Assessing Rumen Biohydrogenation and Its Manipulation in Vivo, in Vitro, in Situ

740
DOI 10.1002/ejlt.200700033
Veerle Fieveza
Bruno Vlaemincka
Tom Jenkinsb
Francis Enjalbertc
Michel Doreaud
Assessing rumen biohydrogenation and its

manipulation in vivo, in vitro and in situ
Laboratory of Animal Nutrition

and Animal Product Quality,
Ghent University,
Melle, Belgium
b
Department of Animal and
Veterinary Sciences,
Clemson University,
Clemson, USA
c
INRA, UMR1289 Tissus
Animaux, Nutrition, Digestion,
Ecosystme, Mtabolisme,
Castanet-Tolosan, France;
INP-ENSAT, Castanet-Tolosan,
France;
ENVT, Toulouse, France
d
INRA, URH, Theix,
Saint-Gens Champanelle,
France
Reference techniques to study rumen biohydrogenation (BH) rely on the comparison of

intake and duodenal or (ab)omasal flows of polyunsaturated fatty acids (PUFA),
whereas the net BH of PUFA to their saturated end-products gives a quantitative
measure of accumulating BH intermediates. The current review paper aims at evaluating alternative in vivo, in vitro and in sacco techniques to simulate reference in vivo
results of unprotected PUFA sources, as well as strategies for overcoming or manipulating BH. In vivo rumen sampling approaches show potential but require further
investigation, whereas in sacco results are inappropriate. In vitro 24-h batch incubations and continuous cultures approach in vivo BH of unprotected C18 PUFA sources
and are useful to assess the pH value at which dissociation of calcium salts of fatty
acids occurs, but overestimate the degree of rumen inertness of formaldehyde-treated
oil(seeds) and marine products. Batch or continuous cultures provide an accurate
estimate (0.6) of the proportion of hydrogenated C18 PUFA that are converted into their
saturated end-product, except for incubations with high levels of fermentable substrate (.1.0 g/100 mL) in combination with high amounts of C18 PUFA (.0.5 mg/mL) or
in incubations with EPA and DHA.
Keywords: Rumen biohydrogenation, in vivo, in vitro, in sacco, lipolysis.
1 Introduction
Review Article
Eur. J. Lipid Sci. Technol. 109 (2007) 740756
The process of biohydrogenation (BH) reduces the rumen

outflow of polyunsaturated fatty acids (PUFA) and contributes to accumulation of cis and trans isomers in ruminant products, including conjugated linoleic acid (CLA)
and trans monoenes. Hence, the extent and type of the
rumen BH process will determine both the amounts and
structures of fatty acids leaving the rumen. As the fatty
acid structure determines its physiological features,
interest has grown in the process of rumen BH. Consequently, information is needed on (i) the disappearance of
PUFA, (ii) the production of the saturated end-products of
the BH process, and (iii) the nature and the amount of the
accumulating BH intermediates. Measures and experimental techniques discussed in the current paper are
limited to the former two. Experimental techniques discussed include in vivo reference techniques as well as in
sacco and in vitro approaches.
An increasing number of novel technologies and strategies to control and/or prevent rumen BH are currently
being investigated. These are aimed at by-passing
Correspondence: Veerle Fievez, Laboratory for Animal Nutrition
and Animal Product Quality, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium. Phone: 132 9 2649002, Fax: 132
9 2649099, e-mail: Veerle.Fievez@UGent.be
rumen PUFA metabolism or ensuring the accumulation

of desired intermediate compounds. However, the response to protection technology often is variable and
over-processing might reduce digestibility. Hence, routine methods are required to assess the effectiveness of
these technologies or strategies, both in terms of absolute data extrapolation to in vivo values as well as their
ranking. In terms of protection technology, three main
process types can be considered: (1) chemical protection, e.g. through encapsulation in a protein matrix followed by aldehyde treatment, direct formaldehyde treatment of oilseeds or formation of a whey gel complex;
(2) formation of calcium salts and amides of fatty acids;
and (3) technological treatments of oilseeds, such as
extrusion, roasting, cracking, etc. Strategies to manipulate the extent of rumen BH and the accumulation of
BH intermediates mostly deal with antimicrobial additives, including fish oil. The reliability of in vitro techniques to assess the effectiveness of strategies for
overcoming or manipulating BH by ruminal microorganisms is evaluated.
Due to data availability, BH of linoleic (18:2n-6) and
linolenic (18:3n-3) acid is the main focus of this
paper, although some aspects of BH of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid
are addressed.
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2 Terminology and calculations

2.1 Rumen lipolysis
Lipolysis is the hydrolysis (release) of fatty acids from the
glycerol backbone of triacylglycerols, galactolipids or
phospholipids. Consequently, separation of esterified
(EFA) and non-esterified fatty acids (NEFA) is a prerequisite for the distinct evaluation of lipolysis and BH.
The extent of rumen lipolysis is calculated from the maximal loss of EFA over a specific time period. This method
of expression can be used to calculate the mean lipolysis
of all fatty acids as well as the extent to which an individual fatty acid (FAi) is released from its EFAi fraction. Calculations based on in vivo data, effluents of continuous
fermentors and batch in vitro results are similar, but the
amounts of EFAi in the inoculum should be taken into
account in the latter case.
Lipolysis EFAi 100
EFAi0h EFAith
EFAi0h
with EFAi 0h = EFA intake or in vitro supply of EFAi at the

start of the incubation, and EFAi th = duodenal or (ab)
omasal flow of EFAi, amount of EFA in continuous fermentor effluents or amount of EFA recovered in batch in
vitro incubation flasks after t hours of incubation.
2.2 Rumen BH of PUFA

Assessment of rumen BH in the forestomach of ruminants is analogous to the determination of apparent
rumen carbohydrate or protein digestibility [1]. Hence,
rumen BH is calculated as the disappearance of PUFAi
relative to their intake. It should be noted that this represents a combined measure of two different processes,
i.e. the release of fatty acids from the esterified lipid and
the first step of PUFA BH, i.e. isomerization for 18:2n-6
and 18:3n-3. This calculation can be applied both to in
vivo flow data and effluent data of continuous fermentors
as well as to batch in vitro measurements, but inoculum
fatty acids have to be included in the latter calculations.
Alternatively, fatty acids that do not originate from the
test product can be excluded by subtracting the
amounts of fatty acids in blank (without the test product)
incubation flasks (e.g. [2]):
Rumen in vivo, in vitro and in situ biohydrogenation
741
2.3 Production of BH intermediates and

saturated end-products
Calculation of rumen BH as shown above might be appropriate to assess protection of PUFA, but it does not
provide information on the accumulation of BH intermediates. The net duodenal flow or in vitro production of
the saturated BH end-product (e.g. 18:0, 20:0 or 22:0)
proportional to the amount of PUFA precursors lost during
rumen BH (i.e. 18:2n-6 1 18:3n-3, EPA and DHA,
respectively) gives a quantitative measure of the overall
PUFA BH efficiency. This approach is most appropriate
for C20 and C22 fatty acids, as dietary C20 and C22 PUFA
are almost exclusively EPA and DHA, respectively, while it
is hampered for C18 PUFA due to the production of 18:0
from dietary oleic acid. The net amount of 18:0 resulting
from BH of C18 PUFA can be calculated as the difference
of the duodenal or in vitro gain of 18:0 and the amount of
oleic acid lost. This requires the assumption that the
duodenal flow or in vitro production of trans monounsaturated fatty acids originating from isomerization of
oleic acid is of minor importance [3].
Nevertheless, this approach does not provide information
on the nature of the accumulating intermediates. Indeed,
rumen fatty acid metabolism is a multistep process,
requiring measures to distinctively assess the conversion
efficiency of each step. Detailed knowledge on the predominant BH pathways allows assessing the inhibition or
stimulation of specific intermediate BH steps. Calculations are complicated due to the movement of fatty acids
among pools, which is illustrated by e.g. Boeckaert et al.
[4], Ribeiro et al. [5] and Harvatine and Allen [6]. However,
limited knowledge on possible (secondary) BH pathways
as well as an insufficient number of data in most experiments limits the current development of multi-compartment models including all intermediates [5]. As a consequence, these approaches exclude the identification of
possible shifts of BH routes, which is further considered
out of the scope of the current paper.
3 Statistical analysis
with PUFAi 0h = PUFA intake or in vitro PUFA supply (in g)

