The quality properties (amino acids and chemical composition) of meat that are important for consumers could be enhanced by including pulses (peas and faba beans) in broiler diets.

A feeding experiment was done on cross Ross 308 broiler chickens (n = 240). The stocking density complied with the rules of Council Directive 2007/43/EC; the max live weight density was 33 kg per m² or 12 chicks per m². The broilers were placed on the floor and sawdust was used for their bedding; the broilers were provided free access to feed and water. In the first week, the temperature in the poultry building was set at 31 °C; from the second week, the temperature was gradually reduced to 28 °C, and from the fourth week onwards, the temperature was set at 22 °C. The fattening period was 42 days and divided into three stages: from 1 – 10 days (starter), 11 – 26 days (grower) and 27 – 42 days (finisher), following the standards for the cross Ross 308 (Aviagen, 2018). Additions of faba beans and peas were included in the feed of experimental groups only at the grower and finisher stages. On day 10, the broiler chickens (n = 240) having a similar body weight (BW) 276.00 ±23.2 g standard deviation (SD) were divided into 8 groups, and the experiment had two replications per treatment for each group (n = 30).

During the starter period, all the groups of broiler chickens were fed the same diet: wheat – 635.80 g.kg^-1; soybean meal (SBM) – 255.60 g.kg^-1; sunflower – 6.00 g.kg^-1; rapeseed cake – 13.00 g.kg^-1; technological, nutritional, physiological and zootechnical additives 89.60 g.kg^-1. The starter feed contained 24.93% of dry matter (DM) CP, 3.27% of DM crude fat, 2.16% of DM crude fiber, 5.70% of DM crude ash, 0.88% of DM calcium (Ca), 0.68% of DM phosphorus (P), 2980 kcal.kg^-1 metabolizable energy (ME), – 1.00% of DM lysine (Lys), 0.40% of DM methionine (Met) and 0.31% of DM cysteine (Cys).

From the 11^th day of the experiment, the diets were as follows: for the control group (C) – the basal diet (control, without peas and faba beans); for the seven experimental groups (group PB200 to group PB100 + FBL150) – the basal diet with SBM was partially replaced with different amounts of peas and faba beans. The peas ‘Bruno’ (PB) developed by the Institute of Agricultural Resources and Economics as well as the peas ‘Pinochio’ (PP) and the faba beans ‘Lielplatone’ (FBL) grown in Latvia were tested in the feeding experiment (Table 1). Crude protein contents in the pulses (dry matter) were as follows: PB – 300.00 ±23.62 g.kg^-1, PP – 284.26 ±61.54 g.kg^-1, FBL – 330.71 ±41.11 g.kg^-1, while in SBM it was 587.95 g.kg^-1. The pea and bean varieties studied contained equivalent amounts of crude fat, crude fiber, crude ash, calcium, and phosphorus. As is known from the results of the previous researches, the amount of pea and faba beans in broiler diets is limited due to the tannins they contain, which are antinutritive compounds and affect the nutritional flavor as well as the digestive system of the birds, thus limiting the number of peas and beans the amount of feed (Crépon, 2006). This indicates that peas and beans cannot completely replace SBM in poultry feed not only because of antinutritional factors but also of their lower crude protein contents.

The feed mixtures for broilers were granulated appropriate for the grower and finisher stage 2.5 – 4.0 mm diameter, 4.0 – 8.0 mm in length, reaching a temperature of up to 84 °C during the granulation process. The granulation process did not significantly affect the chemical composition of peas and beans, even though the proportion of soluble protein increased by 0.61% and that of undegradable intake protein decreased by 5.97% in the total crude protein content of peas during the granulation process, compared with the nongranulated peas. Such a change in crude protein components may contribute to the digestibility and absorption of crude protein of peas in the bird organism.

The AA contents of peas, faba beans and SBM are reported in Table 2. The Lys content in peas was in the range of 1.40 – 1.50%, 1.71% in faba beans, and 2.80% in SBM, while the amount of Met in SBM was threefold higher than that in the peas and beans (Table 2). The amounts of other essential AA in peas and beans were also about twofold lower than that in SBM. This means that the amounts of AA most important for metabolism in the broiler organism were significantly lower in peas and faba beans than in SBM.

