Supplementation of Molasses and Branched-Chain Amino Acids to Increase In Vitro Digestibility of Ammoniated Corn Cob in Ruminants Feed

Puastuti W, Yulistiani D, Handiwirawan E. 2017. Supplementation of molasses and branched-chain amino acid to increase in vitro digestibility of ammoniated corn cob in ruminants feed. JITV 22(4): 179-187. DOI: http://dx.doi.org/10.14334/jitv.v22i4.1664 Corn cob contains high fiber and lignin which causes low nutritive value. The objective of the study was to improve the digestibility of ammoniated corn cob (CC) by supplementation of molasses and branched-chain amino acid (BCAA: valine, leucine and isoleucine). CC was processed by addition 3% urea. The first stage of in vitro test was done with 4 levels of molases 0, 5, 10 and 15% of dry matter (DM) of CC. The experiment was carried out using complete randomized design with 4 treatments and 4 replications. The second stage was also in vitro study of supplementation of two levels each for valine = V, leucine = L and isoleucine = I namely 0.1 and 0.2% of DM of ammoniated CC. There were 8 treatments combination of the BCAA as follow: A = V0.1 L0.1 I0.1; E = V0.2 L0.1 I0.1; B = V0.1 L0.1 I0.2; F = V0.2 L0.1 I0.2; C = V0.1 L0.2 I0.1; G = V0.2 L0.2 I0.1; D = V0.1 L0.2 I0.2; H = V0.2 L0.2 I0.2 and 1 control (V0.0 L0.0 I0.0). The experiments were done using completely randomized design with 9 treatments and 3 replications. The results showed that treated CC with urea was able to increase protein content by 78% (increased from 3.34% to 5.95%) while neutral detergent fiber (NDF) decreased by 15.4%, acid detergent fiber (ADF) by 7.9% and lignin 16.7%. Addition of molasses in ammoniated CC increased DM digestibility (P <0.05) by 7.5% (41.9 vs 43.51-46.26%) and NDF by 17.7% (38.41 vs 43.76 – 46.31%). Supplementation of BCAA resulted in the highest DM, OM and NDF digestibility (P <0.05) in the treatment of A, C, D and G. Compare to treament I, the digestibility of DM, OM and NDF in G treatment increased by 31.4%, 27.5% and 36.5%, respectively and produced the highest total population of rumen bacterial of 12.4 x 10 colonies /ml. It can be concluded that the digestibility of ammoniated CC increased by the supplementation of 5% molases and BCAA combination consisted of 0.2% valine, 0.2% leucine and 0.1% isoleucine.


