Effect of soybean meal replacement by slow-release urea on ruminal parameter, blood metabolites, and microbial protein synthesis in Zel ram

The objective of this study was to determine the effect of soybean meal replacement by slow-release urea on feed intake, ruminal parameters, blood metabolites, dry matter, nutrients digestion, and microbial protein synthesis (MPS) in ram. Rams were used for four 21-day periods. The experimental design was a 4×4 Latin square design. The experiment has consisted of the following four rations: 1) basic diet with soybean meal as a protein source, 2) basic diet with soybean meal plus 0.5 % of DM slow-release urea (SRU), 3) basic diet with soybean meal plus 1 % of DM SRU, and 4) basic diet with soybean meal plus 1.5 % of SRU, respectively. Soybean meal replacement by SRU decreased ruminal ammonia and blood urea. SRU increase in diets significantly increased acetic acid 3 hours after morning feeding, blood glucose, total purine, uric acid excretion, microbial nitrogen, and microbial protein. The results showed that soybean meal replacement by SRU sources had no negative effect on rams performance.


Introduction
Urea is rapidly hydrolyzed to NH 3 in the rumen in the first-hour post-ingestion to improve the utilization of ammonia released from urea. Urea is cheap and its can improve the utilization rate of carbohydrates and the feed conversion rate (Ding, Lascano, & Heinrichs, 2015). Slow-release sources have been designed to promote the constant availability of ammonia (NH 3 -N) over long periods (Taylor-Edwards et al., 2009). Rumen bacteria break down true protein to peptide, amino acids, and often NH 3 -N. Ruminants can utilize non-protein nitrogen (NPN) sources such as urea and biuret and synthesize microbial protein (Hristov & Jouany, 2005). Urea can produce NH 3 -N which exceeds the ruminal bacterial capacity (Galo, Emanuele, Sniffen, White, & Knapp, 2003). The NH 3 -N/urea high concentrations in blood have multiple negative effects on ruminants including reduced feed consumption, changes in liver metabolism, reduced performance, induced NH 3 -N toxicity which may all result in the animal's death (Huntington, Harmon, Kristensen, Hanson, & Spears, 2006). The SRU resources can synchronize the availability of N and carbohydrate fermentation (Highstreet, Robinson, Robison, & Garrett, 2010;Huntington et al., 2006), which improves microbial protein production and meet the amino acid requirements for animals. Microbial growth depends on protein degradation rate and NH 3 -N availability in rumen (Brooks, Harvey, Johnson, & Kerley, 2012). SRU in the ruminant diet can decrease N pollution and diets cost, therefore, the objective of this research was to investigate the effect of slow-releasing NPN replacement by SBM on feed intake, ruminal pH and volatile fatty acid (VFA), blood metabolites, dry matter and nutrients digestion, and MPS.

Ethics statement
All experimental procedures described in this study were reviewed and approved by the research ethical committee of University of Sari Agriculture and Neutral Science.

Sampling
Lambs were weighed biweekly and dry matter intake (DMI) was measured daily. Total feces of all lambs from day 14 to 20 (5 days) were collected, weighed and 100 g of the fecal samples were frozen for further analysis. On day 20 the frozen samples were mixed and a 100 g final fecal sample was submitted to the lab for further analysis.
On day 21 of each period, rumen fluid samples (50 mL) were collected from the ventral sac at 0 (before the morning feeding), 3, 6, and 12h after the morning feeding using the rumenocentesis technique to measure pH, NH3-N and VFA. Ruminal pH was measured immediately after ruminal fluid collection using a pH meter (model 632, Metrohm, Herisau, Switzerland), and samples were frozen at -20°C to analyze NH3-N and VFA. Urine volume was measured daily between days 15 and 20. To prevent purine derivatives (PD) precipitation in urine during storage, 10% of the daily amount was sampled, diluted 5-fold with distilled water, and then stored at -20°C to estimate PD and N.

