Partial substitution of fish oil for linseed oil enhances beneficial fatty acids from rumen biohydrogenation but reduces ruminal fermentation and digestibility in growing goats

Abstract

This study was performed to investigate effects of partial replacement of fish oil (FO) for linseed oil (LO) on digestibility, ruminal fermentation and biohydrogenation in growing goats. Experiment 1 was carried out in four growing male goats aged 6 months in a 4 × 4 Latin square design. Goats were fed a basal diet supplemented with 25 g/kg dry matter either LO alone or in combination with tuna FO. Treatments were developed by replacing FO for LO at ratios of 0, 5, 10 and 15 g/kg DM corresponding to FO-0, FO-5, FO-10 and FO-15, respectively. Experiment 2 was carried out in an in vitro incubation system including 12 fermenters with the same four treatments. Each fermenter consisted of 40 mL goat ruminal fluid, 160 mL warm buffer, 2 g mixed substrates, and 50 mg FO-0, FO-5, FO-10 or FO-15. Fish oil inclusion reduced (P < 0.05) digestibility and nitrogen retention in Exp. 1. Increasing doses of FO in the diet induced a strong drop (P < 0.001) in ruminal total volatile fatty acid (VFA) concentration and protozoa population at 3 h post incubation, but did not affect individual VFA proportions. Substitution of FO for LO decreased mean concentrations of C18:0 (P = 0.057), c-9,c-12 C18:2 and C18:3n-3 (P < 0.001), but increased (P < 0.001) C20:5n-3 and C22:6n-3. Feeding FO-10 enhanced formation of ruminal c-9,t-11 conjugated linoleic acid (CLA) concentration compared with FO-0. Overall, combined data suggest that to improve ruminal concentrations of C20:5n-3, C22:6n-3, and c-9,t-11 CLA for deposition in tissues or milk with minimal risk of affecting digestibility and ruminal fermentation, a dietary supplementation of 15 g/kg LO and 10 g/kg FO would be suitable.

Full text

1
2Corresponding author: phuocthanh@ctu.edu.vn
Received May 3, 2021.
Accepted July 5, 2021.
Partial substitution of sh oil for linseed oil enhances benecial fatty acids from
rumen biohydrogenation but reduces ruminal fermentation and digestibility in
growinggoats
LamPhuocThanh,†,1,2, NoppharatPhakachoed,‡ WisitipornSuksombat,|| JuanJ.Loor,$, and
TranThiThuyHang¶
†Department of Animal Sciences, Can Tho University, Ninh Kieu, Can Tho 94000, Viet Nam ‡Department of
Animal Production Technology, Kalasin University, Mueang, Kalasin 46000, Thailand ||Technopolis, Suranaree
University of Technology, Muang, Nakhon Ratchasima 30000, Thailand $Department of Animal Sciences,
University of Illinoi at Urbana Champaign, Urbana, IL 61801, USA ¶Department of Agricultural Technology,
Can Tho University, Phung Hiep, Hau Giang 95000, Viet Nam 1Present address: Department of Animal
Sciences, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
ABSTRACT: This study was performed to in-
vestigate effects of partial replacement of sh oil
(FO) for linseed oil (LO) on digestibility, ruminal
fermentation and biohydrogenation in growing
goats. Experiment 1 was carried out in four
growing male goats aged 6months in a 4×4 Latin
square design. Goats were fed a basal diet supple-
mented with 25g/kg dry matter either LO alone
or in combination with tuna FO. Treatments were
developed by replacing FO for LO at ratios of 0, 5,
10 and 15g/kg DM corresponding to FO-0, FO-5,
FO-10 and FO-15, respectively. Experiment 2 was
carried out in an in vitro incubation system includ-
ing 12 fermenters with the same four treatments.
Each fermenter consisted of 40mL goat ruminal
uid, 160mL warm buffer, 2g mixed substrates,
and 50 mg FO-0, FO-5, FO-10 or FO-15. Fish
oil inclusion reduced (P<0.05) digestibility and
nitrogen retention in Experiment 1. Increasing
doses of FO in the diet induced a strong drop
(P < 0.001) in ruminal total volatile fatty acid
(VFA) concentration and protozoa population at
3h post incubation, but did not affect individual
VFA proportions. Substitution of FO for LO de-
creased mean concentrations of C18:0 (P=0.057),
c-9,c-12 C18:2 and C18:3n-3 (P<0.001), but in-
creased (P < 0.001) C20:5n-3 and C22:6n-3.
Feeding FO-10 enhanced formation of ruminal
c-9,t-11 conjugated linoleic acid (CLA) concen-
tration compared with FO-0. Overall, combined
data suggest that to improve ruminal concentra-
tions of C20:5n-3, C22:6n-3, and c-9,t-11 CLA for
deposition in tissues or milk with minimal risk of
affecting digestibility and ruminal fermentation, a
dietary supplementation of 15g/kg LO and 10g/
kg FO would be suitable.
Key words: fatty acid biohydrogenation, sh oil, growing goat, linseed oil, ruminal
fermentation
© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society
of Animal Science.
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribu-
tion, and reproduction in any medium, provided the original work is properly cited.
