Coated organic acids with essential oils in Japanese quail’s fed restricted during the 2^nd week of age: effects on performance, carcase traits, blood profile, antioxidants status, and caeca microbiota

Abstract

This study aimed to evaluate the effect of Coated Organic Acids with Essential Oils (COAWEO) supplementation on performance, carcase traits, blood biochemistry, antioxidant status, and gut microbiota of Japanese quail (JQ) fed restricted during the 2^nd week of age. Five hundred and forty, 7-days old JQ were distributed into 6 groups with six replicates/groups. The feed restriction (FR) was applied only at days 7, 9, 11, and 13 of age. Two levels of FR were used: −12.5 and −25.0% of the amount of feed consumed by the control group (feeding ad libitum), on the previous day. The diets (control, FR12.5 and FR25.0) were fed with or without 100 g/ton of COAWEO. The results indicate that FR and COAWEO-supplemented diets did not affect weight gain, feed intake, and carcase weight. The feed conversion ratio (FCR) was improved by FR during 7–42 days of age. The total bacterial and Lactobacilli counts in the caeca were, respectively, decreased and increased due to FR and COAWEO. Japanese quail can tolerate 12.5% FR without adverse effects on growth performance, carcase traits, blood biochemistry, and gut microbiota. FR25.0 group supplemented with COAWEO showed improved growth performance than the control without any adverse effect on carcase traits, blood biochemistry, and gut microbiota.

Highlights

1. Feed restriction can represent a way to improve the sustainability of poultry farms.

2. The concerns about animal welfare can be overcome by applying feed restrictions for a short period.

3. The combination short-period feed restriction/antioxidant supplementation can further improve animal welfare.

Keywords:

• Feed restriction
• organic acids
• essential oils
• Japanese quail
• growth performance
• carcase
• blood biochemistry
• antioxidants
• gut microbiota

Introduction

Feed restriction (FR) is a strategy to overcome the shortage of feed availability in animal production. According to Belaid-Gater et al. (2022) the quantitative feed restriction at 10 and 20% of the daily feed intake, provides a significant reduction in feed costs, a reduction in the carcase cost price and, consequently, a significant improvement in farmers' income. However, FR can compromise animal welfare by modifying the behaviour: in fact, the application of FR decreases sitting or lying and comfort behaviour, while increasing the walking activities of birds (Trocino et al. 2020). In addition, under a restricted-feeding regimen, starvation induces the birds to intake the litter (Soltanmoradi et al. 2014; Jahanpour et al. 2015) exposing the animals to possible infections; starvation per se can affect the gut microbiota (Siegerstetter et al. 2018; Yan et al. 2021). Therefore, it would be necessary to identify possible solutions to avoid health and welfare problems in animals subjected to feed restrictions.

Organic acids and salts have been used in poultry diets and drinking water for decades and seem to elicit positive responses in growth performance (Petruška et al. 2012). However, many previous studies have been conducted to evaluate the effect of organic acid supplements on the growth of poultry species, including quail instead of antibiotics, for the reduction of feed cost and profitable quail production (Attia et al. 2013; Zaman 2018). The effect of organic acids includes i10m-proved gut health and reduced pathogens; thus, the bird can maximise nutrient utilisation (Attia et al. 2019). The organic acids also stimulate the pancreatic secretion and provide better intestinal villus integrity, which improves the surface area of absorption in the intestine and endogenous digestive enzymes in addition to antioxidants capacity and lipid metabolism activities (Dibner and Buttin 2002; Botsoglou et al. 2005).

Essential oils have shown potential effects on growth performance, gut health, and control of pathogens, according to recent evidence (Attia et al. 2018; Attia et al. 2019; Irawan et al. 2021; Puvača et al. 2022). However, studies addressing the effect of the combination of FR with an organic acid with essential oils supplementation on Japanese quail are little available in the current literature. Hence the present study investigated coated organic acids with essential oils supplementation on performance, carcase traits, blood biochemistry, antioxidant status, and gut microbiota of Japanese quail fed restricted during the 2^nd week of age.

Material and methods

The trial was carried out at the Poultry Unit, Agricultural Researchers and Experiments Station, Faculty of Agriculture, Mansoura University. The experimental protocol does not cause pain, suffering, distress, or lasting harm. The experimental procedures were according to the Royal Decree number M59 from 14/9/1431H and were approved by the Animal Care and Use Committee office of King Fahd Medical Research Centre, under the approval code ACUC-22-1-2.

Five hundred and forty, 1-day-old Japanese Quails (JQ), weighing an average 8.73 g, were randomly distributed in a factorial (3 × 2) design to six equal groups with six replicates in each group (15 birds/replicate). In the first week, the JQ in all groups were fed the basal diet (Table 1) ad libitum. From 7 to 42 days of age, the control group was fed ad libitum; the second and third groups were submitted to feed restriction at 12.5 and 25% of the feed intake recorded in the control group on the previous day, respectively. The 4^th, 5^th and 6^th groups received, respectively, the same diets as the control, 12.5FR and 25FR groups but their diets were supplemented at 0.01% with coated organic acids and synergetic essential oils (COAWEO). The feed restriction was applied at only 7, 9, 11, and 13 days of age. The experimental diets were fed without any antibiotics or anticoccidial drugs. Chemical-nutritional characteristics of the basal diet were calculated (NRC, 1994).

Table 1. Ingredients and calculated analysis of the basal diet.