Laboratory analyses
Feeds, feces and orts samples were dried at 55°C and ground through a Wiley mill (1-mm screen). Samples were analyzed for DM, Kjeldahl nitrogen, ether extract, organic matter and ash at 605°C for 3h (Association of Official Analytical Chemists [AOAC], 2002), NDF and ADF (Van Soest, Robertson, & Lewis, 1991); with α-amylase being added and without sodium sulfite. The NDF was expressed without residual ash. The NFC was calculated using following formula: 100-(% CP+ %NDF+ %Ash+ %EE).
The chemical components of TMR and feces were used to calculate intake and nutrients digestibility. Table 1 displays a feed analysis. The NH3-N concentration was measured using Kjeltec Auto Analyser (Model 1030, Tecator Co. Sweden;Fenner, 1965).
Blood samples were collected from the jugular vein on day 21 in each period. Samples were collected just before the morning feeding (time 0). Approximately 10 mL of blood was collected by evacuated collection tubes containing Ethylenediaminetetra-Acetic Acid, transferred to the laboratory on ice, and centrifuged (3,500 × g at 4°C for 10 min), and plasma was stored at -20°C until analysis. The concentrations of glucose, triglycerides, cholesterol, Low-Density Lipoprotein, High-Density Lipoprotein, and blood urea were measured using the auto analyzer system (270 -CA FURUNO).
Urinary PD was estimated by spectrophotometric methods (Chen & Gomes, 1995). Allantoin was measured in urine using a calorimetric method at 522 nm after its conversion to phenylhydrazine. The sum of xanthine and hypoxanthine was calculated by conversion to uric acid with xanthine oxidase (X-1875; Sigma Aldrich, St. Louis, MO) and measuring subsequent optical density at 293 nm. Uric acid was measured from the reduction in optical density at 293 nm after degradation of uric acid to allantoin with urease (U-9375; Sigma-Aldrich). Total PD excretion per day was calculated as the sum of all 4 compounds (allantoin, uric acid, and xanthine plus hypoxanthine). The daily absorbed exogenous purines were estimated, and MPS was predicted. The urinary N concentration was estimated using the Kjeldahl method (AOAC, 2002), and N retention was calculated as daily N excretion (urinary N plus fecal N) subtracted from the daily N intake

Calculations and statistical analysis
Estimated MPS and urinary purine derivative excretion were calculated using the equations of Chen and Gomes (Chen & Gomes, 1995).
Data were analysed using the 4 × 4 Latin Square design by Glimmix procedure of (Hermenegildo & Puebla, 2002), based on following model: where, Y(i)jk was the dependent variable of each treatment in period j of rams; µ, was the overall mean; T(i) was the effect of treatment i (i=1, …, 4); Pj was the effect of period j (j = 1, …, 4); Ak was the effect of rams k (k = 1, …, 4); and e(i)jk was a random error associated with each observation. The P-value was set at 0.05.

Nutrients intake and digestibility
Dry matter, nutrients intake and digestibility are shown in Table 2. The SBM replacement by SRU did not affect DMI, CPI, EEI, NDFI, NFCI, Ash (p ≥ 0.11).

Ruminal pH and ammonia
The rumen fluid pH did not vary for different levels of SRU in the ration at 0, 3, 6, 12 h (p ≥ 0.10; Table3). There were significant effects between ruminal ammonia before feeding at 0 time (linear p = 0.02; quadratic p = 0.01; p = 0.02; Table3). When SRU increased in diet NH 3 -N decreased. Treatment 4 showed the lowest NH 3 -N concentration. Concentration of ruminal NH 3 -N did not differ at 3, 6, and 12h (p = 0.09), after feeding.

Blood metabolite
The blood glucose increased by SBM replacement with SRU in the diet (linear p = 0.02; quadratic p = 0.01; p =0.02; Table 4), which had no effect on cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein (p ≥ 0. 07; Table4), There were significant differences among treatments on plasma urea concentration (linear p = 0.01; quadratic p = 0.01; p = 0.01). The results were achieved due to the increase in the SRU level of the diet which lowered blood urea.

Dry matter intake and digestibility
In this experiment, it was hypothesized that SBM replacement with SRU in sheep diets would not affect the synchrony between N and energy in the ruminal environment and ruminal MPS. Consequently, SBM replacement with SRU would maintain the diet digestibility and DM intake.
The DMI and total track digestibility were not significantly affected by experimental diets ( Table 2). The absence of the effects on DMI can be explained by the fact that the diets were isoproteic and that the SRU has no effect on diet palatability. Treatment 4 had the highest SRU percentage of SRU and DMI. Some studies showed that SRU in the ruminant diet did not demonstrate significant differences in DMI (Eweedah, Bassuony, EL-Razik, & El-Baky, 2016;Pinos-Rodríguez, Peña, González-Muñoz, Bárcena, & Salem, 2010;Taylor-Edwards et al., 2009). Previous studies showed that the absence of the effects on DMI as a consequence of urea plus SRU supplementation has been observed in finishing lambs fed with a 50:50 forage and concentrated diet and in steers fed a finishing diet with 70% concentrate (Castañeda-Serrano, Ferriani-Branco, Teixeira, Garcia-Diaz, & Diego-Sofiati, 2013; Pinos-Rodríguez et al., 2010). The SRU replacement may improve the DMI and nutrients due to a higher fibrolytic bacteria activity. The rumen ability to hold rumen contents and the microbial biomass increase results in the higher digestion rate and more postruminal amino acids flow. The SRU in rations had no effect on DM, NDF, and ADF digestibility (Valério Geron et al., 2016;Xin, Schaefer, Liu, Axe, & Meng, 2010). Besides, Alves et al. (2014) showed that SBM replacement by SRU did not change the CP intake. The SRU improves nutrient imbalance for rumen bacteria by increasing the energy availability from carbohydrates. Also, the hepatic urea cycle may indirectly trigger the oxidative metabolism in the liver, which would increase the production of ATP affecting the animal feed intake (Santiago et al., 2015). Changes in productivity and/or energy efficiency can be partially be explained by improvements in nutrient synchrony between N and carbohydrate compounds in the rumen and greater N retention (decreases in ruminal ammonia concentration and increases in the flow of microbial N to the duodenum). However, this is not to be confused with the popular notion that the rate of soluble feed N release to the rumen be in synchrony with carbohydrate fermentation. The provision of adequate ruminal available N, regardless of the rate at which it is degraded or solubilized within the rumen, is the relevant factor affecting microbial protein synthesis. The above researchers have argued that their results can be partially explained by the possible synchrony of ruminal degradation rates between urea and starch.

