Effect of urea and molasses fermented cassava pulp on rumen fermentation, microbial population and microbial protein synthesis in beef cattle

ABSTRACT This study aimed to investigate the effects of fermented cassava pulp with urea and molasses on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis. All brahman crossbred beef cattle were randomly assigned according to a 4 × 4 Latin Square Design to receive four dietary treatments; cassava pulp (control), cassava pulp fermented with urea 4%, cassava pulp fermented with molasses 4%, and cassava pulp fermented with urea 4% and molasses 4%. All animals were fed concentrate pellet at 1.5% BW and supplemented 0.5% BW of cassava pulp. The results showed that rumen NH3-N concentration was increased in cattle fed with both cassava pulp fermented with urea and/or molasses (P < 0.05). The total VFA was not affected by dietary treatments while there was an increase in propionic acid when cattle were supplemented with fermented cassava pulp. However, methane production and protozoal population were lower in treatments with fermented cassava pulp (P < 0.05). Feeding of fermented cassava pulp resulted in an increase in microbial protein synthesis (P < 0.05). Based on the present findings, it could be concluded that fermented cassava pulp with urea and molasses can increase propionic acid, microbial protein synthesis while reducing protozoal population and CH4 production.


Introduction
Cassava (Manihot esculenta Crantz) is an annual crop grown widely in tropical and subtropical regions. It thrives well in sandy-loam soils with low organic matter, and in climates with low rainfall and high temperature. Cassava roots have high levels of energy (75-85% of soluble carbohydrate) and minimal levels of crude protein (2-3% CP); they have been used as a source of readily-fermentable energy (Polyorach et al. 2013;Wanapat and Kang 2013a;Kang et al. 2015). The top growth could be harvested at four months initially and at 2-3-month intervals subsequently. The leaves and green stems are chopped and sun-dried to reduce their moisture and hydrocyanic acid (HCN) content to produce cassava hay (CH), which has been used as a ruminant feedings successfully (Wanapat 2000(Wanapat , 2003Lunsin et al. 2012). Harvesting of cassava leaves at an early growth stage (3 months) to make hay could reduce the condensed tannin (CT) content and increase the protein content (25% of DM) resulting in a higher nutritive value (Wanapat et al. 1997). Root yield is about 50 t/h and when processed into a dried chip, it will be about 15-20 t/h. If the whole plant is harvested for hay making after 4 months after planting, it can be harvested when reaching one year, in this case, the root yield will be reduced by about 15% from the original yield .
Cassava pulp, a fibrous by-product of the cassava processing industry, has recently become attractive as a cellulosic biomass due to its nature as a cheap, abundant and renewable agricultural product. At present, cassava pulp is generally used as low-value animal feed. In Thailand, most of the cost of production is the cost of feeds. There are many attempts to reduce the cost of feeds through the utilization of cheap raw materials, such as agro-industrial by-products. Cassava pulp offers an alternative to high-starch grains, and can be used as an energy source in ruminant diets, but is low in crude protein (CP) (Lounglawan et al. 2010). High levels of urea have been found to be a useful mechanism for increasing crude protein (Ferreiro and Preston 1976;Silvestre et al. 1977a). Ensiling is a crop preservation method based on natural lactic acid fermentation under anaerobic conditions where anaerobic microbes build up organic acids mainly lactic acid by using fermentable carbohydrates (Gollop et al. 2005;Ki et al. 2009). To improve silage preservation and guarantee animal feed quality, silage additives such as chemicals, enzymes and bacterial agents can be employed. Addition of carbon and nitrogen sources could improve the quality of silage. Urea has been utilized as a supplemental nitrogen source for beef cattle rations when protein sources are expensive. Adding urea is a common and cheap method of increasing nitrogen supply; however, urea decreases the fermentation quality of silage by increasing pH with the release of ammonia (Panchol et al. 1994). Kuikui et al. (2017) reported that molasses treated silages was increase crude protein. So, it is considered that the addition of different combinations of urea and molasses may improve both the protein content and fermentation quality of the silage. Norrapoke et al. (2018) reported that supplementation of urea and molasses could improve the quality of fermented cassava pulp in terms of nutritive value and rumen degradation. Addition of carbon and nitrogen sources could improve the quality of silage and have subsequent effects on rumen degradation characteristics and production in ruminants. Therefore, the objective of this study was to study the effects of urea and molasses fermented cassava pulp on feed intake, nutrient digestibility, rumen fermentation, microbial population and microbial protein synthesis in beef cattle.

Animals, diets, experimental design and animal management
Four brahman crossbred beef cattle (75% brahman with 25% Thai native breed) with an average body weight of 300 ± 30 kg were used in this experiment in a 4 × 4 Latin square design. All animals were housed in individual pens and given feed twice a day and fed rice straw ad libitum with concentrate pellet at 1.5% body weight (BW) and cassava pulp/cassava pulp fermented at 0.5% BW. By fed to the cattles separately and separate container to fed two kinds of feed. Clean, freshwater, and mineral salt block were offered freely. The four treatments were un-supplementation (control), supplementation with cassava pulp fermented urea 4% (CU), supplementation with cassava pulp fermented molasses 4% (CM), and supplementation with cassava pulp fermented urea 4% and molasses 4% (CUM). Chemical compositions of dietary feed were shown in Table 1. The experiment was conducted for four periods and each experimental period lasted for 21 days. During the first 14 days, all animals were fed with their respective diets, and samples were collected during the last 7 days of each period.

