This research was carried out under real field conditions at an altitude of 822 m at the coordinates 40°42'53.62" N, 31°31'29.82", in modern R&D test house with capacity for 12,000 broilers/period from a company located in Bolu, Turkey, this being one of the most important aspects of the research. There were 60 ground pens in the house, and each of the pens measured 13.0 m² (6.5 m × 2.0 m) and were equipped with two nipple drinker line kits (SPARKcup, Roxell NV, Belgium), a pan feeder (MINIMAXline, Roxell NV, Belgium), and a ceramic radiant heater (Rd 3 FA, SBM Int., France). In addition, a digital scale connected to the central computer was set in each of the pens to monitor the live weight of the broilers throughout the study.

Numerous concerns were considered during the study design. To begin, the primary limiting factor is the number of replicates required and the number of compartments in the test house in which the broilers were reared. The rearing phase of the study was conducted in a test house with 60 compartments measuring 13 m², each capable of housing 200 broilers at a standard stocking density.

Dissolved air floatation sludge was produced by dewatering it in a belt press following physical, chemical, and biological treatment processes in the water treatment unit of a poultry slaughterhouse plant located where the experiment was conducted. The average dry matter content of DAFS increased from 5 to 25% during the process using a belt press, and this value was recorded as 21.54% during the experiment. The obtained DAFS was then added at the determined ratios into the trial design with other slaughterhouse byproducts (head, blood, feather etc.) to batch-cookers (HM 5000, HAARSLEV A/S, Denmark) at the rendering plant for POM production. All the byproducts were processed in these batch-cookers according to a process that takes approximately 4 h, in which a high temperature of 135 °C and a high pressure of 2.5 bars are used to completely deactivate viruses, spore-forming bacteria, and microorganisms (Fransen et al., 1998; El Boushy and Van Der Poel, 2000), and consists of cooking, hydrolysing, and drying stages. All the processes in POM production, such as cooking, pressing, and loading, are carried out in accordance with the regulations set for EU and EU candidate countries (EC, 2004a,b,c; EFSA, 2007). The poultry offal meals with/without DAFS produced in this way were put through an oil press and sent to the feed plant.

Chemical (moisture, crude protein, crude oil, crude ash, calcium, magnesium, potassium, iron, manganese, copper, mercury, lead, nickel, and potassium permanganate) analyses of DAFS used in POM production and POM with/without DAFS used in feed production were carried out using classical methods (the method numbers are, respectively: 930.15, 988.05, 920.39, 942.05, 968.08, AOAC (2000); atomic absorption method; and TSE 8336 based on Walckley and Black method, Anonymous, 2023) (Tables 1 and 2).

Table 1

Content	DAFS
Moisture (%)	21.54	0.00
Crude protein (%)	7.38	34.24
Crude fat (%)	1.92	6.12
Crude ash (%)	2.04	9.46
Calcium (mg/kg)	1842.00	8546.88
Magnesium (mg/kg)	167.00	774.88
Potassium (mg/kg)	46.00	213.44
Ferrous (mg/kg)	6217.00	28846.88
Manganese (mg/kg)	26.82	124.44
Copper (mg/kg)	10.58	49.09
Mercury (mg/kg)	0.01	0.046
Lead (mg/kg)	4.20	19.489
Nickel (mg/kg)	1.06	4.91
Potassium permanganate (mg/kg)	94.58	438.85

Table 2

	DAFS usage rate in POM1 (%)
	0.00	11.00	22.00
Dry matter (%)	96.28	96.28*	96.28*
Crude protein (%)	62.09	60.12	58.02
Crude fat (%)	20.72	19.94	19.07
Crude ash (%)	9.62	9.80	10.00

¹ These values are revised for the dry matter content of POM without DAFS.

