Dietary n-6:n-3 Fatty Acid Ratios Alter Rumen Fermentation

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DIETARY N-6:N-3 FATTY ACID RATIOS ALTER RUMEN FERMENTATION PARAMETERS AND MICROBIAL POPULATIONS IN GOATS Mahdi Ebrahimi, Mohamed Ali Rajion, Kazeem Dauda Adeyemi, Saeid Jafari, Mohammad Faseleh Jahromi, Ehsan Oskoueian, Yong Meng Goh, and Morteza Hosseini Ghaffari J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04704 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Journal of Agricultural and Food Chemistry

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DIETARY N-6:N-3 FATTY ACID RATIOS ALTER RUMEN FERMENTATION PARAMETERS AND MICROBIAL POPULATIONS IN GOATS

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Mahdi Ebrahimi 1*, Mohamed Ali Rajion 1, Kazeem Dauda Adeyemi2,4, Saeid Jafari1, Mohammad Faseleh Jahromi3, Ehsan Oskoueian3, Goh Yong Meng1,3, Morteza Hosseini Ghaffari5

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Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Malaysia; Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, 43400 UPM, Serdang, Malaysia; Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Malaysia Department of Animal Production University of Ilorin, PMB 1515, Ilorin, Nigeria.

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, T6G 2P5 Canada

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Abstract

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Revealing the ruminal fermentation patterns and microbial populations as affected by

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dietary n-6:n-3 PUFA ratio would be useful for further clarifying the role of the rumen in

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the lipid metabolism of ruminants. The objective of the present study was to investigate

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the effects of dietary n-6:n-3 PUFA ratios on fermentation characteristics, fatty acid (FA)

31

profiles and microbial populations in the rumen of goats. A total of twenty-one goats

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were randomly assigned to three dietary treatments with different n-6:n-3 PUFA ratios of

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2.27:1 (Low ratio, LR), 5.01:1 (Medium ratio, MR) and 10.38:1 (High ratio, HR). After

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100 days of feeding, all goats were slaughtered. Dietary n-6:n-3 PUFA ratios had no

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effect (P> 0.05) on rumen pH and NH3N concentration. Goats fed HR diet had lower (P
0.05) on the ruminal populations of F. succinogenes, total

44

bacteria, methanogens, total protozoa, Entiodinium and Holotrich. The population of B.

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fibrisolvens was lower (P < 0.05) in the LR goats compared with the MR and HR goats.

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It was concluded that HR would increase the concentration of cis-9 trans-11 CLA and

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C18:1 trans-11 in the rumen. However, LR whould decrease the B. fibrisolvens

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population, which is involved in BH process in the rumen. Further research is needed to 2 ACS Paragon Plus Environment

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evaluate the potential role and contribution of rumen microbiome in the metabolism of

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FA in the rumen.

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Key words: Boer goat, Fatty acid, Microbial population, Rumen fermentation.

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Introduction

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Fat supplements are included in the diet of ruminants to increase energy density,

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improve nutrient utilization1 and modify the fatty acid composition of milk2 and meat.3

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Nonetheless, dietary fat could affect rumen metabolism, which could have production

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and economic consequences. The n-6 to n-3 ratio of fatty acids in ruminant tissue is

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influenced by dietary fatty acids and can influence the concentration of conjugated

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linoleic acid and vaccennic acid in the rumen, milk, and meat.3,4 A lowered ratio of n-6

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:n-3 FA in the diet is desirable due to its potential to reduce the incidence of chronic

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diseases in humans.

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dietary n-6 :n-3 PUFA ratios of ruminant products.6.7

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Goats are gaining acceptance as a set up model for biomedical research and for

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surgical preparing and teaching.8 They are utilized as a part of medicinal, orthopedic,

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mental, chemotherapeutic, and physiologic research.8 Compared with cows, their little

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size grants goats to be kept up in a generally little range. Goats also play an important

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role in food production systems in developing countries. Their popularity can be clarified

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by their great adjustment to a wide range of atmospheres (biological adjustment) and

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the many uses for which they can be kept.

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The rumen microbial ecosystem is highly responsive to changes in the type and

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composition of the diet, which are critical factors that affect rumen microbial activity and

5

This has stimulated research interest in decreasing the ratio of



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rumen function. 9 Thus, a general understanding of the intricate ruminal microbial

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populations, their interactions, and their response to different diets is essential. 10, 11

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The impact of dietary fat differing in n-6 :n-3 PUFA ratios on rumen microflora have

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been examined in sheep

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in vivo studies in goats, whose rumen microflora might have a different response to

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dietary n-6 :n-3 PUFA ratios based on the suggested interspecies differences in lipid

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metabolism.17 This underscores the need for further studies in different production

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systems to allow cognizant choices and tailored decisions in the use of dietary n-6 :n-3

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PUFA ratios in ruminants. Thus, the present study was conducted to determine the

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effects of modifying the dietary n-6 :n-3 PUFA ratios on FA composition, fermentation

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kinetics and microbial populations in the rumen of goats.

