Impact of Ecklonia stolonifera extract on in vitro ruminal fermentation characteristics, methanogenesis, and microbial populations

Objective This study was conducted to evaluate the effects of Ecklonia stolonifera (E. stolonifera) extract addition on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations. Methods One cannulated Holstein cow (450±30 kg) consuming timothy hay and a commercial concentrate (60:40, w/w) twice daily (09:00 and 17:00) at 2% of body weight with free access to water and mineral block were used as rumen fluid donors. In vitro fermentation experiment, with timothy hay as substrate, was conducted for up to 72 h, with E. stolonifera extract added to achieve final concentration 1%, 3%, and 5% on timothy hay basis. Results Administration of E. stolonifera extract to a ruminant fluid-artificial saliva mixture in vitro increased the total gas production. Unexpectedly, E. stolonifera extracts appeared to increase both methane emissions and hydrogen production, which is contrasts with previous observations with brown algae extracts used under in vitro fermentation conditions. Interestingly, real-time polymerase chain reaction indicated that as compared with the untreated control the ciliate-associated methanogen and Fibrobacter succinogenes populations decreased, whereas the Ruminococcus flavefaciens population increased as a result of E. stolonifera extract supplementation. Conclusion E. stolonifera showed no detrimental effect on rumen fermentation characteristics and microbial population. Through these results E. stolonifera has potential as a viable feed supplement to ruminants.


INTRODUCTION
Macroalgae are economically important and an under exploited plant resources, provid ing integral biomass for human foods and animal feed in recent years. Macroalgaederived compounds have a broad range of biological activities such as antibiotic, antiviral, antiox idant, antifouling, antiinflammatory, cytotoxic, antiadipogenic, and antimitotic and thus confer potential health benefits [1]. In addition, macroalgaederived compounds have been shown to increase growth rates and feed efficiency in ruminants [2]. However, they have counterintuitively also been shown to impair fiber digestibility; thereby limiting diet di gestibility [3].
Phaeophyta or brown algae are predominantly greenish brown in color due to the pres ence of the carotenoid fucoxanthin, and contain primary polysaccharides such as alginates, laminarins, fucans, and cellulose [4]. Ecklonia stolonifera (E. stolonifera) is a brown algae belonging to the Laminariaceae family that is commonly found in the sea forests off the coasts of Korea and Japan, growing on rocks near and below the lowtide mark on rough open coasts [5]. E. stolonifera has traditionally been utilized as an edible product and contains high levels of diverse phlorotan nins, which are polymers of phloroglucinol found only in brown algae that have diverse biological activities, including antioxidative, antibacterial [5], and antiinflammatory [6] properties. Moreover, E. stolonifera contains polyphenolic compounds that have been suggested to deter the grazing and growth of the seaweed' s predators [7]. However, a few studies reported that algae have potential effect on rumen fermenta tion characteristics and methane reduction [8,9]. Identification of feed additives that can modify the rumen microbial system to manipulate ruminal fermentation characteristics and in crease the efficiency of feed utilization is an effective strategy for inhibiting ruminal methanogenesis for reducing methane emissions without an adverse effect on rumen function.
To this end, we evaluated the potential effect of E. stolonifera on rumen fermentation using in vitro gas production techni que. It has previously been applied to study the fermentation kinetics of feed composition. In addition, it can allow for the rapid screening of a large number of feed additives that may have effects on gas production [10].
Therefore, this study was conducted to evaluate effects of E. stolonifera extracts on in vitro ruminal fermentation, gas profile, and changes in microbial populations. These results could help to promote E. stolonifera as a natural alternative for improving ruminal fermentation.

MATERIALS AND METHODS
All experimental protocols were approved by the Animal Care and Use Committee of Gyeongsang National University (GNU 180130A0007, Jinju, Gyeongsangnamdo, Korea).
Ecklonia stolonifera extract preparation E. stolonifera extract was obtained from the Jeju Biodiversity Research Institute (JBRI, Jeju, Korea). In brief, the plant ma terial was washed and cut into small pieces, freezedried, and crushed. The plant powder was extracted with 80% metha nol at room temperature (20°C) using an ultrasonic cleaner (Branson Ultrasonics Corporation, Danbury, CT, USA). After extraction, the methanol eluate solutions were filtered through Whatman No. 1 filter (Whatman International Ltd, Maidstone, UK) paper and concentrated under a vacuum.