Ingredients (as fed basis), %
Yellow corn	56.2
Soybean meal	28.9
Corn gluten	10.0
DCP	1.7
Limestone	2.0
Salt	0.5
Premix	0.3
Methionine	0.1
Lysine	0.3
Calculated analysis
Metabolizable energy (ME), kcal/kg	2914
Crude protein (CP), %	24.03
Ether extract (EE), %	2.62
Crude fibre (CF), %	3.39
Calcium, %	1.21
Total P %	0.71
Avail. P%	0.45
Lysine, %	1.26
Methionine, %	0.53
Methionine + Cystine, %	0.93

* Each 3 kg premix contains: Vit. A,12,000,000 IU; Vit. D3 2,500,000 IU; Vit. E,10 g; Vit. K, 2.5 g; Vit. B2,5g; Vit. B6,1.5g; Vit. B12,10mg; Biotin,50mg; Folic acid,1.0g; Nicotinic acid,30mg; Pantothenic acid,10g; Antioxidant,10g; Mn,60g; Cu,10g; Zn,55g; Fe,35g; I, 1.0 g; Co,250mg and Se,150mg.

The supplement was added to the top of the diet and mixed well with the diet to assure homogeneity. The COAWEO contained coated citric acid at 115 g, coated lactic acids 72 g, coated malic acids 83 g, coated sorbic acid 72, coated formic acid 184 g, thymol extracts 25 g, berberine extract 30 g and 5′-methoxyhydnocarpin and hydrogenated palm oil up to 1000 g (Biofeed, https://www.biofeedtech.com/en/products.html produced by the DACIDS G2P).

The basal diet was administered in a mash form. The quails were housed in battery brooders, and each cage was equipped with one tube feeder and two still-stain nipple cub waters. The chicks were submitted to 23:1 light-dark cycle during the 1st week and 20: 4 after that.

Growth performance and carcase traits

Weekly feed intake (FI), body weight (BW), and weight gain (BWG) were recorded, whereas feed conversion ratio (FCR) was calculated weekly and for the whole experimental period (7-42 days of age). In addition, the mortality rate was recorded during the testing period, and the survival rate was calculated during 7–13, and 7–42 days of age.

At 42 days of age, 10 quails from each experimental group were chosen randomly to represent all treatment replicates and slaughtered after 8 h of feed withdrawal. The quails were slaughtered according to the Islamic method; the feathers were bulked, and the carcases were eviscerated to calculate the yield, totally edible and inedible parts, and dressing percentage relative to live body weight. The internal organs were separated and expressed relative to live body weight.

Blood parameters

At slaughtering, blood samples were collected from 1 animal per replicate (a total of 36 samples, 6 per group) in test tubes. The serum was separated by centrifugation at 3000 rpm x g for 20 min, and the serum was then separated and stored at −18 °C for analysis. Serum total protein (TP, Henry 1964) and albumin (Alb, Doumas et al. 1971) were determined, while globulin (Glb) was calculated by subtracting Alb from TP and used to calculate the albumin/globulin ratio.

Total lipids (TL) were measured according to Chabrol and Charonnat (1973). The serum lipid profile such as triglycerides (TG, Tietz, 1995), total cholesterol (TC, Allain et al. 1974), and high-density lipoprotein cholesterol (HDL, Sawle et al. 2002) were determined using diagnostic kits. The serum low-density lipoprotein-cholesterol (LDL) was also estimated according to Friedewald et al. (1972) as follows: LDL-C = Total Cholesterol–(HDL-C + VLDL), where very low-density lipoprotein (VLDL) = serum triglycerides/5.

The indices of liver leakage enzymes, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured according to Reitman and Frankel (1957). Total antioxidant capacity (TAC), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined according to Koracevic et al. (2001), Mihara and Uchiyama (1978) and Yi-Sun et al. (1988), respectively. Corticosterone (Cort) concentration in blood serum was determined according to Jezová et al. (1994). Alkaline phosphatase, catalase in red blood cells, antioxidant status (TAC/MDA), tri-iodine threonine (T3), thyroxine (T4), immunoglobin G (IgG), M (IgM), and (IgA) were assayed using an automatic analyser with commercial kits from Bio-diagnostic Company (Giza, Egypt) according to the manufacturing procedures. The Hemagglutination inhibition (HI) test for infectious bursa disease (IBD) and avian influenza (AI, H5N1 subtype Re-5 strain) was determined as outlined by Attia et al. (2019).

Gut microbiota

The gut microbiota was evaluated using ten samples of each treatment collected from the caeca of the slaughtered birds at day 42 of age. Salmonella spp. was isolated on 1 g of each sample homogenised in 9 ml of Buffered Peptone Water (BPW) and incubated at 37 °C for 20 h; the incubated samples were gently shacked and added to Rappaport-Vassiliadis broth (Oxoid, Basingstoke, UK) at 1:100 and incubated at 42 °C for further 24 h followed by streaking on Xylose Lysine Desoxycolates at 37 °C for 24 h. The plates were examined after incubation for the presence of typical and suspected colonies. Up to five colonies from each sample were further identified based on biochemical characterisation (Indole test, motility, nitrate reduction, methyl red, Voges Proskauer, citrate utilisation, and urease) according to the guidelines of ISO 6579:2002. Biochemically typical salmonella isolates were serotyped according to Kauffman–White–Le–Minor technique using slide agglutination technique with polyvalent somatic (O) and flagellar (H) antisera (Welcome Diagnostic, UK), which were performed at the faculty of agriculture, Mansoura university following the White–Kauffmann- Le Minor scheme (Grimont and Weill 2007).

For the isolation of E. coli, 1 gram of each caeca sample was aseptically added to 9 mL of Tryptic Soya Broth (TSB/CM129, Oxoid, Hants, UK) into a stomacher bag for at least 2 min, and then they were incubated at 37 °C for 24 h according to De Boer and Heuvelink (2000). Loopful from the incubated broth was streaked onto the surface of EMB agar (CM69, Oxoid, Hants, UK). After 24 h at 37 °C, the colonies of E. coli were confirmed biochemically followed by O-serotyping. On the other hand, one gram was aseptically added to 9 mL of 0.1% sterile peptone solution to prepare a ten-fold serial dilution of up to 107 ml of each sample.

