Sunflower Oil and Nannochloropsis oculata Microalgae as Sources of

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Sunflower Oil and Nannochloropsis Oculata Microalgae as Sources of Unsaturated Fatty Acids for Mitigation of Methane Production and Enhancing Diets’ Nutritive Value Ali S Gomaa, Ahmed E. Kholif, A.M. Kholif, Reda Salama, Hamza A. El-Alamy, and Olurotimi Olafadehan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04704 • Publication Date (Web): 04 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Journal of Agricultural and Food Chemistry

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Running head: Dietary fat sources affect diets’ nutritive value

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Sunflower Oil and Nannochloropsis Oculata Microalgae as Sources of Unsaturated

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Fatty Acids for Mitigation of Methane Production and Enhancing Diets’ Nutritive

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Value

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Ali S. Gomaa†, Ahmed E. Kholif

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Alamy†, Olurotimi A. Olafadehan§

†,*

, Abdelkader M. Kholif †, Reda Salama‡, Hamza A. El-

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†

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‡

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Egypt

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§

Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, Giza, Egypt Department of Animal Production, Faculty of Agriculture, Al-Azhar University, Cairo,

Department of Animal Science, University of Abuja, Abuja, Nigeria

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Corresponding author: Ahmed E. Kholif. Address: Dairy Science Department, National

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Research Centre, 33 Bohouth St. Dokki, Giza, Egypt. E-mail: [email protected]; Tel:

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+201114012306.

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ACS Paragon Plus Environment


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ABSTRACT: The objective of this assay was to investigate the effect of adding sunflower

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oil and Nannochloropsis oculata microalgae, and their mixture at 0, 1, 2, 3, 4, and 5% to

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three total mixed rations (TMR) with different concentrate:forage ratios (40C:60F, 50C:50F,

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and 60C:40F) on in vitro gas production (GP), methane (CH4) production and nutrient

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degradability. Asymptotic GP, GP rate, CH4 concentration/g acid detergent fiber (ADF), dry

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matter (DM) degradability (DMD), short chain fatty acids (SCFA), and ruminal bacteria

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population increased, but neutral detergent fiber (NDF) degradability (NDFD), ADF

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degradability (ADFD), and protozoa count decreased with increasing concentrate level in the

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TMR. Methane production/g DM and NDF was higher for 50C:50F TMR. Sunflower oil

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reduced asymptotic GP, lag time, CH4 production/g ADF, ammonia-N (NH3-N), and SCFA.

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Compared to the control treatments, additives decreased GP rate while sunflower oil/N.

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oculata mixture increased DMD and NDFD. All additives at 5% increased GP rate and lag

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time and decreased CH4 production/g DM, ADF and NDF, ruminal NH3-N, and protozoa

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count. All additives at 2% increased DMD, NDFD and ADFD, SCFA, and bacteria

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population. Supplementation of TMR, containing different concentrate:forage ratios, with

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sunflower oil, N. oculata, and sunflower oil/N. oculata mixture at different doses modified in

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vitro GP, CH4 production, and nutrient degradability.

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KEYWORDS: Dietary fats, in vitro gas production, in vitro methane production, microalgae,

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sunflower oil.

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INTRODUCTION

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Manipulation of ruminal microbial ecosystems and fermentation to mitigate methane (CH4)

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emission and improve nutrient utilization for improved and sustainable ruminant production

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is one of the major concerns of ruminant nutritionists. Enteric CH4 from ruminant livestock

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accounts for 3.3 and 17% of global greenhouse and CH4 emissions, respectively,1 and also

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represents a loss of up to 15% of gross energy intake.2 It thus becomes imperative to develop

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abatement strategies that reduce enteric CH4 production and improve feed utilization, diet

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digestibility, and livestock productivity.3 Nutritional strategies, including yeast,4 organic

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acids salts,5 exogenous enzymes,6 and essential oils,7 have been used to mitigate ruminal CH4

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production from ruminants.

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Both plant oils and microalgae are rich in unsaturated fatty acids (UFA) which have

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proved effective in suppression of methanogenesis and improvement of ruminal fermentation

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and nutrient degradability.6,8,9 Vegetable oils and microalgae are rich sources of UFA,

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including docosahexaenoic acid and conjugated linoleic acid fatty acids. However, very few

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studies have compared the efficacy of these two additives in CH4 abatement and feed

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utilization in ruminants. Sunflower oil is an excellent source of polyunsaturated fatty acids

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(PUFA) and conjugated linoleic acid (66% of total fatty acids),10 and is therefore considered

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as a strategy to alter the proportion of saturated (SFA) and UFA in animal products through

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extensive ruminal biohydrogenation.8 Unlike other major oilseeds (soybeans, cottonseeds and

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rapeseeds), sunflower does not have anti-nutritional factors; therefore, it is a safe feedstuff for

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all livestock species.8 Morsy et al.8 and Kholif et al.9 observed that feeding vegetable oils to

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lactating goats modified the fatty acid profile without negative effects on ruminal

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fermentation or nutrient digestibility.



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Microalgae have been used to improve growth rates and feed efficiency in ruminants11

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and mitigate enteric CH4 emission.6,12 Some microalgae have been reported as rich sources of

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n-3 PUFA, such as α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid.12

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Microalgae species, such as Nannochloropsis sp., have been considered as sources of these

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fatty acids.13 Nannochloropsis oculata, a marine microalga, has an excellent composition and

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contains all the essential amino acids required for animal feed.14 It contains a high

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concentration of eicosapentaenoic acid (215 g/kg total fat) and some docosahexaenoic acid

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(32 g/kg fat).15 It can therefore be used to inhibit the in vitro biohydrogenation of fatty acids,

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resulting in reduction in the amount of SFA and an increase in UFA.16 Inclusion of dietary

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lipids rich in docosahexaenoic acid and eicosapentaenoic acid can enhance the nutritive value

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of the end products of ruminant production (e.g., milk and meat) and improve animal

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performance with several beneficial effects on human health.9 Polyunsaturated fatty acids

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have been shown to possess defaunating property due to their toxicity to rumen

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methanogens17 and ability to disrupt microbial cell membranes18 and improve ruminal

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fermentation.8 Some awareness about the negative effects of high inclusion levels of plant

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oils19 and microalgae20 should be considered, because the high levels may reduce feed intake

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and fiber digestion.

