Adding Maja leaves (Crescentia cujete) to ruminant ration affects rumen fermentation characteristics, gas production and Levels of undegraded rumen protein

M Akbar¹, Rohmiyatul Islamiyati² and Syahriani Syahrir²

¹ Animal Science and Technology, Faculty of Animal Science, Hasanuddin University, Makassar 90245, South Sulawesi, Indonesia
nanisyahrir@gmail.com
² Departement of Animal Feed and Nutrition, Faculty of Animal Science, Hasanuddin University, Makassar 90245, south Sulawesi, Indonesia

Abstract

This study aims to analyze the effect of adding maja leaves to ruminant livestock rations on rumen fermentation characteristics, gas production, and undegraded rumen protein. The study was conducted in vitro using a completely randomized design with four treatments (maja leaf levels of 0, 10, 20, and 30 g/100 g of feed) with four replicates and analyzed using a One-Way ANOVA, followed by the Duncan test. The results indicated that adding maja leaves at 30 g/100 g with a tannin content of 2.38% had no significant effect on NH³, pH, or partial VFA concentration. However, it significantly influenced gas production and undegraded rumen proteins. Gas production decreased with increasing levels of maja leaves in the ration. The highest level of undegraded rumen protein, 34.8%, was obtained by adding maja leaves at 20 g/100 g, while the lowest level was found in the ration treatment without any maja leaves. It was concluded that the recommended treatment was CC20, which involved adding maja leaves at 20 g/100 g in the ration. In this treatment, the percentage of undegraded proteins that had the potential to be bypassed was 34.8%, the highest among all treatments.

Keywords: Crecentia cujete, gas production, rumen ecosystem, undegraded rumen protein

Introduction

The efficiency of feed utilization in ruminant livestock is still a significant challenge in the livestock industry. The problem that often occurs is the high degradation of proteins in the rumen, which causes low availability of by-pass proteins. One way to do this is to manipulate fermentation by rumen microbes by using available and sufficient materials (Sun et al 2023). Continuous exploration to find feed ingredients suitable for the content of certain compounds that can maximize nutrient absorption by ruminant livestock is a must. One of the feed ingredients that have the potential to be used is Maja leaves (Crecentia cujete L.)

Maja leaves are a typical plant of tropical regions whose availability is relatively abundant and has a tannin content in its leaves of 7.92%. Tannins are known to be used as agents that can manipulate rumen ecosystems. Tannins can increase the bypass of rumen proteins and reduce losses caused by excessive protein fragmentation in the rumen (Besharati et al 2022). According to their structure and concentration, these compounds can benefit or harm ruminant livestock (Min et al 2020; Fonseca et al 2023). Tannins in low concentrations can support rumen microbial activity. On the other hand, tannins in high concentrations can reduce ration consumption due to their spicy taste, reduce digestibility, and reduce toxic effects on rumen microbes because they can inhibit enzyme activity (Besharati et al 2022; Verma et al 2021).

Tannins will protect proteins from rumen microbial degradation so that they can increase proteolysis in the abomasum. Tannins-bound proteins have a positive impact on protein degradation in the rumen, thereby increasing protein flow to the duodenum, nitrogen utilization efficiency, maximizing microbial protein synthesis as well, and reducing the toxic effects of high concentrations of NH₃ in the rumen (Brutti et al 2023). The purpose of this study is to analyze the effect of the addition of maja leaves containing tannins in ruminant rations on the characteristics of rumen fermentation, gas production, and undegraded rumen protein levels in vitro, so it is hoped that this study will be able to provide a theoretical basis for making and developing the use of maja leaves in animal feed in the future.

Materials and methods

Preparation and nutritional composition of feed treatment

Feed ingredients used to make ruminant rations include corn tumpi, rice bran, corn mill, dregs, soybean meal, molasses, and mineral mix. Rations are formulated regarding the Indonesian National Standard (SNI 3148.2, 2024). The maja leaves used have the composition of nutrients and tannins in Table 1.

The addition of Maja leaf flour is carried out at four levels of treatment, namely :

CC0:Rations without the addition of Maja leaf flour.

CC10: Ration + 10 g/g maja leaf flour.

CC20: Ration + 20 g/g maja leaf flour.

CC30: Ration + 30 g/g maja leaf flour.

All feed ingredients were tested for nutritional content at the Feed Chemistry Laboratory, Hasanuddin University, with the results as shown in Table 2.

