INTRODUCTION

The increasing broiler production around the world has led to the application of nutritional strategies that improve nutrient utilization. The supplementation of exogenous enzymes to corn- and soybean meal-based broiler diets allows supplying nutrient deficiencies and to reduce endogenous losses, thereby optimizing performance, particularly during early life.

The dietary supplementation of the exogenous phytase reduces nutrient variability of feedstuffs, and counteracts the antinutritional effects of phytate, increasing the accuracy of feed formulation.

Exogenous phytase is included in feed formulations not only to reduce phosphorus supplementation, but also to release minerals, particularly calcium, as well as amino acids and carbohydrates by the hydrolysis of phytate, improving nutrient utilization (^{Oluyinka et al., 2007}; ^{Slominski, 2011}). However, nutrient utilization can also be affected by other factors, including dietary calcium, phosphorus, protein, and energy levels, intestinal pH, environmental temperature, etc.

Dietary calcium content may negatively influence phosphorus utilization, particularly phytic phosphorus, due to the formation of insoluble complexes with calcium in the digestive tract, counteracting the effect of phytase, which is not able to hydrolyze such complexes (^{Leeson et al., 1996}). ^{Nelson et al. (1964)} reported that dietary calcium levels higher than 0.70% at pH 6.0 promote the reaction of calcium with phytic acid, resulting in calcium phytate, which precipitates and cannot be broken down by phytase, consequently reducing phosphorus bioavailability. High dietary calcium levels change the pH of the upper sections of the digestive tract, inactivating phytase. Phytase activity is optimal at pH between 5.0 and 6.5, and it is reduced when pH is lower or equal to 3.0 (^{Casey & Walsh, 2004}).

Broilers fed diets with low inorganic calcium and phosphorus levels have higher capacity to hydrolyze phytate than those fed high levels of these minerals (^{Denbow et al., 1995}). Therefore, minimal calcium and phosphorus levels need to be maintained in the diet; however, their interaction should not be overlooked, particularly considering phytase activity (^{Qian et al., 1997}).

Protein utilization and amino acid absorption may also be influenced by dietary phytic acid levels (^{Lehnen et al., 2011}; ^{Walk et al., 2012}). These authors observed that phytic acid is a potent chelating agent. Its negative charges react with the positive charges of some amino acids (lysine, arginine, histidine), some proteins (including those involved in protein digestion, such as pepsin and trypsin), and carbohydrates (α-amylase), forming insoluble complexes, thereby reducing their availability and digestibility (^{Maenz, 2001}) and consequently affecting metabolism and organ biometry.

Therefore, the objective of this study was to evaluate the supply of feeds containing reduced amino acid, energy, calcium, and phosphorus levels and supplemented or not with phytase on the performance, gastrointestinal pH, relative organ weight, bone mineral content, and blood biochemistry of broilers.

MATERIALS AND METHODS

The experiment was carried out at the Poultry Sector of the Department of Animal Science of the Federal University of Viçosa. In the study, 1,120 male Cobb 500^(r) broilers, with 161±1g average weight and between eight and 21 days of age, were used. A completely randomized experimental design in a 3x2+1 factorial arrangement (Ca and P reduction, energy and amino acid reduction, and Ca, P, energy, and amino acid reduction; supplementation or not with phytase; and a positive control treatment) with eight replicates of 20 broiler each. Until eight days of age, all broilers were reared in a conventional poultry house according to the recommendations of the genetic company's (Manual, 2009). On day 8, broiler were individually weighed and distributed into 56 pens, measuring 1,5 m² and equipped with a nipple drinker and a tube feeder each.

The increase in nutrient and energy bioavailability obtained with phytase supplementation was considered when formulating the experimental diets (Table 1). The phytase product was derived from Escherichia coli, and presented 500 FTU/kg of feed. It was added at a dose of 100g/ton of feed.

The experimental diets were based on corn and soybean meal and were formulated to supply the nutritional requirements of male broilers, as recommended by ^{Rostagno et al. (2011)} for the starter phase.

Birds, feed offer, and feed residue were weighed per pen to calculate weight gain, feed intake, and feed conversion ratio. Mortality was also recorded to correct performance data.

On day 21, two broiler per stall, with body weight close to the average stall weight (±100 g), were selected, identified, and had their blood collected by heart puncture. Blood was placed in test tubes, centrifuged for 5 min at 1,500 rpm, and the serum was then placed in duly identified Eppendorf tubes and immediately frozen until analyses. Serum calcium, phosphorus, albumin, and total protein were analyzed using specific commercial kits.