Transl. Anim. Sci. 2021.5:1-13
https://doi.org/10.1093/tas/txab116
INTRODUCTION
Conjugated linoleic acids (CLA) are known to
have anti-carcinogenic, anti-obesity, antioxidant
and anti-inammatory effects (Kim etal., 2016).
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2Thanh etal.
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Ruminant animal products are the richest sources
of CLA (Alfaia etal., 2017), a fact that has led to
vast amount of research in terms of feed formula-
tions to enhance the production of these fatty acids
(Siurana and Calsamiglia, 2016; Cabiddu et al.,
2017). Biohydrogenation (BH) of linoleic acid (LA)
and alpha-linolenic acid (ALA) takes place natur-
ally in the rumen to form CLA, followed by for-
mation of vaccenic acid (t-11 C18:1, VA) and then
stearic acid (SA) as the nal product (Bauman
etal., 2003). The t-11 C18:1 isomer, which is used
for further synthesis of c-9,t-11 CLA in adipose tis-
sues and mammary gland, is derived from incom-
plete BH of unsaturated fatty acids (UFA) in the
rumen. Linseed oil is one of the richest sources of
ALA (Thanh and Suksombat, 2015). Regarding
health effects of very long chain n-3 fatty acids
(FA) including eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA), Calder (2014) com-
piled evidence indicating that these FA could reduce
risk of cardiovascular morbidity and mortality, en-
hance mental development, reduce the burden of
psychiatric illnesses in adults and help maintain im-
portant roles in the eye and brain structure.
Docosahexaenoic acid, a main FA in marine
oil, is responsible for inhibiting ruminal BH of VA
into SA, resulting in an increase of trans C18:1
available for incorporation in tissue lipids (Lee
etal., 2008). Ferreira etal. (2016) concluded that
only a small amount of FO inclusion (2.5g/kg dry
matter, DM) in lamb diets was necessary to opti-
mize ruminal concentration of CLA, whereas Lee
et al. (2005) found a linear increase in duodenal
ow of total CLA with increasing FO up to 40g/
kg DM. Finding a proper amount of FO to replace
LO could increase concentrations of c-9,t-11 CLA,
ALA, EPA and DHA in the rumen as well as ru-
minant meat and milk. Dietary supplementation
of FO in combination with LO has been tested in
dairy and beef cattle (Brown etal., 2008; Shingeld
etal., 2011), but data in goats are scarce. Thus, the
aim of this study was to investigate how partial sub-
stitution of FO for LO affects feed intake, nutrient
digestibility, ruminal fermentation and ruminal FA
BH in growing goats fed a diet based on guinea
grass.
MATERIALS ANDMETHODS
All experimental procedures were conducted
following the Ethical Principles and Guidelines for
the Use of Animals issued by National Research
Council of Thailand. The study was performed at
Experimental Farm and Center for Scientic and
Technological Equipment, Suranaree University of
Technology, Thailand.
Experiment1
Animals, experimental design and diets. Four
growing male goats (Saanen breed), aged 6months
and weighing 18.13 ± 0.25 kg, were used in this
study. Goats were kept in individual wooden cages
(1.5m × 1.0 m × 1.4 m, L × W × H) and had free
access to water and a mineral block. The basal diet
consisted of concentrate fed in pelleted form and
chopped fresh guinea grass offered ad libitum (C:F
35:65). Diets were offered in equal amounts twice
daily at 07:00 and 17:00h. Goats were assigned to
treatments according to a 4× 4 Latin square design.
The treatment diet consisted of the basal diet sup-
plemented (DM basis) with 25g/kg DM either LO
alone or in combination with tuna FO. Treatments
were developed by replacing FO for LO at ratios of
0, 5, 10 and 15g/kg DM corresponding to FO-0,
FO-5, FO-10 and FO-15, respectively. Diets (Table
1) were formulated to meet nutrient requirements
of growing male goats (NRC, 2007). Pelleted con-
centrate was weighed daily into plastic bottles, oil
blends added, and then mixed well prior to feeding.
Goats were then offered fresh guinea grass for ab
libitum intake. Oil supplement was daily monitored
to conrm that the goats were supplemented with
2.5% DM of added oils in the total ration. Each
period lasted for 21days including 14days for ad-
justment and 7days for sample collection.
Sampling and measurements. Dry matter in-
take (DMI) was determined by weighing daily feed
offered and refused during the experiment and cor-
recting for the DM content of each dietary com-
ponent. Feed samples were pooled and stored at
−20°C for further analysis. From d15 to d19, total
feces and urine were collected to calculate ap-
parent nutrient digestibility and nitrogen balance.
