Nitrogen Balance, Microbial Protein Synthesis and Blood Metabolites in Fattening of Male Bali Cattle Fed Ration with Different Protein Levels in Smallholder Farms

Research was aimed to determine nitrogen balance, microbial protein synthesis, and blood metabolites of male Bali cattle fattening fed ration with different protein level in smallholder farms North Central Timor, Province of East Timor Tenggara, Indonesia. The cattle used were 18 heads aged 2 to 2.5 years with initial body weight of 229.86±12.46 kg. The cattle were randomly divided into three treatment groups. The T0 group was given feed the same as traditional fattening cattle practices by farmers,T1 group fed ration containing 12% crude protein (CP) and 72% total digestible nutrients (TDN), andT2 group fedration containing 15% CP and 72%TDN. Cattle were fed individually for 90 days and drinkingwater ad libitum. The data were analyzedby analysis of variance.Results of research indicated the nitrogen balance, and blood urea nitrogen between T1 and T2 were relatively similar, but those were higher (P<0.05) than T0 . In contrast, microbial proteins synthesis, and blood glucose at 0, 4, and 6 hours before and after feeding were relatively similar between the groups. Blood glucose of T2 at 2 hours after intake were higher (P <0.05) than T0, but was not different with T1 . It can be concluded, that the fattening maleBali cattle fed ration containing 12% CP and 72% TDNimprovedthe nitrogen balance and blood metabolites, butit was no positive effect on the microbial proteins and N synthesis.


INTRODUCTION
Productivity of beef cattle during fattening period was influenced by the feed quality and quantity given by farmers. When the adequately of feed covered during the growth phase, the synthesis of body tissue increases. Such conditions have a positive effect on the body weight gain and carcass production of the cattle.
In West Timor, the fattening of Bali cattle was mostly done in smallholder farms traditionally (Tahuk and Dethan, 2010). As a result, the guarantee of adequate protein and energy to meet the needs of livestock for optimal production is not achieved, especially in the dry season. These was indicated by the low average daily gain (ADG) as well as the poor of feed conversion ratio .
The strategy to ensure the good growth of Bali cattle during fattening period in smallholder farms is improvement of the feed quality. Protein and energy are two important nutrients, which need to be considered in balancing the dietary livestock to increase productivity. When cattle were given high protein with inadequate energy, the inefficient protein utilization may occur (Valente et al., 2016), and microbial protein synthesis is not optimum (Bach et al., 2005). The current concept in ruminant nutrition is focused on maximizing rumen microbial protein synthesis (Zadeh et al., 2013).
The enhancement of microbial proteins synthesis in the fattening ruminant is important, as it contributes greatly to the increase of livestock productivity. Mullik (2006) reported, about 60 to 80% of the total protein required by ruminants originates from the rumen microbial protein. On the other hand, the maximum potential of rumen microbes to produce microbial protein and degraded nutrients in the rumen may be explored only by the provision of high quality diet (Verbic, 2002).
Improving the feed quality on fattened beef cattle is very important to be done by farmers. In vitro observation in the Bali cattle rations that have different levels of protein-energy indicated that microbial protein synthesis has not been maximal (Tahuk et al., 2016); similarly, microbial protein synthesis in Bali cattle fed different levels of protein and energy were relatively similar (Setiawan et al., 2016).
Nitrogen balance was an indicator to evaluate the adequacy of protein in livestock based on the amount of protein consumed, and which is excreted by livestock (Utomo, 2012). This is due to live weight gains may not be related to protein stored, and a more accurate evaluation of a protein may be obtained by using the results of nitrogen balance experiments (Mc Donald et al., 2011). Therefore, the data of nitrogen balance generally indicates nutrient status in livestock. According to the above description, then the aims of study was to determine the balance of nitrogen, purine derivates, microbial protein synthesis and efficiency, as well as blood metabolites of male Bali cattle fattened in smallholder farms fed rations with different protein levels.