Rumen fluid collection

The procedure for collecting rumen liquid involves preparing a thermos filled with warm water (39ºC) to suit the natural condition of the rumen. The thermos is emptied for a while before filling the rumen liquid. The rumen liquid is filtered using a filter cloth and then put into the thermos through a funnel. The rumen liquid is then taken to the laboratory for in vitro testing experiments.

Ethical approval

The livestock that became the donors of rumen liquid in this study have been examined and approved by the Indonesian Ulema Council of South Sulawesi Province, with the number 06020013030319

Preparation of Mc Dougall

The Mc Dougall solution used in this study was made by adding as much as 1000 ml of distilled water to 0.25 ml of micro mineral solution (CaCl₂.2H₂O (13.2 g), MnCl₂.4H₂O (10 g), CoCl₂.6H₂O (1 g), FeCl₃.6H₂O (8 g)), and stirred until homogeneous, then adding 500 ml of buffer solution (NH₄HCO₃ (4.0 g), NaHCO₃ (35 g)), 500 ml of macro mineral solution (Na₂HPO₄ (5.7 g), KH₂HPO₄(6.2 g), MgSO₄.7H₂O (0.6 g)) and 100 ml of reducing solution (NaOH 1N (4 g). Homogenize with a magnetic stirrer; once the solution is well mixed, add distilled water until the solution reaches 2500 ml while continuing to stir. The solution is then put into a water bath at a temperature of 39ºC and filtered with CO₂ gas overnight so that the solution does not contain O₂ gas and is at pH 7.

In vitro incubation and determination of rumen fermentation characteristics

A 100 ml fermenter tube was filled with 0.5 grams of sample, then 50 ml of Mc Dougall solution and rumen liquid in a ratio of 4:1 (Tilley and Terry 1963) were added to each fermenter tube. The fermenter tube is covered with ventilated rubber, placed in a shaker water bath at 39ºC, and incubated for 4 hours. After the incubation, the fermenter tube is shaken, and the pH is checked (6.5-6.9). Put the centrifuge fermenter tube at 10000 rpm for 15 minutes. Supernatants were then taken for further analysis. NH₃ was measured using the Conway micro diffusion method. Partial VFA was measured using the gas chromatography method (Galyean 2010), the total protozoan population was measured using the Ogimoto and Imai (1981) method, and the fermentation efficiency was calculated by the following equation:

A is acetate, p is Propionate and B is butyrate (Baran et al 2002).

Gas production measurements: accumulation and kinetic equations

Fermentation was carried out in vitro using the Theodorou et al method (1994). With gas intake at the 2nd, 4th, 6th, 8th, 10th, 12th, 24th and 48th hours. The amount of gas production was measured using a syringe made of plastic with a volume of 50 ml. The kinetics of gas production is calculated by the exponential equation approach initiated by Orskov and McDonlad (1979) as follows:

Where P is the cumulative gas production at time t hours. Meanwhile, a, b, and c are the constants of the exponential equation. It is interpreted as the production of gases from a fraction that is young (a), the production of gases from an insoluble but fermentable fraction (b), and the rate of gas formation reaction (c). The sum of a+b can be interpreted as the maximum gas production formed during fermentation when t is close to infinity.

Determination of undegraded rumen protein

The results of in vitro fermentation for 48 hours and 96 hours were filtered using a 50 μm nylon cloth to produce fermentation residues. Then, it is placed in the oven for 3 days at 75ºC. After that, the residue is separated from the nylon fabric, which is tested for proximate.

Data analysis

The study utilized a completely randomized design with one-way ANOVA and the Duncan test to identify significant differences in the mean treatment. Analytical tools were applied using IBM SPSS Statistics 25.

Results and discussion

Characteristics of rumen fermentation

The fermentation characteristics of the rumen are evaluated based on the observation of pH value, NH3 concentration, total protozoa population and production of VFA (Volatile fatty acids), as shown in Table 3. The results showed that the pH value did not differ significantly (p>0.05) across all treatments at 7.08. This value corresponds to the ideal conditions for optimal fermentation by rumen microbes, specifically a pH range of 6.0-7.0 (Makmur et al 2020). Extreme fluctuations in pH values within the rumen can lead to high mortality of microorganisms, adversely impacting the rumen metabolic process (Pazla et al 2023).

The concentration of NH₃ also did not differ significantly (p>0.05). The highest value was 18.85 mM in the CC10 treatment, and the lowest was 16.17 mM in the CC30 treatment. The range of NH₃ concentrations is still within the ideal range of NH₃ required in rumens, which ranges from 85-300 mg/l or 6-21 mM (Weiner et al 2017). NH₃ is the result of amino acid deamination and hydrolysis of non-protein nitrogen. NH₃ serves as the primary precursor for microbial protein synthesis in ruminants. Although it does not have a significant effect, there is a tendency for NH₃ concentration to decrease along with the addition of Maja leaf flour. This is due to tannin compounds contained in maja leaves that bind to proteins. Ammonia concentrations indicate the number of feed proteins degraded by rumen microbes through N balance in ruminants (Zain et al 2023).