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Materials and methods

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Plant materials, animals, diets, and management

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Fresh oil palm frond (OPF) used in this experiment were harvested in the fields of the

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Malaysian Agricultural Research and Development Institute (MARDI) located in

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Serdang, Selangor, Malaysia (3°00′18.88″N, 101°42′15.05″E). This study was

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conducted at UPM under the guidelines of the Research Policy on Animal Ethics of the

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Universiti Putra Malaysia. Twenty-one male Boer goats (Five-month-old; 13.66 ± 1.07 kg

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of body weight, BW) were used in a completely randomized design in which animals

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were randomly assigned to 1 of the following 3 diets differing in n-6:n-3 PUFA ratios:

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2.27:1 (LR), 5.01:1 (MR), and 10.38:1 (HR). Animals were initially drenched against

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parasites, and housed individually in wooden pens measuring 1.2 m × 1 m of each, built

12,13

and cattle.14, 15,16 Nonetheless, there is a dearth of similar


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inside a shed with slatted floor 0.5 meters above ground. The percentage of FA contents

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of oil sources used to adjust the n-6:n-3 PUFA ratios ratios are shown in Table 1.

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The concentrate (70% DM basis) and OPF silage (30% DM basis) were mixed and

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offered in 2 equal meals at 8:00 AM and 5:00 PM. The experimental diets were fed daily

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at 3.7% of BW (DM basis), with adjustments made weekly according to the changing

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BW. All goats had free access to water and mineral blocks. The feeding trial lasted 100

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days with three weeks of adaptation period. At the end of the 100 day trial, the goats

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were slaughtered in accordance to the Halal slaughter procedures outlined in the MS

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1500:2004.18

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The ingredients and fatty acid contents of the experimental diets are shown in Table 2.

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The ingredients of LR, MR and HR were; OPF silage (30%), corn grain (17%), soybean

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meal (13.30%), palm kernel cake (25.11%), rice bran (8.18%). The diets were

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formulated to be isonitrogenous and isocaloric to meet the protein and energy

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requirements of growing goats.19 Oleic acid (OA) and linoleic acid (LA) were the

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predominant FA in all the diets. The LR diet showed a more balanced supply of the

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three main FA of plant origin namely OA, LA and linolenic acid (LNA), although LA was

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quantitatively the most important (Table 2). Intakes of feed (TMR) were measured daily

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and individual BW was recorded using an electronic balance every month throughout

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the experiment. Amounts of feed offered and refused were recorded daily for each

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individual goat. Overall means of average daily gain (ADG) and gain to feed ratio (kg of

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BW gain/kg of total DMI) were also calculated.

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Sampling and chemical analyses

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Oil palm frond samples were oven dried at 55 oC for 2 days, and stored at -80 oC for

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further analysis and animal trial. Subsamples of feeds (concentrate and OPF silage,

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500g) were collected weekly and stored at -20 °C until analysis. The proximate

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composition of the diet was determined following the protocol of AOAC.20 Samples were

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thoroughly mixed and dried at 60 °C for 48 h and then ground to pass a 1-mm screen in

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a grinder before chemical analyses for DM content (method 934.01; AOAC)

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protein (CP, method 988.05; AOAC) 20, ether extract (EE, method 920.39; AOAC) 20 and

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ash (AOAC; method 942.05).

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ash, were determined with the inclusion of heat-stable α-amylase (100 mL/0.5 g of

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sample) and without sodium sulfite following the protocol of Van Soest et al.21The

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chemical composition of experimental diets (LR, MR and HR) were; CP (13.00%), EE

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(7.00%), NDF (48.90%), ADF (33.00%), Calcium (0.68%), Phosphorous (0.36%).

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Metabolizable energy (LR, MR and HR) of the experimental diets was also 2.51

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(Mcal/kg).