at the start of the incubation and PUFAi th = duodenal or
(ab)omasal flow of PUFA (g), PUFA (g) in continuous fermentor effluents or PUFA (g) recovered in batch in vitro
incubation flasks after t hours of incubation.
Where sufficient literature information is available, we

have attempted to formulate quantitative models. Two
major principles were taken into account in this statistical
approach: (1) Observations within a given study have
more in common than observations across studies, and
(2) inference is to future, unknown studies. This means
that the factor study was considered random as the
studies included in this review only represent a random
sample of a larger set of potential studies and the interest
is not in evaluating the effect of a specific study but rather
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Biohydrogenation PUFAi 100
PUFAi0h PUFAith
PUFAi0h
742
V. Fievez et al.
to derive models that are applicable to all potential studies within a population. Hence, relations and regression
equations presented in the current paper were developed
using a mixed-model regression analysis as described by
St-Pierre [7]. Data were analyzed according to the following model: Yij = B0 1 B1Xij 1 si* 1 bi*Xij 1 eij; with i = the ith
study, j = the jth observation within the ith study, B0 1 B1Xij
= the fixed effect part of the model, si* 1 bi*Xij 1 eij = the
random effect part of the model and with the means of si*
and bi* equaling 0.
To illustrate effects, it was further attempted to graphically
present regression results. However, results from a
mixed-model regression cannot simply be presented as a
Y vs. X graph as the observations come from a (j 1 2)dimensional space, which should first be collapsed into a
two-dimensional space. To account for this loss of
dimensions, adjusted observations should be calculated.
The latter is done by adding the residual from each individual observation to the predicted value of the study
regression [7]. These adjusted observations for study
effects were used to calculate determination coefficients.

form. This dataset is too limited to allow comparisons
between markers and flow calculations. However, an
overview of the use of flow markers has been made by
Owens and Hanson [14] who did not draw conclusions on
the superiority of any marker.
4.1 Rumen lipolysis

Although lipolysis is a prerequisite of rumen BH, in vivo
studies reporting dietary and digesta EFA and NEFA
fractions are scarce [1517]. Omasal or abomasal sampling offers the greatest opportunity for the correct
assessment of rumen lipolysis, without the risk of intestinal hydrolysis. A duodenal cannula should be inserted
cranial to the common bile and pancreatic duct to provide
evidence that the production of the unesterified fatty
acids did not take place in the small intestine, as specified
by Atkinson et al. [15].
4.2 Rumen BH of PUFA
As with digestibility studies, determination of rumen PUFA

BH is typically assessed through the use of digestibility
markers and the collection of digesta via sampling from
the omasal canal or from abomasal or duodenal cannulas.
Almost all published experiments with data on fatty acid
ruminal metabolism used duodenal cannulas. Omasal
canal measurements were used by Shingfield et al. [8] and
Lundy et al. [9], whereas an abomasal cannula was used
in five experiments in Australia and Canada between
1972 and 1987 [1012]. To correct for non-representative
digesta sampling, a double-marker method is suggested
that allows recombination of the fluid and particle phase
[13]. Apparently, the choice of appropriate markers,
representative of rumen fatty acid outflow, and their
accurate analysis are essential. However, the number and
nature of markers and the flow calculations largely vary
among experiments. In experiments with measurements
on digestive lipids, Ru, Yb, Dy, La (radioactive or as chloride and acetate), Cr (as Cr2O3, mordanted, or 51Cr), lignin,
C31 and/or C33 alkanes and acid-insoluble ash have
been used as particle phase marker; Cr-EDTA, Co-EDTA
(radioactive or not) and PEG have been used as liquid
phase marker. Out of 81 published studies and 7 unpublished experiments from INRA-Theix (either partially published or submitted for publication) estimating in vivo fatty
acid flows, there were 49 that used two markers. When
one marker was used, it was mostly chromic oxide in any
Jenkins [18] estimated the average in vivo BH of unprotected PUFA sources from slopes of the regression lines,
estimating ruminal loss of dietary PUFA per unit of PUFA
consumed, after adjustment for the random effect of
study. Slopes were 828 6 17 and 875 6 22 g/kg for
18:2n-6 and 18:3n-3, respectively. However, 18:2n-6 and
18:3n-3 BH data in recent studies are calculated using
data obtained with improved analytical methods allowing
the chromatographic separation of numerous 18:2 and
18:1 isomers. It appears now that 18:2n-6 only represent
a part of the 18:2 fatty acids. Different conjugated
isomers (CLA) and non-conjugated isomers, especially
18:2t11c15, account for a large proportion of the total
18:2, depending on the diet. Loor et al. [19] separated
15 isomers of 18:2 and showed that the percentage of
18:2n-6 in total 18:2 in duodenal chyme varied between
77% for a forage-based diet and 34% for a concentraterich diet supplemented with linseed oil. Obviously, this
might result in a bias in the calculated BH when isomers
are not separated. Indeed, in all determinations of fatty
acid flows published before 2001, one chromatographic
peak was evidenced in the 18:2 area, which was considered as 18:2n-6 whereas it represented in fact a sum of
co-eluted isomers. This explains why in recent studies the
18:2n-6 BH is often higher than in earlier studies (800 and
650 g/kg on average for diets containing less or more
than 60% of concentrate, respectively; [20]). We have
evaluated this bias using 60 dietary treatments from
16 experiments published from 2001 and two unpublished experiments in which the separation between 18:2
isomers was made. The disappearance of 18:2n-6 was
857 g/kg, which is slightly higher than the formerly
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4 Reference in vivo techniques: Duodenal,

omasal and abomasal flows
743
reported value obtained by Jenkins [18]. However, the BH

calculated based on the disappearance of total 18:2
isomers was 805 g/kg. A relationship between BH of
total 18:2 and 18:2n-6 was established: BH18:2 n-6 =
0.450(SE = 0.0503; p ,0.001) 1 0.506(SE = 0.06151; p ,0.001)6BH18:2
(R2 = 0.748; n = 60). As the proportion of 18:3n-3 in total
18:3 duodenal fatty acids is higher than 0.90, it can be
expected that the bias on BH is of lesser importance for
this fatty acid.
4.3 Production of BH intermediates and

saturated end-products
Calculation of net 18:0 production from hydrogenated
18:2n-6 and 18:3n-3 is hampered when based on inadequate separation of 18:1 isomers. As dietary 18:1 almost
exclusively represents oleic acid, some attempts to estimate the net 18:0 production could be made assuming a
constant ruminal loss of oleic acid per unit of oleic acid
consumed. Hence, duodenal flow of 18:0 has been integrated as dependent variable in a multiple linear model
(Fig. 1) with the amount of hydrogenated C18 PUFA and
oleic acid intake as independent variables. The proportional conversion of hydrogenated 18:2n-6 and 18:3n-3 to
18:0 might then be estimated from its respective coefficient in this multiple linear regression, which suggests
that on average 0.599 of the hydrogenated C18 PUFA were
completely hydrogenated to 18:0. The data considered
here include all published experiments from 1987 until
now that quantified intake and duodenal flow of C18 fatty
acids, but excluding dietary treatments where protected
fats, fish oil or fermentation modifiers (e.g. monensin)
were used. Variation within the dataset, such as forage/
concentrate ratio (wt/wt, minmax, 500 : 500820 : 180 in
dairy cattle diets), experimental animal (e.g. dairy cattle,
finishing beef steers, sheep), dry matter intake (DMI) (min
max, 1.127.0 kg/day), dietary PUFA content (minmax,
8.330.5 g/kg DM) or variation in BH of oleic acid (not
quantified), might be the reason for some deviation of the
observed values from the trend line in Fig. 1. Two observations by Loor et al. [19], with dairy cattle fed high-concentrate diets (350 : 650 forage/concentrate, wt/wt) supplemented with linseed or soybean oil, were excluded
from the multiple linear regression. Nevertheless, these
data are presented in Fig. 1 and illustrate a possible inhibitory effect of increased concentrate levels on the production of the saturated BH end-product.
Fish oil is another well-known inhibitor of BH to 18:0 as
end-product [21]. Effects of fish oil on rumen metabolism
were suggested to be attributed to the action of nonesterified EPA and DHA [22], but to our knowledge,
attempts to quantitatively assess their overall effect on
duodenal flow of 18:0 have not yet been made [21].
Fig. 1. Net duodenal gain of 18:0 (18:0 0.643618:1