The EAA content of PB and PP was in the range of 8.70 – 9.38%, and 11.99% in FBL. SBM had an EAA content of 20.52%. This means that SBM had a higher EAA content than peas and faba beans by 11.14 – 11.82% and 8.53%, respectively. The NEAA content in pea protein was in the range of 8.16 – 9.62%, in faba bean protein – 11.57%, but 21.62% in SBM protein. This means that the NEAA content of SBM protein was 11.98 – 13.44% higher than that of pea protein and 10.03% higher than that of faba bean protein (Table 2).

The grower stage feed contained 23.63 – 25.64% of DM CP, while the finisher stage feed had a CP content of 22.01 – 24.48% of DM. At the grower stage, the 8^th group was fed a lower CP content diet (0.8% lower than that for the C group) that contained a mixture of 150 g.kg^-1 FBL and 100 g.kg^-1 PB. However, at the finisher stage, such a mixture of 150 g.kg^-1 FBL and 100 g.kg^-1 PB in the diet had a lower CP content (by 2.47%), compared with the diet for the C group; however, the CP content in the diets for the experimental groups, compared with that for the C group, was not significantly different (p >0.05) at both feeding stages (Table 3). Using SBM, PB, PP, and FBL, for the experimental groups, a ME of 750 – 1196 kcal.kg^-1 was provided at the grower stage and 664 – 1158 kcal.kg^-1 at the finisher stage, exceeding the ME provided for the C group by 94 – 494 kcal.kg^-1, which might be explained by a 4 – 5 times higher ST content of peas and faba bean, compared with that of SBM. Such a difference in the CP content could not negatively affect the live weight of broilers at the selling age, compared with the C group. The contents of crude fat, crude ash, fiber, Ca and P in the broiler diets were equal for all the groups.

A test of the feed, peas, and faba beans were performed to calculate a precise feed ratio for the relevant period. During the experiment, feed tests for all the groups were performed on the 1^st day, 10^th day, and 27^th day. At the final stage of the experiment (on day 42 of rearing), 10 birds from each group were slaughtered and dissected.

The biochemical analyses of feed and broiler breast samples were carried out at the Latvia University of Life Sciences and Technologies (LLU) Research Laboratory of Biotechnology (LATAK, 1999). 100 g of each experimental diet was collected and sent for analysis.

Dry matter, crude protein, and ash were analyzed in the diets according to the method AOAC (Herlich, 1990). The samples of meat were analyzed for DM (ISO 6496, 1999), CP (ISO 5983-2, 2009), crude fat (ISO 6492, 1999), crude ash (ISO 5984, 2002/COR 1:2005), P (ISO 6491, 1998), yet the cholesterol level was determined using the method described by Sperry and Webb (1950). AA contents in feed and meat were determined by FTIR spectroscopy.

A chemical score (C_S) for broiler breast meat was calculated for each AA using the following formula:

(1) C s  = A  x /A  s

where A_x is the content of an AA in the investigated protein (%) and as is the content of an AA in the standard protein (the same whole egg protein) (Alfaig et al., 2014).

An AA score was determined by comparing the essential amino acid (EAA) contents of the samples to the AA contents suggested for humans (Straková et al., 2009). The AA content recommended for children aged 2 – 5 years is used to calculate an AA score of the samples (Table 7) (WHO, 1985; Straková et al., 2009). The essential AA index (EAAI) is a geometrical average of CS values (Písaříková, Kráčmar and Herzig, 2005; Straková et al., 2009).

A biological value (B_y) for the experimental food materials was calculated from the EAAI using Oser’s method (Oser, 1959) as follows:

(2) B y  = 1.09×(EAAI) - 11.7

Statistical analyses were performed with SPSS for Windows version 20.0 statistical software package. The dietary energy and other variables were compared statistically by ANOVA. Statistical significance was calculated using a t-test. Differences between the control and experimental groups were considered significant at p ≤0.05. Data were reported as mean ± SD.

