Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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INFLUENCE OF GRADED LEVELS OF NIGELLA SATIVA SEEDS
AND ROSMARINUS OFFICINALIS LEAVES ON IN VITRO
RUMEN FERMENTATION AND APPARENT
BIOHYDROGENATION
Kifah Jumaah Odhaib*,** , Awis Qurni Sazili*,***,****
*Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
** Department of Physiology, College of Veterinary Medicine, University of Basrah,
61004 Basra, Iraq
*** Laboratory of Sustainable Animal Production and Biodiversity, Institute of
Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
**** Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
Keywords: y NS seeds, Fatty acids, in vitro.
Corresponding Author:kifahodhaib@gmail.com.
ABSTRACT
The results in present study are illustrated that the effects of different levels of NS
seeds and RO leaves on in vitro gas production, rumen fermentation, fatty acids
composition and the apparent biohydrogenation of oleic, linoleic and linolenic acids
using rumen liquor from Dorper lambs. The NS seeds and RO leaves were
supplemented at the rate of 0, 0.5, 1, 1.5 and 2% (w/w) DM of basal substrate [60%
forage (urea treated rice straw) and 40% concentrate] and incubated for 24 h at 39°C.
Substrates containing RO and NS had greater (P<0.05) gas production than the control
substrates. The volume of gas produced increased as the levels of RO and NS
increased up to 1.5% and decreased afterwards. Supplementation of RO and NS did
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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not affect (P>0.05) in vitro dry matter digestibility, in vitro organic matter
digestibility, rumen pH, CH4 and NH3-N, total volatile fatty acids (VFA) and the
molar proportion of acetate, propionate and butyrate. The RO supplements reduced the
ruminal concentration of C18:0 and increased the ruminal concentration of C18:1n-9
in a dose dependent manner. The supplementation of RO leaves reduced (P<0.05) the
apparent biohydrogenation of C18:1n-9 but had no effect (P>0.05) on the apparent
biohydrogenation of C18:2n-6 and C18:3n-3.
INTRODUCTION
Herbs are used in animal feeds as the growth promoters. They play a major role as
antibacterial, antioxidant, anthelmintic and anticoccidial. Majority of medicinal plants
do not have the residual effects. It has been shown that phytochemicals and plant
secondary metabolites could increase protein flow to the duodenum (1). The plants
containing saponins have been found to suppress or eliminate protozoa from the
rumen and decrease ammonia and methane production (2).
Plant polyphenols are commonly employed in ruminant nutrition and their usage could
have positive and/or detrimental effects on rumen metabolism (3). The effects of plant
polyphenols on rumen metabolism depends on the chemical nature of the polyphenols,
the quantity in the feed, the abundance and diversity of rumen microbes and the
response of rumen microbiota to the polyphenols (4;5). However, their primary
sources are mainly the medicinal herbs (6).They are not always well characterized
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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because there are differences in their composition among various plant species and
parts. Moderate concentration of these polyphenols in many plants may possibly
reduce ruminal protein breakdown and increase duodenal protein flow (7).
Nonetheless, when given to animal at higher doses they may also adversely affect the
animal performance. Higher concentration in the diets has been known to cause a
negative effect on digestibility of feed and nutrients in ruminants (8). This is due to
their ability to bind nutrients particularly proteins and carbohydrates which in turn
modifies the rate and extent of their digestion (9).
In some cases, presence of very high levels of polyphenols, particulary tannins can
produce toxicity and eventually lead to death of the animals (10). Several feeding
strategies have been introduced such as addition of antimicrobial constituents which
modifies the rumen environment thereby improving animal’s health and productivity
these will increase yield and improve quality of products. Digestibility of herbs has
been tested in vitro (11). Tannins have been used to reduce rate of biohydrogenation in
vitro (12). Oil rich in polyunsaturated fatty acids (PUFA) have been supplemented in
the diet of sheep (13) and alter to the rumen fermentation characteristics in cattle (14).
However, there is no information on the use of the herbs on biohydrogenation of fatty
acids.
There are new approaches to increase the effectiveness of digestion and metabolism of
nutrients that will improve productivity and product quality and environment. One
economical, sustainable and safer approach is the use of herbs such as Nigella sativa
(NS) and Rosmarinus officinalis (RO) which contain diterpenoids, and polyphenols
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such as flavonoids, tannins and essential oils (15; 16). These PSMs are among the
other potent anti-microbial agents present in Nigella sativa seeds and Rosmarinus
officinalis leaves such as alkaloids, saponine, thymo, luteolin , carnosic acid, and
rosmarinic acid were the major phenolic compounds in these two herbs (17; 18 ). It is
possible that the presence or combination of these plant bioactives could improve the
effectiveness of nutrient utilization in ruminants. The in vitro study is a relatively
simple, cheap and direct measurement of microbial activities in vitro and reflects all
nutrients fermented. Thus, the purpose of this finding was to examine the suitable
level of Nigella sativa seeds and Rosmarinus officinalis leaves from in vitro rumen
metabolism using an in vitro gas production technique.
