Cannabidiol as a Water Additive: Impact on Performance, Egg Quality, Stress Reduction, and Gut Health in Laying Hens*

Groups	Description
C (Control)	Water without additive
B (Blank)	Water + emulsifier
I	Water + CBD 20 mg/kg BW¹ + emulsifier
II	Water + CBD 40 mg/kg BW + emulsifier
III	Water + CBD 80 mg/kg BW + emulsifier

1 Milligram per kilogram of body weight.

Table 2.

CBD solution sampling time and analytical method (HPLC method)

Time point	Notes¹	Calculated concentration³ (mg/mL)			Analyzed concentration³ (mg/mL)
Time point	Notes¹	group I	group II	group III	group I	group II	group III
1st week	Freshly prepared	0.144	0.288	0.576	0.169	0.318	0.581
2nd week	Stored sample²	0.136	0.272	0.544	0.168	0.319	0.581
13th week	Freshly prepared	0.144	0.272	0.544	0.171	0.321	0.583
14th week	Stored sample²	0.144	0.272	0.544	0.170	0.320	0.582

1 50 mL of solution collected in sterile falcon tubes.

2 Stability check after 7 days.

3 Group I: CBD 20 mg/kg BW; group II: CBD 40 mg/kg BW; group III: CBD 80 mg/kg BW.

Table 3.

Actual CBD intake per kilogram of body weight at the 1st and 13th week

Groups	Description	1st week	13th week
I	CBD 20 mg/kg BW	25 mg/kg¹	20 mg/kg
II	CBD 40 mg/kg BW	48 mg/kg	39 mg/kg
III	CBD 80 mg/kg BW	82 mg/kg	73 mg/kg

1 Milligram per kilogram of body weight.

Table 4.

Feed composition and nutritional value of the diet during the first laying period

Item	Amount (%)
Corn	29.91
Wheat	15.8
Triticale	13
Post-extraction rapeseed meal	12
Post-extraction soybean meal	9.8
Calcium carbonate (limestone)	8.89
Corn distillers dried grain	5
Wheat bran	2.2
Soybean oil	1.9
Blood products – dried poultry hemoglobin	0.5
Sodium chloride	0.24
Sodium bicarbonate	0.13
Monocalcium phosphate	0.12
Premix¹	0.51
Calculated nutritional value
Item²	Value
ME³ for layers (kcal/kg) – calculated	2748
Crude protein (g/kg) – calculated	167.79
Crude fiber (g/kg) – calculated	42.91
Crude ash (g/kg) – calculated	117.52
Crude fat (g/kg) – calculated	45.33
Lysine (g/kg) – calculated	8.11
Methionine (g/kg) – calculated	3.74
Total Ca (g/kg) – calculated	35.98
Total P (g/kg) – calculated	4.79

1 Composition of the 1 kg of premix: vitamin A, 10000 IU; vitamin D₃, 3000 IU; vitamin E, 14 IU; copper (copper sulfate 5-hydrate), 10 mg; iron (ferric sulfate monohydrate), 30 mg; manganese (manganese oxide), 80 mg; zinc (zinc sulfate monohydrate), 80 mg; iodine (calcium iodine anhydrous), 1 mg; selenium (sodium selenite), 0.25 mg; BHT (E321) 1b, 1.04 mg; BHA (E320) 1b, 0.57 mg; phytase, 900 OUT, endo 1,4 beta-xylanase, 1500 VU.

2 The nutrient composition was calculated based on CVB norms + enzyme (xylanase).

3 Metabolizable energy.

Table 5.

Performance of laying hens over the 15-week experimental period

group	mean	SD	median	P-value
C²	3.34	0.38	3.51	0.994
B	3.33	0.40	3.51
I	3.35	0.45	3.51
II	3.33	0.42	3.54
III	3.32	0.44	3.55
Water intake¹ (L/bird)
group	mean	SD	median	P-value
C	6.04	1.07	6.35	0.442
B	5.89	0.98	6.25
I	6.26	1.16	6.60
II	6.26	1.22	6.80
III	6.16	1.21	6.75
FCR³
group	mean	SD	median	P-value
C	1.65	0.16	1.6	0.083
B	1.63	0.16	1.6
I	1.68	0.21	1.6
II	1.64	0.14	1.6
III	1.59	0.08	1.6
Egg production (%)
group	mean	SD	median	P-value
C	93.08	6.73	95.2	0.260
B	93.97	7.23	96.4
I	92.19	9.22	95.2
II	93.21	7.00	95.2
III	95.37	4.98	96.4

1 Performance data represents monthly averages based on cumulative measurements provided for each group.

2 C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier

3 FCR was calculated based on kg feed per dozen eggs.

Feed intake changes from 1st to 15th week
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier. a, b – values with different letters differ significantly (P≤0.05).

