Research Article

In vitro Evaluation of Mangrove Leaves (Rhizophora apiculata) and Native Grass Based on Phytochemical, Nutritional and Fiber Degradability, Rumen Liquid Characteristics and Gas Production

Rani Winardi Wulan Sari1, Novirman Jamarun2*, Suyitman3, Khasrad4, Elihasridas2, James Hellyward5, Gusri Yanti1

1Graduate School of Animal Nutrition and Feed Technology, Faculty of Animal Science, Andalas University Kampus Limau Manis, Padang-25163, Indonesia; 2Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, Andalas University, Kampus Limau Manis, Padang, 25163, Indonesia;3Departement of Pasture and Forage Crops, Faculty of Animal Science, Andalas University, Kampus Limau Manis, Padang, 25163, Indonesia;4Departement of Animal Production, Faculty of Animal Science, Andalas University, Kampus Limau Manis, Padang, 25163, Indonesia;5Departement of Social Economic, Faculty of Animal Science, Andalas University, Kampus Limau Manis, Padang, 25163, Indonesia.

Received | July 28, 2022; Accepted | August 28, 2022; Published | October 20, 2022

*Correspondence | Walaa Yehia El-Sayed, Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, Andalas University, Kampus Limau Manis, Padang, 25163, Indonesia; Email: novirman55@gmail.com

Citation | Sari RWW, Jamarun N, Suyitman, Khasrad, Elihasridas, Hellyward J, Yanti G (2022). In vitro evaluation of mangrove leaves (rhizophora apiculata) and native grass based on phytochemical, nutritional and fiber degradability, rumen liquid characteristics and gas production. Adv. Anim. Vet. Sci. 10(11): 2412-2420.

DOI | http://dx.doi.org/10.17582/journal.aavs/2022/10.11.2412.2420

ISSN (Online) | 2307-8316

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Rhizophora apiculata are dominant plants that can grow in mangrove ecosystems (Dahuri, 2003). Rhizophora apiculata has characteristics of modified roots as a support for standing, with root color dark brown, brownish yellow speckled; the color of the main stem is grayish white; brownish old branch color (Rizki et al., 2016). Mangrove’s potential can be feed for ruminants, especially the leaves part. The nutritional composition of mangrove leaves contain 11.40% crude protein and 63.50% NFE (Sari et al., 2020). Mangroves contain high crude protein and play a role in rumen microbial growth. There is a breakdown of protein into ammonia which will be used as the main nitrogen resource for microbial synthesis in the livestock’s rumen (Russell et al., 2009). The mangrove plant lived in the littoral area are considered to have the potential become forage source (Yanti et al., 2021a; Sari et al., 2021).

Anti-nutritional substances liked polyphenol compounds and tannin are contain in mangrove’s leaves (Sari et al. 2022). Hay mangrove leaves processing aims to reduce water content due to harvesting carried out in large quantities, so it is hoped that the nutritional content is maintained. In addition, the tannin content will decrease so that it is not harmful to livestock. Hay processing function also increasing durability for stored and does not rot easily (Suherman, 2019).

Fiber feeds source was native grass which is quite likely by ruminants, especially sheep and goats. Field grass is abundant and easy to obtain, but the nutrient content of native grass is relative low. Rusdin et al. (2009) stated that the content of native grass produces crude protein 3.10-5.89%, crude fiber 34.89-40.68%, and nitrogen free extract 40.35-46.35%. This causes native grass to be combined with other feed ingredients such as mangrove leaves to fulfilled livestock nutritional needs, especially in the coastal area.

The nutrients supply mainly protein of native grass was limited compared to other plants (Rosadi et al., 2018). Exploration of forage resources needed to overcome this problem by utilizing the leaves of Rhizophora apiculata as feed source. Indonesia has the second largest mangrove forest in the world with an area of 42,550 km² (Sridahr, 2004), while the area of West Sumatra’s mangrove forest is 39,832 hectares, with the largest density in the Mentawai Islands Regency (Noegroho, 2013). In Indonesia, mangrove forests often serve as partial range-lands. Ruminant’s livestock liked Buffaloes, cattle, goats, and sheeps naturally known to feed mangrove leaves (Kusmana and Sukristijiono, 2016). Based on nutritive value, mangrove leaves get the best forage’s cathegory (Hamilton and Snedaker 1984). In other hand, tannins of mangrove leaves affect to methanogenic microbes as anti-methanogenic agent (Makkar et al., 2007). The impact of synergy between feed which contain tannin with fiber as carbohydrate source increasing fiber digestibility and nutrient utilization (Arowolo et al., 2018). This study aimed to evaluate the combination of mangrove leaves (Rhizophora apiculata) and native grass based on phytochemical contents, nutrient and cells fiber digestibility, rumen fluid characteristic, and gas production using an in vitro methodology.

