Effect of reducing dietary protein level on performance responses and some microbiological aspects of broiler chickens under summer environmental conditions

Hot environmental conditions are a problem for poultry producers in many areas of the world, particularly in the humid and hot tropics and sub-tropics. Some studies investigated the influence of thermal environment and dietary nutrient level on the productive traits of broiler chickens (Gu et al., 2008; Attia et al., 2011). The nutrient requirements of poultry are altered as the environmental temperature changes. Dietary protein is a crucial regulator of poultry growth and reproductive performance, but also of the gastro-intestinal tract features (Laudadio et al., 2012). Previous findings have demonstrated that diets with low concentrations of crude protein worsen the performance and affected growth parameters and chemical composition of broilers’ carcass when the birds were reared under heat conditions (Dozier et al., 2000). However, Mujahid et al. (2009) reported that increasing the dietary protein at particular metabolisable energy concentrations had little or no effect on the feed intake and growth rate of birds kept at high temperatures. Moreover, considerable attention has recently been shown in the use of low dietary protein to influence the bacterial ecology of their intestinal tract in different livestock species (Canibe et al., 2008; Li et al., 2011) Reduction of pathogens on farms is necessary if cleaner products are expected during processing, in fact there are many challenges presented when attempting to reduce human exposure to foodborne pathogens, especially when these microorganism are associated with animal origin foods (Normanno et al., 2007). Cross contamination in a poultry processing plant generally result when faecal contents are released from the birds (Rasekh et al., 2005).

There is a great deal of interest in the possibility of altering the intestinal microflora in a favourable way for the health of the host. However, this concern has not been widely applied to poultry. We hypothesised that the unfavourable influence during the summer season of hot environmental conditions on broiler chickens would be decreased by lower dietary protein level. Therefore, the present study investigated the effect of different dietary protein levels on the growth performance and microbiological characteristics of broiler faeces. 

The present trial was conducted during the summer season in a research facility situated in Apulia region in Southern Italy (41°N and 15°E). The climate is predominantly of the semi-arid Mediterranean type with hot and dry summers and mild and rainy winters.

One hundred fifty one-day-old Hubbard broiler chicks were randomly divided into 15 experimental units of 10 chicks each on arrival at the research facility in an open-sided house having thermostatically controlled (33°C and 70% relative humidity) curtains and crossventilation. Each pen was equipped with a pan feeder, a manual drinker and wood shavings. On day 14, birds were individually weighed and divided among pens and randomly assigned to one of three dietary groups. Pens were randomly assigned to three dietary treatments with each having five replicates of 10 birds (0.12 m² /bird). Up to slaughtering age (42 days), birds were fed three diets containing different levels of crude protein (CP) that were obtained by replacing wheat middlings with soybean meal and formulated to meet or exceed broiler amino acid requirements (NRC, 1994). These diets (Table 1) were: (1) low-protein diet (LCP – 18.5% CP); (2) medium-protein diet (MCP – 20.5% CP); and (3) high-protein diet (HCP – 22.5% CP). The metabolisable energy (ME) of the diets was estimated using the Carpenter and Clegg equation (Leeson and Summers, 2001): 

 

The diets were based on wheat middlings obtained from durum wheat (Triticum durum cultivar Appulo). The wheat middlings were previously sieved to separate the fibrous components and so obtain a raw ingredient with an average crude fibre level less than 3% (Laudadio and Tufarelli, 2011). Feed, in pelleted form, and water were provided ad libitum throughout the whole trial.

Body weight (BW, g) and feed consumption by replicate were determined weekly for all birds. Average daily gain (ADG, g·day^–1), average daily feed intake (ADFI, g·day^–1), and feed conversion ratio (FCR, g g^–1) were then calculated. Diet samples were ground in a hammer mill with a 1 mm screen and analysed in triplicate for dry matter (DM), ash, CP (N × 6.25), crude fiber and ether extract according to the methods of AOAC (2000). The amounts of Ca and P determined by an auto analyser (Cobas Mira; Roche, Basel, Switzerland) using standard commercial kits.

Samples of faecal material were collected on days 14, 28 and 42 of the trial. Faecal pH was measured from each bird in the trial using pH meter with a glass electrode (Ingold, Mettler Toledo, Greifensee, Switzerland) previously calibrated in the laboratory using known buffers (Merck, KGaA, Darmstadt, Germany).

