Introduction

In the economics of poultry farming, feed cost accounts for 65–70% from which 30% is composed of protein supply (Moosavi et al. 2011). Improvement in broiler nutrition and continuous genetic selection resulted in a faster growth rate in modern strains, which ultimately has increased the requirement of CP and amino acids (Hartcher and Lum, 2020). Predominantly, the CP is derived from soybean meal (SBM) due to well-balanced amino acids (AAs) coupled with a higher rate of digestibility. However, SBM meets about 80% of the protein and amino acid requirements (Ndazigaruye et al. 2019). The ideal balance of AA and nitrogen would be necessary for poultry rather than enduring the CP (NRC, 1994). To achieve the maximum growth potential of fast-growing broilers, the importance of a balanced AA pattern in the diet is inevitable. When the dietary AAs are precisely balanced, the rate of body tissue synthesis and efficiency to achieve the desired growth rates is quite practicable (Wecke and Liebert, 2013). Thus, a systematic addition of AA not only enhances broiler feed efficiency but also provides a surplus benefits of suitable reduction in CP levels to curtail the feed cost as well as to decrease ammonia emission in the litter (Hernández et al. 2013).

Lysine is used as a reference AA to which all other essential AAs are balanced in the ideal AA patterns, and its analysis in feedstuffs is simple as compared to others (Farkhoy et al. 2012; Abdel-Maksoud et al. 2010). Higher dietary lysine content also improves protein retention, increases feeding efficiency, and minimizes fat retention of broiler carcass (Saima et al. 2010). The absorbed lysine, in the best way, is used for protein accretion and is essential for the breast meat yield (Garcia and Batal, 2005). Thus, the essential AA balanced at an ideal lysine ratio paves a path to formulate a low CP diet while sustaining the broiler performance (Macelline et al. 2020).

In certain circumstances, the ideal digestibility of protein may get hindered due to excessive dietary CP, imbalance of AA, certain anti-nutritional factors, as well as lower availability of endogenous enzymes (Parsons et al. 1997). Protein digestion in monogastric animals occurs mostly by endogenous proteases released in the gastrointestinal tract (GIT), and the quantity released is frequently enough to optimize the utilization of feed protein (Ndazigaruye et al. 2019). Conversely, a considerable amount of protein (~18 to 20%) passes through the GIT without being digested (Lemme et al. 2004). Therefore, exogenous proteases have been added to broiler feed to enhance its nutritive value (Cowieson and Roos, 2016). The dietary exogenous enzyme can facilitate the protein utilization that is otherwise unavailable to the animal, particularly when feed ingredients vary in quality or bioavailability (Kocher et al. 2002; Law et al. 2018). Furthermore, the use of exogenous protease may also help to reduce the CP contents of the broiler diet (Mehmood et al. 2018).

However, to our knowledge, the studies uniting the aspects of AA balanced at ideal lysine ratio, reducing CP with added exogenous protease in broiler diet are quite limited. Therefore, the current study is conducted to investigate the interactive effect of amino acids balanced at ideal lysine ratio with exogenous protease supplemented to low CP diets in broilers and impact on growth performance, carcass traits, gut morphology, and serum metabolites.

Materials and methods

This study was carried out at Poultry Research and Training Centre, University of Veterinary and Animal Sciences, Lahore, Pakistan. The experimental procedures used in this study were approved by the Animal Ethical Review Committee (AERC) of UVAS, Lahore.

Enzyme composition

A concentrated acid protease (Product of VTR, Bio-Tech Co., Ltd. YIDUOZYME X-3001, China) was used in the current study. This enzyme is produced through a combination of both submerged solid-state fermentation and liquid-state fermentation technologies derived from Aspergillus niger with a PROT activity of 2000 units/g. One PROT unit was defined as the amount of enzyme that releases 1 μmol of P-nitroaniline from 1 μM of substrate per minute at pH 9.0 and 37 °C.

