b

0.10

0.033

Energy Retention, MJ/day

0.97

1.16

0.16

0.292

Energy Efficiency %

22.4

24.8

2.74

0.426

a bmeans in the same row for each parameter with different superscripts are significantly different   *(P< 0.05), **(P<0.01), ***(P< 0.001)


However, the overall metabolizable N was lower in EG than CG (P<0.05). There was no difference in DE or ME between both the groups. The q-value of the diet (ME/GE) was 0.82 and 0.83 in CG and EG respectively. ME available for each gram of metabolizable N (MN) was 0.75 and 1.48 MJ in CG and EG, respectively. Although the ratio between MN and ME was narrow on EG (P< 0.05), heat increment was 9 per cent more (P< 0.05) in EG than CG.

 

Discussion

Alianthus excelsa tree leaves are one of the widely used and good protein sources among top feeds in semi -arid regions (Kumawat and Chaudhary 2004). These tree leaves are important component of the diet of small ruminants during November to July in the environmentally challenged, underdeveloped semi-arid regions. Apart from shortage of grazing resource in these regions, decrease in the availability of protein from top feeds as a result of binding to cell wall polysaccharides with maturity may further affect the productivity of sheep. Like many other tree leaves, CP content in Alianthus excelsa leaves were comparable with CS. However, even with higher CP content in leaves, earlier studies reported negative N balance (Aganga and Tshwenyane 2003, Salem et al 2005). This was mainly due to cell wall bound fraction that was around 1/3 of the total protein in Alianthus excelsa. Normally cell wall bound fraction may range between 9 to 43% of total  CP content of tree leaves used for livestock feeding in India (Bhadauria et al 2002). 

 

In order to improve the protein solubility in tree leaves, trypsin has been selected owing to two reasons; 1. Formation of peptides by trypsin hydrolysis has been reported to be more extensive than other proteolytic enzymes (Pintado et al 1999) which may effectively hydrolyze at the least 8% of all peptide bonds present (Pintado et al 1999, Kananen et al 2000) and, 2. Trypsin has been used effectively without any adverse affects in poultry feeds and in some dairy whey products consumed by human beings.  Increase in NPN fraction in TFM was due to improvement in the solubility of protein.  Such improvements have been observed in the cheese whey proteins also with the application of trypsin (Pouliot et al 1997, Kim et al 2004). However, as per the objective of the investigation, it is important that the increased solubility of protein should come from the bound fraction of CP rather than other cellular fractions whose availability has been reported to 92% (Nag and Matai 1991). Trypsin treatment decreased bound fractions of protein such as protein soluble in NDS and ADS and, indigestible fractions though the level of significance was 20%. Such variation within the leaves is also possible as maturity may vary within the leaves or even between plant parts. This however, was indicative of possibility in disassociation of bound protein in the tree leaves by trypsin.

 

Feeding tree leaves along with CS increased their acceptability by sheep and decreased the wastage.  Reducing the wastage of diet is also necessary in semi-arid regions for careful use of limited feed resources. Contrary to expectation, CP digestibility was reduced after trypsin treatment but hemicelluloses digestibility was enhanced. Partial hydrolysis of cell wall bound protein in TFM improved the availability of hemicelluloses which may also presumably have influence on disassociation of bound protein.  However, decrease in CP digestibility may probably due to HCl used in the stock solution of the enzyme that might have reduced the potential degradability of CP in the rumen besides the reduced MBP production. Higher DMI in EG with 50% lower average daily gains were also indicative of restriction in energy utilization as a result of low metabolizable N which is actually available for synthesis of organic body constituents and secretions. (Kebreab et al 2002).

 