In vitro fermentation design
One cannulated Holstein cow (450±30 kg) was used as rumen fluid donors and provided with ad libitum access to a mineral vitamin block and water. Twice daily (09:00 and 17:00), cows were fed 2% of their body weight in timothy hay and com mercial concentrate at a 60:40 (w/w) ratio. Rumen fluid was collected before morning feedings and filtered through four layers of cheesecloth. Next, it was diluted with artificial saliva and stored at 39°C.
The rumen fluid was mixed with McDougall's buffer in a 1:2 ratio. Next, 15 mL of the mixture was dispensed anaero bically into 50mL serum bottles containing 0.3 g of timothy for CON and E. stolonifera extract for treatments (TRTs) (3 mg for TRT1, 9 mg for TRT2, 15 mg for TRT3). The serum bottles were sealed anaerobically with an aluminumcapped butyl rubber stopper in pure N 2 gas, and incubated in a shak ing incubator (Jeio Tech, SI900R, Daejeon, Korea; 120×rpm) at 39°C for 72 h. The in vitro fermentation experiment was a completely randomized block design and performed in trip licate, using 60 serum bottles (4 treatments × 5 incubation times × 3 replicates times).

Determination of gas profiles and ruminal fermentation characteristics
Total gas production in the samples was measured with head space gas chromatography using a detachable pressure trans ducer and a digital readout voltmeter (Laurel Electronics, Inc., Costa Mesa, CA, USA). The transducer was connected to the inlet of a disposable Luerlock threeway stopcock. Gas pressure in the headspace above the culture medium was read from the light emitting diode display unit after inserting a hypodermic syringe needle. Methane and carbon dioxide content was measured using a TCD detector with a Carboxen 1006 Plot capillary column (30 mm×0.53 mm, Supelco, Belle fonte, PA, USA), after connecting another stopcock outlet to a gas chromatograph (HP 5890, Agilent Technologies, Santa Clara, CA, USA).
In vitro DM disappearance rate was determined following a modified Ørskov' s method, using nylonbag digestion. After incubation, the nylon bag containing serum bottles was washed twice in a waterbath equipped with a Heidolph Rotamax 120 (Heidolph Instruments, Nuremberg, Germany) at 100×rpm for 30 min and then oven dried at 60°C to a constant weight. The DM disappearance was the difference in serumbottle weight before and after incubation.

Microbial growth rate
At the end of each fermentation period, samples were cen trifuged at 3,000×rpm for 3 min to remove feed particles. The supernatant was then recentrifuged at 14,000×rpm for 3 min to obtain a final supernatant for protein and glucose analysis. Some of the supernatant was dyed with Coomassie Blue G250 for spectrophotometrically measuring protein content as OD at 595 nm (Model 680, BioRad Laboratories, USA) [11]. For measuring glucose, 200 μL of supernatant was mixed with 600 μL of DNS solution and incubated for 5 min in a boiling water bath. Glucose concentration was the OD at 595 nm, determined with a microplate reader (Model 680, BioRad Laboratories, USA) [12]. Pellets from the centrifugation were washed with sodium phosphate buffer (pH 6.5) four more times and then subjected to OD measurements at 550 nm (Model 680, BioRad Laboratories, USA) to evaluate micro organism growth rates.
Quantitative polymerase chain reaction DNA was extracted from the incubated rumen samples using a QIAamp mini kit (QIAGEN, Valencia, CA, USA) according to the modified beadbeating protocol. Total nucleic acids were extracted by a high speed reciprocal shaker (TissueLyser; QIAGEN, USA), which retains the samples in screwcapped tubes containing ceramic and silica beads. In brief, 1 mL aliquots were taken from 15 mL of the incubated culture so lution and centrifuged at 3,000 rpm for 5 min; 1 μL of the supernatant was used for nucleic acid concentration deter mination using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Quantitative realtime PCR assays (CFX96 RealTime sys tem; BioRad, USA) were conducted using the SYBR Green Supermix (QPK201, Toyobo Co., Ltd., Tokyo, Japan) accord ing to the methods described by Denman and McSweeney [13] and Denman et al [17]. The relative abundance of mi crobes was expressed according to the cycle threshold (Ct) difference as: 2 -ΔCt (target) -ΔCt (control) . All quantitative PCR mix tures consisted of a 20 μL volume, containing forward and reverse primers, DNA template, and DNA dye SYBR Green Supermix. The PCR amplification conditions for the target DNA, including the primer annealing and extension tempera tures, were the same as those reported in the corresponding reference for each primer (Table 1).