Feces were collected in wire-screen baskets placed
under the oor of the cages, and urine was collected
through a funnel into plastic buckets containing
50mL of 10% H2SO4 to keep the nal pH below
3.After recording the weight, 10% proportions of
24h feces were collected and dried in a forced-air
oven at 60°C for 48h, milled through a 1-mm mesh
and stored at −20°C for subsequent chemical ana-
lysis. On d21, ruminal uid samples were collected
at 0 and 3h post morning feeding using a 100-mL
syringe. Aportion of ruminal uid was immedi-
ately xed with 10% formalin solution in sterilized
0.9% normal saline (1:9, v:v) for direct counting of
protozoa (Galyean, 1989). Another portion was
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Fish oil and biohydrogenation ingoats
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Table 1.Chemical composition and fatty acid prole of feed, oil, rumen uid + buffer and treatment
Item1
Feed ingredients
Rumen uid + buffer
Treatment3
Concentrate2Guinea grass Linseed oil Fish oil FO-0 FO-5 FO-10 FO-15
Chemical composition (%DM unless otherwise noted)
DM 91.00 21.08 46.91 46.91 46.91 46.91
CP 21.15 11.14 14.28 14.28 14.28 14.28
Lipid 3.52 1.15 4.43 4.43 4.43 4.43
Ash 10.66 10.16 10.08 10.08 10.08 10.08
NDF 35.96 61.47 51.23 51.23 51.23 51.23
ADF 19.11 34.62 28.46 28.46 28.46 28.46
NFC428.71 16.08 19.99 19.99 19.99 19.99
ME, Mcal/kg DM53.20 2.09 7.74 7.74 2.61 2.61 2.61 2.61
Fatty acid prole (μg/mg for feeds and oils, μg/mL for rumen uid + buffer)
C12:0 6.91 0.12 0.10 0.71 3.09 2.44 2.44 2.44 2.45
C14:0 5.00 0.09 0.60 38.63 9.11 1.78 1.97 2.16 2.35
C16:0 5.17 2.33 55.20 221.75 60.02 4.62 5.45 6.29 7.12
C18:0 1.47 0.35 32.20 63.20 104.27 1.53 1.68 1.84 1.99
t-9 C18:1 nd6nd nd nd 8.95 – – – –
c-9 C18:1 8.28 0.68 178.60 127.31 6.40 7.72 7.47 7.21 6.95
c-9,c-12 C18:2 7.09 1.83 165.30 16.99 4.12 7.71 6.97 6.23 5.49
C18:3n-3 nd 5.70 557.50 nd 2.67 17.55 14.76 11.97 9.19
C22:0 0.29 0.06 0.73 10.06 nd 0.16 0.20 0.25 0.30
C20:5n-3 nd nd nd 82.61 nd – 0.41 0.83 1.24
C22:6n-3 nd nd nd 373.74 nd – 1.87 3.74 5.61
1DM: dry matter; CP: crude protein; NFC: non-ber carbohydrate; NDF: neutral detergent ber; ADF: acid detergent ber, ME: metabolizable energy.
2Contained: 32% cassava distillers dried meal, 20% soybean meal, 17.5% corn distillers dried grains with solubles, 10% rice bran, 10% wheat bran, 8% molasses, and 2.5% mineral and vitamin supplement.
Mineral and vitamin supplement provided per kg of concentrate including 5,000 IU vitamin A; 2,200 IU vitamin D3; 15 IU vitamin E; 8.5g Ca; 6g P; 9.5g K; 2.4g Mg; 2.1g Na; 3.4g Cl; 3.2g S; 0.16mg Co;
100mg Cu; 1.3mg I; 64mg Mn; 64mg Zn; 64mg Fe; 0.45mgSe.
3FO-0, FO-5, FO-10 and FO-15: sh oil replaced for linseed oil at ratios of 0, 5, 10 and 15g/kg, respectively.
4Calculated as 100− (CP + NDF + lipid + ash).
5Calculated using values from NRC (2001) tables.
6Not detectable.
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4Thanh etal.
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immediately used to determine pH using a digital
pH meter (HI-5522, Hanna Instruments, Inc.,
US). Asubsample was also ltered through a clean
double layer of cotton cloth, and the liquid fraction
was acidied with 1M H2SO4 (9:1 w/w), centrifuged
at 10,000× g for 15 minutes and stored at −20°C for
analyses of volatile fatty acids (VFA) and NH3-N
concentrations.
Experiment2
Experimental design and treatments.Assessment
of FA BH was carried out in vitro using an incu-
bation system with 12 continuous fermenters. The
experiment was a completely randomized design
including the same four treatments as in Experiment
1.Total added oil alone or in the mixtures was 2.5%
DM in each fermenter.
Substrates, added oil, and inoculum. Feeds
including guinea grass and the concentrate mix col-
lected from Experiment 1 were used as substrates.
Guinea grass and concentrate were mixed at a 65:35
ratio (wt:wt, DM basis). Feed samples were ana-
lyzed for FA proles before conducting the in vitro
experiment. Oils were prepared and added into
in vitro fermenters mixed with tween 80 solution
(P1754, Sigma-Aldrich, USA). Ruminal contents
were obtained before the morning feeding from
four male goats, aged 6months, fed a diet based
on fresh guinea grass and 21% crude protein (CP)
concentrate (C:F 35:65) twice daily at 07:00 and
17:00 h for a 1-week prior to sampling. Ruminal
uid was transported in four pre-warm thermos
asks to the laboratory within 30min of collection.
Ruminal uid was ltered through a metal sieve
with a pore size of 1-mm to retain small particles
under continuous ushing with CO2 at 39°C. Fatty
acid proles of mixed substrates, oils and inoculum
are presented in Table 1.
In vitro incubation. Strained ruminal uid
(40mL) from each goat was added to the fermenter
containing warm buffer (160mL) and mixed sub-
strates (2.0 g). After 30 min, oil solutions were
directly added into the fermenters. Cultures were
continuously mixed in slow-shaking water bath
at 39°C under continuous ushing with CO2 gas.
