Dietary supplementation of hemp oil in teddy dogs: Effect on apparent nutrient digestibility, blood biochemistry and metabolomics

Figures & data

Table 1. Fatty acid composition of hemp seed oil

Fatty acid composition		Content %
C4:0	Butyrate	0.05
C14:0	Myristic acid	0.03
C15:0	Pentadecanoic acid	0.23
C16:0	Palmitic acid	6.33
C16:1	Palmitoleic acid	0.09
C17:0	Margaric	0.05
C17:1	Margaroleic	0.03
C18:0	Stearic	2.88
C18:1n9t	Trans-elaidic	0.00
C18:1n9c	Oleic	13.05
C18:2n6c	Linoleic	58.71
C20:0	Arachidic	0.69
C18:3n6	γ-Linolenic	0.67
C18:3n3	α-Linolenic	16.81
C20:2	Eicosadienoic	0.05
C22:0	Behenic	0.24
C24:0	Lignoceric	0.08
UFA Unsaturated fatty acid		89.42
SFA Saturated fatty acid		10.58

Table 2. Composition and nutrient level of experimental diets (Dry matter basis)

Items	Content %
	A	B	C	D	E
Ingredients
Chicken bone meal	10.20	10.21	10.24	10.27	10.29
Poultry by-product meal	15.01	14.99	14.97	14.93	14.92
Chicken liver powder	1.00	1.00	1.00	1.00	1.00
Corn flour	18.95	18.96	19.09	19.12	19.13
Wheat	20.04	20.02	19.86	19.78	19.68
Oat	22.99	23.00	23.01	23.05	23.10
Yeast extract	1.00	1.00	1.00	1.00	1.00
Chicken oil	9.81	9.32	8.83	7.85	5.88
Hemp seed oil	0.00	0.50	1.00	2.00	4.00
2% Premix 2%^1)	1.00	1.00	1.00	1.00	1.00
Total	100.00	100.00	100.00	100.00	100.00
Nutrient levels^2)
CP	27.49	27.49	27.49	27.49	27.49
EE	14.36	14.36	14.35	14.35	14.35
CF	2.17	2.17	2.17	2.16	2.16
Ash	3.43	3.43	3.43	3.43	3.43
Ca	0.93	0.93	0.93	0.93	0.93
P	0.76	0.76	0.76	0.76	0.76
ME (Kcal/Kg)	4023.38	4023.37	4023.43	4023.45	4023.32

CP: crude protein, EE: crude fat, CF: crude fiber, Ca: Calcium, P: Phosphorus, ME: Metabolizable energy. A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.

Table 3. The effect of hemp oil on the apparent nutrient digestibility of teddy dogs

Index	Treatments
	A	B	C	D	E
Apparent Digestibility of Dry Matter (DMD %)	86.32 ± 1.37	87.02 ± 0.83	87.43 ± 1.36	88.08 ± 1.71	87.03 ± 2.99
Apparent digestibility of crude protein (CPD %)	86.87 ± 2.04	88.27 ± 0.56	88.77 ± 1.15	88.87 ± 2.01	88.71 ± 1.82
Apparent digestibility of crude fat (EED %)	96.76 ± 1.11	97.50 ± 0.37	96.35 ± 1.02	97.43 ± 0.86	96.92 ± 0.69

A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.

