Inuence of Kappaphycus Alvarezii and Gracilaria Salicornia Supplementation on in Vitro Fermentation Pattern, Total Gas and Methane Production of Mixed Substrates

This experiment was conducted to study the effect of supplementation of Kappaphycus alvarezii (KA) and Gracilaria salicornia (GS) in vitro fermentation pattern, total gas and methane production of mixed substrates. Basal substrate comprising of concentrates and wheat straw (50:50) was supplemented with either 0% (control), 1 (KA 1 ), 2 (KA 2) , 4 (KA 4 ), 6 (KA 6 ), and 8 % (KA 8 ) of Kappaphycus; and, 1 (GS 1 ), 2 (GS 2 ), 4 (GS 4 ), 6 (GS 6 ), and 8 (GS 8 ) of Gracilaria, respectively. Asymptote, rate constant of gas production and t-half, concentration of total volatile fatty acids (TVFA), and in vitro dry matter digestibility (IVDMD) was not affected up to 2% level KA supplementation, beyond which asymptote, and rate constant of gas production, TVFA, and IVDMD decreased and t-half increased (P<0.001). Asymptote, rate constant of gas production, TVFA and IVDMD was not affected at 1% level of inclusion, beyond which a steady decline in these parameters was observed (P<0.001). Methane production (ml/g DM) was higher (P<0.001) in CON, followed by KA 1 and KA 2 , and lower values were observed in by KA 4 , KA 6 and KA 8 . Methane production (ml/kg DM) declined (P<0.001) steadily with increased level of GS in the substrates. From the results it was concluded that inclusion of Kappaphycus alvarezii and Gracilaria salicornia at 2 and 1%, respectively in the fermentation substrate can reduce in vitro methane production without any adverse impact on total gas production and in vitro dry matter digestibility.


Introduction
Seaweeds are renewable natural resource of macroscopic marine algae found growing in large quantities along the coasts of India. Mainly they are used as sources of phycocolloids, fodder, fertilizers and for direct human consumption. Generally, seaweeds are markedly rich in minerals, complex carbohydrates, proteins and low molecular weight nitrogenous compounds, lipids, vitamins, volatile compounds, polyphenols and pigments. However, only limited data are available on the effect of seaweeds on ruminant production (Makkar et al., 2016). Li et al., (2016) reported that supplementation of Asparagopsis in Merino cross sheep fed with high bre diet resulted in dose dependent reduction in methane emission. Dietary supplementation of A. taxiformis reduced enteric CH 4 emissions in steers without any adverse impact on production performance (Roque et al., 2021). Feeding of tropical seaweed based formulation (Kappaphycus alvarezii, Gracilaria salicornia and Trbinaria conoides at 1:1:1) improved antioxidants, immunity and milk yield in lactating Murrah buffaloes (Maheswari et al., 2021). As compared to in vivo experiments there are many more in vitro experiments that demonstrated the effect of seaweeds supplementation on fermentation characteristics. Supplementation of ve different brown seaweed extracts reduced methane emission in vitro, but the response varied depending upon the species (Choi et al., 2021). Machado et al. (2014b) reported that supplementary feeding of Asparagopsis inhibited total gas production by 61.8% and CH 4 production by 98.9%. Maia et al., (2016) reported that on incubation with meadow hay, Ulva sp., Gigartina sp. and G. vermiculophylla decreased methane production, but with corn silage, methane production was only decreased by G. vermiculophylla. Thus, it is evident that the capability of macroalgae to alter fermentation characteristics depend on species, active component present, and level of inclusion (Molina-Alcaide et al., 2017). Kappaphycus alvarezii and Gracilaria salicornia, are the two most abundantly available cultivated species of red seaweeds of India. To, the best of our knowledge the in uence of these two species on rumen fermentation parameters is not yet tested. It was hypothesized that both the tropical seaweeds, Kappaphycus alvarezii and Gracilaria salicornia, have anti-methanogenic activity, that may differ in magnitude, and if supplemented in an appropriate amount would reduce enteric methane emission without any adverse impact on rumen fermentation.Taking all these points into account this research work was carried out to study the effect of graded levels of Kappaphycus alvarezii and Gracilaria salicornia supplementation of mixed substrates on in vitro total gas and methane production, dry matter digestibility, total volatile fatty acid production, fractions of VFA and NH 3 -N production.

Materials And Methods
All protocols and procedures followed in this study were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of Ministry of Environment, Forests and Climate Change, Government of India, New Delhi.

