Can cactus (Opuntia stricta [Haw.] Haw) cladodes plus urea replace wheat bran in steers’ diet?

Objective The study aimed to evaluate the effect of replacing wheat bran for cactus cladodes plus urea (0%, 25%, 50%, 75%, and 100%) on the intake of nutrients, nitrogen balance, microbial protein synthesis, and rumen fermentation for steers. Methods Five crossbred steers (1/2 Holstein-Zebu), with rumen cannula and an average body weight of 180±5.3 kg, were assigned to a 5×5 Latin square design. Dietary treatments consisted of the replacement of the total of wheat bran in basal diet by cactus cladodes using the following proportions: 0% for basal diet, 25%, 50%, 75%, and 100% cactus cladodes replacing wheat bran. Urea was added to the diets to adjust the crude protein (CP) content to 130 g/kg dry matter. Results Maximum dry matter intake (5.73 kg/d) and maximum nitrogen balance (103 g/d) were estimated for 54.6% and 70.8% replacement levels of wheat bran. The maximum microbial protein production (44.6 g/d) was obtained at a replacement level of 49.7%, and a medium value (125 g CP mic/kg total digestible nutrients) of microbial protein efficiency was observed. The rumen pH increased linearly according to cactus cladodes inclusion, while the ammonia nitrogen medium value was 24.5 mg/dL. Conclusion The replacement of 55% wheat bran for cactus cladodes plus urea in the diet of crossbred steers is recommended.


INTRODUCTION
In semiarid regions, the occurrence of frequent droughts determines the success of economic activities such as livestock and agriculture, due to the seasonality of forage production. To overcome this problem, farmers are forced to import grains from other regions to meeting the animals' nutrient requirements, representing an increase in production cost. A greater feed ingredient in ruminant nutrition is the wheat bran, a byproduct with a high energy content and relevant crude protein (CP) [1], however its inclusion in diet represents increases in the cost, once US$ 0.22 per kg of dry matter (DM).
In this scenario, the use of local alternative feeds as cactus cladodes encourages the live stock sector [2] been an essential to sustainability of the productive system in arid and semiarid regions [3]. The cactus cladodes present adaptive agronomic characteristics, such as being tolerant to hydric stress, and being suitable as forage in diets for dairy goats, dairy cattle, and sheep [46], respectively. It also has a high content of nonfibrous carbohydrates (NFC), and consequently significantly high energy content compared with other forage plants [7,8]. Nonetheless, the cactus cladodes are poor in protein, and thus the nonprotein nitrogen (NPN) compounds, such as urea, should be used to increase dietary protein content, due to its low cost compared to true protein sources such as soybean meal [9].
The urea can totally replace the protein ingredient in bovine diets confined fed medium concentrated meal for 1 kg/d of average daily gain [10]. The elevations in the hepatic ureagene sis could increase the losses of urine nitrogen, compromising the utilization efficiency of the dietary nitrogen compounds [11]. It has long been recognized that supplemental NPN is most efficiently utilized in rations low in protein and relatively high in digestible energy. However, the maximum level of inclusion of urea in ruminant diet that compromising the performance it not clear.
In these assumptions, it was speculated that due to the high content of carbohydrates rapidly fermentation in the rumen in diets based on cactus cladodes, it was possible to raise the level of urea without compromising animal intake and meta bolic condition, since a greater amount of ammonia could be assimilated by the rumen microbiota. Thus, this study aimed to evaluate the effect of replacing wheat bran for cactus cladodes plus urea on the N balance, microbial protein synthesis, and rumen fermentation in crossbred steers.

Animal care
The management and care of animals were performed in ac cordance with the guidelines and recommendations of the Committee of Ethics on Animal Studies at the Federal Rural University of Pernambuco (License N°009/2015), Recife, Brazil. This study was carried out in the Department of Animal Sci ence at the Federal Rural University of Pernambuco, located in Recife, Pernambuco State, Brazil.

Animals and diests
Five rumen fistulated steers (1/2 HolsteinZebu) with an average initial body weight (BW) of 160±5.3 kg were assigned in a 5×5 Latin square design. The experiment lasted 80 days, corresponding to five 16day periods. The first seven days were allocated to the adaptation of the animals to the experimental diets according to [12,13], followed by nine days of sample collections. The animals were weighed, identified and vermi fuged prior to the experiment and housed in individual pens equipped with feeders and drinkers.

