What happens in the bag? : development and evaluation of a modified in situ protocol to estimate degradation of nitrogen and starch in the rumen

Research output: Thesisinternal PhD, WU

Abstract

The most widely used method to estimate the rumen degradation of dietary components in feedstuffs is the in situ or in sacco method. This method is based on rumen incubation of substrate (feed) in nylon or dacron bags followed by rinsing and analysis of the residue. Small pores in the bag allow microbes to enter the bag whilst a variable portion of the feed is retained in the bag. The results are used to estimate the ruminal effective degradation (ED) that is used in several protein evaluation systems. The weaknesses of the in situ method are its low precision, the lack of standardization, and its inaccuracy. The accuracy can be divided in a bias with respect to the in situ method itself, and the difference between the in situ and in vivo degradation. The bias of the in situ method itself is related to several assumptions regarding the size and degradation rate of the washable fraction, secondary particle loss, and microbial contamination. The aim of this thesis was to examine possibilities to reduce this bias by modification of the in situ methodology.

The bias related to the assumptions regarding the washout fraction was successfully reduced by developing and using a modified rinsing method that involves less vigorously shaking conditions and a solvent which mimics the rumen pH and osmolality. This modified rinsing method markedly reduced the soluble (S) fraction of N, especially for legume seeds, and the non-soluble washout (W-S) fraction, especially for starch, compared to the conventional method. Consequently, the estimation of the ED became less dependent on the assumptions regarding the degradation of the S and W-S fraction. In vitro results did not support the assumption of a much faster degradation of the W-S fraction of starch than that of the non-washout fraction of starch. The modified rinsing method also allowed measuring the in situ degradation of products that contain mainly small particles such as wheat yeast concentrates.

The bias related to the breakdown of particles was also successfully reduced by development and application of a combination of the modified rinsing method and an in vitro method that simulates particulate matter loss during incubation. This in vitro method was based on in situ results obtained with an inert marker (i.e., silica gel) which showed that particulate matter loss during incubation was moderate and limited to particles smaller than approximately 40 μm. Correction for these losses decreased the estimated ED of feed ingredients used. This study also showed that the fractional degradation rate of starch in grains was strongly affected by the process of secondary particulate loss when using the conventional method, and applying the modified rinsing method markedly reduced the error due to secondary particulate loss.    

In comparison to the conventional method, the modified method resulted in an increase of  the bias related to microbial contamination of the residues. The ratio between diaminopimelic acid (DAPA), a marker for bacterial protein, and N in the residues was higher when using the modified method than that in the conventional method. The results obtained for the modified method also indicated lysis of bacterial cells during rinsing. This bias led to a lower ED when using the modified method with the impact greatly depending on the degree of lysis of bacterial cells.

In summary, the modified method increased the non-washout fraction of N and starch of various feed ingredients, which offers the possibility to use it for a larger range of feed ingredients, and reduced the bias related to assumptions on the washout fraction and the breakdown of particles compared to the conventional method. On the other hand, the modified method increased the bias related to microbial contamination and enlarged the difference between the in situ and in vivo degradation. 

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Hendriks, Wouter, Promotor
  • Dijkstra, Jan, Co-promotor
  • van Laar, H., Co-promotor, External person
Award date2 Oct 2015
Place of PublicationWageningen
Publisher
Print ISBNs9789462574519
Publication statusPublished - 2015

Fingerprint

starch
degradation
nitrogen
evaluation
in situ
protocol
method
incubation
lysis
particulate matter
protein

Keywords

  • ruminants
  • rumen fermentation
  • nitrogen
  • starch digestion
  • rumen digestion
  • protocols
  • evaluation
  • methodology
  • estimation
  • nutrition physiology