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Treatment of diets containing different concentrate:forage ratio with varying doses of

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different UFA sources could be a viable option for improving in vitro gas production (GP)

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and nutrient degradability and mitigating enteric CH4 production. Unfortunately, to our

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knowledge, no study has considered GP, nutrient degradability and CH4 production

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abatement of concentrate:forage diets treated with UFA additives at different doses. The

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present assay thus aimed to compare sunflower oil and N. oculata microalgae, as UFA

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sources, to alter in vitro ruminal fermentation, CH4 production, and nutritive value of three

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different total mixed rations (TMR) with different concentrate:forage ratios (40C:60F,


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50C:50F, and 60C:40F). The hypothesis was that different diets (substrates) with different

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concentrate:forage ratios and characteristics (different energy and fiber contents), and

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different sources of dietary fats would make some changes in the ruminal microorganisms

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and fermentation, resulting in changed dietary nutritive value and fermentation patterns.

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MATERIALS AND METHODS

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Nannochloropsis oculata microalgae

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Lyophilized N. oculata biomass was obtained from the Algal Biotechnology Unit, National

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Research Centre, Egypt. Inoculum was prepared using BG-II growth medium.21 Production

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of microalgae was in an artificial seawater growth medium containing 9.9 mmol N in the

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form of KNO3. The macronutrients growth medium contained (per 1 L): 1 g KNO3 [KNO3

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was substituted for urea as a nitrogen source at 0.297 g/L based on the initial N concentration

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(9.9 mmol N)], 0.07 g KH2PO4, 6.6 g MgSo4.7H2O, 1.5 g CaCl2.2H2O, 27 g NaCl, 0.014 g

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FeCl3.6H2O, 0.019 g EDTA disodium salt, and 1.0 mL trace elements. The trace elements

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solution contained per 1 L of distilled water: 2.86 g H3BO3, 1.81 g MnCl2.4H2O, 0.222 g

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ZnSO4.7H2O, 0.39 g NaMoO4.2H2O, 0.079 g CuSO4.5H2O, and 49.4 mg Co(NO3)2.6H2O.

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Continuous light illumination was provided from daylight lamps (10 × 40 w). Aeration

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was achieved using an oil-free air compressor (Hiblow Air Pump, SPP-100GJ-H, Techno

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Takatsuki Co. Ltd., Japan) through a 3-mm polyethylene tube. Room temperature was

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adjusted to 27 ± 2 °C during the whole incubation period. Incubation was carried out using

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fully transparent polyethylene bags (75 × 5 cm2 and 100 µm thickness) containing 2.5 L of

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algal broth. Mass production of N. oculata was performed within a 1200-L Zigzag

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photobioreactor. For harvesting and cleaning of the obtained biomass, a series of precipitation



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and washing was performed using tap water and a cooling centrifuge (Runne, Hideberg,

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RSV-20, Germany).

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Chemical composition of N. oculata microalgae showed that it contained 910 g dry

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matter (DM)/kg and 807 g organic matter, 290 g crude protein, 105 g total carbohydrates

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content, and 102 g oil/kg DM.

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Substrates and chemical analysis

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Berseem clover (Trifolium alexandrinum) forage was combined with a concentrate diet at

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different ratios to produce TMR. Thus, three TMR with different concentrate:forage ratios:

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(1) 40% concentrate + 60% berseem clover, (2) 50% concentrate + 50% berseem clover, and

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(3) 60% concentrate + 40% berseem clover) were prepared and used as substrates (Table 1).

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N. oculata microalgae and sunflower oil were included individually or their mixture (1:1 on

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DM basis) was added to each TMR at 0, 1, 2, 3, 4, and 5% on DM basis [n= 3 replicates

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(bottles) for each level]. The individual fatty acids (g/kg total fatty acids) of sunflower oil

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were: 54 g myristic acid (C14:0), 46 g stearic acid (C18:0), 210 g oleic acid (C18:1) [ω9],

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and 690 g linoleic acid (C18:2) [ω6].

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Samples of the ingredients, TMR, and microalgae were analyzed for DM (method ID

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934.01), ash (method ID 942.05), N (method ID 954.01), and ether extract (method ID

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920.39) according to AOAC22. The ingredients and TMR were analyzed for neutral detergent

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fiber (NDF), acid detergent fiber (ADF), and lignin according to Van Soest et al.23 with the

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use of an alpha amylase and sodium sulfite.

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For dry weight measurement, 5 mL of the algal broth was separately filtered over a pre-

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weighed Whatman sterile membrane filters (pore size 0.45 µm, 0.47 mm diameter and white

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grade). After filtration, filters were left to dry for 30 minutes at 105°C in a circulated oven,


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kept over anhydrous calcium chloride until room temperature was attained, and re-weighed.

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The difference between weights mirrored the net dry weight of the grown alga within a

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defined sampling time. Dry weight was calculated as g/L.

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To determine fatty acids of N. oculata, fatty acid methyl esters of the total lipids were

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prepared by transemethylation using 2% sulfuric acid in methanol.24 The fatty acid analysis

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was done by a Perkin Elmer Auto System XL gas chromatography (Perkin-Elmer, Norwalk,

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CT) equipped with flame ionization detector and a DB5 silica capillary column (60 m × 0.32

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mm i.d.). The oven temperature was maintained initially at 45oC, programmed to 60°C at a

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rate 1oC/min, and further programmed from 60°C to 240°C at a rate of 3°C /min. Helium was

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used as the carrier gas at flow rate 1 ml/min. The injector and the detector temperatures were

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set at 230°C and 250°C, respectively.

