Abstract: Background and Objective: Coleus amboinicus L., also known asbangun-bangun in Indonesia, is a herb that is often used to increase digestibility and milk production, facilitate metabolic processes in the body and increase VFA. This study aimed to determine the effect of Coleus amboinicus L. supplementation on in vitro digestibility. Methodology: The study used a completely randomized design with 4 treatments and 4 replications: B0 = control (70% forage+30% concentrate), B1 = B0+0.3 g kg–1, B2+0.6 g kg–1 and B3 = B0+0.9 g kg–1 supplementation with C. amboinicus. The observed variables were dry matter digestibility (DMD), organic matter digestibility (OMD), acid detergent fiber (ADF), neutral detergent fiber (NDF), ammonia (NH3), pH, volatile fatty acid (VFA), acetic acid but yric acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, bacterial count and rumen protozoa. Results: The results demonstrated that supplementation with C. amboinicus significantly increased DMD, especially in treatment B1 (74.62±11.30%) and B2 (74.79±6.45%) and increased OMD in B1 (77.74±10.80%) and B2 (78.26±7.76%). However, supplementation with C. amboinicus did not affect ADF, NDF, In vitro gas production, pH of rumen, valeric acid, isovaleric acid, isobutyric acid, bacterial count or rumen protozoa amount. Supplementation with C. amboinicus significantly increased N-NH3 (33.19±1.54 nM), VFA (106.39±6.20 mM), acetic acid (59.49±1.94 mM), propionic acid (26.50±2.70 mM) and butyric acid (11.49±1.88 mM) in treatment B1. Conclusion: Supplementation with C. amboinicus may increase DMD, OMD, VFA, acetic acid, propionic acid and butyric acid but does not affect ADF, NDF, pH, gas production, isobutyric acid, valeric acid, isovaleric acid, bacterial count or rumen protozoa.
INTRODUCTION
In vitro measures of digestion are conducted by mimicking the processes that occur in the rumen via a fermentor tube. This is a description of the digestibility of feed by rumen microbes that can be done in the laboratory. For the digestion process to take place, it is necessary to note the quality and quantity of the feed1,2. One of the plants that could potentially be used as a supplement to improve digestibility and production is Coleus amboinicus L (C. amboinicus).
The local name of this plant in Indonesia is torbangun or bangun-bangun and it is a herb that is often used in the North Sumatra Province, Indonesia. The benefit of this plant is that it increases milk production and restores the uterus after delivery. According to Rumetor et al.3 the leaves of C. amboinicus can improve the metabolic processes in the body, increase the VFA from 111.15-167.10 mM in feeds with 9%. VFA is the final product of carbohydrate fermentation and is the main energy source of ruminants of rumen origin4,5. The production of VFA in the rumen becomes the benchmark of feed fermentability process in ruminants.
Coleus amboinicus has the potential to exhibit antioxidant, diuretic, immunostimulant, anti-inflammatory and anti-infertility effects6-9. Coleus amboinicus can increase feed consumption, growth and efficiency of dietary use in pigs10 and can increase reproductive performance11. Coleus amboinicus defends against worm infection because C. amboinicus contains a volatile oil, which can act as an antiseptic12,13, thereby improving the digestion process. According to Duke14, C. amboinicus contains carvacrol, thymol and forskolin. Coleus amboinicus has high content of Fe, carotenoids and potassium to help create a sense of calm, cleanse the blood and relieve pain. The calm conditions of the cattle would optimize the cattle production.
Coleus amboinicus leaves contain flavonoids, saponins and polyphenols7. They may increase the hormones prolactin and oxytocin6. Lactogogum is a component that can stimulate and increase milk production. For rats, it could increase milk production by approximately 30%15. In goats, it could increase milk production by approximately 8.65-67.22%16, increase dairy goat milk production to 9 g kg–1 body weight17 and restore stamina18. Increased production is closely related to the digestive process in the rumen.
Thus, we can determine the effect of C. amboinicus on In vitro digestibility. An increase in the digestibility of C. amboinicus is expected to increase nutrient absorption into cells, thereby increasing milk production. Based on these conditions, it is necessary to do a study to determine the effect of Coleus amboinicus L. supplementation on In vitro digestibility.