Viable counts of total aerobes, coliforms, and lactobacilli were performed using the spread-plate technique. Total aerobes were enumerated on nutrient agar (Oxoid, Hants, UK) and coliform was enumerated on Mac Conkey agar, the plates were incubated aerobically at 37 °C for 24 h. For lactobacilli culture, deMan-Rogosa-Sharpe (MRS) agar (Biolife, Milan, Italy) was used; the plates were incubated in 5% CO₂ for 48 h. The media plates were inoculated with 0.1 ml of the sample dilutions. After incubation, colonies were counted according to colony morphology. Numbers of CFU per gram of digesta content were recorded (Mahdavi et al. 2010).

For the isolation of Clostridium, the inoculation of samples was carried out in a cooked meat medium (Becton, Dickinson and Company, USA). The samples were then anaerobically incubated at 37 °C for 24 h in an anaerobic jar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) containing GasPakTM (Becton Dickinson and Company, USA) according to Dar et al. (2017). Enriched samples were streaked on Sulphite Polymixin Sulphadiazine (SPS) agar plates (Hi-Media Laboratories, Mumbai) and were anaerobically incubated as previously mentioned. Suspected colonies were stained with Gram’s stain and sub-cultured on brain heart infusion (BHI) agar plates until they were free from contaminating bacteria. The pure colonies, suggestive of Clostridium were further streaked on the 5% sheep blood agar and egg yolk agar plates and anaerobically incubated for 24 h. The colonies producing characteristic double zone of haemolysis around them on blood agar and producing zone of opalescence around the colonies on egg yolk agar were tentatively identified as Clostridium.

Statistical analysis

The normal distribution of the data was checked by SAS (2004). Data were analysed using a two-way analysis of variance of the GLM procedure of SAS (2004). The following statistical model was used: Yijk = µ + Fi + Aj + FAij + eijk. Where: Yij = observed traits; µ = the overall mean; Fi = effect of feed restriction (i = 0, 12.5 and 25.0); Aj = effect of feed additive (j = 0, 1); FAij = effect of interaction between feed restriction and feed additive; eijk = experimental random error. The experimental unit was the replicate. The difference among means was evaluated using the Tukey’ test (SAS, 2004). Differences between means were considered significant at p < 0.05.

Results

Growth performance

Table 2 showed that FR significantly (p < 0.05) affected quails’ FI during all experimental periods except for 1–6 and 7–42 days of age periods. Quails’ FI during 14–27 days of age was significantly (p < 0.05) affected by the FR regimen, showing an inconstant effect depending on the period. During 14–27 days of age, feed intake of the 25% group was significantly increased by 17.7% compared to the control. In the 28–42 days of age, there were no significant differences in feed intake among different FR groups.

Table 2. Effects of feed restriction and coated organic acids with essential oils on feed intake (g/chick/period) of Japanese quail.

	Days
	1-6	7-13	14-27	28-42	7-42
Effect of feed restriction
0	31.1	66.5^a	250^b	444^a	761
12.5	30.4	61.7^b	269^ab	390^b	721
25.0	30.9	58.0^c	272^a	396^b	726
Effect of feed additive
0	30.9	66.9^a	263	417	746
COAWEO	30.7	63.2^b	265	403	732
Interactions between feed restriction and feed additive
0 × 0	31.1	71.4^a	246	442	760
0 × COAWEO	31.0	61.5^b	254	445	761
12.5 × 0	30.6	67.5^ab	273	399	739
12.5 × COAWEO	30.3	65.1^ab	266	381	712
25.0 × 0	31.0	61.8^b	269	410	740
25.0 × COAWEO	30.8	61.8^b	276	383	722
RMSE	0.819	3.87	17.2	40.3	42.5
P-values
FR	0.770	0.046	0.024	0.015	0.164
COAWEO	0.237	0.021	0.787	0.526	0.475
Interaction	0.815	0.022	0.513	0.565	0.682

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error FR: feed restriction; COAWEO: coated organic acid with essential oil.

Feed intake during only 7–13 days of quail supplemented with COAWEO was considerably (p < 0.05) decreased by 13.9% than the control group; in addition, there was a significant (p < 0.05) interaction between the FR regimen and COAWEO for feed intake, during only 7–13 days of age.

Table 3 showed that feed restriction did not significantly affect quails’ BWG except during 7-13, and 14–27 days of age. Weight gain for 7–13 days was similar in the 12.5% FR group to the control group, but better by 23% (p < 0.05) than the 25.0% FR group. This indicates that JQ tolerates the 12.5% FR treatment without adverse effects on growth as no difference in growth from the control was observed during the FR and following periods.

Table 3. Effects of feed restriction and coated organic acids with essential oils on body weight gain (g/chick/period) of Japanese quail.

	Days
	Initial weight	1-6	7-13	14-27	28-42	7-42
Effect of feed restriction
0	25.9	17.3	23.6^ab	87.5^b	102.0	213
12.5	25.4	16.6	26.2^a	95.8^ab	92.8	215
25.0	25.7	17.0	21.3^b	103^a	93.6	217
Effect of feed additive
0	25.7	16.9	24.6	91.5	98.3	214
COAWEO	25.7	17.0	22.8	99.0	94.2	216
Interactions between feed restriction and feed additive
0 × 0	26.0	17.2	24.6	78.1	108	210
0 × COAWEO	26.0	17.5	22.6	96.9	96.8	216
12.5 × 0	25.5	16.6	27.9	93.9	91.6	213
12.5 × COAWEO	25.4	16.6	24.5	97.7	94.1	216
25.0 × 0	25.7	17.0	21.2	103	95.4	219
25.0 × COAWEO	25.7	17.0	21.4	102	91.8	216
RMSE	0.311	0.312	3.38	10.1	9.71	7.60
P-values
FR	0.064	0.098	0.012	0.015	0.067	0.561
COAWEO	0.723	0.815	0.142	0.072	0.427	0.440
Interaction	0.902	0.882	0.438	0.149	0.380	0.314

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil.

