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In vitro digestibility and ruminal fermentation profile of ruminant diet in response to substitution of mixture feedstuff protected

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

Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur,Yogyakarta - 55281, Indonesia


Excess degradation of high protein feed in the rumen can reduce the efficiency of post-rumen nutrient utilization. The protected feed is expected to increase the protein absorption in the intestine. Meanwhile, supplementation of oil or fat in the ration has a negative effect due to biohydrogenation of fat in the rumen. Protection of fat is expected to reduce these negative effects in the rumen. This in vitro study was conducted to determine the rumen fermentation parameters of a mixture of soybean meal, saponified crude palm oil (CPO) and Agromix® mineral premix which was protected with formaldehyde. The crude palm oil used is protected by the saponification method (saponification) and mixed with soybean meal and Agromix® mineral premix. The ratio of saponified CPO with soybean meal is 1: 4 with the addition of Agromix® mineral premix 0.3% of the total production of protective feed. The mixture of the three ingredients is then protected with formaldehyde (formalin 0.8%). Independent sample T-test was used to compare 2 treatments (CTL/ control = commercial rations; 7.6PF = commercial rations with 7.6% protected feed substitution). All treatments were incubated for 48 and 96 hours according to the 2-stage in vitro technique.

Protected feed substitution can increase the efficiency of ruminant feed that contains high protein and reduce the negative effect of fat on rumen microbes. This is evidenced by the increase in IVDMD, IVOMD, and IVCPD of ration in post-rumen.

key words: substitution, protection, in vitro


High quality protein provides amino acids to support ruminant productivity. On the other hand, excessive degradation of feed in the rumen, especially high protein feed can reduce the efficiency of nutrient utilization in the post-rumen gastrointestinal tract so that feeding protein sources must consider its fermentation ability and resistance to rumen degradation. The level of feed protein degradation in the rumen is known to be different. One of the high protein feeds with high nutritional value is soybean meal, but has a rumen degradation rate of 71-79% (Stern et al 2006). Feed protein protection is important to inhibit protein degradation in the rumen and to increase the amount of protein digested in the intestine, which is often considered as “Rumen Undegradable Protein” (Boucher et al 2009). The protein protection method using formaldehyde (HCHO) is known to protect feed protein from rumen microbial degradation (Varvikko et al 1983).

The decrease in feed efficiency also occurs in the degradation of fats, especially unsaturated fatty acids because there will be hydrolysis and biohydrogenation processes in the rumen. In addition, the provision of unsaturated fatty acids has a negative effect on the degradation of feed fiber. Crude palm oil (CPO) is one of Indonesia's local feeds that contain unsaturated fatty acids and abundant production (Directorate General of Estate Crops 2016). The components of free fatty acids in CPO are palmitate (40-45%), and oleic (39-45%) (Setyopramono 2012). Feed protection is one way to reduce negative effects and increase feed efficiency for ruminants. The saponification method with sodium hydroxide (NaOH) and calcium salt (CaCl2) is able to protect feed fat from rumen microbial degradation (Pramono et al 2013). CPO protection with 5% NaOH using the saponification method is the optimal protection of fat against rumen degradation (Wulandari et al 2020). This research was conducted to determine digestibility and rumen fermentation parameters of dairy cattle rations with in vitro protected feed substitution.

Materials and methods

The soybean meals, CPO, rice bran, copra meal, wheat pollard, and cassava pulp used in this study were obtained from PT Sari Rosa Asih Feedmill located in Yogyakarta, Indonesia. King grass was obtained from the experimental field of the Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia. Premix mineral Agromix® was obtained from CV Agromix Lestari located in Yogyakarta, Indonesia. Rumen fluid was derived from 2 Bali bulls with rumen fistula. All proximate analysis followed the AOAC (2005) procedures. Instruments used were a set of in vitro test (Tilley and Terry 1963), analytical scales (Ohaus, New Jersey, USA with precision 0.0001), grinder (Thomas Willey Laboratory Mill, Philadelphia, USA), ovens (Memmert, Schwabach, Germany), and digital scales (Shanghai Yamato, Shanghai, China with precision 0.1).

Sample preparation and chemical analysis

All feed material samples were dried in an oven at 55 °C until the weight was constant and ground using a Willey mill sieved through 1 mm screen. The sample was analysed for chemicals composition (dry matter (DM), ash, crude protein (CP), crude fat (Extract Ether, EE), and crude fiber (CF) by AOAC method (AOAC 2005).