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Rumen fermentation parameters

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After slaughter at the end of the 100 day trial, approximately 100 mL of ruminal fluid

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were collected by sampling the rumen contents from the ventral, caudal, and central

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areas of the rumen and squeezing it through four layers of cheesecloth. Rumen pH was

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measured immediately with a pH meter (Mettler-Toledo Ltd., England). An aliquot (4

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mL) of the rumen fluid was acidified with 1 mL of 25% meta-phosphoric acid and stored

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(‒20°C) until analysis for VFA. For the rumen microbial populations, the rumen fluid

20

20

, crude

Concentrations of ADF and NDF inclusive of residual


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sample was mixed properly and 10 mL of rumen fluid was taken, snap-frozen in liquid

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nitrogen and stored at -80 oC until DNA extraction. The frozen rumen fluid was thawed

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and centrifuged at 10000 × g at 4°C for 20 min, and the supernatant was collected to

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measure the concentrations of NH3N and volatile fatty acids (VFA). The ruminal NH3N

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was determined using an alkaline phenol hypochlorite method as described by

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Solorzano.22 The VFA was determined by using gas-liquid chromatography with

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Quadrex 007 Series (Quadrex Corporation, New Haven, CT 06525 USA) bonded phase

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fused silica capillary column (15m, 0.32mm ID, 0.25 µm film thickness) in an Agilent

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7890A gas-liquid chromatography (Agilent Technologies, Palo Alto, CA, USA) equipped

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with a flame ionization detector (FID). The injector/detector temperature was

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programmed at 220/230 oC respectively. The column temperature was set in the range

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of 70 oC -150 oC with temperature programming at the rate of 7 oC/ min increment to

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facilitate optimal separation. Peaks were identified by comparison with authentic

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standards of acetic, propionic, butyric, isobutyric, valeric, isovaleric and 4-methyl-n-

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valeric acids (Sigma, St. Louis, Mo., USA).

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Fatty acid analysis of experimental diets and rumen fluid

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The total fat was extracted from the experimental diet and rumen fluid samples by using

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chloroform: methanol (2:1 v/v) containing butylated hydroxytoluene to prevent oxidation

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during sample preparation as described by Ebrahimi et al.23 The extracted fat was

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saponified with methanolic KOH for 10 min at 90°C. Fatty acids were converted to FA

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methyl esters (FAME) by transesterification with methanolic boron trifluoride (BF3, 14%)

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at 90°C for 20 min. The FAME were then analyzed by gas–liquid chromatography

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(Agilent 7890A). The FAME (1μL) was injected by an auto sampler into the 7 ACS Paragon Plus Environment


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chromatograph, equipped with a flame ionization detector (FID). Helium was the carrier

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gas used at a flow rate of 1 mL × min-1. The FAME were separated on a 100 m × 0.32

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mm × 0.25 μm film thickness using Supelco SP-2560 capillary column (Supelco, Inc.,

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Bellefonte, PA, USA). The injector and detector temperature was maintained at 250°C

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and 300°C respectively. The column was operated isothermally at 120°C for 5 min, then

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programmed to 250°C at 4°C/min, increased by 2°C/min up to 170°C, held at 170°C for

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15 min, increased again by 5°C/min up to 200°C, and held at 200°C for 5 min and then

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increased again by 2°C/min to a final temperature of 235°C and held for 10 min. Peak

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identification was performed by using known standards (mix C4–C24 methyl esters;

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Sigma-Aldrich, Inc., St. Louis, Missouri, USA) and relative quantification was

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automatically carried out by peak integration.

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Quantification of microbial population by real-time polymerase chain reaction

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(PCR)

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Primers used to quantify the population of different groups of microorganisms were

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shown in Table 3. The DNA was extracted from the rumen fluid using the QIAamp DNA

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Stool Mini Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s protocol. The

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extracted DNA was stored at -20 oC until used. Plasmid DNA from each group of

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microorganisms was used for the preparation of a standard curve. The purity and

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concentration

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spectrophotometer and the number of copies of a template DNA per mL of elution buffer

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was calculated using the following formula that is available online in URL Genomics and

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Sequencing

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(www.uri.edu/research/gsc/resources/cndna.html).

of

plasmid

DNA

in

each

sample

Center

were

web-based


measured

using

a

calculator

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Standard curves were constructed using CT values that were obtained from a serial

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dilution of plasmid DNA of each bacterial group. Real-time PCR was performed with the

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Bio-Rad CFX96 Touch (Bio-Rad Laboratories, Hercules, CA, USA) using optical grade

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plates. The PCR reaction was performed on a total volume of 25 μL using the

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iQTMSYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Each reaction

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included 12.5 μL SYBR Green Supermix, 1 μL of each primer, 1 μL of DNA samples

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and 9.5 μL RNAse free waters. To confirm the specificity of amplification, a melting

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curve analysis was carried out after the last cycle of each amplification.

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Quantification of rumen ciliates

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Rumen ciliates were identified according to the method of Hungate.