Partial VFA production, including acetate, propionate, iso butyrate, butyrate, isovalerate, and valerate, also showed no significant difference (p>0.05).This indicates that adding maja leaf flour up to 30 g/g does not affect the fermentation activity of carbohydrates by rumen microbes. The stable VFA content ensures energy availability for rumen microbes and livestock. Fermentation efficiency (FE), acetate: propionate (A:P), and Propionate: butyrate (P:B) ratios and NGR were also not affected by the treatment, indicating that the fermentation process was efficient at all levels of maja leaf flour addition. This study found that the total protozoan population had no significant effect, possibly due to the addition of Maja leaves containing tannins at a low level. Protozoa can stabilize the fermentation process because they can consume lactic acid faster than bacteria, so the pH of the rumen increases and prevents acidosis (Newbold & Ramos Morales 2020).

Gas production and gas production kinetics

In this study, adding maja leaves to the diets significantly affected total gas production, fraction potential to decompose, degradation rate of fraction b, and maximum gas production from a+b during 48 hours of incubation (p<0.05).This is possible because the tannins in maja leaves can bind proteins, so rumen microbes’ accumulation of fermentation processes can be suppressed. Gas production will decrease along with adding maja leaf levels in the treatment. The production of fractions that have the potential to decompose decreases along with the increase in maja leaf levels, which is possible because tannins bind to proteins that will have an impact on rumen microbes such as protozoa that will break down hemicellulose and cellulose.

In this study, adding maja leaf levels to the treatment did not significantly affect the decomposed young fraction (a). This is possible because the value of a has not been detected. After all, the lag phase affects rumen microbes by degrading nutrients contained in feed (Permatasari et al 2024).

Figure 1. This chart displays the cumulative gas production over the 48 hours of incubation. Gas production declines with the inclusion of Maja leaves in the ration.

Figure 1. Cumulative gas production for 48 hours with CC0 treatment = ration without the addition of Maja leaves (control), CC10 = ration with the addition of Maja leaves 10 g/100g, CC20 = ration with the addition of Maja leaves 20 g/100g, CC30 = ration with the addition of Maja leaves 30 g/100g

Undegraded rumen protein

In this study, adding maja leaves to the ruminant rations significantly affected (p<0.05) the level of undegraded rumen protein in vitro. The analysis results shown in Figure 2 indicate that the highest levels of undegraded protein are present in the CC20 treatment, with UDP at 34.8%. At the same time, the lowest is found in the treatment without adding maja leaves, with UDP at 22.23%. This indicates that the tannin content in maja leaves at this level effectively binds proteins to prevent degradation by rumen microbes without disturbing the rumen ecosystem.

Figure 2. Total undegraded rumen protein from rations supplemented with maja leaves containing tannins: CC0 treatment = rations without maja leaves; CC10 = rations with the addition of 10 g/100g of maja leaf flour; CC20 = rations with the addition of 20 g/100g of maja leaf flour; CC30 = rations with the addition of 30 g/100g of maja leaf flour.

The presence of tannins at the level of 2% in the feed does not interfere with the activity of rumen microbes. Instead, it inhibits the degradation of actual proteins by rumen microbes. Adding maja leaves to the ration of 20 g/100g of feed can increase the efficiency of the ration. The difference in UDP values in the treatment showed that adding feed ingredients containing tannins could form protein bonds and create insoluble complex compounds, thereby reducing rumen microbial degradation. With the tannin content in the feed, the protein becomes protected from rumen microbes, which in turn become a by-pass protein to improve the efficiency of nutrient utilization (Chuzaemi et al 2023). Rumen Undegraded Dietary Protein (RUDP) supports the amount of protein digested and absorbed in the abomasum and intestine for livestock productivity (Cahyani et al 2012).

Conclusion

This study concludes that adding maja leaf at a level of 30 g/100 g with a tannin content of 2.38 g/100 g of feed does not significantly affect the fermentation characteristics of the rumen but does have a significant impact on gas production and levels of undegraded rumen protein. Gas production decreased with the addition of Maja leaf, and the highest level of undegraded rumen protein 34.8% was achieved by including 20 g/100 g of maja leaves in the ration.

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