The analysis results are consistent with those reported by other researchers (Lee, 2002) and fell within the specified limits. This shows that DAFS has an average content of 78.46% total solids, 21.54% total moisture, and 9.46% ash of total solids. These findings from the current research are consistent with previous studies that have shown that poultry DAFS content has a high density of organic matter consisting of soluble proteins, carbohydrates, and fibres (Ghaly and MacDonald, 2012; Hu et al., 2017; Cui et al., 2019; Ozdemir et al., 2020). However, some of these values differ from the values reported in previous studies (Ferreira et al., 2018; Irshaid et al., 2021). It is thought that the differences in sludge compositions and properties are attributable to differences in poultry type, production process, feed, age of the poultry, and the cleaning process applied at the facility.

In addition, the most abundant component in DAFS samples is crude protein, accounting for approximately 34.24% of the dry weight of DAFS. Crude fat is the second most common component of DAFS, accounting for approximately 6.12% of its dry weight. Recent studies on the nutritional values and chemical composition of poultry DAFS have revealed that sludge is a good source of protein, fat, and minerals that can be incorporated into animal diets in agricultural practices (Ghaly and MacDonald, 2012; Hu et al., 2017; Adhikari et al., 2018).

Accordingly, POM usage rates, which are generally recommended in diet formulas of integrated feed plants, were considered and, thus multiple control groups were created. Treatment groups, on the other hand, were simply generated by adding DAFS at two different rates to the control groups. As a result, the final trial plan was designed considering the above-mentioned poultry house conditions and possibilities, along with an attempt to consider all possible alternatives.

In this study, test diets were formulated by adding POM with/without the DAFS at determined proportions. This study included 12 treatment groups that were designed based on their diet contents (Table 3).

Table 3

Treatment group	Group code		DAFS in POM production (%)	POM (%)
				Starter feed	Grower feed	Finisher and pre-slaughter feeds
		C24			2.0	4.0
Control	C	C25	0.0	0.0		5.0
		C34			3.0	4.0
		C35				5.0
		D124			2.0	4.0
	D1	D125	11.0	0.0		5.0
		D134			3.0	4.0
Treatment		D135				5.0
		D224			2.0	4.0
	D2	D225	22.0	0.0		5.0
		D234			3.0	4.0
		D235				5.0

POM - poultry offal meal; DAFS - dissolved air flotation sludge.

The feeds were prepared with corn-soya and manufactured in the feed plant of a private company located where the experiment was carried out. Poultry offal meal with/without DAFS was added to the basal diet based on the trial design. Diets were formulated in accordance with international standards (NRC, 1994) and according to the recommendations of grandparent company ROSS (2.0-2.5 kg average live weight [ALW]) (Aviagen, 2014) were taken into consideration for the trial design. Diets were prepared in four periods (starter, grower, finisher, and pre-slaughter). The starter feeds were in crumble, while the other feeds were manufactured in pellet form (3.5 mm in diameter; Tables 4 and 5) and the prepared basal diets were analysed with AOAC (2000) (Table 6).

Table 4

	Starter1	Grower2
DAFS3 (%)	0.00	0.00	0.00	11.00	11.00	22.00	22.00
POM (%)	0.00	2.00	3.00	2.00	3.00	2.00	3.00
Anticoccidial4	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Bonkalite	20.00	20.00	20.00	20.00	20.00	20.00	20.00
Broiler starter vitamin	1.00	0.00	0.00	0.00	0.00	0.00	0.00
Broiler chick vitamin	0.00	1.00	1.00	1.00	1.00	1.00	1.00
Canola meal-35	15.00	15.00	15.00	15.00	15.00	15.00	15.00
Choline chloride-75	0.48	0.28	0.28	0.28	0.28	0.28	0.28
DCP-185	6.30	4.40	4.20	4.40	4.20	4.40	4.20
Corn	559.32	573.66	583.18	573.66	583.18	573.66	583.18
Corn gluten-60	20.00	0.00	0.00	0.00	0.00	0.00	0.00
Fish meal	25.00	20.00	20.00	20.00	20.00	20.00	20.00
Full fat soybean meal	167.96	144.94	135.32	144.94	135.32	144.94	135.32
Lysine-996	3.36	1.64	1.82	1.64	1.82	1.64	1.82
Marble powder	11.90	9.10	8.80	9.10	8.80	9.10	8.80
MDCP5	6.00	4.50	4.20	4.50	4.20	4.50	4.20
Methionine (dry)7	3.30	2.52	2.44	2.52	2.44	2.52	2.44
Phytase enzyme8	0.30	0.30	0.30	0.30	0.30	0.30	0.30
POM	0.00	20.00	30.00	20.00	30.00	20.00	30.00
Poultry trace elements	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Salt	2.00	2.00	2.00	2.00	2.00	2.00	2.00
Sodium bicarbonate	2.20	1.80	1.80	1.80	1.80	1.80	1.80
Soybean meal-48	154.38	156.36	148.16	156.36	148.16	156.36	148.16
Vegetable oil (soybean)	0.00	21.00	20.00	21.00	20.00	21.00	20.00
Total	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00

