Evaluation of perilla seeds (Perilla frutescens) based on nutrient digestibility and pH in vitro tilley and terry

The study was carried out to evaluate the provision of perilla seeds (Perilla frutescens) with the addition of tannins to pH, IVDMD (in vitro dry matter digestibility), and IVOMD (in vitro organic matter digestibility) in the rumen and post rumen. The research used according Completely Randomized Design with four treatments and four replications: T1 = 100% perilla seeds, T2 = 100% perilla seeds + 1% tannin, T3 = 90% basal ration + 10% perilla seeds, T4 = 90% basal ration + 10% perilla seeds + 1% tannin. The method used in vitro two-stage digestibility to the observed of stage I (rumen) and stage II (total). The variables evaluated in this study included IVDMD stage I, IVDMD stage II, IVOMD stage I, IVOMD stage II, and pH. The results showed that giving perilla seeds with the addition of tannin had a pH value obtained in accordance with the pH standard in the rumen (P>0.05); IVDMD stage I and stage II respectively 22.91-35.85% and 36.01-44.15% (P<0.05); IVOMD stage I and stage II were 38.15-48.36% (P<0.05) and 50.04-55.35% (P<0.05), respectively. The conclusion of this research was carried out to give perilla seeds at a level of 10% and 1% tannin in the ration had diverse nutrient digestibility in the rumen and post-rumen. However, it does not affect the pH of the rumen.


Introduction
Animal feed has a very important role in the process of growth and productivity of livestock in producing products (milk, meat, and others).Livestock can grow accordingly expected, if the type of feed given is of good quality and meet the nutritional needs of livestock [1].Feed given in ruminants, is generally in the form of forage and reinforcing feed or concentrate.Good quality feed gives effect livestock that can increase livestock productivity [2].The productivity of livestock is affected by the value of feed digestibility and feed consumption, if the value of digestibility is low, the feed degradation will also be low so it is not able to offset rumen microbial activity resulting in low microbial growth and low feed consumption.
Feed given to livestock must be of good quality with energy and nutrient content balanced, Perilla seed (Perilla frutencens) is a feed ingredient that contains a high protein source and is rich in amino acids.Therefore in this study using perilla seeds which are rich in nutrient content and the use of perilla seeds in livestock rations can improve the digestibility of nutrients of the feed so that it can increase livestock productivity and not cause an impact negative in ruminant.Perilla seeds come from the perilla plant, which is one of the traditional plants and perennial plants native to the mainland Southeast Asia and India [3].Perilla seeds have superiority as a source of unsaturated fatty acids and efficacy as antimicrobial, antioxidant, cytotoxic, anti-inflammatory, and anti-allergic activity [4].
Perilla seeds are also rich in protein content the protein content of defatted perilla seed is 36% and the core cake perilla is 46% [5].The protein content of whole perilla seeds is in the range of 24.18-25.01%[6,7], 3457.55Kcal/kg metabolic energy, 43.1% crude fat, 29.83% crude fiber, 2.49% calcium, and 2.031% available phosphorus [8].High-quality protein feed will experience degradation in the rumen by rumen microorganisms.Feed degradation involves proteolytic enzymes produced by rumen microbes.This process produces amino acids and peptides, ammonia (NH3) as the end product of fermentation [9].Protein in the rumen will undergo a process of degradation into ammonia, peptides, and amino acids while the protein that will be absorbed in the small intestine is what escapes the process of degradation [10] [11].If perilla seed protein is not protected it will be degraded by microbes rumen so it is not effective in feeding.Protein does not degrade and needs to be protected by a technology that is by using tannins.According to [12] the addition of 1% tannin in livestock rations is able to protect feed protein.
Tannins are compounds anti-nutrients that play a role in reducing the quality of the material by forming complexes with proteins.Complex compounds between tannins and proteins insoluble in the rumen, in an acidic environment in the abomasum, complex undergo enzymatic digestion so that the protein can be utilized by ruminants [13].The method used is a two-stage in vitro method as a technique [14] digestibility measurement.According to [15,16] the in vitro method is a digestive process outside the body as it happens in the rumen.Based on the background above, the aim of this study was to determine the effect of using perilla seeds with the addition of tannins to pH, IVDMD (in vitro dry matter digestibility), and IVOMD (in vitro organic matter digestibility) in the rumen and post rumen.

