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Article

Altering Methane Emission, Fatty Acid Composition, and Microbial Profile during In Vitro Ruminant Fermentation by Manipulating Dietary Fatty Acid Ratios

by
Xiaoge Sun
1,2,
Qianqian Wang
1,
Zhantao Yang
1,
Tian Xie
1,
Zhonghan Wang
1,
Shengli Li
1 and
Wei Wang
1,*
1
State Key Laboratory of Animal Nutrition, Beijing Engineering Technology Research Center of Raw Milk Quality and Safety Control, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
2
Institute of Agricultural Sciences, Department of Environmental and Systems Science, ETH Zurich, 8092 Zurich, Switzerland
*
Author to whom correspondence should be addressed.
Fermentation 2022, 8(7), 310; https://doi.org/10.3390/fermentation8070310
Submission received: 3 June 2022 / Revised: 28 June 2022 / Accepted: 28 June 2022 / Published: 30 June 2022
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
This study evaluated the effects of different dietary n-6/n-3 polyunsaturated fatty acid (PUFA) ratios on in vitro ruminant fermentation. Methane production, fatty acid composition, and microbial profiles were compared after the in vitro fermentation of rumen fluid collected from cows that had been fed isoenergetic and isoproteic experimental diets at three different n-6/n-3 ratios: 3.04 (HN6, high n-6 source), 2.03 (MN6, medium n-6 source), and 0.8 (LN6, low n-6 source). The fermented rumen fluid pH and total volatile fatty acid (VFA) levels were significantly decreased (p < 0.05) in the HN6 group as compared with those in the MN6 and LN6 groups. Additionally, the HN6 group produced a significantly lower (p < 0.05) proportion of methane than the MN6 group during in vitro fermentation. The MN6 and LN6 groups had significantly increased (p < 0.05) levels of C18:2n6 and C18:3n3 in the fermented rumen fluid, respectively, as compared with the HN6 group. The Chao 1 diversity index value was lower (p < 0.05) in the HN6 group than in the MN6 and LN6 groups. The observed species richness was significantly lower (p < 0.05) in the HN6 group than in the MN6 group. The reduced relative abundances of Lachnospiraceae UCG-006 and Selenomonas in the HN6 group resulted in lower pH and VFA levels (i.e., acetate, propionate, butyrate, and total VFA) during in vitro fermentation. Furthermore, n-6 and n-3 PUFAs were toxic to Butyrivibrio_2 growth, resulting in high levels of incomplete biohydrogenation. Taken together, the study findings suggest that supplementation of high-forage diets with high levels of n-6 PUFAs could reduce methane emissions, whereas both VFA concentration and pH are reduced.

1. Introduction

Meat and dairy products from ruminants are sought-after sources of nutrients for human diets. Fatty acids (FAs) are a major component of lipids in animal products, among which polyunsaturated fatty acids (PUFAs) comprise the main components of cell membranes and play a key role in various biological processes such as the immune system [1,2], enzyme activities, cell proliferation and differentiation, and receptor expression [3]. The two major PUFA families include the n-3 (omega-3) and n-6 (omega-6) FAs.
Studies using a variety of animal models have demonstrated that n-6 and n-3 PUFAs regulate the expression of several genes associated with carbohydrate and lipid metabolism as well as hormonal responses [4,5]. For example, dietary supplementation with octadeca carbon FA (including n-3 and n-6 PUFAs) seemed to reduce methane emissions from ruminants due to biohydrogenation and the toxic effects of free FAs on both methanogens and protozoa [6]. In ruminants, biohydrogenation transformed PUFAs into saturated FAs and thereby decreased the amount of PUFAs for absorption, which in turn affected the microbial community associated with rumen fermentation. However, some studies have reported that despite the biohydrogenation of unsaturated FAs, the remaining n-6 and n-3 PUFAs that escape from the rumen and are absorbed by the intestine can still increase the n-3 or n-6 PUFAs in meat FA and play a key role in cellular function and animal performance [7,8]. Additionally, supplementation with n-3 or n-6 PUFAs has been found to reduce the methane output of dairy cows [9] and to increase the levels of conjugated linoleic acid or vaccenic acid in the rumen [10,11], which are beneficial to human health [12]. The negative effects of dietary fat supplementation depend on the fat type (chain length, degree of unsaturation), process (oil, coated, raw, or processed oilseeds), basal diet composition (forage ratio), and dosage [13,14,15].
The rumen simulation technique is widely used to precisely control the conditions and factors that affect fermentation in dairy cows and to measure end-product formation [16,17]. Previous studies have evaluated the effects of different fat sources on ruminant fermentation in vitro [18,19]. However, these studies mainly investigated the effects of the addition of oil sources on ruminant fermentation. Thus far, the effects of different ratios of n-3 to n-6 PUFA on in vitro rumen fermentation and microorganism-associated changes in methane production have rarely been explored. Therefore, the effects of specific fat sources on digestibility, rumen fermentation, and the microbial population need to be assessed.
The objectives of the current study were to evaluate the effects of dietary fat sources with different PUFA ratios (high saturated FA source, high n-6 source, low n-6 source) on in vitro ruminant fermentation patterns, including pH, gas production, FA profile, and microbial diversity.

2. Materials and Methods

All experimental procedures were approved by the Ethical Committee (Grant number: AW1110202-2, 25 August 2019) of the College of Animal Science and Technology at China Agriculture University (Beijing, China).

2.1. Fermentation Substrates

Three isoenergetic and isoproteic experimental diets, referenced to the dry dairy cow diet containing high forage, were designed as fermentation substrates (Table 1). Forage has a relatively higher n-3 fatty acid content than grain feed. Therefore, the n-6/n-3 ratio was relatively low compared to other studies using low-forage diets for lactating dairy cows. Normally, the n-6/n-3 ratio in a dry cow diet is approximately 2:1, therefore we chose the ratios of 2.03:1, 3.04:1, and 0.8:1 for our study. The diets with high n-6 PUFA levels (HN6, n-6/n-3 ratio = 3.04), moderate saturated FAs levels (MN6, n-6/n-3 ratio = 2.03), and low n-6 PUFA levels (LN6, n-6/n-3 ratio = 0.8) contained 9.67% extruded soybean, 1.51% hydrogenated FAs, and 4.47% extruded flaxseeds, respectively, on a dry matter (DM) basis. Hydrogenated FAs were obtained from Menergy (Yihai Kerry Group, Wuhan, China). Extruded soybeans enriched with n-6 PUFAs and extruded flaxseeds enriched with n-3 PUFAs were obtained from the Henan Shennong Feed Technology Company (Zhengzhou, China).
All samples were dried in an oven at 65 °C for 48 h until they reached a constant weight; they were subsequently ground in a small hammer mill (Thomas Scientific, Swedesboro, NJ, USA) and passed through a 1 mm sieve (40 mesh). The DM content of the total mixed ration (TMR) was determined by oven drying at 135 °C for 2 h, as previously described [20]. The crude protein (CP) content was determined using a Rapid N Exceed nitrogen and protein analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany) according to the manufacturer’s instructions [21]. The crude fat content was determined by ether extraction (EE) according to method 920.39 of the Association of Official Analytical Chemists [21]. Feed samples were also analyzed for acid detergent fiber (ADF) and neutral detergent fiber (NDF) content using the ANKOM 2000i automatic fiber analyzer (Beijing Anke Borui Technology Co., Ltd., Beijing, China), as described by Van Soest et al. [22].

2.2. Rumen Fluid Preparation

Samples of rumen fluid were collected from three multiparous Holstein cows (daily milk yield: 25.3 ± 2.1 kg) via permanent rumen fistulas during the mid-lactation period. The cows were provided free access to water and TMR twice daily (6:00 am and 6:00 pm), with ad libitum consumption. The TMR diet was formulated using the CPM-Dairy software to meet the nutritional requirements recommended by NRC [23]. The dry matter intake (DMI) of the cows was 19.8 kg/d. The net energy of lactation and the crude protein content of the feed was 1.68 Mcal/kg and 17.3%, respectively. Two hours after the morning feeding, the rumen content was obtained from each cow and squeezed into a plastic thermos through four layers of cheesecloth. The collected rumen fluids were combined in a 5 L beaker, mixed thoroughly, blanketed with CO2, and heated to 39 °C in a preheated water bath.

2.3. Equipment and In Vitro Incubation

Buffer solution (pH 6.87) was prepared according to Menke [24], and CO2 was continuously injected into the buffer solution for approximately 30 min prior to inoculation. For each incubation bottle (capacity: 120 mL), a total of 0.5 g substrate, 25 mL filtered rumen fluid, and 50 mL pre-heated buffer were added. All bottles (3 treatments × 6 replicates × 5 time points = 90 bottles) were purged with N2 to create anaerobic conditions, rapidly sealed with butyl rubber stoppers and Hungate’s screw caps, and then immediately connected to the AGRS-III automated trace gas recording system (China Agricultural University, Beijing, China) via medical transfusion tubes. The AGRS-III system can be used to record cumulative gas production (GP) in real time, according to Yang et al. [25]. Samples were collected after incubation at 39 °C for 3, 6, 12, 24, and 48 h. Meanwhile, bottles (3 treatments × 6 replicates = 18 bottles) prepared according to the same procedures were separately connected to pre-emptied air bags and incubated at 39 °C for 48 h for further gas composition analysis.

