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Effect of lignosulphonate treatment of groundnut and mustard cake on ruminal protein degradability in cattle

G Mondal, T K Walli and A K Patra*

Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal, Haryana 32001, India
goutam_mondal@rediffmail.com
* Department of Animal Nutrition, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, Kshudiram Bose Sarani, Belgachia, Kolkata 700037, India
patra_amlan@yahoo.com

Abstract

The objectives of the study were to determine the effect of lignosulphonate (LSO3) treatment of groundnut (GNC) and mustard cake (MC) on in sacco ruminal degradability and in vitro post-ruminal digestibility. The samples of GNC and MC were treated with LSO3 at 0, 5, 6 and 7 percent (w/w) levels. Three fistulated animals were used to conduct the in sacco experiment and the nylon bags were incubated for 3, 6, 12, 24, 36 and 48 h to estimate dry matter (DM) and crude protein (CP) degradability. Residues after 24 h of incubation in the rumen were taken for determination of in vitro protein digestibility by pepsin and pancreatin method.

 

The LSO3 treatment decreased phosphate buffer soluble nitrogen and increased acid detergent insoluble nitrogen of GNC and MC. For both the cakes, B2 fraction increased whereas in case of GNC only B3 increased. The effective DM degradability (EDMD) of GNC decreased (P<0.05) from 73.8 to 60.6 percent at 5 percent LSO3 treatment. No statistical difference (P>0.05) was observed at 5, 6 and 7 percent levels. However, for mustard cake EDMD decreased at 7 percent level, not at 5 or 6 percent levels. The effective protein degradability (EPD) of GNC decreased (P<0.05) from 74.9 at 0 percent level to 56.0 percent at 5 percent level, but 5, 6 and 7 percent levels were not significant (P>0.05). The EPD of MC decreased (P<0.05) from 74.1 at 0 percent level to 60.6 percent at 7 percent level, but 0, 5 and 6 percent levels were not significant (P>0.05). As a result, at 5 and 7 percent levels of LSO3 treatment undegradable protein content in GNC and MC increased by 19 and 13 percent, respectively. In vitro post ruminal digestibility indicated that for GNC, 5 and 6 percent level of treatment did not affect the digestibility, but at 7 percent level digestibility decreased. The post ruminal digestibility of rumen undegradable protein from MC was not affected by LSO3 treatment.

 

From this study it can be concluded that GNC and MC can be treated with LSO3 at 5 and 7 percent levels, respectively, to reduce the ruminal CP degradability without affecting the post ruminal protein digestibility.

Key words: nitrogen fractions, post-ruminal digestibility, rumen undegradable protein


Introduction

Groundnut cake (GNC) and mustard cake (MC) are two most commonly used protein sources in the diet of ruminants in India. However, their degradability in rumen is very high (Chatterjee and Walli 2003; Mondal et al 2008), resulting in the depreciation of protein value for ruminants. Different methods have been evolved to reduce the rumen degradability, which in turn increase outflow and balance of amino acids to intestine, such as heat treatment (Mustafa et al 1998; Petit et al 1999), tannic acid treatment (Bhagwat and Srivastata 1991), formaldehyde treatment (Gupta and Walli 1987; McAllister et al 1992; Kumar and Walli 1994; Chatterjee and Walli 2003) and coating of protein by fat (Dhiman et al 2001; Monterola et al 2001). 