Experimental procedure and chemical
The chemical composition of the ingredients and proportion of the ingredients in the concentrate mixture and the chemi cal composition of the diets are shown in Table 1   treatments consisted of the replacement of the total of wheat bran in basal diet by cactus cladodes using the following pro portions: 0% for basal diet, 25%, 50%, 75%, and 100% cactus cladodes replacing wheat bran. Urea was added to the diets to adjust the CP content to 130 g/kg DM. The fresh sugar cane and cactus cladodes were cut and chopped daily and then provided to animals. The DM con tent of sugarcane and cactus cladodes was evaluated weekly to adjust the amount of feed allowed to the animals. The mix ture of ingredients was performed manually in the feeders, highlighting that the cactus cladodes mucilage allowed a uni form aggregation of urea. The diets were supplied ad libitum, allowing approximately 100 g/kg as orts. The animals were fed twice daily in equal portions at 06.00 h and 18.00 h. Water was provided ad libitum throughout the experimental period.
The intake of DM and nutrients from the diets was calcu lated as the difference between the total nutrients in the feed offered and the total nutrients present in the orts. Forage pro vided and orts were sampled daily during the collection period and subjected to partial drying in a forced ventilation oven set at 60°C for 72 h. The ingredients that comprised the con centrate were sampled directly from the feed mill silos on the days that they were mixed. All samples were processed in a Wiley mill to pass through a 2mm screen sieve. After that each sample was homogenized and divided in two portions. Half of each sample was processed again in the same mill to pass through a 1mm screen sieve.
The NFC contents were quantified according to [15] as follows: NFC = OM-[(CP-CPu+U)+NDF+EE +MM]; where CPu = CP content from urea, U = urea content, and NDF = NDFap corrected for residual ash and protein. The other terms were previously defined and all of them are expressed as g/kg DM.
For three days in each experimental period, after provid ing the morning diet, total feces and urine collection (24 h) were performed, and the pH (the urine) was measured every 6 h to make sure that it is below 3.0. To collect urine samples, funnel collectors were coupled to animals and attached to hoses used to conduct urine to a container containing 500 mL of 20% sulfuric acid. At the end of each collection period, the weight and total volume of urine were measured, and the total N content was determined using the Kjeldahl method INCT CA N004/1 [14]. The urinary purine derivatives (PD) were quantified using the colorimetric [17]. Urea (blood and urine) levels were measured from commercial kits, using a colori metric system in a semiautomatic biochemical analyzer D250 (Labtest, Lagoa Santa, MG, Brazil). Blood samples were col lected after 11 days in each experimental period, 4 h after the morning feeding, through a puncture in the jugular vein using vacutainer tubes containing a separation gel with a coagulant activator (SST II Advance, BD Vacutainer, Curitiba, PR, Brazil). The samples were immediately centrifuged (5,000 rpm for 20 min) to remove plasma and analyze the urea content.
The N balance estimate was obtained by subtracting the fecal and urinary excretion values from ingested N. To deter mine the efficiency of dietary N compound utilization, the following indicators were used: Nurea in plasma, urinary excretion of Nurea, and N balance. The ureaN from plasma and urine was estimated using the factor 0.466 [18].
The total excretion of PD was calculated as the sum of the quantities of allantoin and uric acid excreted in the urine. Absorbed purine concentrations (X, mmol/d) were calculated using the equation Y = 0.85X+0.385BW 0.75 [19].
Rumen fluid was collected from the 11th to 13th day of each experimental period, before feeding and two, four, and six hours after feeding. The manual collection was performed at several locations in the ruminal environment, taking a repre sentative sample of the content (100 mL), which was filtered through cotton fabric. After collection, pH was measured with a digital potentiometer. Immediately after collection, the liquid was frozen (-15°C) for later analyses. The ammonia nitrogen (NNH 3 ) concentration was determined after samples were centrifuged at 3,000 rpm/15 min, using the supernatant for analysis by Kjeldahl method INCTCA N007/1 [14].

Statistical analyses
The variables studied were analyzed with the PROC MIXED option in SAS software (version 9.4), adopting 0.05 as the criti cal level of probability for type I error, according to the following model: Y ijk = μ+T i +a j +p k +ε ijk Where, Yijk is the dependent variable measured in animal j that was subject to the i treatment in period k; μ is the gen eral mean; T i is the fixed effect of treatment i; a j is the random effect of animal j; p k is the random effect of period k; and ε ijk is the unobserved random error assuming normal.
After analysis of variance, the significance of the linear and quadratic effects of the replacement of the total of wheat bran in the basal diet by cactus cladodes was evaluated. Rumen pH and NH 3 N were considered the effect of sampling time as repeated measures in time.