intake) as estimated from the multiple linear regression
model [18:0 (g/kg DMI) = 3.22(SE = 1.37; p = 0.023) 1
0.599(SE = 0.061; p ,0.001)6C18 PUFAlost (g/kg DMI) 1
0.643(SE = 0.061; p ,0.001)618:1 intake (g/kg DMI), R2 = 0.890,
n = 48] in relation to the amount of 18:2n-6 and 18:3n-3 lost
by rumen BH (C18 PUFA lost) [6, 8, 9, 24, 25, 42, 44, 45, 57,
7586]. Observations represented by grey squares [43]
were not included in the multiple linear model development
and illustrate a possible inhibition of complete 18:2n-6 and
18:3n-3 BH at higher dietary concentrate levels.
Introduction of an interaction term (amount of C18 PUFA

lost * EPA 1 DHA intake) in the former regression (Fig. 1)
allowed assessment of the effect of EPA and DHA (g/kg
DMI) on the conversion efficiency of unesterified C18 PUFA
to 18:0. As the BH of dietary 18:1, and hence the coefficient
of 18:1 in the multiple linear regression, might be lower for
Megalac or formaldehyde-treated linseed, fish oil supplementation data using these sources were not included
in the database, e.g. [23] and some treatments of [24] and
[25]. The negative coefficient of the interaction term confirms the inhibitory effect of EPA and DHA on the production of 18:0 from C18 PUFA and suggested the conversion
of C18 PUFA to 18:0 to be reduced by 20% for each unit of
EPA 1 DHA intake (g/kg DMI). The latter value assumes
negligible effects of EPA and DHA on the BH of oleic acid
and accumulation of its isomers. A negligible effect of EPA
and DHA on the BH of oleic acid seems justified [21],
whereas the effect on the accumulation of its isomers has
not yet been described. Moreover, this approach assumes
EPA and DHA to exert an equally inhibitory effect, which is
unlikely (e.g. [26]). However, the limited number of fish oilsupplemented duodenal flow data (n = 7) does not allow a
more detailed evaluation.
5 Alternative in vivo approaches

5.1 Calculations based on concentrations
instead of flows
The need for quantitative flow measures was suggested
to be redundant in rumen fatty acid studies, as rumen
fatty acid metabolism is essentially limited to hydrolysis
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744
V. Fievez et al.
and BH. Hence, total fatty acid intake can be assumed to

equal ruminal fatty acid outflow, which would allow to
apply the former equation (see Section 2.2.) to calculate
BH, but expressing fatty acid intake and flow relative to
the total fat or to the total C18 fatty acids [27]. However,
Doreau and Ferlay [28] showed evidence of some rumen
loss of fatty acids through oxidation and/or absorption,
whereas bacterial de novo synthesis of fatty acids also
occurs [29]. In addition, quantification of total C18 fatty
acids has been tedious due to extensive rumen fatty acid
metabolism resulting in the formation of a wide range of
identified as well as unidentified C18 fatty acids. This suggests that estimations of BH based on fatty acid concentrations or total 18-carbon fatty acids might be
biased. From a database of experiments in which fatty
acid duodenal flows were determined (n = 218), we
assessed the importance of this bias through the comparison of BH calculated from quantitative measurements
of intake and duodenal flow with calculations based on
18:2n-6 and 18:3n-3 concentrations in total fatty acids
and proportional to the sum of C18 fatty acids. As the ratio
between fatty acid duodenal flow and fatty acid intake
depends on the fatty acid content of the diet (see for
example [28]), dietary fatty acid content was included in
the regression equation. From the equations presented in
Tab. 1, BH can be predicted from data obtained using
concentrations in total fatty acids or in C18 fatty acids,
with good accuracy (R2 between 0.85 and 0.97).
5.2 BH estimates through rumen sampling

From an animal welfare point of view, use of experimental
animals with duodenal cannulas should be minimized.
(Ab)omasal sampling through the rumen cannula might

offer an alternative, but direct rumen sampling is the least
demanding technique. However, continuous lipid intake
hampers calculation of rumen BH, as variations in fatty
acid concentrations are insufficient [30]. Alternatively,
Doreau and Gachon [31] ruminally infused a pulse dose of
300 g of a linseed product into cows and then followed
the fatty acid kinetics in consecutive samples of rumen
juice and total contents. This method was used for linseed oil, rolled linseeds and extruded linseeds together
with duodenal flow measurements [21]. The mean proportions of the various C18 fatty acids in the rumen could
be estimated from the areas under the curves of these
fatty acids during 24 h of sampling and was shown to be
similar to values obtained from sampling the duodenal
chime effluent. Differences between treatments were also
similar to duodenal flow measurements and ruminal
kinetics. This shows the potential of this technique, but
estimation of PUFA BH requires a dynamic model, to
adjust for factors such as (i) time of lipid infusion into the
rumen, (ii) lipid hydrolysis, and (iii) the liquid outflow rate
of the rumen. Alternatively, PUFA BH can be estimated
from rumen fatty acid pools determined during a fasting
period (16 h) as described by Lourenco et al. [32]. BH was
estimated from 18:3n-3 intake and its hydrogenation rate
based on rumen pool sizes of 18:3n-3 and acid detergent
lignin clearance rates. In this approach, acid detergent
lignin clearance rates were assumed to equal rumen
passage rates of fatty acids, which is justified as fatty
acids are adsorbed onto particles. Despite the low number of rumen evacuations (n = 3), this approach apparently generated reliable BH measures, but no reference
duodenal flow measurements were available to unequivocally assess this technique. However, both rumen
Tab. 1. Prediction of the 18:2n-6 and 18:3n-3 BH calculated based on total intake and duodenal flows (BH18:2 flow and
BH18:3 flow) from calculations based on (i) 18:2n-6 [BH18:2 (%FA)] and 18:3n-3 [BH18:3 (%FA)] concentrations in total fat
and (ii) proportional to the sum of 18-carbon fatty acids [BH18:2 (%C18) and BH18:3 (%C18)] (n = 218).
Intercept
Estimate
SE
p$
BH18:2flow
NS
BH18:2flow
NS
BH18:3flow
20.101
0.0202
,0.001
BH18:3flow
20.127
0.0239
,0.001
$
#
R2
Independent variable
Variable
Estimate
SE
p$
BH18:2 (%FA)
dietary fat content
BH18:2 (%C18)
dietary fat content
BH18:3 (%FA)
dietary fat content
BH18:3 (%C18)
dietary fat content
0.930
0.009
0.928
0.010
1.075
0.005
1.102
0.006
0.0092
0.0013
0.0105
0.0015
0.0223
0.0009
0.0263
0.0010
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
0.896
0.857
0.961
0.970
SE, standard error of the regression coefficient.

p, p-value of the regression coefficient.
NS, the intercept is not significantly different from zero.
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sampling techniques might have some limitations due to
massive perturbation of the substrate pool size during
fasting and/or effects of unsaturated fatty acid overload
on rumen BH enzymes or pathways when supplying a
pulse dose of PUFA.
6 Batch in vitro techniques

Rumen metabolism of PUFA has been studied in vitro for
several decades at different laboratories using slightly
different methods. Most often, 24-h incubations are carried out. However, as effects on rates of lipolysis and BH
might be masked after 24 h of incubation, some
researchers perform time series of in vitro incubations to
allow estimations of kinetic parameters such as lag time
and rate of lipolysis or BH. An overview of the in vitro
incubation characteristics of the studies used in this
review is given in Tab. 2.
6.1 In vitro conditions