The effect of various protein diets on meat quality parameters is shown in Table 5. The CP content was significantly (p <0.05) higher in breast meat of broilers fed diets FBL100 and PB100 + FBL100 (21.34 and 21.4% respectively), compared with the C group.

Increased fat content was found in broilers from the C group (1.85% of DM) and diet PB300 (1.84% of DM), but a significant decrease was observed in the broilers from groups PP300 and PB100 + FBL100. No significant difference in the crude ash content was recorded among the treatments (p >0.05).

There was a significant reduction (p <0.05) in the cholesterol concentration of broiler breast meat from the broilers fed diet PP200 supplemented with 200 g.kg^-1 PP (110.5 mg.100g^-1) and FBL100 – with 100 g.kg^-1 FBL (110.6 mg.100g^-1). In general, cholesterol concentration in meat decreased in all the experimental groups (except PB300) in comparison with the C group (Table 5).

The meat energy value contents were higher in the breast of broilers fed PB100 + FBL100 diets supplemented with 100 g.kg^-1 FBL with 100 g.kg^-1 PB (107.84 kcal.100g^-1), but the lowest – in the C group – from the dietary treatment with SBM (95.26 kcal.100g^-1). No significant difference (p >0.05) in meat energy value was recorded among the treatments.

The effects of dietary treatments on the AA contents of broiler breast meat from the eight experimental groups are shown in Table 6.

The results indicated that in most cases, the average level of total AA (TAA) was increased in meat from the broilers fed diets PB100 + FBL100 and PB100 + FBL150. The Met content was significantly higher in muscle from diet PB100 + FBL150, while Cys – in broiler meat from groups PB200, PB100 + FBL100, and PB100 + FBL150. Compared with the C group diet, diets PB300 and FBL100 significantly decreased (p <0.05) the TAA, whereas diet PB100 + FBL150 increased (p <0.05) the TAA content.

The highest values in the EAA group were recorded for Arg (3.14 g.kg^-1 DM) and Leu (3.81 g.kg^-1 DM), but the lowest value – Cys (0.58 g.kg^-1 DM). In the NEAA group, Gln was found to be the most abundant (7.54 g.kg^-1 DM), but Tyr had the lowest value – (1.4 g.kg^-1 DM). In total, the highest average EAA level (23.15 g.kg^-1 DM) was recorded in experimental diet PB100 + FBL150 (with 150 g.kg^-1 FBL with 100 g.kg^-1 PB), but the lowest (20.13 g.kg^-1 DM) – in diet PB200. On average, diets PP300, PB100 + FBL100 and PB100 + FBL150 increased the total EAA content in breast muscle, but the TAA level was increased by only diets PB100 + FBL100 and PB100 + FBL150 (41.94 and 42.59 g.kg^-1 DM, respectively).

The calculated indicators of breast meat samples are set out in Table 7. The chemical score, the AA score, the EAA index and the biological value of all the tested AA were significantly different between the control and the treatment groups.

A comparison of the protein-supplemented diet groups revealed that the chemical and AA scores were increased in the meat of groups PB100 + FBL100 and PB100 + FBL150 (supplemented with faba beans and peas), but were decreased in the breast meat of broilers fed diets PP200 and PB300 (supplemented with 200 g.kg^-1 PP and 300 g.kg^-1 PB, respectively).

Regarding the EAAI and biological value, there were significant increases between the control (C) group and groups PB100 + FBL100 and PB100 + FBL150, respectively.

The results of the use of faba beans in the poultry diet have been contradictory (Crépon, 2006). Faba beans have a quite high level of protein (about 26%), but the presence of antinutritional constituents, such as vicine and convicine, and the low level of Sulphur AA content reduce their nutritional value in poultry meat.

Some studies showed adverse effects on poultry performance or growth performance similar to control diets at various inclusion levels (Dal Bosco et al., 2013). Some results indicated that replacing SBM with alternative protein sources had a positive effect on the dietetic value of poultry meat, whereas other data indicate something opposite (Milczarek, Osek and Pachnik, 2016). Parameters used to determine protein quality included chemical score, AA score, essential AA index (EAAI), and biological value (Aronal, Huda and Ahmad, 2012). Proteins with a high content of essential AA are the most important components of poultry meat (Straková et al., 2002).