Samples for FA analysis (5 mL) were taken at 0,
1, 2, 4, 6, 12 and 24h. Reactions were immediately
stopped by cooling in an ice bath.
Chemical Analysis
Feed and fecal samples were analyzed for DM,
organic matter (OM), CP, ether extract (EE), and ash
using standard methods (AOAC, 1998). Crude pro-
tein (N×6.25) was determined by the macro-Kjeldahl
method (Kjeltec™ 8100, Foss, Denmark), pro-
cedure 928.08 of AOAC (1998). Ether extract was
determined using petroleum ether in a Soxtec ex-
traction system (Soxtec 8000, Foss, Denmark), pro-
cedure 948.15 of AOAC (1998). Neutral detergent
ber (NDF) and acid detergent ber (ADF) were
analyzed following the methods of Van Soest etal.
(1991), adapted for the ber analyzer (FibertecTM
8000, Foss, Denmark). The concentration of N in
acidied urine samples and ruminal NH3–N concen-
tration were analyzed by the micro-Kjeldahl method
(AOAC, 1998). VFA concentration was determined
using a gas chromatograph (Filípek and Dvořák,
2009) equipped with a 30 m × 0.32mm × 0.15μm
lm fused silica capillary column (HP Innowax, AB
002, Agient, USA). Injector and detector temperat-
ures were 250°C. The column temperature was set
as follows: 80°C for 5min followed by increased at
10°C/min to 170°C, then increased at 30°C/min to
250°C and held at 250°C for 5min. VFA peaks were
identied based on their retention times, compared
with external standards (acetic acid, propionic acid
and butyric acid; Sigma-Aldrich, USA). To ana-
lyze FA composition, samples were trans-esteried
to methyl esters via a base-catalyzed step followed
by an acid-catalyzed step as described by De Weirdt
etal. (2013). The FA methyl esters (FAME) were ex-
tracted twice with 3 and 2mL of hexane and pooled
extracts were evaporated under N2 stream until dry-
ness. The residue was dissolved in 1mL of hexane
and analyzed by gas chromatography (HP 7890A
series, Agilent Technology, Palo Alto, CA, USA)
equipped with a 100 m × 0.25mm × 0.2 μm lm
fused silica capillary column (SP 2560, Supelco Inc,
Bellefonte, PA, USA) and a ame ionization de-
tector. The column temperature was kept at 70°C
for 4min, then increased at 13°C/min to 175°C and
held for 27min, then increased at 4°C/min to 215°C
and held for 17min, then increased at 4°C/min to
240°C and held for 10min. Fatty acids were identi-
ed by comparison of retention times with external
FAME standards (Supelco 37-Component FAME
Mix, Supelco Inc, Bellefonte, PA, USA). The CLA
mixture (Sigma–Aldrich, Louis, MO, USA) con-
tained c-9,t-11 CLA, t-10,c-12 CLA, c-9,c-11 CLA,
and t-9,t-11 CLA.
Statistical Analysis
Data in Experiment 1 were analyzed using
the GLM procedure. The statistical model was
Yijk=µ+ Oi + Aj + Pk+ εijk. Where Yijk observation
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Fish oil and biohydrogenation ingoats
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from animal j, receiving diet i in period k; µ, the
overall of mean; Oi, the effect of sh oil level (i=1 ,
2, 3, 4); Aj, the effect of animal (j=1, 2, 3, 4); Pk,
the effect of period (k=1, 2, 3, 4); εijk, the residual
effect. Mean amounts of FA in the Experiment
2 were statistically analyzed by a PROC MIXED
procedure with the statistical model Yijk= µ + Oi
+ Tj + (O×T)ij + εijk, where Yijk=the dependent
variable; µ=the overall mean; Oi=the xed ef-
fect of added oil; Tj=the xed effect of incuba-
tion time; (O×T)ij=the xed effect of interaction
between added oil and incubation time; εijk=the
random residual error. Goat inoculum source
was considered as a random factor. Orthogonal
polynomial contrasts (linear and quadratic) were
used to examine treatment effects on response
variables. Signicant differences among treat-
ment means were statistically compared using
Tukey. Statistical tests were performed using SAS
University Edition 2019 (SAS Institute Inc., Cary,
NC, USA). Signicant effect of treatment on least
squares means was declared at P<0.05 and ten-
dency was declared at 0.05≤ P<0.1.
RESULTS
Experiment1
Linseed oil was rich in ALA (557.50μg/mg),
whereas FO contained (µg/mg) high amounts of FA
including EPA (82.61) and DHA (373.74) that were
not present in feeds or LO. However, compared
with LO, FO also contained high amounts (µg/mg)
of some saturated FA consisting of C14:0, C16:0
and C18:0, thus, replacement of FO for LO in the
diet also increased their concentrations. Ruminal
uid + buffer contained high amounts (μg/mL) of
C16:0 andC18:0.