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Ruminal inoculum and incubation

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The inoculum was collected before morning feeding from three Barki sheep fed a standard

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diet consisting of berseem clover and CFM containing 158 g crude protein and 316 g NDF/kg

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DM at 1:1 DM basis ad libitum, with free access to water. Sheep were fed twice daily at

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08:00 and 16:00 h and managed under the conditions stipulated in the Guide for the Care and

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Use of Agricultural Animals in Agricultural Research and Teaching.25 Rumen content was

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placed in a plastic thermos preheated at 39°C and transported to the laboratory where it was

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flushed with CO2, mixed, and strained through four layers of cheesecloth into a flask with O2-

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free headspace. The rumen content was maintained at a temperature of 39°C with a

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continuous flow of CO2. The three different inoculums were first pooled and then distributed.

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Samples (0.5 g DM) of the substrates were weighed into 120 mL-bottles with

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appropriate addition of the additives (i.e., N. oculata microalgae, sunflower oil or their



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mixture)/g DM. Additives were added on top of the samples before adding the incubation

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medium. The incubation solution was prepared following the Menke and Steingass26 method,

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and warmed at 39°C under a continuous flow of CO2. Exactly 40 mL of the buffer solution

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together with 10 mL of ruminal liquor were added to each bottle, maintained at constant CO2

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flow for 30 sec, capped with neoprene plugs, and sealed with aluminum rings. As previously

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noted, additives were added at 0, 1, 2, 3, 4, and 5% on DM basis (n = 3 bottles for each level)

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for each individual TMR. Additionally, three bottles as blanks (rumen fluid only) were

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incubated for 72 h. The bottles were placed in an incubator (Thermo Fisher Scientific, TX,

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USA) at 39°C for 72 h. Incubation runs were performed three times in three different weeks,

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with an inoculum collected before morning feeding from the same sheep.

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In vitro gas production protocol and methane determination

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Gas production was determined as described by Menke and Steingass26 at 2, 4, 6, 8, 10, 12,

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24, 36, 48, 60, and 72 h of incubation, using a glass-calibrated syringe. After each gas

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reading, the bottles were vented to release the gas pressure, shaken, and returned to the

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incubator. Accumulated gas volumes (mL/g DM) were fitted using the NLIN procedure of

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SAS (SAS Inst. Inc. Cary, NC, USA) according to France et al.27 model as:

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y = A × [1 − e−c (t−Lag)]

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where y is the volume of GP at time t (h), A is the asymptotic GP (mL/g DM), c is the

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fractional rate of fermentation (/h), and Lag (h) is the discrete lag time prior to any gas

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formation.

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After recording the final gas volume at the end of incubation at 72 h, 4 mL of NaOH

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(10 M) was introduced to each bottle using a 5-mL capacity syringe. Mixing of the contents


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with the NaOH solution allowed the absorption of CO2, with the gas volume remaining in the

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headspace of bottles considered to be CH4.28

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Fermentation parameters and nutrient degradability

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After 72 h of incubation, bottles were opened, and samples of the supernatant (5 mL) from

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each bottle were collected in glass tubes for short chain fatty acids (SCFA) and ammonia-N

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(NH3-N) determinations. A subsample of 3 mL was preserved in 3 mL of 0.2 mol

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hydrochloric for NH3-N analysis according to AOAC22. Another subsample (0.8 mL) was

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mixed with 0.2 mL of a solution of metaphosphoric acid (250 g/L) for SCFA analyses by

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titration, after steam distillation.

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Four mL of the medium was mixed with 1 mL of 10% formaldehyde, shaken slightly,

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and placed in a refrigerator at 4°C until bacterial and protozoal counts according to the

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method of Galyean,29 based on the use of hemocytometer (Boeco, Hamburg, Germany) under

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optical microscope.

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After termination of incubation at 72 h and sampling the supernatant, the contents of

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each bottle were filtered under vacuum through glass crucibles with a sintered filter (coarse

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porosity no. 1, pore size 100 to 160 µm; Pyrex, Stone, UK), washed with distilled water, and

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dried at 105°C overnight to estimate apparent DMD. Both NDF and ADF were determined in

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the residues, after DMD determinations, for estimation of the degradability of NDF (NDFD)

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and ADF (ADFD) after correcting values for blanks.

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Statistical analyses



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Data were analyzed using the GLM procedure (SAS Inst. Inc. Cary, NC, USA) for a

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complete randomized design using the model: Yijkl = µ + Ai + Rj + Dk + (A × R)ij + (A × D)ik

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+ (R × D)jk + (A × R × D)ijk + εijkl where: Yijkl is the observation, µ is the population mean, Ai

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is the additive type effect, Rj is the ration type effect, Dk is the additive dose effect, (A × R)ij

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is the interaction between additive type and ration type, (A × D)ik is the interaction between

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additive type and additive dose, (R × D)jk is the interaction between ration type and additive

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dose, (A × R × D)ijk is the interaction between additive type, ration type and additive dose,

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and εijkl is the residual error. Tukey test was used to separate means. Polynomial contrasts

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were used to examine dose responses to increasing levels of concentrate in the rations (linear

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and quadratic) and for increasing doses of the additives (linear, quadratic, and cubic). The

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interactions were non-significant (i.e., P > 0.05) for most of the measurements; thus, only the

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main effects of ration types, additive types, and additive doses were reported.

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RESULTS

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In vitro gas production kinetics, methane production and nutrient degradability

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In vitro GP (expressed as mL/g DM) of the three TMR with different treatments is shown in

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Fig. 1. Asymptotic GP was highest and lowest (P < 0.001) for N. oculata and sunflower oil,

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respectively, while the additives increased (P = 0.046) rate of GP relative to the no-additive

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control. Lag time was highest and lowest (P = 0.032) for the control and sunflower oil,

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respectively (Table 3). Increasing the concentrate portion in the TMR linearly and

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quadratically increased (P < 0·001) asymptotic and rate of GP, but had no effect on lag time.

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Asymptotic GP was highest for 2% additive dose, resulting in linear, quadratic, and cubic

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trends (P < 0.001). Additive dose at 5% increased GP rate (P < 0.001) and lag time (quadratic

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effect, P = 0.005).