Materials and Methods

Materials

The study was conducted in December 2021 until February 2022. Sample of mangrove leaves (Rhizophora apiculata) was carried out from Sasak Village, West Pasaman Regency, West Sumatra. Native grass collected from Limau Manis Village, Padang, West Sumatra used plot method with 1 m X 1 m size. Rumen liquid for in vitro was obtained from slaughterhouse from Kacang Goats (a native Indonesian goat) in Sungai Sapih Village, Padang, West Sumatra. Whole rumen is taken directly from the slaughterhouse to a laboratory where in vitro fermentation equipment has been prepared. The whole rumen is tied at one end so that the liquid does not spill, then put in a clean and airtight plastic bag,put in a bucket during the trip so it doesn’t shake and maintains the rumen temperature. Analysis of in vitro, proximate analysis, van soest analysis, rumen liquid analysis, gas production already implemented in Animal Nutrition Laboratory, Andalas University. Phytochemical contents analysis was carried out in Biotechnology Laboratory, Andalas University. The mangrove leaves are taken from the top (3-4 top leaves) like pick tea leaves methods. Mangrove leaves was dried as hay using oven with 30° C temperature for 3 days. The sample of hay mangrove leaves and native grass dried using oven with a set on 60° C during 48 h, then the sample was cut and mashed with grinding machine. The chemical composition of hay mangrove leaves and native grass is presented in Table 1. The experi­ment used randomized block design with 4 treatments and 5 replications. The treatments consisting of P1= 60% Hay Mangrove Leaves (HML)+ 40% Native Grass (NG), P2=50% HML+50% NG, P3=40% HML+ 60% NG, and P4=30% HML+70% NG. The chemical composition of each treatment diet is given in Table 2.

Phytochemical

Samples combination HML and NG of each treatments was mashed into powder. Then determined the total phenol using folin ciocalteu reagents (Singleton and Rossi, 1965), as was performed by (Marinova et al., 2005). The standard was made with 1 ml gallic acid with four different concentrations (120, 140, 160 and 180 ppm) and added with 1 ml of 9% folin ciocalteu, 9 ml H2O and boiled. Incubated the mixture at room temperature for 5 minutes, then 4 ml H2O and 10 ml of Na2CO3 7% were added. The mixture was stirred and incubated at room temperature for 90 minutes. The absorbance was measured using a spectrophotometer (UV-1800 Shimadzu A11455008975) with wavelength of 750 nm. Gallic acid was used as a standard and the result was expressed as milligram of gallic acid equivalents (GAE) per gram of defatted sample.

 

Table 1: The chemical composition of feed material (% DM)

Composition (%)	Feed Material
	Hay Mangrove Leaves	Native Grass
Dry Matter	89.80	87.90
Organic Matter	94.81	88.52
Crude Protein	9.10	4.68
Crude Fiber	11.09	24.74
Crude Fat	3.00	1.24
Ash	5.19	11.48
NFE	71.62	57.86
TDN	73.40	49.99
NDF	34.96	57.58
ADF	23.57	36.72
Cellulose	11.45	20.85
Hemicellulose	14.98	30.79
Lignin	7.30	4.25

 

Table 2: The chemical composition of the treatments (% DM)

Composition (%)	Treatment
	P1	P2	P3	P4
Dry Matter	89.04	88.85	88.66	88.47
Organic Matter	92.29	91.67	91.04	90.41
Crude Protein	9.06	9.05	9.03	9.02
Crude Fiber	16.55	17.92	19.28	20.65
Crude Fat	2.30	2.12	1.94	1.77
Ash	7.71	8.34	8.96	9.59
NFE	64.39	62.59	60.78	58.97
TDN	68.55	67.17	65.78	64.40
NDF	44.01	46.27	48.53	50.79
ADF	28.83	30.15	31.46	32.78
Cellulose	15.21	16.15	17.09	18.03
Hemicellulose	21.30	22.89	24.47	26.05
Lignin	6.08	5.78	5.47	5.17

 

Tannin in the dry extracts was determined with hide-powder method that had modified by (Seigler et al., 1986). The number of dry extracts was dissolved in distilled water, filtered and the obtained filtrate then divided into two parts. The first part was dried to see the level of water soluble extract. The second part was treated with the hide powder to withhold the tannin content. The level of tannin was calculated by the formula: Tannin (%) = level of water soluble extract (%) - level of water soluble extract without tannin (%).