Table 4. The effect of hemp oil on dog blood biochemical indexes

Parameters	Treatments
	A	B	C	D	E
IgM (g/L)	0.88 ± 0.45^b	1.07 ± 0.32^ab	1.39 ± 0.12^a	1.52 ± 0.12^a	1.49 ± 0.12^a
IgG (g/L)	4.00 ± 0.83	4.00 ± 0.65	4.38 ± 0.44	4.46 ± 0.40	4.49 ± 0.40
IgE (kU/L)	203.60 ± 83.42	321.63 ± 89.62	321.67 ± 58.10	347.88 ± 78.97	246.08 ± 33.74
C4 (g/L)	0.06 ± 0.01	0.08 ± 0.02	0.08 ± 0.01	0.08 ± 0.01	0.07 ± 0.01
γ-IFN (ng/L)	23.04 ± 5.14^b	24.63 ± 6.78^b	31.32 ± 5.73^a	31.78 ± 9.30^a	25.78 ± 6.64^b
TP (g/L)	61.33 ± 2.97^b	68.10 ± 3.04^a	69.54 ± 4.13^a	69.30 ± 2.73^a	65.60 ± 4.25^ab
ALB (g/L)	35.08 ± 3.90^b	38.76 ± 2.58^a	39.20 ± 2.39^a	38.66 ± 0.43^a	40.38 ± 2.06^a
GLB (g/L)	26.53 ± 3.18	27.93 ± 1.42	31.63 ± 2.50	29.78 ± 3.00	27.48 ± 2.93
T4 (nmol/L)	27.11 ± 3.45^b	31.30 ± 3.93^ab	32.23 ± 4.60^ab	36.75 ± 2.91^a	33.98 ± 4.11^a
T3 (nmol/L)	0.94 ± 0.07^b	1.24 ± 0.15^ab	1.67 ± 0.51^a	1.50 ± 0.17^a	1.49 ± 0.16^a
ACTH (pg/ml)	7.80 ± 0.45^b	8.24 ± 1.49^b	8.41 ± 1.80^b	9.20 ± 1.89^ab	11.41 ± 0.78^a
TG (mmol/L)	0.76 ± 0.06^a	0.75 ± 0.17^a	0.47 ± 0.08^b	0.53 ± 0.06^b	0.55 ± 0.16^b
TC (mmol/L)	5.55 ± 1.46	4.83 ± 0.31	4.53 ± 1.25	4.58 ± 1.35	4.55 ± 1.35
LDL-C (mmol/L)	1.49 ± 1.24^a	1.23 ± 0.55^a	0.34 ± 0.16^b	0.26 ± 0.08^b	0.31 ± 0.06^b
GLU (mmol/L)	5.03 ± 0.58	4.76 ± 0.65	4.65 ± 0.15	4.57 ± 0.24	4.62 ± 0.24

IgM: immunoglobulin M, IgG: immunoglobulin G, IgE: immunoglobulin E, C4: complement 4, γ-IFN: γ-interferon, TP: total protein, ALB: albumin, GLB: globulin, T4: thyroxine, T3: triiodothyronine, ACTH: adrenocorticotropic hormone, TG: triglycerides, TC: total cholesterol, LDL-C: low-density lipoprotein cholesterol, GLU: blood sugar. A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.

Table 5. The effect of hemp oil on the anti-oxidation indexes of dog serum

Parameters	Treatments
	A	B	C	D	E
SOD (U/mL)	26.47 ± 2.65^c	27.83 ± 2.94^bc	28.99 ± 1.69^bc	30.63 ± 1.29 ^ba	33.02 ± 3.25^a
T-AO (mM)	0.38 ± 0.00	0.39 ± 0.02	0.40 ± 0.02	0.38 ± 0.05	0.39 ± 0.04
CAT (U/ml)	3.72 ± 0.43	4.08 ± 0.62	4.23 ± 0.54	4.20 ± 1.10	3.84 ± 0.38
MDA (nmol/ml)	5.30 ± 1.04^a	4.85 ± 1.02^a	3.83 ± 0.39^ab	3.28 ± 0.71^b	4.95 ± 1.13^a

SOD: superoxide dismutase, T-AO: total antioxidant capacity, CAT: catalase, MDA: malondialdehyde. A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.

Figure 1. The principal component analysis (PCA) between control A and treatment B (Figure 1a), C (Figure 1b), D (Figure 1c), E (Figure 1d), and the orthogonal partial least square discriminant analysis (OPLS-DA) score (Figure 1e) and model verification (Figure f). The abscissa represents the model accuracy and the ordinate represents the frequency of model classification effect. The prediction parameters R²X and R²Y represent the interpretation rate of X and Y matrix, and Q² represents the prediction ability of the model. When Q² > 0.5 means effective model, and when Q² > 0.9 represents excellent model.