The objectives of this study were:
1- To determine the suitable level of Nigella sativa seeds and Rosmarinus
officinalis leaves from in vitro gas production and rumen fermentation
parameters.
2- To determine the suitable level of Nigella sativa seeds and Rosmarinus
officinalis leaves from in vitro fatty acid composition and apparent
biohydrogenation using rumen liquor from Dorper lambs.
MATERIALS AND METHODS
Animal welfare and source of medicinal herbs
The finding was carried out in line with the guidelines approved by the Universiti
Putra Malaysia Institutional Animal Care and Use Committee. For the Care and Use of
Animals Scientific Purposes (Research Policy, Universiti Putra Malaysia). Nigella
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sativa seeds and Rosmarinus officinalis leaves were purchased from a local market in
Selangor, Malaysia. The seeds and leaves were ground into powder, packaged in airfree
polyethylene bags and stored in a cool, dry place prior to analysis.
Treatments and experimental design
The experimental design was a completely randomized design, with three replications
per treatment including triplicates of blank in three incubation runs. The basal
substrate was 60% forage (urea treated rice straw) and 40% concentrate. The effect of
NS seeds and RO leaves were examined in a separate incubation runs. The following
diets were formulated:
1. Basal diet an additive (control).
2. Basal diet + 0.5% (w/w) DM NS seeds or RO leaves.
3. Basal diet + 1% (w/w) DM NS seeds or RO leaves.
4. Basal diet + 1.5% (w/w) DM NS seeds or RO leaves.
5. Basal diet + 2% (w/w) DM NS seeds or RO leaves.
Upon completion of the in vitro digestibility trial, the levels of NS and RO were
increased to 2% (w/w) DM and for biohydrogenation following the procedures of
Hassim et al. (19). The results generated through the in vitro studies were then used as
baseline data for the conduct of the subsequent feeding trial.
Estimation of total phenol, tannins and non-tannin phenol
Determination of total polyphenol and tannins were executed following the procedure
of Makkar et al. (21, 22). The phytochemical contents of the substrates are as
presented in Table 3.1.
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1.1.1 Proximate analyses
The (DM) dry matter, (CP, total nitrogen x 6.25) crude protein, crude fat and ash
content of the treatments were evaluated in line with to the protocol of (23). The
(NDF) Neutral detergent fiber were estimated according to the method of Van Soest
et al (24) as well as and (ADF) acid detergent fiber. The nutrient compositions of the
substrates are shown in Table 3.1.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Table . 1.خطأ! لا یوجد نص من النمط المعین فی المستند Chemical composition of in vitro experimental diets
Parameter (%) DM Substrates
control 0.5% RO 1% RO 1.5% RO 2% RO 0.5% NS 1% NS 1.5% NS 2% NS
DM 91.95 92.23 91.87 91.92 91.67 92.98 92.36 92.06 92.37
Crude protein 16.81 16.96 16.66 16.53 16.73 16.72 16.85 17.04 16.78
Crude fat 3.39 3.46 3.44 3.27 3.39 3.53 3.24 3.24 3.35
Ash 8.08 8.43 8.35 8.18 8.37 8.06 8.06 8.32 8.45
ADF 27.93 27.66 27.81 28.94 28.07 28.21 28.60 29.58 29.30
NDF 58.75 56.49 58.01 57.83 58.28 56.80 57.40 57.04 56.18
Phytochemical
compounds
Total polyphenol (g/kg) 3.46 25.11 46.75 68.40 90.04 22.31 41.15 59.99 78.80
Non-tannin polyphenol
(g/kg)
1.02 6.38 11.73 17.89 22.44 2.10 3.18 4.26 5.34
Tannin polyphenol (g/kg) 2.96 19.25 35.54 51.05 68.12 20.73 38.49 56.26 74.02
DM = dry matter. ADF=acid detergent fiber. NDF= neutral detergent fiber. RO = Rosmarinus officinalis leaves. NS=Nigella sativa seeds.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Rumen liquor collection
The rumen liquor was collected from three fistulated Dorper sheep fed diet containing
40% concentration and 60% forage (urea treated rice straw) for 2 weeks before feeding
the animal in the morning into a thermo flask and continuously flushed with CO2 while
the liquor was transported to the laboratory. The rumen liquor was pooled together and
sieved with the aide of cheese cloth in four layers.