In vitro Method

Tilley and Terry method (Tilley and Terry, 1963) used for in vitro experiment to determine the feed digestibility, rumen liquid characteristics, and gas production. Fresh rumen liquid was filtered using nylon (100 µm sieve) and filled into pre-warmed (39°C) thermos flasks. Filtered rumen liquid was diluted with the buffer solution suggested by McDougall Fresh rumen liquid was filtered using nylon (100 µm sieve), then filled into pre-warmed 39°C thermos flasks. Rumen liquid was diluted with buffer solution recommended by McDougall (1947), with ratio rumen fluid: buffer solution (1:4). Incubated 2.5 g sample in each Erlenmeyer flask with 250 mL solution (rumen fluid and buffer solution mixed), an aerobical condition by inserting carbon dioxide gas into the flask, then sealed it with a rubber lid. All flask was placed in a shaking incubator with 39° C for the temperature, and 100 rpm for speed during 48 hours. Gas production is measured by withdrawing all the fermented gas accommodated using a syringe and reading the volume scale. Methane gas volume measurement was done used indirect method (Fieves et al., 2005) modified by (Ramaiyulis, 2018). The gas that has been drawn into the syringe for measurement of the total gas sprayed into a vacuum bottle with a rubber cap which already contains 20 ml of 10M NaOH and is plugged in an empty syringe. Methane gas will come out through an empty syringe and read the scale volume, while part of the CO2 gas will be captured by NaOH.

After incubation, immersing the flask into ice water for stopping microbial activity, then pH was measured. After that, the supernatant was separated by moving the content of each flask in centrifuge tubes at 4° C with 3000 rpm for 5 minute. The result of supernatant was stored inside bottles on freezer with -18° C temperature until NH3 and total Volatile Fatty Acid (VFA) analysis completed. Conway and O’ Malley method (Conway and O’Malley, 1942) used for determined NH3 levels. The residue was filtered using whatman No. 41 filter paper, then dried in oven at 60° C for 24 hours (AOAC, 2005).

Chemical Analyses

Each treatment’s sample combination and residue was analyzed using proximate analysis (AOAC, 2005) to determine the contents of dry matter, organic matter, protein, fat, and crude fiber. Van soest analysis (Van soest et al., 1991) to determine the contents of NDF, ADF, cellulose, hemicellulose, and lignin.

Statistical analysis

The obtained data were analyzed using General Linear Model by IBM SPSS Statistics 26 (Yanti et al., 2021a). Data groups that showed statistical significance (p<0.05) were further analyzed using Duncan’s Multiple Range Tests (DMRT).

Results and Discussion

Effect Combination of Hay Mangrove Leaves and Native Grass on Phytochemical

Data of total phenol and tannin of the experimental are summarized in Table 3.

 

Table 3: Total Phenol and Tannin

Phytochemical	Treatment				S.E
	P1	P2	P3	P4
Total Phenol (mg GAE/gr)	18.65a	15.52b	11.52c	9.71d	0.200
Tannin (%)	5.69a	4.83b	4.22c	3.56d	0.033

Description : Different letters (a,b,c) in the same rows indicate significant differences (p<0.05); S.E : standard error

 

Combination of hay mangrove leaves and native grass is significantly different (p<0.05) on total tannin and total phenol. Tannin of mangrove leaves ranged from 5.69-3.56%, then total phenol ranged from 18.65%-9.71%. The results of statistical analysis showed that total phenol and tannin in P1 was higher than in other treatments (p<0.05). Based on the table above, it was found that the higher used of mangroves in the treatment resulted in a high total phenol and tannin combination, this is based on previous research by Sari et al. (2022) anti-nutritional substances contained in mangrove leaves in the form of tannins and another polyphenol compounds. The total phenol and tannin content are related because tannins are included in secondary metabolites synthesized on plants and classified as polyphenolic compounds and had ability to form complex compounds with another macromolecule. In other hand, tannins also interact with proteins derived, so that reducing their availability for rumen microorganism (Egea et al., 2016). Tannin protected protein from the degradation of rumen microorganism, that will make positive effect to the forage digestibility increase. These metabolites substances are mainly found in the post rumen gastrointestinal tract. On abomasum tannin and protein can release from complex binding because the pH condition was low which enables protein degrade by the enzyme, like pepsin. Hence, amino acids it contains are readily available to livestock requirement (Yanti et al., 2021a).