Researh Location and Bali cattle
The study was conducted in the Nekmese Farmer Group, Usapinonot Village, West Insana District, Regency of Timor Tenggah Tenggara, East Nusa Tenggara, Indonesia for 90 days. The chemical composition of feed and feces were analysis in the Nutritional Biochemistry Laboratory, Animal Science Faculty, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Male Bali cattle used were 18 heads aged 2-2.5 years with initial body weight (BW) of 229.86±12.46 kg. Cattle were randomized and placed in individual stall equipped with different eating and drinking places. Fattening was done for 90 days, including the adaptation period for 14 days. Feeding was done three times a day at 7:00 am, at 12:00 and 17:00 pm. Drinking water was available ad libitum. Feeding of forage and concentrates were carried out separately. Forage give first, while concentrate was given 60 minutes later after the cattle get forages.

Design of Research and Animal Feed
Exprimental design used was completely randomized design with three treatments and six replications. The treatments was T 0 group (control), T 1 , and T 2 groups. Cattle in the T 0 group obtained varied rations according to the traditional practice of farmers in fattening the cattle. Rations for the group T 1 and T 2 were prepared with different levels of crude protein (CP) with isoenergy in the form of total digestible nutrients (TDN). The group of T 1 get ration containing 12% of CP and 72% of energy (TDN), and the group of T 2 get ration 15% CP, and 72% energy (TDN). Rations used for T 1 dan T 2 group include of native grass, Gliricidia sepium, corn meal and rice bran (

Variable Measurement and Data Collection
In this study, the observed variables included the nitrogen intake, excretion, and nitrogen balance, purine derivates, microbial protein synthesis, rumen organic matter digestible, and blood metabolites (total plasma protein, hemoglobin, leucocytes, erythrocytes, packed cell volume, blood glukosa and blood urea nitrogen) of male Bali cattle.
Feces collection for determining feed digestibility was conducted over 10 days by using the technique of the total collection (Harris, 1970). Feces were collected daily, weighed, and sprayed with a solution of 10% formalin to avoid decomposition of feces and lost of N feces. The sample was dried until the weight constant; furthermore the samples collected during 10 days was mixed thoroughly, then 10% of the samples were milled with willey mill of 1 mm diameter for laboratory analysis.
Urine collection for the determination of N balance and microbial protein synthesis of purine derivatives were using a harness in conjunction with the implementation of the feces collection (Harris, 1970). The Urine collected from each animal was filtered with a filter cloth to avoid a three-layer mixed with other debris, then the volume weight was weighed and calculated every morning. Urine samples was taken 120 mL and placed in a plastic bottle size of 120 cc/mL, added with 10% of H 2 SO 4 until the pH became acidic (pH 2-3), then stored in a freezer at -20°C while waiting for laboratory analysis.
Nitrogen consumption was calculated from the amount of N in the ration reduced by the remaining N of the ration. The N excretion of feces was calculated by the total feces multiplied by the N content of feces (%). The nitrogen balance was calculated by reducing the N consumption with the N feces and N urine (Mc Donald et al., 2011).
Estimates of microbial protein synthesis in the rumen were measured by the purine derivative method of urine as directed by Chen et al. (1992). The calculation of estimation of microbial of nitrogen supply (EMNS) was based on the amount of urine absorbed (X=mmol/day), where the X value was estimated based on excretion of purine derivatives in urine (Y = mmol/day). The supply of N microbial calculation in Bali cattle used the equation according to Yusiati (2005): EMNS (g/day) = (X x 70) / (0.83 x 0.195 x 1000). The value of X was estimated from the equation Y = 0.145 W0.75 + 0.86 X mmol/day (X = purine derivative). The N Purine content was 70 mg/mmol, microbial purine digestibility was 0.83, and ratio N purine with N microbial of Bali cattle was 0.195.
The blood collection was conducted to determine glucose levels and blood urea. Blood sampling was conducted at 0 hours before feeding, and 2, 4 and 6 hours after feeding, which was carried out in the last week of the study. The examination of both variables using spectrophotometer method with reagent kit make DiaSys (according to manufacturer's instructions of DiaSys System GmbH, Holzheim, Germany). Blood glucose level was calculated by the equation: (Abs. Sample / Abs. Standard) x 100 mg/dl, at wavelength 546 nm, while the blood urea nirogen level was calculated with the equation: (Abs. Sample / Abs. Standard) x 50 mg/dl , at a wavelength of 365 nm). The variable hemoglobin was analyzed by Sahli method, the amount of erythrocytes and leucocytes using neubauer count method with hemacytometer, total plasma protein with refractometer method, and hematocrit/packed cell volume (PCV) using Wintrobe method.
Identification of chemical feed composition was carried out by proximat analysis according to the AOAC procedure (2005).