Feed protected preparation and total mixed ration

The saponification method of CPO used NaOH and CaCl2 (Shelke et al 2012). The ratio of CPO : NaOH : CaCl2 was 4:1:1 (Hartati 2014). The concentration of NaOH used was 5%, while CaCl2 concentration was 0.25% (Wulandari et al 2020). A cream is a form of soap produced from the saponification process. Cream was added to soybean meal and Agromix® then mixed until homogeneous. The ratio between SBM and CPO is 4 : 1, while Agromix® was added as much as 0.3%. The mixture of the three feed ingredients was protected by spraying 0.8% formaldehyde (Wulandari et al 2017). Thus, there were 2 treatments added into commercial ration: CTL/ control = commercial ration, and 7.6PF = commercial ration + 7.6% protected feed substitution. Total mixed ration ingredients and chemical composition is presented in Table 1.

Ruminal fluid preparation

The rumen fluid used for in vitro digestibility and fermentation analysis was derived from two male Bali cattle (approximately 243 to 327 kg). The cattles were adapted with diet carried out a week before the research. The adaptation diet consist of forages and concentrates (80:20) and given free access to water. The diet adaptation given twice a day at 07.00 am and 02.00 pm. The ruminal fluid collection was carried out in the morning before the cattles were fed.

In vitro digestibility and ruminal fermentation parameters

In vitro digestibility analysis was measured using a 2-stage in vitro method (Tilley and Terry 1963), carried out in 6 replications for each dietary treatment. The sample (0.50 g dry weight) was put into an 100 mL in vitro tube. Artificial saliva (McDougall’s solution) was mixed with rumen fluid in a ratio of 4:1. Rumen fluid was filtered with a PeCap screen to remove the filtrate before mixing (Noviandi et al 2014). A mixture of artificial saliva and rumen fluid (50 mL) was put into a sample tube, flowed with CO2 gas and sealed with a rubber cork equipped with a gas relief valve. After 48 h of incubation, the sample substrate was filtered apart with rumen fluid. The substrate used for analysis of in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), in vitro crude protein digestibility (IVCPD), as well as the remaining ruminal fluid used for measurements of pH, volatile fatty acids (Filipek and Dvorak 2009), ruminal NH3 (Chaney and Marbach 1962), microbial protein (Plummer 1987), and carboxy methyl cellulose enzyme activity by chromatography gas. At the end of the first incubation stage (48 h), 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, IVOMD, and IVCPD (Tilley and Terry 1963).

Table 1. Total mixed ration ingredients and chemical composition




Ingredients (% of DM)

King grass



Rice bran



Copra meal



Wheat pollard



Cassava pulp



Soybean meal









Feed protected



Nutrient composition (%)
















Nitrogen-free extract (NFE)



Total digestible nutrients (TDN)



Statistical analysis

Data obtained were analyzed using T-test of Windows IBM SPSS 25.0 (IBM Corporation, New York, USA), and significance was set at p<0.05 (Steel et al 1997).

Results and discussion

In vitro digestibility

Table 2 showed that substitution of protected feed in the ration resulted in decreased digestibility of dry matter, organic matter, and crude protein ration in the first stage (in the rumen), but experienced an increase in the second stage digestion (post-rumen).

Table 2. Digestibility of ruminant ration in response to protected feed substitution






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
















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
















CTL = commercial ration, 7.6PF = commercial ration with 7.6% protected feed substitution.
IVDMD = in vitro dry matter digestibility, IVOMD=in vitroorganic matter digestibility, IVCPD = in vitro crude protein digestibility.
Different superscripts at the same row showed significant effects (p<0.05).

In general, both IVDMD, IVOMD, and IVCPD in rations with protected feed substitution (7.6PF) showed a significant decrease (p<0.05) when compared to rations without protected feed substitution (CTL). The opposite occurs in post-rumen digestion (second stage), that IVDMD, IVOMD, and IVCPD in the 7.6PF ration showed an increase compared to the CTL ration. This can be interpreted that some feed nutrients in the ration are successfully protected from rumen microbial degradation, and can be digested and absorbed in the post-rumen so that it can be of direct benefit to livestock.

Decreased digestibility of 7.6PF nutrients in the rumen is caused by the substitution of protected feed. The protection method used was saponification for the protection of fatty acids from CPO, and formaldehyde for protein protection from SBM. Saponification of CPO with calcium salts causes the bonding of fatty acids with calcium salts which are considered inert (not easily changed) in the rumen because calcium soaps are not soluble in rumen pH but dissolve in abomasum which have a pH of 2-3 (Bayourthe et al 1994). This is consistent with the results of the study, that the digestibility of post-rumen 7.6PF has increased compared to CTL. The results of the formation of fat saponification are calcium soap deposits that can be used as supplements or mixed in concentrated ration components (Ansar et al 2014).