2.4. Sampling

After incubation, the bottles were disconnected from the AGRS-III system and air bags. The pH value of the fermented material was measured immediately using a Starter 300 handheld pH meter (Ohaus Instruments Co., Ltd., Shanghai, China). The fermented material was filtered through a pre-dried and weighed nylon bag (300 mesh, 9 cm × 14 cm) to collect the fermented rumen fluid. The nylon bag was rinsed under tap water, squeezed manually, and dried in the oven at 65 °C for 48 h to determine the in vitro DM disappearance (IVDMD). The filtered and fermented rumen fluid was divided among five 2 mL sterile tubes. Two tubes were centrifuged at 4000× g for 15 min at 4 °C, the supernatants were mixed with 0.2 mL meta-phosphoric acid solution (250 g/L) at 4 °C for 30 min, and the mixtures were centrifuged at 10,000× g for 10 min at 4 °C. Subsequently, the supernatants were collected for ammonium nitrogen (NH3-N) and volatile fatty acid (VFA) analyses. Two tubes were stored at −20 °C for further FA profile analysis and microbial protein (MCP) analysis. The remaining tube was stored at −80 °C for further microbial community analysis. Gas samples (1 mL), obtained from the air bags using a gas-tight Hamilton syringe, were used for gas profile analysis.

2.5. Chemical Analysis and Calculations

NH3-N and MCP levels in the fermented rumen fluid were determined by spectrophotometry, as described by Verdouw et al. [26] and Cui et al. [27], respectively. VFA concentrations in the filtered rumen sample were measured by gas chromatography using the 6890N GC system (Agilent Technologies, Avondale, PA, USA), as previously reported by Zhang and Yang [28]. CH4, CO2, and H2 concentrations in the gas sample were analyzed using the 6890N GC (Agilent Technologies) equipped with a thermal conductivity detector, as described by Cui et al. [27]. The reference gas contained 1% H2, 8% CH4, and 40% CO2 (PanGas, Dagmersellen, Switzerland).
Samples (50–500 mg) of freeze-dried TMR were accurately weighed and transferred into the incubation tubes. Each of the tubes were slowly filled with 1 mL of benzene-containing internal standard, 1 mL of benzene, and 3 mL of freshly made 5% methanolic HC1 (prepared by slowly adding 10 mL of acetyl chloride to 100 mL of anhydrous methanol) so that the solvents would fall over the material without touching the side walls of the tube. After being tightly capped, the culture tubes were vortexed for 1 min at a slow speed in order for the material to remain 2–3 cm from the bottom. The tightly sealed tubes were heated in a water bath at 70 °C for 2 h; if the solvent escaped, 2 mL of benzene was added after cooling, and the tube was returned to the water bath to ensure complete methylation. FA methylation in the fluid was performed as previously described [29]. Either the fatty acid methyl esters obtained from the feed or the rumen fluid was determined by gas chromatography using a fused silica capillary column (DB-23, 60.0 m × 0.25 mm × 0.25 μm, US) and the 6890N GC (Agilent Technologies) equipped with a flame ionization detector. Results for each FA were expressed as mg/g of rumen culture sample.
IVDMD was calculated by determining the weight change of the substrate (DM basis) before and after in vitro incubation.
The cumulative GP data were fitted to a nonlinear model [30] as follows:
GP48 = A/(1 + (C/48)B)
where GP48 represents the total gas production (mL/g dietary DM) at time 48(h); A represents the asymptotic gas production (mL/g dietary DM) at a constant fractional rate (c) per unit time; B represents a parameter reflecting the shape of the curve; C represents the time (h) at which the maximum gas production rate reaches ½, and t represents the time at which the gas is recorded.
The maximum rate of substrate degradation (RmaxS,/h) was calculated as follows [31]:
TRmaxS = C × (B−1)(1/B)
RmaxS = (B × TRmaxS(B−1)/(CB + TRmaxSB)

2.6. DNA Extraction and Determination

Ruminal fluid DNA was extracted using the TIANGEN® TIANamp Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). Total DNA concentration and purity were determined using the NanoDropND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The V3-V4 region of the 16S rRNA gene was amplified according to the method described by Hao et al. [32]. The forward primer sequence 341F (CCTACGGGNGGCWGCAG) and the reverse sequence 806R (GGACTACHVGGGTATCTAAT) were used, as described by Guo et al. [33]. Amplicons were extracted from 2% agarose gels, purified using the AxyPrepDNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR green chemistry (SuperReal PreMix Plus, Tiangen Biotech Co., Ltd., Beijing, China).
Purified amplicons were pooled in equimolar amounts and sequenced using the Miseq PE300 platform (Illumina, Inc., San Diego, CA, USA) to generate paired-end (2 × 300) sequences. The original data were stored in Fastq format. Quality control processing was performed on the measured Fastq data using Trimmomatic v.0.36 and Pear v.0.9.6 software. A sliding window strategy was adopted when using the Trimmomatic software, with the window size set to 50 bp, the average quality value set to 20, and the minimum reserved sequence length set to 120. Pear software was used to remove N’ sequences. The high-quality raw reads were further filtered using fastp criteria (https://github.com/OpenGene/fastp accessed on 10 May 2021). Reads with unknown nucleotides (>10%) were removed. Paired-end clean tags were merged as raw tags using the FLASH v. 1.2.11 software, as described by Magoč and Salzberg [34]. The minimum overlap was set to 10 bp, and the mismatch rate was set to 0.1 to obtain Fasta sequences. Chimeric Fasta sequences were removed using UCHIME against a known database, and a de novo method was used to remove undesirably short sequences at the same time. A total of 1,679,967 raw tags and 1,653,042 clean tags were obtained. On average, 93,332 raw tags and 91,836 clean tags were obtained for each sample. The clean tags were further purified using the QIIME v.1.9.1 pipeline under specific filtering conditions [35]. After removing noise, the high-quality tags were clustered into operational taxonomic units (OTUs) with ≥97% similarity using the Vsearch v.2.7.1 software, according to the UPARSE pipeline [36]. Finally, 467,244 tags and 35,417 OTUs were obtained. On average, 25,958 tags and 1968 OTUs were obtained for each sample. Representative sequences (those with the highest relative abundance in each cluster) were classified using the RDP classifier v. 2.2 software, which is based on a naïve Bayesian model [37], along with the SILVA V.138.1 database (https://www.arb-silva.de/, accessed on 18 May 2021) [38], with confidence threshold values ranging from 0.8 to 1. The abundance statistics of each taxonomy were visualized using the Krona v. 2.6 software [39]. Alpha diversity indices (i.e., Chao 1, observed species, and Shannon) were calculated using QIIME. Sequences were aligned using the MUSCLE v. 3.8.31 software [40] (http://www.drive5.com/muscle/ accessed on 20 May 2021).

2.7. Statistical Methods

The relative abundances of bacterial taxa among the three groups were analyzed by Kruskal–Wallis test using the qvalue package in the R software. Data on bacterial beta-diversity were analyzed based on the unweighted UNIFRAC distance metric in the R software. The data from the bacterial alpha diversity indexes (i.e., Chao 1, observed species, and Shannon), total gas production, CH4 production, CO2, and O2 were analyzed by one-way analysis of variance (ANOVA) using the SAS version 9.4 software (SAS Institute Inc., Cary, NC, USA). Other data (i.e., IVDMD, pH, VFA, MCP, NH3, gas kinetics) were analyzed by two-way ANOVA using the SAS version 9.4 software. The data were presented as the mean ± standard error of the mean (SEM). Multiple comparisons were further analyzed using Tukey’s test for ANOVA (0.05 ≤ p ≤ 0.10) and Dunk’s test for Kruskal–Wallis. Statistical significance was considered when p-value ≤ 0.05, whereas tendencies were assumed for 0.05 ≤ p ≤ 0.10.

3. Results

3.1. Effects of Dietary Fat Source on In Vitro Ruminant Fermentation and N-Metabolism

The fermented rumen fluid of the HN6 tended to decrease the pH (p < 0.10) when compared with the other two groups (Table 2). In addition, the pH significantly increased (p < 0.01) over the incubation period. The IVDMD values did not differ significantly among the three dietary groups and tended to increase linearly (p < 0.01) with increasing incubation time (Table 2). No interaction effect was detected between the pH and the IVDMD.
The total VFA production was the lowest in the HN6 group. The total VFA production in the LN6 group was greater than that in the MN6 group after incubation for 48 h and that in the HN6 group after 12 h (Figure 1). The HN6 group had lower levels of acetate (p < 0.10), propionate (p < 0.05), and butyrate (p < 0.05) than the LN6 and MN6 groups (Table 2). However, the acetate to propionate ratio did not differ significantly (p > 0.05) among the three dietary groups. The total VFA concentration in fermented rumen fluid was affected (p = 0.04) by the interaction effect of treatment and time, but no interaction effects were detected for individual VFA concentrations.
NH3-N levels were lower (p < 0.10) in the HN6 and LN6 groups than in the MN6 group, and NH3-N accumulated over the incubation period (Table 2). MCP synthesis was significantly increased (p < 0.05) in the HN6 and LN6 groups as compared to that in the MN6 group. MCP levels increased (p < 0.05) over the incubation period, reaching a maximum level at 6 h before significantly decreasing (p < 0.05) to the lowest level at 48 h (Table 2). NH3-N and MCP levels did not display interaction effects with treatment and time.