Effect Combination of Hay Mangrove Leaves and Native Grass on Nutrient Digestibility

Data of nutrient digestibility of the experimental combination hay mangrove leaves and native grass are summarized in Table 4.

 

Table 4: Nutrient Digestibility

Nutrient Digestibility (%)	Treatment				S.E
	P1	P2	P3	P4
Dry Matter	59.12b	58.04c	62.82a	56.54d	0.549
Organic Matter	61.72b	60.90b	65.69a	59.69c	0.657
Crude Protein	64.24b	63.54c	66.17a	59.74d	0.125

Description : Different letters (a,b,c) in the same rows indicate significant differences (p<0.05);

S.E : standard error

 

Dry matter, organic matter, and protein digestibility were also found to be significantly different (p<0.05). The dry matter digestibility ranged from 62.82-56.54%, whereas organic matter digestibility ranged from 65.69-59.69%, and the protein digestibility ranged from 66.17-59.74%. The results of statistical analysis showed that the digestibility of dry matter, organic matter, and crude protein in P3 was higher than in other treatments (p<0.05). The variations on the feed digestibility show the contribution of the feed for livestock requirements and the synthesis of microbial proteins for producing energy. This is appropriate with the research of Nolan and Dobos (2005) they ferment the ingredients of feed (proteins, sugars, polysacharides) to generate the ATP molecules required to keep the homeostatic and maintaining their growth. The number of microorganisms on rumen may have been increased, therefore the distribution area for feed degradation was present. All of this leading to an increased the TDN values (Zebeli et al., 2007).

The higher organic matter, dry matter and protein digestibility on P3 (p<0.05). The higher number of dry matter digestibility affect to the higher chances of good nutrition can be used by livestock for growth. P3 had higher digestibility might be happened because the tannin content at treatment on Table 3 is relatively low. Strong bond between protein and tannins, carbohydrates or another substance will affect the digestibility of fiber. Carbohydrate consist of crude fiber and other polysaccharides are non-soluble substances such as hemicellulose, cellulose, and lignin. The decrease in active tannin levels will binding carbohydrates and can be well degraded by microbe’s rumen, the effect of digestibility will also be increased (Yanti et al., 2021b).

The average of dry matter and organic matter in vitro digestibility on this study was lower than in the research of Jamarun et al. (2021) with the ranges from 60.50%-73.36%. Organic matter digestibility consists of feed ingredients digestibility in the form of organic substances like protein, carbohydrate, fat, and vitamin. The organic ingredients present in feed is available in the form of insoluble, therefore a process of breaking down substances is required it becomes easy substances late. High digestibility of organic matter hay mangrove leaves combine with native grass might happened cause rumen microorganism’s activity caused in the treatment feed used contained high nutritional content (Suardin, 2014).

In this study organic matter produced ranged from 90.00%-91.11%. The content of this organic material is not too different from research Sari et al. (2022) results of proximate analysis organic matter of Mangrove Leaves (Rhizhopora apiculata) soaking with lime water around 91.14%-92.07%. High digestibility of organic matter in this study, it is suspected because dry matter digestibility in the study higher. Based on Sutardi (1980), material degradation organic matter is closely related to dry matter degradation because some ingredients of dry matter consist of organic matter.

The protein digestibility on P3 is higher than other treatments (p<0.05). This combination protein source might result a better biological value of protein composition and increased protein synthesis of rumen microbes (Atmojo et al., 2019). Protein protect can also using tannins. Tannins are compounds that can be used to protect proteins from rumen microbial degradation, because tannin are able to bind proteins by forming complex compounds that resistant to protease, resulting in decreased protein degradation in the rumen (Cahyani et al., 2012).

Effect Combination of Hay Mangrove Leaves and Native Grass on Fiber Fraction Digestibility

Data of fiber fraction digestibility of the experimental combination hay mangrove leaves and native grass are summarized in Table 5.