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fluid sample was transferred into a screw-capped test tube containing 5 mL formalinized

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physiological saline (containing 20 mL formaldehyde in 100 mL saline, which contained

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0.85 g sodium chloride in 100 mL distilled water). Thereafter, two drops of brilliant green

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dye (2 g brilliant green and 2 mL glacial acetic diluted to 100 mL with distilled water)

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were added to the test tube, mixed thoroughly and allowed to stand overnight at room

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temperature. Total and differential counts of protozoa were carried out by the use of a

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haemocytometer and a phase contrast light microscopy (Olympus BX51, Olympus, and

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Melville, NY) at 40 × magnification in a haemocytometer (Neubauer improved,

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Marienfeld, Germany).


24

Two mL of rumen


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Statistical Analysis

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Data on feed intake, ADG, and gain to feed ratio were analyzed using a repeated-

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measures mixed model (PROC MIXED) of SAS (version 9.1; SAS Institute Inc., Cary,

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NC).25 Data was subjected to ANOVA using the MIXED procedure of SAS.Goat was the

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experimental unit and random effect with animals. The statistical model used is

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presented below. Y ijk = µ + Ti + Fk +eijk

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where µ was the overall mean, T was the different dietary n-6 :n-3 Ratios, F was the

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animal effect and e was the residual error. Before analyses, all data was screened for

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normality using the UNIVARIATE procedure of SAS, any parameter that was not

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normally

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adjustment was used. Means were separated using the “pdiff” option of the “lsmeans”

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statement of the MIXED procedure. Significance was declared when P ≤ 0.05 and

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trends were declared when P < 0.10.

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Results

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Growth performance

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The growth performance of goats fed diets with varying n-6:n-3 PUFA ratios are shown

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in Table 4. The dietary treatments did not affect (P> 0.05) final BW, and total feed intake.

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The n-6:n-3 PUFA ratios had no effect (P>0.05) on ADG, mean total weight gain, and

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gain to feed ratio over the 100-days of the feeding trial.

distributed

was

logarithmically

transformed

226


and

the

Kenward-Roger

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Rumen fermentation characteristics

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The rumen fermentation parameters of goats fed diets different in n-6 :n-3 ratios are

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presented in Table 5. Rumen pH increased (linear, P=0.04) with increasing the n-6:n-3

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PUFA ratios. The dietary treatments did not affect (P>0.05) NH3-N concentrations in the

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rumen. Total VFA concentration (linear, P 0.05) by the different n-6:n-3 PUFA ratio..

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Rumen fatty acid profile

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The FA composition of the rumen fluid of goats fed different n-6:n-3 PUFA ratios are

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presented in Table 6. Proportions of C10:0, C12.0, C14:0, C14:1, C15:0, and C15:1 in

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the rumen were not affected (P> 0.05) by the different n-6:n-3 PUFA ratios. The

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proportions of trans-11 C18.1, C18.2n-6, cis-9 trans-11 CLA, C18.3n-3, and C20.4n-6 in

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the rumen of goats linearly increased (P < 0.05) with increasing the n-6:n-3 PUFA ratios.

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In contrast, the proportions of C18.0 and C18.1n-9 in the rumen linearly decreased (P
0.05) by

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the different n-6:n-3 PUFA ratios. While the proportions of MUFA and n-3 PUFA in the

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rumen linearly decreased, the proportions of n-6 PUFA, total trans FA, total CLA, and n-

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6:n-3 ratio in the rumen linearly increased (P < 0.05) with increasing the n-6:n-3 PUFA

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ratios. Proportions of C22:5n-3 and C22:6n-3 in the rumen of goats did not differ (P>

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0.05) due to treatments. 11 ACS Paragon Plus Environment


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Rumen microbial Population

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The rumen microbial populations of goats fed diet different n-6 :n-3 PUFA ratios are

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presented in Table 7. The populations of R. albus and R. flavefaciens in the rumen

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linearly decreased (P < 0.05) with increasing the n-6:n-3 PUFA ratios. The populations

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of F. succinogenes, total bacteria, methanogens, total protozoa, Entiodinium and

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Holotrich in the rumen fluid of goats were not affected (P> 0.05) by the different n-6:n-3

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PUFA ratios.

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Discussion

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Performance

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The present study, BW, feed intake, ADG, and gain-to-feed ratio of goats were

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unaffected by the different dietary n-6:n-3 PUFA ratios. These results could be due to

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the same amount of fat in the experimental diets or the similar feed intake of goats

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during the trial. Generally, the effects of dietary lipid supplements on the growth

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performance of goats and lambs are indecisive.26,27,28 Our results are in agreement with

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Kim et al.29 who showed that different n-6:n-3 PUFA ratios did not alter BW, dry matter

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intake, and ADG of lambs. The intake of experimental feed averaging 3.7% of the BW

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daily indicated that the goat consumed all the feed offered. It had already been shown

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that different dietary fat sources had no detectable effects on growth rate of steer and

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growing lambs.30,31

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Rumen fermentation characteristics

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The rumen pH obtained in this study was within the range (5.8 - 7) for normal rumen

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function. 32,33 Increasing the n-6:n-3 ratios increased the rumen pH in goats in this study, 12 ACS Paragon Plus Environment

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which is in line with the results of Kim et al.29 where rumen pH numerically increased

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with increasing the ratio of n-6 to n-3 from 2.3:1 to 12.8:1 of growing lambs. In the

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current study, the lack of dietary n-6 :n-3 ratios effects on the concentration of NH3-N in

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the rumen is consistent with those of Kim et al.