In vitro rumen fermentation of substrate
The method of Menke and Steingass (25) was adhere to during the in vitro gas
production experiment. The strained rumen liquor was included inside the media in a 1:2
(v/v) ratio under nonstop CO2 flushing in a water bath at a temperature of 39 ºC. The
media is made up of 200 mL macro mineral solution, 0.1 mL micro mineral solution,
200 mL buffer solution, 40 mL reducing solution, 1 mL Resazurin solution and 400 mL
distilled H2O
pH determination
After 24 h incubation the pH of the rumen fluid was measured using (Mettler- Toledo,
Ltd England) pH meter.
In vitro dry matter digestibility (IVDMD)
The IVDMD was measured in accordance with the method of Tilley and Terry (26).
Determination of volatile fatty acids (VFA)
After 24 h incubation, 25% metaphosphoric acids in the of ratio 4:1 (v/v) was added to
the rumen fluid for the evaluation of the VFA profile with the aide of Gas Liquid
Chromatography (Hewlett Packard 6890).
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Determination of ammonia nitrogen (NH3-N)
The NH3-N concentration of the treatment were evaluated with following the procedure
of Solorzano (27).
Calculations
The Neway software program was employed to fit the data from gas production into the
model V = a + b (1 – e^(-ct)) has explained by Ørskov and McDonald (28). Each of the
variables in the model are detailed below.
V = Gas quantity at a specific period t, a = quantity of gas produced from the soluble
portion, b = Gas quantity produced from the insoluble portion, c = rate of gas production
for the insoluble portion and t = time of incubation. However, ME, IVOMD and
methane were calculated using the formular below Menke and Steingass (25) and Moss
et al. (2000)
ME=.0.0029.CF + 0.057.CP+ 0.136V + 2.20,
IVOMD%=.14.88+ 0.651AS +.0.45CP +.0.889 V
CH4=.0.45C2 -.0.275 C3 +.0.4 C4.
CF (crude fiber), CP (crude protein),.V total quantity of gas produced, AS (ash).
1.1.2 Determination of FA composition of substrates and rumen liquor
The mixture of chloroform: methanol (2:1, v/v) was employed as the extraction solvent
for the total FA from each substrate diet (1 g) or rumen liquor (10 mL) in accordance
with Folch et al (29) altered by Rajion et al (30). The fatty acid composition of the
treatment are presented in Table 3.2.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Table . 2.خطأ! لا یوجد نص من النمط المعین فی المستند Fatty acid composition (% of total fatty acid) of substrates
Substrates
Parameter Control 0.5 % RO 1% RO 1.5 %RO 2% RO 0.5 %NS 1% NS 1.5 %NS 2% NS
C14:0 0.45 0.56 1.26 0.24 0.21 1.12 0.470 0.24 0.43
C16:0 15.97 17.36 16.09 16.37 14.07 14.07 15.018 15.08 15.82
C16:1 0.55 0.65 0.29 0.55 0.43 0.81 0.322 0.43 0.66
C18:0 5.15 6.31 6.24 4.83 5.24 5.32 5.169 5.43 4.78
C18:1n-9 21.52 25.76 19.99 21.25 26.27 24.35 24.26 26.82 20.49
C18:2n-6 10..27 10.02 10.8 11.15 11.50 10.5 10.57 11.23 11.45
C18:3n-3 6.73 3.94 5.47 5.37 5.63 3.72 4.132 4.63 5.61
C20:4n-6 0.90 1.81 0.94 1.03 0.79 1.79 0.88 0.75 0.77
C20:5n-3 0.91 1.42 1.03 0.88 0.41 1.28 0.63 0.39 0.67
C22:5n-3 0.52 1.02 0.35 0.97 0.58 0.74 0.41 0.43 0.79
C22:6n-3 0.95 1.52 1.01 2.15 1.83 1.95 2.16 1.83 2.48
ΣSFA 21.58 24.25 23.60 21.45 19.53 20.51 20.65 20.76 21.04
ΣUFA 42.38 46.14 40.00 43.39 47.46 45.18 43.37 46.54 42.93
ΣMUFA 22.08 26.41 20.28 21.81 26.70 25.17 24.58 27.2 21.15
ΣPUFA 20.30 19.73 19.68 21.57 20.75 20.01 18.79 19.20 21.78
Σ n-3 9.12 7.90 7.88 9.39 8.46 7.71 7.340 7.29 9.56
Σn-6 11.17 11.82 11.79 12.18 12.29 12.30 11.45 11.98 12.22
n-6/n-3 1.22 1.49 1.49 1.29 1.45 1.59 1.55 1.64 1.27
UFA:SFA 1.96 1.90 1.70 2.02 2.43 2.20 2.10 2.24 2.04
PUFA:SFA 0.94 0.81 0.83 1.00 1.06 0.97 0.90 0.92 1.03
RO = Rosmarinus officinalis leaves. NS=Nigella sativa seeds. ΣSFA = (C14:0 + C16:0 + C18:0), ΣMUFA = (C16:1+ C18:1), ΣUFA = (C16:1+
C18:1+Σn-3 + Σn-6), ΣPUFA = (Σn-3 + Σn-6), Σn-3 = (C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3), Σn-6 = (C18:2n-6 + C20:4n-6) n-6:n-3 =
(C18:2n-6 + C20:4n-6) ÷ (C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3).