INTRODUCTION
National production of corn grain in 2017 was 27.957 million tons (Pusdatin 2017).The potential of corn cob availability calculated based on ratio of grain, straw, husk and corn cob, was 55.0, 20.3 12.3 and 12.3%, respectively (Yulistiani et al. 2012).Corn cob production in 2017 was 6.251 million tons.The utilization of corn cob as feed was still limited.Some of dry corn cob was used as fuel, while most leaves were piled up and rotten in the field.Corn cob had low nutrient quality due to low protein (1.5-3.5%),high fiber (35-45%) high lignin (5.2%), and high cellulose (30%) contents (Yulistiani et al. 2012;Hastuti et al. 2011).Lignin in corn cob caused low nutrient availability and low dry matter digestibility (45.5%) and organic matter digestibility (42.5%) (Yulistiani et al. 2012).Lower digestibility (29.34%) was also reported by Setyadi et al (2013).
To increase the nutrient quality of fibrous feed, many processing methods have been done such as physical, chemical and biological or combination of these methods.Those treatments were reported able to increase the utilization and digestibility of agricultural by-products feedstuff (Van Soest 2006;Zain et al. 2010;Nurhaita 2010).
Physical treatment by grinding resulted in easier feed handling and for consumption, however it did not significantly increase feed digestibility.Increasing quality of roughage was reported by using chemical treatment of sodium hyadroxide (Adeniji 2010) or urea (Oji et al. 2007;Dean et al. 2008;Yalchi et al. 2009;Puastuti et al. 2010;Yulistiani et al. 2012, Ubwa et al. 2014).Urea treatment on rice straw was able to increase nutrient content increasing animal growth through improving rice straw palatability (Huyen et al. 2012), increased nutrient digestibility in dairy cattle (Wanapat et al. 2009) and increased NDF and ADF digestibility in goat and bull (Gunun et al. 2013).The increased of nutrient content and digestibility due to urea treatment was also reported in Triticale straw (Yalchi et al. 2009), cacao pod (Puastuti et al. 2010;Laconi & Jayanegara 2015), corn cob (Yulistiani et al. 2012), oil palm frond (Febrina 2012) and rice husk (Ubwa et al. (2014).The mechanism of urea treatment in improving nutrient content of low quality feed is through hemicellulose solubilization, swollen of plant cell wall and disruption of lignin and cellulose linkage (Yalchi et al. 2009).Consequently the digestibility of cellulose and hemicellulose was increased.This increased digestibility was not followed by the loss of lignin (Bata 2008).Differed to biological treatment, urea treatment in low quality feed is simple and applicative for small holder farmers.
The nutritive value of fibrous feed beside affected by processing method also affected by enzymatic digestion produced by rumen microbes.Various rumen microbes have important role in digesting feed in the rumen.Beside that rumen microbes also be used as protein source in ruminant.Rumen microbes at least supplied two third or 70-80% of amino acid required by ruminants (Chumpawadee et al. 2006;Pathak 2008).The higher rumen microbes population particularly cellulolytic bacteria produced more enzyme consequently increased fiber digestibility.Feeding low quality feeds with low protein and high fiber contents would limit its nutrient availability for microbial.(Puastuti 2009;Nurhaita & Ningrat 2011).
Fermentable energy source (sugar, starch) was needed for the growth of rumen microbials.Molases was fermentable source of carbohydrate and could be used as energy source for rumen microbes (Bata 2008).Amino acid was main nitrogen source for microbial synthesis in the rumen.Although cellulolytic rumen microbial was able to use ammonia, but amino acid and protein had better advantage compared to urea for microbial synthesis (McAllen & Smith 1983;Clark et al. 1992).High degradable protein will be fast degraded by rumen microbes in the rumen into amino acid and partly will be further degraded and produced ammonia.This ammonia is used as nitrogen source and as carbon skeleton source for rumen microbial synthesis.Therefore amino acid composition of feed protein is important for ruminant animals.Methionin, lysine, fenylalanin and threonine were amino acid needed by ruminants (Scholljegerdes et al. 2005).Other amino acid also needed by ruminants were valin, isoleusin, dan leusin (Volden 1999;Zain 2007).These three amino acids were known as branched chain amino acid (BCAA) in the rumen which would be decarboxylated and oxidative deaminated into branched-chain volatile fatty acid (BCVFA) which had important role in the activity of cellulolytic bacteria (Tedeschi et al. 2015).BCVFA could increase dry matter digestibility, increase rumen microbial growth and increase microbial function and enzyme activity in the rumen of sheep (Moharrery 2004).Considering results from the previous studies, this study was aimed to increase ammoniated corn cob digestibility through supplementation of molasses and combination of branch-chain amino acid valin, leusin, and isoleusin.

MATERIALS AND METHODS
Corn cob was obtained from Majalengka District, West Java Province.The cob was ground, then processed using ammoniation by urea addition at 3% of cob dry matter (DM) (Yulistiani et al. 2012).Corn cob and urea mixture was then kept anaerobically condition for 21 days.Ammoniated corn cob then dried and ground for chemical analysis and evaluated for its in vitro digestibility.First in vitro digestibility study was conducted to evaluate the requirement of energy source on the utilization of ammoniated corn cob as fiber source.Molasses supplementation as energy source was allocated in 4 levels namely 0, 5, 10, dan 15% of corn cob DM.The study was conducted through randomized complete design with four replications.Substance samples were incubated for 72 hour, which was previously prepared according to the method of Menke and Steingass (1988) and digestibility was measured according to the methods of Blummel et al (1997).Rumen fluid for in vitro study was taken from ram fed on fresh chopped elephant grass, supplemented with concentrate feed containing crude protein 16%.Parameter recorded were digentibility of DM, organic matter (OM) and neutral detergent fiber (NDF).The best molasses level in this in vitro digestibility was used in the second experiment in which the ammoniated corn cob and molasses mixture was supplemented by branch-chain amino acid (BCAA).
The second in vitro evaluation was carried out to study the effect of BCAA supplementation.BCAA supplemented were valin (V), leusin (L) and isoleusin (I) (L-Valine product by Himedia Laboratory Pvt., Ltd., and L-Leucine by Appli Chem).Levels of BCAA supplementation were 0.1 dan 0.2% of DM ammoniated CC.There were 8 combinations of the BCAA of treatment, which were: 2) and I (V0.0 L0.0 I0.0).The study was conducted in randomized complete design with 9 treatments and 3 replications.The method used in vitro evaluation was similar to the first experiment.Parameter recorded were digestibility of DM, OM, and NDF, and number of population bacteria and protozoa.
DM, OM, crude protein (CP) and gross energy (GE) content of the samples were analysed using method of AOAC (1990).Whereas NDF, acid detergent fiber (ADF) and lignin were analyzed according to the methods of Van Soest et al. (1991).Bacterial and protozoal population were determined using total plate count method (Ogimoto & Imai 1981).Data obtained were analysed using analysis of variance.The difference between treatment mean were analysed using Duncan multiple range test.Statistical analysis was done using soft ware of SAS 9.0 (SAS 2002).