Statistic Analysis
The data collected was analyzed according to the one way analysis of variance procedure.  Further trials with Duncan's multiple range test (DMRT) do when there were differences among the treatments (Quinn and Keough, 2002).

Nitrogen Intake, Excretion and Balance
Nitrogen intake (N) (g/head/day; g/kg BW 0.75 ) of cattle in T 1 group was higher than those in T 0 (P<0.05), but those in T 2 was relatively similar to T 1 and T 0 . The N feces of T 0 , T 1 , and T 2 groups was relatively similar. In contrast, the excretion of N in urine (g/head/day) from T 1 and T 2 roup was relatively the same, but those were lower (P<0.05) than the T 0 groups ( Table 3). The high of N intake in T 1 group was related to the DM and CP intake which is quite high if compared to the T 0 group. This allows cattle to increase their productivity.
The excretion of N feces was relatively similar between the T 0 , T 1 and T2 goups. This was related to CP digestibility which was also not different between the cattle groups . In contrast, urinary N excretion of T 0 cattle group was higher than those of the T 1 and T 2 . This condition was caused by inefficient utilization of N because the lack of energy derived from digestible carbohydrates (Gehman and Kononoff, 2010). The cattle of T 0 groups only obtain energy source from structural carbohydrates which have a lower digestibility, so it does not sync with the high N supply of legumes intake. This study differs from Jetana et al. (2010) study which mentions the increased synchronization of carbohydrates and N causes decreased fecal N in Thai Brahman cattle. This condition was probably caused by the differences of livestock genetic and feed quality used in the study.
In the T 0 group the amount of N excretion reached 87.01% of the total N consumed by cattle, where 30.07% N came from the feces, and 56.94% of the urine. Thus, only 12.99% N was utilized to meet livestock needs. This condition illustrates that in T 0 group, the utilization of N feed by livestock for synthesis of body tissue was not maximal. Low digestible carbohydrates as the source of the carbon skeleton is thought to be the trigger for this condition. This was evident from the lower of growth performance on T 0 cattle than the other two treatments. According to Soeparno (2009), the high intake of protein and energy on the livestock will result in a faster of growth rate.
Nitrogen total excretion of T 1 group reached 59.37% from total N intake, consisted of N feces excretion 26.53% and N urine 32.84%. Similarly, total N excretion of T 2 group cattle reached 50.82% of total N intake, which came from N feces 25.37% and N urine 25.45%. This means that in the T 1 and T 2 cattle groups, the use of N by livestock for performance improvement was 40.63 and 49.18%, respectively. The results showed that the use of N feed on the male Bali cattle of T 1 and Nitrogen Balance and Microbial Protein Synthesis of Male Bali Cattle (P.K. Tahuk et al.) 47 1.00± 0.14 b 1 Data is presented in average ±SD 2 T 0 =control (according to the traditional raising of farmers); 3 T 1 = ration with 12% CP and TDN 72%; 4 T 2 = ration with 15% CP and 72% TDN; ns = not significantly different; a,b Different superscript in the same row shows differences at P<0.05. T 2 group was better if compared to T 0 group. The lower N excretion value of T 1 and T 2 , as well as the growth performance of the two treatments was higher than those of T 0 , where the ADG of T 1 and T 2 were 0.70 and 0.47 kg/head/day, respectively .
Nitrogen balance in male Bali cattle fattening groups of T 1 and T 2 were not significantly differ, but those were higher (P<0.05) than T 0 group (Table 3). The results of this study showed that the improvements feed in T 1 and T 2 groups can increase consumption and digestibility of CP or N, which in turn had a positive impact towards N balance. According to Yusiati (2005), the low N intake, contributes to the decrease of N balance in male Bali cattle.
The N balance of the three groups was positive in the study. This can be seen from the increase of ADG of T 1 and T 2 groups, each of which reaches 0.70±0.16, and 0.47±0.47 kg/head/day . The T 0 group showed a smaller balance value and tended to approach a value of 0. This situation illustrated the use of CP or N was less than the maximum for the cattle growth, indicated by ADG which only reaches 0.30±0.05 kg/head/day . Instead, the group of T 1 and T 2 had the equilibrium value of N that was higher from value 0, which described high deposit of N to improve the performance of cattle production. According to Howard et al. (2007), rations composed of carbohydrates and N synchronized have a positive effect on improving N balance. When nitrogen balance means was positive, it means cattle will increase body weight due to the addition of the woven tendons (Mc Donald et al., 2011).