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1.1.3 Rate of biohydrogenation
The rate of disappearance or apparent biohydrogenation of oleic acid (C18:1n-9)
linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6) were calculated from the
difference in the concentration of the fatty acids between 0 h and 24 h in vitro incubation
as: Apparent biohydrogenation (%) = [100*[(CFA)i – (CFA)f ]/ (CFA)i]
Where (CFA)i= % concentration of unsaturated fatty acid at 0 h incubation,
(CFA) f = % concentration of unsaturated fatty acid at 24 h incubation (19).
1.1.4 Statistical analysis
The gas production parameter was examined in a completely randomized design using
the MIXED procedure of SAS (31) with sampling time as a repeated measure. Data for
fatty acid composition, VFA and rumen fermentation were analyzed in a completely
randomized design using the generalized linear model (GLM) procedure of SAS (SAS,
2003). For all parameters, differences between treatments means were separated by
Duncan Multiple Range test. Mean differences were considered significant at (P< 0.05).
Results
1.1.5 In vitro gas production and rumen fermentation parameters
The in vitro gas production and rumen fermentation parameters of substrates
supplemented with graded levels of Rosmarinus officinalis leaves and Nigella sativa
seeds are as presented in Tables 3.3, 3.4 and 3.5. Supplementation of graded levels of
RO and NS influenced (P<0.05) gas production throughout the 24 h incubation (Table
3.3). Substrates containing RO and NS had greater (P<0.05) gas production than the
control substrates. There were significant differences (P<0.05) among substrates
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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containing RO and NS. The volume of gas produced increased as the levels of RO and
NS increased up to the 1.5% and decreased afterwards (Table 3.3).
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Table . 3.خطأ! لا یوجد نص من النمط المعین فی المستند In vitro gas production of substrates containing graded levels of Rosmarinus
officinalis and Nigella sativa seeds during 24 h incubation
Treatment 3 h 6 h 9 h 12 h 15 h 18 h 21 h 24 h SEM P value Diet*Time
0% RO 11.95cz 14.62cz 17.84cy 22.05by 26.24bx 28.66bx 32.27bw 34.95bw 1.039 <.0001
0.5% RO 20.62aby 21.63bxy 25.80abxy 28.95abwx 32.70abwx 34.77abw 37.34abv 39.39abv 1.76 1% RO 21.40aby 32.25abxy 27.20abxy 31.40awx 35.39aw 38.96aw 41.44aw 43.50av 3.31 0.001
1.5% RO 24.03ay 27.92axy 30.63axy 34.56awx 37.54aw 39.72aw 41.54av 43.04abv 2.07 <.0001
2% RO 15.10bcy 18.58bcy 22.83bcxy 30.24axy 32.16abwx 35.40abwx 38.79abw 40.99abv 1.73 <.0001
P value 0.012 0.004 0.004 0.037 0.023 0.031 0.107 0.162
0% NS 11.95dz 14.62cz 17.84cy 22.05dy 26.24cx 28.66cx 32.27cw 34.95bw 1.039 <.0001
0.5% NS 19.30bcy 21.81aby 24.70by 28.81bcy 31.35bxy 34.13abwx 36.86abcw 38.48abv 0.96 1%NS 24.03az 26.13ayz 29.07ayz 32.83abxy 35.92awx 37.69aw 39.99aw 41.22av 1.92 <.0001
1.5%NS 21.93abx 26.12ax 30.01awx 35.71aw 35.12axw 37.18aw 39.27abv 40.39av 2.36 0.0003
2%NS 15.89cdy 18.40bcy 22.05bw 25.50cdw 29.47bcw 32.47bcw 34.80bv 37.00abv 0.55 <.0001
P value 0.001 0.001 0.0003 0.0008 0.0003 0.003 0.024 0.080
RO = Rosmarinus officinalis leaves. NS=Nigella sativa seeds.
abcd Means there is significant difference between treatment.
