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In sacco degradation characteristics of protected soybean meal with different levels of formaldehyde in Bali cattle

Wulandari, Budi Prasetyo Widyobroto1, Cuk Tri Noviandi1 and Ali Agus1

Research Center for Animal Husbandry, National Research and Innovation Agency, Cibinong Science Center, Jl. Raya Jakarta – Bogor, Cibinong, Kabupaten Bogor, Jawa Barat - 16915, Indonesia
1 Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur, Yogyakarta – 55281, Indonesia


This study was conducted to determine the effect of the concentration of formaldehyde on the manufacture of undegraded protein (UDP) used for feed supplementation on the kinetics of dry matter (DM), organic matter (OM), and crude protein (CP) degradation with in sacco method in Bali cattle. The by-product of agricultural processing is used as a protein source feed material, namely soybean meal. The concentration of formaldehyde used is 0; 0.6; 0.8; 1.0; and 1.2% (volume/weight) to protect soybean meal in the manufacture of UDP. Protected soybean meal was tested for digestibility using in sacco technique in rumen fistulated Bali cattle. 5.0 g of the sample was put into a nylon bag and then incubated in the rumen for 0, 2, 4, 8, 16, 24, and 48 hours, then the feed residue was analyzed for DM, OM, and CP. This study used two cows with rumen fistulated. The research data were analyzed with one-way analysis of variance, the difference between the means were analyzed using Duncan's Multiple Range Test. The results showed that soybean meal protected with formaldehyde could reduce the degradation parameters of DM, OM, and CP (p<0.01). The use of 1.0% formaldehyde level is the optimum level as a soybean meal protection agent, but the use of 0.8% formaldehyde can be used as an efficiency consideration.

Key words: undegraded protein, formaldehyde, in sacco, Bali cattle


Feed is one of the factors that determine the high and low productivity of ruminants. High quality feed is closely related to the protein contained in the feed, so it is necessary to choose feed ingredients that contain quality protein to obtain maximum livestock productivity. However, protein feed given to ruminants will change the structure if it is in the rumen. Feed protein will be degraded into amino acids and deaminated into ammonia by microbes in the rumen, while feed protein that is not degraded will go to the intestine and enzymatic digestion occurs. Rumen microbes can convert low-quality protein into high-quality protein, but they can also convert high-quality feed protein into ammonia. The breakdown of protein in the rumen causes energy loss during the fermentation process in the form of gas (CO2 and CH4) so that it will reduce the biological value of protein (Cheeke 2005)

Undegraded protein is protein feed that is not degraded in the rumen which is given to ruminants to meet protein needs because it can reach the intestines and can be utilized directly by the host animal (Suhartanto et al 2014). Protein protection can reduce degradation in the rumen so that the use of feed will be more efficient and reduce feed costs. Protein protection can be done in several ways, including heating, protection with tannins, and protection with chemicals, one of which is formaldehyde.

Materials and methods

The soybean meals used in this study were obtained from PT Sari Rosa Asih Feedmill 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 nylon bag, formaldehyde, 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

Sample of soybean meal 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 analyzed 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

Protected soybean meal was carried out through a process of coating the soybean meal. Four levels of formaldehyde (0, 0.6, 0.8, 1.0, and 1.2%; volume/weight) were sprayed on soybean meal. After through mixing the material was kept overnight (about 12 h), then dried 2 to 3 days (11-!5% moisture content).