Purine Derivates
The content of creatinine, uric acid, allantoin and purine derivatives (mmol/l) in male Bali cattle fattening were not significantly different between groups (Table 4). Thus, the treatment of CP levels differing by 12 and 15% in 72% TDN, generally did not positively affect to the increase secretion of purine derivatives.
According to Yusiati (2005), purine derivatives are influenced by species of livestock and the amount of feeding. In addition, Bali cattle generally have lower purine derivatives of other local beef cattle in Indonesia, such as PO and PFH cattle. Similarly, purine derivatives increased in Bali cattle that received feed ad libitum than livestock given limited feed .

Estimate Microbial Protein and N Synthesis of Purine Derivatives
Estimation of microbial N synthesis (g/head/day) of purine derivates in group T 0 , T 1 , and T 2 was 7.22±1.94; 8.09±3.23; and 8.51±1.01, respectively; whereas estimation of microbial protein synthesis of T 0 , T 1 , and T 2 was 45.12±12.09; 50.55±20.21, and 53.20±6.29, respectively (Table 4). The estimation of microbial N and protein synthesis of purine derivates in the three groups showed no significant difference. This is related to the secretion of purine derivatives from the three groups of cattle that were also relatively similar. Those indicated that the use of feed containing 12 and 15% CP and 72% TDN had no significant impact on the increase in the N and microbial protein synthesis compared to the use of forage as a single feed on fattening cattle in smallholder farms. The results of this study were not different from in vitro study by Tahuk et al. (2016) which obtained relatively the same of microbial protein synthesis in fattening rations of male Bali cattle based on different protein and energy levels. However, the synthesis of microbial proteins in this study was lower than that of Bali cattle that obtained rations with multivitamins and minerals supplementation (Mudita et al., 2014).
Theoretically, livestock given high CP and TDN can improve the microbial proteins synthesis in the rumen. In fact, microbial protein synthesis in the group T 1 dan T 2 was not optimal. These were allegedly because associated with the lack synchronization of nitrogen and energy in the rumen.
The study were in accordance with the report of Devant et al. (2001), which declared the use of N sources in rations of high concentrate and low protein in beef cattle had sufficient N available for microbial growth, but limited to microbial protein synthesis and digestion of nutrients. Pathak (2008) stated that a crude protein content in rations arranged higher than 11% may be necessary to assist optimal microbial growth; but because of it resistance protein to microbial degradation may result in limited microbial protein synthesis. Therefore, to increase the amount of microbial synthesis, the manipulation of energy and N fermentation in the rumen is necessary to obtain an energy supply pattern appropriate to the rumen conditions.

Rumen Organic Matter Digestible
Rumen organic matter digestible (ROMD) of the T 1 group was higher (P <0.05) than those of T 2 group; similarly, the T 2 was higher (P<0.05) than T 0 . Despite having a lower ROMD, livestock in the T 0 group was more efficient in utilizing the organic matter consumed and digested in the rumen for microbial synthesis. This can be seen from the efficiency of EMNS and estimation microbial of protein supply (EMPS from ROMD of T 0 group (g/kg.ROMD/day) that was higher (P<0.05) than that of T 1 and T 2 groups. In contrast, the efficiency of EMNS and EMPS values of ROMD in the T 1 and T 2 groups were relatively similar.
The feed proportion of the T 0 group consisted of grasses as a source of structural carbohydrates, and forage leguminous as a source of N was suspected of slow digestion. As a result, the duration of staying in the rumen was longer so that the chance of microbes to degrade was greater than T 1 and T 2 group. This condition has a positive impact on the increase of N synthesis and microbial protein rumen of each organic matter (kg). According to Hasson (2004), when the passage rate increase, the time for digestion in the rumen decreased, this results in a less efficient degradation. At higher feeding level the rumen outflow increase and this depresses fibre degradation.
In the T 1 and T 2 groups, the efficiency of protein and N microbial synthesis was lower than T 0 group due to lack of synchronization between proteins and digestible carbohydrates. Microbial protein synthesis can be increased if there is synchronization between protein and energy (Zadeh et al., 2013).