The collection of stool samples (approximately 2 g/ bird) from each bird was performed using a sterile swab containing 10 mL of 0.85% (w/v) sterile tryptone salt solution. Samples were transported under refrigeration in the laboratory where they were serially diluted 10-fold for determination of total aerobic mesophilic counts (TAMC) and Escherichia coli counts (ECC) using the TEMPO® System (bio-Mèrieux, Italy). Then, the collected samples were incubated for 40 hours at 30°C and 24 hours at 37°C for TAMC and ECC, respectively. All analyses were conducted in triplicate. Results were expressed as log₁₀ colony-forming units per gram of faeces (log₁₀ CFU g^–1).

Data recorded for broilers’ growth performance were statistically analysed using the one-way analysis of variance (ANOVA). Three treatments with five replications were used as completely randomised design, and each pen was an experimental unit. If occurred, Duncan’s multiple range test was applied to compare the differences between the means (Steel and Torrie, 1980). Microbiological data (log₁₀ CFU·g^–1) were statistically analysed with two-way ANOVA. Statistics were carried out using Statview (SAS Institute, 2004). 

Broiler body weight did not differ between the experimental treatments during the whole experiment (Table 2). BW gain and feed intake both tended to decrease (P > 0.05) in broiler chickens fed the low CP diet compared to those fed the medium and high CP diets (Table 2). FCR was not influenced (P = 0.089) by the dietary treatment. There were no significant differences between diets regarding mortality that was generally low, averaging 1.8% over the whole experiment.

The mean values of the microbial counts of broiler faeces and faecal pH at 14, 28, and 42 days of age are shown in Table 3. Though all the faecal material groups were acidic, it was found that both the HCP and MCP groups were similar, but they resulted less acidic then LCP group during the whole period (P<0.05 at day 14 and 28; P<0.01 at day 42). In particular, the reduction of CP up to 18.5% decreased faecal pH values of the broilers, at each stage of growth, by nearly 0.4 unit.

Reducing dietary CP level was associated with a significant decrease in faecal microflora (Table 3). The reduction of CP level led to a decrease of number of TAMB and E. coli in broilers excreta, particularly when dietary CP level was reduced up to 18.5%. At day 14, faecal TAMB did not vary among dietary groups, whereas at days 24 and 42 birds receiving the LPC diet resulted in significantly lower (P = 0.039 and P = 0.007, for day 24 and 42 respectively) TAMB compared to the HCP and MCP diets. When birds were fed the LCP (18.5%) diet, the faecal samples resulted in lowest E. coli count at days 14 and 28 (P < 0.05), 42 (P < 0.01) if compared to the other experimental dietary groups. 

Table 1 Ingredients and chemical composition of the diets fed to broiler chickens

Table 2 Effect of dietary protein level on growth performance of broiler chickens

Dietary protein is a important regulator of poultry performance, but also of the development and ecology of gastrointestinal tract. At the end of the feeding trial, broilers achieved an overall live BW of 2.5 kg in the three dietary treatments. The feed to gain ratio was not influenced indicating that the light depression in BW gain of birds receiving the low CP diet was associated with reduced feed consumption rather than with problems of nutrient digestibility. These results agree in part with those reported by Kamran et al. (2004) and Azarnik et al. (2010) who observed no difference in feed consumption or FCR of broilers when dietary CP contents were decreased from 23 to 20% under hot climatic conditions. Conversely, our results disagreed with the findings of Kidd et al. (2001) who reported significant increase in feed intake when broilers were fed a diet with 20% CP and supplemented with amino acids, when compared with those fed a diet with 23% CP. 

Table 3 Effect of dietary protein level on characteristics of broiler chicken faeces (log10 CFU g^–1)Table 3 Effect of dietary protein level on characteristics of broiler chicken faeces (log10 CFU g^–1)

Dozier et al. (2000) stated that heat stress conditions probably necessitate a decrease in growth to enable survival and the high environmental temperatures are known to result in reduced feed intake and meat yield (Oliveira et al., 2006), which would also reduce heat production in its own right (Yang et al., 2009). Our findings are not in accordance with the results of Kamran et al. (2008) who reported a linear decrease in weight gain of broilers fed low-protein diets having constant energy-to-protein ratio, whereas feed intake and FCR were increased linearly as dietary protein and energy decreased during grower, finisher, and overall experimental periods.