Ammoniated corn cob
Physical condition of ammoniated corn cob was changed, after treatment and its texture was softer and had brownish color.The chemical analysis of the cob showed that ammonization increased the CP content by 78% (from 3.34 in untreated into 5.95% in urea treated corn cob).Chemical composition of untreated and urea treated corn cob are presented in Table 1.The increased of CP content in ammoniated corn cob was due to nitrogen fixation from urea catabolism by urease bacterial (Dean et al. 2008).The increase of CP content in this study was in agreement with the study reported by Oji et al. (2007).They reported that processing of corn by-products with 3% urea was able to increased CP content by 77%.Yadete (2014) reported that processing of wheat straw using 4% urea was able to increased CP content by 87.5% (from 3.2% into 6.0%).
The increase of CP content in rice husk due to urea treatment was also reported by Ubwa et al. (2014).Yulistiani et al. (2012) reported higher increased of CP content in corn cob treated with urea 3%, increasing the CP content up to 284.5%.While Ramirez et al. (2007) reported the CP content was increased by 125% and 270% in maize by-products with urea treatment levels of 4.5 and 6.5% respectively.The different in increase of CP content due to urea treatment in corn cob might be caused by the different level of urea used for treatment.Optimum level of urea in the process of ammonization was reported at the level of 3-5% (Gunun et al. 2013;Khejornsart & Wanapat 2010).Urea levels less than 3% only had function as preservative and it would loss as ammonia when higher than 5% (Khejornsart & Wanapat 2010).The length of storage (21 days) might vary the difference in CP content as the urea added was fractionating into ammonia and some evaporate during drying, so that the amount of N calculated as CP would be lower.
NDF content of ammoniated corn cob decreased by 15.5%, while ADF by 7.9% and lignin by 16.7%, whereas GE content decreased only by 2.5%.The results of the present study was in agreement with Nurfeta et al. (2008) who reported that urea  Result from Duncan analysis showed that molassess supplementation up to 5% was not able to increased DM and OM digestibility.The digestibility was increased (P<0.05) at 10 and 15% molasses supplementation compare to control (without molasses supplementation).The NDF digestibility was increased (P<0.05) at 5% molasses supplementation compared to control, but this was not significantly different from 10 and 15% supplementation.DM digestibility of 41.9% of ammoniated CC in this study was similar to the results reported by Prastyawan et al. (2012).Who reported that fermented ammoniated corn cob without starter microbes for 4 weeks incubation resulted in DM digestibility of 41.3%.The value was higher than reported by Setyadi et al. (2013) which was 29.34%.
Compared to control, the average incrase in DM digestibility due to molasses supplementation was 9.2% (41.9 vs 43.51-46.26%),whereas the incrase in NDF digestibility was 17.7% (38.41 vs 43.76-46.31%)(Table 2).The increase digestiility with the increase of urea treatment could be due to urea treatment producing nitrogen in the form of NH3.Similar results was also reported on nitrogen supplementation in rice straw which was able to improve deficiency of CP content, consequently the straw digestibility was increased due to the ammonia (NH3) availability which was needed for the activity of rumen microbial for the fermentation of feed (Wanapat et al. 2009).Microbial protein synthesis required NPN and carbohydrate source from feed (Al Qori'ah et al. 2016).Therefore, the high potential of ammonia had to be synchronized with availability of fermentable energy.Ammoniated feed which directly fed to ruminants will result in imbalanced N caused by high N degradation but lack of fermentable carbohydrate and consequently more N release which in turn caused low feed digestibility.Therefore this feed should be balanced with fermentable carbohydrate which will optimize rumen microbial activity.The available fermentable carbohydrate ammonia will be converted into protein microbial and stimulate multiplication of rumen microbial, which will be able to increase feed digestibility (Bata 2008).
The increase of rumen microbial population which produces more enzymes resulted in the increased feed digestibility.Therefore in the present study ammoniated corn cob as feed supplemented with fermentable carbohydrate such as molasses which was expected to be able to optimize the activity of rumen microbial through the available N in the form of ammonia and energy which needed for rumen microbial multiplication and optimizing microbial activity in  supplementation for ruminants fed on fibrous feed with low quality, resulted in better rumen environment for carbohydrate fermentation due to the availability of ammonia and energy for the growth of rumen microbial.Crude protein degradation in ammoniated corn cob produced intermediate products in the form of ammonia.With the availability molasses as energy source, rumen microbes will incorporate with NH3 as source of N for protein synthesis of the microbes, particularly cellulolytic bacteria which in turn this bacteria will digest fiber.Some of rumen microbial required N for microbial synthesis in the form of ammonia.Minimum concentration required was about 3.57 mM (Satter & Slyter 1974) or in the ranges of 3.57-7.14mM that had to be produced from protein in the diet (Swandyastuti and Rimbawanto, 2015).The increase of molasses levels resulted in non-significant increase of NDF digestibility, this could be caused by excessive energy available wich was not balanced with available N. The simultaneous available energy and nitrogen was essential to be to provide nutrient needed by rumen microbes.
In the present study, though digestibility of ammoniated CC supplemented by molasses increased in DM digestibility, but somehow still lower than of elephant grass digestibility (60.9%) (Santoso et al. 2009).Therefore, the process of treating crn cob with urea, it had to be combined with fermentable carbohydrate supplementation to increase digestibility due to the increase rumen microbial population.Principally fiber digestibility has to be close relation to enzyme produced by rumen microbial.Basic nutrients needed for rumen microbial synthesis was available energy balanced with nitrogen in the form of ammonia.In the present study, molasses containing high glucose was added.As stated by Nurhaita et al (2014) that nutrient required for microbial protein synthesis was energy, nitrogen, mineral and amino acid in the form of BCAA.Therefore it is proper in order to increase digestibility of ammoniated CC can be done by supplementation of branched-chain amino acid (BCAA) to stimulate the growth of rumen microbial.