Cassava pulp fermented
Dry cassava pulp was bought from industrial and ensiled with urea at 4%, molasses at 4% and mix urea 4% with molasses 4% on dry matter basis (DM) (Norrapoke et al. 2018). Dry cassava pulp was mixed with urea, molasses and mix urea 4% with molasses 4% then packed into plastic bags. The fermented bags were kept at ambient temperature (about 25-30°C) for 21 days.

Data collection, sampling procedures and statistical analysis
Roughage and concentrate were sampled daily during the collection period and were composited for analysis. Feed and fecal samples were collected during the last 7 days of each period. Samples of faeces were taken by rectal sampling and samples of urine were taken by using a spot sampling method. Composite samples were dried at 60°C in a forced-air oven for 48 h and ground at 1 mm screen using Cyclotech Mill, Tecator, Sweden for analysis of dry matter, ash, ether extract, crude protein (AOAC 1990), and neutral detergent fibre (NDF), acid detergent fibre (ADF) (Van Soest et al. 1991). During the final day of collection period, rumen fluid and blood samples were collected at 0, 3 and 6-h post-feeding. Approximately 200 mL of rumen fluid were obtained anaerobically via the esophagus using a stomach tube (outside diameter 1 cm, inside diameter 0.8 cm, length 300 cm) connected to a vacuum pump from different sites within the rumen. When the tube was employed, a back and forth action was done to be desirable in obtaining large quantities of rumen contents from different areas of the rumen. Rumen fluid was immediately measured for temperature and pH (Hanna Instruments HI 8424 microcomputer, Singapore). Rumen fluid samples were then kept for analysis of ammonia nitrogen (NH 3 -N) using the micro-Kjeldahl methods AOAC (1990) and volatile fatty acids (VFA) using high-pressure liquid chromatography (HPLC) according to Samuel et al. (1997). A blood sample (about 10 ml) was collected from the jugular vein into tubes containing 12 mg of EDTA, and plasma was separated by centrifugation at 500 × g for 10 min at 4°C and stored at −20°C until analysis of blood urea nitrogen (BUN) according to Crocker (1967), hematocrit according to Kaneko et al. (1997). At the 0, 3 and 6 h post-feeding, rumen fluid was collected and prepared for measuring microbial population by total direct count of protozoa, bacteria and fungi using methods of Galyean (1989) by hemocytometer (Boeco, Hamburg, Germany). Urine samples were analysed for urinary nitrogen (IAEA 1997) and allantoin in urine was determined by HPLC as described by Chen and Gomes (1995). The amount of microbial purines absorbed was calculated from purine derivative excretion based on the relationship derived by Chen and Gomes (1995).

Statistical analysis
All data were statistically analysed according to a 4 × 4 Latin square design by using Proc GLM/Proc Mix (SAS 1996). Differences between treatment means were determined by Duncan's New Multiple Range Test (Steel and Torrie 1980). Differences between means with P < 0.05 were accepted as representing statistically significant differences.

Chemical compositions
Chemical compositions of dietary feed were shown in Table 1. Concentrate diet had a high quality in terms of high CP and low NDF and ADF content. Cassava pulp quality was improved by fermented with urea/molasses in terms of CP and this was also reported in the finding of Norrapoke et al. (2016). Similar to Pilajun and Wanapat (2018), the nutritive value of fermented cassava pulp with yeast and EM was improved by MU supplementation.  Table 2 presents data of daily feed intakes and nutrient digestibility affected by cassava pulp source supplementation. Feed intakes were not significantly different among treatments (P > 0.05). Moreover, based on the observation during the experiments, cassava pulp did not show any depression on feed intake of beef cattles, it is noteworthy that the value of cassava pulp fermented intake was lower than cassava pulp intake, it may be because of this report by dry matter basis. Similarly, Chanjula et al. (2007a) reported that higher levels of U (3%) could be used with high levels of cassava chip as a concentrate without altering feed intake. Apparent digestibility of DM and OM were also increased (P < 0.05) in beef cattle consumed a diet with CM. It is may be due to lower feed intakes. The CP digestibility was increased by both factors CM and CUM. This increased is in agreement with the results of Pal and Negi (1978) when ruminants were fed with alkali-treated straw and resulted in optimal value for fibre and protein digestion (Hoover 1986). Moreover, NDF and ADF digestibility were significantly increased in beef cattle fed cassava pulp fermented (P < 0.05). Highest values were found with feeding CUM.

Feed intake and nutrient digestibility
It is supposed that rumen fermentation and digestibility increases with the urea supplementation due to the increased rumen microorganisms growth rate. As there is more available nitrogen in the form of ammonia from the urea hydrolysis (Khattab et al. 2013;Kang et al. 2015).