Relative proportion of allantoin or uric acid excreted in both the groups was with in the normal range. TFM damaged the MBP production even after precautions were taken to dilute residual activity by adding water to feed mix (Beauchemin et al 1995, Feng et al 1996). Different schools of opinion prevail about the application of exogenous enzymes owing to strong proteolytic activity in the rumen. Many workers opined that the exogenous proteolytic enzymes might not be stable and any excess may be inactivated in the normal rumen pH range of 6.2 to 6.8 (Chesson 1994, Feng et al 1996, Kung 1998). Contrary to it, exogenous trypsin in the feed mix was found to be active in the rumen environment and damaged the production of  rumen MBP. MBP production in rumen would represent both protein and energy supply and utilization in rumen. Decrease in microbial N yield due to trypsin treatment of feed mix was indicative of a poor rumen fermentation and imbalance in the DOM and N in the diet.  Though the DOMI was apparently higher in EG, gram microbial-N synthesized per Kg of DOMI was 54 % lesser (P<.05) than CG. The functional inefficiency of MBP production per kg of DOMI in EG compared to CG was thus, indicative of inefficient utilization of CP in the diet (Tebot et al 2004). Trypsin treatment induced imbalance in the ratio between available N and DOM in rumen. There was no significant difference in the fermentative and fecal heat losses as well as ME between CG or EG.  Higher heat increment however, observed on EG was an indication of increased energy use during metabolism by the animal. Additional heat increment from diet may also be a disadvantage in semi-arid regions owing to high air temperature. Energy losses were therefore, because of more heat increment during post gut absorption than fecal or fermentative energy losses on TFM. This enumerated that the distortion in nutrient utilization occurred with application of trypsin mainly due to imbalance in the absorbed protein and energy across the gut (Blaxter 1989).

 

Conclusions

 

Acknowledgement 

Authors are thankful to the Director, Central Sheep and Wool Research Institute for financial support and extending the facilities.

 

References 

Aganga A A and Tshwenyane S O 2003 Feeding values and anti-nutritive factors of forage tree legumes. Pakistan Journal of Nutrition 2: 170-177 http://www.pjbs.org/pjnonline/fin103.pdf

 

Alpers D H 1986 Uptake and fate of absorbed amino acids and peptides in the mammalian intestine. Federation Proceedings 45: 2261-2267

 

A O A C 1995 Official methods of analysis. Association of Official Analytical Chemists. 16th Edition. Washington, DC.

 

A R C 1990 The nutrient requirement of ruminant livestock.  Commonwealth Agricultural Bureaux. Farnham Royal, England.

 

Beauchemin K A, Rode L M and Sewalt  V J H 1995 Fibrolytic enzymes increase fiber digestibility and growth rate of steers fed dry forages. Canadian Journal of Animal Science 75: 641-644

 

Bedford M R 1993 Mode of action of feed enzymes. Journal of Applied Poultry Research 2: 86-92

 

Bhadauria  K K S, Pailanbhadauri G H, Das M M, Kundu S S,  Singh J P and Lodhibhadauri G N 2002 Evaluation of shrubs and tree leaves for carbohydrate and nitrogen fractions. Indian Journal of Animal Science 72: 87-90

 

Blaxter K L 1989 Energy metabolism in animals and man. P 129, Cambridge University Press. Cambridge, UK.

 

Chalupa W and Sniffen C J 1996 Protein and amino acid nutrition of lactating dairy cattle-today and tomorrow. Animal Feed Science and Technology 58: 65-75

 

Chen X B, Mefia A T,  Kyle D J and Orskov E R 1995 Evaluation of the use of the purine derivative: Creatinine ratio in spot urine and plasma samples as an index of microbial protein supply in ruminants: study in sheep. Journal of Agriculture Science Cambridge 125: 137-143

 

Chesson A 1994 Manipulation of fibre degradation: an old theme revisited. In: Lyons T P, Jacques K A. (eds.) Biotechnology in the feed industry. pp. 83-98. Proceedings  Alltech 10th Annual Symposium, Nottingham University Press, Loghborough, UK.

 

Daniel H 2003 Perspectives in post-genomic nutrition research. In: Zampelni J,  Daniel H. (eds) Molecular Nutrition, CABI publishing, CAB international, Wallingford, Oxon OX10 8DE, UK.

 

Fadda S, Sanz Y, Vignolo G, Concepción Aristoy M, Oliver G and Toldra F 1999 Characterization of muscle sarcoplasmic and myofibrillar protein hydrolysis caused by Lactobacillus plantarum. Applied Environment Microbiology  65: 3540-3546

 

Feng  P, Hunt C W, Pritchard G T and Julien W  E 1996 Effect of enzyme preparations on in situ and in vitro degradation and in vivo  digestive characteristics of mature cool-season grass forage in beef steers. Journal of Animal Science 74: 1349-1357 http://jas.fass.org/cgi/reprint/74/6/1349

 

Foregeding E A, Davis J P, Doucet D and Mc Guffey K 2002 Advances in modifying and understanding whey protein functionality. Trends in Food Science and Technology 13: 151-159

 

Fujihara T, Orskov E R, Reeds P J and Kyle D J 1987 The effect of protein infusion on urinary excretion of purine derivatives in ruminants nourished by intragastric nutrition. Journal of Agricultural Science Cambridge 109: 7-12

 