In sacco degradation procedure

Ruminal degradation measurements using the nylon bag technique were carried out in 2 Bali cattle after one week adaptation period. The body weight of Bali cattle approximately 223 to 316 kg live weight, and fitted with permanent rumen canula. The cattle was fed twice a day, at 7 am and 2 pm with maintenance diet of forage: concentrates (80:20) and free access to water. Evaluation of feed ingredients in sacco using the method developed by Orskov et al (1980), Widyobroto et al (1995) and Soejono et al (1998). The in sacco method used a nylon/polyester bag with a porosity of 45 m measuring 611 cm (Shabi et al 1998), with all three sides clamped with one side still open, then the center of the bag was labeled/marked according to the treatment, incubation time and replication. The bags were then dried in an oven at 55 C for 6 hours and their empty weight was weighed. Five grams of the sample was put into a nylon bag, then folded and pressed on the fourth side. A nylon bag that has been filled with feed samples is attached with a rope to a ring made of iron coated with chrome, then the ring is tied with a plastic rope measuring 40 – 60 cm. Samples were incubated in rumen fistula cattle with different time intervals, namely: 0, 2, 4, 8, 16, 24 and 48 h. Each incubation point was repeated six times. Each sample at the incubation point is tied with a different color of rope to facilitate withdrawal at a certain point. Furthermore, nylon bag samples taken from the rumen according to the incubation time were stored in a freezer at a temperature of -15C or immediately washed in a washing machine for 6 minutes using running water. Washing is carried out to remove feed particles or microbes adhering to the nylon fabric and also rumen microbes bound to feed particles that are still left in the bag. The bag was dried in an oven at 55 C for 72 h until the weight was constant and then the residue was weighed. DM, OM, and CP were analyzed, then disappearance values were calculated as the difference between weight of nutrients before and after incubation of each sample. The degradability data obtained for DM, OM, and CP for each feed were fitted to the equation P = a+b(1-e -ct), where p = degradation potential, t = incubation time, a = rapidly soluble fraction, b = potentially degradable fraction, c = rate of degradation of b fraction (Orskov 1980).

Statistical analysis

Results were analyzed as a one way completely randomized design by 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

Chemical composition of soybean meal

Table 1 showed that soybean meal has nutrient characteristics with high protein content. Therefore, soybean meal deserves to be used as a protein source feed ingredient.

Table 1. Chemical composition of soybean meal

Chemical composition


Dry matter (DM)


Crude protein (CP


Ether extract (EE)


Crude fiber (CF)




Total digestible nutrients (TDN)


Soybean meal was a by-product of soybean oil extraction. The amount of crude protein allowed the soybean meal was classified in feed ingredients of protein source or fifth-grade feed ingredients. Soybean meal contains crude protein up to 49 until 54% with dry matter reaching 88% (Agus 2008). Soybean meal was one of the high quality protein concentrate, but in most ruminant around 80-90% protein was degraded in the rumen (Widyobroto et al 1995). Therefore, it is important to protect soybean meal to reduce digestibility in the rumen. Promkot and Wanapat (2003) found that soybean meal and Leucaena leaf meal were highly degraded in the rumen, while cassava hay, cotton seed meal, and dried brewer gain were less degraded.

Degradability characteristics

In sacco is one way to evaluate the quality of feed biologically using nylon bags that are inserted into the rumen of fistulous cattle. The advantage of testing using the in sacco method is that a perfect physiological process still occurs. In this experiment, the parameters observed were the degradation rate of DM, OM, and CP. Dry matter degradation rate is presented in Figure 1.

Figure 1. Dry matter degradation rate of soybean meal

The results, which are presented in Figure 1, show that for each protected soybean meal with different levels of formaldehyde, there will be an increase in DM loss with increasing incubation time, but the level of degradation of each treatment showed a decreasing trend when compared to DM degradation of unprotected soybean meal (control). Concentration of 0.6% formaldehyde showed a fast DM degradation rate compared to other levels, while 1.2% formaldehyde concentration showed the slowest DM degradation rate. Organic matter degradation rate is presented in Figure 2.

Figure 2. Organic matter degradation rate of soybean meal

The rate of OM degradation in this study is presented in Figure 2 which shows that for each protected soybean meal with different concentrations of formaldehyde, there will be an increase in OM loss with increasing incubation time, this is in accordance with the results shown by DM loss (Figure 1). Concentration of 0.6% formaldehyde showed a fast OM degradation rate compared to other concentrations, while 1.2% formaldehyde concentration showed the slowest OM degradation rate and had almost the same value as OM degradation at 0.8 and 1.0% concentrations of formaldehyde. Crude protein degradation rate is presented in Figure 3.