3.2. Effects of Dietary Fat Source on In Vitro Gas Kinetics

As shown in Figure 2, the total cumulative GP after incubation for 48 h and the asymptotic GP were unchanged by the three dietary treatments, while the maximum rate of substrate degradation (RmaxS) was significantly higher (p < 0.05) in the MN6 group than in the HN6 and LN6 groups.

3.3. Effects of Dietary Fat Source on In Vitro Gas Emission Production

A significantly lower (p < 0.05) CH4 production was observed in the HN6 group than in the MN6 group, while the production of CH4 did not differ between the MN6 and LN6 groups (Figure 3A). The production of O2 was significantly decreased (p < 0.05) in the HN6 and LN6 groups as compared to that in the MN6 group (Figure 3C).

3.4. Effect of Dietary Fat Source on Fatty Acid Profile

The C16:1 level was greater in the MN6 group than in the LN6 and HN6 groups after incubation for 3 h, but it did not differ at other incubation times (Figure 4A). Moreover, the LN6 group had higher C18:3n3 levels after incubation for 3 and 6 h as compared with the HN6 and MN6 groups, but the levels did not differ at 12 and 24 h (Figure 4B). The C20:1 level was significantly greater (p < 0.05) in the LN6 group than in the MN6 group, especially after incubation for 6 h, whereas the C20:1 level did not change over the incubation period in the HN6 group as compared with that in the LN6 and MN6 groups (Table 3, Figure 4C). The HN6 and LN6 groups had significantly higher (p < 0.05) levels of C18:2n6 and C18:3n3, respectively, as well as higher C18:1n9c levels as compared to the MN6 group (Table 3). A significantly higher (p < 0.05) level of the C16:0 FAs was observed in the MN6 group as compared to that in the HN6 and LN6 groups. The C24:0 FAs were higher in LN6 than in HN6 at an incubation time of 3 h; inversely, HN6 FAs were higher at 6 h as compared to those in HN3 (Figure 4E). Other saturated FAs (i.e., C12:0, C14:0, C15:0, C17:0, C18:0, C20:0, C22:0) were not affected by the dietary n-6/n-3 ratio (Table 3, Figure 4D). Both C16:0 and C16:1 levels were significantly greater (p < 0.05) in the MN6 group than in the LN6 and HN6 groups (Table 3). The n-6/n-3 ratio was significantly lower (p < 0.05) in the LN6 group than in the MN6 and HN6 groups.
Incubation time also had an influence on FA fermentation in vitro. Overall, unsaturated FA levels decreased with increasing incubation time such that C18:1n9, C18:2n6, and C18:3n3 levels decreased and achieved relative stability after incubation for 12 h, whereas C16:1, C20:1, and C22:1 levels were reduced to zero after incubation for 48 h. The levels of medium-chain (<C18) saturated FAs (C12:0, C14:0, C:15, C17:0) increased over the incubation period. C16:0, C18:0, C20:0, and C22:0 levels reached a maximum after incubation for 6 h and then decreased to their lowest levels at 48 h. In contrast, the C24:0 level steadily decreased during incubation, from 3 to 48 h.

3.5. Effect of Dietary Fat Source on Microbial Profile

The Chao 1 diversity index value of the HN6 group was significantly lower (p < 0.05) than that of the MN6 and LN6 groups, which did not differ (Figure 5). The observed species richness was significantly lower (p < 0.05) for the HN6 group than for the MN6 group (Figure 5). Shannon diversity index values did not differ among the three dietary groups (Figure 5). The overall picture of the microbial composition of the samples in the three groups was obtained by PCoA (Figure 6), and the p-value of PERMANOVA was 0.210, showing that microbial communities were similar in the tested conditions.
The genera influenced by the three dietary treatments are listed in Table 4. The relative abundances of Ruminococcaceae_UCG-002, Lachnospiraceae_UCG-006, Selenomonas, Ruminiclostridium_6, and Ruminococcaceae_UCG-010 were significantly lower (p < 0.05) in the microbial community of the HN6 group than in the microbial community of the LN6 group. Meanwhile, the relative abundances of Prevotellaceae_UCG-003 and Methanobrevibacter were significantly higher (p < 0.05) in the microbial community of the HN6 group than in the microbial community of the LN6 group (Table 4). The relative abundances of Methanobrevibacter and Butyrivibrio_2 were lower (p < 0.05) in the MN6 group than in the LN6 group. Meanwhile, the relative abundances of Lachnospiraceae_UCG-006, Prevotellaceae_UCG-003, and Butyrivibrio_2 were increased (p < 0.05) in the MN6 group as compared to those in the HN6 group.