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Additives effect on CH4 production, expressed as mL CH4/g DM and mL CH4/g NDF,

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was marginal. Methane production expressed as mL CH4/g ADF was lowest (P = 0.026) for

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sunflower oil additive (Table 3). Additive level of 5% reduced CH4 production expressed as

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mL CH4/g DM and mL CH4/g ADF (linear effect, P < 0.001) and mL CH4/NDF (linear and

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quadratic effects, P < 0.01).

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N. oculata/sunflower oil mixture increased (P < 0.01) DMD and NDFD. Additives did

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not affect ADFD (Table 4). Increasing concentrate level in the TMR linearly and

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quadratically increased (P < 0.01) DMD, but decreased NDF (linear effect, P < 0.001) and

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ADF (linear and quadratic effects, P < 0.001). Compared to the control treatments (treatments

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without additives), sunflower oil, N. oculata, and N. oculata/sunflower oil mixture additive

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dose of 2% increased DMD, NDFD, and ADFD (P < 0.01).

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In vitro fermentation and ruminal microorganisms

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Feed additives had no effect on ruminal total bacteria and total protozoa counts (Table 4).

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Sunflower oil reduced (P < 0.01) GY24, NH3-N, and SCFA. Ruminal SCFA (linear effect, P

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< 0.001) was increased with increasing concentrate level in the TMR (Table 4).

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Concentrate:forage ratio in the TMR did not affect NH3-N concentration. Whereas ruminal

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total bacteria count increased (linear effect, P < 0.001; quadratic effect, P = 0.041) with the

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increasing concentrate:forage ratio, total protozoa count was reduced (P < 0.001).

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Two percent additive dose of individual and combined additives increased (linear,

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quadratic, and cubic effects, P < 0.001), SCFA, and total bacteria count (Table 4). Ammonia-

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N (linear and quadratic effect, P < 0.001) and total protozoa (linear effect, P = 0.017) were

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highest and lowest for 0% and 5% additive levels, respectively.

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DISCUSSION

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In vitro fermentation kinetics, methane production and nutrient degradability

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Higher asymptotic GP of N. oculata compared to the control treatment suggests that the

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microalgae supported increased fermentation of the insoluble but degradable fraction.30 This

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implies that the microalgae improved the availability and fermentation of dietary

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carbohydrate to acetate and butyrate.31 Greater rate of GP of the treatments compared to the

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control indicates enhanced ruminal degradability. Additives supplementation of diets varying

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in concentrate:forage ratio may thus enhance ration fermentability. Lower lag time of

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additives, particularly sunflower oil, implies faster microbial adaptation and activity.30,32 The

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increasing asymptotic, rate, and lag time of GP with increasing concentrate:forage ratio may

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be related to variation in the chemical constituents, particularly protein, energy, and fiber of

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the diets which markedly affect GP and fermentation kinetics.6,32 Generally, a TMR with a

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high concentrate level contains more fermentable organic matter (energy and protein)

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essential for enhanced ruminal fermentation and GP production.33 However, lower rates of

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GP have been reported for rations with high concentrate proportions.6,30 Discrepancy in

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results may be due to different substrates used. Asymptotic GP increased from 0 to 2%

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additive dose and reduced progressively as the dose level was increased to 5%, implying that

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2% additive dose was the optimum dose for improved carbohydrate fermentability. Higher

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GP rate and lag time with 5% additive dose indicate improved ruminal degradation and

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delayed microbial adaptation, respectively, to the substrates.

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Lower CH4 production at 72 h incubation (per g ADF) for sunflower oil additive may

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be attributed to the suppression of methanogens due to anti-methanogenic property of its

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PUFA. Vegetable oils are good sources of PUFA which are toxic to methanogens and thus

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inhibit methanogenesis.17 In the current study, sunflower oil was more effective for CH4


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abatement than N. oculata and the mixture of the two additives. Reduced CH4 production

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with 40C:60F was unexpected because fibrous diets digestion is accompanied by increased

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production of acetate, butyrate, and CH4 compared to concentrate rations.34 Moreover,

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hydrogen gas produced from the ruminal fermentation of carbohydrates can be utilized by

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methanogenic Archaea to synthesize CH4.35 The result contradicts earlier reports where

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increasing forage portion in the TMR increased total gas and CH4 production,6 and did not

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affect CH4 production.33 The inconsistency in results may be due to differences in the dietary

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and chemical composition of substrates, inoculum, and diets fed to the donors of the

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inoculum used. However, improved fermentation of the 60C:40F TMR may be responsible

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for the increased CH4 production since high GP, which comprises H2, CO2, and CH4,

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accompanies intensive ruminal fermentation. Decreased CH4 production with 5% additive

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level may be due to its reduced DMD, in consonance with previous reports6,12 where reduced

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CH4 production was attributed to decreased DMD. It could as well be attributed to reduced

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ruminal protozoa population, since fermentation by protozoa increases CH4 production.

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The observed higher DMD and NDFD with N. oculata/sunflower oil mixture suggests a

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synergy between these two additives in modulating the ruminal environment for improved

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ruminal microbes’ activity. Kholif et al.6 attributed improved ruminal nutrient digestibility of

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TMR diets supplemented with Chlorella vulgaris to enhanced ruminal microbes activity. The

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increasing DMD as the concentrate level increased in the TMR is speculatively due to

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progressive increase in highly digestible non-structural, readily fermentable carbohydrates,

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and decrease in less digestible structural fibers of the rations36 which possibly improved

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ruminal activity for enhanced degradation. This, however, did not translate into improved

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fiber degradability. Thus, the decreased fiber degradability of the high concentrate rations

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may be related to the lower dietary fiber levels relative to the high forage ration. Greater



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DMD, NDFD, and ADFD with 2% additive dose indicates this additive level as the most

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effective for optimal ruminal ecosystem, microflora activity, and thus nutrient degradability.