 

The addition of lignosulfonate (LSO3) to canola screenings in an in situ study showed that LSO3 treatment was effective in reducing ruminal degradation of DM and CP with a corresponding increase in disappearance of DM and CP from the lower gastrointestinal tract (von Keyserlingk et al 2000). It was shown in situ that heat treatment and the addition of LSO3 can reduce the proportion of rumen degradable protein (RDP), thereby increasing the availability of essential amino acids to the mammary gland for milk synthesis (Wright et al 2005). The combination of heat treatment and LSO3 treatment has been effective in reducing the ruminal degradability of canola meal without affecting its digestibility (McAllister et al 1993; Stanford et al 1995). The treatment of soybean meal with LSO3 decreased protein degradability of soybean meal in vivo (Windschitl and Stern 1988a) and in vitro (Windschitl and Stern 1988b). LSO3 treatment of soybean meal decreased ruminal protein degradability and increased the ruminal undegradable CP concentration of full-fat soybeans by up to 173 percent (Petit et al 1999). Moreover, diets supplemented with LSO3-treated soybean (Nakamura et al 1992) and canola meal (Wright et al 2005) increased milk production than diets supplemented with untreated soybean meal (Nakamura et al 1992). However, information related to LSO3 treatment of GNC and MC on protein degradability is scanty.

 

Therefore, keeping positive effects of LSO3 on production response, an investigation was carried out to study the effects of LSO3 treatment of GNC and MC on in sacco rumen degradability and in vitro digestibility of protein in cattle.

 

Materials and methods 

LSO3 treatment of GNC and MC

 

The samples of GNC and MC were procured from the local markets of Karnal, Haryana, India, and were ground to pass 2.5 mm sieve size. They were treated with calcium lignosulphonate (National Chemicals, India, Code No. 19660) at the rate of 0, 5, 6 and 7 percent on fresh basis (91.5% DM) with the addition of 10 percent water (weight basis of fresh samples) and mixed well. Then the treated samples were heated to 95C for 2 h in a hot air oven (Wright et al 2005). The samples of GNC and MC without lignosulfonate treatment (0 percent samples) were also processsed with water and heat in the same manner along with lignosulphonate treated samples. At the end of the treatment period the samples were dried, ground and stored for the study.

 

In sacco study

 

In sacco study was conducted on three mature fistulated male crossbred cattle fed on a diet consisting of concentrate and roughage at the ratio of 35:65 for maintenance requirements. Pre-weighed nylon bags containing 5 g of milled samples (triplicate) were placed inside the rumen of each animal. The samples were taken out at 3, 6, 12, 36, and 48 h interval and thoroughly washed, and the bags were dried in an oven first at low temperature (60C) for 8 h and then at higher temperature (90C) for 12 h to determine DM disappearance in the rumen. Dry and protein disappearance data were fitted to the model of Orskov and McDonald (1979) to estimate the kinetics of degradability of DM and CP such as a, b and c. 
 

Y= a + b(1 - exp-ct)

where,

a = rapidly degradable fraction,
b = insoluble but potentially degradable fraction,
c = rate of degradation

The effective degradability of DM (ED) and CP was calculated using the equation shown below, using rumen fractional outflow rates (r) of 0.05 h-1
 

ED = a + [(b c)/(c + r)]

 

In vitro post ruminal digestibility

 

In vitro post-ruminal digestibility of protein was estimated largely following the procedure of Antoniewicz et al (1992) and modified by Chatterjee and Wali (2000). Briefly, the pooled samples of undegraded nylon bag residues (0.5 g) after 24 h rumen incubation and drying at 65 C for at least 12 h were taken in a vial containing 20 ml of 0.075 M HCI-pepsin (2 g/l, Sigma, USA, P-7000) solution and incubated at 39C for 2 h. The contents were filtered through polyamide cloth having pore size of 40 m and washed with distilled water. The deposit on the filter was quantitatively transferred back into the vessel using 0.1 M phosphate buffer (pH 7.4) and incubated with 20 ml of pancreatin (Sigma, USA, P-1750) solution in the same buffer (final concentration was 6 g/l) at 39C for 2 h. Additional 20 ml of buffer (39C) was added to the incubation mixture and left overnight at 39C. The solid residues were filtered through Whatman filter paper No. 42 and washed 5 times with luke warm distilled water. The residue along with filter paper was subjected to N determination by Kjeldahl’s method as per AOAC (1990). The post-ruminal digestibility of undergraded protein residues was as follows:

X = (Y – Z)/Y 100

where,

X = post-ruminal digestibility (%) of UDP,
Y = rumen undegradable protein amount (in g) (in nylon bag residue), and
Z = pepsin-pancreatin indigestible protein amount (in g; protein residue left on filter paper).