On days 14–27, the 25.0% FR group had a better (p < 0.05) growth rate (+17.7%) than the control group, which was not considerably different from the 12.5% FR group. Body weight gain was not different among the experimental groups for the whole period (7–42 days of age).

Supplementation with COAWEO did not influence the BWG of birds during all periods. The interactions between feed restriction and feed additive (COAWEO) were insignificant for BWG of quails during all periods. Table 4 showed that quails FCR was significantly improved (p < 0.05) by both FR regimen during the whole experimental period (7–42 days of age).

Table 4. Effects of feed restriction and coated organic acids with essential oils on the feed conversion ratio and survival rate of Japanese quail.

	Feed conversion ratio g feed/g gain					Survival rate %
	1-6	7-13	14-27	28-42	7-42	7-13	7-42
Effect of feed restriction
0	1.80	2.83	2.92	4.37	3.58^a	95.0	94.4
12.5	1.84	2.58	2.83	4.20	3.38^b	97.2	95.0
25.0	1.82	2.98	2.68	4.24	3.37^b	97.8	95.0
Effect feed additive
0	1.83	2.76	2.92^a	4.26	3.49	96.6	94.8
COAWEO	1.81	2.83	2.70^b	4.28	3.39	96.6	94.8
Interactions between feed restriction and feed additive
0 × 0	1.81	2.93	3.20	4.13^ab	3.61	94.4	94.4
0 ×COAWEO	1.78	2.73	2.63	4.61^a	3.52	95.5	94.4
12.5 × 0	1.85	2.43	2.91	4.35^ab	3.46	96.6	94.4
12.5 × COAWEO	1.82	2.73	2.74	4.04^b	3.29	97.8	95.5
25.0 × 0	1.82	2.92	2.65	4.31^ab	3.38	98.9	95.5
25.0 × COAWEO	1.82	3.03	2.71	4.17^ab	3.35	96.6	94.4
RMSE	0.048	0.377	0.274	0.272	0.169	2.66	1.48
P-values
FR	0.507	0.052	0.176	0.535	0.042	0.095	0.702
COAWEO	0.361	0.446	0.047	0.949	0.158	0.964	0.993
Interaction	0.922	0.399	0.074	0.014	0.681	0.340	0.275

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil.

Adding COAWEO to JQ diet did not influence FCR during any experimental period. However, there was a significant interaction between COAWEO and FR regimen during 28–42 days of age (p < 0.05). Quails’ feed utilisation was improved during 14–27 days of age due to COAWEO supplementation to the control diet, (p < 0.05) which was connected mainly with the reduction in feed intake. During 28–42 days of age, COAWEO supplementation improved FCR of the 12.5% FR groups by 12.4% compared to the control group supplemented with COAWEO. Survival rates during 7–13 or 7-–42 days of age are displayed in Table 4. The results show that the survival rate was not affected by feed restriction and/or COAWEO.

Carcase traits

Table 5 shows that FR and/or COAWEO did not affect the carcase and most of the internal organs’ percentage except for the gizzard, which was significantly decreased by 10.9% due to COAWEO supplementation. There was a significant effect (p < 0.01) of the interaction COAWEO x FR on only giblets percentage, showing COAWEO increased giblets of 12.5% FR regimen by 9.9% and decreased by 14.3% of 25.0% FR group. FR, COAWEO and their interaction had no significant effects on carcase traits and internal organs.

Table 5. Effects of feed restriction and coated organic acids with essential oils on carcase traits (%) of Japanese quail at 42 days of age.

	Carcase	Liver	Gizzard	Heart	Giblets	TEP	NEP
Effect of feed restriction
0	71.8	2.06	1.59	0.851	4.50	76.3	23.7
12.5	71.6	2.15	1.64	0.870	4.67	76.2	23.8
25.0	70.5	2.05	1.69	0.854	4.60	75.1	24.9
Effect of feed additive
0	70.9	2.08	1.74^a	0.847	4.67	75.6	24.4
COAWEO	71.6	2.09	1.55^b	0.869	4.51	76.1	23.9
Interactions between feed restriction and feed additive
0 × 0	71.6	2.05	1.71	0.837	4.60^ab	76.3	23.7
0 × COAWEO	72.0	2.07	1.47	0.865	4.40^b	76.3	23.7
12.5 × 0	71.1	1.96	1.64	0.847	4.45^b	75.5	24.5
12.5 × COAWEO	72.0	2.35	1.65	0.893	4.89^a	76.9	23.1
25.0 × 0	70.0	2.24	1.85	0.858	4.95^a	75.0	25.0
25.0 × COAWEO	70.9	1.85	1.54	0.850	4.24^b	75.2	24.8
RMSE	2.47	0.432	0.301	0.196	0.553	2.60	2.60
P-values
FR	0.212	0.697	0.553	0.947	0.633	0.261	0.261
COAWEO	0.276	0.957	0.021	0.665	0.285	0.436	0.436
Interaction	0.909	0.021	0.238	0.906	0.007	0.705	0.705

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil; TEP: total edible parts; NEP: non-edible parts.

Serum blood biochemistry

Tables 6–9 show serum blood characteristics as affected by FR and/or COAWEO supplementation. There was no significant effect of FR, COAWEO and their interaction on protein metabolites and different kinds of globulin except for β- globulin (Table 6). The results indicate that COAWEO addition to the control group decreased significantly β- globulin, but COAWEO increased β- globulin of the 12.5% FR group. In addition, when the unsupplemented groups were compared 12.5% FR decreased β-globulin compared to the control group.