vwxyz Means there is significant difference between gas production.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Supplementation of RO did not affect (P>0.05), metabolizable energy, pH, IVOMD,
IVDMD, CH4 and NH4-N, total VFA and the molar proportion of acetate, propionate
and butyrate (Table 3.4). The volume of gas produced from soluble fraction was greater
in the control substrate compared with the substrates supplemented with 0.5% RO and
2% RO. The value of gas production from the soluble fraction in 1% RO and 1.5% RO
did not differ (P>0.05) from that of other treatments. The value of NGP, b, a+b and c did
not differ (P>0.05) among the treatments. The molar proportion of propionate in the
control substrate did not differ (P>0.05) from those supplemented with graded levels of
RO. The 2% RO had lower (P<0.05) ratio of acetate to propionate (A:P) compare with
the control, 0.5% and 1% RO substrates.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Table . 4.خطأ! لا یوجد نص من النمط المعین فی المستند In vitro fermentation, pH, and concentration
of volatile fatty acids in substrates containing graded levels of Rosmarinus officinalis
during 24 h incubation
Substrates
Parameter 0% RO 0.5% RO 1% RO 1.5% RO 2% RO SEM P value
a (mL) 33.53x 30.90y 32.51xy 31.81xy 28.05z 0.75 0.017
b (mL) 69.68 86.89 76.26 62.83 77.54 8.15 0.36
c (mL/h) 0.040 0.023 0.033 0.043 0.036 0.005 0.23
a + b (mL) 103.21 117.79 108.77 94.64 106.59 8.29 0.43
NGP (mL) 37.25 40.50 43.50 43.16 41.66 1.75 0.16
pH (unit) 6.90 6.90 6.76 6.80 6.83 0.035 0.073
ME 7.76 8.23 8.57 8.45 8.02 0.105 0.086
IVOMD 58.78 62.21 64.46 63.53 60.75 0.840 0.119
IVDMD 52.83 50.83 54.00 54.83 58.50 2.98 0.871
CH4 21.74 22.06 22.93 24.03 22.54 0.443 0.318
NH4N 11.92 11.99 12.65 11.34 12.70 0.296 0.364
Total VFA (mmol/L) 78.97 78.82 82.74 87.33 82.83 2.80 0.24
Acetate (A) 55.56 55.89 58.38 61.66 58.14 2.01 0.28
Propionate (P) 16.44 16.21 17.40 18.76 18.09 0.69 0.12
Butyrate 3.15 3.41 3.60 3.62 3.37 0.15 0.26
A:P 3.38xy 3.44x 3.35xy 3.29yz 3.21z 0.025 0.003
RO= Rosmarinus officinalis a=volume of gas produced from soluble fraction; b =volume of gas produced from
insoluble fraction; c=gas production rate constant from the insoluble fraction; NGP= net gas production; pH=
ruminal pH. xyz Means there is significant difference
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Supplementation of NS did not affect (P>0.05), metabolizable energy, pH, IVOMD,
IVDMD, CH4, NH4-N, total VFA and the molar proportion of acetate, propionate and
butyrate (Table 3.5).
Table . 5.خطأ! لا یوجد نص من النمط المعین فی المستند In vitro fermentation, pH, and
concentration of volatile fatty acids in substrates containing graded levels of
Nigella sativa after 24 h incubation
Substrates
Parameter 0% NS 0.5%NS 1% NS 1.5% NS 2% NS SEM P value ns.
a (mL) 33.53 32.10 32.48 30.05 32.22 1.23 0.43
b (mL) 69.68 68.59 75.74 54.95 79.30 7.91 0.30
c (mL/h) 0.040 0.033 0.036 0.056 0.026 0.006 0.07
a + b (mL) 103.21 100.69 108.23 85.00 111.52 7.51 0.19
NGP (mL) 37.25 39.83 41.83 41.50 38.75 1.12 0.07
pH (unit) 6.90 6.89 6.82 6.86 6.87 0.025 0.29
ME 7.76 8.33 8.87 8.83 8.54 0.203 0.159
IV0MD 58.78 62.67 66.38 66.23 64.37 1.303 0.183
IVDMD 52.83 51.83 54.17 56.16 57.00 3.376 0.961
CH4 21.74 22.61 22.19 22.00 21.38 0.636 0.944
NH4-N 11.92 12.71 12.32 12.48 13.01 0.167 0.949
Total VFA (mmol/L) 78.97 78.82 82.74 87.33 82.83 4.41 0.92
Acetate 55.56 58.64 57.95 57.01 55.40 3 0.91
Propionate 16.44 18.42 18.32 17.95 17.54 1.32 0.82
Butyrate 3.15 3.22 2.88 3.20 3.20 0.2 0.77
A:P 3.38 3.20 3.18 3.18 3.16 0.08 0.53
NS=Nigella sativa seeds. a=volume of gas produced from soluble fraction; b =volume of gas produced from
insoluble fraction; c=gas production rate constant from the insoluble fraction; NGP= net gas production pH=
ruminal pH. ns= not significant.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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1.1.6 In vitro biohydrogenation and fatty acid composition of rumen liquor
The fatty acid composition of rumen liquor supplemented with different levels of RO are
presented in Table 3.6. The concentration of C12:0 and C14:0 were lower (P<0.05) in
2% RO compared to other substrates. The concentrations of C15:0, C15:1, C16:0 and
C16:1n-7 did not differ (P>0.05) among the treatments. The 2% RO had greater
(P>0.05) concentrations of C16:1n-9 and C18:1n-9 and lower concentration of C18:0
compared with other treatments. The concentrations of C18:0 in 1% and 1.5% RO did
not differ but was greater (P<0.05) than that of the control and 0.5% RO substrates. The
concentrations of C18:2n-6, C18:3n-3, CLAc9t11, CLAc12t10, C20:4n-6 and C22:6n-3
did not differ (P>0.05) among the substrates. The total UFA was greater (P< 0.05) while
the total SFA was lower (P< 0.05) in 2% RO compared with other substrates. The rate of
biohydrogenation of C18:1n-9 decreased (P<0.05) as the level of RO increased in the
substrate. Supplementation of RO had no significant effect on the apparent
biohydrogenation of C18:2n-6 and C18:3n-3 in the substrate.