MATERIALS AND METHODS
Coleus amboinicus L. is obtained by harvesting stems and leaves and then chopping them to a size of ±1 cm. The leaves that have been chopped are dried in sunlight for about ±2 days. After the leaves are dried, the leaves are smoothed into flour and then mixed into the concentrate.
This research was conducted In vitro by compiling ration for In vitro digestibility of 70% elephant grass (Pennisetum purpureum) and 30% concentrate (15% bran, 2.5% palm kernel meal, 2.5% coconut meal, 8% soybean meal, 0.5% lime, 0.5% salt, 0.5% urea and 0.5% topmix). All ingredients are mashed and mixed according to the treatment condition. The feed quality can be seen in Table 1.
A completely randomized design was used in this study with 4 treatments and 4 replications: B0 = control (without C. amboinicus), B1 = 0.3 g kg–1, B2 = 0.6 g kg–1 and B3 = 0.9 g kg–1 supplementation with C. amboinicus.
Fume rumen fluid fistula was obtained from the Fapet Farm Faculty of Animal Husbandry, University of Jambi and transported to the laboratory in a Thermos. The rumen fluid obtained was squeezed and filtered using gauze and then, the rumen liquid was immediately used for In vitro analysis. The rumen liquid was then mixed with a solution of McDougle 1:4. Then, a maximum of 40 mL was inserted into the fermentor tube that was already present with the feed sample. Then, the sample was incubated for 48 h. After 48 h, the tube was opened and microbial activity was stopped by introducing 1 drop of HgCl2 fluid.
| Table 1: | Quality of research feed |
| Result of Proximate Analysis, Laboratory of Nutrition and Animal Feed, Faculty of Animal Husbandry, University of Jambi | |
Then, the fluid was centrifuged at a rate of 2000 rpm. The obtained supernatant was dried in an oven at 600°C for 24 h or until a constant weight and then, the supernatant was analyzed.
The observed variables were as follows: In vitro dry-matter digestibility (DMD), organic-matter digestibility (OMD) using the Tilley and Terry method19, ADF digestibility and NDF were measured using the Van Soest method20, VFA production was measured using the steam distillation method, N-NH3was measured using microdiffusion according to Conway21, the number of bacteria was measured by enumerating live bacterial colonies and the protozoa of cow rumen fluid were examined according to the method of Ogimoto and Imai22.
An analysis of variance was used to analyze the data and the Duncan multiple range test was used for post hoc analyses23.
RESULTS AND DISCUSSION
Degradation of Dry Material, Organic Material, ADF and NDF: Data regarding the Average Dry Matter Digestibility, Organic Matter Digestibility, NDF and ADF feed supplemented with C. amboinicus are shown in Table 2.
Supplementation with C. amboinicus had a very significant effect on DMD (p<0.01). The treatment of B1 and B2 is significantly higher than B0 and B3. B0 is significantly higher than B3. This result is stronger than that of Rumetor et al.16, who observed the basal baseline DMD was 65.24%. When supplemented with Zn-vitamin E, the DMD yield was 68.23%. Krisnan et al.24 observed that DMD was 58.84% when treated using probion and catalytic supplements. Adriani et al.5 observed that DMD was 65.38-81.56%in the treatment of probiotic and C. amboinicus.
This condition is suspected due to the influence of the active compound, carvacrol, contained in the leaves. Carvacrol compounds play a role in reducing the speed of deamination of amino acids and protein degradation in the rumen. The reduction of deamination of amino acids results in the breakdown of proteins, resulting in an increase in the amount of absorbed proteins, which affects the digestibility of dry matter and organic matter1. According to Garcia et al.25 increased carvacrol administration of 250 and 500 mg L–1 could reduce protein degradation from 72.8-51.5%. Therefore, the protein that escapes degradation is higher and can increase the digestibility of organic matter.