Water intake changes from 1st to 15th week
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

FCR changes from 1st to 15th week
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

Egg production changes from 1st to 15th week
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

Laying hens' body weight
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier. a, b, c – values with different letters differ significantly (P≤0.01).
CI: confidence interval.

Table 6.

Multiple linear regression of the body weight and performance traits

trait	parameter	95% CI¹		P-value
Feed intake (kg/bird)	–0.34	–1.302	0.623	0.49
Water intake (L/bird)	0.097	–0.033	0.228	0.15
Egg production %	–0.002	–0.004	0	0.09
R² (%)	7.1
Middle body weight (1st–7th week)
trait	parameter	95% CI		P-value
Feed intake (kg/bird)	0.158	–0.063	0.379	0.16
Water intake (L/bird)	–0.026	–0.059	0.006	0.12
Egg production %	–0.003	–0.007	0.001	0.22
R² (%)	9.3
Final body weight (1st–15th week)
trait	parameter	95% CI		P-value
Feed intake (kg/bird)	0.293	0.141	0.446	<0.001
Water intake (L/bird)	–0.016	–0.034	0.003	0.09
Egg production %	–0.002	–0.007	0.002	0.33
R² (%)	10.2

1 Confidence interval.

Table 7.

Egg weight at 0 and 90 days of the experiment

Egg weight (g)	Group	Mean	SD	P-value
	C¹	56.23 a	1.63	0.042
	B	54.89 ab	3.11
Day 0	I	54.17 ab	2.30
	II	53.34 b	2.86
	III	55.89 a	2.52
	C	59.96	4.56	0.86
	B	60.01	3.01
Day 90	I	60.25	2.65
	II	59.88	2.87
	III	58.83	3.09

1 C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier

a, b – values in columns with different letters differ significantly (P≤0.05).

Table 8.

Egg shell strength at 0 and 90 days of the experiment

Shell strength (kgf)¹	Group	Mean	SD	P-value
Day 0	C²	4.49	0.79	0.527
	B	4.42	0.79
	I	4.43	0.68
	II	4.08	0.55
	III	4.58	0.85
Day 90	C	4.80	0.93	0.961
	B	4.95	0.55
	I	4.79	0.50
	II	4.83	0.75
	III	4.94	0.66

1 Kilogram force.

2 C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

Table 9.

Egg shell thickness of the three parts of the eggs

Shell thickness Sharp end (mm)	Group	Mean	SD	P-value
Day 0	C¹	0.36	0.02	0.058
	B	0.34	0.03
	I	0.35	0.02
	II	0.33	0.02
	III	0.35	0.03
Day 90	C	0.36	0.02	0.381
	B	0.35	0.02
	I	0.35	0.02
	II	0.34	0.03
	III	0.35	0.02
Shell thickness Equator (mm)	Group	Mean	SD	P-value
Day 0	C	0.36	0.02	0.079
	B	0.35	0.02
	I	0.35	0.02
	II	0.33	0.02
	III	0.35	0.03
Day 90	C	0.35	0.02	0.455
	B	0.35	0.01
	I	0.35	0.02
	II	0.34	0.03
	III	0.35	0.02
Shell thickness Blunt end (mm)	Group	Mean	SD	P-value
Day 0	C	0.35	0.02	0.056
	B	0.34	0.03
	I	0.34	0.03
	II	0.32	0.02
	III	0.35	0.03
Day 90	C	0.35	0.02	0.393
	B	0.35	0.02
	I	0.35	0.03
	II	0.33	0.02
	III	0.34	0.02

1 C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

Table 10.