The success of the protection method is also evidenced by a decrease in the crude protein digestibility of 7.6PF in the rumen, and increase in the post-rumen. Formaldehyde can form bonds with proteins that are stable at rumen pH and become unstable at acidic pH (post-rumen) so that the bonds are released and protein can be digested in the intestine (Kamalak et al 2005). Protein protection is intended to increase the amount of undigested feed protein in the back (intestine) digestive tract (Boucher et al 2009). Amino acids digested in bovine intestines are derived from microbial proteins synthesized in the rumen and feed proteins that are not degraded in the rumen so that substituted feed is expected to increase the intake of amino acids for ruminants and increase feed efficiency.

Ruminal fermentation profile

In general, substitution of protected feed in 7.6PF ration decreases the rumen fermentation profile without affecting rumen pH (Table 3). The pH of the rumen fluid in this study was 7.27 (both CTL and 7.6PF). The pH was still within the normal pH range of Bali cattle, which is 7.04 - 7.34 (Mudita et al 2016). This can be interpreted that the substitution of protected feed in the ration did not have a negative effect on the rumen microbial environment, so rumen microbial performance was not be disrupted.

Table 3. Ruminal fermentation profile of ruminant ration in response to protected feed substitution






Ruminal pH





NH3 (mg/100 mL)*





Microbial protein (mg/mL)*





CMC-ase activity (µmol/g)*





Total VFA (mM)





Acetate (mM)





Propionate (mM)





Butyrate (mM)





A:P ratio





CTL = commercial ration, 7.6PF = commercial ration with 7.6% protected feed substitution. A:P ratio = acetate:propionate ratio.
a,bDifferent superscripts at the same row showed significant effects (p<0.05)

The NH3 concentration of rumen fluids substituted with protected feed was lower (p<0.05) compared to rations that were not substituted with protected feed. This decrease in NH3 concentrations is in line with the decrease in IVCPD (Table 2), which implies that there is successful protection of the protein substrate in the protected feed, so that the NH3 concentration decreases, where ammonia is formed from the true proteins in the feed (Owens and Basalan 2016). The NH3 concentration in this study was still within the normal range of the rumen fermentation process required for rumen microbial growth, namely 10.21 to 35.76 mg / 100 mL (McDonald et al 2011). This means that protected feeding as a substitute for feed in the ration is still safe for rumen ecology because the NH3 concentration is still within the normal range for microbial protein synthesis.

The decrease in microbial protein concentration at 7.6PF was also in line with the decrease in ammonia concentration, because microbial protein production is largely determined by the availability of N-NH3 in the rumen. The rumen microbial population increases when the availability of nutrients in the form of nitrogen and carbon supplies meets the microbial needs so that it will increase microbial protein synthesis. The amount of carbohydrate degradation with protein that is synergistic and in accordance with rumen ecology can increase the efficiency of microbial protein synthesis. The formation of rumen microbial protein is strongly influenced by the availability of NH3, readily available carbohydrate (RAC), and availability of sulfur. In addition, the consumption of dry matter which contains organic matter is also important to support optimal microbial protein synthesis in the rumen (Khampa and Wanapat 2007).

The decrease in endoglucanase activity (p<0.05) at 7.6PF was in line with the decrease in IVDMD. Endoglucanase activity interprets the activity of cellulolytic bacteria, so that when IVDMD decreases, it will also decrease endoglucanase activity. Cellulolytic activity is highly dependent on the type and amount of substrate in the form of fibers. If the fiber content of the feed is high, the level of cellulase enzyme activity will increase, and conversely. The high cellulolytic activity is caused by the ability of microbes to digest cellulose quickly (Qori'ah et al 2016).

The results showed that the presence of protected feed substitution in the ration gave a significant difference (p<0.05) to the total concentration of VFA and partial VFA of rumen fluid (Table 3). These data showed a decreasing trend, this is due to the substitution of protected feed in the ration and consistent with the endoglucanase activity results so that rumen microbial activity decreases in degradation of feed associated with VFA production. The amount of VFA formed is influenced by digestibility and the quality of the fermented ration. Volatile fatty acids are formed from the degradation process of crude fiber (CF) by microorganisms, so that the CF content in the ration will greatly affect the VFA formed (Hapsari et al 2018). This is consistent with the statement of McDonald et al (2011) VFA concentrations in rumen fluid varied, depending on the type of ration given. The resulting VFA concentration can be used as an energy source and a carbon framework for microbial protein formation.



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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