The selected birds were identified and sacrificed according to the norms of the Committee of Ethics for the Use of Animals of the Department of Animal Science of the Federal University of Viçosa process nº 76/2013. Broilers were stunned, sacrificed by neck dislocation, and their gastrointestinal tract (GIT) with the digesta was aseptically removed. Before removing the digesta, the proventriculus, gizzard, duodenum (from the caudal end of the gizzard until the end of the duodenal loop), jejunum (from the end of the duodenum until Meckel's diverticulum), ileum (from Meckel's diverticulum until the ileal-cecal junction), and the ceca were isolated to prevent the digesta from mixing. Then, the pH was measured sing a digital pHmeter gently introducing the sterilized fine tip of the glass electrode in each GIT section. As recommended by ^{Pang & Applegate (2007)}, pH was measured twice in each section and the average of the two measurements was used for the statistical analysis. Subsequently, the intestine, gizzard, liver, heart, bursa, and spleen were removed. The intestine was measured using a measuring tape.

The left tibia bone was collected, dehydrated in ethanol, and de-fatted in petroleum ether in Soxhlet apparatus for four hours. Bones were then burnt in a muffle at 600ºC to determine ash content, according to Gardiner et al. (1961). Ashes were then used to prepare a mineral solution by dissolving the ashes by digestion on a heated plate for four hours at 200ºC. Calcium content in the ashes was determined by atomic absorption spectrophotometry and phosphorus content was determined by colorimetric spectrophotometry, according to the methods described by Detmann.

Data were submitted to Shapiro Wilk's normality test and then to analysis of variance using the General Linear Model (PROC GLM) procedures of SAS statistical package (1998). Means were compared by orthogonal contrasts. Each contrast was independently tested by the F test a 10% probability level.

The following orthogonal contrasts were analyzed: contrast C₁, comparing diets with reduced calcium (Ca) and phosphorus (P) levels supplemented or not with phytase; contrast C₂, comparing diets with reduced amino acid (AA) and energy (ME) contents supplemented or not with phytase; contrast C₃, comparing diets with reduced AA, ME, Ca and P levels supplemented or not with phytase; contrast C₄, comparing the positive-control diet and the diet with reduced AA, ME, Ca and P levels and not supplemented with phytase; contrast C₅, comparing the positive-control diet and the diet with reduced AA, ME, Ca and P levels and supplemented with phytase, and contrast C₆, comparing the diets with reduced nutrient levels supplemented or not with phytase.

RESULTS

Contrast C₁ shows that the performance of broilers fed the diet with reduced Ca and P levels and supplemented with phytase was better (P<0.01) than those fed the same diet with no phytase supplementation, with improvements of 4.40, 11.04, and 7.14% in feed intake (FI), weight gain (WG), and feed conversion ratio (FCR), respectively (Table 3).

The contrast comparing the supplementation or not of phytase of diets with reduced Ca, P, AA, and ME level (Contrast C₃) indicated that broilers receiving phytase presented better FI (p<0.01), WG (p<0.01), and FCR (p<0.10), which improved 4.14, 7.69, and 3.59%, respectively. Interestingly, the improvement in performance with phytase supplementation was more evident in broilers fed reduced Ca and P levels (contrasts C₁ and C₃).

When the positive-control diet was compared with the diets supplemented with phytase (contrast C₅), it was observed that the dietary reduction of nutrients and energy did not influence broiler performance (p>0.05). On the other hand, broilers fed the positive-control diet presented better FI and WG (p<0.01) compared with those fed the diet with reduced nutrient and energy levels and no phytase supplementation (contrast C₄).

When the diets with reduced nutrient and energy levels and supplemented or not with phytase were compared (contrast C₆), phytase supplementation improved WG and FCR (p<0.01).

The addition of phytase to the reduced AA and ME diet (contrast C₂) reduced the pH of the proventriculus (p<0.10) and of the gizzard (p<0.01) in 11.41 and 19.54%, respectively (Table 4).

The pH of the proventriculus (contrast C₄) and of the gizzard (contrast C₆) increased (p<0.10) when phytase was added to the diets.

There was no influence of treatments on gizzard relative weight (Table 5). However, when the phytase supplementation was evaluated in reduced Ca and P diets (contrast C₁), liver (p<0.10) and heart (P<0.01) relative weights increased in 9.06 and 13.33% in broilers fed the diet with no phytase addition.

