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In vitro digestibility and ruminal fermentation profile of pangola grass (Digitaria decumbens) supplemented with crude palm oil protected by sodium hydroxide

Wulandari, Budi Prasetyo Widyobroto, Cuk Tri Noviandi and Ali Agus

Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur, Yogyakarta - 55281, Indonesia
c.t.noviandi@ugm.ac.id

Abstract

Oil or fat supplementation in ruminants ration does not have a positive effect due to the biohydrogenation of fat in the rumen. Fat in ruminants ration also may decrease fiber digestibility in the rumen. Protection of fat is expected to reduce those negative effects in the rumen. This in vitro study was conducted to determine the most effective concentration of NaOH in protecting fat in the ruminant ration, so that it did not interfere with the performance of rumen microbes, as evidenced by ruminal fermentation profile. In this study, crude palm oil (CPO) as the main fat supplementation was protected by saponification method by mixing CPO with NaOH and 0.25% CaCl2 in 4:1:1 ratio (v/v). Four treatments of NaOH concentrations were applied to Pangola grass (Digitaria decumbens), thus the treatments were:CTL/control (Pangola grass + CPO without protection),3NAOH (Pangola grass + CPO protected with 3% NaOH),5NAOH (Pangola grass + CPO protected with 5% NaOH), and 10NAOH (Pangola grass + CPO protected with 10% NaOH). All the experimental diets were then incubated for 48 and 96 h according to the 2-stage in vitro technique.

Protecting CPO with NaOH can increase the efficiency of ruminant feed containing high fat and reduce its negative effects on rumen microbes. CPO protection with 5 to 10% of NaOH by saponification method was the most optimum fat protection against rumen degradation. This was proven by the increased IVDMD and IVOMD of Pangola grass, as well as increasing of CMC-ase activity, total and individual VFA concentration, NH3 concentration, and microbial protein concentration.

Keywords: ammonia, fat, saponification


Introduction

Crude palm oil (CPO) is one of the main agricultural products in Indonesia with 35.4 million tonnes production per year (Directorate General of Estate Crops 2016). Crude palm oil contains lots of unsaturated fatty acids, which may have a positive effects on animal performance. However, excessive use of fat (above 5% of total ration) in ruminant diet may cause negative effects on the physical form of the feed and rumen microbes. In the rumen, fat undergoes into two major processes: hydrolysis of ester bonds in fat derived from feed and biohydrogenation of unsaturated fatty acids into saturated after fat hydrolyzed to free fatty acids (Bauman and Lock 2006). Fat that enters the rumen will occur hydrolysis by rumen bacteria such as Anaerovibrio lipolytica and Butyrivibrio fibrisolvens which will release lipase, galactosidase, and phospholipase enzymes (Wina and Susana 2013).

Protection of fats in feed is important to inhibit the biohydrogenation of unsaturated fatty acids. This is based on the process of hydrolysis and hydrogenation of unsaturated fatty acids in the rumen. Protection is needed to prevent polyunsaturated fatty acids (PUFAs) from hydrogenation of double bonds by rumen microbes into saturated fatty acids (Harvatine and Allen 2006; Lourenc et al 2010), so that unsaturated fatty acids arrive at intestine can increase levels of unsaturated long-chain fatty acids of milk (Gulati et al 2005). In addition, it can reduce the negative impact of fatty acid supplementation on high levels (a maximum of 5% dry matter) in the form of decreased fiber degradability (Aharoni et al 2004). Fat also has a function as energy source that has a high density. The value of fat energy is at least twice as large as carbohydrates (NRC 2001).

One possible fat protection method is saponification with calcium salt (CaCl2). The chemical process occurs by reacting fatty material with a solution of NaOH which is known to form the saponification process. NaOH solution helps in the formation of soap when it is reacted with fat. After that it reacts again with CaCl2 in order to obtain calcium soap, which is insoluble in water (Pramono et al 2013). The addition of NaOH concentration will affect the weight of the soap produced. This shows that the more NaOH reactants, the higher the reaction of oil to produce soap will increase the density of soap (Sari et al 2010). In this study, fat protection was carried out using the saponification method with different concentrations of NaOH at 0.25% CaCl2. This research was conducted to obtain optimal NaOH concentrations for CPO protection, so as to improve the digestibility efficiency of feed, especially fiber due to the reduced negative effects of CPO in feed.