Statistical analysis
All experimental data were analyzed using the general linear model procedure of SAS [18] as a completely randomized block design. The effects of supplementation of E. stolonifera extract on pH, total gas production, DM disappearance, gas profiles, VFA profiles, and methanogen diversity were com pared to those of the CON group, and the data were subjected to polynomial regression to measure the linear and quadratic effects of increasing concentrations of E. stolonifera. Variability in the data is expressed as the standard error of the mean; p<0.05 was considered to be statistically significant, whereas p<0.10 was considered to indicate a tendency. production by mixed ruminal microorganisms as compared to that of the CON group (Table 2). However, there was no effect of E. stolonifera at different concentrations on pH and DM disappearance as compared with those of the CON group, except for an effect on DM disappearance at 24 h detected in the quadratic model.

In vitro fermentation characteristics E. stolonifera extract demonstrated improved cumulative gas
As shown in Table 3, supplementation of E. stolonifera ex tract reduced the total levels of VFAs at 3 h and 48 h, acetate at 48 h, and butyrate at 3 h. Overall, supplementation of E. stolonifera extract decreased the acetic acidto propionic acid ratio (A/P ratio) at 48 h as compared with that of the CON group.
Lastly, supplementation of E. stolonifera extract increased the methane emissions at 3 h and 12 h (linear models only); hydrogen production at 3 h, 12 h, and 72 h; and ammonia production at 72 h. By contrast, ammonia production was reduced at 24 h, respectively, as compared to those of the CON group (Table 4).
Change in ruminal microbial diversity E. stolonifera extract increased the microbial growth rate at 48 h and the glucose concentration at 3 h, while reducing the protein concentration at 12 h and at 24 h as compared with those of the CON group ( Table 5).
The ciliateassociated methanogen and methanogenic ar chaea populations were reduced at 12 h (p<0.0001) and 24 h (p = 0.0164) following supplementation with various con centrations of E. stolonifera extract as compared with those of the CON group. In addition, E. stolonifera extract reduced the abundance of the major fibrolytic microorganisms such as F. succinogenes at 12 h (p = 0.0113) and 24 h (p = 0.0145). The proportion of R. flavefaciens increased at 12 h of incuba tion with E. stolonifera extract (p = 0.0001), whereas the R. albus population remained unchanged or slightly increased as compared with that of the CON group ( Figure 1).