RESULTS
Intake of DM, CP, and nitrogen as excretion of urinary nitro gen and nitrogen balance demonstrated a quadratic pattern, whereas excretion of fecal nitrogen decreased linearly (p<0.01; Table 3). The maximum values of intake of DM (5.73 kg/d), CP (830 g/d), N (132 g/d), N balance (103 g/d) and minimum value of urinary nitrogen (11.5 g/d) were estimated at 54.6%, 56.7%, 56.8%, 70.8%, and 76.7% of replacement of wheat bran for cactus cladodes plus urea, respectively.
The plasmatic concentration of urea and ureaN presented a quadratic pattern (p<0.01; Table 4), whereas the maximum plasma levels of urea (41.9 mg/dL) and ureaN (19.5 mg/dL) were estimated for 55.1% and 54.9% of replacement of wheat bran for cactus cladodes plus urea, respectively. The urea excre tion in urine increased linearly by 3.05 mg/kg BW for every 1% increase in replacement levels ( Table 4).
The microbialN and microbial protein presented a qua dratic response in accordance with the replacements levels (p<0.01; Table 4). The maximum microbialN (49.9 g/d), microbial protein (443.9 g/d) were estimated at 74.0, 49.7 of replacement of wheat bran for cactus cladodes plus urea, re spectively. The microbial protein efficiency (125.2 g CP mic/kg TDN) was not altered by the replacement (p>0.05; Table 4).
The replacement levels influence the pH which increased according to cactus cladodes plus urea increment (p<0.001; Table 5). There was an interaction between time and replace ment for pH, and a deployment was made (p<0.01; Table 5). For the replacements levels of 0% and 25% were observed as a quadratic pattern through the time, where the minimum pH values were 5.92, 6.05 at 3.96, and 5.92 hours, respectively, after feeding ( Figure 1). Instead of it, for all collection time after feeding, the pH increased linearly according to cactus cladodes plus urea inclusion in the diets ( Table 6).
The rumen NH 3 N varied through time, presenting a quad ratic pattern with a maximum value of 31.9 mg/dL estimated