Table 6. Effects of feed restriction and coated organic acids with essential oils on serum blood parameters of Japanese quails.

	TP g/dL	Alb g/dL	Glb g/dL	Alb/Glb	α-Glb g/dL	β-Glb g/dL	ɣ-Glb g/dL
Effect of feed restriction
0	5.58	2.54	3.04	0.863	1.67	1.01	0.730
12.5	5.51	2.61	2.90	0.928	1.61	1.01	0.850
25.0	5.48	2.47	3.01	0.838	1.61	1.01	0.780
Effect of feed additive
0	5.63	2.55	3.08	0.843	1.60	1.00	0.780
COAWEO	5.42	2.53	2.89	0.910	1.66	1.02	0.793
Interaction between feed restriction and feed additive
0 × 0	5.80	2.50	3.30	0.759	1.68	1.10^a	0.720
0 × COAWEO	5.36	2.58	2.78	0.967	1.66	0.92^b	0.740
12.5 × 0	5.54	2.60	2.94	0.903	1.54	0.90^b	0.860
12.5 × COAWEO	5.48	2.62	2.86	0.954	1.68	1.12^a	0.840
25.0 × 0	5.54	2.54	3.00	0.867	1.58	1.00^ab	0.760
25.0 × COAWEO	5.42	2.40	3.02	0.808	1.64	1.02^ab	0.800
RMSE	0.321	0.296	0.412	0.207	0.119	0.116	0.111
P-values
FR	0.777	0.579	0.729	0.606	0.441	0.996	0.072
COAWEO	0.091	0.903	0.211	0.388	0.180	0.642	0.745
Interaction	0.378	0.696	0.315	0.363	0.339	0.003	0.829

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil; TP: total protein; Alb: albumin; Glb: globulin.

Table 7. Effects of feed restriction and coated organic acids with essential oils on serum blood parameters of Japanese quails.

	TL mg/dL	Trg mg/dL	Chl mg/dL	HDL mg/dL	LDL mg/dL	HDL/LDL	vLDL mg/dL	ALT U/L	AST U/L	ALT/AST
Effect feed restriction
0	5.88	179	208	40.7	88.4	0.46	35.9	65.4	57.4	1.15
12.5	5.67	180	210	39.6	90.4	0.44	35.9	65.0	56.8	1.15
25.0	5.79	176	204	41.0	91.7	0.45	35.1	65.7	58.6	1.12
Effect of feed additive
0	6.01	179	207	41.5	88.7	0.47	35.9	66.0	56.9	1.16
COAWEO	5.55	177	207	39.4	91.7	0.43	35.4	64.7	58.3	1.11
Interaction between feed restriction and feed additive
0 × 0	6.18	187^a	204	41.4	85.6	0.49	37.5	67.0	56.0	1.21
0 × COAWEO	5.58	171^b	212	40.0	91.2	0.44	34.3	63.8	58.8	1.09
12.5 × 0	5.82	174^b	213	40.8	89.6	0.46	34.8	65.4	56.4	1.16
12.5 × COAWEO	5.52	185^a	207	38.4	91.2	0.42	37.0	64.6	57.2	1.13
25.0 × 0	6.04	177^ab	204	42.2	90.8	0.47	35.3	65.6	58.4	1.12
25.0 × COAWEO	5.54	175^ab	203	39.8	92.6	0.43	34.9	65.8	58.8	1.12
RMSE	0.920	8.48	10.6	4.18	4.69	0.057	1.70	2.64	2.86	0.0878
P-values
FR	0.878	0.504	0.368	0.735	0.303	0.685	0.504	0.839	0.374	0.781
COAWEO	0.178	0.446	0.878	0.188	0.093	0.080	0.446	0.201	0.215	0.118
Interaction	0.934	0.007	0.331	0.954	0.569	0.947	0.007	0.351	0.610	0.339

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil; TL: total lipids; Trg: triglycerides; Chl: cholesterol; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein-cholesterol; VLDL: very low-density lipoprotein; ALT: alanine ami-notransferase; AST: aspartate aminotransferase.

Table 8. Effects of feed restriction and coated organic acids with essential oils of serum blood parameters of Japanese quails at 42 days of age.

	AP U/100mL	CRB U/mL	TAC Umol/l	MDA Umol/L	AB	SOD U/mg	T3 Ug/dL	T4 Ug/dL	T3/T4	Cor pg/mL
Effect of feed restriction
0	7.64	39.3	422	1.71^b	255^a	313^ab	0.174^ab	7.84	2.22	4.14
12.5	7.51	37.4	428	2.19^a	197^b	313^a	0.157^b	7.60	2.07	3.80
25.0	7.72	37.4	427	2.09^a	208^b	312^b	0.194^a	7.93	2.45	4.01
Effect of feed additive
0	7.54	38.7	425	1.89^b	233	313	0.169	7.66	2.21	3.96
COAWEO	7.71	37.4	427	2.10^a	207	313	0.181	7.92	2.28	4.01
Interaction between feed restriction and feed additive
0 × 0	7.56	38.9	419	1.64	269	312^ab	0.152	7.54	2.02	4.20
0 × COAWEO	7.72	39.7	426	1.78	241	314^a	0.196	8.14	2.42	4.08
12.5 × 0	7.56	38.4	428	2.12	203	314^a	0.154	7.46	2.07	3.62
12.5× COAWEO	7.46	36.4	427	2.26	190	313^ab	0.160	7.74	2.07	3.98
25.0 × 0	7.50	38.8	428	1.92	225	312^b	0.202	7.98	2.54	4.06
25.0× COAWEO	7.94	36.0	426	2.26	191	313^ab	0.186	7.88	2.36	3.96
RMSE	0.334	2.30	6.59	0.257	36.1	0.955	0.0278	0.456	0.383	0.362
P-values
FR	0.380	0.127	0.181	0.001	0.003	0.034	0.023	0.266	0.102	0.128
COAWEO	0.184	0.125	0.430	0.037	0.069	0.233	0.275	0.131	0.618	0.727
Interaction	0.216	0.197	0.266	0.609	0.814	0.013	0.070	0.248	0.246	0.265

^a-b : means within the column with different superscripts are significantly different, at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil; AP: Alkaline Phosphatase; CRB: catalase in red blood cells; TAC: Total antioxidant capacity; MDA: malondialdehyde; AB: antioxidant balance; SOD: superoxide dismutase; T3: Triiodothyronine; T4: Thyroxine.