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dietary n-6 :n-3 PUFA ratios did not affect NH3N concentration in the rumen of sheep. In

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the present study, the concentrations of rumen NH3N were in the range of 7.14-14.28

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mg/dL, wich is still above the minimum concentrations required (≥5 mg/ dL) for rumen

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microbial growth 35 and for optimum fiber degradation in the rumen.36

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In the current study, HR goats had lower concentration of total VFA compared with

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those fed LR and MR diets. This suggests lower microbial degradation efficiency in the

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HR goats. This observation could be due to the lower population of cellulolytic bacteria

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in the HR goats compared with those fed other diets. In contrast, diets differing in n-6 :n-

284

3 PUFA ratios did not affect total VFA in sheep

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dietary n-6 :n-3 PUFA ratios decreased total VFA in sheep 38,39 and goats.40

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Dietary n-6 :n-3 PUFA ratios did not affect the molar proportion of acetate in the rumen

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of goats. Similar observation was observed in sheep.29 The MR and LR goats had

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higher molar proportion of propionate and lower molar proportion of butyrate compared

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with those fed the HR diet. Because of hydrogen sink properties of propionic acid, there

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would be less hydrogen for methanogenic bacteria to use as substrate during

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fermentation for CH4 production41,42 The current observation concurs with those

292

of Ikwuegbu43 and Sutton et al.44 who observed higher propionate and lower butyrate in

293

the rumen of sheep fed LO based diets. Similarly, lowering dietary n-6 :n-3 PUFA ratios

29

and Toral et al.

28,34

34

who observed that

and cattle. 17,37 Whereas decreasing



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increased the molar proportion of propionate and lowered the molar proportion of

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butyrate in the rumen of sheep.29

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Rumen fatty acid profiles

297

Regardless of the treatment, the major FA in the ruminal digesta was C18:0 (46.02-

298

49.95%) due to ruminal biohydrogenation (BH) of unsaturated fatty acids. Similar

299

observation was observed in goats,

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the rumen increased as dietary n-6 :n-3 PUFA ratios decreased indicating that C18:3n-3

301

underwent greater BH than C18:2n-6. This observation agrees with the results of

302

previous studies in sheep

303

proportion of C18:1 trans-11 (13.49-14.32%) was greater than that of C18:1n-9 (2.38-

304

3.47%). This could be attributed to the greater concentrations of PUFA in the diets and

305

because C18:1 trans-11 is a mutual intermediate in BH of unsaturated 18-carbon FA.45

306

The proportion of C18:1n-9 decreased as dietary n-6 :n-3 PUFA ratios increased.

307

Similar trend was observed in sheep.29

308

The linear decrease in C18:2n-6 and increase in C18:3n-3 with decreasing n-6 :n-3

309

PUFA ratios resulted in a linear decrease in the n-6 :n-3 ratios from 11.58 to 2.66, which

310

reflected the decreasing dietary n-6 :n-3 PUFA ratios.

311

The proportion of C18:1 trans-11 increased as dietary n-6 :n-3 PUFA ratios increased.

312

Similarly, dietary plant oils rich in C18:2n-6 increased the flow of trans C18:1 leaving the

313

rumen in sheep

314

the accumulation of all trans C18:1 intermediates in ewes.34 Increasing dietary n-6 :n-3

315

PUFA ratios increased the proportion of cis-9 trans-11 CLA in the rumen fluid of goats.

46,47

44

40

sheep

and goats.

29

40

and cattle.37 The proportion of C18:0 in

Irrespective of dietary treatments, the

and cattle.48 In addition, dietary supplementation of SO increased


Page 15 of 33


29

and cows.49 In addition, Váradyová et al.

44

316

Similar findings were observed in sheep

317

observed that dietary soybean oil or rapeseed oil increased the proportion of cis-9 trans-

318

11 CLA in the rumen fluid sheep compared to LO. However, Beaulieu et al.50 reported

319

that an increased dietary intake of soybean oil did not affect the proportion of cis-9

320

trans-11 CLA in the ruminal contents of steers.