Materials and methods

Crude palm oil used in this study was obtained from PT Sari Rosa Asih Feedmill in Yogyakarta, Indonesia. Pangola grass (Digitaria decumbens) was obtained from the experimental field of the Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia. Rumen fluid was derived from 2 Bali bulls with rumen fistula. All proximate analysis followed the AOAC (2005) procedures. Instruments used were digital scales (Shanghai Yamato, Shanghai, China with precision 0.1), analytical scales (Ohaus, New Jersey, USA with precision 0.0001), ovens (Memmert, Schwabach, Germany), a set of in vitro test (Tilley and Terry 1963), and grinder (Thomas Willey Laboratory Mill, Philadelphia, USA).

Ruminal fluid preparation

Rumen fluid used for in vitro digestion and fermentation analysis was derived from two Bali bulls (weighted approximately 223 and 316 kg). Two weeks prior to rumen fluid collection, bulls were adapted with diet consisted of forages and concentrates in 80:20 ratio offered at 07.00 am and 02.00 pm daily with free access to water. The ruminal fluid collection was carried out in the morning before the bulls were fed.

Crude palm oil protection and sample preparation

The fat protection method adopted was the saponification method. The method of fat saponification used a solution of NaOH and calcium salt CaCl 2 (Shelke et al 2012). The ratio of CPO: NaOH: CaCl2 was 4: 1: 1, CaCl2 concentration was 0.25%, and NaOH was at 3, 5, and 10%. Calcium soap was made by heating the oil and mixing it with NaOH, then slowly mixing with 0.25% CaCl2 with stirring, so that the resulting soap was in the cream form. Similar procedure was done for 3, 5, and 10% of NaOH. Protected CPO in the cream form then was added to Pangola grass as much as 10% of the total proportion of in vitro samples. Thus, there were 4 treatments added into Pangola grass:CTL/control (Pangola grass + CPO without protection),3NAOH (Pangola grass + CPO protected with 3% NaOH),5NAOH (Pangola grass + CPO protected with 5% NaOH), and 10NAOH (Pangola grass + CPO protected with 10% NaOH). Prior to mixed with protected CPO, Pangola grass was dried in a 55░C oven, then ground using a Wiley mill with a 2 mm screen.

In vitro digestibility and fermentation parameters analysis

A total of 0.50 g (dry weight) of the sample was put into an 100 mL in vitro tube. Rumen fluid was mixed with artificial saliva (McDougall's solution) in a ratio of 1:4. Before mixing, rumen fluid was filtered with a PeCap screen to remove the filtrate (Noviandi et al 2014). A total of 50 mL of a mixture of rumen fluid and artificial saliva was put into a sample tube, flowed with CO2 gas and sealed with a rubber cork equipped with a gas relief valve. In vitro digestibility analysis was performed using a 2-stage in vitro method (Tilley and Terry 1963), carried out in 6 replications for each dietary treatment. Samples were then analyzed for in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), as well as the remaining ruminal fluid used for measurements of pH, ruminal NH 3 (Chaney and Marbach 1962), microbial protein (Plummer 1987), and volatile fatty acids (Filipek and Dvorak 2009). At the end of the first incubation period, the in vitro tube was opened, and each tube was added with a 6 mL of 20 per cent HCl and 2 mL of 5 per cent pepsin solution. The tubes were then incubated at 39 ░C for 48 h with occasional shaking. Anaerobic conditions were not necessary during this stage. At the end of the incubation, the liquid samples were filtered by pouring on a crucible which is basically coated with glass wool. The crucible and residue of sample were dried at 105 ░C to constant weight. The dry weight of residue was calculated. The parameter observed in second stage is IVDMD and IVOMD (Tilley and Terry 1963).

Statistical analysis

Data obtained were analyzed with one-way analysis of variance using Windows IBM SPSS 25.0 (IBM Corporation, New York, USA), and significance was set at p<0.05. Differences between means were analyzed using Duncan's new multiple range test (Steel et al 1997).


Results and discussion

In vitro digestibility

Table 1 showed that supplementation of CPO protected with NaOH as a saponification media resulted in increase of in vitro dry matter and organic matter digestibilities of Pangola grass, both at the first stage digestion (in the rumen) and at second stage digestion (post-rumen).