Kananen A, Savolainen J, Makinen J, Perttila U, Myllykoski L and Pihlanto-Leppala A 2000 Influence of chemical modification of whey protein conformation on hydrolysis with pepsin and trypsin. International Dairy Journal 10: 691-697

 

Karabulut A, Canbolat O, Ozkan CO and Kamalak A 2006 Potential nutritive value of some Mediterranean shrub and tree leaves as emergency food for sheep in winter. Livestock Research for Rural Development 18, Article #81 http://www.lrrd.org/lrrd18/6/kara18081.htm

 

Kebreab E, France J, Mills J A and Dijkstra J 2002 Dynamic model of N metabolism in the lactating dairy cow and an assessment of impact of N excretion on the environment. Journal of Animal Science 80: 1248-1259  http://jas.fass.org/cgi/reprint/80/1/248

 

Kim S B, Shin H S and Lim J W 2004 Separation of calcium-binding protein derived from enzymatic hydrolysates of cheese whey protein.  Asian-Australasian Journal of Animal Science 17: 712-718

 

Kumawat J R and Chaudhary J L 2004 Comparative intake and nutrient digestibility of Ardu (Alianthus excelsa) leaves in sheep and goats. Indian Journal of Small Ruminant 10: 71-73

 

Kung L Jr 1996 Direct fed microbial and enzyme feed additives In: Direct fed microbial, enzymes and forage additive compendium (Editor: S Murihead)., pp. 15-20 The Miller publishing company, Minetoenka, Minnesota.

 

Kung  L Jr 1998 Direct-fed microbial and enzyme feed additives, In: Direct-fed microbial, enzyme and forage additives compendium (Editor: S Murihead). The Miller publishing company, Minnetonka, Minnesota, pp. 15-20

 

Licitra G, Hernandez T M and Vansoest P J 1996 Standardization of procedures for nitrogen fraction of ruminant feeds. Animal Feed Science and Technology 57: 347-358

 

Mota M V T, Ferriera I M P L V O, Oliveira M B P, Rocha C, Teixeira J A, Torres D and Goncalves M P 2006 Trypsin hydrolysis of whey protein concentrates: Characterization using multivariate analysis. Food Chemistry 94: 278-286

 

Nag A and Matai S 1991 Ailanthus excelsa Roxb. (Simaroubacae): a promising source of leaf protein. Indian Journal of Nutrition and Dietetics  29: 136-139

 

Pintado M E, Pintado A E and Malcata F X 1999 Controlled whey protein hydrolysis using two alternative proteases. Journal of Food Engineering 42: 1-13

 

Pouliot M, Paquin P, Martel R, Gauthier S F and Pouliot Y 1997 Whey changes during processing determined by near infrared spectroscopy. Journal of Food Science 62: 475-479

 

Russell J B, O’Connor J D, Fox D G, Van Soest P J and Sniffen C J 1992 A net carbohydrate and protein system for evaluating cattle diets. I. Ruminal fermentation. Journal of Animal Science 70: 3551-3561 http://jas.fass.org/cgi/reprint/70/11/3551.pdf

 

Salem H Ben, Nefzaqoui A, Makkar H P S, Hochlef H, Salem I Ben and Salem L Ben 2005 Effect of early experience and adaptation period on voluntary intake, digestion and growth in barbarine lambs given tannin-containing (Acacia cyanophylla Lindl. foliage) or tannin-free (oaten hay) diets. Animal Feed Science and Technology 122: 59-77

 

Singh K K and Srinivas B 1998 Ruminal digestion kinetics of range grasses and legumes in crossbred cattle. Indian Journal of Animal Nutrition 15: 305-307

 

Singh N P and Patnayak B C 1977 Nutritive value of Nutritive value of Ailanthus excelsa Roxb. (Ardu) leaves for sheep. Indian Veterinary Journal 54: 198-201

 

Tebot I, Ibarra A L, Purtscher F and Cirio A 2004 Influence of energy supply on microbial protein synthesis and renal urea handling in Corriedale sheep. Journal of Animal Feed Science 13:  223-226

 

Van Soest P J, Robertson J B and Lewis B A 1991 Methods for dietary fiber, neutral detergent fiber, and non starch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74: 3583-3597 http://jds.fass.org/cgi/reprint/74/10/3583.pdf

 

Wu G 1998 Intestinal amino acid catabolism. Journal of Nutrition 28: 1249-1252 http://jn.nutrition.org/cgi/reprint/128/8/1249



Received 7 May 2008; Accepted 2 September 2008; Published 5 December 2008

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