Figure 3. Crude protein degradation rate of soybean meal

The CP degradation rate presented in Figure 3 shows that each protective treatment of soybean meal with different concentrations of formaldehyde showed a lower CP degradation trend when compared to the control. Concentration of 0.6% formaldehyde showed a faster rate of degradation than other concentrations, while concentrations of 0.8, 1.0, and 1.2% showed almost the same degradation rate of CP. This is in sync with the DM degradation rate in Figure 1. Based on the three degradation rate images (Figures 1 to 3), it shows that the protection of soybean meal with different concentrations of formaldehyde has successfully protected feed ingredients thereby reducing the degradation of DM, OM, and CP in the rumen.

The percentage loss of DM and CP of soybean meal protected with formaldehyde concentration in this study was smaller than the percentage loss of DM and CP in soybean meal that was not protected based on the research of Todorov et al (2016). The percentage loss of unprotected soybean meal DM at 8 hours of incubation reached more than 60% and increased to 98% at 48 hours of incubation. The percentage of CP loss at 16 hours of incubation reached more than 70% and increased to 91% at 24 hours of incubation (Todorov et al 2016). At 4 hours of incubation onwards, the DM loss of unprotected cake was greater than that of protected soybean meal, this indicated the successful bond formed between the aldehyde and protein components in soybean meal so as to protect the protein contained in soybean meal from rumen microbial degradation.

The rapidly soluble fraction (a fraction), potentially degradable fraction (b fraction), rate of degradation of b fraction (c) and potential degradation (a+b) are presented in Table 2.

Table 2. In sacco degradation characteristics and effective degradability of soybean meal protected and unprotected


Level of formaldehyde (%)








DM degradation

a (%)








b (%)








c (%/h)








a+b (%)








EDDM (%)








OM degradation

a (%)








b (%)








c (%/h)








a+b (%)








EDOM (%)








CP degradation

a (%)








b (%)








c (%/h)








a+b (%)








EDCP (%)








a,b,c,d Different superscripts at the same row showed significant effects (p<0.05) DM = dry matter, OM = organic matter, CP = crude protein, EDDM = effective degradability of dry matter, EDOM = effective degradability of organic matter, EDCP = effective degradability of crude protein, a = the rapidly souluble fraction, b = the potentially degradable fraction, c = the rate of degradation of fraction b, a+b = potential degradation

Based on the results of the study, it was shown that the different concentrations of formaldehyde given were able to affect the changes in the degradation of fraction a in the degradation of DM, OM, and CP ( p<0.01). The higher the level of formaldehyde administration, the lower the value of fraction a, fraction b, and c. It can be assumed that the potential degraded soybean meal protein fraction in the rumen is bound to formaldehyde solution which will be released after the rumen. The value of fraction b in the degradation of unprotected soybean meal of DM degradation was 72.85%, while the value of c was 0.026 (Todorov et al 2016). This indicates that the formaldehyde protection treatment against soybean meal will reduce the rate of feed degradation in the rumen.

The basic principle of treating protein with formaldehyde (H2CO) is to form chemical bonds with proteins that are stable at neutral pH such as rumen pH, but become unstable at acidic pH as at abomasum pH. Protein protection by using H2CO causes the formation of methylene bonds with proteins that envelop the outer layer of the protein matrix so that proteins bound to H2CO are not easily soluble in water (Anggraeny and Krishna 2005). The European Food Safety Authority stated that formaldehyde in animal feed at 68-680 ppm was rapidly absorbed in the gastrointestinal tract and joined to formaldehyde pool in the body. The formaldehyde was rapidly oxidized to formic acid and then CO2 and water (Adiveter 2014).



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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