321

Rumen microbial population

322

There is great interest in the study of ruminal microorganisms involved in lipid

323

metabolism due to their effect on the quality of ruminant-derived products. Several

324

studies

325

supplementation on the rumen microbiota in ruminants. In addition, interspecies

326

differences between ruminants concerning their lipid metabolism have been

327

suggested.17 However, there is a paucity of information on the effects of dietary n-6:n-3

328

PUFA ratios on rumen microbial populations.

329

Fibrobacter succinogenes, B. fibrisolvens, R. albus, and R. flavefaciens are the most

330

predominant cellulolytic species in the rumen, and changes in their relative amounts

331

could potentially affect ruminal fermentation.52 In the present study, we observed that

332

some bacterial populations in the rumen associated with BH of PUFA were affected by

333

dietary n-6:n-3 PUFA ratios. The proportion of F. succinogenes, B. fibrisolvens,

334

Methanobacteriales, total protozoa, Holotrich, and Entiodinium were similar across

335

treatments, implying that these microorganisms were not sensitive to dietary n-6:n-3

336

PUFA ratios. The proportion of B. fibrisolvens increased as dietary n-6:n-3 PUFA

337

increased. So far, most rumen bacteria that are actively involved in the rumen BH

338

belonged to the Butyrivibrio group.53,54

using

molecular

techniques51

have

investigated


the

effect

of

lipid


Page 16 of 33

339

Butyrivibrio fibrisolvens has been identified as the most active rumen bacteria in BH of

340

PUFA and forming trans-11-18:1 and CLA as intermediates in the process of BH of

341

PUFA.55,56 The current data suggest that the inhibition of B. fibrisolvens depends at least

342

in part on the degree of unsaturation of dietary fatty acids. In line with our findings, Maia

343

et al.

344

that increasing dietary n-6:n-3 PUFA ratios decrease the inhibitory effects of PUFA on

345

B. fibrisolvens in the rumen.

346

The proportion of R. albus and R. flavefaciens decreased as the n-6:n-3 PUFA ratios

347

increased in the diet. Ruminococcus albus and R. flavefaciens also play a predominant

348

role in the rumen BH of PUFA, where they remove the unsaturated double bonds in

349

order to detoxify the dietary PUFA in the rumen.53 Our findings are consistent with those

350

from the study by Maia et al.

351

albus and R. flavefaciens than other bacteria in the rumen. The inhibitory effect of HR

352

on the proportion of R. albus and R. flavefaciens appears to be associated with the

353

degree of unsaturation of the FA, with higher inhibitory effect by two unsaturated double

354

bonds (LA) compared to three double bonds (LNA).57 However, the reason for the

355

greater antibacterial action of n-6 compare to n-3 against R. albus and R. flavefaciens is

356

not clear. Despite the increase in rumen cellulolytic bacteria as the level of n-6 :n-3

357

PUFA ratios decreased in diet, the total rumen bacterial population was not affected.

358

This suggests that the increase in the rumen population of the cellulolytic bacteria was

359

at the expense of other bacterial populations. Similar observations were found in dairy

360

cows.9

12

reported that LNA was more toxic to B. fibrisolvens than LA or CLA, saggesting

12

who reported that LA was more toxic than LNA for R.


Page 17 of 33


361

The total protozoal population in this study was similar to that observed in dairy cows

362

fed LO

363

had no effect on the population of total rumen protozoa. Kišidayová et al.

364

that the rumen ciliates have no uniform response to oil supplements. The current

365

observation is in tandem with that of Loor et al.

366

also did not change the protozoal population in dairy cows. In contrast, Váradyová et al.

367

44

368

in sheep. The population of Holotrich and Entodinium protozoa was not affected by

369

dietary n-6 :n-3 PUFA ratios. Regardless of the diet, the protozoa of the genus

370

Entodinium were found in absolute larger population. Similar finding was found in sheep

371

fed soybean oil.13

372

The results of this study showed that dietary n-6 :n-3 ratios had no detrimental effects

373

on rumen fermentation parameters in goats. High dietary n-6 :n-3 FAR increased the

374

concentration of CLA, VA and C18:2n-6 and decreased the concentration of C18:0 in

375

the rumen of goats. Low dietary n-6 :n-3 decreased the B. fibrisolvens population, which

376

is involved in BH process in the rumen while increasing the proportion of the cellulolytic

377

bacteria in the rumen. Further research to evaluate the potential role and contribution of

378

rumen microbiome in the metabolism of FA in the rumen is suggested.

379

CONFLICT OF STUDY

380

The authors declare that there is no conflict of interests regarding the

381

publication of this paper.