Because culture conditions affect microbial metabolism
in vitro, factors characterizing batch in vitro incubations
and their effect on the extent and kinetics of rumen lipolysis and BH are discussed below.
745
bacteria and rumen microbial BH (e.g. [2]), as earlier

studies showed that BH took place on the surfaces of
feed particles [36]. In most incubations, particles are provided either by the inoculum or by the fermentation substrate, which is required as a carbon source because
anaerobes do not generate energy (ATP) from PUFA. In
this respect, the entire removal of inoculum particles
through centrifugation should not be considered and
might explain the reduced BH activity in incubations by
Choi et al. [37].
6.1.3 Introduction of PUFA in incubation

medium
Several methods have been described for the introduction of water-insoluble PUFA into the incubation medium.
Fellner et al. [38] proposed the preparation of 18:2n-6
emulsions through sonication. Martin and Jenkins [39]
described this technique as unsuccessful for unesterified
18:2n-6, but emulsions of soybean oil triglycerides were
homogeneous and evenly distributed. In the laboratory at
Ghent University, fatty acids in oil or fat are added as a
solvent-oil/fat solution, with the solvent being evaporated
by N2 flushing. Deswysen et al. [40] ensured fatty acid
dispersion in an aqueous environment through the nonionic surfactant Tween-80. The latter group illustrated a
possible effect of the mode of PUFA introduction on the in
vitro rumen fatty acid metabolism.
6.1.1 Rumen inoculum from adapted or

non-adapted donors
6.1.4 Gas pressure
Experiments evaluating the effect of adaptation of the
donor animal to dietary PUFA on in vitro BH are available
for 18:2n-6 sources only [33]. From this study, it was
concluded that dietary supplementation of soybean oil to
the donor cows did not affect in vitro BH of 18:2n-6, which
is consistent with the overall conclusions from experiments summarized in Tab. 2. Adaptation of the donor
animal might be of minor importance for 18:2n-6- and
18:3n-3-rich sources since rumen microbes of donor animals are continuously exposed to lower amounts of these
fatty acids through ingestion of forages or grains. However, this basal diet generally does not contain any of the
longer PUFA such as EPA and DHA. Based on milk CLA
concentration after fish meal supplementation, Abu Ghazaleh et al. [34] and Roy et al. [35] suggested that the
rumen microbial population required a gradual adaptation
of several weeks.
Among laboratories, a multitude of devices are used for

batch in vitro simulation of the rumen environment, with
some allowing gases produced during fermentation to
accumulate in the incubators, while others permit gas
escape to maintain gas pressure at low levels. Supplementation of non-esterified 18:2n-6 or 18:3n-3 in incubators where gases were continuously collected in syringes
and accumulated for 24 h resulted in enhanced production of 18:0 and lower amounts of BH intermediates
accumulated (J. P. Jouany, B. Lassalas, T. T. Chow, D.
Demeyer, V. Fievez, unpublished results). This is in accordance with the increased 18:0 concentrations in incubations
with sunflower oil under a 50 : 50 H2/CO2 atmosphere
compared to a 100% CO2 atmosphere [4]. This stimulating
effect of gas pressure accumulation has also been suggested for other hydrogen-consuming reactions, such as
methane production [41].
In a few in vitro studies, undegradable particles have been

added to create optimal conditions for solid-adherent
Obviously, in vitro conditions might affect estimates of

rumen BH and their kinetics, which underlines the need
for a detailed description of the incubation characteristics
and the inclusion of a control treatment under the same
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6.1.2 Particulate material
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V. Fievez et al.
Tab. 2. Overview of characteristics of 24-h batch in vitro incubations used in the current review and indication whether the
study resulted in appropriate (3) simulation or not () of in vivo 18:2n-6 or 18:3n-3 BH and production of the BH end-product
(18:0). The 18:0 cell is empty when the reported results did not allow evaluation of 18:0 production. In the final row, some
characteristics are summarized that impair appropriate in vivo simulation of both 18:2n-6 and 18:3n-3 BH as well as 18:0
production. IND (independent) is mentioned when the whole range of the above-mentioned features ensures appropriate
simulation.
Substrate
Incubated PUFA
main
[2]
18:2
18:3
[3] 18:2
18:3
[4] 18:2
18:3
[26] 18:2
18:3
[27] 18:2
18:3
[33] 18:2
[37]
[40]
[40]
[47]
[51]
Gas
Inoculum
ani- adap- time{{ pre-treatment{
mal## tation{ [h]
pH
start/
24
Total BH 18:0
volume
[mL]
source concentration
[mg/mL]
intro$
type
concentration#
[mg/mL]
seed
0.5 mm
C1U
10
12
0.8-mm sieve
1:1
6.9/UN 120
weight
18.8
1.6-mm sieve
1:1
7.0/6.4 160
hexane evapor. F
16
SH
15
1-mm sieve
1:4
7.0/5.8
25
finely ground
TMR
10
cheese-cloth (2) 1 : 4
UN/UN
52
weight
10
ST
12
UN/UN
50
weight
9.6
12
UN/UN 208
3
3
3
3
3
3
3
3
3
albumin
tween 80
hexane evapor.
hexane evapor.
weight
grinder
hexane evapor.
none
F
F
F
F
8
8
16
15
3
3
3
3
semi
C
C
C
SH
SH
5
1
1
15
15
particle free
0.5-mm sieve
0.5-mm sieve
1-mm sieve
cheese-cloth (4)
}
1:4
1:4
1:4
1:9
6.8/UN 10
7.1/6.4 50
7.1/6.4 50
7.0/5.7 25
6.7/UN 200
10
SH
15
1-mm sieve
1:4
7.0/6.0
weight
ethanol
UN
tween 80
albumin?
FC
TMR
SM
none
9.6
9.6
10
15
3
3
IND
C
C
ST
UN
3
IND
2
3
12
IND
cheese-cloth (2)
cheese-cloth (2)
cheese-cloth (2)
IND
1 : 4
18:2
1:4
no
buffer
UN/UN 52
UN/UN 52
UN/UN 50
,6.0 IND
oil
oil
corn
oil
oil or
NEFA
NEFA
NEFA
NEFA
oil
oil or
seed
oil
18:2
18:2
18:2
18:2
18:2
18:3
[60] 18:2
18:3
[87] 18:2 oil
[88] 18:2 NEFA
[89] 18:2 oil
Impaired simulation
0.24
0.50
0.31.9
0.04
0.20.6
0.10.5
0.3
0.01
0.03
0.06
0.10.5
0.2
0.040.2
0.040.2
0.32.7
0.2
1.0
0.20.6
0.3
0.30.5
0.10.3
0.7
0.5
dilution{{
50
}}
}}
3}}
3
3
3
3
3
3
3
3
3
3
}}
}}
3}}
3
3
3
In vitro gas accumulation (3) or not (); semi indicates that gas is allowed to accumulate for a certain period (3, 6 or 9 h)
after which it is released.
Initial concentration in incubation flask of predominant PUFA.