pH and NH3-N
The SBM replacement by the SRU did not affected on ruminal pH and ammonia concentration in ruminal environment (Table 3) reported no difference in the rumen pH in sheep and goat fed NPN. Van Soest (1994) reported that cellulolytic organisms grow optimally at pH 6.7 and pH level below 6.2 inhibits the digestion, decreases acetic acid, and depresses cellulolytic activity. Ruminal fermentation products and, rumen pH are highly affected by factors such as digestibility, DMI, forage, diet concentration, feed physical structure and feeding method (Van Soest, 1994). In this study, there was no difference in sheep ruminal pH and feeding SRU had no detrimental effect on ruminal pH and metabolism. In addition, ruminal pH range of 6.22 to 6.99 was suitable for cellulolytic bacteria and fiber digestion.
After feeding there was a significant effect between different treatments and NH 3 -N concentration (Table 3). Treatment 4 had the lowest NH 3 -N concentration (14.46 mg dL -1 ). Treatments with SRU had significant effect on 'control' treatment. The ruminal NH 3 -N concentrations range from 14.84 to 23.74 mg dL -1 and were close to the optimal ruminal NH 3 -N range (15 to 30 mg dL -1 ) (Anantasook, Wanapat, Cherdthong, & Gunun, 2013). The NH 3 -N was absorbed across the rumen wall into the blood as ammonium. When rumen pH increased, especially above pH 7.0, NH 3 -N absorption increased. The high concentrate rations typically result in lower ruminal pH, hence, decreasing the rate of NH 3 -N absorption into the blood and consequently the risk of toxicity. Furthermore, there is more energy available for NH 3 -N conversion to MPS in the high fermentable concentrate, especially when the urea and carbohydrate degradation rate were synchronized. There are 2 ways N losses can be managed; either by decreasing protein degradation or by increasing the N use by the rumen microbes (Bach, Calsamiglia, & Stern, 2005). The SRU utilization results in a better synchrony between the urea hydrolysis and ammonia utilization by ruminal bacteria. (Holder et al., 2013), demonstrated higher N and microbial CP values for diets with SRU. The ruminal NH 3 -N concentration and blood urea nitrogen were similar among sheep fed different SRU levels. However, the NH 3 -N concentration in treatment 4 was greater at 3 and 6 h after feeding. It could be inferred that SRU diets prolong microbial utilization of additional N sources during ruminal fermentation. Thus, the synchronization between rumen NH 3 -N release and carbohydrate availability may be improved, therefore resulting in higher microbial protein synthesis. Future research is required to 1) investigate the N balance effect on animal performance; 2) to ensure the SRU inclusion level and replacement by SBM 3) to improve tissue deposition and animal performance.

Blood metabolites
Glucose was significantly different between treatments including the SRU and 'control'. Control group had the lowest glucose concentration among treatments (Table 4). Increase in glucose concentration may be due to more protein bypass and increased availability of glucogenic amino acid for gluconeogenesis. This is probably due to increased blood glucose in diets including the SRU, the deamination and glucose synthesis from amino acids has decreased, and consequently, since the amine group of amino acids has not been released, urea production has been reduced by the liver, hence resulting in decreased urea plasma concentration. (Huntington et al., 2006) suggested that feeding urea, decreases insulin and increases plasma glucose concentration due to increased gluconeogenesis in the liver which this result was agree with our results. This experiment showed that feeding high rumen undegradable protein resulted in increased blood glucose concentration due to more glucogenic amino acids availability for gluconeogenesis.
Replacement the SBM by SRU had significant effect on blood urea (Table 4). Treatment 4 had the lowest blood urea. Other studies have reported that the SRU reduced blood urea concentration when compared to feed grade urea (Cherdthong, Wanapat, & Wachirapakorn, 2011;Taylor-Edwards et al., 2009). Huntington et al. (2006) reported no difference in blood urea concentration between the feed grade urea and a slow release urea-calcium. However, the blood urea nitrogen concentration was lower for lactating dairy cows fed by the SRU and SBM rather than regular urea (Gonçalves et al., 2015). Probably, more urea was produced in the liver and then excreted in urine for cows consuming urea (Xin et al., 2010). The excess NH 3 -N in the rumen is absorbed into blood and transported into liver, where it is converted to urea, but if there is excess NH 3 -N in the rumen, the liver is unable to convert it all, therefore increase in in peripheral blood ammonia results in toxicity. This endogenous urea is then excreted into urine or milk, around 10 to 40% of N consumed in the feed is recycled back to the digestive tract as urea from saliva where it can be used again for microbial synthesis or if the needs are already met, the urea is excreted into urine (Bach et al., 2005). In this study, degradable protein intake from the SBM in the diets was effectively replaced by up to 1.5% SRU without any adverse effects on intake or production. In addition, 1.5% urea inclusion in the diet did not increase blood urea and had no negative effect on blood metabolism. The SBM replacement by SRU allows better urea recovery in liver, saliva and rumen. The SRU provides higher urea concentrations that SBM.