Housing management

A total of 480 straight run Ross-308 broiler chicks (procured from 45 weeks old parents) were evaluated between the periods of 1 to 35 days old. All birds were maintained to the standard management practices, according to the genetic company’s guidelines (Ross, 2019). Before the chick’s arrival, the experimental house was washed with freshwater and then disinfected with (Virkon® S, USA [potassium peroxymonosulfate 21.41%, sodium chloride 1.50%] used as 2% solution]). Fumigation was performed using a mixture of formalin and KMNO4 (35 ml 40% formaldehyde added to 10g KMNO4/ft3) a couple of days before placing the chicks. The temperature of the house was maintained at 35 °C during brooding and reduced to 3 °C on weekly basis by using installed brooders and exhaust fans. Rice husk was used as litter material placed around 3–4 inches on the floor. Birds were immunized against Newcastle disease on day 4 (intraocular) and day 20 (drinking water) and against infectious bursal disease on day 8 (intraocular) and day 24 (drinking water). The photoperiod applied during the experimental period was 23L:1D, with 5 lux of artificial lighting intensity.

Experimental design and diets

A completely randomized design in a 2 × 2 factorial arrangement was applied. The 4 treatments consisted of two lysine ratios (100% and 110%; factor 1) without or with exogenous protease supplementation (200 g/ton; factor 2) with 6 replicates; the study unit was 20 birds.

For all the treatments, the diet with a 20% reduction of CP (from given standards of Ross-308, 2019; Table 1) was formulated for both starter and grower phases. In the current study, the 20% reduction of CP in diets was deduced following the work of Law et al. (2019) and Wang et al. (2020). Among four dietary treatments, the first was T1 designated as LR100-ve, being offered with formulated diet at CP level of 18.4% (reduced from 23% as per standards of Ross-308) in starter phase and at CP level of 17.2% (reduced from 21% as per standards of Ross-308) in grower phase, whereas AA balanced at 100% LR without protease. T2 designated as LR100+ve was offered the same diet as T1 with the supplementation of exogenous protease (at 200 g/ton; Yiduozyme X-3001, VTR, China). T3 designated as LR110-ve with dietary CP of 18.4% and 17.2% in starter and grower phases respectively with AAs balanced at 110 % LR without protease. T4 designated as LR110+ve was offered the same diet as T3 with the supplementation of exogenous protease (at 200 g/ton; Yiduozyme X-3001, VTR, China).

Table 1 Ross-308 nutrient specification
Full size table

Broiler starter diet was offered from days 1 to 21, whereas grower diet was offered from days 22 to 35 (Table 2). Provision of ad libitum feed was ensured for the whole experiment. An uninterrupted supply of clean and freshwater was provided through a nipple drinking system.

Table 2 Ingredients and nutrient composition of diets (as-fed basis)
Full size table

Sample analysis

Chemical evaluation of feed ingredients was performed by proximate analysis following standard methods of AOAC (2019) in the lab of the Department of Animal Nutrition, University of Veterinary and Animal Sciences, Lahore, Pakistan. The samples of individual feed ingredients and formulated feed were ground through Willey mill (Arthur H. Thomas Co) in which ground particles pass through the 2-mm screen, and these were further ground through the 1-mm screen of the cyclone mill (CT 293 CYCLOTEC™). These samples were further dried in a hot air oven (Universal oven UF260, Memmert GmbH + Co. KG) at 105 °C for 4 h to attain a constant weight. Grounded and dried samples were analyzed for ether extract (EE), crude protein (CP), crude fiber, and total ash estimation. Determination of crude protein was done by micro-Kjeldahl method. The total ash content of feed ingredients was estimated through a muffle furnace (Box-Type Resistance Furnace, SX-2.5-10). Crude fiber was determined by using ANKOM fiber analyzer (Ankom Technology, AOAC International, 2019). Ankom fat extractor was used to determine the ether extract by using petroleum ether (AOAC International, 2019).

Parameters evaluated

For growth performance, feed intake, weight gain, feed conversion ratio, and livability were evaluated throughout the experimental period as adapted by Yameen et al. (2020).

At the age of 35 days, three birds from each replicate (18 birds from each treatment; total of 72) were randomly selected and slaughtered (PS3733: 2016) for the evaluation of carcass traits, gut health, and serum metabolites. Parameters regarding carcass traits were recorded as live weight, carcass weight, carcass yield, heart, liver, gizzard, thigh, breast weight, and abdominal fat. Carcass yield and abdominal fat weight were calculated as a percentage of live body weight (Ojewola et al. 2001).