There are numerous challenges presented when attempting to reduce human exposure to food borne pathogens, especially when these pathogens are associated with foods of animal origin (Rasekh et al., 2005). The reduction of pathogens on farms is necessary if cleaner products are expected after processing. Reduction of faeces contamination provides a direct measure of microbial safety for at least the risk from pathogens likely to be present in digestive tract contents (Russell and Walker, 1997). Furthermore, the control of poultry contamination by digestive tract contents is one of several regulatory standards that have used a visible product standard (Zhao et al., 2010). In our study, the difference in faecal pH between animals receiving low protein diet and those receiving medium or high protein diets was nearly the same at all production stages. The decrease in faecal pH with low CP regimens provides a more favourable pH environment for digestive activity, allowing for greater digestion and absorption of nutrients, particularly in the small intestine. In a previous trial, Forbes and Shariatmadari (1994) reported that faecal pH is a valuable indication of the amount of nutrients available for bacterial fermentation in the intestine.

The reduction of dietary CP level caused significant effects on faecal microflora. The effects of reducing dietary CP level on decreasing TAMB in broiler faeces might be due to the lower faecal pH from birds fed a lower amount level of CP. Results for the E. coli count showed a clear alteration of faecal microflora, in fact reducing the level of CP a decrease (in particular on day 42) of E. coli was found in faeces especially when dietary CP level was reduced up to 18.5%. As an alternative dietary regimen, low protein diet (18.5% CP) had beneficial effects during the whole growth stage of the poultry, most likely due to the optimal intestinal environment as confirmed by the faecal pH. In this regards, from another point of view, the microbiological safety and quality of poultry production are very important for producers and consumers, and both involve microbial contaminants on the product (Mead, 2004).

In conclusion, our findings indicate that reducing the dietary protein level in the broilers’ rations that could be advantageous compared to the conventional feeding programs. Furthermore, our dietary low protein regimen (18.5% CP) had little impact on growth performance of broilers and promoted beneficial effects on faecal microflora. However, further research is needed to evaluate the effects of dietary CP density on other intestinal bacteria populations. 

The present research was part of the project founded and supported by the Apulia Region, Italy (Programma Integrato per la Prevenzione e la Riduzione dell’Inquinamento da Nitrati di Allevamenti Avicoli - Asse 6, Programma per la Tutela dell’Ambiente). The trial was conducted observing the animal welfare Legislative Decree 116/92, Council Directive 98/58/EC received in Italy by Legislative Decree 146/2001, Council Directive 2007/43/CE received in Italy by Legislative Decree 181/2010 and Legislative Decree 267/2003. 

AOAC (2000) Official methods of analysis, 17th edn. Association of Official Analytical Chemists, Arlington, Virgina, USA.

Attia, Y.A., Hassan, R.A., Tag El-Din, A.E. and Abou-Shehema, B.M. (2011) Effect of ascorbic acid or increasing metabolizable energy level with or without supplementation of some essential amino acids on productive and physiological traits of slow-growing chicks exposed to chronic heat stress. J. Anim. Physiol. Anim. Nutr., 95, 744–755.

Azarnik, A., Bojarpour, M., Eslami, M., Ghorbani, M.R. and Mirzadeh, K. (2010) The effect of different levels of diet protein on broilers performance in ad libitum and feed restriction methods. J. Anim. Vet. Adv., 9, 631-634.

Canibe, N., Miettinen. H. and Jensen, B.B. (2008) Effect of adding Lactobacillus plantarum or a formic acid containing product to fermented liquid feed on gastrointestinal ecology and growth performance of piglets. Livestock Sci., 144, 251- 262.

Dozier, III W.A., Moran, Jr E.T. and Kidd, M.T. (2000) Threonine requirement of broiler males from 42 to 56 days in a summer environment. J. Appl. Poult. Res., 9, 496-500.

Forbes, J.M. and Shariatmadari, F. (1994) Diet selection for protein by poultry. World Poult. Sci. J., 50, 7-24.

Gu, X.H., Li, S.S. and Lin, H. (2008) Effects of hot environment and dietary protein level on growth performance and meat quality of broiler chickens. Asian-Austral. J. Anim. Sci., 21, 1616-1623.