4. Discussion

Previous researchers have reported that the addition of dietary lipids trigger various changes in rumen fermentation depending on the amount and type of FA supplemented and the diet composition [41,42,43]. Moreover, unsaturated FAs are reportedly toxic to many species of rumen bacteria, particularly those that are involved in fiber digestion [44]. Furthermore, relatively higher dietary levels of vegetable oil (5.8 or 7% DM) induced the decrease of DMI and organic matter digestibility [9,45]. However, several studies did not report the negative effect of dietary C18 PUFAs on fiber degradation, especially at relatively lower levels of oil addition (≤5% dietary DM) [46,47,48]. Thus, the similar IVDMD values obtained among dietary groups in the current study could be due to the relatively low level of fat supplementation (4% DM). In addition, previous studies have suggested that C18 PUFA supplementation has little effect on rumen pH [49,50,51] and total VFA concentration [51,52,53], which contradicts the results of the current study, in which the HN6 diet induced lower pH and VFA concentrations (i.e., acetate, propionate, butyrate, and total VFA) as compared to the other diets; however, no difference was observed between the LN6 and MN6 diets. These inconsistencies may be due to differences in the fat type and the proportion of dietary forage used among the studies since a TMR containing 70% forage and 30% concentrate was used in the current study. Nevertheless, the study results concur with previous reports, in which high-forage diets supplemented with oil enriched with n-6 PUFAs (soybean oil) interfered with ruminal fermentation in dairy cows [54]; on the other hand, oil enriched with n-3 PUFAs (flaxseed oil) [53] and saturated FAs (palm oil) [52] had no effects on ruminal pH and total VFAs, respectively. Notably, the acetate to propionate ratio was not affected by any of the dietary treatments, which concurred with another study demonstrating that the FA type did not affect the acetate to propionate ratio in the rumen [55].
The n-6 PUFAs were hypothesized to decrease some microorganism activities associated with high-forage diets. Ruminococcaceae_UCG-002 was previously reported to be negatively correlated with glucose production [56], while Ruminococcaceae_ UCG-010 was associated with digestive fibers [57]. Prevotellaceae UCG-003 belongs to the genus Prevotella, members of which play a key role in the metabolism of carbohydrates such as sugar, starch, and xylan, and protein [58]. The relative abundances of Ruminococcaceae_UCG-002 and Ruminococcaceae_ UCG-010 were decreased in the HN6 group, but those of Prevotellaceae UCG-003 were increased. These results imply that a high n-6 PUFA diet could enhance the metabolism of carbohydrates and reduce fiber digestibility. Lachnospiraceae UCG-006 was reportedly negatively correlated with acetic acid and butyric acid levels, but positively correlated with the mRNA expression of adenosine monophosphate-activated protein kinase-α (AMPK-α) [59]. These results might explain the lower VFA concentrations observed in the HN6 group, which may have been caused by the inhibited growth of Lachnospiraceae UCG-006. Selenomonas spp. are obligate saccharolytic bacteria that participate in the fermentation of soluble sugars and lactate in the rumen [60]. Lactic acid is a major metabolic product of the genus A. furcosa [61]. These results could partially explain the lower pH in the rumen fluid of the HN6 group, in which the high n-6 PUFA diet decreased the relative abundance of Selenomonas. Taken together, these results indicate that oil with a high ratio of n-6/n-3 added to the high-forage diet had detrimental effects on the pH and VFAs during in vitro ruminant fermentation as compared with oil enriched with a relatively high n-6/n-3 ratio.
VFA concentrations increased with increasing incubation time. Increased pH after incubation for 3 h could be explained by the accumulation of NH3-N in fermented rumen fluid over the incubation period. Additionally, lower NH3-N concentrations and higher MCP concentrations were observed in the HN6 and LN6 groups. These results could indicate that the addition of unsaturated fat sources decreased the NH3-N concentration in the rumen [62] and enhanced the effectiveness of microbial nitrogen synthesis [63]. This may be due to the fact that the addition of high n-3 or n-6 PUFA can reduce the protozoa numbers [64]. This is in line with a previous study, which reported that plant oil (enriched with n-3 or n-6 C18 PUFA) supplementation in ruminant diets could depress the rumen ammonia concentration [65]. Ammonia is usually assumed to be produced by the decomposition of bacterial protein [66], which is often associated with the presence of protozoa [67]. According to Hristov et al. [68], the number of protozoa has a positive correlation with ammonia concentrations; reducing the number of protozoa could inhibit bacterial protein breakdown in the rumen. MCP levels were the highest after incubation for 6 h and decreased to their lowest levels at 48 h, which could be due to decreased substrate levels and the decomposition of MCP over the incubation period. Moreover, IVDMD values were not affected in the current study, which is likely due to the low FA concentration (<5%) used to supplement the diet. A previous study reported that lipid blends in the diet reduced IVDMD values when FA concentrations were more than 5%, but not at lower concentrations [69].
Generally, the addition of oil enriched with n-6 or n-3 PUFAs to a high-forage diet has been shown to reduce the production of fermentation gases [70]. Despite the cumulative GP and the asymptotic GP not being affected in the current study, the maximum rates of substrate digestion in the HN6 and LN6 groups were lower than that in the MN6 group. Additionally, the HN6 and LN6 groups produced less CH4 than the MN6 group, particularly the HN6 group. These results were consistent with the majority of studies reporting that dietary unsaturated fat supplementation can depress enteric methane emissions, although the extent of inhibition varied [71,72]. In particular, studies have reported that supplementation with C18 PUFA-rich oils inhibited methane production in vitro [73] or in vivo [74], and the extent differed according to the degree of unsaturation and the inclusion level [6]. This indicates that the n-6/n-3 ratio can also depress methane production as the different ratios will modify the degree of unsaturation of the dietary fat. In our study, it seems that the high n-6/n-3 ratio of additional PUFA is more efficient in decreasing CH4. Moreover, some studies have reported that high-forage diets might increase the inhibitory effect of PUFAs on CH4 production. For example, Martin et al. [9] reported that dairy cows fed high-forage diets supplemented with various forms of oilseed enriched with n-3 PUFAs exhibited a 10% reduction in CH4 production (g/kg DMI) for unprocessed seeds and a 49% reduction for crude oil from the seeds. Other studies demonstrated that the supplementation of a forage-based diet with oil enriched with n-6 PUFAs reduced the CH4 production of beef cattle (g/kg of DMI) by 17% [75] or 15% [76]. Taken together, these results indicate that supplemental PUFAs could reduce CH4 emissions, and the extent of CH4 inhibition may be influenced by the proportion of forage in the diet.
The biohydrogenation of unsaturated FAs in the rumen and propionate production are considered to be a competitive pathway for hydrogen use that inhibits CH4 production. However, the results of the current study indicate that CH4 and propionate levels were both decreased in the HN6 group. This inconsistency may be due to the small amount of total metabolizable hydrogen used in the biohydrogenation of endogenous PUFAs [77], but it may also reflect the direct effect on methanogenesis performed by rumen microorganisms [78]. However, a decreased relative abundance in methanogenic bacteria was not detected in the HN6 group, possibly because only bacterial relative abundance was measured and the ciliate protozoal population, considered to be one of the main methanogenesis microorganisms, was not detected [79]. Interestingly, the relative abundance of Methanobrevibacter, an anaerobic archaean that produces methane [80], was decreased in the HN6 group. These results suggest that higher n-3 PUFA levels might reduce CH4 emission.
The unexpected high O2 production may be due to the rumen fluid collected from cannula cows. Wang et al. [81] reported that the laparotomy incision is usually bigger than the diameter of the cannula, and that the muscle and skin support around the cannula can be compromised, leading to leakage of fermentation gases and rumen content. The leakage of fermentation gases from the rumen could then result in the increases in concentrations of N2 and O2. The influx of O2 into the headspace of the rumen has the potential to alter its strictly anaerobic microbial ecosystem, consequently increasing O2 concentration. This probably has little effect on the fermentation process. However, the specific reason is still unclear, and we intend to focus on a study regarding O2 in the future.
The highest levels of C16:0, C18:2n6, and C18:3n3 were detected in the MN6, HN6, and LN6 groups, respectively, which reflected the higher amounts of these FAs in the diets. This finding indicates that the n-6/n-3 ratio in the diet affects the FA composition in the ruminal fluid. Additionally, relatively high levels of C18:1n9c (and cis-C18:1 isomers), intermediaries of the biohydrogenation of unsaturated FAs [82], were detected among the three dietary groups, although the C18:1n9c concentrations in the HN6 and LN6 groups were higher than that in the MN6 group. However, the amount of stearic acid (C18:0), the end product of the biohydrogenation of unsaturated FAs [82], was not influenced by the diet. These results could reflect the incomplete biohydrogenation of unsaturated C18 FAs, which concurs with unsaturated C18 FA (C18:1n9c, C18:2n6, and C18:3n3) concentrations decreasing after incubation for 12 h but remaining stable at 24 and 48 h. However, unsaturated C16:1 and C22:1 were not detected at 48 h, suggesting that the complete biohydrogenation of these unsaturated FAs occurred. Notably, Kalscheur et al. [83] reported that incomplete biohydrogenation occurred to a greater extent in cows supplemented with C18 FA-enriched fat. Furthermore, the HN6 and LN6 groups displayed decreased relative abundances of Butyrivibrio_2 as compared with the MN6 group. Butyrivibrio_2 is reportedly involved in biohydrogenation [84]. The results suggest that n-6 and n-3 PUFAs were toxic to Butyrivibrio_2 growth, resulting in the incomplete biohydrogenation of unsaturated FAs.
For the rumen fermentation, the first 12 h of fermentation activities were very fast, and the biohydrogenation process mainly occurred during this period. The fatty acid composition is highly related to biohydrogenation. Therefore, the effect of fat addition on the rumen fluid fatty acid will be different between the beginning and end stages. This is the reason for the interaction effect in some FAs. This can also explain why some saturated fatty acids such as C22:0 increased (C22:1 biohydrogenation) in the first couple of hours but decreased in the last few hours after a breakdown in the rumen. In addition, fatty acids < 16 carbon mainly come from de novo synthesis using VFA [85]. As a result, C12:0 and C14:0 both increased with time. As for the fatty acids ≥ C16, which mainly come from feed, these would partially catabolize to VFA or CO2 [86]. Thus, the C16:0 decreased with time.
Beyond the aforementioned microorganisms, the Chao 1 diversity index value and the observed species richness were the lowest in the HN6 group, indicating that an increase in the n-6/n-3 ratio decreased bacterial community diversity and abundance. In addition, the HN6 group displayed the lowest relative abundance of Lachnospiraceae NK4A136 and the highest relative abundance of Ruminiclostridium_6. Lachnospiraceae NK4A136 is considered to be a potential probiotic and exerts anti-inflammation effects [87]. Higher Ruminiclostridium_6 abundance was positively correlated with cytokine levels [88]. Therefore, n-6 PUFA supplementation might have pro-inflammatory effects on ruminants, whereas n-3 PUFAs might have anti-inflammatory effects.

5. Conclusions

In conclusion, the study findings suggest that manipulating the n-6/n-3 ratio of dairy cow feed by adding extruded flaxseeds or soybeans could affect rumen fermentation, fatty acid composition, and the microbial community. Furthermore, the results indicate that supplementation with a high n-6 PUFA source decreased bacterial community richness and diversity, specifically reducing the relative abundance of Lachnospiraceae UCG-006 and Selenomonas, which resulted in lower ruminant pH and VFA (i.e., acetate, propionate, butyrate, and total VFA) levels in vitro. The results also suggest that n-6 and n-3 PUFAs are toxic to Butyrivibrio_2, resulting in a higher level of incomplete biohydrogenation in the rumen. Taken together, the study findings suggest that supplementing high-forage diets with high levels of n-6 PUFAs could reduce CH4 emissions.

Author Contributions

Conceptualization, S.L. and W.W.; methodology, W.W.; software, X.S.; validation, S.L.; formal analysis, X.S.; investigation, X.S., Z.Y., Q.W., Z.W. and T.X.; writing—original draft preparation, X.S.; writing—review and editing, W.W.; visualization, X.S.; supervision, S.L. and W.W.; project administration, W.W.; and funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32130100), and Beijing Municipal Science and Technology Project (Z191100004019023).