Intestinal samples of 2 cm (from duodenum and jejunum) were taken from three birds of each replicate (n = 18 birds/treatment; total 72); the preserved duodenal tissue sample were washed, fixed in embedded paraffin, and dehydrated in different dilutions of alcohol. Luminal contents of the intestinal section were flushed with normal saline via sterile syringe and fixed in 10% buffered formalin for 48 h for histological measurements. Histological slide preparation passed through the processes of drying, clearing, and execution with modified paraffin wax (paraffin with 5% ethylene vinyl acetate). Five-micrometer-thick tissue sections were cut on a rotary microtome (at 44 °C). The samples were further rinsed in the water bath (50–60 °C) and stained with hematoxylin and eosin (H-E) method. At least 5-well aligned intestinal villi and associated crypts were selected for morphometric analysis. The villus height was defined from their tip to the base, and the crypt depth was measured as the depth of the invagination between adjacent villi. The ratio of villus to crypt was calculated by dividing the villus height by the crypt depth in all measured villus and crypt (Marchewka et al. 2021). Thereafter, the morphological indices were observed at 0× magnification using a fluorescent microscope (Labomed, T121100), and pictures were taken using a digital camera (Euromex, D C. 1355 F050) and were then calibrated by using software (Labomed PixelPro™).

Two milliliters of blood was collected from the wing vein of 3 birds of each replicate (18 birds from each treatment; total 72) through centrifugation serum that was separated and sent to CSP Laboratory Lahore, Pakistan, for the analysis of total protein (TP), albumin (ALB), and uric acid (UA) using Automatic Chemical Analyzer (Merck kit, Germany) by the method adopted by Gunes et al. (2002).

Statistical analysis

The data were analysed through factorial ANOVA using PROC GLM in SAS software (Version 9.1). Lysine ratio and protease were considered as main effects in the model and their interaction were tested too. For comparison of significant treatment means, Duncan’s multiple range test was applied considering significance at p ≤ 0.05. The following mathematical model was used:

$${Y}_{ij k}=\mu +{\alpha}_i+{\beta}_j+{\left(\alpha \times \beta \right)}_{ij}+{\upepsilon}_{ij k}$$

where:

  • Yijk = Observation of dependent variable recorded on ith and jth treatment groups

  • μ = Population means

  • αi = Effect of ith treatment (i = 1, 2)

  • βj = Effect of jth treatment (j = 1, 2)

  • (α × β)ij = Interaction effect of ith and jth treatment groups

  • ϵijk = Residual effect associated with ith and jth NID ~ 0, σ2

Results and discussion

Growth performance

In the current study, the interaction of lysine ratio (LR) and protease did not influence feed intake among all the treatments during the whole experimental period (0–35 days) as shown in Table 3. This corresponds with the findings of Rada et al. (2013) who added exogenous mono-component serine protease at dose 15,000 PROT/g feed added into low protein diet (4% CP reduction), and it did not affect the feed intake of broilers from days 0–35. Similarly, Van Harn et al. (2019) reported that feed intake of broilers was not affected by low crude protein (CP) diet (i.e., 1, 2, and 3%, respectively) with supplementation of free amino acids over the whole experimental period (0 to 35 days). Results of the current experiment are also in agreement with the findings of Law et al. (2019) who concluded that the addition of exogenous protease at a dose rate of 300 units/g of feed to low CP diets (19.75, 18.50, 17.24%) did not affect feed intake during both the starter (0–21 days) and finisher phase (22–42) of broiler. Similarly, Angel et al. (2011) revealed that fortification of exogenous protease at a dose rate of (200, 400, and 800 g) along with a low CP diet (20.52%) did not impact the feed intake of broilers as compared to those fed a 23% CP diet. In this study, this non-significant impact on feed intake can be attributed to the diets being iso-caloric and iso-nitrogenous among all the treatments. Furthermore, the improvement of body weight and FCR can be the consequence of better nutrient digestibility rather than the impact of feed intake.

Table 3 Effects of level of lysine ratio and exogenous protease with reduced CP diet on growth performance of commercial broiler
Full size table