 

Chemical and statistical analyses

 

Dry matter contents of the samples were determined by drying in a forced air oven at 100C for 24 h (AOAC 1990). CP content of treated and untreated GNC and MC, and residues after ruminal incubation and pepsin-pancreatin treatment was estimated by Kjeldahl’s method (AOAC 1990). Fractionation of protein was done as per Chalupa and Sniffen (1996). The samples of GNC and MC were treated with phosphate buffer, neutral detergent and acid detergent solution to get fraction of phosphate buffer insoluble N (PBIN), neutral detergent insoluble N (NDIN) and acid detergent insoluble N (ADIN), respectively. Accordingly, different protein fractions were calculated:

(A + B1):  Corresponds to non-protein nitrogen and rapidly degradable true proteins (all globulin and some albumin). This fraction is soluble in phosphate buffer (PBSN).

B2:  (PBIN - NDIN) is rest of albumin and all glutelins. This true protein has intermediate degradation rate.

B3: (NDIN - ADIN) is prolamins, extension proteins and denatured protein, and is slowly degradable.

C: It is derived from acid detergent insoluble nitrogen (ADIN). This fraction corresponds to Maillard products and N bound to lignin.

Data were subjected to one way analysis of variance using SPSS (1996) to find out the differences among levels within feed.

 

Results and discussion 

Effect of LSO3 treatment on GNC

 

The N fractions of different feeds are shown in Table 1.


Table 1.  Effect of lignosulphonate treatment of groundnut (GNC) and mustard cake (MC) on protein fractions (percent of total N)

Feed

Level*

PBSN

NDIN

ADIN

B2

B3

GNC

0

74.2a

20.8c

5.17c

5.35b

15.3c

 

5

54.9b

38.9b

8.92b

6.25ab

30.0b

 

6

47.5c

42.6b

9.31b

8.95a

33.3ab

 

7

45.1c

48.5a

12.3a

6.48ab

36.2a

 

SEM

1.58

1.22

0.78

0.96

1.55

MC

0

76.2a

13.2c

3.23b

10.6d

9.96

 

5

68.0b

15.0bc

7.11a

17.0c

7.85

 

6

59.6c

17.7b

8.24a

22.7b

9.49

 

7

43.2d

19.2a

8.42a

37.6a

10.8

 

SEM

1.98

1.39

1.16

1.35

0.93

Means followed by different letters in the same column within feed differ significantly (P<0.05).

*GNC and MC were treated with lignosulphonate at 0, 5, 6 and 7 percent levels.

PBSN, phosphate buffer soluble N (A + B1 fraction); NDIN, neutral detergent insoluble N; ADIN, acid detergent insoluble N (C  fraction); PBIN, phosphate buffer insoluble nitrogen; B2 fraction = (PBIN - NDIN); B3 fraction = (NDIN - ADIN)

SEM, standard error of mean


In case of GNC, LSO3 treatment decreased the content of phosphate buffer soluble nitrogen (PBSN), but increased the content of NDIN, ADIN, B2 and B3 fractions. The effect of LSO3 treatment on digestion kinetics of DM is presented in Table 2.


Table 2.  Effect of lignosulphonate treatment of groundnut (GNC) and mustard cake (MC) on digestion kinetics of dry matter in cattle

Feed

Level*

a, %

b, %

c, /h

EDMD, %

GNC

0

46.4a

42.63

0.091a

73.8a

 

5

41.8b

55.7a

0.026c

60.6b

 

6

40.1bc

58.1a

0.027c

60.4b

 

7

37.40

37.8c

0.050b

56.3b

 

SEM

1.37

0.86

0.008

2.12

MC

0

68.4a

31.6a

0.033c

81.0a

 

5

65.9ab

22.5b

0.095b

80.6a

 

6

59.2bc

34.9a

0.149a

79.8a

 

7

51.8bc

32.2a

0.044c

70.3b

 

SEM

2.60

2.61

0.0047

1.85

Means followed by different letters in the same column within feed differ significantly (P<0.05).