Rumen liquor sampling
Four rumen stulated animals kept on standard diets was used as donor animals. Rumen liquor was collected before feeding in the morning. After collection rumen liquor samples were screened with 4 layers of muslin cloth in a preheated thermos at 39 0 C and were transported to laboratory for inoculation of substrates. In vitro gas production was studied as per the procedure of Menke and Steingass (1988).Accurately weighed 200 mg of each substrates in triplicate was put into the graduated 100 ml calibrated glass syringe (Haberle Labortechnik, Lonsee-Ettenchie, Germany). The medium consisting of buffer and rumen liquor in 2:1 ratio (Menke and Steingass, 1988) was dispensed (30 ml) into the syringes by an automatic dispenser (OPTIFIX, Walter Graf & Co., Wertheim, Germany). Fermentation syringes were then kept in an incubator at 39°C with periodical shaking. Thus, total numbers of samples incubated were 2 (seaweeds)×6 (treatments) 7 (incubations)×3 (triplicate)=252. Besides, four blank syringes containing no substrate were also run during each incubation. The fermentation was terminated after 96 h of incubation.

Proximate and nutrient composition
Samples of wheat straw, fodder maize, Kappaphycus alvarezii and substrates were analyzed for proximate principles, bre constituents and minerals. Proximate analysis (dry matter (DM), organic matter (OM), crude protein (CP), crude ber (CF), and nitrogen free extract (NFE)) of the samples were estimated as per AOAC (2005) and cell wall constituents (neutral detergent ber (NDF), acid detergent ber (ADF), hemicellulose and cellulose) were estimated as per Van Soest et al. (1991). For determination of the concentration of minerals (calcium, magnesium, iron, copper, zinc, manganese and Iodine), samples of feedstuffs and seaweeds were subjected to wet digestion (HNO 3 : HClO 4 : H 2 SO 4 -3:2:1) using Kelplus-KES 12L R system till it became clear and the digested samples were diluted with double distilled water (DDW) and passed through Whatman lter paper No. 42 and nal volume was made to 25 ml. The contents of different minerals in the feed sample were analyzed using atomic absorption spectrophotometer (Hitachi-5000 series). The calibration curve for determination of different minerals was prepared using a blank and working standard solution of different minerals. The calibration was periodically veri ed by analyzing a standard at the frequency of 20 readings. If the recovery was outside the limits, the analysis was stopped and the system was recalibrated. Content of P in feedstuff and seaweed was determined by using a colorimetric method (AOAC, 2005) Measurement of total gas and methane production For each substrate, cumulative gas production (CGP) was calculated as the amount of gas production (ml) from the substrate minus gas production from blank divided by the weight of substrate. Gas production (ml/g DM) after 6, 12, 24, 48, 72 and 96 h of incubation was recorded by piston displacement. For methane estimation, 100 μl of gas sampled from headspace (after 24h of incubation) of the syringe was injected into a Nucon-5765 gas chromatograph equipped with Porapak Q column and ame ionization detector (Agarwal et al., 2008). A mixture of 50% carbon dioxide and 50% methane (Spancan; Spantech Products Ltd, Godstone, UK) was used as standard.

Estimation of volatile fatty acids (VFA)
Estimation of volatile fatty acids was done by using Nucon-5765 gas chromatograph (AIMIL, New Delhi, India) equipped with a double ame ionization detector and the glass column (4 ft length and 1/8 inch diameter) packed with chromosorb 101 as per method described by Cottyn and Boucque (1968) and later modi ed by Agarwal et al. (2008). The gas ows for nitrogen, hydrogen and air were 30, 30 and 320 ml/min, respectively. Temperature of injector oven, column oven and detector was 270 0 C, 172 0 C and 270 0 C, respectively. Standard VFA mixture was prepared by mixing stock solutions (each of 25 mg/ml concentration) of standard VFAs and water (acetic acid, 1.68 ml; propionic acid, 0.48 ml; butyric acid, 0.24 ml; distilled water, 7.24 ml) to obtain nal concentration of acetic acid, 7.0; propionic acid, 1.62; butyric acid, 0.68 mM/100 ml. Fermentation liquor samples were prepared by adding 0.2 ml of 25 % metaphosphoric acid per ml of liquor, allowing it to stand for 2 h followed by centrifugation at 7000 ×g for 10 min.
Supernatant was used for estimation of VFA.

