Animal care and handling in the present study was approved by the Animal Ethics Committee of Gyeongsang National University, South Korea (GNU-191011-E0050). Rumen fluid was collected from two rumen-cannulated Hanwoo heifers (Average body weight = 432.63 kg) fed rice straw (CP = 5.40%, NDF = 63.85%) and concentrate (CP = 12.51%, NDF = 47.51%) mixture at a 8:2 ratio of dry matter (DM) weight. The concentrate mainly consisted of corn meal, soybean meal, soybean hull, and cotton seed pellet. This fluid was collected before morning feeding, filtered with double cheese cloths, stirred to obtain a uniform fluid, and then mixed with anaerobic culture medium at a 1:2 ratio to make rumen buffer. The detail protocol to prepare a rumen buffer was described by Adesogan et al. (2005). Carbon dioxide (CO₂) gas was flushed into rumen buffer continuously to maintain anaerobic conditions (Carriquiry et al., 2008; Jayanegara et al., 2015). A fat-free synthetic diet was prepared using as a substrate, containing 411 g cellulose (Sigma, C6413), 411 g starch (Sigma, S4180), and 178 g casein (Sigma, C3400) per kg of DM (Carriquiry et al., 2008). The treatments consisted of pure C18:2n-6 FA (Sigma, L1376; LA), pure C18:3n-3 FA (Sigma, L2376; LNA), or a mixture of these FAs (Combo) at 1:1 ratio (w/w). Five hundred milligrams of LA and LNA was dissolved with 5 mL methanol (A 4524, Fisher Chemical), respectively. After then, 2 mL from each dissolved FAs were mixed for Combo. Before ruminal incubation, 20 μL of dissolved FA (2 mg of FA) was applied into 150 mg of synthetic diet. This synthetic diet applied FA was stored at room temperature to evaporate methanol for 12 h. In the present study, the application rate of each FA treatment was 1.3% DM, which demonstrate the general C18:2n-6 FA and C18:3n-3 FA concentrations in ruminant diet (Kim et al., 2007, 2016). Ruminal incubation was performed in the 50-mL glass serum bottle containing 15 mL of rumen buffer with synthetic diet applied FA at 39°C for 0, 1, 2, 4, and 8 h. Each FA treatments used five incubation bottles as replications along with three blanks for each hour. Thus, total 90 incubation bottles were used in the present study.

In an assigned hour (0, 1, 2, 4, and 8 h, respectively), all bottles were withdrawn from the incubator, the gas pressure was quickly measured by a pressure transducer (Fisher Scientific, Traceable^TM, Friendswood, TX, United States), and then, the bottles were placed into ice to stop microbial activity (Honkanen et al., 2012). All incubated rumen samples including blank were subsampled 1 mL for microbial quantification using PCR. The remain incubated rumen samples were transferred to 50 mL conical tube to separate sample residue and supernatant of incubated rumen buffer through centrifugation at 2,568 × g for 15 min (Supra 21k, Hanil Electric Corporation, Seoul, South Korea, with rotor A50S-6C No.6). The supernatant of incubated rumen buffer from centrifugation was subsampled 10 mL and stored for further analyses of rumen fermentation indices. For FA analysis, 2 mL rumen buffer was frozen at −70°C for 2 days and freeze-dried using Cascade Console Freeze Dry System (LABCONCO, FreeZone Plus 12 Liter, MO, United States) as the procedure to sample preparation according to previous studies (Paradhipta et al., 2020).

The experiment was a completely randomized design. All data were analyzed using the general linear model procedure of Statistical Analysis System (SAS) ver. 9.3 (SAS, 2000). Its model was Y_ij = μ + T_i + e_ij, where Y_ij = response variable, μ = overall mean, T = effect of treatment i, and e_ij = error effect. In addition, rumen pH, NH₃-N, acetate, propionate, CH₄, CO₂, rumen microbes, and major 18-carbon FAs were analyzed using the PROC MIXED procedure of SAS to test the significant levels of supplementary treatment, incubation hour, and the interaction between supplementary treatment and incubation hour. Its model was Y_ijk = μ + α_i + β_j + (αβ)_ij + e_ijk, where Y_ijk = response variable, μ = overall mean, α_i = the effect of supplementary treatment, β_j = the effect of incubation hour, (αβ)_ij = the interaction effect between supplementary treatment and incubation hour, and e_ijk = error effect. Mean differences were tested for significance using Tukey’s test at P ≤ 0.05.