Sensory evaluation of the eggs

Parameter	Group	N²	Mean	SD	P-value
1	2	3	4	5	6
Yolk texture	C¹	60	4.97	2.28	0.099
	B	60	5.37	2.22
	I	60	5.53	2.56
	II	60	6.02	2.24
	III	60	5.03	2.39
Unusual taste	C	60	2.62	2.27	0.179
	B	60	3.05	2.46
	I	60	3.13	2.45
	II	60	2.48	2.00
	III	60	2.30	1.90
Unusual smell	C	60	2.70	1.97	0.789
	B	60	2.88	2.28
	I	60	2.85	2.39
	II	60	2.47	2.00
	III	60	2.55	2.13
Yolk color	C	60	4.32 B	2.00	0.001
	B	60	5.57 A	2.29
	I	60	5.72 A	2.26
	II	60	5.80 A	2.14
	III	60	5.38 A	2.23

1 C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

2 N = number of panelists.

A, B – values in columns with different letters differ significantly (P≤0.01).

Corticosterone levels
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier. a, b, c – values with different letters differ significantly (P≤0.01).

Table 11.

Duodenum, jejunum and ileum morphology after 15 weeks

Parameter	Group	Duodenum			Jejunum			Ileum
Parameter	Group	mean	SD	P-value	mean	SD	P-value	mean	SD	P-value
Villi surface area (μm²)	C¹	596692.7 D	8597.54		230707.2 C	3378.26		737787.1 A	5880.13
	B	678855 C	8645.92		330212.6 A	7400.45		571801.9 C	2434.71
	I	892653.8 A	4731.11	<0.001	328623.4 A	3808.11	<0.001	542386.6 D	5906.26	<0.001
	II	333471.1 E	2141.69		231102.3 C	2495.33		684701 B	2394.76
	III	844503.2 B	3611.66		236472.8 B	2628.21		44728.5 E	550.72
Villi height (μm)	C	1900.2 B	34.99		1050.44 C	2.35		1277.3 C	16.85
	B	1637.2 D	9.41		1107.11 B	1.62		1339.2 B	5.37
	I	1987.3 A	7.72	<0.001	1346.3 A	4.76	<0.001	987.1 D	3	<0.001
	II	1062.1 E	7.62		783.4 E	1.51		1407 A	2.4
	III	1681.3 C	9.19		886.1 D	1.79		950 E	3.06
Crypt depth (μm)	C	123.3 C	1.49		101 A	1.56		88.1 D	0.88
	B	71.3 E	1.34		68.2 D	1.03		107.2 A	2.39
	I	131.4 B	1.9	<0.001	68 D	0.82	<0.001	64.3 E	1.89	<0.001
	II	79.2 D	2.1		95 B	0.82		102.4 B	1.26
	III	141.1 A	7.75		85 C	5.44		98.4 C	5.34
Villi height/Crypt depth	C	15.41 B	0.31		10.39 C	0.16		14.5 B	0.26
	B	22.97 A	0.51		16.23 B	0.25		12.5 D	0.3
	I	15.13 B	0.21	<0.001	19.8 A	0.26	<0.001	15.36 A	0.43	<0.001
	II	13.42 C	0.31		8.25 D	0.08		13.74 C	0.16
	III	11.95 D	0.64		10.46 C	0.65		9.68 E	0.53

1 C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

A, B, C, D, E – values in columns with different letters differ significantly (P≤0.01).

Small intestine morphology of laying hens at the end of 15 weeks.
C (control): water without additive; B (blank): water + emulsifier; I: water + CBD 20 mg/kg BW + emulsifier; II: water + CBD 40 mg/kg BW + emulsifier; III: water + CBD 80 mg/kg BW + emulsifier.

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DOI: https://doi.org/10.2478/aoas-2026-0016 | Journal eISSN: 2300-8733 (formerly 1642-3402) | Journal ISSN: 1642-3402

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Language: English

Submitted on: Jun 27, 2025

Accepted on: Feb 6, 2026

Published on: Oct 6, 2026

Published by: National Research Institute of Animal Production

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

eggs,

CBD,

performance,

corticosterone,

intestinal morphology

Related subjects:

Medicine,

Veterinary medicine

© 2026 Sepideh Fallahi, Łukasz Bobak, Anna Lipińska, Muhammad Umair Asghar, Katarzyna Olejnik, Sebastian Opaliński, published by National Research Institute of Animal Production
This work is licensed under the Creative Commons Attribution 3.0 License.

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Cannabidiol as a Water Additive: Impact on Performance, Egg Quality, Stress Reduction, and Gut Health in Laying Hens*

Figures & Tables

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Figure 5.

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Figure 6.

Table 11.

Figure 7.

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