 

Table 5: Fiber Fraction Digestibility

Fiber Fraction Digestibility (%)	Treatment				S.E
	P1	P2	P3	P4
ADF	48.82b	46.10c	50.67a	44.87d	0.264
NDF	52.60b	50.82c	57.22a	46.77d	0.440
Cellulose	50.58b	47.72c	55.84a	45.37d	0.352
Hemicellulose	61.31b	59.48c	63.27a	56.52d	0.332

Description : Different letters (a,b,c) in the same rows indicate significant differences (p<0.05); S.E : standard error

 

Fiber fraction digestibility were found to be significantly different (p<0.05) on ADF, NDF, cellulose, and hemicellulose digestibility (Table 5). ADF, NDF, cellulose, hemicellulose digestibility ranged from 50.67-44.87%; 57.22-46.77%; 55.84-45.37%; 63.27-56.52%. The results of statistical analysis showed that digestibility of ADF, NDF, cellulose and hemicellulose in P3 was higher than in other treatments (p<0.05). The number of fiber fraction digestibility was affected by many factors, including the composition of fiber fraction, amount fiber in the feed, and microorganism activity (Maynard et al., 2005). Based on Jamarun et al. (2018) and Yanti et al. (2021c) low crude fiber in feed content made easier to digest because the microbes also easily to penetrate the cell walls. Beside of that, the higher number of crude fiber in a feed content will affect to the cell walls thicker and resistant to microbes digesting. That can result in a decrease of feed digestibility. It can be seen from the lowest. Another substance affected the fiber fraction digestibility is anti-nutritional compound. The higher fiber digestibility on P3 (p<0.05) was found that tannin content on Table 3 was low. The lowest fiber digestibility on P4 (p<0.05), even the tannin content on P4 was lowest, but the crude fiber was higher than other treatments (Table 2), crude fibre had in general the lowest digestibility (De-Oliveira et al., 2011). Tannin might bind with rumen microorganism’s cell wall and can inhibit microorganism growth and enzyme activity (Smith et al., 2005). Tannins also the defaunation agent and functions to protect protein, the weakness in their function is the phenol compound. Tannins also have antibacterial properties. Gram-positive bacteria are sensitive to polyphenols certain (Smith et al., 2003) whereas some fiber fraction digesting bacteria including gram bacteria positive. Tannins in high doses will decrease the digestibility of fiber in the rumen.

Effect Combination of Hay Mangrove Leaves and Native Grass on Rumen Liquid Characteristics

Data of rumen liquid characteristics of the experimental combination of hay mangrove leaves and native grass are summarized in Table 6.

 

Table 6: Rumen Liquid Characteristics

Rumen Liquid Characteristics	Treatment				S.E
	P1	P2	P3	P4
pH	6.58a	6.59a	6.56a	6.62a	0.007
VFA (mM)	142.25b	138.70c	156.20a	129.60d	1.017
NH3 (mg/100ml)	8.94b	8.47b	9.84a	7.38c	0.123

Description : Different letters (a,b,c) in the same rows indicate significant differences (p<0.05);

S.E : standard error

 

The result show that pH found to be not significantly differet (p>0.05) on Table 6. This study has normal range pH of the rumen fluid for fermenting feed is still within 6.56-6.62. Nor­mal rumen condition has pH for 5.5-6.9, dry material content of 10-13%, and temperature of 38-41°C for optimum rumen microbes (Puniya et al., 2015). Rumen pH is important because plays role in the capability of microbial rumen to degrade feed protein (Buckner et al., 2013).

Total Volatile Fatty Acid (VFA) in this study ranged from 156.20-129.60 mM and significantly different (p<0.05). The results of statistical analysis showed that VFA in P3 was higher than in other treatments (p<0.05). The higher number of VFA in P3 correlated with higher fiber fraction digestibility of P3 in Table 5. Fiber fraction contained in feed because crude fiber contains carbohydrates which is one of the important factors that can affect VFA production. This is in accordance with the opinion of Pamungkas et al. (2008) which stated that ruminant rations mostly consist of forage containing structural carbohydrates in the form of fiber fraction (cellulose and hemicellulose) and simple carbohydrates that are easily fermentable (sugars, starches), both of which will then be fermented into Volatile Fatty Acids (VFA), methane and CO2.