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Table . 6.خطأ! لا یوجد نص من النمط المعین فی المستند Fatty acid composition (% of total fatty
acids) of rumen fluid and rate of biohydrogenation at 24 h incubation of substrates
containing graded levels of Rosmarinus officinalis
Fatty acids Diet
0% RO 0.5% RO 1% RO 1.5% RO 2% RO SEM P-value
C12:0 1.15ᵃ 1.10ᵃ 1.20ᵃ 0.84ᵃᵇ 0.58ᵇ 0.14 0.04
C14:0 1.60ᵃ 1.99ᵃ 1.50ᵃᵇ 0.96ᵇᶜ 0.79ᶜ 0.17 0.005
C15:0 4.21 3.77 5.21 4.04 4.09 0.31 0.06
C15:1 0.87 0.65 0.29 0.63 0.17 0.25 0.18
C16:0 22.39 25.90 28.80 23.78 18.02 3.37 0.29
C16:1n-7 1.06 0.77 0.67 0.63 0.53 0.11 0.09
C16:1n-9 1.40ᵇ 1.25ᵇ 1.83ᵇ 1.93ᵇ 2.93ᵃ 0.27 0.01
C18:0 29.32ᵃᵇ 32.78ᵃ 22.04ᶜ 24.98ᵇᶜ 15.65ᵈ 1.76 0.0004
Trans-11 C18:1 3.59 4.28 2.89 2.49ᵃᵇ 2.28 0.48 0.08
C18:1n-9 13.20ᶜ 10.37ᶜ 21.52ᵇᶜ 26.67ᵇ 43.58ᵃ 4.66 0.003
C18:2n-6 13.60 7.68 8.40 6.84 4.50 1.81 0.060
C18:3n-3 2.72 2.07 2.58 2.66 2.67 0.4 0.78
CLAc9t11 0.92 2.15 0.55 0.55 0.80 0.40 0.09
CLAc12t10 1.37 1.60 0.78 0.55 1.08 0.28 0.14
C20:4n-6 1.10 1.26 1.07 1.13 0.39 0.25 0.20
C22:6n-3 1.43 2.31 1.32 0.97 1.79 0.4 0.24
ΣSFA 58.68b 65.55a 58.76b 54.61b 39.14c 3.85 0.007
ΣUFA 41.32ᵇ 34.44ᵇ 41.23ᵇ 45.38ᵇ 60.86ᵃ 3.85 0.007
ΣMUFA 20.15ᵇ 17.34ᵇ 26.51ᵇ 32.67ᵇ 49.62ᵃ 4.87 0.006
UFA:SFA 0.67ᵇ 0.52ᵇ 0.82ᵇ 0.80ᵇ 1.55ᵃ 0.08 0.0001
Apparent biohydrogenation (%)
C18:1n-9 68.21a 64.43b 61.23C 54.78d 52.11d 3.67 0.04
C18:2n-6 83.04 84.34 86.03 80.23 80.45 4.79 0.21
C18:3n-3 89.20 89.00 90.25 85.44 86.56 4.85 0.10
RO = Rosmarinus officinalis leaves. ΣSFA = (C14:0 + C16:0 + C18:0), ΣMUFA = (C16:1+ C18:1+ C18:1
trans-11), ΣUFA = (C16:1+ C18:1+Σn-3 + Σn-6), Σn-6 = (C18:2n-6 + C20:4n-6) n-6:n-3 = (C18:2n-6 +
C20:4n-6) ÷ (C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3). SFA= saturated fatty acids; UFA= unsaturated
fatty acids; MUFA= monounsaturated fatty acids.