The total FAs before incubation from LA, LNA, and Combo were 4.11, 3.87, and 3.77 mg/mL, respectively (Table 2). The concentrations of C18:2n-6 was highest (P < 0.001; 18.4 vs. 11.02 vs. 3.07%) in LA, followed by Combo and LNA, while the concentrations of C18:3n-3 was highest (P < 0.001; 6.78 vs. 3.72 vs. 0.26%) in LNA, followed by Combo and LA. The concentrations of C14:0, C14:1, C16:0, C16:1, C18:0, SFA, monounsaturated fatty acid (MUFA), and PUFA were higher (P < 0.05) in LNA than LA. The ratio of SFA to PUFA was higher (P = 0.005; 7.65 vs. 3.74 and 4.92) in LNA than LA and Combo. In addition, the ratio of n-6 to n-3 was higher (P < 0.001; 35.76 vs. 0.48 and 2.84) in LA than LNA and Combo.

After 8 h of incubation, the pH was lower (P = 0.034; 6.52 vs. 6.65) in LA than in Combo, while the pH in LNA was not different compared with that in the other treatments (Table 3). The NH₃-N concentration was higher (P = 0.002; 33.46 vs. 30.38 and 30.38 mg/100 mL) in LA than in LNA and Combo after 8 h of incubation. The total VFA concentration was higher (P = 0.039; 85.22 vs. 78.93 mmol/L) in LA than in Combo, while the concentration in LNA did not differ compared with that in the other treatments. Among the individual VFAs, the concentrations of acetate, propionate, butyrate, isovalerate, and valerate remained unaffected. Only the concentration of isobutyrate was found to be higher (P < 0.001; 0.93 vs. 0.83 and 0.74%) in LA than in LNA and Combo. The total gas volume in LA was higher (P < 0.001; 35.43 vs. 31.13 and 30.29 mL/g) than that in LNA and Combo. The CH₄ emissions were highest (P = 0.001; 55.50 vs. 46.60 vs. 40.20 mg/g) in LA, followed by LNA and then Combo. The CO₂ gas concentration was higher in Combo than in LA and LNA (P = 0.042; 2.05 vs. 1.61 vs. 1.80 mg/g).

The rumen pH was decreased by increasing incubation hour, but supplementary treatment had no effect from 0 to 4 h of incubation (Figure 1A). The NH₃-N concentration was increased by incubation hour in all supplementary treatments, which Combo presented higher concentration than LA at 0 h (P = 0.043; 20.44 vs. 18.90 mg/100 mL) and 1 h (P = 0.032; 23.94 vs. 22.40 mg/100 mL) of incubation (Figure 1B). The acetate and propionate concentrations were affected by the supplementary treatment at 1 and 2 h of incubation, but the patterns of concentration change over hour were cubical pattern (Figures 1C,D). Furthermore, LA resulted in highest concentrations of acetate (P = 0.001; 45.41 vs. 41.01 and 41.49%) and propionate (P < 0.001; 25.14 vs. 21.86 and 21.92%) at 1 h, then had lowest (P < 0.01) concentrations of acetate (P < 0.001; 44.22 vs. 46.86 and 47.00%) and propionate (P = 0.001; 22.74 vs. 23.61 and 24.42%) at 2 h of incubation. There were interaction effects between the supplementary treatment and hour on rumen pH (P = 0.041) and NH₃-N (P < 0.001), acetate (P < 0.001), and propionate (P < 0.001) concentrations, which might be a reason for the cubical patterns of these results during incubation. The concentration of CH₄ was increased by increasing incubation hour in all supplementary treatments (Figure 2A). LA had highest (P < 0.01; 3.68 vs. 2.68 and 2.60 mg/g) CH₄ concentration at 2 h of incubation, while LA and Combo presented higher (P < 0.05; 4.50 and 4.80 vs. 3.85 mg/g) concentration than LNA at 4 h of incubation. Similar to CH₄, concentration of CO₂ was also increased by increasing incubation hour, which Combo and LA presented higher (P < 0.05; 0.83 and 0.92 vs. 0.76 mg/g) concentration than LNA at 1 h or incubation (Figure 2B). Additionally, there were interaction effects between the supplementary treatment and hour on the CH₄ (P < 0.001) and CO₂ (P = 0.005) concentrations.