Ammonia nitrogen estimation
Ammonia nitrogen was estimated by the method of Weatherburn (1967). Brie y, to the sample (suitable quantity), 5.0 ml of solution A (1 g phenol and 5 mg sodium nitroprusside) was added to which 5.0 ml of solution B (0.5 g sodium hydroxide 0.84 ml sodium hypochlorite) was added and mixed thoroughly. The tubes were incubated at 39 o C for 15 min for colour development. Samples were then read spectrophotometrically at 625 nm against a reagent blank. In a similar way standard tubes (ammonia nitrogen concentration ranging from 1.0 to 10 μg/ml) were processed and a calibration curve was plotted.
Concentration of the unknown sample was calculated by the standard curve.

In vitro true digestibility (IVTD) of substrates
After 24 h of incubation, the content of the syringes was transferred to spout-less beaker by repeated washings with 100 ml of neutral detergent solution. The ask content was re uxed for 1 h and ltered through pre-weighed Gooch crucibles (Grade 1). The dry matter content of the residue was weighed and in vitro true digestibility of feed was calculated as follows: (Initial DM of feed taken for incubation -DM residue) (Initial DM of feed taken for incubation) 2.6. Statistical analysis Cumulative gas production recorded at different hours of incubation were tted to the following model to determine gas production kinetics: Y = b × (1-e -c * t ); where 'y' is the cumulative volume (ml) of gas produced at time 't' (h), 'b' the asymptotic gas volume (ml) and 'c' the rate constant of gas production (% h -1 ). The constants b and c were determined using the nonlinear procedure of SAS (2001). Substrate speci c times were de ned by the half time (t -1/2 ) of asymptotic gas production. Halftime (h) of gas production (t -1/2 ) [i.e., the time (h) when half of the asymptotic gas volume (b; ml) was produced] was calculated as: t1/2 = ln 2/c. Data of the study were subjected to analysis of variance using the General Linear Model (GLM) procedure of the Statistical Software Package (SPSS for windows, V21.0; Inc., Chicago, IL, USA). The effect of green fodder replacement with corn silage on blood biomarker was tested using the following model: Where; Yijk is the dependent variable, μ is the overall mean of the population, Si is the mean effect of the source of seaweed, Dj is the mean effect of dose of seaweed, (S×D)ij is the effect of the interaction between source and dose, and eijk is the unexplained residual element assumed to be independent and normally distributed. Individual animals were used as the experimental unit for all data. The pair-wise comparison of means was carried out using "Tukey's honest signi cant difference (HSD) test". Signi cance was determined at P<0.05 and the values are presented in the tables. Treatment means were separated using Duncan's Multiple range Test and were considered signi cant at P<0.05.

Chemical composition of feed ingredients and substrates
Chemical composition of wheat straw, fodder maize, Kappaphycus alvarezii and concentrate mixture are presented in Table 1. Nutritional composition of wheat straw, maize fodder and concentrate mixture was within the normal range reported for these feed ingredients (Ranjhan, 1988). Both the seaweeds were characterized by higher acid soluble and insoluble ash content. The OM component of the seaweeds comprised mostly of NFE and cell content with lower CP, CF and EE content.

In vitro gas production kinetics
Data pertaining to in vitro gas production (IVGP) at different hours of incubation are presented in Figure 1. In vitro cumulative gas production was affected by both hour of incubation (P<0.001) and level of inclusion of seaweed (P<0.001). The interaction between period x treatment was signi cant (P<0.001) for Gracilaria salicornia but not for Kappaphycus alvarezii. This would imply that dose response of inclusion of Gracilaria salicornia differed at different hours of incubation. Rate constant of gas production decreased and t-half increased when Kappaphycus alvarezii was included at a rate higher than 2%. Asymptote, IVGP (P<0.001) and rate constant of gas production decreased and 't'half increased when Gracilaria salicornia was included at a rate higher than 1%. Interestingly, at lower level of inclusion (up to 2%), rate constant of gas production increased and t-half decreased in KA 1 and KA 2 as compared to control ( Table 2).

In vitro gas and methane production
In vitro gas production (IVGP) and methane production at 24 h of incubation of substrates containing various proportions of Kappaphycus alvarezii and Gracilaria salicornia has been presented in the Table 3. Proportion of methane in gas was highest (P<0.001) in CON, followed by KA, KA 1 and was lowest in KA 2 , KA 4 and KA 4 . Similar trend was also observed when substrates containing different proportions of Gracilaria salicornia. In the case of Kappaphycus alvarezii based substrates methane production (ml/g DM) was higher (P<0.001) in CON, KA 1 , and KA 2 as compared to other 3 groups. Methane production (ml/kg DM) differed (P<0.001) among all Gracilaria salicorniacontaining substrates (ml /kgDM or DOM) declined steadily with increased level of Gracilaria salicornia in the substrates (Figure 2).