Preparation of feed samples
The feed samples used in this research were perilla seed samples as a single ingredient, mixed feed between concentrate and tannin feed.Perilla seeds were washed and dried in a drying oven at 55-60 o C for 2-3 days until the weight was constant.The feed samples used consisted of pangola grass as standard, bengal grass, perilla seed, and concentrate consisting of soybean husk, pollard, rice bran, and soybean meal.Feed samples were mixed according to the treatment and ration composition (tables 1 and 2).The feed samples were ground using a disk mill with a sieve measuring ± 2 mm to measure the digestibility value in vitro.Furthermore, the feed samples were analyzed for their nutritional value and digestibility in vitro in the rumen and post rumen.

Treatment feed composition
The nutritional content of the feed ingredients used in the study is presented in Table 1.Meanwhile, the composition and nutritional content of the research rations are presented in Table 2.The study used the composition of the ratio between Panicum maximus grass and concentrate with a ratio of 25%:75%.Concentrate feed ingredients in the form of 5% soybean meal; soybean husk 15%; pollards 30%; rice bran 48%; and 2% vitamin-mineral premix with 100% of the total ration.

Chemical compotition analysis
Nutrient analysis of feed samples [17] was carried out in a drying oven at 55 o C (moisture content, crude protein, extract ether, and crude fiber) and oven at 105 o C for ash analysis.The difference in organic matter obtained was ash = 100% -% ash.

pH of rumen fluid measurement
The process of measuring the initial and final rumen fluid pH for in vitro digestibility test using a LUTRON PH-201 digital pH meter.

Preparation of rumen fluid
Rumen fluid was obtained from 3 bulls slaughtered at the abattoir in the morning, then flowed with CO2, filtered using 4 layers of nylon cloth to get rumen fluid without rumen content particles, and mixed with McDougall's buffer solution (1:2, v/v) anaerobically.In addition, no ethical clearance was issued for these experiments since animals were not treated directly.

McDougall's solution preparation
The artificial saliva (buffer) utilized was McDougall's solution.Prior to dissolving in 500 ml of aquadest in a beaker (1000 ml), weigh the following ingredients carefully: 9.8 g of NaHCO3, 0.57 g of KCl, 10 g of Na2HPO4.12H2O,0.12 g of MgSO4 7H2O, and 0.47 g of NaCl.At 39 °C, stirring was done with a magnetic stirrer.Then, 100 ml of aquadest was added to 5.3 g of CaCl2 and mixed to form Solution 2 (Solution 2).Solution 3 is created by adding up to 1 cc of Solution 2 to Solution 1 and stirring until it is thoroughly combined.McDougall's solution is created by adding aquadest to solution 3 until the volume reaches 1000 ml.The solution was mixed with 0.1 M HCl to bring the pH level back to neutral.By mixing 44.25 ml of aquadest with 455.75 ml of concentrated HCl (normality 11.3), a 0.1 M HCl solution was created.

HCl-pepsin solution preparation
To produce 50 ml of pepsin solution, 0.2 g of pepsin must be dissolved in 0.1 N HCl then added to distilled water to a volume of 50 ml.

IVDMD (In vitro dry matter degradability) and IVOMD (in vitro organic matter digestibility)
Measuring the rumen digestibility of DM (IVDMD) and the rumen digestibility of OM (IVOMD) was carried out using [14] method.The first procedure is the fermentative digestion process with the procedure described previously, only the incubation time that is applied is 48 hours for fermentative digestion, HCl and pepsin are introduced right away without first screening the remnants from the rumen phase to the post-rumen phase.It takes 48 hours for the first stage of incubation (in the rumen).The 50 mL in vitro fermenter tube was filled with rumen fluid and artificial saliva at a ratio of 1:4 v/v, along with 0.5 g of the treatment sample.After in vitro administration of rumen CO2 gas to create anaerobic conditions, incubated at 39-40 °C in a water bath.Every two hours, the fermenter tube was manually shaken to mimic rumen-like movements.To stop rumen microbial activity after 48 hours, 1-2 drops of saturated HgCl2 were applied to each tube of the fermentor.
The stage of the post-rumen is created in this second phase by adding 1 mL (given in stages 0.5, 0.5, 1, and 1 mL) of 5% pepsin and up to 3 mL of 20% HCl to each test tube for the rumen.The 48-hour post-rumen (abomasum) incubation period.Five times were given to each incubation point from the treatment, the blank, and the standard.Blank treatment, which serves as a corrective factor, is a tube that is filled without a treatment sample.Pangola grass, which is also present in the fermenter tube, is used in the standard feed sample.Every four hours, manual shaking is performed.