POM - poultry offal meal; DAFS - dissolved air flotation sludge.

Table 5

	Finisher1						Pre-slaughter1
DAFS2 (%)	0.00	0.00	11.00	11.00	22.00	22.00	0.00	0.00	11.00	11.00	22.00	22.00
POM (%)	4.00	5.00	4.00	5.00	4.00	5.00	4.00	5.00	4.00	5.00	4.00	5.00
Anticoccidial3	0.50	0.50	0.50	0.50	0.50	0.50	0.00	0.00	0.00	0.00	0.00	0.00
Bonkalite	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00	20.00
Broiler vitamin	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Canola meal-35	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00
Choline-75	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30
DCP-184	3.90	3.20	3.90	3.20	3.90	3.20	3.90	3.20	3.90	3.20	3.90	3.20
Corn	638.98	649.42	638.98	649.42	638.98	649.42	639.48	649.92	639.48	649.92	639.48	649.92
Corn gluten-60	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Fish meal	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00	15.00
Full fat soybean meal	99.40	88.08	99.40	88.08	99.40	88.08	99.40	88.08	99.40	88.08	99.40	88.08
Lysine-995	1.88	2.06	1.88	2.06	1.88	2.06	1.88	2.06	1.88	2.06	1.88	2.06
Marble powder	8.40	8.20	8.40	8.20	8.40	8.20	8.40	8.20	8.40	8.20	8.40	8.20
MDCP4	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00
Methionine (dry)6	2.06	1.98	2.06	1.98	2.06	1.98	2.06	1.98	2.06	1.98	2.06	1.98
Phytase enzyme7	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30
POM	40.00	50.00	40.00	50.00	40.00	50.00	40.00	50.00	40.00	50.00	40.00	50.00
Poultry trace elements	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Salt	2.00	1.80	2.00	1.80	2.00	1.80	2.00	1.80	2.00	1.80	2.00	1.80
Sodium bicarbonate	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60
Soybean meal-48	116.68	109.56	116.68	109.56	116.68	109.56	116.68	109.56	116.68	109.56	116.68	109.56
Vegetable oil	29.00	28.00	29.00	28.00	29.00	28.00	29.00	28.00	29.00	28.00	29.00	28.00
Total	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00	1000.00

POM - poultry offal meal; DAFS - dissolved air flotation sludge.