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305

In vitro fermentation kinetics and ruminal microorganisms

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Reduced NH3-N and SCFA with sunflower oil additive confirms the anti-microbial activity of

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the oil on ruminal microbes’ activity and consequently fermentation. The reduced ruminal

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fermentation must have lowered the cumulative GP and SCFA. Sunflower oil is a rich source

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of PUFA8 which have defaunating property or anti-methanogenic potential.17 Reduced NH3-

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N may be due to reduced population and activity of ruminal protozoa37 which play a major

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role in ruminal feed protein degradation38 or reduced peptidolytic activity of ruminal

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bacteria39 which inhibit the activity of hyper-NH3 producing bacteria. The reason for the

313

unaffected ruminal total bacteria and protozoa counts is unknown since both N. oculata and

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sunflower oil are rich sources of PUFA, which have anti-microbial activity. However, it

315

appears that the anti-microbial property of the two additives was not potent enough to

316

significantly reduce the numbers of the microbes.

317

Increased SCFA for the 60C:40F compared to the other diets may be due to increased

318

nutrient availability for rumen microflora growth and activity to stimulate the degradability of

319

the ration. Higher bacteria counts for the 60C:40F may be related to its greater non-structural

320

fermentable carbohydrates, whereas the increased protozoa counts for the 40C:60F may be

321

due to its high forage level and thus structural carbohydrates. Diet plays a prominent role in

322

influencing or changing rumen microbial composition and population. Effect of varying

323

concentrate:forage ratio on ruminal microbiota is demonstrated by the higher bacteria and

324

protozoa counts for the 60C:40F and 40C:60F, respectively. Diet has been reported to

325

influence rumen microbial composition within ruminant species and an individual.40 The


Page 14 of 44

Page 15 of 44


326

higher bacteria population and lower protozoa population of 60C:40F is desirable as it allows

327

for a lower CH4 production and higher bacteria numbers, but this was not the case in the

328

present study. Similar observations were made in buffaloes fed increasing level of legume

329

hay supplementation.41

330

Results of effect of additive dose on fermentation parameters and ruminal bacteria and

331

protozoa numbers show that they were additive dose dependent. Additive dose at 2%

332

improved ruminal ecosystem and thus fermentation, resulting in increased SCFA and total

333

bacteria counts relative to other additive doses. The decreased NH3-N concentration and

334

protozoa count of the treatments compared with the control is desirable as it indicates less

335

proteolysis of dietary crude protein and increased by-pass protein42 and the propensity for

336

lower CH4 production. Ruminal fermentation by protozoa is accompanied by CH4 production

337

which accounts for as much as 15% of dietary gross energy loss.2 Therefore, 5% additive

338

dose with lowest NH3-N concentration and protozoa count could reduce ruminal protein

339

degradation and CH4 production.

340

The TMR with a high concentrate proportion improved ruminal fermentation efficiency

341

and bacteria count, but increased CH4 production. Sunflower oil additive appears more

342

efficient in improving ruminal fermentation by increasing gas yield, NH3-N, and SCFA and

343

reducing CH4 production/g ADF. Additive dose at 5% increased GP rate and lag time and

344

decreased CH4 production, ruminal NH3-N, and protozoa population, while 2% dose

345

increased DMD, NDFD, ADFD, SCFA, and total bacteria count.

346

347

In summary, supplementation of TMRs with unsaturated fatty acids from sunflower oil, N.

348

oculata, and the equal mixture of the two unsaturated fatty acid sources at different doses

349

altered ruminal fermentation, in vitro GP, CH4 production, and nutrient degradability.



350

351

FUNDING SOURCES

352

None.

353

Conflict of interest

354

All authors declare that there are no present or potential conflicts of interest among them and

355

other people or organizations that could inappropriately bias their work.

356

Abbreviations

357

ADF, acid detergent fiber expressed exclusive of residual ash; ADFD, ADF degradability;

358

CH4, methane; DM, dry matter; DMD, DM degradability; GP, gas production; NH3-N,

359

ammonia-N; NDF, neutral detergent fiber expressed exclusive of residual ash; NDFD, NDF

360

degradability; PUFA, polyunsaturated fatty acids; SCFA, short chain fatty acids; SFA,

361

saturated fatty acids; TMR, total mixed rations; UFA, unsaturated fatty acids.

362

363

Manuscript’s significance

364

To the best of our knowledge, no study has tested ruminal fermentation and digestion of

365

diets with different concentrate and forage proportions supplemented with unsaturated fatty

366

acid additives at different doses.

367

Comparing different sources of dietary fat at different levels may define the best source and

368

the optimal dose for diets with different concentrate and forage proportions. This could be a

369

guide for animal nutritionists to formulate diets with the ability to alter fatty acids profile in

370

animal products (milk and meat) to the favor of human consumption.


Page 16 of 44

Page 17 of 44


371

Results of the present experiment may be applied in milk and meat production from goats and

372

cows to produce healthier animal products. Such feed additives may affect the chemical

373

composition of animal products. Moreover, the study also provides useful information on the

374

effect of such feed additives on methane production during ruminal fermentation of feeds.

375

Molecular studies of the effect of the tested feed additives on ruminal microbiota are

376

recommended.

377

378

REFERENCES

379

(1) Knapp, J.R.; Laur, G.L.; Vadas, P.A.; Weiss, W.P.; Tricarico, J.M. Invited review: Enteric

380

methane in dairy cattle production: Quantifying the opportunities and impact of reducing

381

emissions. J. Dairy Sci. 2014, 97, 3231-3261.

382

(2) Patra, A.K.; Saxena J. A new perspective on the use of plant secondary metabolites to

383

inhibit methanogenesis in the rumen. Photochemist. 2010, 71, 1198–1222.

384

(3) Grainger, C.; Beauchemin. K.A. Can enteric methane emissions from ruminants be

385

lowered without lowering their production? Anim. Feed Sci. Technol. 2011, 166, 308–320.

386

(4) Elghandour, M.M.Y.; Vázquez, J.C.; Salem, A.Z.M.; Kholif, A.E.; Cipriano, M.M.;

387

Camacho, L.M.; Márquez, O. In vitro gas and methane production of two mixed rations

388

influenced by three different cultures of Saccharomyces cerevisiae. J. Appl. Anim. Res. 2017,

389

45, 389-395

390

(5) Elghandour, M.M.Y.; Kholif, A.E.; Salem, A.Z.M.; De Oca, R.M.; Barbabosa, A.;

391

Mariezcurrena, M.; Olafadehan, O.A. Addressing sustainable ruminal methane and carbon

392

dioxide emissions of soybean hulls by organic acid salts. J. Clean Prod. 2016, 135, 194–200.