The fatty acid composition of rumen liquor supplemented with graded levels of NS after
24 h incubation is as presented in Table 3.7. Supplementation of graded levels of NS had
no significant effect on the fatty acid composition of rumen liquor after 24 h incubation.
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The apparent biohydrogenation of C18:1n-9, C18:2n-6 and C18:3n-3 was not influenced
by the supplementation of NS.
Table . 7.خطأ! لا یوجد نص من النمط المعین فی المستند Fatty acid composition (% of total
fatty acids) of rumen fluid and rate of biohydrogenation at 24 h incubation of
substrates containing graded levels of Nigella sativa seeds
NS=Nigella sativa seeds. ΣSFA = (C14:0 + C16:0 + C18:0), ΣMUFA = (C16:1+ C18:1+ C18:1 trans-11),
ΣUFA = (C16:1+ C18:1+Σn-3 + Σn-6); Σn-6 = (C18:2n-6 + C20:4n-6) n-6:n-3 = (C18:2n-6 + C20:4n-6)
÷ (C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3); SFA= saturated fatty acids; UFA= unsaturated fatty
acids; MUFA= monounsaturated fatty acids. ns=not significant.
Fatty acids Substrate
0% NS 0.5% NS 1 % NS 1.5% NS 2% NS SEM P value ns.
C12:0 1.15 1.22 1.64 1.32 0.78 0.17 0.07
C14:0 1.60 1.93 2.20 1.62 1.26 0.64 0.86
C15:0 4.21 4.90 5.04 4.53 3.46 0.47 0.21
C15:1 0.87 1.27 1.28 0.21 1.55 0.80 0.79
C16:0 22.39 24.40 27.25 25.25 26.68 1.70 0.33
C16:1n-7 1.06 0.81 0.90 0.88 0.72 0.11 0.44
C16:1n-9 1.40 2.56 1.19 1.51 1.99 0.37 0.15
C18:0 29.32 23.19 31.45 28.96 22.61 3.10 0.24
Trans-11 C18:1 3.59 2.90 3.88 4.34 1.81 0.69 0.16
C18:1n-9 13.20 22.72 7.46 14.61 25.81 5.94 0.25
C18:2n-6 13.60 6.57 12.05 8.20 7.06 1.78 0.06
C18:3n-3 2.72 2.80 0.91 1.76 2.31 0.80 0.47
CLAc9t11 0.92 0.97 0.74 1.41 0.37 0.30 0.26
CLAc12t10 1.37 0.96 0.84 0.81 0.51 0.24 0.24
C20:4n-6 1.10 1.27 0.33 1.73 1.36 0.4 0.25
C22:6n-3 1.43 1.48 2.80 2.76 1.69 0.47 0.15
ΣSFA 58.68 55.65 67.59 61.69 54.80 4.53 0.33
ΣUFA 41.32 44.34 32.40 38.30 45.19 4.53 0.33
ΣMUFA 20.15 30.27 14.71 21.58 31.88 5.29 0.19
UFA:SFA 0.67 0.86 0.48 0.64 0.83 0.12 0.34
Apparent biohydrogenation (%)
C18:1n-9 69.00 67.25 65.12 66.08 66.21 0.83 0.21
C18:2n-6 78.21 80.23 76.21 75.32 75.17 0.24 0.11
C18:3n-3 86.15 87.23 87.45 87.24 88.16 0.95 0.09
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DISCUSSION
Rumen fermentation and gas production has close association, therefore gas-measuring
techniques were believed to be used as a routine method for feed evaluation (32). Many studied
have reported a high correlation between gas production in vitro and in vivo apparent
digestibility (33; 34). It has been proved that the inclusion of NS and RO increased the
cumulative in vitro gas production over the 24 h incubation period.
The in vitro gas production was greater in substrates containing 1% and 1.5% concentrations of
NS and RO than the substrate containing higher concentration of NS and RO (2%). This
observation suggests that the effects of NS and RO on in vitro gas production were
concentration-dependent. This was particularly true in RO supplements when where in the 2%
RO had lower value of a (gas produced from soluble fraction) than other supplements (table 3.4).
This observation could be due to the higher concentration of polyphenols present in the
supplements. This finding is in agreement with the results of Soycan-Önenç (35) who observed
that the supplementation of Vitex agnus-castus improved cumulative in vitro gas production up
to 8 h, and then the GP-reducing effect occurred from 8 to 12 h of incubation in all the treatments
compared with the control, it was shown that, the concentrate feed with Vitex agnus-castus
addition reduced degradation of storage polymers such as starch. Contrarily, Moujahed et al, (36)
found that rosemary (Rosmarinus officinalis) EOs at doses (0, 5, 10, 20, and 40 μl/50 ml of
buffered rumen fluid) did not affect cumulative in vitro gas production, while thyme (Thymus
capitatus) EOs decreased (P<0.0001) gas production starting from 10 μl dose.