Supplementation with C. amboinicus had a significant effect on OMD (p<0.01). Treatment B1 and B2 did not differ from each other but they were significantly higher than B0 and B3. Treatment B0 was significantly higher than B3. This result is relatively similar to Rumetor et al.16, who stated that OMD feed supplemented with C. amboinicus ranged from 60.30-76.25%. The increase of OMD in the B1 and B2 treatments may be due to the active substance of carvacrol in C. amboinicus, which helps the reduction of the deamination process to increase the amino acid, thereby increasing the digestibility of organic matter1.
Variability analysis showed that supplementation with C. amboinicus did not affect ADF and NDF (p>0.05). The average ADF is 40.08±1.54% and the average NDF is 70.18±0.89%. This result is higher than that of Garcia et al.25, who stated that NDF was decreased by 24.2 and 21.1% after supplementation with carvacrol at approximately 250 and 500 mg L–1, respectively. This result is due to C. amboinicus improving the conditions that are conducive to the digestive process, thus yielding better ADF and NDF.
In vitro digestibility: Data regarding In vitro digestibility, N-NH3 and pH after supplementation with C. amboinicus are shown in Table 3.
Supplementation with C. amboinicus significantly affected N-NH3 In vitro (p<0.01). The B0 and B1 treatments had significantly higher N-NH3 In vitro than B2but relatively similar to B3. These results are similar to those of other studies that obtained amino In vitro content in sheep0.45 g kg–1 carvacrol; in those studies, the ammonia yield decreased from 36.55/100-23.05/100 mL26. This result is relatively similar to those of Rumetor et al.3, who found that increased administration of C. amboinicus tends to decrease the production of N-NH3 In vitro. This condition is thought to be due to the influence of carvacrol, an active substance contained in C. amboinicus13. Ammonia is used by microbes as a source of nitrogen in the synthesis of cell proteins and thus, the presence of ammonia is absolutely necessary.
| Table 2: | Average dry matter digestibility, organic matter digestibility, ADF and NDF In vitro supplemented by Coleus amboinicus |
Different capitalized superscripts in one line showed significant differences (p<0.01), DMD: Dry matter digestibility, OMD: Digestibility of organic matter, ADF: Acid detergent fiber, NDF: neutral detergent fiber |
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| Table 3: | In vitro digestibility, N-NH3 and rumen gas production after supplementation by Coleus amboinicus |
| Different lowercase superscripts in a row show a marked difference (p<0.05). Different capillary superscripts in a row showed significant differences (p<0.01) | |
The N-NH3 content in the rumen is an illustration of the process of degradation and the process of protein synthesis by microbes27.
Supplementation with C. amboinicus affected VFA content (p<0.05). Treatment B0 did not differ from B1 but did differ from B2 and B3 did not differ from B0, B1 or B2. The highest VFA was observed in treatment B1. This result is relatively similar to those of Noirot and Bayourthe28, who stated that administering carvacrol at low doses does not affect the rumen VFA but higher doses of carvacrol decrease the VFA yield. This result is similar to that of Garcia et al.25, who observed that the administration of carvacrol at 250 and 500 mg L–1 decreased VFA from 17.6-7.87 mmol L–1. This result is due to C. amboinicus being antimicrobial; thus, its administration needs to be closely monitored. The use of plant extracts for digestion engineering is carried out in controlled doses29.
Coleus amboinicus leaves affected the resulting acetic acid concentration (p<0.05). Treatment B0 and B1 differed from B2 and B3 but B0 and B1 did not differ significantly (p>0.05). This finding is similar to the results for In vitro VFA, where treatment B1 is higher than other treatments. This result is higher than what Prihartini28 observed-that rumen acetic acid ranged from 20.01-47.25 mM. Acetic acid describes the digestibility of crude fiber in the rumen.
Coleus amboinicus treatment influenced propionic acid concentration (p<0.05). Treatment B1 was significantly higher than B2 and B3 but similar to B0, while treatment B2, B3 and B1 did not differ (p>0.05). The highest content of propionic acid was found in treatment B1. This result is thought to be due to the presence of other compounds that create a barrier in the form of antimicrobial thymol, such that higher doses of thymol can reduce propionic acid. Castilejos et al.1 found that administration of thymol compounds could decrease the production of propionic acid but do not affect the In vitro VFA content.