$
Mode of introduction of the PUFA source into incubation flask in case of oils or NEFA, with evapor. indicating that solvent has been evaporated prior to incubation, albumin indicating the use of bovine serum albumin to ensure that fatty
acids remain in suspension and tween 80 indicating the use of the non-ionic surfactant Tween-80 to disperse fatty acids in
the aqueous buffer solution. All seeds were weighed into the incubation flasks and the mode of grinding (coffee grinder or
through 0.5 mm mesh width) has been indicated as grinder and 0.5 mm, respectively.
$$
Type of fermentation substrate added (forage, F; concentrate, C; total mixed ration similar to donor cows diet, TMR;
soybean meal, SM; undegradable straw or hay particles, U).
#
Concentration of fermentable substrate; without undegradable straw when added.
##
Type of donor animal: sheep (SH), steer (ST) or dairy cow (C).
{
Adaptation of animal (3) or not () to the incubated PUFA source.
{{
Time of inoculum sampling after last meal or concentrate feeding.
{
Numbers between brackets indicate layers of cheesecloth. Particle-free inoculum obtained through centrifugation at
50006g.
{{
Rumen incolum/buffer (vol/vol).
pH at start (ST) of the incubation and minimum pH after 24 h of incubation; UN indicates values were not reported.
Reducing solution was added together with buffer and inoculum (0.04 of final volume).
}
Washed cell suspensions were added to sterile rumen contents in ratio 3 : 10, no buffer solution added.
}}
6-h [37] and 9-h [40] instead of 24-h in vitro incubation.
www.ejlst.com

conditions, to allow correct interpretation of data on the
accumulation of BH intermediates.
6.2 In vitro simulation of BH of unprotected

PUFA sources
The amount (mg/mL) of 18:2n-6 or 18:3n-3 lost during
24-h incubations from NEFA, oils or oilseeds that have not
been subjected to lipid protection technology is proportional to the amount (mg/mL) incubated (Fig. 2). The
overall linear relationship includes both NEFA and EFA,
indicating that 24-h in vitro BH is not limited by lipolysis or
impaired by the presence of EFA in the oleosomes of oilseeds. The linear relationship further illustrates 24-h in
vitro BH to be independent of the amount of fat added to
the incubation flask, although the rate of lipolysis and BH
has been suggested to decrease with increasing amounts
of soybean oil or PUFA in the culture substrate [3, 33]. The
slopes of these overall linear equations reveal that the in
747
vitro BH of 18:3n-3 (882 g/kg) exceeds the BH of 18:2n-6

(816 g/kg), in agreement with in vivo observations.
Moreover, 24-h in vitro and in vivo (825 and 875 g/kg for
18:2n-6 and 18:3n-3, respectively; [18]) slopes were
similar.
Average in vitro BH of EPA (367 6 295 g/kg, n = 28) and

DHA (225 6 242 g/kg, n = 52) are not consistent with in
vivo results (781 6 123 g/kg, n = 17 and 763 6 134 g/kg,
n = 17 for EPA and DHA, respectively) following fish oil
and microalgae supplementation [2325, 4245]. Large
differences between in vitro and in vivo BH data of EPA
and DHA might be attributed to the absence of these fatty
acids in the diet of the inoculum donor animals. Moreover,
an exponential decline in the 24-h BH of EPA and DHA
has been suggested by Gulati et al. [46] from their doseresponse study. Although inter-experimental variation
hampered the establishment of a global dose-response
relation, the exponential decline holds within most in vitro
experiments (Fig. 3). Variable composition of marine
sources as well as a lack of information on EPA and DHA
Fig. 2. Disappearance of 18:2n-6 and 18:3n-3 during 24h batch in vitro incubations with pure unesterified C18:2n6, oils or oilseeds [24, 26, 27, 33, 47, 51, 60, 61, 8789].
Fermentation substrates were mainly hay or lyophilized
grass or alfalfa, but total mixed rations (TMR) have been
added in some experiments n check TMRn. [18:2 lost
(mg/mL) = 0.816(SE = 0.0111; p ,0.001)618:2 initial (mg/mL),
n = 43;
18:3
lost
(mg/mL)
=
R2 = 0.986,
0.882(SE = 0.0054; p ,0.001)618:3 initial (mg/mL), R2 = 0.999,
n = 19].
Fig. 3. Biohydrogenation of EPA and DHA during 24-h

batch in vitro incubations relative to the initial amount of
EPA or DHA added through supplementation of pure
unesterified EPA or DHA, fish oil or microalgae [4, 26, 47,
51, 60, 61, 87, 90]. Data from dose-response studies are
connected with lines.
www.ejlst.com
748
V. Fievez et al.
lipolysis might contribute to inter-experimental differences. Indeed, accumulation of non-esterified EPA [4, 47]
and to a larger extent DHA [26] has been shown to exert a
negative feedback on the BH of EPA and/or DHA.
6.3 In vitro simulation of production of BH

intermediates and the saturated BH
end-product from unprotected PUFA sources
The adequate identification of oleic acid in the batch in
vitro incubations under consideration allowed the calculation of the net gain of 18:0 [3] which has been related to
the amounts of C18 PUFA lost. Oleic acid concentrations
in 24-h incubations of Akraim et al. [2] were not reported
and in this case an average BH value of oleic acid (491 g/
kg) has been assumed to calculate the oleic acid remaining after 24 h. The linear relationship revealed that a constant proportion (0.666; Fig. 4) of hydrogenated C18
PUFA was transformed into the saturated end-product,
18:0 (Fig. 4), which was not different from the in vivo conversion of hydrogenated C18 PUFA to 18:0 (0.599). The
similarity of the coefficients refutes the opinion that batch
in vitro conditions generally stimulate the accumulation of
BH intermediates. Nevertheless, accumulation of BH
intermediates is stimulated under in vitro conditions with
high substrate levels (.1.0 g/100 mL) in combination with
relatively high (.0.5 mg/mL) C18 PUFA supplementation
as NEFA or oils (Fig. 4). This might have been provoked by
a pH effect, which was on average 5.8 at the end of the
24-h incubations of Boeckaert [4]. Indeed some compre-

hensive studies by Van Nevel and Demeyer [48] and
Troegeler-Meynadier et al. [3] showed that a lower rumen
pH was associated with increased accumulation of BH
intermediates. However, incubations with relatively high
PUFA supplementation in combination with higher substrate levels all have been performed with sheep inocula
(Tab. 2). Hence, a higher sensitivity of microbes from
sheep compared to cow or steer inocula in terms of
accumulation of BH intermediates cannot be excluded.
6.4 In vitro assessment of protection

technology and additives
Formaldehyde-treated seeds, calcium salts and fish oil
supplementation are used as prototypes to assess the
reliability of batch in vitro incubations to estimate rumen
BH and production of the saturated end-products of protected PUFA sources or when supplementing antimicrobial BH modifiers. The choice for these prototypes
was based on the number of available in vitro and in vivo
data. To our knowledge, the effect of heat treatment, i.e.
extrusion, on PUFA BH has been assessed using both in
vitro and in vivo approaches for one seed only [2, 49].
Comparison of other in vitro and in vivo data is limited as
different technological processes and different oilseeds
were used, which illustrates the need for further in vitro-in
vivo comparisons of processed oilseeds.
6.4.1 Assessment of effectiveness of protection

technology: Formaldehyde treatment
Fig. 4. Net gain of 18:0 in relation to 18:2n-6 and 18:3n-3

lost by rumen BH (C18 PUFA lost) during 24-h batch in
vitro incubations with pure unesterified 18:2n-6, oils or
oilseeds and with substrate concentrations and C18
PUFA from oils below (diamonds) [net 18:0 produced (g/
kg DMI) = 0.666(SE = 0.054; p ,0.001)6C18 PUFA lost (g/kg
DMI), R2 = 0.968, n = 15] and beyond (triangles) 1 g/
100 mL and 0.5 mg/mL, respectively [24, 26, 27, 51, 60,
87, 88]. Observations represented by grey squares [3]
were not included in the linear model development and
illustrate a possible inhibitory effect of increasing
amounts of C18 PUFA as oils at higher substrate concentrations (1.6 g/100 mL) on the production of the saturated BH end-product from hydrogenated 18:2n-6 and
18:3n-3.
A procedure to protect polyunsaturated oil droplets from