Table 6

	Starter (0-10th day)	Grower (11-25th day)		Finisher and pre-slaughter (26-36th and 37-41st day)
Poultry offal meal (%)	0.00	2.00	3.00	4.00	5.00
Dry matter (%)	88.30	88.40	88.39	88.38	88.37
Metabolic energy (kcal/kg)	3035.00	3170.00	3170.00	3250.00	3250.00
Crude protein (%)	22.00	21.10	21.10	19.10	19.10
Crude fat (%)	5.62	7.55	7.46	8.00	7.88
Dry fibre (%)	3.16	3.06	3.02	2.87	2.83
Dry ash (%)	6.03	5.46	5.42	5.00	4.93
Calcium (%)	1.05	0.90	0.90	0.85	0.85
Phosphorus (%)	0.76	0.70	0.70	0.65	0.65
Phosphorus (available) (%)	0.50	0.45	0.45	0.42	0.42
Sodium (%)	0.17	0.17	0.17	0.17	0.17
Chlorine (%)	0.19	0.19	0.19	0.19	0.19
Methionine (%)	0.70	0.58	0.58	0.51	0.50
Met + cyst (%)	1.07	0.95	0.95	0.86	0.86
Lysine (%)	1.43	1.26	1.26	1.11	1.11
Choline (%)	1.62	1.51	1.51	1.42	1.43

Twelve thousand broiler chicks obtained from middle-aged (36 weeks) Ross 308 broiler breeders were used in the present study. The chicks were sexed, vaccinated, and transferred to the test house. They were then weighed using a scale (EC-130, Bizerba SE/Co. KG, Germany) and randomly distributed to the 60 pens as 200 chicks per pen (stocking density of 15.38 chicks/m²) with each subgroup consisting of 50% female + 50% male. Five replications were used per treatment during the growing period.

The animals were fed in four phases namely starter, grower, finisher, and pre-slaughter, and these four phases were between 0-10th, 11-25th, 26-36th, 37-41th days, respectively. Water and feed and water were given ad libitum during the study.

The ALW of the broilers were determined for each subgroup at the end of the growing period before loading for transport and weighing all animals. The FI levels of the subgroup were determined by weighing the feed left in the feeders at the beginning and end of the growing period. Feed conversion ratio (FCR) values were determined using the equation given below (Aviagen, 2018):

The number of chicks that died in the subgroups was daily recorded and the mortality and liveability rates were calculated. European Production Efficiency Factor (EPEF) values were calculated by using liveability, live weight, age and FCR as follows (Aviagen, 2018):

The DAFS usage rate (control/0, 11, and 22%) in POM production; and the POM usage rate in grower (2 and 3%), finisher and pre-slaughter feeds (4 and 5%) were used in a 3 × 2 × 2 factorial arrangement. The quantity of replicates was calculated via power analysis with PASS 11 (Hintze, 2011), and the replication quantity for each group was determined as four. However, the repetition number was finally determined as five to improve trial reliability and to account for deviations that may occur due to field conditions considering that the test house contained 60 pens. Additionally, the available treatment number could have been increased by re-dividing the compartments in the test house. However, considering the potential difficulties throughout the rearing period, it was preferred to work with 200 birds in total.

The results were based on actual data since the differences were not significant (P>0.05) between the data with and without arc sinus transformation. The statistical analyses of the results were performed using IBM SPSS 22 (SPSS, 2013), by first obtaining the skewness and kurtosis values, and subsequently confirming the normal distribution by Shapiro-Wilk test. After this process, an analysis of variance (ANOVA) was conducted for the experiment using the GLM procedure of SPSS (2013) suitable for two-way designs.

The two-way ANOVA model is as follows:

in which Y_ijkl is the dependent variable; μ denotes the overall mean; DAFS_i is the effect of the DAFS usage rate in POM (i = control/0, 11 or 22% in the experiment); POMg_j is the effect of the POM usage rate in grower diets (j = 2 and 3% in the experiment); POMf_k is the effect of the POM usage rate in finisher and pre-slaughter diets (k = 4 and 5% in the experiment); DAFSPOMgPOMf_ijk is the effect of the interaction between DAFS, POMg, and POMf; and e_ijkl is the random error term. The two-way ANOVA and post-hoc Tukey test were employed to analyse the differences in the investigated parameters in relation to the DAFS usage rate in POM, POM usage rate in grower, finisher, and pre-slaughter feed, as well as their interaction (Kocabas et al., 2013).