DISCUSSION
Denis et al [19] reported that algae contain candidate com pounds with potential to assist in ruminants feeding for improved gas production and fermentation management, within the context of dietary fiber provision. In this study, dietary fiber, as determined through the dose response of E. stolonifera, induced an increase in total gas production without any accompanying change in DM loss. DM disap pearance only showed an effect with the addition of 1%, 3%, and 5% E. stolonifera extract at 24 h incubation, whereas the total gas production under all levels of E. stolonifera extract was higher as compared to that under incubation with Timo thy hay alone at 24, 48, and 72 h, indicating the potential of this extract for improved feed efficiency [20]. The pH also remained consistent in the range of 6.49 to 7.48 for all doses of E. stolonifera applied during microbial fermentation, sug gesting that ruminal microbial activity was not negatively affected since it was greater than the minimal pH of 5.0 to 5.5 [21]. By contrast, Wang et al [3] and Dubois et al [20] reported that brown algae species resulted in lower gas production than that of the control sample during in vitro ruminal fermenta tion. Therefore, some bioactive compounds of certain brown algae species might reduce the utilization of nutrients, there by directly inhibiting microbial activity or indirectly by forming complexes with the nutrients [22]. Interestingly, the E. stolon ifera extract caused a decrease in the total VFA and acetate concentrations, and resulted in a lower A/P ratio than those of the CON group at 48 h incubation, demonstrating that fermen tation was affected. Secondary metabolites from E. stolonifera extracts have been reported to contain phlorotannins and polyphenolic compounds, which have strong antimicrobial properties and can deter the growth of the seaweed's preda tors [7]. Thus, it is possible that these secondary metabolites may have induced a reduction in the total VFA concentra tion and altered the acetate and propionate concentrations, which are common characteristics often associated with anti nutritional factors that interfere with ruminal fermentation [23].
With regards to emission gases, E. stolonifera extracts ap peared to increase the in vitro methane emissions, and hydrogen and ammonia production, while carbon dioxide production did not increase under in vitro ruminal fermentation. As such, these results do not demonstrate a clear consensus trend, given that a mixed outcome was observed under different condi tions. Rumen ammonia production may vary depending on the proportion of feed protein and the degradation rate; there fore, it was difficult to observe any difference in ammonia production except at 24 h and 72 h of fermentation, since   timothy hay was the only substrate utilized. Wang et al [3] and Machado et al [23] reported a reduction of methane emissions when experimenting with brown algae extracts under in vitro fermentation conditions. Brown algae species generally show the ability to reduce methane emissions, which is most likely attributed to their phlorotannins and a range of other natural products [22,24]. However, the results from our study are in disagree with those of Wang et al [3] and Machado et al [23] as the E. stolonifera extracts appeared to actually increase methane emissions and hydrogen production at 3 h. This finding is in line with the results of Mitsumori and Sun [25], who suggested that ruminal methanogens utilizing mainly hydrogen would be the main source of an increase in methane emissions.
The effects of E. stolonifera on microbial diversity also ini tially appeared to be counter intuitive with the observed increase in methane and hydrogen production. E. stolonifera extracts reduced the populations of the ciliateassociated methanogens, methanogenic archaea, and F. succinogenes, while increasing the R. flavefaciens population as compared with those of the CON group. However, the R. albus popula tion was left unchanged. Ciliateassociated methanogens may generate up to 37% of the methane produced in the rumen [26], and most methanogenic archaea can reduce CO 2 with H 2 to produce methane [27]. However, F. succinogenes is a nonH 2 producing species [28]. Therefore, given the major reduction in the ciliateassociated methanogens and methano genic archaea populations, a consequent reduction in methane  24 h. Dietary treatments were as follows: CON, basal diet (without Ecklonia stolonifera extract); TRT 1, 1% Ecklonia stolonifera; TRT 2, 3% Ecklonia stolonifera; TRT 3, 5% Ecklonia stolonifera, on a substrate (timothy hay) basis. a,b Means with different superscripts in the same row indicate significant differences (p<0.05). production would be expected; however, this was not the case. R. albus and R. flavefaciens are two of the three major mem bers of the fibrolytic microorganism population, the third being F. succinogenes. R. albus has shown great promise in the production of H 2 from energy forage, with potential for utilizing cellulosic and hemicellulosic biomass [29]. In addi tion, R. flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H 2 , and CO 2 [30]. As such, the increase in the R. flavefaciens population along with the unchanged R. albus population may have contributed to the observed increase in hydrogen produc tion. Therefore, even with reductions in the ciliateassociated methanogens and methanogenic archaea populations, the increase in hydrogen availability may have allowed for in creased methane emissions. ChaucheyrasDurand et al [28] showed that methane emissions clearly reduced when the dominant fibrolytic species was a nonH 2 producing species such as F. succinogenes, without significantly impairing fiber degradation and fermentation in the rumen. This suggests that H 2 is the critical factor for the microbial ecosystem in ruminants. The H 2 produced during enteric fermentation is the precursor of methane emissions from ruminants, and thus the regulation of H 2 , rather than methane appears to be the key to controlling ruminant methane emissions.
Lastly, the E. stolonifera extract doses that led to higher mi crobial growth rates also caused higher total gas production as compared to the CON group; therefore, the rumen mi croorganism growth rate appears to be closely related to the total gas production and fermentation process, as suggested by Hungate [31]. In particular, the E. stolonifera extracts sig nificantly increased microbial growth at 48 h as compared to that of the CON group. Moreover, our results confirmed that rumen fermentation with E. stolonifera extracts did not result in any negative side effects on protein or glucose concentra tions throughout the experimental period. In fact, E. stolonifera extracts appeared to reduce the protein concentration at 12 h and 24 h. However, the protein concentration does not ap pear to be correlated with ammonia concentration, as Mehrez et al [32] reported that the optimal ammonia concentration could lead to maximal protein synthesis by microorganisms.
In conclusion, we demonstrated the effects of E. stolonifera on in vitro ruminant fermentation characteristics. E. stolonifera extracts also appear to be capable of mitigating a series of ef fects throughout the period of in vitro rumen fermentation, some of which may not be desirable. For example, E. stolon ifera extracts could increase methane emissions and hydrogen production, which disagrees with previous observations on brown algae extracts under in vitro fermentation conditions. However, the changes in ruminal microbial diversity were able to partially explain the observed increase in methane and hydrogen observed with treatment of E. stolonifera ex tracts. More research is required to elucidate the potential of E. stolonifera for improving growth performance and meth ane emissions of ruminants.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.