Table 1. Pangola grass digestibility as a response to NaOH-protected CPO supplementation

Item

Treatments

SEM

p

CTL

3NAOH

5NAOH

10NAOH

1st stage in vitro digestibility (in the rumen, %):

IVDMD

16.6a

20.4b

26.1c

27.7d

0.94

<0.01

IVOMD

16.0a

18.8b

25.2c

27.6d

0.99

<0.01

2nd stage in vitro digestibility (post-rumen, %):

IVDMD

22.1a

22.1a

26.9b

27.7c

0.56

<0.01

IVOMD

18.7a

18.8a

25.5b

27.7c

0.84

<0.01

CTL = Pangola grass + CPO without protection, 3NAOH = Pangola grass + CPO protected with 3% NaOH, 5NAOH = Pangola grass + CPO protected with 5% NaOH, 10NAOH = Pangola grass + CPO protected with 10% NaOH. IVDMD= in vitro dry matter digestibility, IVOMD= in vitro organic matter digestibility.
a,b,c,d
Means in ame roow without common superscripts differ at p<0.05

In general, both IVDMD and IVOMD in the 2nd stage (post-rumen) showed greater values compared to those in the 1st stage (in the rumen). This greater in vitro digestibilities of post-rumen proved that the NaOH protection on CPO worked well. Additional digestible feed ingredients in post rumen, which is a portion of the calcium soap bond resulting from CPO protection was released due to the addition of HCl after passing abomasum in acidic conditions, so that calcium soap was decomposed in the form of calcium and fatty acids. Bonding calcium soap with fatty acids did not break down easily in rumen liquids that have a neutral pH, but when it passed through the abomasum, the bond was released to produce Ca ions and free fatty acids because the conditions in the abomasum have an acidic pH, then it entered the small intestine and absorbed in the small intestine (Schaefer 2000).

Results in Table 1 showed that protecting CPO with NaOH reduced the negative effects of oil supplementation, which was indicated by an increase in the IVDMD and IVOMD of Pangola grass (p<0.05); those digestibilities also showed an increasing trend as the concentration of NaOH was increased. Optimal rumen conditions caused DM degradation by microbes to be more maximal, so that IVDMD of Pangola grass becomes more optimal. This is in accordance with Schaefer (2000) that the saponification method with calcium salt (CaCl2) aims to reduce the negative impact of the use of fat at a high level on the rumen microbial ecosystem. In their in vivo test, Manso et al (2006) reported that adding 4% calcium fat in sheep diet did not cause any negative effect on fiber digestibility of feed.

Post-rumen digestibility of Pangola grass increased (p<0.05) when CPO was protected with 5 and 10% NaOH. The increasing in vitro digestibility in post-rumen at 5NAOH and 10NAOH (5 and 10% NaOH) implied that NaOH protection in those levels still can be degraded by HCl in post-rumen. Schaefer (2000) reported that digestible feed materials that is a part of the calcium soap bond resulting from CPO protection can be released due to the addition of HCl after passing abomasum in acidic conditions, so that the calcium soap will decompose in the form of calcium and fatty acids. In addition, protein also breaks down into amino acids due to the addition of the pepsin enzyme after passing through the small intestine. Saponification of fat with calcium salt, causing bonding of fatty acids and calcium salt is inert (not easily changed) in the rumen because calcium soap is not soluble in ruminal pH (6.2 to 6.8) but dissolves in abomasum which have a pH of 2 to 3 (Ansar et al 2014).

Ruminal fermentation profile

In general, CPO supplementation with NaOH protection improved rumen fermentation profiles without affecting ruminal pH (Table 2). The ruminal fluid pH in this study ranged from 7.27 to 7.32, which was that were within still in the normal pH range of Bali bulls (7.04 to 7.34; Mudita et al 2016). This can be interpreted that the addition of NaOH-protected CPO had no negative effects on the rumen microbial environment, so rumen microbial performance was not be disrupted.

Table 2. Ruminal fermentation profile of Pangola grass as a response to NaOH-protected CPO supplementation

Item

Treatments

SEM

p

CTL

3NAOH

5NAOH

10NAOH

Ruminal pH

7.27

7.32

7.29

7.31

0.01

0.35

CMC-ase activity (Ámol/g)

4.29a

5.00b

5.99c

6.17c

0.21

<0.01

Total VFA (mM)

18.6a

26.3b

43.0c

73.2d

6.32

<0.01

Acetate (mM)

14.8a

20.3b

35.0c

57.2d

4.94

<0.01

Propionate (mM)

2.92a

4.62b

6.30c

12.6d

1.10

<0.01

Butyrate (mM)

0.87a

1.37b

1.78c

3.42d

0.29

<0.01

A:P ratio

5.06b

4.39a

5.55c

4.55a

0.14

<0.01

NH3 (mg/100 mL)

5.71a

6.62b

7.07c

7.12c

0.15

<0.01

Microbial protein (mg/mL)

3.28a

4.74b

5.51c

5.63c

0.24

<0.01

CTL = Pangola grass + CPO without protection, 3NAOH = Pangola grass + CPO protected with 3% NaOH, 5NAOH = Pangola grass + CPO protected with 5% NaOH, 10NAOH = Pangola grass + CPO protected with 10% NaOH.A:P ratio = acetate:propionate ratio.
a,b,c,d Means in same row without common superscripts differ at p<0.05