Cite this

@phdthesis{aa0e3683ecf9441eaf36b924b6e7919a,
title = "What happens in the bag? : development and evaluation of a modified in situ protocol to estimate degradation of nitrogen and starch in the rumen",
abstract = "The most widely used method to estimate the rumen degradation of dietary components in feedstuffs is the in situ or in sacco method. This method is based on rumen incubation of substrate (feed) in nylon or dacron bags followed by rinsing and analysis of the residue. Small pores in the bag allow microbes to enter the bag whilst a variable portion of the feed is retained in the bag. The results are used to estimate the ruminal effective degradation (ED) that is used in several protein evaluation systems. The weaknesses of the in situ method are its low precision, the lack of standardization, and its inaccuracy. The accuracy can be divided in a bias with respect to the in situ method itself, and the difference between the in situ and in vivo degradation. The bias of the in situ method itself is related to several assumptions regarding the size and degradation rate of the washable fraction, secondary particle loss, and microbial contamination. The aim of this thesis was to examine possibilities to reduce this bias by modification of the in situ methodology. The bias related to the assumptions regarding the washout fraction was successfully reduced by developing and using a modified rinsing method that involves less vigorously shaking conditions and a solvent which mimics the rumen pH and osmolality. This modified rinsing method markedly reduced the soluble (S) fraction of N, especially for legume seeds, and the non-soluble washout (W-S) fraction, especially for starch, compared to the conventional method. Consequently, the estimation of the ED became less dependent on the assumptions regarding the degradation of the S and W-S fraction. In vitro results did not support the assumption of a much faster degradation of the W-S fraction of starch than that of the non-washout fraction of starch. The modified rinsing method also allowed measuring the in situ degradation of products that contain mainly small particles such as wheat yeast concentrates. The bias related to the breakdown of particles was also successfully reduced by development and application of a combination of the modified rinsing method and an in vitro method that simulates particulate matter loss during incubation. This in vitro method was based on in situ results obtained with an inert marker (i.e., silica gel) which showed that particulate matter loss during incubation was moderate and limited to particles smaller than approximately 40 μm. Correction for these losses decreased the estimated ED of feed ingredients used. This study also showed that the fractional degradation rate of starch in grains was strongly affected by the process of secondary particulate loss when using the conventional method, and applying the modified rinsing method markedly reduced the error due to secondary particulate loss.     In comparison to the conventional method, the modified method resulted in an increase of  the bias related to microbial contamination of the residues. The ratio between diaminopimelic acid (DAPA), a marker for bacterial protein, and N in the residues was higher when using the modified method than that in the conventional method. The results obtained for the modified method also indicated lysis of bacterial cells during rinsing. This bias led to a lower ED when using the modified method with the impact greatly depending on the degree of lysis of bacterial cells. In summary, the modified method increased the non-washout fraction of N and starch of various feed ingredients, which offers the possibility to use it for a larger range of feed ingredients, and reduced the bias related to assumptions on the washout fraction and the breakdown of particles compared to the conventional method. On the other hand, the modified method increased the bias related to microbial contamination and enlarged the difference between the in situ and in vivo degradation. ",
keywords = "herkauwers, pensfermentatie, stikstof, zetmeelvertering, pensvertering, protocollen, evaluatie, methodologie, schatting, voedingsfysiologie, ruminants, rumen fermentation, nitrogen, starch digestion, rumen digestion, protocols, evaluation, methodology, estimation, nutrition physiology",
author = "{de Jonge}, L.H.",
note = "WU thesis 6139",
year = "2015",
language = "English",
isbn = "9789462574519",
publisher = "Wageningen University",
school = "Wageningen University",

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TY - THES

T1 - What happens in the bag? : development and evaluation of a modified in situ protocol to estimate degradation of nitrogen and starch in the rumen

AU - de Jonge, L.H.

N1 - WU thesis 6139

PY - 2015

Y1 - 2015

N2 - The most widely used method to estimate the rumen degradation of dietary components in feedstuffs is the in situ or in sacco method. This method is based on rumen incubation of substrate (feed) in nylon or dacron bags followed by rinsing and analysis of the residue. Small pores in the bag allow microbes to enter the bag whilst a variable portion of the feed is retained in the bag. The results are used to estimate the ruminal effective degradation (ED) that is used in several protein evaluation systems. The weaknesses of the in situ method are its low precision, the lack of standardization, and its inaccuracy. The accuracy can be divided in a bias with respect to the in situ method itself, and the difference between the in situ and in vivo degradation. The bias of the in situ method itself is related to several assumptions regarding the size and degradation rate of the washable fraction, secondary particle loss, and microbial contamination. The aim of this thesis was to examine possibilities to reduce this bias by modification of the in situ methodology. The bias related to the assumptions regarding the washout fraction was successfully reduced by developing and using a modified rinsing method that involves less vigorously shaking conditions and a solvent which mimics the rumen pH and osmolality. This modified rinsing method markedly reduced the soluble (S) fraction of N, especially for legume seeds, and the non-soluble washout (W-S) fraction, especially for starch, compared to the conventional method. Consequently, the estimation of the ED became less dependent on the assumptions regarding the degradation of the S and W-S fraction. In vitro results did not support the assumption of a much faster degradation of the W-S fraction of starch than that of the non-washout fraction of starch. The modified rinsing method also allowed measuring the in situ degradation of products that contain mainly small particles such as wheat yeast concentrates. The bias related to the breakdown of particles was also successfully reduced by development and application of a combination of the modified rinsing method and an in vitro method that simulates particulate matter loss during incubation. This in vitro method was based on in situ results obtained with an inert marker (i.e., silica gel) which showed that particulate matter loss during incubation was moderate and limited to particles smaller than approximately 40 μm. Correction for these losses decreased the estimated ED of feed ingredients used. This study also showed that the fractional degradation rate of starch in grains was strongly affected by the process of secondary particulate loss when using the conventional method, and applying the modified rinsing method markedly reduced the error due to secondary particulate loss.     In comparison to the conventional method, the modified method resulted in an increase of  the bias related to microbial contamination of the residues. The ratio between diaminopimelic acid (DAPA), a marker for bacterial protein, and N in the residues was higher when using the modified method than that in the conventional method. The results obtained for the modified method also indicated lysis of bacterial cells during rinsing. This bias led to a lower ED when using the modified method with the impact greatly depending on the degree of lysis of bacterial cells. In summary, the modified method increased the non-washout fraction of N and starch of various feed ingredients, which offers the possibility to use it for a larger range of feed ingredients, and reduced the bias related to assumptions on the washout fraction and the breakdown of particles compared to the conventional method. On the other hand, the modified method increased the bias related to microbial contamination and enlarged the difference between the in situ and in vivo degradation. 