The results of statistical analysis showed that NH3 in P3 was higher than in other treatments (p<0.05). The higher number of NH3 in P3 correlated with higher protein digestibility of P3 in Table 4. Degradation in the rumen will produce amino acids which will then undergo deamination to produce NH3, VFA and CO2 (Kozloski et al., 2000). This VFA production was enough for rumen microbial protein synthesis, VFA needs for microbial rumen growth about 80-160 mM (Van Soest, 1994). Increased microbial production correlated with increased of VFA concentration. Quickness of feed degraded by rumen microbes could be show on increased of the VFA number. Anggraeni et al. (2017) VFA production is the energy adequacy for ruminant. The difference of feed composition, physical form, and feed processing can alter the composition of VFA in rumen. NH3 of all treatments was enough for microbial protein production, based on McDonals et al. (2002) NH3 6-21 mM production increasing the microbial protein production. Putri et al. (2013) high concentrations of ammonia might be reason of the faster degradation protein feed process than the formation of microbial protein. The rumen fluid ammonia concentration is influenced by the protein consumed and protein degradation process on rumen. Rahmadi et al. (2010) the low of NH3 on rumen influenced by N availability on feed. NH3 affects the growth of microorganisms because it is used in protein synthesis.

Effect Combination of Hay Mangrove Leaves and Native Grass on Gas Production

Data of gas production of the experimental combination hay mangrove leaves and native grass are summarized in Table 7.

Total gas production and methane gas found to be significantly different (p<0.05) on Table 7. Total gas production ranged from 167.20-133.80 ml/hours, whereas methane gas ranged from 20.57-41.00 ml/gr Dry Matter. The highest total gas production digestibility was found in P3 (p<0.05). Gas production is the result of a fermentation process that occurs in the rumen which can show microbial activity in the rumen and describes the amount of organic matter digested (Ella et al., 1997). The higher gas production mean better quality of the feed ingredients, this can be seen in Table 4 digestibility of nutrients and Table 5 digestibility of crude fiber that P3 has the highest digestibility. Methane production is correlated with considerable energy losses, and decreasing energy gain and productivity.

 

Table 7: Gas Production

Gas Production	Treatment				S.E
	P1	P2	P3	P4
Total Gas Production (ml/hours)	151.70b	141.80c	167.20a	133.80d	2.350
Methane Gas (ml/gr Dry Matter)	28.75c	38.59b	20.57d	41.00a	0.049

Description : Different letters (a,b,c) in the same row indicate significant differences (p<0.05); S.E : standard error

 

The highest methane gas production on P4 (p<0.05). P4 contain higher crude fiber (Table 2) and received at least tannin protection because the use of HML was lowest than the other treatment and. Crude fiber content has a relationship with the production of methane gas, the higher crude fiber content increased potential for methane gas production (Beauchemin et al., 2008).Crude fiber is Tannin decreasing the protozoa’s population in the rumen (Patra and Saxena, 2010). The indirectly inhibitation mechanism of methane gas production using tannin via fiber digestion with reduces the production of hydrogen, and directly mechanism via the activity and growth of methanogens (Tavendale et al., 2005). This indicates that tannin of hay mangrove leaves was affecting the ruminal methanogenesis pathway with decreasing protozoa ecology (Goel et al., 2008) and had toxic effect for methanogenic bacteria (Carulla et al., 2005). High methane gas production is not good because methane gas having a negative impact on the environment and also energy losses which should be converted into livestock nutrient absorption.

Conclusion

This research concludes that P3 (40% hay mangrove leaves + 60% native grass) had the best resulted effected on nutrient and fiber in-vitro digestibility, rumen fluid characteristic and gas production. Future research can be conducted to determine the effect combination of 40% hay mangrove leaves and 60% native grass on ruminant livestock production.

Acknowledgments

This research was supported by PMDSU Scholarship Grant by The Ministry of Education, Culture, Research And Technology Republic of Indonesia 2022 with grant number T/18/UN.16.17/PT.01.03/PPS-PMDSU-Pangan/2022. This research would not have been possible without the technical assistance of the staff in The Laboratory of Ruminant Nutrition and Biotechnology, Animal Science Faculty of Andalas University.

Conflict of Interest

The authors declare that they have no competing interests

Authors Contributions

Novirman Jamarun, Suyitman, Khasrad, Elihasridas, and James Hellyward formulated the experimental design and experimental work at the lab­oratory. Rani Winardi Wulan Sari and Gusri Yanti drafted the manuscript, did experimental work at the lab­oratory and data analysis. All the authors read and approved the final version of the manuscript.

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