Analysis of data
The data obtained in this research was examined using one-way ANOVA to determine the effect of treatment on variables that were observed.The Duncan's New Multiple Range Test (DMRT) was used to examine the mean differences between treatments with a level of significance of (p<0.05).

Results and discussion
The average values of pH, DM digestibility, and OM digestibility can be seen in Table 3 based on the results of a two-stage in vitro test for nutrient digestibility and degree of acidity (pH) in the rumen and post-rumen.The pH value obtained from the treatment between 7.00-7.02(P> 0.05) is still within the normal range of rumen pH so this pH value can make the rumen environment during the feed fermentation process run optimally.According to the research results of [15], which used acacia plants that contain high tannins, the rumen pH was 6.58-7.02.According to previous research [18], for optimum fermentation, the rumen's pH should be between 6.5 and 7.5.According to [19], pH levels outside of the range of 5.7-7.8 will negatively affect the rumen environment and feed fermentation in the rumen.For this reason, the pH in this study is still within the normal range so as not to affect rumen function.A high-grain diet can have an impact on pH, fermentation, and the makeup of the bacterial population in the rumen and reticulum, according to [20],.After consuming large amounts of grains, the pH of the rumen and reticular tissues dropped, and changes in bacterial composition, similarity, and copy number were also seen.These modifications may have an impact on the ability of the rumen and reticulum.
There are differences in the nutritional content of IVDMD and IVOMD at each stage of fermentation in the rumen and post-rumen between stages.The results showed that fermentation values in the rumen (IVDMD and IVOMD) were lower than post-rumen (P<0.05)(Table 3).This difference in numbers is caused by the feed ingredients making up the ration (perilla seed meal, grass, concentrate feed, and tannins) and the nutrient content of each treatment therein.The good quality of the feed given and the value of fermentation in the digestive system of ruminants is shown by the digestibility of dry matter and organic matter in the rumen and post-rumen.The amount of feed nutrients that can be utilized by livestock to meet their nutritional needs increases along with the digestibility value of dry matter.This is in accordance with the opinion [21,22], stating that the digestibility value of feed is influenced by the nutrient composition of the feed, the type of ration, and the quantity of feed provided to livestock.The dry matter digestibility value from a single feed, a mixture of both forage (hay and silage) and concentrate feed ingredients (soybean meal and soybean hulls) is 49.4-75.4% [23].In addition, the content of NDF and ADF in the feed will affect the feed flow rate and the dry matter digestibility in the rumen [24].
The result of the IVOMD is consistent with the IVDMD, this is because of the reason that the dry matter portion of the treatment ratio contains the organic matter component (Table 2).The difference in the values of IVDMD and IVOMD perilla seed meal both as single feed and mixed feed (concentrate, grass, and tannins) is due to the number of tannins, the secondary metabolism of plants in perilla seeds.The greater the secondary metabolite compounds, Organic matter digestibility will be decreased in the rumen and higher in the post-rumen (Table 3).The content of phenolic compounds, flavonoids, and tannins can affect the digestibility value of ration organic matter [25,26].According to [27], differences in the total level of feed mixtures can affect the value of ruminant livestock products.

Conclusion
The conclusion of this study was carried out to give perilla seeds at a level of 10% and 1% tannin in the ration had diverse nutrient digestibility in the rumen and post-rumen.However, it does not affect the pH of the rumen.

Table 1 .
Chemical compotition of feed ingredients

Table 3 .
The result of pH, DM digestibility, and OM digestibility