The chick weight (CW) values of the treatment groups (C024, C025, C034, C035, T124, T125, T134, T135, T224, T225, T234, and T235) were 42.3 g (CV_CW = 1.51), 41.9 g (CV_CW = 2.76), 42.0 g (CV_CW = 1.28), 41.8 g (CV_CW = 1.67), 42.0 g (CV_CW = 1.15), 41.6 g (CV_CW = 0.62), 42.3 g (CV_CW = 0.78), 42.2 g (CV_CW = 0.42), 41.8 g (CV_CW = 0.46), 41.9 g (CV_CW = 1.01), 42.1 g (CV_CW = 2.08), and 41.9 g (CV_CW = 1.92), respectively (Table 7).

The uniformity of the chicks utilised in the research was high, and the variations between the CW values of the treatment groups were not statistically significant (P>0.05).

A decrease in FI was found when POM was administered with DAFS as opposed to POM alone (Table 8). This is particularly evident in grower FI, in which the difference between some treatment groups was significant (P<0.05). Depending on the grower FI, the difference between the total FI of some groups was also found to be statistically significant (P<0.05).

Table 8

Treatment group	DAFS in POM (%)	POM in starter and grower diets (%)	POM in finisher and pre-slaughter diets (%)	Feed intake (g/broiler)
				Starter (0-10th day)	Grower (11-25th day)	Finisher (26-36th day)	Pre-slaughter (37-41st day)	Total
C024		2.00	4.00	324±4	1090±1ab	2185±24	542±2	4141±21
C025	0.00		5.00	325±5	1093±2ab	2220±18	544±3	4182±11
C034		3.00	4.00	344±4	1102±2ab	2235±24	541±1	4222±28a
C035			5.00	325±1	1100±1ab	2235±14	528±13	4188±27
T124		2.00	4.00	325±2	1013±17cd	2231±26	527±16	4096±26c
T125	11.00		5.00	331±4	987±21d	2251±7	531±14	4099±26bc
T134		3.00	4.00	327±4	1051±14bc	2238±4	514±17	4130±25
T135			5.00	330±5	1056±14abc	2208±23	546±4	4141±35
T224		2.00	4.00	339±6	1084±14ab	2255±19	543±1	4221±15ab
T225	22.00		5.00	333±4	1105±4a	2226±125	499±16	4163±19
T234		3.00	4.00	332±4	1109±4a	2225±25	527±15	4193±23
T235			5.00	331±6	1105±2a	2191±22	530±14	4157±32
P-value				0.151	0.000	0.276	0.196	0.009

C - control; T - treatment; first digit after the letter (0, 1, and 2) - DAFS usage percentage in POM; second digit after the letter (2 and 3): POM usage percentage in grower diets; third digit after the letter (4 and 5) - POM usage rate in finisher and pre-slaughter diets.

When ALW results were analysed, it was found that there were numerical differences between the treatment groups, and ALW decreased as the DAFS rate increased, being more evident among various groups (Table 7) and significant in certain groups (P<0.05). For example, the ALW in the treatment group fed POM without DAFS in 2% grower and 5% finisher feeds were significantly higher than the group fed POM with the highest level (22.0%) in 3% grower and 5% finisher feeds (2472±12 g and 2377±26 g, respectively).

When DWG results were analysed (Table 7), we found numerical variations across the treatment groups, and the DWG decreased as the DAFS rate increased, with this effect being more evident in some groups and significant (P<0.05).

There were numerical differences among the treatment groups (Table 7). These numerical differences among some of the groups were more evident, and the differences between these groups were statistically significant (P<0.05). For example, the FCR in the treatment group fed POM without DAFS in 2% of grower and 5% of finisher feeds was significantly higher than in the group fed POM with level of 22.0% in 3% of grower and 5% finisher feeds (1.757±0.010 and 1.814±0.015, respectively).

Numerical differences were found between EPEF values (Table 7). These numerical differences were more evident in some groups and some were found to be statistically significant (P<0.05). For example, the EPEF values in the treatment group fed POM without DAFS in 2% of grower and 5% finisher feeds were significantly higher than in the group fed POM with highest level (22.0%) in 3% grower and 5% finisher feeds (329.46±8.162 versus 308.87±0.46 and 311.39±7.08, respectively).