DISCUSSION
Replacement of 54.6% wheat bran with cactus cladodes al lowed a greater nutrient intake, which suggests that cactus cladodes, owing to its chemical characteristics (Table 1), im proved the flow of digestion through the gastrointestinal tract, resulting in increased intake [20]. The negative effect of greater levels of cactus cladodes in diets on voluntary intake could be associated with the high moisture content in this feed, which increases its capacity to occupy space in the rumen envi ronment [21]. According to [22], water content in forages exceeding 700 g/kg can compromise voluntary intake. Moisture of the greater level of cactus cladodes diet was estimated at 840.3 g/kg.
In parallel to the above, it should be considered the ani mals' urea intake amount calculated at 83 g/100 kg BW for 54.6% of replacement level is considered high up in the recom mendations for ruminants (0.45 to 0.50 g/kg BW) [23]. High level of urea in diet decreases the palatability and rises the intoxication risk due to its greater solubility in the rumen and consequent absorption [24]. Concomitantly, the increases in ruminal pH facilitates the rapid transport of ammonia across the rumen epithelium, resulting in a rapid increase in blood ammonia [25]. In accordance to the exposed in Table 5, a lin ear increase was noted due to the replacement, insofar there was an increase in plasma urea concentration and urea ex cretion in urine (Table 4), confirming that those aspects may contributed to the DM intake drop for the replacement level from 54.6%.
The maximum recommendation of urea seems to be some  Figure 1. pH values for different replacement levels (0%, 25%, 50%, 65%, 100%) of wheat bran for cactus cladodes plus urea through the time. what conservative, since, despite being associated to DM intake depression, the animals did not show symptoms of intoxica tion over the experimental period, even considering that the urea intake was twice of the amount recommended by the cited authors. Thus, the observed values for urea inclusion in the diets ratify the recommendation of [10] who suggested that urea can totally replace the protein ingredients in bovines' diets, guaranteeing moderated daily gains (0.8 to 1.0 kg/d). At replacement levels above 54.6%, the input of N rumen degradable (urea) increased significantly, promoting an excess, which is also evidenced by the increase of plasma ureaN (Table 4). According to [26], concentrations between 14 and 16 mg/dL of plasma ureaN in Zebu steers allows for maxi mal microbial efficiency, and above these levels, as observed in this study (19.5 mg/dL for 54.9% replacement; Table 3), dietary protein losses begin [27]. The excess urea concentra tions, as demonstrated by increased excretion in the urine [28] and plasma (Table 3), indicate the impairment of diet palat ability (bitter flavor of urea) and rumen fermentation. In this case, it may be inferred that the energy released by NFC fer mentation was not sufficient to assimilate the large amount of readily available N (NPN) from rumen microorganisms.
The reduction in the loss of N in the feces (Table 3) can likely be explained by the increased use of NH 3 N by rumen microorganisms (Table 5). Furthermore, the smaller N loss in the urine (estimated at 76.5% replacement of wheat bran) suggests the largest conversion of N to urea, which is seen in the increased urinary excretion of urea (Table 3).
According to [29], when the rumen protein degradation rate exceeds the rate of carbohydrate degradation, an increase in the excretion of nitrogenous compounds and urea produc tion occurs. Due to the higher energy costs for urea synthesis in animals [30], a reduction of the energy available for micro bial protein synthesis occurs, confirming the results obtained for this variable above the 50 % replacement level of wheat bran ( Table 4).
The maximum N balance (NB) estimated at a 70.8% re placement level of wheat bran with cactus cladodes (Table 3) complements the results obtained for N excretion in urine and feces in that the lower excretion of this nutrient indicates higher retention in the organism. The protein utilization in animal metabolism at replacement levels close to 75% of the DM of diets justifies microbial efficiency pattern and no re placement influence (medium value of 125.2 g microbial CP/kg TDN; Table 4), consistent with results proposed by [31] for Zebu crosses where 120 g microbial CP/kg TDN was used as a reference for microbial efficiency in tropical conditions.
There was a linear increase in rumen pH, demonstrating an improvement in the rumen environment equilibrium with the cactus cladodes plus urea inclusion ( Table 5). The muci lage in cactus cladodes may stimulate salivation, thus preventing pH decrease [2]. Also, the greater pH values may be associated with the biggest amount of urea input in rumen according to increased replacement levels, as mentioned above, due to its rapid solubilization with consequent ammonia amassing, which has a wellknown alkaline potential [32,33].
To highlight the difference in carbohydrate profiles for the diets, it is seen that those with more inclusion of wheat bran present a more content of starch versus cactus cladodes diets, which directly impacts the rumen fermentation pattern and is confirmed in Figure 1. The diet with no cactus cladodes in clusion shows a rumen pH minimum of 5.92 estimated at 3.96 hours after feeding vs a linear decreasing (7.10 to 6.8; Table  6) in ruminal pH for diets with 50.51% of cactus inclusion.
The concentration of NH 3 N in the rumen is important for microbial growth, and is largely dependent on the amount of substrate and the fermentation of the OM present in the rumen. In this study, an increase in the intake of NPN, de rived from urea, probably resulted in a higher concentration of rumen NH 3 N (24.5 mg/dL), as found by [34]. The maxi mum rumen NH 3 N presented is accordance with the amount (10 to 23 mg/dL) suggested by [35] for maximum for maxi mum rumen fermentation activity and microbial growth.
When urea is supplied in the diet of ruminants, the peak rumen ammonia is usually 1 or 2 h after a meal, as results proposed by [36]. In this study, the maximum concentration of rumen NH 3 N (31.9 mg/dL) occurred only 3.82 h after feeding. This result may be related to the quality of the fiber used in the diets, derived from sugar cane and wheat bran, where the association made the microbial fermentation slower, thus promoting greater retention of food and delaying the peak in NH 3 N.
On the basis of the results of this study, replacement of 55% wheat bran with cactus cladodes in the diet of steers is recom mended to promote a better DM intake and microbial protein synthesis. In semiarid regions, the cactus cladodes can be considered as an alternative to wheat bran without altering microbial synthesis efficiency, which contributes to animal production in periods of forage restriction.

IMPLICATIONS
The paper provides brand new about animal production and nutrition information, relating to cactus cladodes plus urea inclusion in cattle diets. Due to semiarid climate condition, there is no expressive grains production, raising animal´s feeds cost. Thus, the information presenting proposes the use maxi mization of cactus cladodes plus urea in replacement to wheat bran, which guarantees lower prices to feed the cattle and the system production sustainability in semiarid areas.