Intakes of DM, OM, and CP did not differ
across diets (Table 2). Replacement of FO for LO
in the diet resulted in increased (P<0.01) intakes
of C14–C18, EPA and DHA, but linearly decreased
(P<0.01) intakes of LA and ALA. Quadratic de-
creases (P<0.05) in apparent digestibility of DM,
OM and CP were observed with increasing levels
of FO in the diet. There was a remarkable drop
(P < 0.01) in OM digestibility when goats were
fed FO-15. Increasing dietary FO decreased lin-
early (P<0.05) nitrogen retention, accounting for
−12.90% in FO-15 as compared toFO-0 (Table 3).
Replacement of FO for LO in diets had
no effect on ruminal pH, NH3-N, VFA, and
protozoa population at 0h before feeding (Table
4). However, at 3h after feeding, total VFA con-
centration was inuenced (P< 0.01) by feeding
FO, with the lowest value observed in the FO-15
group relative to FO-0. Surprisingly, replace-
ment of FO for LO did not cause any shifts in
the proportions of individual VFA (P > 0.05).
Table 2.Effect of treatment diets on intakes
Item1
Treatment2
SEM P-value
Contrast3
FO-0 FO-5 FO-10 FO-15 L Q
Feed and nutrient intake, g/d
DM 666.37 642.32 650.14 635.33 48.43 0.822 0.615 0.646
OM 596.01 575.32 582.55 567.27 42.88 0.809 0.642 0.631
CP 99.38 97.17 98.25 95.70 4.13 0.652 0.674 0.552
EE 30.05 29.19 29.38 28.44 1.26 0.418 0.579 0.302
EE/DM, % 4.52 4.55 4.53 4.49 0.22 0.986 0.802 0.844
Fatty acid intake, g/d
C12:0 1.76 1.76 1.77 1.77 0.01 0.709 0.877 0.320
C14:0 1.29d1.41c1.53b1.64a0.02 <0.001 0.002 <0.001
C16:0 3.14b3.59b4.16a4.55a0.20 <0.001 0.083 <0.001
C18:0 1.04c1.12bc 1.11ab 1.28a0.05 0.003 0.216 0.001
c-9 C18:1 5.31a5.02ab 4.88bc 4.59c0.16 0.004 0.158 0.002
c-9,c-12 C18:2 5.25a4.63b4.19c3.62d0.15 <0.001 0.008 <0.001
C18:3n-3 11.60a9.34b7.62c5.57d0.48 <0.001 0.004 <0.001
C20:5n-3 0.00d0.27c0.54b0.77a0.04 <0.001 0.001 <0.001
C22:6n-3 0.00d1.20c2.42b3.46a0.18 <0.001 0.001 <0.001
Total FA 30.06 29.19 29.38 28.44 1.26 0.417 0.580 0.302
1DM: dry matter; OM: organic matter; CP: crude protein; EE: ether extract; FA: fatty acid.
2FO-0, FO-5, FO-10 and FO-15: sh oil replaced for linseed oil at ratios of 0, 5, 10 and 15g/kg, respectively.
3Linear (L) and quadratic (Q) effects of supplemented treatments.
a-dMeans within a row with different superscripts are signicantly different (P<0.05).
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Concerning ruminal microbes, compared with
those fed only LO, protozoa population de-
creased signicantly (P<0.01) in goats fed the
blended FO and LOdiets.
Experiment2
Dietary replacement of FO for LO resulted in
time-dependent shifts in ruminal FA concentrations
characterized by decreases in C18:0, LA and ALA
and concomitant increases in t-9 C18:1, EPA and
DHA (Table 5). Compared with LO inclusion alone,
FO and LO blends decreased (P<0.001) mean con-
centration of C18:0. Further analysis of FA changes
during incubation revealed that concentration of
C18:0 remained unchanged until 12h of incubation;
however, a different change was detected at 24h in-
cubation (Figure 7) with the highest amount in FO-0
(175.61 μg/mL) versus the lowest value in FO-15
(102.20 μg/mL). Relative to the amounts at 0 h
Table 4.Ruminal fermentation characteristics
Item
Treatment1
SEM P-value
Contrast2
FO-0 FO-5 FO-10 FO-15 L Q
0 h
pH 6.95 6.82 6.89 6.76 0.21 0.597 0.563 0.575
NH3-N, mg/dL 14.12 13.67 13.94 14.14 2.76 0.994 0.810 0.923
Total VFA, mM 83.10 82.71 80.88 82.12 4.32 0.893 0.813 0.540
Acetate, % 73.94 75.11 73.24 73.36 1.23 0.228 0.267 0.094
Propionate, % 17.41 16.08 17.79 17.28 1.09 0.239 0.199 0.198
Butyrate, % 8.64 8.82 8.97 9.35 0.75 0.611 0.934 0.293
Acetate/propionate 4.29 4.70 4.15 4.25 0.37 0.268 0.224 0.164
Protozoa3, ×106/mL 1.93 2.00 1.90 1.92 0.13 0.711 0.515 0.409
3 h
pH 6.66 6.66 6.55 6.66 0.18 0.777 0.822 0.565
NH3-N, mg/dL 22.07 21.06 20.90 17.92 2.72 0.261 0.996 0.164
Total VFA, mM 116.09a107.85ab 102.84b98.68b4.26 0.006 0.075 0.002
Acetate, % 70.26 71.39 70.29 70.48 0.95 0.373 0.185 0.390
Propionate, % 18.97 18.03 19.05 18.49 0.80 0.332 0.252 0.522
Butyrate, % 10.77 10.58 10.66 11.03 0.31 0.285 0.224 0.312
Acetate/propionate 3.71 3.96 3.70 3.83 0.20 0.318 0.225 0.494
Protozoa, ×106/mL 1.86a1.22b1.15b1.08b0.15 0.001 0.002 0.002
1FO-0, FO-5, FO-10 and FO-15: replacement of sh oil for linseed oil at ratios of 0, 5, 10 and 15g/kg DM, respectively.