*GNC and MC were treated with lignosulphonate at 0, 5, 6 and 7 percent levels.

EDMD, effective DM degradability; SEM, standard error of mean


The LSO3 treatment of GNC decreased the ‘a’ value of DM with increasing levels of LSO3. Up to 6 percent level, there was an increase in ‘b’ value of DM for GNC and thereafter a considerable decrease in ‘b’ value of DM for GNC at 7 percent level of LSO3 treatment was noted. The potential degradability (a + b) of DM was highest at 5 and 6 percent levels of LSO3 treatment (97.49 and 98.11, respectively), which decreased considerably (P<0.05) at 7 percent (75.16). It indicates that the potential degradability of DM may increase at 5 and 6 percent levels of LSO3 treatment. This result suggests that greater levels of LSO3 might affect the degradability of nutrients other than protein of GNC. Windschitl and Stern (1988a) and Wright et al (2005) also noted a significant decrease in ruminal disappearance of DM of 5 percent LSO3 treated soybean or canola meal, respectively. In their experiments, total tract digestibility of DM was not affected by LSO3 treatment for canola meal (Wright et al 2005), but total OM digestibility decreased due to LSO3 treatment for soybean meal (Windschitl and  Stern 1988a). The rate of degradation (‘c’ value) was highest in untreated GNC, followed by 7 percent LSO3 treated GNC, whereas in 5 and 6 percent levels, ‘c’ values were 0.0261 and 0.0275/h, respectively. The effective degradability of DM (EDMD) decreased (P<0.05) due to LSO3 treatments and the value was 13.2 percentage lower at 5 percent level of application compared with untreated GNC, which did not further decrease statistically beyond 5 percent levels.

 

The results obtained for digestion kinetics of crude protein, rate of degradation and effective protein degradability (EPD) has been shown in Table 3.


Table 3.  Effect of lignosulphonate treatment of groundnut (GNC) and mustard cake (MC) on digestion kinetics of crude protein in cattle

Feed

Level*

a, %

b, %

c, /h

EPD, %

GNC

0

27.0c

70.4a

0.107a

74.9a

 

5

31.4b

54.0b

0.042c

56.0b

 

6

36.5a

45.2c

0.044c

57.7b

 

7

30.2bc

46.8bc

0.059b

55.4b

 

SEM

1.16

2.24

0.002

0.80

MC

0

46.7a

53.0

0.060b

74.1a

 

5

44.6ab

54.0

0.045b

70.2ab

 

6

41.0b

54.5

0.048ab

67.6ab

 

7

28.5c

52.4

0.079a

60.6b

 

SEM

1.29

1.49

0.011

3.28

Means followed by different letters in the same column within feed differ significantly (P<0.05)

*GNC and MC were treated with lignosulphonate at 0, 5, 6 and 7 percent levels

EPD, effective CP degradability; SEM, standard error of mean


In case of GNC, ‘a’ value of CP was lowest in untreated GNC (26.98) and highest (36.53) in 6 percent LSO3 treated GNC. The ‘b’ value of CP was highest in untreated GNC, and lowest (45.23) at 6 percent LSO3 treated GNC with no difference between 6 and 7 percent level of LSO3 treatment. The potential degradability (a + b) of this nutrient was highest in untreated GNC and decreased linearly with the increasing levels of LSO3 treatment. The ‘c’ value of CP was highest (0.107/h) in untreated GNC and lowest in 5 and 6 percent LSO3 treated GNC. The EPD of GNC decreased (P<0.05) at 5 percent LSO3 treatment by 18.9 percentage unit, but no further decrease in EPD was evident beyond 5 percent levels. Stanford et al (1995) reported that LSO3 treatment decreased EPD significantly in soybean cake which resembles with the present study. Similar observation also reported by Petit et al (1999) in an in vitro experiment with full fat soybean meal. The RDP content was highest in untreated GNC, which decreased (P<0.05) at 5 percent level of LSO3 treatment on GNC. However, no significant differences (p>0.05) were noted among different levels of LSO3 treatment. The results of the LSO3 treated MC and GNC on the post-ruminal digestibility have been given in Table 4.