57

but was higher than that reported for sheep fed SO.13 Dietary n-6 :n-3 PUFA

49

58

suggested

who observed that feeding LO or SO

showed that LO decreased the protozoal population compared to SO or rapeseed oil

382 17 ACS Paragon Plus Environment


Page 18 of 33

383

AUTHOR INFORMATION

384

Corresponding Author

385

*Tel. +60386093401 - Fax: +603-89471971- Email: [email protected].

386

Acknowledgments

387

The authors would like to thank the Faculty of Veterinary Medicine, Universiti Putra

388

Malaysia and the Malaysian Government for providing the E-Science Grant No. 05-01-

389

04-SF0200.

390

Abbreviations

391

ADF: acid detergent fiber; BW: body weight; CAE: catechin acid equivalents; CT:

392

Condensed tannins; DM: dry matter; EE: ether extract; GAE: gallic acid equivalents;

393

GLM: general linear model; NDF: neutral detergent fiber; OM: organic matter; PL:

394

papaya leaf; VFA: volatile fatty acid.

395

REFERENCES

396 397 398 399

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400 401 402 403

2. Adeyemi, K. D.; Sabow, A. B.; Ebrahimi, M.; Samsudin, A. A.; Sazili, A. Q. Fatty acid composition, cholesterol and antioxidant status of infraspinatus muscle, liver and kidney of goats fed blend of palm oil and canola oil. Italian J. Anim. Sci. doi.2016, org/10.1080/1828051X.2016.1158081.

404 405 406 407

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411 412 413

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550 551 552 553

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565 566 567 568

53. Devillard, E.; Andant, N.; Wallace, J. R. Increased expression of a molecular chaperone GroEL in response to unsaturated fatty acids by the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. FEMS Microbiol Lett. 2006, 262, 244–248.

569 570 571 572

54. Paillard, D.; McKain, N.; Chaudhary, L. C.; Walker, N. D.; Pizette, F.; Koppova, I.; McEwan, N. R. Relation between phylogenetic position, lipid metabolism and butyrate production by different Butyrivibrio-like bacteria from the rumen. Antonie van Leeuwenhoek, 2007, 91, 417–422.

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56. Benchaar, C.; Romero-Pérez, G. A.; Chouinard, P. Y; Hassanat, F., Eugene, M., Petit, H. V.; Côrtes, C., 2012: Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition. J. Dairy Sci. 2012, 95, 4578–4590.

581 582 583

57. Hristov, A. N.; Ivan, M.; McAllister, T. A. In vitro effects of individual fatty acids on protozoal numbers and on fermentation products in ruminal fluid from cattle fed a high-concentrate, barley-based diet. J. Anim. Sci. 2004, 82, 2693–2704.

584 585 586 587

58. Kišidayová, S.; Mihaliková, K.; Váradyová, Z., Potkański, A.; SzumacherStrabel, M.; Cieślak, A.; Čertík, M.; Jalč, D. Effect of microbial oil, evening primrose oil, and borage oil on rumen ciliate population in artificial rumen (Rusitec). J. Anim. Feed Sci. 2006, 15, 153–156.

588 589 590 591

59. Liu, S. J.; Bu, D. P.; Wang, J. Q.; Liu, L.; Liang, S.; Wei, H. Y.; Zhou, L. Y. Effect of incremental levels of fish oil supplementation on specific bacterial populations in bovine ruminal fluid. J. Anim. Physiol. Anim. Nutri. 2012. 96, 916.

592 593 594

60. Lane, D.J.16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematic. 1991. In: Stackebrandt, E., & Goodfellow, M., (Eds), John Wiley & Sons, New York, 329 pp.

595 596

61. Koike, S.; Kobayashi, Y. Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus 23 ACS Paragon Plus Environment


Page 24 of 33

597 598

albus and Ruminococcus flavefaciens. FEMS Microbiol. Lett. 2001. 204, 361366.

599 600 601

62. Sylvester, J.T.; Karnati, S.K.R.; Yu, Z.; Morrison, M.; Firkins, J.L. Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR. J. Nutri. 2004.134, 3378-3384.

602

603

604

605

606

607

608

609

610

611

612

613

614 615 24 ACS Paragon Plus Environment

Page 25 of 33

616 617


Table 1. The fatty acid contents (% of total identified fatty acids) of oil sources used to adjust the n-6:n-3 fatty acid ratios Oil

C14:0

C16:0

C18:0

C18:1

C18:2n-6

C18:3n-3

SFO1

-

6.40

3.66

28.32

61.23

0.39

PKO2

26.55

22.20

3.36

26.62

18.47

2.80

LO3

1.85

5.15

3.17

16.62

16.12

57.09

source

618 619 620 621

1

SFO, sunflower oil (Lam Soon Edible Oils Sdn. Bhd.); 2PKO, palm kernel oil (Malaysian Palm Oil Board); 3LO, linseed oil (Brenntag Canada, Inc.), C14:0, myristic acid; C16:0, palmitic acid; C18:0, stearic acid (SA); C18:1, oleic acid; C18:2n-6, linoleic acid (LA); C18:3n-3, linolenic acid (LNA).