The present findings revealed a significant interaction of LR × protease on body weight gain (BWG) of broilers at the starter phase (0–21 days) and the overall period from 0 to 35 days as shown in Table 3. LR110+ve treatment resulted in the highest BWG both in the starter and overall growth phases, respectively, as compared to other dietary treatments. It might be due to the higher dietary lysine content that improved protein retention in broilers. As explained by Corzo et al. (2002), the absorbed lysine, in the best way, is used for protein accretion. Furthermore, it has been reported that dietary exogenous protease supplementation would have facilitated the utilization of proteins that were otherwise unavailable to animals (Kocher et al. 2002). Current findings agreed with the results of Abdel-Maksoud et al. (2010) who revealed that low CP levels plus supplementation of EAA showed higher BWG of broilers as compared to those fed a high level of CP during the whole experiment. The findings of the present study are also in line with the previous studies (Ndazigaruye et al. 2019; Jabbar et al. 2020) who reported that supplementation of exogenous protease with low CP diets improved the BWG of broilers. The exogenous protease may help to enhance weight gain by increasing the availability of AA in the intestine and decreasing the amount being excreted in feces. Furthermore, the AAs absorbed in the free form are metabolized at a higher rate in the enterocytes than that in the form of short-chain peptides. Thus, this enhanced body weight may be the consequence of the enzyme-mediated increased nutrient digestibility. However, Namroud et al. (2008) observed that crystalline AA supplementation, even 10% exceeding the NRC recommendations, is of no use when CP is extensively reduced below 17% and BWG gets decreased. This might be due to a deficiency of dietary crude protein, which may increase the body fat deposition and reduce the efficiency of feed utilization in broilers.

In the current study, LR × protease interaction for the FCR was significant for days 1–21 as well as for an overall period from days 0 to 35 as shown in Table 3. The LR110+ve depicted improved FCR of 1.40 and 1.73 during d 1–21 and days 0–35, respectively. The possible reason for this betterment in FCR may be that the LR110+ve diet contains higher levels of essential AA supplemented with exogenous protease. Higher dietary lysine content also improves protein retention as well as feeding efficiency as elaborated by Corzo et al. (2002). These findings are in accordance with Han et al. (1992) who claimed that fortification of amino acids with a low protein (LP) diet (19%) improved the FCR than those fed with standard protein (SP) (23%) throughout the experimental period. Similarly, Odetallah et al. (2003) reported that keratinase protease enzyme supplementation (0.1%, 0.15%) with a low CP diet (18%) improved the FCR of broiler chicken at 21 and 26 days of age as compared to those fed high protein diet (21.39%). This betterment may be attributed to the phenomenon of reduced viscosity of the jejunal contents, exogenous enzymes activity, and protein breakdown to small peptides. Furthermore, these peptides become easier to degrade by the digestive enzymes. Similar outcomes were recorded in the study of Favero et al. (2009) who reported that FCR was improved significantly (p ≤ 0.01) by lowering the crude protein with protease supplementation as compared to non-supplemented groups. However, Cardinal et al. (2019) reported that an extreme reduction of CP (6% to that of standard, though with added AA in diet) the FCR of broilers was poor during the starter phase (0–21 days) being associated that starter diet requires high protein to meet the demands of rapidly developing muscles.

The current study indicated a non-significant interaction of LR × protease for the livability of broilers throughout the trial (days 1–35) as shown in Table 3. Similarly, van Harn et al. (2019) revealed that lowering the CP from 2 to 3%, along with the addition of AA, showed a non-significant effect on the mortality of commercial broilers (days 0 to 35). However, Khan et al. (2011) revealed higher mortality rates, varying between 2 and 6% in response to a low CP diet and this may be due to extremely low levels of CP 15 to 16% in the diet.

Carcass characteristics

The interaction of LR × protease for different carcass parameters remained significant in the current study including live weight, carcass, and breast weight and for carcass yield (%) and abdominal fat (%) as shown in Table 4. Higher dietary LR with protease (LR110+ve) produced the highest output in relevance to carcass parameters. Present findings are in consonance with Summers et al. (1988) who reported 17% CP level with the fortification of AA improved the breast meat yield of broiler birds as compared to higher dietary protein levels (20% and 23%). The increasing yield of breast meat might be due to higher AA availability and utilization by muscles. Higher dietary lysine contents also help to improve protein retention and minimize the fat retention of broiler carcass while considered essential for the yield of boneless breast meat (Si et al. 2001; Saima et al. 2010). The results are also consistent with the findings of Xu et al. (2017) who observed that fortification of coated protease (150 mg/kg) increased the breast weight of broiler chickens during both the starter days 1–21 and finisher phases days 22–42. Another study reported by Mehmood et al. (2018) revealed that exogenous protease supplementation improved the carcass yield % in broilers fed poultry by-product meal-based diet (PBM), whereas the remaining carcass parameters were unaffected. In contrast with current results, Faria Filho et al. (2005) concluded that leg quarter yield remained unaffected by different dietary CP (21.5%, 20%, 18.5%) levels but a significant decrease in the breast yield was observed during the entire experimental period (0–35 days). They attributed this decreased yield output to a difference in energy to protein ratio in the diet which is not the case in the current study. The results of the current study show that both protease and LR did not influence the weight of the heart, liver, gizzard, and thigh as shown in Table 4. Similar findings were reported by Raju et al. (1999) who revealed that feeding low dietary protein levels (23% and 20%) had no impact on the giblet weight of broilers when fortified with essential AA at the age of 8 to 49 days.