Table 4.  Effect of lignosulphonate treatment of groundnut (GNC) and mustard cake (MC) on concentration (percent of DM basis) of rumen degradable (RDP), undegradable (UDP) protein and digestible UDP, and post ruminal digestibility (percent) of UDP

Feed

Level

CP

RDP

UDP

Post ruminal digestibility

Digestible UDP

GNC

0

43.2

32.4a

10.8b

88.3a

9.55b

 

5

43.1

24.1b

18.9a

89.32

16.9a

 

6

43.1

24.9b

18.2a

83.5ab

15.2a

 

7

43.4

24.0b

19.4a

80.6b

15.6a

 

SEM

2.68

1.68

1.13

1.88

1.22

MC

0

33.2

24.5a

8.69b

85.7

7.43b

 

5

33.5

23.5ab

9.97ab

85.4

8.51ab

 

6

33.2

22.4ab

10.8ab

86.7

9.36ab

 

7

33.2

20.1b

13.1a

83.6

10.9a

 

SEM

2.05

1.32

1.48

1.90

1.27

Means followed by different letters in the same column within feed differ significantly (P<0.05)

*GNC and MC were treated with lignosulphonate at 0, 5, 6 and 7 percent levels

SEM, standard error of mean


The post ruminal digestibility of GNC decreased (P<0.05) at a 7 percent level of LSO3 treatment, but remained statistically similar (P>0.05) at 0, 5 and 6 percent levels of LSO3 application. The LSO3 which contains xylose may react with some amino group of protein at higher levels, therefore, affecting digestibility of protein. Mansfield and Stern (1994) also reported that LSO3 treatment of soybean decreased ruminal degradability of protein by 15 percent in lactating dairy cattle.

 

Effect of LSO3 treatment on MC

 

Similar to GNC, the LSO3 treatment of MC decreased the content of PBSN, but increased the content of NDIN, ADIN, B2 and B3 fractions. The response of LSO3 treatment of MC on digestion kinetics of DM revealed that ‘a’ value decreased (P<0.05) with increasing levels of LSO3 treatment. However, ‘b’ value of DM in MC was lowest at 5 percent level with no significant differences among 0, 6 and 7 percent levels of treatments. The potential degradability decreased with the LSO3 treatment up to 7 percent level. Stanford et al (1995) reported that the LSO3 treatment increased the acid detergent and neutral detergent insoluble N and thereby decreased the soluble DM of soybean cake and canola cake, but the potentially digestible fractions remained unaffected. The ‘c’ value of DM in this cake was highest at 6 percent level (0.1448/h), followed by 5 percent (0.149/h), 7 percent (0.044/h) and lowest in untreated MC (0.033/h).

 