Heart relative weight increased (p<0.01) when broilers were fed the reduced nutrient diets with no phytase additions (contrasts C₄ and C₆).

Intestinal length was different (p<0.10) in broiler fed diets containing phytase, as shown in contrast C₅.

Tibial ash content (p<0.01, % DM) and phosphorus deposition (p<0.10; % DM) increased 9.53 and 13.56%, respectively, when broilers were fed the diet with reduced Ca and P levels supplemented with phytase (contrast C₁; Table 6).

Tibial ash content (%DM) was significantly different (p<0.05), as shown by contrast C₃. Broilers were fed the reduced nutrient diets containing phytase than those fed the positive-control diet (contrasts C₄ and C₅) presented higher tibial ash content (% DM). In addition, when the positive-control diet was compared with the reduced Ca and P diet containing phytase, there was higher Ca and P deposition (% DM), with 5.32 and 13.77%, respectively. Contrast C₅ did not reveal any differences (p>0.05) in tibial Ca or P content (% DM).

As determined by contrast C₆, tibial Ca and P deposition (% DM) and Ca to P ratio were higher (p<0.05) when the diets were supplemented with phytase.

Blood albumin levels (g/dL) were higher (p<0.10) in birds in the treatments with no phytase supplementation, as shown by contrasts C₁ and C₂ (Table 7).

Blood P levels (mg/dL) were different (p<0.10) as determined by contrasts C₃ and C₄, as well as Ca/P ratio detected by C₁ (p<0.01), C₃ (p<0.01)and C₄ (p<0.05).

DISCUSSION

Literature studies show differences in the capacity of phytase of releasing P from phytate due to influence of dietary Ca content. The better performance of broilers fed reduced Ca and P levels and phytase obtained in the present study suggest that phytase promoted better Ca and P utilization. This is consistent with the results of ^{Powell et al. (2011)}, who found that it phytase is used more efficiently when dietary Ca levels are reduced.

These results also confirm that the use of enzymes in corn- and soybean meal-based broiler diets promote better performance (p<0.01) by breaking down the antinutritional factors present in the cell wall of plants (^{Graham et al., 2002}), and hence, better nutrient absorption and animal performance (^{Costa Leite et al., 2011}; ^{Powell et al., 2011}; ^{Walk et al., 2012}).

On the other hand, ^{Alvarenga et al. (2011)} obtained lower broiler weight gain when the diets contained phytase. Also, in his review, ^{Slominsk (2011)} found little effect of phytase supplementation on performance and the utilization of energy and amino acid utilization in broilers. These differences may be related to the different source of phytase currently available in the market (^{Elkhalil et al., 2007}). The positive results obtained in the present study may be possibly attributed to a better efficiency phytase derived from E. coli.

Another possible cause of the improved responses may have been the lower dietary Ca levels. Inadequate dietary Ca levels may impair Ca, P, Zn, and Mg absorption. However, Ca reduction in phytase-supplemented diets must be proportional do total phosphorus (tP) reduction in order to maintain the required Ca:P ratio. According to ^{Pereira et al. (2012)}, the inclusion of phytase in feed formulation matrices provides economic and environmental benefits in broiler production.

The lower pH values observed in the gizzard (p<0.01) and the proventriculus (p<0.10) of broilers fed the reduced nutrient and energy level and no phytase can be attributed to the attempt of birds to balance nutrient intake and energy utilization to supply their requirements.

Phytate is hydrolyzed mainly in the upper gastric tract (proventriculus and gizzard), where the pH favors the action of phytase and its substrate is more water soluble (^{Selle & Ravidran, 2007}). According to ^{Penz et al. (1993)}, GIT pH reduction enhances enzyme activity, thereby increasing dietary nutrient absorption and improving animal performance.

The observed increase in liver (p<0.10) and heart (p<0.01) relative weights in the broilers fed diets with reduced Ca and P levels and no phytase supplementation is probably due to low phosphorus supply. Heart hypertrophy (increase of heart muscle mass beyond the usual limit) may be caused by hypophosphatemia due to low phosphorus supply (^{Rosário et al., 2004}; ^{Temprano et al., 2004}). Hearts with hypertrophy usually present reduced contraction due to reduced myosin ATPase activity and lower cyclic adenosine monophosphate (cAMP), which may result from hypophosphatemia. This may lead to heart failure, reducing the systolic volume and increasing myocardial fiber diameter (^{Riedesel, 2006}).