Kamran, Z., Mirza, M.A., Haq, A.U. and Mahmood, S. (2004) Effect of decreasing dietary protein levels with optimal amino acids profile on the performance of broilers. Pak. Vet. J., 24, 161-164.

Kamran, Z., Sarwar, M., Nisa, M., Nadeem, M.A., Mahmood, S., Babar, M.E. and Ahmed, S. (2008) Effect of low-protein diets having constant energy-to-protein ratio on performance and carcass characteristics of broiler chickens from one to thirtyfive days of age. Poult. Sci., 87, 468-474.

Kidd, M.T., Gerard, P.D., Heger, J., Kerr, B.J., Rowe, D., Sistani, K. and Burnham, D.J. (2001) Threonine and crude protein responses in broiler chicks. Anim. Feed Sci. Technol., 94, 57-64.

Laudadio, V., Passantino, L., Perillo, A., Lopresti, G., Passantino, A., Khan, R.U. and Tufarelli, V. (2012) Productive performance and histological features of intestinal mucosa of broiler chickens fed different dietary protein levels. Poult. Sci., 91, 265-270.

Laudadio, V. and Tufarelli, V. (2011) Dehulled-micronised lupin (Lupinus albus L. cv. Multitalia) as the main protein source for broilers: influence on growth performance, carcass traits and meat fatty acid composition. J. Sci. Food Agric., 91, 2081-2087.

Leeson, S. and Summers, J.D. (2001) Scott’s nutrition of the chicken. University Books, Guelph, Ontario.

Li, P.F., Xue, L.F., Zhang, R.F., Piao, X.S., Zeng, Z.K. and Zhan, J.S. (2011) Effects of fermented potato pulp on performance, nutrient digestibility, carcass traits and plasma parameters of growing-finishing pigs. Asian-Austral. J. Anim. Sci., 24, 1456-1463.

Mead, G.C. (2004) Microbiological quality of poultry meat: a review. Braz. J. Poult. Sci., 6, 135-142.

Mujahid, A., Akiba, Y. and Toyomizu, M. (2009) Progressive changes in the physiological responses of heat-stressed broiler chickens. J. Poult. Sci., 46, 163-167.

Normanno, G., La Salandra, G., Dambrosio, A., Quaglia, N.C., Corrente, M., Parisi, A., Santagada, G., Firinu, A., Crisetti, E. and Celano, G.V. (2007) Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. Int. J. Food Microbiol., 115, 290-296.

NRC (1994). Nutrient requirements of poultry. 9th rev. edn. Natl. Acad. Press, Washington, DC.

Oliveira, G.A., de Oliveira, R.F.M., Donzele, J.L., Cecon, P.R., Vaz, R.G.M.V. and Orlando, U.A.D. (2006) Effect of environmental temperature on performance and carcass characteristics of broilers from 22 to 42 days old. R. Bras. Zootec., 35, 1398-1405.

Rasekh, J., Thaler, A.M., Engeljohn, D.L. and Pihkala, N.H. (2005) Food safety and inspection service policy for control of poultry contaminated by digestive tract contents: a review. J. Appl. Poult. Res., 14, 603–611.

Russell, S.M. and Walker, J.M. (1997) The effect of evisceration on visible contamination and the microbiological profile of fresh broiler chicken carcasses using the Nu-Tech evisceration system or the conventional streamlined inspection system. Poult. Sci., 76, 780–784.

SAS (2004) SAS/STAT 9.13 User’s Guide. Statistical Analysis System Inst, Cary, NC.

Steel, R.G.D. and Torrie, J.H. (1980) Principles and procedures of statistics, a biometrical approach, 2nd edn. McGraw-Hill Book Co., New York.

Yang, Y.X., Guo, J., Yoon, S.Y., Jin, Z., Choi, J.Y., Piao, X.S., Kim, B.W., Ohh, S.J., Wang, M.H. and Chae B.J. (2009) Early energy and protein reduction: effects on growth, blood profiles and expression of genes related to protein and fat metabolism in broilers. Brit. Poult. Sci., 50, 218-227.

Zhao, X. , Li, Y., Wang, L., You, L., Xu, Z., Li, L., He, X., Liu, Y., Wang, J. and Yang, L. (2010) Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples. Mol. Biol. Rep., 37, 2183–2188.