In spite of the changes in the cumulative gas production, net gas production and gas produced
from the insoluble fraction were not affected by the supplementation of RO and NS after 24 h
incubation. This observation could be responsible for the lack of significant changes observed
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among the supplements for IVDMD, IVOMD, pH, total and individual VFA. Natural feed
additives such as RO and NS and their product can be regarded has been beneficial in ruminant
nutrition when there is a positive impact on the propionic acid and total VFA by increasing their
production and less impact by reducing the acetic/propionic ratio (37; 38). Many findings have
attested to the damaging impact of this compound on the total VFA production along with a
decrease in the digestion of feed, particularly with high concentration. Some plant extract have
been examined which include (cove, anise, oregano, fenugreek, yucca, cinnamon, garlic, bud,
ginge and tea tree) included at varied quantity for 24 h in vitro ruminal fermentations (39). The
author of this research noticed a reduced total VFA production in nearly all the treatment as the
quantity increased in the feed and this could be an indication of low feed digestion. In addition,
Moujahed et al, (36) reported that rosemary (Rosmarinus officinalis) EOs at doses (0, 5, 10, 20,
and 40 μl/50 ml of buffered rumen fluid) rosemary EO increased NH3-N concentration
(P<0.001) when administered at the doses of 20 and 40, while there is no significantly diffirent
of VFA accumulation. Cobellis et al. (40) indicated that rosemary (Rosmarinus officinalis L.)
and oregano (Origanum vulgare L.) essentials oil at doses (0, 0.5, 1, 1.5, 2 g/L) decreased total
VFA by oregano but was unaffected by rosemary, while the yield of ammonia was greatly
decreased among the treatments with exclusion of rosemary at the minimum quantity. Also
nearly all the treatment neutral detergent fiber and dry matter degradability was greatly
decreased.
Even though, IVDMD, IVOMD, total and individual VFA did not differ statistically (P>0.05),
the slightly higher values observed in the (1% and 1.5%) in both NS and RO could partly be due
to the concentration of polyphenols at accepted level to improve rumen fermentation (41; 42).
Hence, the concentration of total polyphenols observed in this study, could have altered the
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433
fermentation characteristics (9). It is due to the presence of those polyphenol (43; 41; 44) which
could be responsible for the slightly higher dry matter, organic matter digestibility, total and
individual VFA as compared to the control diet.
This study demonstrated that, supplementation of RO reduced the concentration of C12:0 and
C14:0 in a dose dependent manner. This observation could be attributed to the phenolic
compounds such as triterpenes, phenolic acids, and flavonoids (45; 46) in the supplements,
which reduced the activities of lipogenic enzymes responsible for the synthesis of medium chain
fatty acids (47). Supplementation of RO increased the concentration of C18:1n-9 and reduced the
concentration of C18:0 in a dose dependent manner. This observation is consistent with the
reduced biohydrogenation of C18:1n-9 in substrates containing RO. Similarly, supplementation
of Andrographis paniculata leaves reduced the concentration of C18:0 and increased the
concentration of C18:1n-9 in rumen liquor from goats. Supplementation of RO reduced the
apparent biohydrogenation of C18:1n-9 but no impact was noticed in the biohydrogenation of
C18:2n-6 and C18:3n-3. The decrease in the apparent biohydrogenation of C18:1n-9 could be
due to the presence of polyphenolic compounds, which selectively inhibit the activities of
microbes responsible for the biohydrogenation of C18:1n-9.
Contrarily to the observations in RO supplements, the supplementation of NS did not have a
significant effect on the fatty acid composition of rumen liquor. Nonetheless, there were
numerical changes in fatty acid composition similar to the trend observed in the RO
supplements. Regardless of the supplement, the apparent biohydrogenation of C18:3n-3 was
greater than that of C18:2n-6, which was in turn greater than that of C18:1n-9. Similar trends
were observed in in vitro (48; 47) rumen fatty acid composition in goats.
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CONCLUSION
Irrespective of the level, the inclusion of RO leaves and NS seeds in the diets indicate no
deleterious effects on rumen fermentation but reduced rumen gas production at 2%. From this
result, it is believed that the supplementation of RO leaves and NS seeds up to 1% DM of
substrate had no deleterious effect on in vitro ruminal fermentation and improve the digestibility.
Generally, the results from the present study suggested that RO leaves and NS seeds can be
efficiently utilized to manipulate rumen fermentation characteristics without adversely affecting
or compromising the production of VFAs, dry matter digestibility and some desirable fatty acids
in the product.
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