Table 4 Effects of level of lysine ratio and exogenous protease with reduced CP diet and on carcass characteristics of commercial broiler
Full size table

Gut morphology

The current study revealed the significant interaction between LR and protease for duodenal villus height (VH) and ratio of VH to crypt depth (CD) as shown in Table 5 (Fig. 1). The results for the LR110+ve diet expressed the highest VH (1506.25) and VH/CD (4.63) ratio, whereas CD was not influenced (p > 0.05; Table 5) by any of the dietary treatments. An increased villus length is associated with increased digestion and absorption of nutrients and an increase of brush border enzymes and nutrient transport systems (Cowieson et al. 2016). Similarly, Cowieson et al. (2017) revealed that jejunal VH and CD were higher in broilers fed a soybean-based diet supplemented with protease due to greater villus integrity and absorptive capacity. Results of the current study supported the findings of Macelline et al. (2020) who reported a significant increase in the VH and CD when broiler fed LP diet (18% in starter and 17% in grower) fortified with synthetic amino acids that were similar to those of broiler fed high protein (HP) diet. They attributed it to an optimal balance of AAs which is essential for the development of gut epithelium and the production of digestive secretions and mucin. Similarly, Abbasi et al. (2014) revealed that reduction of dietary CP levels (18.89%) with threonine supplementation at the rate of 110% resulted in a significant increase in the VH and CD in jejunal epithelial cells, attributing it to higher threonine metabolism enhancing the intestinal absorptive surface. However, Hussain et al. (2019) and Ndazigaruye et al. (2019) reported that supplementation of exogenous protease and a combination of mannanase and xylanase enzymes with a low CP diet did not improve the VH, CD, and VH/CD ratio of broilers.

Table 5 Effects of level of lysine ratio and exogenous protease with reduced CP diet on gut health and serum metabolites of commercial broiler
Full size table
Fig. 1
figure 1

Intestinal morphology. A LR100-ve. B LR100+ve. C LR110-ve. D LR110+ve. The numbers indicate morphometrics (1) villus height and (2) crypt depth measured at 0–X scale

Full size image

Serum metabolites

The present findings revealed a non-significant interaction between LR and protease for all the serum metabolites (total protein (TP), albumin, and uric acid (UA)) as shown in Table 5. The present results are sharp in line with the findings of Hernández et al. (2012) who observed no change in serum TP when broilers were fed with a low-CP diet. Similarly, Corzo et al. (2009) reported that TP will only be affected when diets ingested by the broilers are deficient in AA. Thus, it appears that meeting the AA requirement could be more important than the CP per se. On the contrary, the study outcome presented by Law et al. (2018) reported a significant decline in serum Alb and TP by reducing the dietary crude protein in both diets with or without exogenous protease supplementation and was attributed to a deficiency in AA intake by the birds, as measured by the lower feed intake. Elshafey et al. (2019) and Wang et al. (2020) also depicted a decrease in the serum UA level with the decrease of CP concentration in the diet. The UA is a by-product of protein catabolism and turnover in the body, that is why the reduction in UA was attributed to insufficiency of ingested AA with subsequent reduced catabolism of AA. However, Ndazigaruye et al. (2019) reported a significant increase in serum UA level by lowering the dietary CP supplemented with exogenous protease in the diet.

Conclusions

In current study, broiler diet balanced for AA at LR 110% supplemented with exogenous protease with a net reduction of 20% CP from Ross-308 standards (2019) resulted in similar feed intake yet an improved body weight gain, feed conversion ratio, intestinal morphology, breast weight yield, and reduced fat deposition. Our findings signify that AA escalation at 110% LR with added exogenous protease can help to formulate 20% lower CP broiler diets with enhanced production efficiency and zero detrimental impact.