rumen BH through their encapsulation in formaldehydetreated casein has been described decades ago [50].
Published reports on both in vitro and in vivo BH of the
same oil sources are limited ([51] vs. [45] and [52] vs. [53];
Tab. 3), but indicate that in vitro procedures tend to overestimate the degree of rumen inertness. From this, Gulati
et al. [54] suggested that effective in vivo protection only
can be guaranteed for products showing in vitro BH
values of 250 g/kg or lower. A further comparison of in
vitro and in vivo studies (Tab. 3), although not with the
same products, suggests oilseed pretreatment to be
essential for the formation of the inert formaldehyde-protein matrix, as extensive PUFA BH has been observed
both in vitro and in vivo when using formaldehyde-treated
oilseeds without pretreatment. In conclusion, batch in
vitro screenings might provide useful information on the
extent of protection of chemically treated fat supplements, but should include both a negative (unprotected)
as well as a positive control of which in vivo data are
available, to enable ranking of products for efficacy and
some speculation on the expected in vivo results.
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749
Tab. 3. Overview of extent of in vivo or 24-h in vitro BH of the predominant C18 PUFA of formaldehyde-treated (FT) oil
sources and their experimental control treatment.
Reference
Oil source
Pretreatment
Control
PUFA
(donor)
animal
BH [g/kg]
Con
FT
In vivo
[23]
[45]
[95]
[53]
[50]
linseed
linseed
canola seed
canola/soybean
linseed oil
no
HCOOH
alkaline
emulsification
emulsification
linseed oil
crushed canola seed
yellow grease
linseed oil
18:3
18:3
18:2
18:2
18:3
sheep
sheep
steer
steer
sheep
920
750
714
981$
927
852
648
570
425$
In vitro
[52]
[51]
[51]
[51]
[52]
[52]
[52]
[96]
canola/soybean
linseed
linseed
linseed
canola{
canola/soybean{
canola/soybean
cottonseed oil
no
no
HCOOH
NaOH
unknown
emulsification
emulsification
emulsification
ground linseed
ground linseed
ground linseed
sunflower oil
18:2
18:3
18:3
18:3
18:2
18:2
18:2
18:2
sheep
sheep
sheep
sheep
sheep
sheep
sheep
sheep
850
850
850
960
800
721
482
333
232
200
160
150
Procedure prior to formaldehyde treatment of protein. Emulsification has been mentioned when no information has
been provided on the chemical compounds used to prepare the emulsions.
$
Indicative data, as dietary 18:3 was not reported and has been assumed as 550 g/kg fatty acids, based on average 18:3
concentrations of linseed oil [6] and alfalfa hay [97].
#
Source used in vivo by Sinclair et al. [45].
{
Source used in vivo by Tymchuk et al. [98], without in vivo BH information.
{
Source used in vivo by Zinn et al. [53].
6.4.2 Assessment of effectiveness of protection

technology: Formation of calcium salts
The level of protection from rumen metabolism for calcium salts has been investigated extensively, with in
vivo reports varying from no protection to about 40% of
the PUFA being protected against rumen BH. Variation
could be related to dissociation of Ca salts, which is
determined both by the rumen pH as well as the degree
of unsaturation of the PUFA, as pKa values generally
seem related to unsaturation of the soaps [55]. Indeed,
in the same manner as lipolysis from glycerides is necessary, dissociation of calcium salts is needed before
BH. Moreover, the extent of dissociation of calcium
salts is usually much lower than the extent of lipolysis,
so that dissociation is the limiting factor of BH of PUFA
salts. In their comprehensive in vitro study, Van Nevel
and Demeyer [56] illustrated that the formation of calcium salts to protect PUFA against rumen BH is ineffective at rumen pH of 6.2 or lower. This threshold pH
value seems to hold in vivo. Indeed, significant reductions in BH of 18:2 and 18:3 were observed in pH ranges above 6.2 only, both under in vitro and in vivo conditions (Fig. 5). Data are presented relative to their control due to relatively high experiment-to-experiment
variation in BH of the controls (ranging from 750 to

990 g/kg). The study by Ferlay et al. [57] has been
excluded because of aberrantly low BH estimates,
whereas a more recent study of Lundy et al. [9] was not
included due to the lack of rumen pH information.
Based on dietary concentrate (500 g/kg) and maize
silage (450 g/kg) proportions in the latter study, an
average rumen pH below 6.2 is expected, which could
explain minor effects on BH when feeding soybean fatty
acids as calcium salts or as soybean oil.
As specific (conjugated) fatty acids exert a number of
physiological effects, efforts are intensified to formulate
rumen-protected lipid supplements enriched in these
fatty acids (e.g. [58]). With calcium salts being one of the
major commercially applied rumen protection formulations, attempts are made to apply this technology to
develop supplements containing these specific (conjugated) fatty acids (e.g. [59]). However, as no information
is available on pKa values of calcium soaps containing (a
mixture of) these specific positional and geometric isomers, in vitro screenings of the effectiveness of these
supplements at resisting rumen BH should include incubations at different pH values as well as the NEFA supplement as a negative control.
www.ejlst.com
750
V. Fievez et al.
Fig. 5. Effect of formation of Ca salts on BH of

18:2n-6 (triangles) and 18:3n-3 (diamonds),
expressed relative to the control treatment
within the experiment. Open symbols indicate
that BH of Ca salts does not differ from control; full symbols indicate significant differences between Ca salts and control. Grey
symbols are in vitro data from Van Nevel and
Demeyer [56]; black symbols represent in vivo
data from Fotouhi and Jenkins [91], Enjalbert
et al. [86, 92], Aldrich et al. [93], Klusmeyer
and Clarck [94], and Wu et al. [76].
6.4.3 Fish oil as a BH modifier

Net 18:0 production from C18 PUFA was negligible during
24-h batch in vitro incubations (Fig. 6), irrespective of the
amount of EPA 1 DHA incubated, although some 18:0
has been produced in incubations with 9.2 g EPA/kg DMI
[26]. This suggests batch in vitro incubations to be inappropriate to simulate the dose-dependent inhibition of BH
by EPA 1 DHA as observed in vivo (see Section 4.3), with
a complete inhibition of the 18:0 production from C18
PUFA for EPA 1 DHA intakes above 5 g/kg DMI. However, it should be noted that EPA 1 DHA intakes were
below the in vivo threshold of 5 g/kg DMI in the study of
Chow et al. [60] only and the in vitro EPA 1 DHA supply
ranged from 3 to 37 g/kg DMI. Accordingly, more in vitro
studies with lower amounts of EPA and DHA are needed
to appropriately evaluate the in vitro potential to simulate
in vivo fish oil supplementation. Results reported by
Wasowska et al. [61] have been omitted from this study,
as BH of 18:2n-6 and 18:3n-3 and the accumulation of
intermediates were aberrant, most probably due to the
incubation of rumen inoculum alone, without buffer.
Fig. 6. Effect of fish oil supplementation (triangles) on net

gain of 18:0 from C18:2n-6 and C18:3n-3 lost by rumen
BH (C18 PUFA lost) during 24-h batch in vitro incubations
with substrate concentrations below 1 g/100 mL [26, 60,
87]. Net 18:0 gain under circumstances without fish oil
supplementation (diamonds) has been added as a comparison.
BH calculations based on daily effluent data are similar to

those based on in vivo duodenal flows, whereas BH has
been calculated from differences in PUFA concentrations

of the input and the steady-state rumen fluid for studies
reported by Fellner et al. [38, 67] and Martin and Jenkins
[39]. Aberrantly low 18:2n-6 BH values (0650 g/kg) have
been calculated from the latter study upon supplementation of soybean oil, although pH (5.0 and 6.7), substrate
concentrations (0.05 and 0.1 g/100 mL) and dilution rates
(0.05 and 0.1 h1) were in line with the other studies
included in the dataset. However, both rumen inoculum
(particle-free fluid obtained after centrifugation of the
squeezed rumen contents instead of squeezed rumen
contents), fermentation substrate (mixed soluble carbohydrates instead of complete feeds) and PUFA-to-fermentable substrate ratio considerably differed. Particularly the absence of particulate material might have
impaired normal rumen BH. Hence, this study was further
excluded from the dataset. Observations by Jenkins et al.
[68] were not included in this dataset as samples were
taken from the rumen vessel 2 h after the morning feeding, which impaired the calculation of the BH.
www.ejlst.com
7 Continuous-culture in vitro techniques