It was observed that methanogenic archaea were less abundant (P = 0.03; 0.74 vs. 0.96 and 0.96) in LNA than in LA and Combo (Table 4). Rumen ciliates were less abundant (P = 0.03; 0.20 vs. 0.41 of fold change) in LA than in LNA, respectively, while the population in Combo had no difference compared with that in the other treatments. Among fibrolytic bacteria, R. albus was observed to be more abundant (P = 0.005; 0.17 vs. 0.09 and 0.10 of fold change) in LA than in LNA and Combo. F. succinogenes and R. flavefaciens remained unaffected by the treatments at 8 h of incubation.

It was observed that the population of methanogenic archaea was not affected by supplementary treatment during 4 h of incubation (Figure 3A). The population of rumen ciliates dropped drastically from 0 to 2 h of incubation in all supplementary treatments (Figure 3B). The population of F. succinogenes seemed to decrease by increasing incubation hour (Figure 3C). Furthermore, LA presented higher F. succinogenes population than LNA and Combo at 2 h (P = 0.003; 1.31 vs. 0.88 and 0.80) and 4 h (P = 0.001; 1.01 vs. 0.72 and 0.86) of incubation. In general, R. flavefaciens population increased during 4 h of incubation in all supplementary treatments, then decreased after that (Figure 3D). The R. flavefaciens population was higher (P = 0.007; 1.08 vs. 0.77 and 0.63) in LA and presented higher population than in LNA and Combo at 2 h of incubation. The population of R. albus was higher in LNA than in LA and Combo at 1 h (P = 0.015; 0.87 vs. 0.71 and 0.57) and 4 h (P = 0.008; 0.84 vs. 0.52 and 042) of incubation (Figure 3E). Generally, all supplementary treatments seemed to decrease the population of R. albus after 8 h of incubation. An interaction between the supplementary treatment and hour was reported for rumen ciliates (P = 0.019), F. succinogenes (P = 0.003), and R. albus (P < 0.001), which caused cubical patterns of those microbes during incubation.

The concentrations of total FAs and MUFAs were found to be unaffected (P > 0.05) by the treatments after 8 h of incubation (Table 5). The concentrations of C14:1 (P = 0.001; 1.45 and 1.44 vs. 1.30%) and C16:1 (P = 0.001; 0.89 and 0.87 vs. 0.77%) were higher in LNA and Combo than in LA. Otherwise, the concentrations of C18:0 (P = 0.008; 56.62 vs. 55.45 and 55.28%) and C20:3n-6 (P = 0.001; 0.36 vs. 0.33 and 0.33%) were higher in LA than in LNA and Combo. On the other hand, the C18:2n-6 concentration was higher (P < 0.001; 3.05 and 3.66 vs. 1.82%) in LA and Combo than in LNA. The C18:3n-3 concentration was higher (P < 0.001; 1.88 vs. 1.26 vs. 0.20%) in LNA, followed by Combo and then LA. LA and LNA produced higher concentrations of C20:0 (P = 0.001; 0.80 and 0.78 vs. 0.76%) and C24:1 (P = 0.005; 0.46 and 0.46 vs. 0.42%) than did Combo. LA had a higher SFA concentration (P = 0.005; 86.87 vs. 85.11%) than that in Combo, while the concentration of SFA in LNA was not different compared with that in the other treatments. Combo had the highest PUFA concentration (P = 0.002; 5.58 vs. 3.96 and 4.37%), but it had the lowest ratio of SFAs to PUFAs (P = 0.002; 15.25 vs. 21.93 and 19.69) compared with those of the other treatments.

Generally, applications of LA and Combo decreased concentration of C18:2n-6 during incubation, while applications of LNA and Combo decreased concentration of C18:3n-3. At 1 h (P = 0.012; 7.61 vs. 5.61 vs. 2.68%), 2 h (P = 0.019; 3.33 and 3.48 vs. 1.96%), and 4 h (P = 0.001; 4.24 and 4.81 vs. 1.69%) were observed that the C18:2n-6 concentration was higher in LA and Combo than in LNA (Figure 4A). However, the C18:3n-3 concentration was higher in LNA and Combo than in LA at 1 h (P = 0.015; 2.51 and 1.98 vs. 0.27%), 2 h (P = 0.012; 2.13 and 2.49 vs. 0.23%), and 4 h (P = 0.008; 1.74 and 2.00 vs. 0.20%) (Figure 4B). All supplementary treatments seemed to increase C18:1cis-9 concentration during 2 h of incubation, and then decreased it after that (Figure 4C). The concentration of C18:1cis-9 was higher (P = 0.028; 7.64 and 747 vs. 7.06) in LNA and combo at 1 h. At 2 h, LNA had the highest (P = 0.008; 7.65 vs. 7.03 and 7.04%) concentration of C18:1cis-9. The C18:0 concentration was not affected by the supplementary treatment during incubation (Figure 4D). The concentration of C18:0 was increased by incubation hour in all supplementary treatments. Interaction effects between the supplementary treatment and hour were observed on C18:2n-6 (P < 0.001), C18:3n-3 (P < 0.001), and C18:0 (P = 0.036).