Table 9. Effects of feed restriction and coated organic acids with essential oils on selected immune indices of Japanese quails at 42 days of age.

	IgG mg/100mL	IgM mg/100mL	IgA mg/100mL	HIIBD Log^2	HIAI Log^2
Effect of feed restriction
0	997.2	254.8	80.10	3.70	3.90^a
12.5	1000.2	254.4	83.30	3.30	2.50^c
25.0	999.1	246.8	81.50	4.30	3.20^b
Effect of feed additive
0	997.6	250.4	81.93	3.80	3.27
COAWEO	1000.1	253.6	81.33	3.73	3.13
Interaction between feed restriction and feed additive
0 × 0	990.4	253.0	79.20	3.80	4.20
0 × COAWEO	1004.0	256.6	81.00	3.60	3.60
12.5 × 0	998.4	252.2	82.00	3.20	2.40
12.5× COAWEO	1002.0	256.6	84.60	3.40	2.60
25.0 × 0	1004.0	246.0	84.60	4.40	3.20
25.0× COAWEO	994.2	247.6	78.40	4.20	3.20
RMSE	11.90	12.39	4.943	1.041	0.500
P-values
FR	0.851	0.285	0.364	0.118	<0.001
COAWEO	0.576	0.486	0.743	0.862	0.472
Interaction	0.109	0.967	0.110	0.885	0.198

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil; IgG: Immunoglobulin G; IgM: Immunoglobulin M; IgA: Immunoglobulin A; HI IBD: Infectious Bursal Disease; HIAI: High-Pathogenic Avian Influenza.

Most lipid metabolites were not significantly affected by FR, COAWEO and their interaction except for serum triglycerides and vLDL (Table 7). The results showed that serum triglyceride and vLDL were decreased considerably due to COAWEO supplementation in the control group but increased when COAWEO was added to the 12.5% FR group. There were insignificant effects of FR, COAWEO and their interaction on the liver leakage enzymes, showing no harmful effects on liver function.

The current results showed that the FR, COAWEO and their interaction had no adverse effects on protein and lipid metabolites and liver function. Table 8 shows that serum MDA was considerably increased, antioxidant balance decreased by FR, and MDA increased due to COAWEO supplementations, while the difference in antioxidant balance was not significant (p = 0.069) due to COAWEO. Serum SOD concentration was significantly decreased due to 25.0% FR regimen compared to 12.5, but serum T3 concentration showed the opposite trend. However, the contents of T3 of 12.5 and 25.0% FR groups were not different from that of the control.

Table 9 showed that different types of immunoglobulins and HIIBD were not significantly affected by the FR regimen and/or COAWEO. However, HIAI titre was significantly decreased by FR treatment compared to the control group.

Caecal microbiota

Table 10 shows the effects of feed restriction and COAWEO supplementation on microbial counts in JQ caeca. Feed restriction and COAWEO significantly reduced the total bacterial count while increasing Lactobacillus. In addition, FR at 25.0% significantly decreased E coli, Clostridia and Salmonella compared to the control group.

Table 10. Effects of feed restriction and coated organic acids with essential oils on the microbial count of Japanese quails at 42 days of age.

	TBC, CFU/mL	E. coli, CFU/mL	Clostridia, CFU/mL	Salmonella, CFU/mL	Lactobacilli, CFU/mL
Effect of feed restriction
0	6.82^a	4.87^a	3.21^a	2.66^a	4.23^b
12.5	6.46^b	4.62^ab	3.19^a	2.44^b	4.98^a
25.0	6.33^b	4.39^b	2.82^b	2.51^b	4.87^a
Effect of feed additive
0	7.06^a	4.89^a	3.47^a	3.03^a	3.98^b
COAWEO	6.01^b	4.36^b	2.67^b	2.04^b	5.41^a
Interactions between feed restriction and feed additive
0 × 0	7.15^a	5.11	3.62	3.21	3.97^b
0 × COAWEO	6.48^ab	4.63	2.79	2.11	4.50^ab
12.5 × 0	7.06^a	4.80	3.68	2.88	4.19^ab
12.5 × COAWEO	5.85^b	4.44	2.70	2.00	5.77^a
25.0 × 0	6.96^a	4.77	3.12	3.01	3.78^b
25.0 × COAWEO	5.70^b	4.01	2.53	2.00	5.96^a
RMSE	0.243	0.310	0.329	0.138	0.313
P-values
FR	0.0005	0.0084	0.0244	0.0041	0.0001
COAWEO	0.0001	0.0001	0.0001	0.0001	0.0001
Interaction	0.0223	0.3393	0.4174	0.2184	0.0001

^a-b : means within the column with different superscripts are significantly different at p < 0.05. RMSE: Root Mean Square Error. FR: feed restriction; COAWEO: coated organic acid with essential oil.