To our knowledge, only eight papers reported the evaluation of rumen BH using the continuous-culture technique,
sampling either the daily effluent [6266] or the continuous-culture vessel after the installation of steady-state
PUFA concentrations [38, 39, 67] or 2 h after a pulse feeding [68] (Tab. 4).
7.1 Continuous-culture simulation of BH of

unprotected PUFA sources
751
Tab. 4. Overview of characteristics of continuous-culture fermentors used in the current review and indication whether the
study resulted in appropriate (3) simulation or not () of in vivo 18:2n-6 or 18:3n-3 BH and production of the BH end-product
(18:0). In the final row, some characteristics are summarized that impair appropriate in vivo simulation of both 18:2n-6 and
18:3n-3 BH as well as 18:0 production. IND (independent) is mentioned when the whole range of the above-mentioned
features ensures appropriate simulation.
Incubated PUFA
main source
[38] 18:2 NEFA

[39] 18:2 oil
[63] 18:2 grass
18:3
[64] 18:2 orchard
18:3 grass
[64] 18:2 red clover
18:3
[65] 18:2 alfalfa
18:3
[66] 18:2 NEFA
18:3 alfalfa
[66] 18:2 NEFA
18:3 alfalfa
[67] 18:2 NEFA
[68] 18:2 oil
Impaired red clover
simulation
Substrate
#
Dilution rate
Inoculum
Adap- pH
tation$
Total BH 18:0
volume
[days]
[mL]
concenintro
tration
[g/kg DMI]
C/F
29
53106
815
57
39
69
28
26
24
2.511
1535
2.3
15
2.3
1530
30
50
emul
emul
50 : 50
24
Soluble carboh. 0.62.4
0 : 100
14
C
ST
C
cheese-cloth (2)
0.068
particle free
0.050.10
cheese-cloth (2) 0.046
0.063
UN
UN
5
6.8
6.5
5.96.3
0 : 10032 : 68 50
0.18
UN
6.87.1 1200
0 : 10032 : 68 50
0.18
UN
6.87.1 1200
0 : 1008 : 92
100
0.10
6.2
1750
emul
62 : 38
120
cheese-cloth (2) 0.040.08 0.12
6.5
1780
emul
62 : 38
120
5.8
1780
emul
feed
IND
50 : 50
24
70 : 30
28
exclusively
IND
soluble carboh.
C
C
UN
cheese-cloth (2)
0.068
cheese-cloth (2)
0.068
particle free
IND
UN
4
IND
6.8
6.8
5.8
700
700
IND
amount
ani- pre-treatment
mal{
[g DM/day]
solid
liquid
[h1]
[h1]
0.12
700
500
700
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
One value for solid and liquid dilution rates indicates single-flow system.
Number of adaptation days of continuous system prior to experimental sampling.
#
Mode of introduction to ensure dispersion of fatty acids in case of oils or NEFA; emul refers to emulsions that are prepared through sonication.
{
Concentrate/forage ratio (C/F); in study [39], soluble carbohydrates (mixture of cellobiose, glucose, maltose and xylose)
were used; in study [65], the concentrate was sucrose.
{
Type of donor animal: steer (ST) or dairy cow (C).
Particle-free inoculum obtained through consecutive filtering through four layers of cheesecloth and centrifugation at
1506g.
$
Overall, a highly significant linear relation between 18:2n6 or 18:3n-3 inputs and their ruminal loss is observed
(Fig. 7). This suggests a negligible effect on the BH of
18:2n-6 and 18:3n-3 of varying continuous-culture conditions, such as the type of the continuous-culture system
(single vs. dual flow), sampling mode (from rumen vessel vs.
effluent), solid (0.040.08 h1) and liquid (0.0630.18 h1)
dilution rates, pH (6.07.25), 18:2n-6 (235 mg/g DMI) or
18:3n-3 (211 mg/g DMI) concentrations, forage/concentrate ratio of the substrate (100 : 030 : 70, wt/wt) and
substrate concentrations (0.10.24 g/100 mL). However,
continuous-culture simulations seem to overestimate in
vivo BH, as suggested from the slope of the linear
regressions (Fig. 7) (948 and 955 g/kg for the 18:2n-6 and
18:3n-3 regression, respectively). Nevertheless, this
might be due to the low number of continuous-culture
studies in combination with the high amounts of unester-
ified fatty acids in the majority of these experiments.

Indeed, the 27 observations used in the linear regression
of 18:2n-6 included six infusions of unesterified 18:2n-6
[38, 66, 67] and 16 NEFA-rich forages, either as silage
(n = 4) [63] or frozen fresh grass (n = 12) [64]. Both ensiling
as well as freezing followed by thawing of forage has been
shown to increase the amount of forage NEFA [69,
70].The linear regression of 18:3n-3 included 25 observations, of which 4 stem from ensiled forages [63], 12 from
thawed grass after frozen storage [64] and 9 from hay [65,
66]. Oilseed sources were completely absent in the continuous-culture database.
www.ejlst.com
Significantly less 18:2n-6 has been lost in continuous

cultures at pH 5.8 [66], which is in accordance with the
protective effect of low pH as suggested from the metaanalysis of duodenal fatty acid flows by Glasser et al. [21].
752
V. Fievez et al.
Fig. 8. Formation of 18:0 in single- (n = 3) and dual-flow

(n = 25) continuous-culture fermentors with pure unesterified 18:2n-6, oil, fresh, ensiled or dried forage [38, 6367]
in relation to 18:2n-6 and 18:3n-3 lost by rumen BH (C18
PUFA lost). Substrate concentrations varied between 0.1
and 0.24 g/100 mL. Grey diamonds indicate observations
at pH 5.8 [66] and cultures with red clover [64] and have
not been included in the linear regression. [18:0 produced
(g/kg DMI) = 0.533(SE = 0.054; p ,0.001)6C18 PUFA lost (g/kg
DMI) 1 0.860(SE = 0.071; p ,0.001)618:1 intake (g/kg DMI),
R2 = 0.809, n = 18].
Fig. 7. Disappearance of 18:2n-6 and 18:3n-3 in single(n = 3) and dual-flow (n = 25) continuous-culture fermentors with pure unesterified 18:2n-6, fresh, ensiled or dried
forages [38, 6367]. Substrate concentrations varied between 0.1 and 0.24 g/100 mL. Grey diamond indicates
observation at pH 5.8 [66], which has not been included
in the linear regression. [18:2 lost (g/kg DMI) =
0.948(SE = 0.0115; p ,0.001)618:2 initial (g/kg DMI), R2 = 0.999,
n = 27; 18:3 lost (g/kg DMI) = 0.955(SE = 0.0182; p ,0.001)618:3
initial (g/kg DMI), R2 = 0.995, n = 25].
However, the deviation from the linear curve of this lowpH observation is greater than suggested from the in vivo
meta-analysis (reduction of BH of 109 g/kg for each pH
unit decrease).
7.2 Continuous-culture simulation of

production of BH intermediates and the
saturated BH end-product from unprotected
PUFA sources
The production of 18:0 is a linear function of the amount of
C18 PUFA lost, with its slope (0.533; Fig. 8) being similar to
the slope observed under in vivo conditions (Fig. 1).
However, in the experiment by Qiu et al. [66], this linear
relation was not observed at a rumen pH of 5.8 (Fig. 8).
Similarly, Fig. 8 also shows that a smaller proportion of
hydrogenated 18:2n-6 and 18:3n-3 from red clover was
completely saturated to 18:0 [64]. Hence, these observa-
tions were not taken into account for the linear regression
development. No 18:1 was detected in gamagrass studied by Eun et al. [63], which resulted in aberrantly low 18:1
intake values when no or low levels of corn were added.
Accordingly, observations of these treatments were
excluded. From the limited number of observations, continuous-culture conditions could be suggested to simulate in vivo conversion of hydrogenated C18 PUFA to 18:0
relatively well. Moreover, this conversion was independent of the continuous-culture conditions, which seems an
improvement compared to the batch in vitro systems.
However, supplementation of fatty acids in continuousculture studies were less than the amounts provoking
accumulation of BH intermediates during in vitro batch
incubations (at least 1.5 g/100 mL substrate in combination with at least 0.5 mg/mL C18 PUFA as NEFA or oils). On
the other hand, fish oil supplementation (2 g EPA 1 DHA/
kg DMI) to continuous cultures with solid dilution rates of
0.03 and 0.06 h1 completely inhibited 18:0 formation
from hydrogenated C18 PUFA of soybean and linseed oil
[62]. Indeed, under these continuous-culture conditions,
the net 18:0 formed (18:0 formed minus oleic acid lost) did
not significantly differ from 0. This would indicate that
continuous cultures are no improvement over batch in
vitro cultures to quantitatively assess the in vivo dose response of fish oil supplementation. However, as the
amount of currently available experimental data from
continuous cultures is limited, further studies are needed
to evaluate the full potential of this methodology.
8 In sacco technique
The in sacco methodology is considered a convenient
compromise between the expensive in vivo approach
www.ejlst.com