Microbial protein synthesis
There was a significant difference among uric acid, excreted purine derivatives, microbial N and microbial protein (Table 5). Ttreatment 1 and treatment 2 had the lowest levels of purine derivatives, microbial N and microbial protein. The SRU replacement at higher levels increased those levels in treatments 3 and 4. Furthermore, excreted purine derivatives in treatment 1 (without SRU) were lower than treatments 2, 3 and 4. Treatment 4 had the highest percentage of excreted purine derivatives, microbial N, and microbial protein.
(Herrera-Saldana, Gomez-Alarcon, Torabi, & Huber, 1990) reported that microbial growth and microbial protein increased by energy and nitrogen synchronization in diet. Excreted purine derivative in urine is an index to measure synthesized rumen microbial protein (Chen & Gomes, 1995) which increased in sheep urine in treatment 4. Experimental rations increased microbial nitrogen and protein. Treatment 4 had the most microbial nitrogen and protein. This research showed that carbohydrate fermentation synchronization with non-protein nitrogen is essential for more efficient microbial protein synthesis. When energy is available, the final products of protein breakdown (peptide, amino acid and NH 3 -N) can be used to make the microbial protein (Bach et al., 2005). It seemed that microbial protein increase in treatment 4, is due to slow and continuous release of nitrogen from SRU (Corte et al., 2018). Therefore, the ruminal NH 3 -N concentration has an impact on MPS, and a synchrony of these processes would improve the efficiency of NPN incorporation into microbial protein and thereby improving the overall efficiency of N use. Hence, the excretion of nitrogen into the environment is reduced and the environmental pollution caused by it is decreased. Lastly, SBM replacement with SRU in the diet can also reduce the final price of the diet, which would be more cost effective than true protein.

Volatile fatty acids
This study demonstrated that acetic acid concentration significantly increased 3 h after feeding, and continued up to 12 hours. It is worth noting that the diet did not play a role on other VFA components. This might be explained by a possible selective effect of urea sources on ruminal microorganisms. Several ruminal microorganisms, especially the fibrolytic bacteria, hold a greater relationship with NPN. Fiber digesting bacteria in the rumen have a preference for NH3-N while producing more acetate than propionate (Van Soest, 1994). Therefore, the supplementation with NPN sources may cause bacteria to pick additional NPN. Volatile fatty acids are the end product of rumen microbial fermentation and represent energy supply for ruminants (Van Soest, 1994). Gardinal et al. (2017) suggested that SRU have no negative effect on ruminal fermentation. Additionally, no change in the total VFA or VFA proportions were reported using urea compared to the SRU. Researchers observed comparable urea effects and recommended that greater acetate and lower butyrate concentrations in diets containing urea produced a lower acetate transformation in conformity with ruminal butyrate (Gardinal et al., 2016;Xin et al., 2010). Khattab, Salem, Abdel-Wahed, &and Kewan (2013) observed greater acetate concentration after feeding urea, and an expansion in microbial protein synthesis. It is possible that adding SRU to high-forage diets provides the NH3-N for fiber digesting bacteria, and induces their growth and population. The results showed that partial SBM replacement by SRU in rations, did not have any negative effect on VFA concentration but, increased the levels of acetic acid.

Conclusion
Replacement of slow-release products has no negative effects on sheep. In addition, in many cases, SRU functioned like SBM, and the released nitrogen increased ruminal microbial protein synthesis compared to SBM The SBM replacement with SRU resulted in the improvement in total purine, allantoin and uric acid excretion, microbial nitrogen, and microbial protein, blood glucose, acetic acid and reduced blood urea. Treatment 4 appeared to perform better but still needs further investigation. Slow release sources of N perform similarly to protein meal and therefore will reduce costs without affecting the livestock production performance.