Institutional Review Board Statement

All experimental procedures were approved by the Ethics Committee of the College of Animal Science and Technology at China Agriculture University (Beijing China).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Moallem, U. Invited review: Roles of dietary n-3 fatty acids in performance, milk fat composition, and reproductive and immune systems in dairy cattle. J. Dairy Sci. 2018, 101, 8641–8661. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Sordillo, L. Nutritional strategies to optimize dairy cattle immunity. J. Dairy Sci. 2016, 99, 4967–4982. [Google Scholar] [CrossRef] [PubMed]
  3. Clandinin, M.; Cheema, S.; Field, C.; Garg, M.; Venkatraman, J.; Clandinin, T. Dietary fat: Exogenous determination of membrane structure and cell function. FASEB J. 1991, 5, 2761–2769. [Google Scholar] [CrossRef] [PubMed]
  4. Jump, D.B. N-3 polyunsaturated fatty acid regulation of hepatic gene transcription. Curr. Opin. Lipidol. 2008, 19, 242. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Palmquist, D. Omega-3 fatty acids in metabolism, health, and nutrition and for modified animal product foods. Prof. Anim. Sci. 2009, 25, 207–249. [Google Scholar] [CrossRef]
  6. Zhang, C.; Guo, Y.; Yuan, Z.; Wu, Y.; Wang, J.; Liu, J.; Zhu, W. Effect of octadeca carbon fatty acids on microbial fermentation, methanogenesis and microbial flora in vitro. Anim. Feed. Sci. Technol. 2008, 146, 259–269. [Google Scholar] [CrossRef]
  7. Silvestre, F.; Carvalho, T.; Francisco, N.; Santos, J.; Staples, C.; Jenkins, T.; Thatcher, W. Effects of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: I. Uterine and metabolic responses, reproduction, and lactation. J. Dairy Sci. 2011, 94, 189–204. [Google Scholar] [CrossRef] [Green Version]
  8. Silvestre, F.; Carvalho, T.; Crawford, P.; Santos, J.; Staples, C.; Jenkins, T.; Thatcher, W. Effects of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: II. Neutrophil fatty acids and function, and acute phase proteins. J. Dairy Sci. 2011, 94, 2285–2301. [Google Scholar] [CrossRef] [Green Version]
  9. Martin, C.; Rouel, J.; Jouany, J.P.; Doreau, M.; Chilliard, Y. Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil. J. Anim. Sci. 2008, 86, 2642–2650. [Google Scholar] [CrossRef] [Green Version]
  10. AbuGhazaleh, A.; Jacobson, B. The effect of pH and polyunsaturated C18 fatty acid source on the production of vaccenic acid and conjugated linoleic acids in ruminal cultures incubated with docosahexaenoic acid. Anim. Feed. Sci. Technol. 2007, 136, 11–22. [Google Scholar] [CrossRef]
  11. Luna, P.; Bach, A.; Juárez, M.; de la Fuente, M.A. Influence of diets rich in flax seed and sunflower oil on the fatty acid composition of ewes’ milk fat especially on the level of conjugated linoleic acid, n-3 and n-6 fatty acids. Int. Dairy J. 2008, 18, 99–107. [Google Scholar] [CrossRef] [Green Version]
  12. McIntosh, F.M.; Shingfield, K.J.; Devillard, E.; Russell, W.R.; Wallace, R.J. Mechanism of conjugated linoleic acid and vaccenic acid formation in human faecal suspensions and pure cultures of intestinal bacteria. Microbiology 2009, 155, 285–294. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Chalupa, W.; Rickabaugh, B.; Kronfeld, D.; Sklan, S.D. Rumen fermentation in vitro as influenced by long chain fatty acids. J. Dairy Sci. 1984, 67, 1439–1444. [Google Scholar] [CrossRef]
  14. Doreau, M.; Ferlay, A. Digestion and utilisation of fatty acids by ruminants. Anim. Feed. Sci. Technol. 1994, 45, 379–396. [Google Scholar] [CrossRef]
  15. Dewhurst, R.J.; Shingfield, K.; Lee, M.R.; Scollan, N.D. Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems. Anim. Feed. Sci. Technol. 2006, 131, 168–206. [Google Scholar] [CrossRef]
  16. Czerkawski, J.; Breckenridge, G. Design and development of a long-term rumen simulation technique (Rusitec). Br. J. Nutr. 1977, 38, 371–384. [Google Scholar] [CrossRef] [Green Version]
  17. Czerkawski, J.; Breckenridge, G. Experiments with the long-term rumen simulation technique (Rusitec); use of soluble food and an inert solid matrix. Br. J. Nutr. 1979, 42, 229–245. [Google Scholar] [CrossRef]
  18. Chalupa, W.; Vecchiarelli, B.; Elser, A.E.; Kronfeld, D.; Sklan, D.; Palmquist, D.L. Ruminal fermentation in vivo as influenced by long-chain fatty acids. J. Dairy Sci. 1986, 69, 1293–1301. [Google Scholar] [CrossRef]
  19. Vargas, J.; Andrés, S.; Yáñez Ruiz, D.R.; López, S. The effect of olive, sunflower or linseed oils on the fermentation pattern and methane production in the rumen simulating technique. Options Méditerr. A 2011, 99, 163–168. [Google Scholar]
  20. AOAC. Official Methods of Analysis of the Association of Official Analytical Chemists, 19th ed.; Association of Official Analytical Chemists, Inc.: Arlington, VA, USA, 2012; pp. 1048–1049. [Google Scholar]
  21. AOAC. Official Methods of Analysis, 15th ed.; AOAC: Arlington, VA, USA, 1990; Volume I. [Google Scholar]
  22. Van Soest, P.v.; Robertson, J.; Lewis, B. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  23. NRC. Nutrient Requirements of Dairy Cattle: 2001; National Academies Press: Washington, DC, USA, 2001. [Google Scholar]
  24. Menke, K.H. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 1988, 28, 7–55. [Google Scholar]
  25. Yang, H.; Song, Z.; Zhu, S.; Mao, E. Yizhong Fajiao Weiliang Qitichanshengliang Shuju Zidong Caiji Cunchu Zhuangzhi Ji Fangfa; Science and Technology at the China Agricultural University: Beijing, China, 2006. [Google Scholar]
  26. Verdouw, H.; Van Echteld, C.; Dekkers, E. Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 1978, 12, 399–402. [Google Scholar] [CrossRef]
  27. Cui, J.; Yang, H.; Yu, C.; Bai, S.; Wu, T.; Song, S.; Sun, W.; Shao, X.; Jiang, L. Effect of urea fertilization on biomass yield, chemical composition, in vitro rumen digestibility and fermentation characteristics of straw of highland barley planted in Tibet. J. Agric. Sci. 2016, 154, 151–164. [Google Scholar] [CrossRef]
  28. Zhang, D.; Yang, H. In vitro ruminal methanogenesis of a hay-rich substrate in response to different combination supplements of nitrocompounds; pyromellitic diimide and 2-bromoethanesulphonate. Anim. Feed. Sci. Technol. 2011, 163, 20–32. [Google Scholar] [CrossRef]
  29. Loor, J.J.; Herbein, J.H. Alterations in blood plasma and milk fatty acid profiles of lactating Holstein cows in response to ruminal infusion of a conjugated linoleic acid mixture. Anim. Res. 2001, 50, 463–476. [Google Scholar] [CrossRef] [Green Version]
  30. Groot, J.C.; Cone, J.W.; Williams, B.A.; Debersaques, F.M.; Lantinga, E.A. Multiphasic analysis of gas production kinetics for in vitro fermentation of ruminant feeds. Anim. Feed. Sci. Technol. 1996, 64, 77–89. [Google Scholar] [CrossRef]
  31. Yang, H.; Tamminga, S.; Williams, B.A.; Dijkstra, J.; Boer, H. In vitro gas and volatile fatty acids production profiles of barley and maize and their soluble and washout fractions after feed processing. Anim. Feed. Sci. Technol. 2005, 120, 125–140. [Google Scholar] [CrossRef]
  32. Hao, Y.; Huang, S.; Si, J.; Zhang, J.; Gaowa, N.; Sun, X.; Lv, J.; Liu, G.; He, Y.; Wang, W. Effects of paper mulberry silage on the milk production, apparent digestibility, antioxidant capacity, and fecal bacteria composition in Holstein dairy cows. Animals 2020, 10, 1152. [Google Scholar] [CrossRef]
  33. Guo, M.; Wu, F.; Hao, G.; Qi, Q.; Li, R.; Li, N.; Wei, L.; Chai, T. Bacillus subtilis improves immunity and disease resistance in rabbits. Front. Immunol. 2017, 8, 354. [Google Scholar] [CrossRef] [Green Version]
  34. Magoč, T.; Salzberg, S.L. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011, 27, 2957–2963. [Google Scholar] [CrossRef]
  35. Bokulich, N.A.; Subramanian, S.; Faith, J.J.; Gevers, D.; Gordon, J.I.; Knight, R.; Mills, D.A.; Caporaso, J.G. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat. Methods 2013, 10, 57–59. [Google Scholar] [CrossRef]
  36. Edgar, R.C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 2013, 10, 996–998. [Google Scholar] [CrossRef] [PubMed]