2Linear (L) and quadratic (Q) effects of supplemented treatments.
3Protozoa counts were calculated based on cells per g rumen content.
a-cMeans within a row with different superscripts are signicantly different (P<0.05)
Table 3.Digestibility and nitrogen balance
Item1
Treatment2
SEM P-value
Contrast3
FO-0 FO-5 FO-10 FO-15 L Q
Digestibility, %
DM 71.19a67.59a,b66.45a,b63.12b2.13 0.010 0.169 0.005
OM 73.90a69.85a,b69.22b66.01b1.73 0.004 0.061 0.003
CP 80.52a78.67a,b77.20a,b74.91b2.12 0.045 0.521 0.016
Nitrogen balance, g/d
Intake N 15.90 15.55 15.72 15.31 0.66 0.652 0.674 0.552
Fecal N 3.09 3.30 3.58 3.85 0.43 0.167 0.713 0.052
Urine N 1.80 1.90 1.92 1.87 0.52 0.987 0.766 0.873
Retention N 11.01a10.34a,b10.22a,b9.59b0.45 0.023 0.214 0.014
1DM: dry matter; OM: organic matter; CP: crude protein; N: nitrogen.
2FO-0, FO-5, FO-10 and FO-15: sh oil replaced for linseed oil at ratios of 0, 5, 10 and 15g/kg, respectively.
3Linear (L) and quadratic (Q) effects of supplemented treatments.
a,bMeans within a row with different superscripts are signicantly different (P<0.05).
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Fish oil and biohydrogenation ingoats
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Table 5.The changes in mean amounts (μg/mL) of fatty acid during 24h incubation
Item
Treatment1
SEM
P-value2Contrast3
FO-0 FO-5 FO-10 FO-15 Trt T Trt × T L Q
Saturated FA
C12:0 58.99 56.66 55.77 53.53 11.93 0.552 <0.001 0.037 0.169 0.986
C13:0 3.67 3.08 2.36 2.36 2.10 0.161 <0.001 0.743 0.037 0.516
C14:0 34.96 35.30 35.32 35.45 7.45 0.997 <0.001 0.528 0.849 0.953
C15:0 6.31b6.59ab 6.91ab 7.08a0.96 0.092*0.002 0.119 0.015 0.803
C16:0 124.27 131.46 133.55 136.70 22.08 0.404 <0.001 0.143 0.112 0.701
C17:0 13.80 13.33 15.31 16.55 4.44 0.105 <0.001 0.986 0.028 0.374
C18:0 175.61a148.03b114.39c102.20c11.03 <0.001 <0.001 <0.001 <0.001 0.344
C20:0 5.13 5.33 5.32 5.25 1.00 0.867 <0.001 0.077 0.693 0.484
Monounsaturated FA
C14:1 11.05 10.15 9.75 9.68 2.15 0.190 0.159 0.636 0.049 0.391
C15:1 7.73 7.50 7.22 7.28 1.93 0.782 <0.001 0.291 0.364 0.718
C16:1 3.81 3.33 3.61 3.91 0.87 0.195 0.001 0.240 0.510 0.061
t-9 C18:1#24.61b31.87a32.72a29.28ab 7.34 0.010 <0.001 0.003 0.046 0.004
c-9 C18:1 109.22 115.93 108.72 102.09 24.12 0.420 <0.001 0.271 0.276 0.258
Polyunsaturated FA
t-9,t-12 C18:2 3.68c5.76a5.96a4.22b1.21 <0.001 <0.001 <0.001 0.240 <0.001
c-9,c-12 C18:2 70.12a69.42a65.39a52.96b16.70 0.021 <0.001 0.306 0.005 0.135
C18:3n-3 83.09a87.13a65.29b47.54c14.65 <0.001 <0.001 0.001 <0.001 0.002
c-9,t-11 CLA 1.40 1.70 1.97 1.67 0.83 0.154 <0.001 0.440 0.162 0.088
t-10,c-12 CLA 0.50 0.54 0.55 0.58 0.45 0.950 <0.001 0.796 0.582 0.955
C20:3n-6 1.26 1.27 1.50 1.32 1.33 0.921 <0.001 0.684 0.754 0.738
C20:5n-3 0.00d2.49c3.82b5.06a0.72 <0.001 <0.001 <0.001 <0.001 0.013
C22:6n-3 0.00d8.87c18.73b24.42a2.66 <0.001 <0.001 <0.001 <0.001 0.026
1FO-0, FO-5, FO-10 and FO-15: replacing sh oil for linseed oil at ratios of 0, 5, 10 and 15g/kg DM, respectively.
2Trt: treatment; T: time.
3Linear (L) and quadratic (Q) effects of supplemented treatments.