Coleus amboinicus treatment had a significant effect on butyric acid (p<0.05). Treatment B1 was significantly higher than B0, B2 and B3 (p<0.05) but B0, B2 and B3 did not differ (p>0.05).
| Fig. 1: | In vitro gas production supplemented with Coleus amboinicus leaves |
This result is higher than what Prihartin30 observed, where butyric acid ranged from 3.46-7.84 mM in feed treated with lignochloritic inoculum. However, this finding is lower than what Koyuncu and Canbolat26 observed, where butyric acid ranged from 12.23-13.09 mM in the feed supplemented with carvacrol.
Coleus amboinicus leaves did not affect the content of isobutyric acid, valeric acid or isovaleric acid (p>0.05). The average content of isobutyric acid, valeric acid and isovaleric acid are 1.98±0.15, 3.29±1.09 and 5.50±0.45, respectively. The results of this study are higher than what Xia et al.27 observed, where isobutyric acid ranged from 0.43-0.53 mmol L–1, valeric acid ranged from 0.61-0.86 mmol L–1 and isovaleric acid ranged from 0.74-1.02 mmol L–1 in rations with molasses.
Coleus amboinicus treatment did not affect gas production for 48 h (p>0.05). Average gas production for 48 h was 43.11±4.98 mL. This result is higher than those of Njidda and Nasiru31, who observed that In vitro gas production ranged from 1.16-25.50 mL per 200 mg DM. The production of gas produced during the In vitro process in Fig. 1.
Figure 1 shows that the production of In vitro gas produced between the second hour and the sixth hour is relatively constant before increasing at the 12th, 24th and 48th h. This result is due to the action not affecting the activity of rumen microbes, such that the resulting gas yield remains relatively constant.
| Table 4: | Number of bacteria, protozoa and pH of rumen supplemented with Coleus amboinicus |
Number of microbes and rumen pH: The number of bacteria, protozoa and pH of rumen supplemented with C. amboinicus are shown in Table 4.
Coleus amboinicus treatment did not affect the pH of rumen fluid (p>0.05). The mean pH of rumen fluid is 6.63±0.09. The results are similar to Rumetor et al.16, who stated that C. amboinicus supplementation yielded pH rumen of 6.14-6.25, a pH between 5.5-7.032 and a pH between 6.76-6.9424. The average pH of the normal rumen is 6-733, while the ideal pH of the digestome is 6.4-6.834. Suitability of pH rumen can help the process of bacterial colonization on the cell wall and encourage cellulase bacterial activity. If the rumen pH is higher than 7.3, the ammonia absorption process will be accelerated. This condition occurs because the formation of unionized ammonia more easily passes through the rumen wall.
Coleus amboinicus treatment did not affect the amount of rumen bacteria (p>0.05). The rumen bacterial averages were 8.65±0.45×108. These results were higher than other studies3which found that rumen microbials were between 46 -52 x 105, 0.8×1011 in concentrated 3% BW35.
Coleus amboinicus treatment did not affect the number of rumen protozoa (p>0.05). The average rumen protozoa is 18.09×105. This result is higher than that of Wanapat and Khampa35, who observed that rumen protozoa fed 3% BW concentrate at 4.1×105. Protozoa in the rumen is functioning to digest fermentable carbohydrates and breakdown the hard-to-digest carbohydrates.
The results of this study can improve the digestibility of dry matter, organic matter and VFA. This increase in digestibility will increase the availability of nutrients in the blood, thereby increasing milk production.
CONCLUSION
Coleus amboinicus supplementation may increase DMD, OMD, VFA, acetic acid, propionic acid and butyric acid but has no effect on ADF, NDF, pH, gas production, isobutyric acid, valeric acid, isovaleric acid, bacterial count and rumen protozoa.
ACKNOWLEDGMENTS
We would like to thank the Directorate of Research and Community Service, Directorate General for Research and Development, Ministry of Research, Technology and Higher Education, who funded this activity, according to Research Contract No.22/UN21.17/PP/2017.