Rumen fermentation and blood metabolites
Ruminal temperature, pH, Hct and BUN were similar among treatments and the values were quite stable at 38.01-38.17°C, pH (6.7-6.82), Hct (28.42-29.17) and BUN at 8.18-8.63 mg/dl, respectively (Table 3). These values were optimal for normal rumen fermentation (Wanapat 1990). However, more fermentation means more acid production and an expectation of lower rumen pH may observe. Mould et al. (1983) suggested that rumen pH should be maintained above 6.0-6.1 to avoid inhibition of cellulolysis. Rumen pH, together with microbial population, nature of substrates, environmental factors such as temperature and existence of cations and soluble carbohydrates, were the factors governing bacterial attachment (Miron et al. 2001). Ruminal pH is one of the most important of these factors because fibrolytic bacteria are very sensitive dependent on pH change. Fibre digestion decreases at low rumen pH, especially below pH 6.0, as observed previously in studies using continuous culture of mixed ruminal microorganisms (Slyter 1986). Treatments with CM were found lower in the concentration of ruminal NH 3 -N than with CU. Both treatments with urea have a higher concentration of NH 3 -N (T2 = 12.96 and T4 = 16.03 mg/dl) than in the treatments control and with molasses (T1 = 8.64 and T3 = 10.94 mg/dl). The available rumen NH 3 -N would be used in microbial protein synthesis by the rumen microbes. It is well known that cellulolytic bacteria utilize NH 3 for their growth (Bryant 1973). They were unable to grow on other nitrogen sources in the absence of NH 3 (Russell et al. 2009). Cassava root contains high levels of readily fermentable energy and could be used in combination with readily available NPN sources such as urea in ruminant rations and this could improve the growth of rumen bacteria (Wanapat and Kang 2013;Wanapat and Kang 2013). It is a potential approach to exploiting the use of local feed resources. Moreover, Chanjula et al. (2007b) also reported that cassava was a good source of ruminal degradable starch in replacing corn grain and had the potential to improve goat performance. In this study, there were differences (P < 0.05) in acetic acid (C 2 ) and propionic acid (C 3 ) concentrations when beef cattle were fed with different treatments. Moreover, Wanapat et al. (2011b) compared from four sources of protein in concentrate diets, soybean meal, cassava hay, Leucaena leucocephala (LL)  and yeast fermented cassava chip in lactating dairy cows and found that propionic acid were found the highest in cows receiving CH and YEFECAP. As shown, the value of Total VFA and C 4 were found no difference (P > 0.05) among treatments. Moreover, CH 4 production was lower than control went supplement with cassava pulp fermented urea/molasses.

Rumen microorganism population
As shown in Table 4, protozoa counts were found different (P < 0.05). Treatments with CUM had the lowest at 4.4 × 10 5 cell/ml and the highest was in treatment with control at 4.9 × 10 5 cell/ ml. Total counts of bacteria and fungal zoospore population were not different. Similarly with Khampa et al. (2009) reported that supplementation with YEFECAP also decreased the population of Holotrich and Entodiniomorph protozoa in the rumen of dairy steers.

Nitrogen utilization and efficiency of microbial protein synthesis
The effect of cassava pulp fermented feeding on microbial protein synthesis in beef cattle is presented in Table 5. The microbial protein synthesis in terms of purine derivatives absorbed, microbial nitrogen supply and efficiency of microbial protein synthesis ranged from 76.4 to 92.5 mmol/d, 55.5-68.5 gN/d and 22.9-32.9 gN/kgOMDR, respectively, and were significantly (P < 0.05) increased with CUM in feed; meanwhile, PD excreted was not different among treatments (P > 0.05). Which prolonged microbial utilization of a more continuous supply of NH 3 -N during ruminal fermentation (Xin et al. 2010). Hungate (1966) concluded that most rumen microbes could use NH 3 -N as an N source for their protein synthesis.
There was no difference in purine derivative excretion among treatments. Moreover, Chanjula et al. (2007b) also reported that cassava was a good source of ruminal degradable starch in replacing corn grain and had the potential to improve goat performance. The optimal inclusion of cassava in replacing corn is suggested to be between 25 and 75% of cassava chip (CC) (12.5-37.50 kg) in concentrate. There was no evidence of any adverse effects on nitrogen balance or animal health when fed fresh elephant grass. Microbial CP synthesis in the rumen often provides the majority of the protein supplied to the small intestine of ruminants, accounting for 500-800 g/kg of total absorbable protein (Robinson et al. 2004a;Firkins et al. 2007). The higher EMNS with the soybean meal versus the urea diet might be due to the use of peptide or amino acid N, which can enhance microbial growth (Galo et al. 2003;Xin et al. 2010).

Conclusion
Based on the present study, it is concluded that fermented cassava pulp with urea and molasses can increase propionic acid, microbial protein synthesis while reducing protozoa population and CH 4 production.

Disclosure statement
No potential conflict of interest was reported by the author(s).