There were numerical differences between the mortality values of the treatment groups. The mortality values increased numerically as the DAFS rate increased, but the differences were statistically significant (Table 7 and Figure 1).

Figure 1 C - control; T - treatment; first digit after the letter (0, 1, and 2) - DAFS usage percentage in POM; second digit after the letter (2 and 3) - POM usage percentage in grower diets; third digit after the letter (4 and 5) - POM usage rate in finisher and pre-slaughter diets.

The uniformity of the chicks utilised in the research was high, and the measured CW were found to be slightly lighter (42.0 < 43.0 g) compared with the specs (Aviagen, 2019). The variations between the CW values of the treatment groups were not statistically significant (P>0.05). It is thought that this is an expected result, that it is due to the fact that the chicks were placed in each replication pen as 50% male and 50% female at the beginning of the trial, so that the chick weights, or in other words the starting weight, would not affect subsequent performance, and that the trial was planned correctly in this respect.

Feed intake decreased when POM was added to DAFS as opposite to POM alone (Table 8). The FI values were slightly different (>304 g, <1369 g, >1807 g, and <1009 g for starter, grower, finisher, and pre-slaughter, respectively) compared with the specs (Aviagen, 2019), and this is thought to be due to diet specifications and their changes due to the addition of DAFS. Also, it was observed that the colour of POM and feed with DAFS darkened slightly, probably due to the chemicals used in DAFS production, and this darkening became more evident as the amount of DAFS added increased, consistent with Fransen et al. (1995).

Average live weight values achieved in the trials (Table 7) were slightly lighter (<2821 g) compared with the specs (Aviagen, 2019), and this is thought to be due to diet specifications and growing conditions. The ALW decreased as the DAFS rate increased, being more evident among various groups. While the results of the study were not consistent with the findings of De Vries and Mudler (1986), who stated that there was no negative effect when using 7% or even 15% DAFS, they were consistent with the results of El Boushy et al. (1984), who reported a decrease in growth rate when DAFS was used at 2-7%. It is thought that the differences between the results are due to differences in the DAFS production system and DAFS content.

The DWG of the treatment groups (Table 7) was numerically lower than the specified values (Aviagen, 2019). Similar to the findings for ALW, the DWG showed a more pronounced decrease as the DAFS rate increased. This result is likely attributable to the feed content and growing conditions. However, the results of the present study were consistent with those of some authors in the literature (El Boushy et al., 1984), who reported a decrease in growth rate when DAFS was used at a concentration of 2-7%, but not with others (De Vries and Mudler, 1986), who stated that there was no negative effect when DAFS was incorporated at 7% or even 15%. It is thought that the differences between the results obtained in similar studies and our samples are due to differences in POM and DAF contents as well as diet specifications.

The FCR value increased extremely as the DAFS rate increased (Table 7). The results were supported by the findings of El Boushy et al. (1984), who reported that growth rate decreased when 2% DAFS was used. However, the results were not supported by the study of De Vries and Mudler (1986), who found no negative effect when using 7% or even 15% DAFS. It is thought that the differences between the results obtained in similar studies and our samples are due to differences in DAF contents and DAF usage levels.

The EPEF values, which were used to evaluate the total performance obtained at the end of the experiment period, decreased as the DAFS rate increased (Table 7). It is thought that the differences between the results are due to differences in the DAF production system and DAFS content. In addition, since the EPEF value is calculated using ALW and FCR data, a similar result is obtained as in these criteria.

The results were compatible with the results of De Vries and Mudler (1986), although they were not consistent with other research (El Boushy et al., 1984) results. This result is thought to be due to differences in the production and processing of DAFS and the elimination of possible harmful organisms that may be present in the DAFS through thermal processes similar to our process, especially sterilization.