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 95^°C 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 (60^°C) for 8 h and then at higher temperature (90^°C) 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 39^°C 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 39^°C for 2 h. Additional 20 ml of buffer (39^°C) was added to the incubation mixture and left overnight at 39^°C. 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 100^°C 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.

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.

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.

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.

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 30^oC up to 130^oC. 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 

• The effective ruminal degradability of GNC protein decreased at 5 percent LSO₃ treatment and post-ruminal digestibility of GNC protein decreased beyond 5 percent LSO₃ treatment. 

  
  

• The effective degradability of protein of LSO₃ treated MC decreased at 7 percent treatment and post-ruminal protein digestibility was not affected up to 7 percent level. 

  
  

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

References 

Antoniewicz A M, Van Vuuren A M, Van der Koelen C J and Kosmala I 1992 Intestinal digestibility of rumen undegraded protein of formaldehyde-treated feedstuffs measured by mobile bag and in vitro technique. Animal Feed Science and Technology 39: 111-124

AOAC 1990 Official methods of analysis. Association of Official Analytical Chemists, 15^th edition, Washington, DC.

Beauchemin K A, Bailey D R C, Mcallister T A and Cheng K J 1995 Lignosulphonate treated canola cake for nursing beef calves. Canadian Journal of Animal Science 75: 387‑395

Bhagwat S R and Srivastava A 1991 Comparative study on digestibility of nutrients in crossbred calves fed soybean cake treated with heat, formaldehyde and tannic acid. Indian Journal of Animal Nutrition 8: 305-311

Blummel M and Becker K 1997 The degradability characteristics of fifty-four roughages and roughage neutral-detergent fibres as described by in vitro gas production and their relationship to voluntary feed intake. British Journal of Nutrition 77: 757-768

Calsamiglia S, Stern M D and Firkins J L 1995 Effect of protein source on nitrogen in continuous and intestinal digestion in vitro. Journal of Animal Science 73: 1819‑1827  http://jas.fass.org/cgi/reprint/73/6/1819

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

Chatterjee A and Walli T K 2000 Effect of formaldehyde treatment on ruminal and post ruminal protein digestibility of mustard cake. Indian Journal of Animal Production and Management 16: 91-95

Chatterjee A and Walli T K 2003 Effect of feeding formaldehyde treated mustard cake on milk yield, milk composition and on economics of milk production in Murrah buffaloes. Indian Journal of Dairy Science 56: 299-306

Dhiman T R, MacQueen I S and Luchini N D 2001 Milk response of dairy cow fed fat along with protein. Animal Feed Science and Technology 90: 169-184

Gupta H K and Walli T K 1987 Influence of feeding formaldehyde treated groundnut cake and its partial replacement with urea on growth and feed utilization in crossbred kids. Indian Journal of Animal Nutrition 4: 94‑99

Kumar V and Walli T K 1994 Effect of feeding urea treated wheat straw supplemented with formaldehyde treated groundnut cakes on growth performance of crossbred calves. Indian Journal of Animal Nutrition 11: 29-33

Mansfield H R and Stern M D 1994 Effects of soybean hulls and lignosulfonate-treated soybean meal on ruminal fermentation in lactating dairy cows. Journal of Dairy Science 77: 1070-1083 http://jds.fass.org/cgi/reprint/77/4/1070.pdf

McAllister T A, Cheng K J, Beauchemin K A, Bailey D R C, Pickard M D and Gilbert R P 1992 Effect of formaldehyde treated barley or escape protein on nutrient digestibility, growth and carcass traits of feed lot lambs. Canadian Journal of Animal Science 72: 309‑316

McAllister T A, Cheng K J, Beauchemin K A, Bailey D R C, Pickard M D and Gilbert R P 1993 Use of lignosulphonate to decrease the rumen degradability of Canola cake protein.  Canadian Journal of Animal Science 73: 211‑215

Mondal G, Walli T K and Patra A K 2008 In vitro and in sacco ruminal protein degradability of common Indian feed ingredients. Livestock Research for Rural Development 20 (4), http://www.lrrd.org/lrrd20/4/mond20063.htm

Monterola H B, Cerda D A and Mira J J 2001 Protein degradability of soybean cake coated with different lipid substances and its effect on ruminal parameters hen included in steer rations. Animal Feed Science and Technology 92: 249-257

Mustafa A F, Christensen D A and McKinnon J J 1998 Effect of moist heat treatment on crude protein composition and degradability of field peas. Canadian Journal of Animal Science 78: 453‑456

Nakamura T, Klopfenstein T J, Owen F G, Britton R A and Grant R J 1992 Nonenzymatically browned soybean meal for lactating dairy cows. Journal of Dairy Science 75: 3519-3523 http://jds.fass.org/cgi/reprint/75/12/3519.pdf

Orskov E R and McDonald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage.  Journal of Agricultural Science (Cambridge) 92: 499-505

Petit H V, Tremblay G F, Marcotte M and Audy R 1999 Degradability and digestibility of full fat soybeans treated with different sugar and heat combinations.  Canadian Journal of Animal Science 79: 213‑220

SPSS 1996 Statistical Packages for Social Sciences, version 7.5, SPSS Inc., Illinois, USA

Stanford K, McAllister T A, Xu Z, Pickard M and Cheng K J 1995 Comparison of lignosulphonate treated Canola cake and soybean cake as rumen undegradable protein supplements for lambs. Canadian Journal of Animal Science 75: 371‑377

von Keyserlingk M A G, Weurding E, Swift M L, Wright C F, Shelford J A and Fisher L J 2000 Effect of adding lignosulfonate and heat to canola screenings on ruminal and intestinal disappearance of dry matter and crude protein. Canadian Journal of Animal Science 80: 215-219

Windschitl P M and Stern M D 1988a Evaluation of calcium-lignosulphonate treated soybean cake: a source of rumen protected proteins for dairy cattle.  Journal of Dairy Science 71: 3310‑3322 http://jds.fass.org/cgi/reprint/71/12/3310

Windschitl P M, Stern M D 1988b Effects of urea supplementation of diets containing lignosulfonate-treated soybean meal on bacterial fermentation in continuous culture of ruminal contents. Journal of Animal Science 66: 2948-2958 http://jas.fass.org/cgi/reprint/66/11/2948.pdf

Wright C F, von Keyserlingk M A G, Swift M L, Fisher L J, Shelford J A and Dinn N E 2005 Heat- and lignosulfonate-treated canola meal as a source of ruminal undegradable protein for lactating dairy cows. Journal of Dairy Science 88: 238-243 http://jds.fass.org/cgi/reprint/88/1/238.pdf

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

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