The increases of NH₃-N and total VFA concentrations and total gas volume with a decrease of rumen pH in LA indicated a higher relative rumen fermentation rate by LA compared with LNA or Combo. The reason for the high VFA concentration and total gas volume in LA could be supported by the higher total amount of fibrolytic bacteria in LA than in LNA and Combo (Table 4), which increased rumen digestion. It was found in Martin et al. (2006) that supplementary treatment with C18:3n-3 at either 100 or 50% could depress degradations of organic matter and fiber in the rumen. This previous study also reported that the population of total fibrinolytic bacteria was lower in LNA and Combo than in LA. In addition, LA had the highest acetate and propionate concentrations at 1 h of incubation, and then presented the lowest at 2 h of incubation. The reason of these results was unknown in the present study. However, on the other side, LA increased the CH₄ emissions. In agreement with the present study, Zhang et al. (2008) observed higher total gas production and CH₄ concentration by supplementary C18:2n-6 compared with C18:3n-3. Combo had the lowest CH₄ emissions in the present study, even though it presented a high population of methanogenic archaea. In addition, supplementary n-9 FA such as oleic acid was reported to reduce methane production in an in vitro study (Wu et al., 2016). The reason for the low CH₄

emissions by Combo was not clear. However, this observation supported the result of the highest CO₂ concentration occurring in Combo followed by LNA and LA numerically, which could indicate a low conversion rate of CO₂ to CH₄ by methanogenic archaea (Wolin et al., 2007; Tapio et al., 2017). Combination of n-3 and n-6 FAs might effectively inhibit methanogenic archaea to utilize free CO₂ during methanogenesis process, which could reduce methane gas emission without decrease its population. However, the further investigations need to be conducted.

Supplementary PUFAs are considered to depress methanogenesis (Fievez et al., 2003). Microbial DNA analysis showed that methanogenic archaea were suppressed only by LNA, while ciliates were suppressed by both LA and Combo. Hristov et al. (2013) reported that both C18:2n-6 and C18:3n-3 were effective to decrease rumen ciliate. Supporting the result of the present study, other previous study reported that C18:2n-6 was more effective than C18:3n-3 to decrease ciliates. However, effectiveness of C18:2n-6 against C18:3n-3 to inhibit ciliates could be influenced by supplementation levels on rumen fluid (Zhang et al., 2008). Combo also contained C18:2n-6 that could present a similar result of ciliates with LA in the present study. In the present study, the observed lower population of methanogenic archaea in LNA than in other treatments (Table 3) is in agreement with other findings (Fievez et al., 2003; Zhang et al., 2008). Methanogenic archaea and rumen ciliate might had different sensibility with each pure essential FA that caused the decrease of methanogenic archaea in LNA and rumen ciliate in LA and Combo. Along with methanogenic archaea, rumen ciliates are responsible for CH₄ emissions in the rumen (Whitelaw et al., 1984). Rumen ciliates are associated with ecto- and endosymbiotic methanogenic archaea (Finlay et al., 1994). Therefore, the degradation of fiber and suppressive effects of FAs on methanogens and ciliates might have occurred and contributed to the CH₄ emissions in the present study. Increased fiber degradation in the rumen is also responsible for an increase of enteric CH₄ emission (Hristov et al., 2013). Decreased methanogenesis sometimes shifts rumen fermentation from acetogenic to propionigenic (Wettstein et al., 2000) because of increased hydrogen availability for propionate synthesis. However, this relation was only observed at 4 h of incubation, when CH₄ production was lowest but propionate production was numerically highest in the LNA treatment (Figures 1D, 2A).