AB - The most widely used method to estimate the rumen degradation of dietary components in feedstuffs is the in situ or in sacco method. This method is based on rumen incubation of substrate (feed) in nylon or dacron bags followed by rinsing and analysis of the residue. Small pores in the bag allow microbes to enter the bag whilst a variable portion of the feed is retained in the bag. The results are used to estimate the ruminal effective degradation (ED) that is used in several protein evaluation systems. The weaknesses of the in situ method are its low precision, the lack of standardization, and its inaccuracy. The accuracy can be divided in a bias with respect to the in situ method itself, and the difference between the in situ and in vivo degradation. The bias of the in situ method itself is related to several assumptions regarding the size and degradation rate of the washable fraction, secondary particle loss, and microbial contamination. The aim of this thesis was to examine possibilities to reduce this bias by modification of the in situ methodology. The bias related to the assumptions regarding the washout fraction was successfully reduced by developing and using a modified rinsing method that involves less vigorously shaking conditions and a solvent which mimics the rumen pH and osmolality. This modified rinsing method markedly reduced the soluble (S) fraction of N, especially for legume seeds, and the non-soluble washout (W-S) fraction, especially for starch, compared to the conventional method. Consequently, the estimation of the ED became less dependent on the assumptions regarding the degradation of the S and W-S fraction. In vitro results did not support the assumption of a much faster degradation of the W-S fraction of starch than that of the non-washout fraction of starch. The modified rinsing method also allowed measuring the in situ degradation of products that contain mainly small particles such as wheat yeast concentrates. The bias related to the breakdown of particles was also successfully reduced by development and application of a combination of the modified rinsing method and an in vitro method that simulates particulate matter loss during incubation. This in vitro method was based on in situ results obtained with an inert marker (i.e., silica gel) which showed that particulate matter loss during incubation was moderate and limited to particles smaller than approximately 40 μm. Correction for these losses decreased the estimated ED of feed ingredients used. This study also showed that the fractional degradation rate of starch in grains was strongly affected by the process of secondary particulate loss when using the conventional method, and applying the modified rinsing method markedly reduced the error due to secondary particulate loss.     In comparison to the conventional method, the modified method resulted in an increase of  the bias related to microbial contamination of the residues. The ratio between diaminopimelic acid (DAPA), a marker for bacterial protein, and N in the residues was higher when using the modified method than that in the conventional method. The results obtained for the modified method also indicated lysis of bacterial cells during rinsing. This bias led to a lower ED when using the modified method with the impact greatly depending on the degree of lysis of bacterial cells. In summary, the modified method increased the non-washout fraction of N and starch of various feed ingredients, which offers the possibility to use it for a larger range of feed ingredients, and reduced the bias related to assumptions on the washout fraction and the breakdown of particles compared to the conventional method. On the other hand, the modified method increased the bias related to microbial contamination and enlarged the difference between the in situ and in vivo degradation. 

KW - herkauwers

KW - pensfermentatie

KW - stikstof

KW - zetmeelvertering

KW - pensvertering

KW - protocollen

KW - evaluatie

KW - methodologie

KW - schatting

KW - voedingsfysiologie

KW - ruminants

KW - rumen fermentation

KW - nitrogen

KW - starch digestion

KW - rumen digestion

KW - protocols

KW - evaluation

KW - methodology

KW - estimation

KW - nutrition physiology

M3 - internal PhD, WU

SN - 9789462574519

PB - Wageningen University

CY - Wageningen

ER -