Blood Metabolites
Blood components status (hemoglobin, leukocytes, erythrocytes) of male Bali cattle in T 2 group was higher (P<0.05) compared to T 0 , but T 1 was relatively similar to both groups. The packed cell volume (PCV) of T 1 and T 2 was relatively similar, but it was higher (P<0.05) than those of T 0 ; whereas the variable of total plasma protein did not significantly differ between the treatments.
Although there were differences, the general status of blood components was under normal conditions. Thus, the body's physiological function was in a good condition, both from the aspect of metabolism, O 2 transport, and nutrients, as well as optimal immune function. The main function of blood is to maintain the body's physiological balance (Radkowska and Herbut, 2014). Therefore, the hematological blood indicators are the main determinant of the animals' environmental adaptation and thus can improve welfare livestock (Sattar and Mirza, 2009).

Blood Glucose
Blood glucose concentrations of male Bali cattle showed no significant difference in 0 hours before feeding, as well as 4 and 6 hours afterwards. However, at 2 hours after feeding, blood glucose of T 2 group was higher (P<0.05) than those of T 0 , while between T 0 and T 1 , and T 1 and T 2 showed no significant difference (Table 5). Increased blood glucose status at 2 hours after feeding was closely related to the level of feed digestibility and availability of precursor glucose in the form of volatile fatty acids (VFA) that were absorbed into the blood which would then be converted into blood glucose in the liver (Mc Donald et al., 2011). Glucose status at 0 hours before feeding as well as 4 and 6 hours after feeding was normal because of the guard system blood glucose levels in ruminants through a mechanism or process of glycogenesis, glycogenolysis, and gluconeogenesis (Soeparno, 2011).
The results of this study were not much different from the reports of  who obtained blood glucose levels in male Bali cattle fattening in the smallholder farms with forage was 58.62 mg/dL. Nevertheless, the status of blood glucose in this study was lower than Kendran et al. (2012)  29.73± 2.32 b 1 Data were presented in average ±SD 2 T0=control (according to the traditional raising of farmers); 3 T1= ration with 12% CP and TDN 72%; 4 T2=ration with 15% CP and 72% TDN; 5 TPP = total plasma proteins; 6 PCV = packed cell volume ns = not significantly different; a.b Different superscript in the same row shows differences at P<0.05. respectively.

Blood Urea Nitrogen
Concentration of blood urea nitrogen (BUN) (mg/dL) of male Bali cattle at 0 hours before feeding or 2, 4, and 6 hours after feeding in T 1 and T 2 groups was higher (P<0.05) compared to those in T 0 (Table 5). Cattle in T 0 group had lower blood urea concentration than the normal range. It was presumably related to low N-NH 3 concentration, so the conversion to blood urea nitogen was lower. The normal range of blood urea in cattle were in accordance to Hungate (1966) cited by Munzaronah et al. (2010) was 26.6-56.7 mg/dL. On the other hand, blood urea in T 1 and T 2 groups was higher than those in T 0 . because of the high concentration of N-NH 3 rumen, while the conversion to microbial protein was lower.
The efficiency of N-NH 3 utilization for protein synthesis in the rumen depended on the availability of energy. If there was a shortage of energy, then the protein would be excessive and cannot be utilized by rumen microbes. The excess of crude protein can increase the concentration of urea in the plasma (Mc Donald et al., 2011). The BUN content in these study was lower than those in Madura cattle fed ration containing different energy levels (Umar et al., 2015). In contrast, the content of BUN in the T 1 and T 2 treatment was higher than those in .

CONCLUSION
The fattened male Bali cattle fed ration with level 12% CP, and 72% TDN can improve the nitrogen balance and blood metabolites, but it was no positive effect on the microbial proteins and N synthesis.