Regarding CP degradability of MC, there was a decrease in ‘a’ value with the increasing levels of LSO3.  There was no effect (P>0.05) on ‘b’ value of MC protein, but potential degradability (a + b) decreased at 7 percent level of LSO3 treatment. The ‘c’ value was higher in 7 percent LSO3 treated MC protein (0.079) and lowest in 5 percent LSO3 treated MC protein. The increased ‘c’ value of MC protein at 7 percent level compared with 6 percent level might has been due to the substantial decrease in solubility of MC protein (as indicated by PBSN and also ‘a’ value). However, this insoluble MC protein was probably degraded by rumen micobiota at increased rate. The LSO3 treatment of MC decreased the EPD value from 74.1 to 60.6 percent, at 7 percent level. Windschitl and Stern (1988a) also found a decrease in EPD due to LSO3 treatment of soybean protein. McAllister et al (1993) has reported the similar observations for canola cake after subjecting it to LSO3 treatment. The undegradable protein content of MC increased linearly with increasing LSO3 treatment. However, there was no difference (P>0.05) in post-ruminal CP digestibility between untreated and LSO3 treated MC up to 7 percent application. Therefore, the digestible undegradable CP content of MC increased linearly with increasing levels of LSO3 treatment. A decreased OM digestibility in continuous culture fermenters (Windschitl and  Stern 1988b) or total tract OM digestibility in dairy cattle (Windschitl and  Stern 1988a) has been reported when 5 percent LSO3 was treated to soybean cake, but Stanford et al (1995) reported a non-significant effect on DM and OM digestibility in lambs. Petit et al (1999) reported that in vitro DM digestibility depends on time of LSO3 treatment but not with temperature above 30oC up to 130oC. Calsamiglia et al (1995) reported that LSO3 treatment did not affect DM intake, ADG or feed efficiency in cattle. Similar observations were also noticed by Beauchemin et al (1995) in beef calves. Stanford et al (1995) also reported that canola cake and soybean cake treated with LSO3 decreased degradability, and N solubility but the treatment did not have any positive effect on protein digestibility in the lower tract and there was no effect on lamb performance. Conversely, McAllister et al (1992) reported that LSO3 treated canola cake and soybean cake improved the lamb performance. The variations in the results could be due to the type of cake used, the type of animal used for the experiment and heating time, which modifie the amino acid residues of protein through reaction with other compound or through cross linking.

 

Effect of LSO3 treatment on GNC versus MC

 

It has been noted that the B2 fraction of MC increased whereas B3 fraction of GNC increased considerably due to LSO3 treatment. The GNC and MC have different amino acid profile. As for example, GNC is deficient in essential amino acids such as lysine, methionine and cysteine whereas MC protein is rich in methionine and lysine. This difference in amino acid profile between the cakes might have responded differently on N fractions due to LSO3 treatment. In case of MC, the DM degradation rate increased with the treatment except the 7 percent treatment. The effective degradability of DM remained also unaffected till 6 percent level. This is in contrast to the GNC where the degradation rate and the ED of DM both declined with treatment at the first level (5 percent) itself. The same trend for degradation characteristics of GNC protein was noted and 5 percent level was optimum for reducing the rumen degradability of GNC protein. The extent of degradation of MC protein was not affected at any level of LSO3 treatment and rate of degradation increased until 7 percent level. However, the EPD of MC protein was lowest at 7 percent level of LSO3 treatment. Therefore, it appears from this experiment that a level of 7 percent LSO3 treatment on MC can be applied to reduce degradability of MC protein. Although PBSN decreased with the treatment, it has been observed that ‘a’ value for GNC protein increased due to LSO3 treatment, which could be an artifact, and is likely due to a deviation from an exponential course of degradation, and it does not agree with the concept directly reflecting the soluble protein of feed (Blummel and Becker 1997). The B3 fraction (slowly degradable) in GNC increased substantially while B2 fraction (intermediately degradable) in MC increased due to LSO3 treatment. Therefore, commencement of degradation of GNC protein compared with MC protein can be delayed or an increased lag can occur for GNC protein, which has presumably showed different pattern of ‘a’ value of the cakes. The LSO3 treatment increased undegradable protein content by 19 percent at 5 percent level in GNC, by 13 percent at 7 percent level in MC. This indicates that different levels of LSO3 treatment could be required to protect the protein in oil cakes from ruminal degradation.

 

Conclusions 

 

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Received 10 June 2009; Accepted 16 July 2009; Published 1 October 2009

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