622 623 624 625 626 627 628 629 630 631 632 633 634 25 ACS Paragon Plus Environment


635

Page 26 of 33

Table 2. Ingredients and fatty acid contents of the experimental diets Dietary n-6:n-3 PUFA ratio 1

Item

LR

MR

HR

Linseed oil (%)

1.30

0.40

0.00

Palm kernel oil (%)

0.10

1.00

1.10

Sunflower oil (%)

2.00

2.00

2.30

SFA 2

27.18

32.99

32.97

MUFA3

20.45

27.99

27.86

n-3 PUFA4

13.63

6.47

3.44

n-6 PUFA5

30.92

32.40

35.68

n-6 / n-3 FAR6

2.27

5.01

10.38

Ingredient (as-fed)

Fatty acid composition (% of total identified FAs)

636 637 638 639 640 641 642 643

1

LR: low n-6:n-3 PUFA ratio (2.27:1); MR: medium n-6:n-3 PUFA ratio (5.01:1); HR: high n-6:n-3 PUFA ratio (10.38:1). 2 SFA= (C10:0 + C12:0 + C14:0 + C16:0 + C17:0 + C18:0) 3 Total monounsaturated FA (MUFA, C16:1 + C18:1n-9) 4 n-3 PUFA = (C18:3n-3) 5 n-6 PUFA = (C18:2n-6) 6 n-6:n-3 FAR = (C18:2n-6)/ (C18:3n-3).

644 645 646 647 648


Page 27 of 33

649 650


Table 3. Microorganisms, sequences and references of the primers used for the quantification of microbial population. Microorganism Sequence 5’ – 3’ Product Reference size (bp) Total bacteria

F1

CGGCAACGAGCGCAACCC

145

R2 CCATTGTAGCACGTGTGTAGCC

(Koike and Kobayashi, 2001)

Methanobacteriales F

CGW AGGGAAGCTGTTAAGT

R

TACCGTCGTCCACTCCTT

Ruminococcus

F

CCCTAA AAGCAGTCTTAGTTCG

albus

R

CCTCCTTGCGGTTAGAACA

343

(Yu et al., 2005)

175


Ruminococcus

F

TCTGGAAACGGATGGTA

flavefaciens

R

CCTTTAAGACAGGAGTTTACAA

259


651

Fibrobacter

F

GTTCGGAATTACTGGGCGTAAA

succinogenes

R

CGCCTGCCCCTGAACTATC

Butyrivibrio

F

TAACATGAGTTTGATCCTGGCTC 417

(Liu et al.,

fibrisolvens

R

CGTTACTCACCCGTCCGC

2012)

Total protozoa

F

GCTTTCGWTGGTAGTGTATT

R

CTTGCCCTCYAATCGTWCT

1

F: forward; 2R: reverse

652 653


122

223

(Lane, 1991)

Sylvester et al., 2004)


654

Table 4. Effect of dietary n-6:n-3 PUFA ratios on growth performance of goats Item

655 656 657

Page 28 of 33

Dietary n-6:n-3 PUFA ratios1 LR

MR

HR

Initial BW (Kg)

14.74

13.97

13.66

Final BW (Kg)

26.38

26.19

Total feed intake (Kg)

68.93

Average daily gain (g/d)

SEM

P-value Linear

Quadratic

0.24

0.619

0.172

25.68

0.41

0.311

0.828

67.58

66.56

0.94

0.885

0.367

116.43

119.86

120.13

5.60

0.526

0.399

Total weight gain (Kg)

11.64

11.99

12.01

0.56

0.526

0.399

Gain to feed ratio

0.17

0.18

0.18

0.01

0.753

0.377

LR: low n-6:n-3 PUFA ratio (2.27:1) ; MR: medium n-6:n-3 PUFA ratio (5.01:1); HR: high n-6:n-3 PUFA ratio (10.38:1).

658 659 660 661 662 663 664 665 666 667 668 669


Page 29 of 33

670


Table 5. Effect of different n-6:n-3 fatty acid ratios on rumen fermentation characteristics P-value

Dietary n-6/ n-3 Ratios Item

LR

MR

HR

SEM

Linear

Quadratic

Rumen pH

6.08

6.16

6.29

0.03

0.04

0.54

Rumen NH3N (mg/dL)

11.00

9.74

10.51

0.41

0.49

0.12

Total VFA (mM)

87.30

86.70

70.02

2.52

Dietary n-6:n-3 Fatty Acid Ratios Alter Rumen Fermentation

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