In vitro dry matter digestibility
Data pertaining to the in vitro true dry matter digestibility at 24 h of incubation is presented in Table 4. There was improvement (P<0.001 in true dry matter digestibility in KA 1 and KA 2 as in case of Gracilaria salicornia treatment in vitro true dry matter digestibility was similar in control and GS 1 while it was found signi cantly lower in all other treatment groups (GS2, GS3 and GS4).

Discussion
Cumulative gas production (CGP), asymptote and rate constants of IVGP decreased at higher levels of  (Targett and Arnold, 1998). Lower IVGP in substrates having higher doses of Kappaphycus alvarezii and Gracilaria salicornia are thus explicable. An interesting observation was that at lower level of inclusion (upto 2%) of Kappaphycus alvarezii in the substrata resulted increased IVGP, IVDMD and TVFA production (Table 5 and 6). Considering the stimulation of fermentation at lower level of inclusion, it is advocated to explore the additive as a mean to improve e ciency of rumen function. Earlier research indicates that 9 out 20 algal extract improved overall fermentation over controls in vitro (Dubois et al., 2013). The positive interaction that we have observed between doses and seaweed sources imply that dose response of the seaweeds are not similar. At higher levels of Inclusion of both the seaweed products resulted in reduction of IVGP, however, the effect of Kappaphycus alvarezii on IVGP was less pronounced as compared to that of Gracilaria salicornia. This could be due to higher content of phenolics in Gracilaria salicornia than Kappaphycus alvarezii. Total phenolics content (mg gallic acid equivalent) was 2.7 times (1.5 vs 4.1 mg/g) higher in Gracilaria salicornia as compared to Kappaphycus alvarezii (Ganesan et al., 2008).The signi cant interaction that was observed between seaweed sources and doses imply that inhibitory effect of seaweed on IVGP was observed at different doses for Kappaphycus alvarezii and Gracilaria salicornia. Inclusion of Kappaphycus alvarezii and Gracilaria salicornia at 2 and 1% respectively did not show any adverse impact on IVGP and rate constants of gas production. This is evident that inclusion of these seaweeds beyond the aforesaid levels may hamper the microbial activity in the rumen and utilization of nutrients.  (Ganesan et al., 2008), it is not possible to attribute the anti-methnaogenic response observed in this experiment to a singular component. A comparison between the two seaweeds revealed that substrates containing Gracilaria salicornia were more effective in reducing methane emission as compared to substrates containing Kappaphycus alvarezii. This observation could be linked to higher pheonolics and PSM content of Gracilaria salicornia (Ganesan et al., 2008). Data pertaining to dose response reveal that supplementation of both Kappaphycus alvarezii and Gracilaria salicornia are effective in reducing methane emission at a level as low as 1% of the substrates. This level is still lower than the level of 2% of Asparagopsis in the fermentation substrate that was effective in reducing methane emission (Kinley et al., 2016b). This is remarkable because these two seaweeds show anti-methanogenic activity at a very low level of inclusion, thus making them more suitable for use as an anti-methanogenic agent without compromising the nutritional quality of the feed.
In vitro fermentation pattern at 24 h of incubation.
Ruminants derive most of their energy through metabolism of volatile fatty acids (VFA) that are produced as an end-product of microbial fermentation. Any reduction in production of VFA could be seen in a negative context. Considering the vast anti-microbial agents present in red seaweed (Paul et al., 2006), it was suspected that inclusion of red seaweeds may decrease the TVFA production at higher levels of inclusion. The results of this experiment indeed testify this assumption. Both Gracilaria salicornia and Kappaphycus alvarezii reduced in vitro VFA production. This nding corroborates well with that of Machado et al.(2016a) who observed a signi cant reduction in TVFA production due to supplementation of red seaweed at a rate between 1-2% to a low quality Rhode grass based substrate. It was suggested that supplementation of red seaweed is more responsive to poor quality roughages because of higher bre and lower protein content. In this experiment, we used standard mixed substrata having roughage: concentrates of 50:50. Yet at higher level of inclusion, both Kappaphycus alvarezii and Gracilaria salicornia reduced VFA production. However, inclusion level up to 2 and 1 % respectively for Kappaphycus alvarezii and Gracilaria salicornia showed no adverse impact on TVFA production. This nding is in agreement with that of Kinley et al. (2016a) who reported that inclusion of Asparagopsis between 1 to 2 % of the DM did not decrease VFA production. However, VFA production decreased consistently at higher level of inclusion