The COAWEO significantly reduced different pathogenic bacteria but increased beneficial bacteria i.e. lactobacillus. There was a significant interaction between feed restriction and COAWEO on total bacterial count and Lactobacillus. The current results show that COAWEO significantly decreased the total bacterial count of 12.5 and 25.0% FR groups and increased Lactobacillus of 25.0% group.

Discussion

Effect of feed restriction

Even if poultry meat production is, in general, characterised by a fast growth rate, feed restriction has been applied in broilers also the last few years (Ebeid et al. 2022; Ogbuagu et al. 2023). The effect of FR on feed intake depended on the severity and duration of feed restriction (Tůmová et al. 2022; Ghanima et al. 2023). Ocak and Erener (2005) and Boostani et al. (2010) reported that broilers exposed to FR significantly decreased feed intake during the restricted period only. On the same line, Ye et al. (2022) observed that the feed intake of broilers progressively decreased when the FR changed from 90 to 70% of the feed intake observed in the control group. However, Abbas et al. (2015) showed that quails on FR regimen of 3 h per day had a higher feed intake compared to the control group. On the same line, Abd et al. (2015) found that quails restricted at 10, 20, and 30% than the control group increased FI at 6 weeks of age. In our trial, the feed intake of restricted groups was higher than the control only during the first two weeks post-restriction, then in the period 28–42 days the feed intake was again lower in the restricted groups, even if the feed restriction regimen was not active. The effect during the first two weeks post-restriction can be ascribed to the compensatory growth of quail. Some researchers (Zubair and Leeson 1994) observed that after feed restriction, broilers achieve a higher feed consumption compared to the birds fed ad libitum, consuming a larger amount of energy, mandatory for achieving compensatory growth. Increased feed intake after free restriction period can be explained by the adaptation of the birds’ digestive tract to restrictive dietary conditions, as the increase in the weight of the digestive organs, especially the stomach, glandular and muscular stomach, pancreas, and liver were observed during and after the restriction period (Radulovic et al. 2021). In addition, feed restricted birds exhibit a certain degree of metabolic adaptation, which is reflected in lower metabolic heat production, because of slowing metabolism. Lower metabolic heat production has a positive effect on the feed conversion ratio. The metabolic adaptation continues even after the period of restriction, but it does not last long. The decreased metabolic heat production during the restriction period is associated with a decrease in serum triiodothyronine (T3) concentration (Spaulding et al. 1976) and lower sympathetic nervous system activity in birds (Jung et al. 1980). It should be noted, however, that low circulating T3 during energy restriction could be the consequence rather than the cause of low basal metabolism (Hornick et al. 2000). However, our results are only partially line with the described mechanisms because T3 was significantly reduced only at 12.5% of FR and only in comparison to the 25.0 restricted groups, but both are no different from the control. Plasma T3, T4, and T3/T4 were not affected by FR for the broiler chickens (Attia et al. 2017). Furthermore, quail submitted to FR had lower plasma T3, and this may be due to quiet metabolism (Kobayashi and Ishii 2002). Similarly, Rønning et al. (2009) found that quails on 2 days FR markedly decreased plasma T3 and T4 while the quails on 5 days FR increased T3. However, Decuypere et al. (2005) reported that quails on FR regimen reduced plasma T3, and T4. Recently, Ghanima et al. (2023) found no differences in T3 levels between feed-restricted broilers and the broilers fed ad libitum; however, feed-restricted birds showed a significant reduction in T4. It is clear, according to Radulovic et al. (2021), that the mechanisms responsible for these processes require further studies to be better elucidated. Surely, our records are measured at the end of the experimental period and when 28 days are passed from the feed restriction: this can explain why the changes in the digestive system above described may no longer be evident. The decrease in HIAI titre indicates that moderate FR induces a stress effect on antibody titre to AI compared to the serve FR, suggesting the need for further research.

In general, FR regimen did not affect the blood profiles of broilers but significantly increased plasma corticosterone according to other authors (Sherif and Mansour 2019). However, Abd et al. (2015) found that quails exposed to FR treatments (10, 20, and 30%) significantly increased cholesterol while reducing triglycerides. On the other hand, plasma protein metabolites, cholesterol, and liver index enzyme AST were significantly reduced due to the FR regimen for JQ (Mahrose et al. 2020).

The results indicate FR regimen during the 2nd week of age had no harmful effect on Japanese quails’ survival rate during the restriction period and the whole experimental period, too. These results agree with those of Saleh et al. (1996), Attia et al. (1995) and Soomro et al. (2019).

The current results are partially like those by Lunedo et al. (2019), who found that Enterococcus and Enterobacteriaceae were reduced while Lactobacillus count was increased during FR of broiler chicks. However, Seifi et al. (2015) found that E. coli count was not influenced by feed withdrawal time for 8, 10 h/day; however, Lactobacilli count was enhanced due to exposing broiler chicks to 12 h feed withdrawal. Also, Siegerstetter et al. (2018) reported that lactobacillus improved and Escherichia/Shigella decreased by broilers on serve FR regimen. In addition, increased beneficial bacteria in broiler chickens indicate improved digestion in the digestive tract due to the FR regimen (Yan et al. 2021).

Effect of COAWEO

The addition of feed additives to the diets decreased the feed intake without negative effects on birds' weight gain: consequently, the FCR was improved when COAWEO was added to the diets. Ahmad et al. (2018) and Matty and Hassan (2020) reported that a quail diet supplemented with 300 g/ton of COAWEO reduced FI. Feed intake was similar for the whole experiment period among different experimental groups. Our results agree with those of Sacakli et al. (2005), Ozdogan and Ustundag (2015), and Ustundag and Ozdogan (2019) found that a quails diet supplemented with organic acids did not significantly affect FI.