Low ATP production stimulates the synthesis of liver enzymes, increasing liver volume (^{Reis et al., 1999}). ^{Oliveira et al. (2009)} observed reduced liver and heart relative weights when broilers were fed diets with 70% reduction in non-phytic phosphorus level.

The increase in tibial ash (p<0.01, % DM) and phosphorus deposition (p<0.10; % DM) determined in broilers fed the diet with phytase addition reduced Ca and P levels and in ash content (p<0.05) in broilers fed the reduced Ca, P, AA, and ME diet with phytase supplementation was expected, because phytase increases the release of phosphorus and other minerals from the feedstuffs. According to ^{Nelson & Walker (1964)} and ^{Pereira et al. (2012)}, bone ash content is the most efficient parameter to estimate the amount of phosphorus released by phytase in corn- and soybean meal-based diets.

^{Geraldo et al. (2006)} found 1.88% reduction in tibial ash content for each 1% increase in dietary calcium levels. The dietary supplementation of phytase allows reducing dietary available phosphorus in 0.15% and Ca in 0.15% (^{Gomide et al., 2011}). Consistent with the present study, ^{Schoulten et al. (2003)} observed that reduced P diets supplemented with phytase allowed reducing Ca requirements to lower values than those present in nutritional tables, but recommended that dietary Ca reduction should be proportional to total P reduction, maintaining adequate Ca to P ratio, as also mentioned by ^{Qian et al. (1997)}.

In the present experiment, average total protein levels in the blood ranged between 2.88 (reduced Ca, P, AA, and ME and no phytase) and 3.11mg/dL (reduced Ca, P, AA, and ME and with phytase) are below those considered normal (3.6 mg/dL). According to ^{Viveiros et al. (2002)}, total protein level in the serum may be reduced in case of malnutrition or low intestinal absorption of nutrients, but the treatments applied in the present study did not cause any of these conditions.

Blood Ca level (mg/dL) was not influenced (p>0.10) by the applied treatments, possibly because Ca homeostasis in the extracellular fluid is maintained by the joint effects of the parathyroid hormone (PTH), calcitonin, and 1,25 di-hydroxy vitamin D (1,25(OH)₂D₃) in the intestines, kidneys, and bone. Those hormones regulate Ca flow between these organs and the extracellular fluid, resulting in less than 5% variation in Ca blood levels under normal circumstances, and only occurs when hormonal mechanisms fail, secondarily to reduced intestinal absorption (^{Macari et al., 2002}). In the present study, mean Ca serum values were lower than those obtained ^{by Vieites et al. (2011)}, of 6.72 and 6.89mg/dL.

The higher albumin values (p<0.10) found in broilers fed reduced nutrient and energy levels and no phytase supplementation are considered normal (1.6-2.0 g/dL; ^{Reece & Swenson, 2006}).

Albumins are synthesized in the liver and are very abundant in blood plasma, where they play an essential role in the maintenance of the osmotic pressure (^{Junqueira & Carneiro, 2011}). Differences in serum albumin levels, according to ^{Macari et al. (2002)} are probably due to acid-base imbalance (metabolic acidosis or alkalosis), because of the buffering action of these proteins that capture or donate H⁺ ions by their carboxylic terminals (COOH or COO^-) attempting to reestablish normal blood pH. This was possibly the case in the present study, and this hypothesis is corroborated by the increase in liver relative weight,

Consistent with this result, the Ca/P ratios presented in contrasts C1, C3, and C4 show higher concentration of P relative to Ca, causing osmotic pressure imbalance (^{Reece & Swenson, 2006}). This difference in the Ca/P ratio is mainly due to P variation (mg/dL), which was different in contrast C3 and C4 (p<0.10). According to ^{Vargas Jr. et al. (2003)}, the Ca to P ratio must be constant; any imbalance may affect body functions.

The difference in the glob/alb ratios obtained in contrasts C1 and C6 may be explained by the reduction of this ratio in the treatments with phytase, as shown in the contrasts between treatments with or without phytase and Ca and P reduction (contrast C1). This was caused by variations in the albumin fraction, demonstrating the effect of Ca and P reduction on the metabolism of the liver, which is the only site of albumin synthesis.

The diet with reduced amino acid and energy levels and phytase addition reduced gizzard and proventriculus pH. Dietary Ca and P reduction increased relative liver and heart weights, as well as albumin blood levels. The bones of broilers fed phytase-supplemented diets presented higher ash content. The results of the present study suggest that broiler nutritional requirements need to be reevaluated when the diet is supplemented with phytase.