#t-11 C18:1 co-eluted with the t-9 C18:1 peak.
a-dMeans within a row with different superscripts are signicantly different (P<0.05).
*Means within a row with different superscripts are signicantly different (P<0.10).
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8Thanh etal.
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incubation, C18:0 concentration differed by 189.68,
137.84, 30.49 and −10.75% in FO-0, FO-5, FO-10 and
FO-15, respectively. Increasing dose of FO in place of
LO linearly decreased (P<0.001) concentrations of
ALA and LA (Table 5). Except at 1, 2 and 24h sam-
pling (Figure 2), the different pattern of ALA con-
centration remained until end of incubations while
the difference (P<0.01) of LA among the treatments
was observed only at 0, 6 and 24h incubation (Figure
3). The FO-10 treatment increased the formation of
c-9,t-11 CLA from the early (1h) to later stages of
incubation. The greatest amounts of c-9,t-11 CLA in
the FO-10 treatment were measured at 6 and 24h of
incubations (P<0.05, Figure 4). Relative to the FO-0
diet, the amounts of c-9,t-11 CLA with the FO-10
diet increased by 2.39- (at 6h) and 2.18-fold (at 24h).
Increasing amount of FO in the diets resulted in an in-
crease of t-9,t-12 C18:2 (quadratic effect; P<0.001),
the highest value in FO-10 (5.96μg/mL) versus the
lower value in FO-0 (3.68μg/mL). Fish oil substitu-
tion for LO notably increased (P=0.01) concentra-
tion of t-9 C18:1 during incubation (Table 5), with
the greatest response (P<0.05) detected with FO-10
at 6h of incubation compared with FO-0 (Figure 5).
Concentration of c-9 C18:1 was not affected by the
replacement of FO for LO, but its concentration was
higher (P<0.01) with FO-10 at 6h incubation rela-
tive to FO-0 (Figure 6). Regardless of FO dose, sh
oil resulted in linear (P<0.001) increases in concen-
trations of EPA and DHA (Table 5) and a linear de-
crease (P<0.001) in DHA concentration over time of
incubation (Figure 1).
DISCUSSION
Although Hassanat and Benchaar (2021) re-
ported that feeding increasing doses of LO linearly
reduced intake in dairy cows, the lack of effect of
treatment diet on DMI in the present study was in
agreement with previous studies in lambs and goats
(Ferreira etal., 2016; Thanh etal., 2018; Büyükkılıç
Beyzi etal., 2020). We speculate that feeding diets
that were iso-lipid in the present study contributed
to the lack of negative impact on DMI. Ferreira
et al. (2016) reported that total digestibility of
DM, OM, and CP was not affected in lambs sup-
plemented with sh oil. Relative to previous study,
the reduction of nutrient digestibility when diets
containing FO were fed in the present study could
have been due to the higher amounts of FO used
to replace LO. For example, Ferreira etal. (2016)
used only 7.5g/kg FO to substitute for soybean oil
in the diet of lambs. Furthermore, supplementation
of sh oil rich in EPA and DHA can be harmful to
microbial membranes in the rumen and led to re-
duced number of total bacteria (Huws etal., 2010),
which contributed to reduced nutrient digestibility.
A transient reduction in ruminal total VFA
concentration after 3h feeding FO diets indicated
that this oil disturbed fermentation by ruminal
microbes, which was in agreement with Ferreira
etal. (2016). The fact that sh oil inclusion caused
a reduction of total VFA concentration without
affecting individual VFA proportions implied
that concentrations of individual VFA including
Figure 1. Temporal change of C22:6n-3 concentration during 24h incubation. Values represent least square means (n=4, SEM=0.89). ***:
P<0.001.
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9
Fish oil and biohydrogenation ingoats
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acetate, propionate and butyrate decreased mark-
edly as sh oil was added into the diet. The lower
ruminal VFA production could have been due to
lower DM and OM digestibility, likely reecting
on the negative inuence of double bonds in EPA
and DHA on microbiota. Alinear drop in ruminal
protozoa population with increasing dose of FO in
the diet underscored that FO is highly toxic to rumi-
nal microbes. The greater mitigation of ruminal
protozoa population in the animals fed FO and LO
blends than those fed only LO seemed a result of
synergistic effect of oil combination (Soliva etal.,
2004). The observed decrease in ruminal protozoa
in this study was a result of oil supplementation
rich in long chain UFA. In fact, dietary lipids are
almost hydrolyzed in the rumen by microbial lip-
ases, releasing free long-chain FA that may inhibit
activity of ruminal microorganisms. Maia et al.
Figure 2. Temporal change of C18:3n-3 concentration during 24h incubation. Values represent least square means (n=4, SEM =3.77). *:
P<0.05; **: P<0.01; ***: P<0.001.
Figure 3. Temporal change of c-9,c-12 C18:2 concentration during 24h incubation. Values represent least square means (n=4, SEM=5.18).
**: P<0.01; ***: P<0.001.
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10 Thanh etal.
Translate basic science to industry innovation
(2007) concluded that microbial toxicity of EPA
and DHA, main FA in FO, was greater than ALA,
predominant FA inLO.