As is known, birds, especially broiler chicks, have an intense growth rate and high genetic potential for productivity. Therefore, broilers need to feed with a high (20 – 25%) crude protein level. Before the start of the feeding test, the chemical composition of peas, beans, and imported soybeans are grown in Latvia was determined. Regarding chemical composition of a pea, faba bean and soybean used in the trial, our results are in agreement with published data of other authors (Koivunen et al., 2016) who reported on significantly lower content of crude protein, crude fat, and ash in faba beans and peas in comparison with soybean meal. However, our results showed a significantly higher content of starch and crude fiber in peas and faba beans. This agrees with the results of Koivunen et al. (2016). Unfortunately, pea starch is less susceptible to enzyme hydrolysis than starch in any of the cereal grains (Laudadio and Tufarelli, 2010). In contrast to the current study, in the study of Laudadio and Tufarelli (2010), the content of crude fiber in soybean was higher as in peas. Faba beans are protein and energy-rich legume seeds well adapted to grow in various climatic zones of Europe and widely used for feed and food (Grosjean et al., 2000).

AA composition is key to the evaluation of a dietary protein source. In the present research study, feeding pulses led to a significantly higher level of EAA, NEAA, and TAA in the diets. Among the analyzed finisher stage samples, basal feed with an addition of 200 g.kg^-1 PP or 300 g.kg^-1 PB or 150 g.kg^-1 FBL with 100 g.kg^-1 PB had a higher content in almost all the groups of AA; however, Sulphur AA (Met and Cys) were the exception in most cases.

AA, termed functional AA, includes Ala, Arg, Asp, Gln, Gly, Pro, and Ser and are important not only for intestinal integrity, nutrient transport, and metabolism but also in immune response (Dai, Wu and Zhu, 2011; Ren et al., 2013). Some authors emphasize the importance of EAA (Lys and Met) in broiler feed. The productivity of broilers, to a great extent, is determined by the EAA content of feedstuffs. This AA is not synthesized in the broiler organism; therefore, the acids must be consumed with feed in a certain quantity and at a certain ratio (Beski, Swick and Iji, 2015). Lys and Met are among the most limiting AA, which is important for the growth of the organism and in the balance of nitrogen.

Met is classified as the first insufficient AA in the organism, as it is available in a limited amount in plant proteins. Met is required to provide the building blocks for immune cells and tissues, therefore, it is an EAA for animals, poultry, including broiler, in particular. Met is required for the growth of feathers and the synthesis of proteins (Bunchasak, 2009).

The second most important EAA needed for the growth of broilers is Lys, which contributes to the growth of chest muscle tissues as well as the growth pace of broilers (Kidd et al., 2004). Deficiency in Lys causes blood circulation problems, a lower level of hemoglobin as well as leads to smaller muscles and bone deterioration (Burnham, 2005) and, consequently, the death rate of broilers increases and an increase in the weight of their muscles is lower, which can cause considerable economic losses.

The research showed that soybean meal had a higher EAA content than peas and faba beans by 11.14 – 11.82% and 8.53%, respectively (Table 3). It has to be taken into consideration that the SBM had such anti-nutritive factors as protease inhibitors – trypsin, chymotrypsin, pectin, goitrogens, and other substances that could affect the digestibility of protein present in the feed, metabolic nitrogen retention, and excretion (Yasothai, 2016).

Our results demonstrated the CP content in the diets for the experimental groups, compared with that for the C group, was not significantly different (p >0.05), yet the overall contents of EAA in groups PP200, PP300, PB100 + FBL150 and TAA in group PP200 were significantly higher than those in the C group at both the grower and the finisher stages. It means that the diet with peas and faba beans contained higher-quality CP than that for the C group (SBM).