As shown in Figure 3, the population of methanogenic archaea in LNA tended to decrease over 8 h of incubation, while the other treatments showed similar populations before and after incubation. LNA has a higher degree of unsaturation than LA. Although most long-chain PUFAs could exert toxic effects on rumen microbes (Maia et al., 2007), greater effects were reported with a higher degree of unsaturation as well as a higher applied dose (Zhang et al., 2008). Both the direct toxic effects of the FA and decreased amount of ruminal hydrogen in LNA could negatively affect methanogens, compared with the effects in the other treatments. A higher degree of unsaturation in LNA consumed more hydrogen for biohydrogenation of the FA (Zhang et al., 2008). However, it was reported that only 1 to 2% of the metabolic hydrogen in the rumen is used for the purpose of biohydrogenation (Jenkins et al., 2008). Therefore, the CH₄ emissions by LNA were reported to be lower than those by LA in the present study. On the other hand, LNA has previously been shown to be toxic to cellulolytic bacteria, particularly F. succinogenes, R. albus, and R. flavefaciens (Maia et al., 2007). This previous study supported the results of the present study, which reported lower populations of R. albus and F. succinogenes (numerically, P = 0.063) bacteria in LNA than in LA after 8 h of incubation. Martin et al. (2006) explained that the negative effects of PUFAs on fibrolytic bacteria were not only the result of direct toxicity of PUFAs themselves but also indirectly related to the inhibition of methanogens. Nevertheless, compared with LA, LNA presented higher R. albus populations at 1 and 4 h of incubation in the present study, but the reason for this result was not clear. The inhibition of methanogens leads to the accumulation of free hydrogen in the rumen, which can negatively affect the growth of cellulolytic bacteria (Wolin et al., 2007). The LNA treatment inhibited methanogenic archaea to a greater extent and consequently exerted maximum negative effects on fibrolytic bacteria overall compared with the effects of the other treatments. Generally, populations of all microbes decreased after 8 h of incubation because substrate was completely degraded and indicated the end of ruminal fermentation. In addition, it also supported the results of major C18 FAs, which also decrease after 8 h of incubation (Figure 4).

It is well known that unsaturated FAs undergo extensive biohydrogenation in the rumen by microbes (Beam et al., 2000; Petri et al., 2014). However, the rate of biohydrogenation might vary depending on the source or form of FA supplied (Carriquiry et al., 2008; Honkanen et al., 2012; Petri et al., 2014). In the present study, pure C18:2n-6 FA and C18:3n-3 FA were used either alone or in combination in equal ratios, and the results suggested less FA degradation in the combined treatment. At 0 h of incubation, the highest concentration of C18:2n-6 in LA and the highest concentration of C18:3n-3 in LNA were expected before considering the respective FAs were added accordingly in those treatments (Table 2). Furthermore, the application of mixed n-6 and n-3 FAs increased the concentrations of both C18:2n-6 FA and C18:3n-3 FA in the Combo treatment. Supplementary C18:2n-6 or C18:3n-3 did not have effect on total FA, but it could presented a minor change on proportion of FA profiles in the diet that could be a reason for results for C14:0, C14:1, C16:0, C16:1, and C18:0 at 0 h. Although the PUFAs were biohydrogenated after 8 h of incubation, the concentration patterns of remained C18:2n-6 FA and C18:3n-3 FA was similar (Table 5) to those in the beginning. This result indicated that the supplementation of these FAs could ensure their existence at an increased rate in the rumen, at least up to 8 h, which is in agreement with Carriquiry et al. (2008). Moreover, the use of these FAs in combination exerted inhibition of biohydrogenation to some extent, in contrast to the individual FA treatments. Lower C18:0 FA and total SFA concentrations and moderate C18:3n-3 concentrations and n-6 to n-3 ratios but higher C18:2n-6 FA and total PUFA concentrations in Combo indicated restricted biohydrogenation in this treatment (Table 5). This indication is more pronounced in Figure 4. This figure shows that from 0 to 2 h of incubation, approximately at 81.6% of linoleic acid was biohydrogenated in the LA treatment, and only 16.2% remained at the end of incubation (8 h). In Combo, 51.4% was biohydrogenated at 2 h, and 32.4% of C18:2n-6 remained at the end of incubation. In the case of C18:3n-3 FA, biohydrogenation was approximately at 64.2 vs. 48.3% in LNA vs. Combo, respectively at the first hour of incubation. And at the end, the remaining C18:3n-3 concentrations were approximately at 26.8 vs. 32.9%. In contrast, the concentration of C18:0 FA, the final end product of biohydrogenation of 18-carbon PUFAs, remained lower in Combo throughout the incubation. These results indicated the partial restriction of biohydrogenation of PUFAs when used in mixtures rather than individually.