Page 18 of 44

393

(6) Kholif, A.E.; Elghandour, M.M.Y.; Salem, A.Z.M.; Barbabosa, A.; Marquez, O.; Odongo,

394

N.E. The effects of three total mixed rations with different concentrate to maize silage ratios

395

and different levels of microalgae Chlorella vulgaris on in vitro total gas, methane and

396

carbon dioxide production. J. Agric. Sci. 2017, 155, 494–507.

397

(7) Hernandez, A.; Kholif, A.E.; Lugo-Coyote, R.; Elghandour, M.M.Y.; Cipriano, M.;

398

Rodríguez, G.B.; Odongo, N.E.; Salem, A.Z.M. The effect of garlic oil, xylanase enzyme and

399

yeast on biomethane and carbon dioxide production from 60-d old Holstein dairy calves fed a

400

high concentrate diet. J. Clean. Prod. 2017, 142, 2384-2392.

401

(8) Morsy, T.A.; Kholif, S.M.; Kholif, A.E.; Matloup, O.H.; Salem, A.Z.M.; Abu Elella, A.

402

Influence of sunflower whole seeds or oil on ruminal fermentation, milk production,

403

composition, and fatty acid profile in lactating goats. Asian Australas. J. Anim. Sci. 2015, 28,

404

1116-1122.

405

(9) Kholif, A.E.; Morsy, T.A.; Abd El Tawab, A.M.; Anele, U.Y.; Galyean, M.L. Effect of

406

supplementing diets of Anglo-Nubian goats with soybean and flaxseed oils on lactational

407

performance. J. Agric. Food Chem. 2016, 64, 6163-6170.

408

(10) Ebrahimi, M.; Rajion, M.A.; Goh, Y.M. Effects of oils rich in linoleic and α-linolenic

409

acids on fatty acid profile and gene expression in goat meat. Nutrients 2014, 6, 3913-3928.

410

(11)

411

Agricultural Science of Biodiversity and Sustainability Workshop, Tune Landboskole,

412

Denmark. pp. 3–7, 1995.

413

(12) Anele, U.Y.; Yang, W.Z.; Mcginn, P.J.; Tibbetts, S.M.; McAllister, T.A. Ruminal in

414

vitro gas production, dry matter digestibility, methane abatement potential and fatty acid

415

biohydrogenation of six species of microalgae. Can. J. Anim. Sci. 2016, 96, 354–363.

Chowdhury,

S.;

Huque,

K.;

Khatun,

M.

Algae


in

animal

production.

Page 19 of 44


416

(13) Hulatt, C.J.; Wijffels, R.H.; Bolla, S.; Kiron, V. Production of fatty acids and protein by

417

Nannochloropsis in flat-plate photobioreactors. PloSone. 2017, 19, e0170440.

418

(14) Archibeque, S.L.; Ettinger, A.; Willson, B.D. Nannochloropsis oculata as a source for

419

animal feed. Acta Agronomica Hungarica 2009, 57, 245–248.

420

(15) Durmic, Z.; Moate, P.J.; Eckard, R.; Revell, D.K.; Williams, R.; Vercoe, P.E. In vitro

421

screening

422

for rumen methane mitigation. J. Sci. Food Agric. 2014, 9(4), 1191-1196.

423

(16) Boeckaert, C.; Vlaeminck, B.; Mestdagh, J.; Fievez, V. In vitro examination of DHA-

424

edible micro algae: 1., Effect on rumen lipolysis and biohydrogenation of linoleic and

425

linolenic acids. Anim. Feed Sci. Technol. 2007, 1, 36: 63-79.

426

(17) Dohme, F.; Machmüller, A.; Wasserfallen, A.; Kreuzer, M. Ruminal methanogenesis as

427

influenced by individual fatty acids supplemented to complete ruminant diets. Let. Appl.

428

Microbiol. 2001, 32, 47–51.

429

(18) Martin, C.; Morgavi, D.P.; Doreau, M. Methane mitigation in ruminants: from microbe

430

to the farm scale. Animal 2010, 4, 351–365.

431

(19) Kucuk, O.; Hess, B.W.; Rule, D.C. Soybean oil supplementation of a high-concentrate

432

diet does not affect site and extent of organic matter, starch, neutral detergent fiber, or

433

nitrogen digestion, but influences both ruminal metabolism and intestinal flow of fatty acids

434

in limit-fed lambs. J. Anim. Sci. 2004, 82, 2985−2994.

435

(20) Burnett, V.F.; Jacobs, J.L.; Norng, S.; Ponnampalam, E.N. Feed intake, liveweight gain

436

and carcass traits of lambs offered pelleted annual pasture hay supplemented with flaxseed

437

(Linum usitatissimum) flakes or algae (Schizochytrium sp.). Anim. Prod. Sci. 2016, 57, 877-

438

883.

of

selected

feed

additives,

plant

essential


oils

and

plant

extracts


439

(21) Stainer, R.Y.; Kunisawa, R.; Mandel, M.; Cohen-Bazire, G. Purification and properties

440

of unicellular blue green algae (order Chroococcales). Bacteriol. Rev. 1971, 35, 171–205.

441

(22) AOAC. Official Methods of Analysis, 16th ed. Association of Official Analytical

442

Chemists, Washington, DC., USA, 1997.

443

(23) Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for dietary ﬁbre, neutral detergent

444

ﬁbre, and non-starch carbohydrates in relation to animal nutrition. J. Dairy Sci. 1991, 74,

445

3583–3597.

446

(24) Christie, W.W. Preparation of ester derivatives of fatty acids for chromatographic

447

analysis. In: Advances in Lipid Methodology - Two, pp. 69-111 (Ed. W.W. Christie, Oily

448

Press, Dundee). 1993

449

(25) FASS. Guide for the Care and Use of Agricultural Animals in Agricultural Research and

450

Teaching. Fed. Anim. Sci. Soc., Champaign, IL. 2010

451

(26) Menke, K.H.; Steingass, H. Estimation of the energetic feed value obtained from

452

chemical analysis and gas production using rumen fluid. Anim. Res. Dev. 1988, 28, 7-55.