Effect of BCAA supplementation on ammoniated CC
Digestibility value of ammoniated CC with supplementation of 5% molasses and BCAA is presented in Table 3. Ammoniated CC feed as fiber source with 5% molasses increased NDF digestibility by 13.9% (Table 2).Ammoniated CC digestibility could be increased by supplementation of branch-chain amino acid through stimulation of protein microbial synthesis.Digestibility value of ammoniated CC is presented in Table 3.The digestibility of DM, OM and NDF of corn cob was increased (P<0.05) by BCAA supplementation.The significant increase of digestibility due to BCAA supplementation was in treatment A, C, D and G.The digestibility of DM, OM and NDF in ammoniated CC (Tabel 2) was improved by 35.03%, 33.38% and 25.42%, respectively.
In the present study supplementation of BCAA might be used as precursor for microbial protein synthesis which was able to increase bacterial population thereby increased fiber digestibility of ammoniated CC.
Bacteria population increased more than 1.5 times compared to control.In contrast, protozoa population (3.50 x 105 cell/ml) in BCAA supplementation was significantly lower compared to control (13.5 x 105 cell/ml).This results indicated that combination of BCAA supplementation in treatment G was able to support the growth of rumen bacterial.
Branched-chain amino acid had significant role in increasing protein synthesis in human and animal (Zhang et al. 2017).Amino acid in the rumen is utilized by rumen microbes to synthesize protein microbial.Rumen bacteria, particularly cellulolytic bacteria required branched-chain fatty acid (BCFA) consisted of isobutyric, 2 metil butiryc, and valeryc acid as source bacterial carbon skeleton.This BCFA produced from decarbocilated and deaminated BCAA.Most BCAA in the rumen produced from fermentation of protein in the diet and lysis of rumen microbial (Zhang et al. 2017).With the low quality of agroindustrial by-products in term of low content in BCAA, therefore BCAA (valin, isoleucin, and leucin) supplementation in agroindustrial by-producs basal diet is needed to increase rumen microbial population and fiber digestibility.
Based on treatment of BCAA supplementation in ammoniated CC, there was indication of negative correlation between bacterial and protozoal population in the rumen (Figure 2).This correlation follows the equation y = -2.1338x+ 27.99; R2 = 0.7756.
Feeding diet containing high fiber such as corn cob with high bacterial population and low protozoal population (Treatment G, Table 3) will be very beneficial due to bacteria population.Fibrolytic bacteria particularly is needed to increase fiber digestibility this fiber digestibility produced energy in the form VFA which is used as energy source for ruminants.------------------------------% ---------------------    Nutrition needed by protozoa is protein and fermentable energy in the form of starch or sugar, on the other hand corn cob contain carbohydrate mostly in the form of fiber this condition retarded the growth of protozoa.Mustofa et al. (2012) reported that fermentation of ammoniated CC which was processed using commercial starter produced 113 mM VFA, increased cellulolytic microbial population increased VFA production as energy source for livestock.The amount of supply protein from microbial biomass into small intestine could be up to 50-80% from total absorbed protein (Bach et al. 2005).Treatment G (V 0.2, L 0.2, I 0.1) produced highest bacterial population among four BCAA combination.Fibrous fermented bacterial such as Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Butyrivibio fibrisolvents required branched-chain fatty acid (BCFA) such as isobutyric, isovaleric, valeric, and 2methylbutyric acids for their growth (Zhang et al. 2013).BCFA could be obtained from feed protein or recycled of rumen bacterial protein through oxidative deamination and decarboxylation from valin, leucin, and isoleucin (Moharrery 2004).The best BCAA combination in the present study was different to the study reported by Zain et al. (2008).They reported higher rumen bacterial population in sheep fed on palm press fiber complete diet supplementd with valin 0.1%, leucin 0.15% dan isoleucin 0.2% than control diet (18.88 x 10 10 col/ml vs 10.9 x 10 10 col/ml).The different results in the present study with Zain et al. (2008) study in term of bacterial population was due to the difference quality nutrient supply to support bacterial synthesis.
The used of BCAA in the field particularly for small holder farmers is quite expensive, this can be as constraint in the used of BCAA.Therefore it is needed to search alternative for BCAA sources which is cheap and easily obtained.Some of forage protein such as leucaena, gliricidia, calliandra and cassava leaves contain high BCAA.According to Hartadi et al. (1997) leucaena contained valin 1.51%, leucin 2.33% and isoleucin 1.3%.Widiawati et al. (2007) reported that leucaena contained valin 637 mg/g N, leucin 504 mg/g N, isoleucin 536 mg/g N, Gliricidia maculate leaves contained valin 646 mg/g N, leucin 521 mg/g N, isoleucin 563 mg/g N. Caliandra leaves contained valin 685 mg/g N, leucin 543 mg/g N, isoleucin 564 mg/g N. Cassava leaves contained valin 1%, leucin 1.3% and isoleucin 0.84% (Puastuti 2013, Unpublished).Therefore in field application utilization of BCAA can be implemented through forage protein source supplementation which is rich in amino acid with BCAA composition closed to treatment G (valin 0.2%, leusin 0.2% dan isoleusin 0.1%).However, attention need to be given type of legume forage source which will affect the composition of total ration and may have different effect, therefore further research on the use of forage as BCAA source need to be done.