using duodenal cannulas and the in vitro procedures
using a non-physiological environment. Traditionally, in
sacco procedures were applied to assess rumen protein
and fiber degradation, and only a limited number of
studies used this technique to assess rumen BH [2, 71
74]. This might not be surprising as the in situ disappearance of PUFA might originate both from physical
losses of (esterified) PUFA from the bag as well as from
BH, which would result in an overestimation of PUFA BH.
In an attempt to correct for these physical losses of
PUFA from the nylon bags, Enjalbert et al. [73] suggested
that calculations should rely on the decrease in PUFA
proportions in the bag residue relative to proportions in
the original substrate. This requires the assumption of an
equal BH of PUFA that have left the bag as those
remaining. Equal losses of 18:3n-3, 18:2n-6 and oleic
acid have been demonstrated with oilseeds, based on in
vitro incubations of nylon bags in sterilized rumen fluid
with added pancreatin [73]. This experiment proved that
all dietary fatty acids were released to the same extent,
but does not demonstrate equal BH of fatty acids
remaining in the bags versus those that have left the bag.
This is unlikely as EFA in intact protective structures such
as oleosomes probably will be predominant in the lipid
residues in the bags, whereas these structures will have
been destroyed in the lipids that have left the bags. This
could explain both the low BH of PUFA and the low 18:0
proportions in 24-h incubated bags, when compared to
in vitro [73] or in vitro and in vivo [2, 49] measurements.
However, it cannot explain the higher 18:1 trans proportions in the bag residues compared to in vitro or in vivo.
These higher amounts of trans fatty acids eventually
might be related to temporarily high concentrations of
non-esterified PUFA within the nylon bag, creating a
microenvironment which might be particularly toxic to
the more sensitive hydrogenating microbes responsible
for the final BH step (conversion of 18:1 trans to 18:0).
Mixing straw with the incubated oilseed might have
decreased the hydrophobicity and diluted the concentrations of non-esterified PUFA in the bags, which
resulted in increased BH rates and production of the
saturated BH end-product [74]. However, in situ BH
remained considerably lower compared to in vivo observations, even when the incubated oilseeds were mixed
with straw or hay and when cows adapted to a PUFArich diet were used. This illustrates the limited applicability of this method to simulate in vivo BH.
753
18:3n-3, 18:2n-6 and production of 18:0 from unprotected fatty acid sources, provided that some considerations are taken into account, which are summarized in the
final rows of Tabs. 2 and 4: (1) The amount of PUFA as
well as the fermentation substrate should be limited to
50 g/kg substrate DM and 10 mg/mL incubation fluid,
respectively. (2) The inoculum should not be treated to
remove all particles, but supplementation of additional
undegradable particulate material is not needed.
(3) Adaptation of the donor animal did not affect in vitro
C18 BH results, but less decisive conclusions could be
made for the type of donor animal, mainly because of the
low number of studies with sheep and steer inoculum.
(4) Weighing, solvent solutions with or without solvent
evaporation, and emulsification through sonication all
seem appropriate techniques to introduce fatty acid
sources into the incubator, and both grinding through a
standard sieve mesh as well as the use of a coffee grinder
are appropriate oil seed pretreatments. (5) Buffer solutions should avoid pH shifts below 6.0, but the inoculum/
buffer ratio is of less importance.
In vitro testing at different pH values is needed to assess
the effectiveness of calcium salts at resisting rumen BH.
However, overestimation of rumen inertness impairs
direct extrapolation of in vitro BH results of technologically protected PUFA sources. Hence, incubation series
of these and other protected sources should include a
negative (unprotected) as well as a positive control of
which in vivo data are available, to enable some speculation on the expected in vivo results.
In vitro BH of EPA and DHA seems dose dependent and
generally underestimates in vivo BH. Moreover, in vitro
supplementation of EPA and DHA most often completely
inhibits 18:0 production, unlike in vivo circumstances
where a dose-dependent inhibition of the trans 18:1 to
18:0 reduction has been suggested. Hence, optimization
of the in vitro methodology to improve simulation of EPA
and DHA rumen metabolism remains challenging. Adaptation of the inoculum donor animal to EPA or DHA supplements should be a first attempt to improve in vitro
simulations.
Acknowledgments
Overall, both batch and continuous-culture in vitro systems show the potential to appropriately simulate BH of
The authors are grateful to the OECD for providing a grant

to V.F. to participate in the biohydrogenation workshop
(an official pre-conference event of the 4th Euro Fed Lipid
Congress). B.V. is a Postdoctoral Fellow of the Fund for
Scientific Research-Flanders (Belgium).
www.ejlst.com
9 General overview on in vitro rumen fatty

acid metabolism and future prospects
754
V. Fievez et al.
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[Received: February 6, 2007; accepted: June 1, 2007]
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Assessing Rumen Biohydrogenation and Its Manipulation in Vivo, in Vitro, in Situ

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Assessing rumen biohydrogenation and its

Laboratory of Animal Nutrition

Reference techniques to study rumen biohydrogenation (BH) rely on the comparison of

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

The process of biohydrogenation (BH) reduces the rumen

rumen PUFA metabolism or ensuring the accumulation

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

2 Terminology and calculations

with EFAi 0h = EFA intake or in vitro supply of EFAi at the

2.2 Rumen BH of PUFA

Rumen in vivo, in vitro and in situ biohydrogenation

2.3 Production of BH intermediates and

with PUFAi 0h = PUFA intake or in vitro PUFA supply (in g)

Where sufficient literature information is available, we

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Biohydrogenation PUFAi 100

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

4.1 Rumen lipolysis

4.2 Rumen BH of PUFA

As with digestibility studies, determination of rumen PUFA

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4 Reference in vivo techniques: Duodenal,

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

Rumen in vivo, in vitro and in situ biohydrogenation

reported value obtained by Jenkins [18]. However, the BH

4.3 Production of BH intermediates and

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Fig. 1. Net duodenal gain of 18:0 (18:0 0.643618:1

Introduction of an interaction term (amount of C18 PUFA

5 Alternative in vivo approaches

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

and BH. Hence, total fatty acid intake can be assumed to

5.2 BH estimates through rumen sampling

(Ab)omasal sampling through the rumen cannula might

SE, standard error of the regression coefficient.

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

6 Batch in vitro techniques

6.1 In vitro conditions

Rumen in vivo, in vitro and in situ biohydrogenation

bacteria and rumen microbial BH (e.g. [2]), as earlier

6.1.3 Introduction of PUFA in incubation

6.1.1 Rumen inoculum from adapted or

Among laboratories, a multitude of devices are used for

In a few in vitro studies, undegradable particles have been

Obviously, in vitro conditions might affect estimates of

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

6.1.2 Particulate material

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

Initial concentration in incubation flask of predominant PUFA.

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

6.2 In vitro simulation of BH of unprotected

Rumen in vivo, in vitro and in situ biohydrogenation

vitro BH of 18:3n-3 (882 g/kg) exceeds the BH of 18:2n-6

Average in vitro BH of EPA (367 6 295 g/kg, n = 28) and

Fig. 3. Biohydrogenation of EPA and DHA during 24-h

2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

6.3 In vitro simulation of production of BH

Eur. J. Lipid Sci. Technol. 109 (2007) 740756

6.4 In vitro assessment of protection