453

(27) France, J.; Dijkstra, J.; Dhanoa, M.S.; López, S.; Bannink, A. Estimating the extent of

454

degradation of ruminant feeds from a description of their gas production profiles observed in

455

vitro: derivation of models and other mathematical considerations. Br. J. Nutr. 2000, 83,

456

143–50.

457

(28) Demeyer, D.; De Meulemeester, M.; De Graeve, K.; Gupta, B.W. Effect of fungal

458

treatment on nutritive value of straw. Int. S. Crop 1988, 53, 1811-1819.

459

(29) Galyean, M. Laboratory procedures in animal nutrition research. New Mexico State

460

University; Las Cruces, NM, USA, 1989.


Page 20 of 44

Page 21 of 44


461

(30) Elghandour, M.M.Y.; Kholif, A.E.; Salem, A.Z.M.; Olafadehan, O.A.; Kholif, A.M.

462

Sustainable anaerobic rumen methane and carbon dioxide productions from prickly pear

463

cactus flour by organic acid salts addition. J. Clean Prod. 2016, 139, 1362-1369.

464

(31) Getachew, G.; Blummel, M.; Makkar, H.P.S.; Becker, K. In vitro gas measuring

465

techniques for assessment of nutritional quality of feeds: a review. Anim Feed Sci Technol.

466

1998, 72, 261–281.

467

(32) Elghandour, M.M.Y.; Kholif, A.E.; Bastida, A.Z.; Martínez, D.L.P.; Salem, A.Z.M. In

468

vitro gas production of five rations of different maize silage and concentrate ratios influenced

469

by increasing levels of chemically characterized extract of Salix babylonica. Turk. J. Vet.

470

Anim. Sci. 2015, 39, 186–194.

471

(33) Elghandour, M.M.Y.; Kholif, A.E.; Hernandez, A.; Salem, A.Z.M.; Mellado, M.;

472

Odongo, N.E. Effects of organic acid salts on ruminal biogas production and fermentation

473

kinetics of total mixed rations with different maize silage to concentrate ratios. J. Clean Prod.

474

2017, 147, 523-530.

475

(34) Kumar, S.; Dagar, S.S.; Sirohi, S.K.; Upadhyay, R.C.; Puniya, A.K. Microbial profiles,

476

in vitro gas production and dry matter digestibility based on various ratios of roughage to

477

concentrate. Ann. Microbiol. 2013, 63, 541–545.

478

(35) Stewart, C.; Flint, H.; Byrant, M.P. The rumen bacteria. In: The Rumen Microbial

479

Ecosystem (Eds PN Hobson, CS Stewart), pp. 10–55. New York, NY: Blackie Academic and

480

Professional. 1997

481

(36) Olafadehan, O.A.; Njidda, A.A.; Okunade, S.A.; Adewumi, M.K.; Awosanmi, K.J.;

482

Ijanmi, T.; Raymond, A. Effects of feeding Ficus polita foliage based complete rations with

483

varying forage:concentrate ratio on performance and ruminal fermentation in growing goats.

484

Anim. Nutr. Feed Technol. 2016, 16, 373-382.



485

(37) Bodas, R.; Prieto, N.; García-González, R.; Andrés, S.; Giráldez, F.J.; López, S.

486

Manipulation of rumen fermentation and methane production with plant secondary

487

metabolites. Anim. Feed Sci. Technol. 2012, 176, 78–93.

488

(38) Jouany, J.P. Effect of rumen protozoa on nitrogen utilization by ruminants: altering

489

ruminal nitrogen metabolism to improve protein utilization. J. Nutr. 1996, 126, 1335–1346.

490

(39) Busquet, M.; Calsamiglia, S.; Ferret, A.; Cardozo, P.; Kamel, C. Effects of

491

innamaldehyde and garlic oil on rumen microbial fermentation in a dual flow continuous

492

culture. J. Dairy Sci. 2005, 88, 2508–2516.

493

(40) Malmuthuge, N.; Guan, L.L. Understanding host-microbial interactions in rumen:

494

searching the best opportunity for microbiota manipulation. J. Anim. Sci. Biotechnol. 2017, 8,

495

doi 10.1186/s40104-016-0135-3.

496

(41) Chanthakhoun, V.; Wanapat, M.; Wachirapakorn, C.; Wanapat, S. Effect of legume

497

(Phaseolus calcaratus) hay supplementation on rumen microorganisms, fermentation and

498

nutrient digestibility in swamp buffalo. Livest Sci. 2011, 140, 17-23.

499

(42) Olafadehan, O.A.; Adebayo, O.F. Nutritional evaluation of ammoniated threshed

500

sorghum top as a feed for growing goats. Trop. Anim. Health Prod. 2016, 48, 785-791.

501


Page 22 of 44

Page 23 of 44


Figure captions

Fig. 1. In vitro rumen gas production (mL/g incubated DM) of three total mixed rations with different concentrate to berseem clover forage ratios in the presence of Nannochloropsis oculata microalgae, sunflower oil or their mixture (1:1 w/w) on (DM basis) added at: 0% ─●─, 1% ─●─, 2% ─●─, 3% ─●─, 4% ─●─, and 5% ─●─ to each ration.