of both Kappaphycus alvarezii and Gracilaria salicornia. This could be linked to overall decrease in fermentation activity as was also re ected in lower IVGP and rate constant of gas production due to presence of antimicrobial activity in red seaweeds (Amorim et al., 2012). Even though proportion of propionate was higher when seaweeds were added at 8% in the substrate, no de nite trend could be established because values varied between the treatments. This nding are not in agreement with those of Kinley et al. (2016b) who reported an increase in propionate at an expense of acetate in response to supplementation of red seaweed extract. Results of this experiment suggests that changes in A;P ratio in response to different doses of Kappaphycus alvarezii and Gracilaria salicornia are minor. Thus, it is unlikely that the anti-methanogenic activity of Kappaphycus alvarezii and Gracilaria salicornia are mediated through a major shift in partitioning of hydrogen among VFAs. Our nding corroborates well with those of Wang et al. (2008). In vitro concentration of NH 3 -N re ects the balance between NH 3 -N produced and it's utilization for microbial protein synthesis. As stated before both Kappaphycus alvarezii and Gracilaria salicornia are rich sources of phenolics (Ganesan et al., 2008) that may hamper microbial degradation of plant protein (Martinez et al., 2006). In a previous report it was demonstrated that crude seaweed extract of Ascophyllum nodosum at a very low level (125 μg phlorotannin /ml) reduced in vitro NH 3 -N production (Wang et al., 2008). A similar response was also observed in this experiment with respect Gracilaria salicornia. The response with respect to Kappaphycus alvarezii, however, was very weak and a signi cant reduction of NH 3 -N was observed only at 6 and 8% level of inclusion. The differences in response between Kappaphycus alvarezii and Gracilaria salicornia could be explained on the basis of their pheonlics content. As stated earlier, Gracilaria salicornia contained 2.7 times more phenolics as compared to Kappaphycus alvarezii.
The anti-methanogenic activity that we have observed in this experiment could be of use only if there is no compromise on the nutritional quality of the substrates. It is evident that at lower level of inclusion Kappaphycus alvarezii stimulates rumen fermentation in vitro. This promising response needs to be ascertained through in vivo experimentation. At higher level of inclusion both Kappaphycus alvarezii and Gracilaria salicornia reduced IVDMD. This corroborates well with our nding of reduced IVGP as both these parameters are strongly correlated (Menke and Steingass, 1988). Further, results are also in agreement with previous report (Wang et al., 2008). Such reduction in IVDMD was often due to decrease in digestibility of bre components and the responses were more pronounced while substrates comprising of poor quality roughages were used (Wang et al., 2008). Results of this experiment demonstrate that at higher levels of inclusion both can reduce the IVDMD of mixed substrates that are typical of ruminant feeding. A comparison between Kappaphycus alvarezii and Gracilaria salicornia revealed that responses are more pronounced while GS was used as supplement. Thus, more caution is required while Gracilaria salicornia was used as animal feed additive. However, the inhibitory effect of the seaweeds on IVDMD was not pronounced while the level of inclusion was restricted to 1 and 2% for Gracilaria salicornia and Kappaphycus alvarezii, respectively. Thus, it is evident that these two seaweeds may not be included in the ruminant diet at dose rate higher than that mentioned above. Present in vitro research on the effect of Red seaweeds on enteric methane emission, setting the stage for further invivo studies to proof them as promising candidates.

Conclusion
Inclusion of Kappaphycus alvarezii and Gracilaria salicornia at 2 and 1% respectively in the fermentation substrate reduced methane production in vitro without any adverse impact on total gas production and in vitro dry matter digestibility. Tropical red seaweed Gracilaria salicornia exhibited more robust antimethanogenic activity than Kappaphycus alvarezii. This project was funded by the CSIR-NMITLI programme (Project title: Kapaphycus alvarezi and Red sea weed based formulations for improving productivity and health of dairy animal and poultry).

Con icts of interest/Competing interests (include appropriate disclosures)
All authors certify that they have no a liations with or involvement in any organization or entity with any nancial interest or non-nancial interest in the subject matter or materials discussed in this manuscript.
Availability of data and material (data transparency) The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.   Figure 1 In vitro cumulative gas production of incubation of substrates containing various proportions of Kappaphycus alvarezii and Gracilaria salicornia