  37. Wang, Q.; Garrity, G.M.; Tiedje, J.M.; Cole, J.R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 2007, 73, 5261–5267. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Pruesse, E.; Quast, C.; Knittel, K.; Fuchs, B.M.; Ludwig, W.; Peplies, J.; Glöckner, F.O. SILVA: A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007, 35, 7188–7196. [Google Scholar] [CrossRef] [Green Version]
  39. Ondov, B.D.; Bergman, N.H.; Phillippy, A.M. Interactive metagenomic visualization in a Web browser. BMC Bioinform. 2011, 12, 385. [Google Scholar] [CrossRef] [Green Version]
  40. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  41. Kim, E.J.; Huws, S.A.; Lee, M.R.; Wood, J.D.; Muetzel, S.M.; Wallace, R.J.; Scollan, N.D. Fish oil increases the duodenal flow of long chain polyunsaturated fatty acids and trans-11 18:1 and decreases 18:0 in steers via changes in the rumen bacterial community. J. Nutr. 2008, 138, 889–896. [Google Scholar] [CrossRef] [Green Version]
  42. Wanapat, M.; Mapato, C.; Pilajun, R.; Toburan, W. Effects of vegetable oil supplementation on feed intake, rumen fermentation, growth performance, and carcass characteristic of growing swamp buffaloes. Livest. Sci. 2011, 135, 32–37. [Google Scholar] [CrossRef]
  43. Marín, A.M.; Gómez-Cortés, P.; Castro, A.G.; Juárez, M.; Alba, L.P.; Hernández, M.P.; De la Fuente, M. Linear discriminant analysis and type of oil added to dairy goat diets. J. Dairy Sci. 2012, 95, 4045–4049. [Google Scholar] [CrossRef]
  44. Jalč, D.; Potkański, A.; Szumacher-Strabel, M.; Kowalczyk, J.; Cieślak, A. The effect of a high forage diet and different oil blends on rumen fermentation in vitro. J. Anim. Feed. Sci. 2006, 15, 141–144. [Google Scholar] [CrossRef]
  45. Palmquist, D.L. The role of dietary fats in efficiency of ruminants. J. Nutr. 1994, 124, 1377S–1382S. [Google Scholar] [PubMed]
  46. Kholif, A.; Morsy, T.; Abdo, M. Crushed flaxseed versus flaxseed oil in the diets of Nubian goats: Effect on feed intake, digestion, ruminal fermentation, blood chemistry, milk production, milk composition and milk fatty acid profile. Anim. Feed. Sci. Technol. 2018, 244, 66–75. [Google Scholar] [CrossRef]
  47. Pi, Y.; Ma, L.; Pierce, K.; Wang, H.; Xu, J.; Bu, D. Rubber seed oil and flaxseed oil supplementation alter digestion, ruminal fermentation and rumen fatty acid profile of dairy cows. Animal 2019, 13, 2811–2820. [Google Scholar] [CrossRef] [PubMed]
  48. Vargas, J.E.; Andrés, S.; Snelling, T.J.; López-Ferreras, L.; Yáñez-Ruíz, D.R.; García-Estrada, C.; López, S. Effect of sunflower and marine oils on ruminal microbiota, in vitro fermentation and digesta fatty acid profile. Front. Microbiol. 2017, 8, 1124. [Google Scholar] [CrossRef] [PubMed]
  49. Adeyemi, K.D.; Sazili, A.Q.; Ebrahimi, M.; Samsudin, A.A.; Alimon, A.R.; Karim, R.; Karsani, S.A.; Sabow, A.B. Effects of blend of canola oil and palm oil on nutrient intake and digestibility, growth performance, rumen fermentation and fatty acids in goats. Anim. Sci. J. 2016, 87, 1137–1147. [Google Scholar] [CrossRef]
  50. Chelikani, P.K.; Bell, J.A.; Kennelly, J.J. Effects of feeding or abomasal infusion of canola oil in Holstein cows 1. Nutrient digestion and milk composition. J. Dairy Res. 2004, 71, 279–287. [Google Scholar] [CrossRef]
  51. Kadkhoday, A.; Riasi, A.; Alikhani, M.; Dehghan-Banadaky, M.; Kowsar, R. Effects of fat sources and dietary C18: 2 to C18: 3 fatty acids ratio on growth performance, ruminal fermentation and some blood components of Holstein calves. Livest. Sci. 2017, 204, 71–77. [Google Scholar] [CrossRef]
  52. Atikah, I.N.; Alimon, A.; Yaakub, H.; Abdullah, N.; Jahromi, M.; Ivan, M.; Samsudin, A. Profiling of rumen fermentation, microbial population and digestibility in goats fed with dietary oils containing different fatty acids. BMC Vet. Res. 2018, 14, 344. [Google Scholar]
  53. Yoshimura, E.; Santos, N.; Machado, E.; Agustinho, B.; Pereira, L.; de Aguiar, S.; Franzolin, R.; Gasparino, E.; dos Santos, G.; Zeoula, L. Effects of dairy cow diets supplied with flaxseed oil and propolis extract, with or without vitamin E, on the ruminal microbiota, biohydrogenation, and digestion. Anim. Feed. Sci. Technol. 2018, 241, 163–172. [Google Scholar] [CrossRef]
  54. Yang, S.; Bu, D.; Wang, J.; Hu, Z.; Li, D.; Wei, H.; Zhou, L.; Loor, J. Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows. Animal 2009, 3, 1562–1569. [Google Scholar] [CrossRef] [Green Version]
  55. Ghasemi, E.; Golabadi, D.; Piadeh, A. Effect of supplementing palmitic acid and altering the dietary ratio of n-6:N-3 fatty acids in low-fibre diets on production responses of dairy cows. Br. J. Nutr. 2021, 126, 355–365. [Google Scholar] [CrossRef] [PubMed]
  56. Huang, A.; Cai, R.; Wang, Q.; Shi, L.; Li, C.; Yan, H. Dynamic change of gut microbiota during porcine epidemic diarrhea virus infection in suckling piglets. Front. Microbiol. 2019, 10, 322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Guo, B.; Li, D.; Zhou, B.; Jiang, Y.; Bai, H.; Zhang, Y.; Xu, Q.; Zhao, W.; Chen, G. Comparative characterization of bacterial communities in geese consuming of different proportions of ryegrass. PLoS ONE 2019, 14, e0223445. [Google Scholar] [CrossRef] [PubMed]
  58. Adeyemi, J.A.; Peters, S.O.; De Donato, M.; Cervantes, A.P.; Ogunade, I.M. Effects of a blend of Saccharomyces cerevisiae-based direct-fed microbial and fermentation products on plasma carbonyl-metabolome and fecal bacterial community of beef steers. J. Anim. Sci. Biotechnol. 2020, 11, 4657–4667. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Li, H.; Liu, F.; Lu, J.; Shi, J.; Guan, J.; Yan, F.; Li, B.; Huo, G. Probiotic mixture of Lactobacillus plantarum strains improves lipid metabolism and gut microbiota structure in high fat diet-fed mice. Front. Microbiol. 2020, 11, 512. [Google Scholar] [CrossRef] [Green Version]
  60. Dworkin, M. Bacteria: Firmicutes, cyanobacteria. In The Prokaryotes; Springer: New York, NY, USA, 2006; Volume 4. [Google Scholar]
  61. Shah, H.N. Anaerorhabdus. In Bergey’s Manual of Systematics of Archaea and Bacteria; John Wiley and Sons: Hoboken, NJ, USA, 2015; pp. 1–2. [Google Scholar]
  62. Egan, A. Regulation of nitrogen metabolism and recycling. In Control of Digestion and Metabolism in Ruminants; Prentice-Hall: Hoboken, NJ, USA, 1986. [Google Scholar]
  63. Oldick, B.; Firkins, J. Effects of degree of fat saturation on fiber digestion and microbial protein synthesis when diets are fed twelve times daily. J. Anim. Sci. 2000, 78, 2412–2420. [Google Scholar] [CrossRef]
  64. Hristov, A.; Ivan, M.; McAllister, T. 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. [Google Scholar] [CrossRef]
  65. Vargas, J.E.; Andrés, S.; López-Ferreras, L.; Snelling, T.J.; Yáñez-Ruíz, D.R.; García-Estrada, C.; López, S. Dietary supplemental plant oils reduce methanogenesis from anaerobic microbial fermentation in the rumen. Sci. Rep. 2020, 10, 1613. [Google Scholar] [CrossRef] [Green Version]
  66. Hobson, P.; Wallace, R.; Bryant, M.P. Microbial ecology and activities in the rumen: Part I. CRC Crit. Rev. Microbiol. 1982, 9, 165–225. [Google Scholar] [CrossRef]
  67. Coleman, G. Rumen ciliate protozoa. Adv. Parasitol. 1980, 18, 121–173. [Google Scholar]
  68. Hristov, A.; Kennington, L.; McGuire, M.; Hunt, C. Effect of diets containing linoleic acid-or oleic acid-rich oils on ruminal fermentation and nutrient digestibility, and performance and fatty acid composition of adipose and muscle tissues of finishing cattle. J. Anim. Sci. 2005, 83, 1312–1321. [Google Scholar] [CrossRef] [PubMed]
  69. El-Sherbiny, M.; Cieslak, A.; Pers-Kamczyc, E.; Szczechowiak, J.; Kowalczyk, D.; Szumacher-Strabel, M. A nanoemulsified form of oil blends positively affects the fatty acid proportion in ruminal batch cultures. J. Dairy Sci. 2016, 99, 399–407. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Kubelková, P.; Jalč, D.; Jančík, F.; Homolka, P. In vitro ruminal fermentation and fatty acid production by various oil seeds. S. Afr. J. Anim. Sci. 2018, 48, 526–534. [Google Scholar] [CrossRef]
  71. Grainger, C.; Beauchemin, K. Can enteric methane emissions from ruminants be lowered without lowering their production? Anim. Feed. Sci. Technol. 2011, 166, 308–320. [Google Scholar] [CrossRef]