CONCLUSION
Processing corn cob using urea treatment at 3% DM of corn cob was able to increase CP content by 78%.Supplemented ammoniated CC with molasses 5% and BCAA combination with valin 0.2%, leusin 0.2% dan isoleusin 0.1% resulted in highest DM, OM and NDF diegestibility of 58.06%, 59.49%, 54.19% respectively as indicated by the highest bacterial population and the lowest protozoa population.The utilization of ammoniated CC is suggested supplemented by molases 5% and BCAA combination valin 0.2%, leusin 0.2% dan isoleusin 0.1%.

Figure 2 .
Figure 2. Corellation between bacteria and protozoa population in the in vitro rumen of amoniated CC supplemented with BCAA

Table 1 .
Nutrient composition of corn cob untreated or ammoniated corn cob Different levels of molasses supplementation significantly (P<0.05)affectedDM,OM and NDF digestibility.Nutrient digestibility of ammoniated corn cob supplemented by molasses is presented in Table2.Digestibility evaluation of DM, OM and NDF of ammoniated CC with molasses addition as energy source, was significantly different (P<0.05)amonglevels of molasses supplementation.Digestibility value of ammoniated CC supplemented by molasses presented in Table2.
DM = Dry matter; NDF = Neutral detergent fiber; ADF= Acid detergent fiber; GE= Gross energy treatment in wheat straw reduced NDF content.

Table 2 .
Effect of molasses supplementation on nutrient digestibility of ammoniated corn cob

Table 3 .
Effect of combination of BCAA valin, leusin and isoleusin supplementation on ammoniated corn cob (CC) digestibility

Table 4 .
Effect of combination of valin, leusin and isoleusin supplementation in ammoniated CC on rumen microbial population