The shift in FA concentrations that occurred
during the 24h incubations (Figure 1–7) indicated
that continuous cultures were an adequate model
of ruminal activity, and BH of FA was compar-
able to what occurs in the rumen (Jenkins etal.,
2008). Replacement of FO for LO positively inu-
enced ruminal concentrations of ALA, LA, c-9,t-
11 CLA and C18:0 likely due to BH of ALA and
LA (Szczechowiak etal., 2016). It is well known
that DHA in FO could inhibit the reductase ac-
tivity of ruminal bacteria responsible for the con-
version of VA to C18:0 (Toral etal., 2010), which
would allow for the production of cis and trans
Figure 5. Temporal change of t-9 C18:1 concentration during 24h incubation. Values represent least square means (n=4, SEM =2.11). *:
P<0.05.
Figure 4. Temporal change of c-9,t-11 CLA concentration during 24h incubation. Values represent least square means (n=4, SEM=0.23). *:
P<0.05.
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11
Fish oil and biohydrogenation ingoats
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C18:1 isomers in the rumen (Laverroux et al.,
2011), and use of VA for c-9,t-11 CLA synthesis
by Δ9 desaturase in mammary gland (Shingeld
etal., 2013). Although concentrations of 18:1n-
9, 18:2n-6 and 18:3n-3 with FO were numerically
lower than those in LO, ruminal concentration of
t-9 C18:1 (Table 5) increased linearly with increas-
ing amounts of FO oil in the diet. Despite FO
leading to higher (nearly two-fold) C18:0 content
than LO, the lower amounts of ruminal C18:0
with FO-10 and FO-15 demonstrated that re-
placement of FO for LO at 10 and 15g/kg DM
reduced VA or other unsaturated-C18 hydrogen-
ation to form stearic acid. Similar results were ob-
served in previous studies (Shingeld etal., 2012;
Toral etal., 2016; Thanh etal., 2018).
A limitation of the FA protocol in this study
was that it could not dene the t-11 C18:1 peak
from other trans isomers of similar elution time;
therefore, it could not explain the changes of all
Figure 6. Temporal change of c-9 C18:1 concentration during 24h incubation. Values represent least square means (n=4, SEM=7.93). **:
P<0.01; ***: P<0.001.
Figure 7. Temporal change of C18:0 concentration during 24h incubation. Values represent least square means (n=4, SEM=5.61). *: P<0.05.
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12 Thanh etal.
Translate basic science to industry innovation
FA involving the process of LA and ALA BH.
The greater amounts of t-9 C18:1 with FO diets
suggested there was an increase in the conversion
efciency of VA to t-9 C18:1 or that the produc-
tion of VA was large enough to co-elute with the
t-9 C18:1 peak. Klein and Jenkins (2011) reported
that DHA (main FA in FO) can elevate trans-18:1
isomers. Kholif etal. (2020) reported that inclu-
sion of EPA-rich microalgae in the diet of dairy
goats increased t-9 C18:1 concentration in milk
fat. The higher amount of c-9,t-11 CLA in FO-10
at 6h after incubation suggested that use of FO
at this level in place of LO not only inhibited the
conversion of VA to C18:0, but also prevented
hydrogenation of c-9,t-11 CLA to VA. However,
feeding FO-15 elicited a modest improvement in
c-9,t-11 CLA compared with FO-10 largely be-
cause LO-15 had a lower amount of LA. Feeding
sh oil in dairy goats increased milk c-9,t-11
CLA (Büyükkılıç Beyzi etal., 2020). A reduced
amount of ruminal EPA and DHA after 24 h
incubation reected extensive BH of these FA
in the rumen (Shingeld et al., 2012; Kairenius
etal., 2015). Alinear increase in amount of rumi-
nal EPA and DHA over time of incubation with
increasing FO level in the diet indicated that BH
of EPA and DHA was partially inhibited when
levels of these FA was high. Ferlay and Chilliard
(2020) reported an increased milk DHA content
in dairy cows fed 2.5% sh oil. This was also in
agreement with the ndings of AbuGhazaleh and
Jenkins (2004), who noted a decrease in the per-
centage disappearance of EPA and DHA in batch
cultures with increased FO supplementation.
CONCLUSION
Substitution of FO for LO decreased nutrient
digestibility and ruminal total VFA concentra-
tion without affecting individual VFA propor-
tions. Replacement of FO for LO from 5 to 15g/
kg DM remarkably decreased ruminal protozoa
populations. Increasing dose of FO in the diet re-
sulted in decreased mean amounts of C18:0, LA
and ALA but increased mean amounts of EPA
and DHA. Mean amount of c-9,t-11 CLA was
not change by feeding FO, but the higher con-
centration of this FA was detected in FO-10 at 6
and 24h incubation. To improve ruminal concen-
trations of EPA, DHA, c-9,t-11 CLA and reduce
C18:0 concentration without or less affecting di-
gestibility and ruminal fermentation, a dietary
supplementation of 15g/kg LO and 10g/kg FO
would be suitable.
ACKNOWLEDGMENTS
This study was nancially supported by Ministry
of Education and Training, Viet Nam (#B2021-
TCT-09). Authors expressed the special thanks to the
Center for Scientic and Technological Equipment
and the Institute of Research and Development,
Suranaree University of Technology, Thailand.
Conict of interest statement. The authors
declare no conicts of interest exist.
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