Supplementing Pangola grass with Na-OH protected CPO increased (p<0.05) endoglucanase (CMC-ase) rumen fluid activity, with the greatest activity noted in the 5NAOH and 10NAOH (5 and 10% NaOH; Table 2). The increase in endoglucanase activity is consistent with the increase of IVDMD and IVOMD in the rumen (Table 1). Supplementation of NaOH-protected CPO may reduce negative effects on rumen microbes, thus it was not affect cellulolytic bacteria in degrading cellulose, which resulted in increasing endoglucanase enzyme activity. One of the enzymes needed to convert cellulose into glucose is endo-1.4 β-gluconase which can be detected by CMC hydrolysis from cellulolytic index values. Endoglucanase is a component of cellulose that is always found in cellulolytic microorganisms (Sirisena and Manamendra 1995).

The total VFA concentration, individual VFA (acetate, propionate, and butyrate), as well as the A:P ratio in this study showed increasing trend as the NaOH used as protection for CPO was increased (Table 2). This data was in corresponding to the increasing IVDMD and IVOMD in the rumen (Table 1). Supplementation of protected CPO had a significant effect ( p<0.05) on ruminal fermentation profile when compared to unprotected CPO (CTL). This is also in line with the activity of the endoglucanase enzyme, so that it can be interpreted that the provision of protected CPO supplements can reduce the negative effect of oil on rumen microbes, so that cellulolytic bacteria can degrade cellulose to glucose which will then be fermented into VFA. The proportion of VFA in rumen fluid varies depending on the type of feed, forage, and concentrate, and its distribution in the rumen (McDonald et al 2011). Total VFA in this study showed a relatively low value due to the low content of water soluble carbohydrates (WSC), but the presence of CPO protection treatment showed an increase in total VFA compared to control. The WSC content in Pangola grass is quite low at 7.4% (Mullik et al 2009). The main nutrient difference between tropical grasslands and temperate climates is that temperate grasslands are usually higher in protein and soluble sugars and lower in cell wall components and therefore easier to digest (Preston and Leng 1987).

The NH3 concentration of ruminal fluid supplemented with NaOH-protected CPO was greater (p<0.05) than the unprotected CPO one (Table 2). Treatments with 5 and 10% NaOH concentrations (5NAOH and 10NAOH) showed the greatest NH3 concentration (7.07 and 7.12 mg/100 mL; p<0.05) compared to the other treatments. This increasing NH3 concentration are in line with the increasing IVOMD of Pangola grass (Table 1), which imply that there were more protein substrates were available for deamination process to become NH3. The NH3 concentration in this study was still within the normal range of the rumen fermentation process or exceeds the minimum NH 3 concentration needed for optimal rumen microbial growth of 5 mg/100 mL (Satter and Slyter 1974), which means that the addition of NaOH-protected CPO in feed was safe for rumen ecology because NH 3 concentration were still in the normal range for microbial protein synthesis. The low concentration of NH3 in this study is consistent with other studies using pangola grass as tropical grass for steers feed, amounting to 5.82 - 6.07 mg / 100 mL (Mullik et al 2009). The low concentration of NH3 is caused by the low CP content in pangola grass which is around 5 to 12% of dry matter (Tikam et al 2013).

Microbial protein concentration in this study were consistent with changes in NH3 concentration (Table 2). Microbial protein synthesis requires a supply of energy and nitrogen to achieve optimal production. The increase in microbial protein synthesis due to supplementation of NaOH-protected CPO in this study occurred due to the synchronization between energy and protein in ruminal fluid. This synchronization of energy and protein availability was indicated by increasing VFA concentration (Table 2); which indicated that there is an increase in the amount of microbial cell biomass that performs substrate degradation activity, and then resulted in increasing of NH3 concentration. Synthesis of microbial proteins in the rumen has an important role in providing about 50 to 80% of the main protein source for livestock. Factors that influence microbial protein synthesis are the consumption of DM, the ratio of forage to concentrate in the ration, fermented energy supply, nitrogen compound supply, nitrogen and energy synchronization, and rumen environment (Pathak 2008).


Conclusions


Acknowledgments

The authors would like to thank the Directorate-General for Science, Technology and Higher Education Resources, Ministry of Research and Technology/National Agency for Research and Innovation of the Republic of Indonesia for financial support of the research through Pendidikan Magister Menuju Doktor untuk Sarjana Unggul (PMDSU) scholarship.


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Received 11 March 2020; Accepted 12 May 2020; Published 1 July 2020

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