  72. Newbold, C.J.; López, S.; Nelson, N.; Ouda, J.; Wallace, R.J.; Moss, A. Propionate precursors and other metabolic intermediates as possible alternative electron acceptors to methanogenesis in ruminal fermentation in vitro. Br. J. Nutr. 2005, 94, 27–35. [Google Scholar] [CrossRef] [Green Version]
  73. Hanaki, K.; Matsuo, T.; Nagase, M. Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process. Biotechnol. Bioeng. 1981, 23, 1591–1610. [Google Scholar] [CrossRef]
  74. Eugène, M.; Massé, D.; Chiquette, J.; Benchaar, C. Meta-analysis on the effects of lipid supplementation on methane production in lactating dairy cows. Can. J. Anim. Sci. 2008, 88, 331–337. [Google Scholar] [CrossRef]
  75. McGinn, S.; Beauchemin, K.; Coates, T.; Colombatto, D. Methane emissions from beef cattle: Effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid. J. Anim. Sci. 2004, 82, 3346–3356. [Google Scholar] [CrossRef]
  76. Beauchemin, K.A.; McGinn, S.M.; Petit, H.V. Methane abatement strategies for cattle: Lipid supplementation of diets. Can. J. Anim. Sci. 2007, 87, 431–440. [Google Scholar] [CrossRef]
  77. Czerkawski, J.W. An Introduction to Rumen Studies; Elsevier: Amsterdam, The Netherlands, 2013. [Google Scholar]
  78. Czerkawski, J.; Blaxter, K.; Wainman, F. The metabolism of oleic, linoleic and linolenic acids by sheep with reference to their effects on methane production. Br. J. Nutr. 1966, 20, 349–362. [Google Scholar] [CrossRef] [Green Version]
  79. Beauchemin, K.; McGinn, S.; Benchaar, C.; Holtshausen, L. Crushed sunflower, flax, or canola seeds in lactating dairy cow diets: Effects on methane production, rumen fermentation, and milk production. J. Dairy Sci. 2009, 92, 2118–2127. [Google Scholar] [CrossRef]
  80. Balch, W.; Fox, G.; Magrum, L.; Woese, C.; Wolfe, R. Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 1979, 43, 260–296. [Google Scholar] [CrossRef]
  81. Wang, R.; Wang, M.; Zhang, X.M.; Wen, J.N.; Ma, Z.Y.; Long, D.L.; Deng, J.P.; Tan, Z.L. Effects of rumen cannulation on dissolved gases and methanogen community in dairy cows. J. Dairy Sci. 2019, 102, 2275–2282. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Ibrahim, N.A.; Alimon, A.R.; Yaakub, H.; Samsudin, A.A.; Candyrine, S.C.L.; Wan Mohamed, W.N.; Md Noh, A.; Fuat, M.A.; Mookiah, S. Effects of vegetable oil supplementation on rumen fermentation and microbial population in ruminant: A review. Trop. Anim. Health Prod. 2021, 53, 422. [Google Scholar] [CrossRef] [PubMed]
  83. Kalscheur, K.; Teter, B.; Piperova, L.; Erdman, R. Effect of dietary forage concentration and buffer addition on duodenal flow of trans-C18: 1 fatty acids and milk fat production in dairy cows. J. Dairy Sci. 1997, 80, 2104–2114. [Google Scholar] [CrossRef]
  84. Boeckaert, C.; Vlaeminck, B.; Fievez, V.; Maignien, L.; Dijkstra, J.; Boon, N. Accumulation of trans C18: 1 fatty acids in the rumen after dietary algal supplementation is associated with changes in the Butyrivibrio community. Appl. Environ. Microbiol. 2008, 74, 6923–6930. [Google Scholar] [CrossRef] [Green Version]
  85. Urrutia, N.; Bomberger, R.; Matamoros, C.; Harvatine, K. Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows. J. Dairy Sci. 2019, 102, 5172–5181. [Google Scholar] [CrossRef] [PubMed]
  86. Jenkins, T. Lipid metabolism in the rumen. J. Dairy Sci. 1993, 76, 3851–3863. [Google Scholar] [CrossRef]
  87. Wu, M.-R.; Chou, T.-S.; Huang, C.-Y.; Hsiao, J.-K. A potential probiotic-Lachnospiraceae NK4A136 group: Evidence from the restoration of the dietary pattern from a high-fat diet. Res. Sq. 2020, in press.
  88. Dou, X.; Gao, N.; Yan, D.; Shan, A. Sodium butyrate alleviates mouse colitis by regulating gut microbiota dysbiosis. Animals 2020, 10, 1154. [Google Scholar] [CrossRef] [PubMed]
Fermentation 08 00310 g001 550
Figure 1. Effects of dietary n-6/n-3 ratio on in vitro total volatile fatty acid (VFA) production at each incubation time. HN6, high n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1); TVFA, total volatile fatty acids. Different superscript letters at the same time point indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM.
Figure 1. Effects of dietary n-6/n-3 ratio on in vitro total volatile fatty acid (VFA) production at each incubation time. HN6, high n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1); TVFA, total volatile fatty acids. Different superscript letters at the same time point indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM.
Fermentation 08 00310 g001
Fermentation 08 00310 g002 550
Figure 2. Effects of dietary n-6/n-3 ratio on in vitro gas production and kinetic parameters. (A) Cumulative gas production (mL/g DM) after incubation for 48 h. (B) Asymptotic gas production (mL/g DM). (C) Maximum rate of substrate digestion (/h). Different superscript letters indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Figure 2. Effects of dietary n-6/n-3 ratio on in vitro gas production and kinetic parameters. (A) Cumulative gas production (mL/g DM) after incubation for 48 h. (B) Asymptotic gas production (mL/g DM). (C) Maximum rate of substrate digestion (/h). Different superscript letters indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Fermentation 08 00310 g002
Fermentation 08 00310 g003 550
Figure 3. Effects of dietary n-6/n-3 ratio on in vitro CH4, CO2, and O2 production. (A) CH4 production (mL/g DM). (B) CO2 production (mL/g DM). (C) O2 production (mL/g DM). Different superscript letters indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Figure 3. Effects of dietary n-6/n-3 ratio on in vitro CH4, CO2, and O2 production. (A) CH4 production (mL/g DM). (B) CO2 production (mL/g DM). (C) O2 production (mL/g DM). Different superscript letters indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
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Fermentation 08 00310 g004 550
Figure 4. Effects of dietary n-6/n-3 ratio on in vitro fatty acid profiles. (A–E): The concentration of C16:1, C18:3, C20:1, C22:0, and C24:0 in the fermentation fluid. Different superscript letters at the same time point indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Figure 4. Effects of dietary n-6/n-3 ratio on in vitro fatty acid profiles. (A–E): The concentration of C16:1, C18:3, C20:1, C22:0, and C24:0 in the fermentation fluid. Different superscript letters at the same time point indicate a significant difference (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Fermentation 08 00310 g004
Fermentation 08 00310 g005 550
Figure 5. Alpha diversity indices for bacterial communities in fermented ruminal fluid. (A) Chao 1 diversity index. (B) Number of observed species. (C) Shannon diversity index. Different superscript letters indicate significant differences among dietary groups (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Figure 5. Alpha diversity indices for bacterial communities in fermented ruminal fluid. (A) Chao 1 diversity index. (B) Number of observed species. (C) Shannon diversity index. Different superscript letters indicate significant differences among dietary groups (p < 0.05), whereas the same or no superscript letters indicate no significant difference. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Fermentation 08 00310 g005
Fermentation 08 00310 g006 550
Figure 6. Beta diversity indices for bacterial communities in fermented ruminal fluid. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Figure 6. Beta diversity indices for bacterial communities in fermented ruminal fluid. Data are represented by the mean ± SEM. HN6, High n-6 fatty acid treatment (n-6/n-3 ratio = 3.04:1); MN6, Moderate n-6 fatty acid treatment (n-6/n-3 ratio = 2.03:1); LN6, Low n-6 fatty acid treatment (n-6/n-3 ratio = 0.8:1).
Fermentation 08 00310 g006
Table
Table 1. Ingredients and chemical composition of the diets.
Table 1. Ingredients and chemical composition of the diets.
IngredientsHN6 (DM %)MN6 (DM %)LN6 (DM %)
Oat hay41.4941.8041.87
Corn silage28.3029.5828.53
Corn fine10.939.129.23
Soybean meal8.6116.9914.90
Hydrogenated FA0.001.510.00
Whole soybean9.670.000.00
Flaxseed0.000.004.47
Mineral vitamin premix 1.001.001.00
Total100.00100.00100.00