From the nutritional aspect, poultry meat is an important source of proteins, vitamins, and minerals, and at the same time has relatively low-fat content, therefore the chemical composition of muscle tissue is an important criterion of broiler meat quality. The biochemical parameters of broiler meat observed in the research indicated that the inclusion of faba beans and peas (100 – 200 g.kg^-1 of the basal diet) in the feed as higher protein diets can induce a higher meat protein content and, at the same time, reduce the fat content of muscles owing to a decrease in the protein-calorie ratio (Bogosavljevic-Boskovic et al., 2010).

Although authors Dal Bosco et al. (2013) reported on a negative effect of faba bean diets due to a lower level of some EAA (Met, Cys, Thr, and Trp) and the presence of vicine, convicine, and tannins as anti-nutritional factors, which reduce energy and protein digestibility by forming tannin-protein complexes, our findings demonstrated that addition to diets of faba bean protein alone or in a mix with peas could elevate the content of dry matter in broiler meat (Dal Bosco et al., 2013).

We found a similar situation regarding the content of CP in dry matter. The highest content was shown by the diets containing 100 g.kg^-1 FBL with 100 g.kg^-1 PB (PB100 + FBL100). Also, the crude ash content of dry matter increased statistically significantly (p <0.05) in the muscle of broilers within all the experimental groups, especially in the group fed basal feed with 200 g.kg^-1 PB of the total diet.

Our results are not in agreement with the data published by other authors Milczarek, Osek and Pachnik (2016) who reported that the introduction of different varieties of faba beans in starter/grower feed for broilers did not affect the content of basic meat components – dry matter, crude ash, and crude protein.

Even though our results demonstrated also an increase in crude fat content within all the experimental groups in comparison with the C group of broilers, Milczarek, Osek and Pachnik (2016) reported that the use of faba beans in the diet of broilers modified the muscle fatty acid profile in a health-enhancing direction.

Concerning Ca, our research demonstrated that the addition of faba beans and peas in various amounts did not affect statistically significantly the level of Ca in broiler muscle meat. We founded the same situation regarding the level of P. Therefore, we would suggest that diets with faba beans and peas do not harm key minerals in broiler meat.

An important indicator of meat quality is the content of cholesterol. As regards this parameter, our experiment showed a statistically significant decrease in all the experimental diets, except for the group of broilers fed 300 g.kg^-1 PP of the total diet. This could be explained by the highest content of CP from peas (21.05% of DM) in this experimental PP300 group in comparison with the other experimental diets. Our results are in agreement with those of researchers Kiczorowska, Samolińska and Andrejko (2016) who also reported on the hypocholesterolemic effect of pea seeds (Pisum sativum L.).

An important criterion of meat quality is the energy value of meat that was higher in all the experimental groups (from 4.25% in group PP200 to 13.20% in group PB100 + FBL100) than in the C group. Our results are not in agreement with the data of authors Dal Bosco et al. (2013) who reported that the introduction of faba beans in starter and grower feeds for broiler chickens did not affect the meat energy value. This probably could be explained by the high value of the dry matter in broiler meat as a result of our experiment. But, the inclusion of 200 – 300 g.kg^-1 peas of the basal diet in the experimental broiler feed demonstrated a lower energy value than that for the PB100 + FBL150 group fed 150 g.kg^-1 FBL with 100 g.kg^-1 PB, which could be explained by an imbalance between energy intake and energy expenditure (Bogosavljevic-Boskovic et al., 2010).

AA are absorbed at the intestinal level and have effects on the physiology of the intestine, and AA might also affect muscle composition and meat quality. Macroorganisms cannot synthesize EAA or indispensable AA and therefore the acids have to be supplied in the diet. Researchers Probst (2009) and Koivunen et al. (2016) inform that various diets can cause significant changes in the content of EAA and NEAA in broiler meat. However, our experiment demonstrated that diets with an addition of faba beans and peas have shown a positive effect on the level of EAA and NEAA in broiler meat.

The nutritional value of meat is usually assessed based on parameters such as the content and composition of proteins, levels of AA, and content of fat (Straková et al., 2002). The calculated indicators in meat samples showed that the chemical score, the AA score, the EAAI, and the biological value of tested AA were significantly increased in the PB100 + FBL150 group broilers fed diets supplemented with 150 g.kg^-1 FBL with 100 g.kg^-1 PB.