Page 24 of 44

Table 1 Ingredients and composition of three rations used as substrates Ingredients Berseem

Rations clover Crushed

yellow Soybean

Wheat 40C:60F 50C:50F 60C:40F

forage

corn

meal

bran

Ingredients Crushed yellow corn

200

250

300

Soybean meal

133.3

166.7

200

Wheat bran

53.4

66.6

80

Calcium carbonate

6.7

8.3

10

Salt

3.3

4.2

5

3.3

4.2

5

600

500

400

Minerals

and

vitamins

mixture1 Berseem clover Chemical composition (g/kg DM)

24


Page 25 of 44


Dry matter (g/kg wet material) 141

866

889

871

423

493

564

Organic matter

882

890

928

852

876

875

874

Crude protein

133

91

408

130

160

166

173

Ether extract

25

45

21

56

30

31

32

Non-structural carbohydrate

301

540

356

204

347

358

370

Neutral detergent fiber

423

214

143

462

340

319

299

Acid detergent fiber

324

89

96

131

232

209

186

Cellulose

276

78

88

93

198

178

159

Hemicellulose

98

126

46

331

108

110

113

1

Contained per kg: 141 g Ca, 87 g P, 45 g Mg, 14 g S, 120 g Na, 6 g K, 944 mg Fe, 1613 mg Zn, 484 mg Cu, 1748 mg Mn, 58 mg I, 51

mg Co, 13 mg Se, 248000 IU vitamin A, 74000 IU vitamin D3, 1656 IU vitamin E.

25



Page 26 of 44

Table 2 Fatty acids profile of Nannochloropsis oculata microalgae (g/kg total fatty acids) Item

Content

Myristic acid (C14:0)

21.8

Pentadecylic acid (C15:0)

11.5

Palmitic acid (C16 : 0)

271.3

Palmitoleic acid (C16:1)

68.4

Hexadecadienoic acid (C16:2)

101.8

Margaric acid (C17:0)

10.4

Stearic acid (C18:0)

21.9

Oleic acid (C18:1)

372.6

Linoleic acid (C18:2) [ω6]

100.6

Alpha linolenic acid (C18:3) [ω3]

19.7

Total C18

514.8

Total saturated fatty acids (SFA)

336.9

Total mono-unsaturated fatty acids

441

Total polyunsaturated fatty acids

222.1

Total unsaturated fatty acids (UFA)

663.1

UFA/SFA

1.97

26 ACS Paragon Plus Environment

Page 27 of 44


Table 3 Effects of feed additives (sunflower oil, Nannochloropsis oculata microalgae, and their mixture (1:1 w/w) administered at six different levels (0, 1, 2, 3, 4, and 5%) to total mixed rations on in vitro gas production kinetics, methane production, and nutrient degradability. Gas production parameters*

Methane production % of gas

Diet

Additive

Dose

A

c

Lag

/g DM

/g NDF

/g ADF

production 40C:60F

Control

0

121

0.026

1.37

32.0

64.4

63.8

74.1

Nannochloropsis oculata

1

140

0.026

0.29

29.7

63.1

65.4

77.1

2

140

0.035

1.69

29.6

66.7

69.3

82.5

3

133

0.030

1.21

29.5

66.7

64.2

76.0

4

109

0.045

1.31

29.4

57.2

58.7

68.9

5

104

0.055

1.73

29.0

54.8

56.1

66.8

1

133

0.034

0.55

30.2

66.6

67.4

80.6

Sunflower oil

27



2

142

0.033

1.02

29.6

76.7

68.8

81.4

3

116

0.050

1.78

29.8

60.1

63.6

74.5

4

115

0.034

0.79

29.8

58.6

62.4

69.6

5

96

0.052

1.36

29.1

54.3

56.8

62.8

1

119

0.052

1.33

30.8

64.6

65.9

77.0

2

133

0.044

0.91

29.0

65.6

67.6

79.7

3

133

0.063

1.96

28.7

65.6

67.5

81.2

4

101

0.040

1.08

28.7

52.3

52.0

60.9

5

95

0.049

1.43

28.2

53.5

51.4

58.7

Control

0

152

0.031

1.30

26.0

62.7

72.7

98.4


1

168

0.028

1.51

24.5

59.1

70.6

91.7

Mixture

50C:50F

Page 28 of 44

28


Page 29 of 44


Sunflower oil

Mixture

2

182

0.026

1.32

24.6

61.2

71.8

98.9

3

189

0.023

0.77

24.9

54.3

76.2

103.0

4

132

0.037

1.74

24.4

53.7

61.9

84.7

5

149

0.023

1.29

24.2

50.3

61.0

81.0

1

158

0.032

1.09

24.3

57.8

68.9

94.3

2

166

0.035

1.43

23.5

59.3

70.7

94.7

3

144

0.031

1.26

22.8

49.3

57.1

79.2

4

133

0.034

0.93

23.3

50.5

61.6

83.1

5

132

0.032

1.33

23.5

51.6

61.2

81.3

1

159

0.037

1.32

24.5

60.0

70.6

95.4

2

182

0.025

0.78

23.5

57.5

68.7

93.8

3

176

0.024

0.79

23.9

56.0

66.5

91.8

29



60C:40F

Page 30 of 44

4

151

0.045

1.75

23.9

57.5

67.8

92.4

5

134

0.026

1.84

23.2

46.8

53.6

75.5

Control

0

168

0.032

1.54

23.0

59.6

72.0

150.7


1

180

0.035

1.73

22.2

60.0

72.2

141.3

2

201

0.029

1.23

21.0

58.3

69.5

139.3

3

158

0.041

1.19

21.0

55.3

67.4

134.3

4

137

0.049

1.77

21.1

50.0

62.8

134.2

5

126

0.046

1.40

21.2

45.9

55.1

112.6

1

171

0.037

1.04

21.7

55.5

68.2

136.9

2

173

0.050

1.24

21.5

56.8

67.9

135.7

3

133

0.045

1.27

21.5

45.9

61.8

119.1

Sunflower oil

30


Page 31 of 44


4

147

0.028

1.50

21.5

48.9

61.6

122.8

5

127

0.043

1.39

21.1

46.4

59.1

120.0

1

183

0.034

1.07

21.1

59.6

67.7

164.1

2

187

0.037

0.95

21.1

57.2

68.6

163.3

3

170

0.028

1.00

20.3

47.8

57.7

157.8

4

153

0.033

1.21

21.0

48.0

58.2

150.2

5

142

0.039

1.21

21.3

49.8

60.6

165.8

SEM

6.2

0.0035

0.200

1.61

3.31

3.51

10.60

Additive effect

Sunflower Oil and Nannochloropsis oculata Microalgae as Sources of

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