Nutrition (DM basis)
DM (kg/d)12.5112.4212.4
CP (%)13.7713.8513.78
NEL(Mcal/kg) 1.491.481.49
NFC (%)33.4634.6033.29
NDF (%)41.9542.0042.41
EE (%)4.013.934.01
UFA (%)2.741.372.73
C16:0 (g/d)50.70175.5546.00
n-6 (g/d)178.23102.03121.48
n-3 (g/d)58.6345.65150.68
n-6: n-33.04:12.03:10.8:1
DM: Dry matter, CP: Crude protein, NEL: Net energy of lactation, a calculated value according to NRC (2001), NFC: Non-fibrous carbohydrate, calculated as: 100—[(NDF–NDFCP) + CP + ether extract + ash]. NDF: Neutral detergent fiber, EE: Ether extract, UFA: Unsaturated fat acid, n-6: C18:2 (Linoleic acid, LA), n-3: C18:3 (α- linolenic acid, ALA). HN6: Higher n-6 fatty acid treatment (n6/n3, 3.04:1), MN6: Moderate n-6 fatty acid treatment (n6/n3, 2.03:1), LN6: Lower n-6 fatty acid treatment (n6/n3, 0.8:1). Premix: 1 kg of premix included vitamin A 440,000 IU, vitamin D3 110,000 IU, vitamin E 4000 IU, niacin 400 mg, Ca 152 g, P 41g, Cu 750 mg, Mn 1,140 mg, Zn 2,970 mg, I 30 mg, Se 36 mg.
Table
Table 2. Effects of dietary n-6/n-3 PUFA ratio on in vitro ruminant fermentation and N metabolism.
Table 2. Effects of dietary n-6/n-3 PUFA ratio on in vitro ruminant fermentation and N metabolism.
Item 1Treatment 2 Time 3 SEM 4 p-Value 5
HN6MN6LN636122448TRTimeINT
PH6.89 B6.94 A6.94 A6.85 c6.94 ab6.92 b6.94 ab6.98 a0.030.09<0.010.12
IVDMD0.600.620.600.48 d0.50 d0.56 c0.72 b0.79 a0.020.49<0.010.16
NH3-N(mg/dL)10.46 b11.69 a10.23 b5.33 d6.58 c7.29 c13.47 b22.49 a0.820.04<0.010.14
MCP(μg/L)640.45 A616.17 B656.62 A640.00 b753.82 a630.03 b614.62 b552.75 c11.770.08<0.010.44
Acetate (mmol/L)25.60 B27.99 A27.65 A20.09 d22.93 c24.08 c30.02 b38.83 a0.830.09<0.010.07
Butyrate (mmol/L)3.99 b4.40 a4.43 a2.73 e3.67 d4.05 c4.77 b6.21 a0.15<0.01<0.010.14
Propionate (mmol/L)10.12 b11.16 a11.12 a7.64 d8.46 c9.11 c12.64 b16.19 a0.40<0.01<0.010.18
TVFA (mmol/L)41.33 b44.27 a45.02 a31.27 d36.12 c38.76 c49.22 b63.25a1.420.02<0.010.04 *
A:P2.572.522.562.63 b2.71 a2.64 b2.38 c2.40 c0.020.18<0.010.56
1 IVDMD: In vitro dry matter digestibility, A:P: Acetic acid: Propionic acid, TVFA: Total volatile fat acid, NH3-N: Ammonia–N, MCP: Microbial protein. 2 HN6: High n-6 fatty acid treatment (n6/n3, 3.04:1), MN6: Moderate n-6 fatty acid treatment (n6/n3, 2.03:1), LN6: Low n-6 fatty acid treatment (n6/n3, 0.8:1), a,b,c: Superscript letters show the multiple comparisons using Tukey’s test, with different lowercase letters indicating significant differences in the indicators among groups (p < 0.05), A,B,C:a row with different uppercase letters indicating that there was a trend difference among groups (0.05 ≤ p < 0.1), whereas those denoted by the same letters or no letters are not significantly different. 4 SEM: standard error of the mean. 3 Time: Incubation time, a,b,c,d,e: Superscript letters show the multiple comparisons using Tukey’s test; a row with different lowercase letters indicates a significant difference under time effect factors (p < 0.05), whereas those denoted by the same letters or no letters are not significantly different. 4 SEM: standard error of the mean. 5 TR: The overall diet treatment effect between groups. Time: The overall effect of incubation time. INT: The interaction effects between diet treatment and incubation time. *: If the interaction effect factor indicates a significant difference, the detailed information of the data will be expressed in the picture (Figure 1).
Table
Table 3. Effects of dietary n-6/n-3 PUFA ratio on fatty acid profiles in fermented rumen fluid in vitro (mg/100 mL).
Table 3. Effects of dietary n-6/n-3 PUFA ratio on fatty acid profiles in fermented rumen fluid in vitro (mg/100 mL).
Fatty Acid 1 Treatment 2Fermentation Time (h) 3SEM 4 p-Value 5
HN6MN6LN6361224TRTimeINT
C12:00.1530.1480.1600.135 b0.129 b0.173 a0.175 a0.0030.133<0.0010.448
C14:00.3630.3670.3770.264 b0.311 b0.440 a0.456 a0.0120.763<0.0010.487
C15:00.3530.3530.3680.2460.3310.4690.3860.0120.746<0.0010.659
C16:05.820 b7.983 a6.092 b10.588 a4.379 c5.875 b5.691 bc0.3470.002<0.0010.165
C16:10.040 b0.056 a0.042 b0.089 a0.091 a0.000 b0.000 b0.0060.012<0.0010.001 *
C17:00.1770.1770.1990.116 d0.161 c0.203 b0.247 a0.0070.490<0.0010.442
C18:011.24711.41413.6696.176 c11.813 a10.401 b10.11 b0.7380.209<0.0010.612
C18:1n9c1.569 a1.523 b1.872 a2.415 a1.844 b1.159 c1.156 c0.0850.019<0.0010.793
C18:2n60.716 a0.697 ab0.606 b0.855 a0.843 a0.459 b0.507 b0.0260.120<0.0010.543
C18:3n30.047 b0.051 b0.136 a0.104 a0.078 b0.072 b0.061 b0.007<0.0010.004<0.001 *
C20:00.1560.1560.1660.082 c0.270 a0.155 b0.130 bc0.0100.859<0.0010.093
C20:10.020 ab0.009 b0.041 a0.045 a0.046 a0.0000.0000.0050.019<0.0010.038 *
C22:00.7070.7080.7370.586 d0.868 a0.788 b0.637 c0.0150.278<0.0010.039 *
C22:1n90.0850.0750.0840.107 ab0.121 a0.099 b0.000 c0.0530.384<0.0010.193
C24:00.0630.0630.0640.142 a0.042 c0.070 b0.000 d0.0060.992<0.0010.001 *
n6/n312.669 a11.062 b4.851c9.42710.8606.9839.2150.551<0.0010.0080.093
1 n6/n3: The ratio of C18:2n6 to C18:3n3. 2 HN6: High n-6 fatty acid treatment (n6/n3, 3.04:1), MN6: Moderate n-6 fatty acid treatment (n6/n3, 2.03:1), LN6: Low n-6 fatty acid treatment (n6/n3, 0.8:1). a,b,c: Superscript letters show the multiple comparisons using Tukey’s test; different superscripts mean significant differences in the indicators among groups (p < 0.05). 3 Time: Incubation time, a,b,c: Superscript letters show the multiple comparisons using Tukey’s test; a row with different superscripts means significant differences under time effect factors exist (p < 0.05), whereas those denoted by the same letters or no letters are not significantly different. 4 SEM: standard error of the mean. 5 TR: The overall diet treatment effect between groups. Time: The overall effect of incubation time. INT: The effects of interaction between diet treatment and incubation time. *: If the interaction effect factor is significantly different, the detailed information of the data will be expressed in the picture (Figure 4).
Table
Table 4. Effects of dietary n-6/n-3 PUFA ratio on the relative abundance of bacterial genes (%).
Table 4. Effects of dietary n-6/n-3 PUFA ratio on the relative abundance of bacterial genes (%).
MicroorganismTreatmentSEMp-Value
HN6MN6LN6
Anaerorhabdus_furcosa0.1360.1310.0740.0090.060
Butyrivibrio_20.573 b0.664 a0.526 b0.0170.024
Lachnospiraceae_NK4A1360.0910.0990.1320.0060.060
Lachnospiraceae_UCG-0060.130 a0.091 b0.131 a0.0050.020
Methanobrevibacter0.035 a0.042 a0.013 b0.0040.001
Prevotellaceae_UCG-0031.264 a0.960 b0.928 b0.1910.025
Prevotellaceae_UCG-0040.4330.2750.2800.0450.053
Ruminococcaceae_UCG-0020.555 b0.666 ab0.714 a0.0210.038
Ruminococcaceae_UCG-0101.366 c1.522 b1.666 a0.0380.001
Ruminiclostridium_60.158 a0.132 ab0.113 b0.0050.033
Selenomonas0.059 b0.100 a0.091 a0.0070.017
HN6: High n-6 fatty acid treatment (n6/n3, 3.04:1), MN6: Moderate n-6 fatty acid treatment (n6/n3, 2.03:1), LN6: Low n-6 fatty acid treatment (n6/n3, 0.8:1). SEM: standard error of the mean. a,b,c: Different superscripts mean significant differences in the indicators among groups (p < 0.05), whereas those denoted by the same letters or no letters are not significantly different.
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Sun, X.; Wang, Q.; Yang, Z.; Xie, T.; Wang, Z.; Li, S.; Wang, W. Altering Methane Emission, Fatty Acid Composition, and Microbial Profile during In Vitro Ruminant Fermentation by Manipulating Dietary Fatty Acid Ratios. Fermentation 2022, 8, 310. https://doi.org/10.3390/fermentation8070310

AMA Style

Sun X, Wang Q, Yang Z, Xie T, Wang Z, Li S, Wang W. Altering Methane Emission, Fatty Acid Composition, and Microbial Profile during In Vitro Ruminant Fermentation by Manipulating Dietary Fatty Acid Ratios. Fermentation. 2022; 8(7):310. https://doi.org/10.3390/fermentation8070310

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Sun, Xiaoge, Qianqian Wang, Zhantao Yang, Tian Xie, Zhonghan Wang, Shengli Li, and Wei Wang. 2022. "Altering Methane Emission, Fatty Acid Composition, and Microbial Profile during In Vitro Ruminant Fermentation by Manipulating Dietary Fatty Acid Ratios" Fermentation 8, no. 7: 310. https://doi.org/10.3390/fermentation8070310

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