The effect of COAWEO on blood metabolites was addressed by Matty and Hassan (2020), who reported that quails supplemented with Gallant+® (organic acids and essential oils) level at 600–900 g/ton and reported a significant reduction in serum triglyceride and an increase in serum total protein, however, decreased triglycerides and total cholesterol and increased serum globulin (El-Shenway and Ali 2016). Al-Harthi and Attia (2016) found that total protein was reduced by the addition 1 and 2 g citric acid in broiamadler diets without any effects on other plasma blood parameters. Ahmad et al. (2018) found that quails supplemented with organic acids decreased cholesterol, HDL, LDL, and vLDL, and cholesterol, LDL, total lipid, T3, T3:T4, and AST of broilers (Naveenkumar et al. 2018).

The effect of organic acids and/or essential oils on immunity and animal health has been addressing in literature for example Ahmad et al. (2018) found that organic acids supplementation for Japanese quails increased IgG, IgA, and IgM. In addition, Naveenkumar et al. (2018) observed that broilers supplemented with organic acids increased globulin and immune response. The positive effect of COAWEO could be attributed to organic acids and essential oils contents and their synergistic effects due to different modes of action (Al-Harthi and Attia 2016; Maty and Hassan 2020).

This result can be ascribed to the improved intestinal environment as elucidated by the reduction of E. coli, Clostridia and Salmonella and the increase of Lactobacilli population in the caeca. The present results demonstrated the benefits of COAWEO on beneficial gut microbiota (Botsoglou et al. 2005; Zaman, 2018; Irawan et al. 2021; Puvača et al. 2022). Our results partially agree with those of Thanh et al. (2009) who showed that adding bacteria and organic acids in broiler diets reduced the colonisation of pathogenic bacteria. However, Ghazalah et al. (2011) observed that organic acid used in quail diets did not affect Lactobacillus count, but decreased Coliforms count in broiler chickens. Along the same line, Král et al. (2014) reported that Lactobacillus was enhanced, and coliform bacteria were reduced due to probiotics addition to broiler diets, but acetic acid had no effect. In addition, Seifi et al. (2015) found that Lactobacillus and Coliforms were not affected by organic acid supplementation to broiler diets. Ustundag and Ozdogan (2019) found that bacteriocin and organic acid used in quail diets increased Coliform in male and decreased total bacteria count in females, suggesting a different gender response. The impact of organic acids and essential oils on the gut bacterial population needs further clarification.

The effect of diet supplement on gizzard may be due to organic acid effects on gizzard erosion (Attia et al. 2013; Al-Harthi and Attia 2016). In general agreement with the current results, Ahmad et al. (2018) found that supplementation of the quail diet with organic acids did not affect carcase weight and giblets percentage. However, organic acids significantly enhanced broiler carcase yield and carcase percentages (Garcia et al. 2007; Mohamed and Bahnas 2009). This effect may be due to the positive impact of organic acids and essential oils on energy and protein utilisation and the antimicrobial effects (Dibner and Buttin 2002).

Effect of the interaction feed restriction x COAWEO

COAWEO had no effect on the growth rate during the FR period, the following periods, and the whole period. In agreement with our results, Ozdogan and Ustundag (2015), Fazilat et al. (2014) and Ustundag and Ozdogan (2019) concluded that quail supplemented with organic acids 3 g/kg diet did not affect BWG compared with the other groups. On the other hand, research with Japanese quail and broilers by Garcia et al. (2007), Mohamed and Bahnas (2009), Maty and Hassan (2020), and Uddin et al. (2020) showed a positive response to organic acids and essential oil (Botsoglou et al. 2005; Zaman, 2018; Irawan et al. 2021; Puvača et al. 2022) and their combination (Maty and Hassan 2020).

The improvements in FCR ranged from 4.9 to 0.89%, for the 12.5 and 25.0% FR groups and intermediate 2.5% for the control group. These demonstrate a beneficial effect of COAWEO on feed utilisation, which relates to the increase in growth and the decreased feed intake in COAWEO-supplemented groups. Similarly, Maty and Hassan (2020) reported that the quails’ diet supplemented by 300 g/ton of COAWEO had improved FCR. Also, Christian et al. (2004) and Uddin et al. (2020) mentioned that a diet supplemented with acetic acid improved the FCR of broilers and Japanese quail due to the antimicrobial function of acetic acid. In this regard, recently Pham et al. (2023) and Hu et al. (2023) stated that the essential oils and organic acids mixture can be used as an effective strategy to ameliorate and alleviate Salmonella enteritidis infection in broilers. In addition, Soomro et al. (2019) found that FR with probiotic supplementation yielded better FCR of quails on FR regimen. Essential oils alone (Botsoglou et al. 2005; Zaman, 2018; Irawan et al. 2021; Puvača et al. 2022) or combined with organic acids (Maty and Hassan 2020) showed a positive effect on feed utilisation due to improve gut health and thus nutrient absorption. Nonetheless, Ozdogan and Ustundag (2015), and Ustundag and Ozdogan (2019) showed that organic acids 3 g/kg diet did not affect quails’ FCR. The current results agree with those reported in broilers’ and quails’ carcase traits and internal organ percentages (Soomro et al. 2019; Gobane et al. 2021).

Conclusions

In conclusion, Japanese quail can tolerate 12.5% FR without adverse effects on growth performance, carcase traits, blood biochemistry, and caeca microbiota. A 25.0% feed re-striction regimen supplemented with COAWEO improved the growth performance of Japanese quails without any adverse effect on carcase traits, blood biochemistry, and gut microbiota. Still, FR negatively affected the antioxidants biomarker and antibody titre for HIAI, which requires further investigations about the safety of feed restriction on immunity on animal health.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data are available upon official request to the corresponding author and after approval of the funding agent.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This research was funded by the Researchers Supporting Project, number (RSPD2023R581), King Saud University, Riyadh, Saudi Arabia.