Figure 2. The statistical of differential metabolites-based VIP value between control A and treatment B (Figure 2a), C (Figure 2b), D (Figure 2c), and E (Figure 2d).

Figure 3. The pathway analysis of the detected differential metabolites between control A and treatment B (Figure 3a), C (Figure 3b), D (Figure 3c), and E (Figure 3d).

Table 6. Statistics of the different metabolites number

Treatments	Total Sig Metabolites	Down regulated	Up regulated
A_VS_B	20	9	11
A_VS_C	18	7	11
A_VS_D	27	12	15
A_VS_E	43	9	34

A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.

Table 7. Differential metabolite Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis

Treatments	Compounds	VIP	FC	Type	Pathway ID
A VS B	^9α,11,15S-^trihydroxythromba−5Z,^13E-^dien−1^−^oic acid	1.29	0.37	down	ko00590, ko04726
	^9α,15S-^dihydroxy−11^−^oxo^−^prosta−5Z,^13E-^dien−1^−^oic acid	1.03	0.31	down	ko00590, ko04080, ko04726, ko05143
	Deoxyguanosine5’-monophosphate (d GMP)	1.11	0.11	down	ko00230
	^5S,12R-^dihydroxy−6Z,^8E,10E,14Z-^eicosatetraenoic ^acid	1.48	4.79	up	ko00590, ko04080, ko04726, ko04750
	Adenosine	1.58	0.03	down	ko00230, ko04080, ko04923, ko04924
A VS C	Taurocholic Acid	1.21	0.14	down	ko00120, ko04976
	Chenodeoxycholic Acid	1.33	2.6	up	ko00120, ko04976
	^9α,15S-^dihydroxy−11^−^oxo^−^prosta−5Z,^13E-^dien−1^−^oic acid	1.58	0.44	down	ko00590, ko04080, ko04726
	Guanine	1.72	0.21	down	ko00230
	Deoxyguanosine5’-monophosphate (d GMP)	1.43	0.08	down	ko00230
	3’-Aenylic Acid	1.42	0.06	down	ko00230
	Ubiquinone	2.07	2.44	up	ko00130, ko00190
	Adenosine	2.08	0	down	ko00230
A VS D	Taurocholic Acid	1.1	0.2	down	ko04976
	Vitamin D3	1.31	2049.29	up	ko05323
	^9α,11,15S-^trihydroxythromba−5Z,^13E-^dien−1^−^oic acid	1.82	0.28	down	ko00590, ko04726, ko04976
	^9α,15S-^dihydroxy−11^−^oxo^−^prosta−5Z,^13E-^dien−1^−^oic acid	1.33	0.25	down	ko00590, ko04080, ko04726
	Deoxyguanosine5’-monophosphate (d GMP)	1.39	0.24	down	ko00230
	γ-Linolenic Acid (C18:3N6)	1.89	2.78	up	ko00591, ko01040
	A-Linolenic Acid (C18:3N3)	1.95	3.04	up	ko00592, ko01040,
	Ubiquinone	1.03	1733.93	up	ko00130, ko00190
	^9-^oxo−11α,15S-^dihydroxy^−^prosta−5Z,^13E-^dien−1^−^oic acid	1.55	0.38	down	ko00590, ko04080, ko04726, ko04750, ko04923, ko04924, ko05323
	Adenosine	1.44	0.10	down	ko00230, ko04080, ko04270, ko04923, ko04924
A VS E	Guanosine	1.59	0.4	down	ko00230
	Ubiquinone	1.03	1733.93	up	ko00130, ko00190
	Deoxyguanosine 5’-monophosphate (d GMP)	1.34	0.07	down	ko00230
	^20-^hydroxy−5Z,^8Z,11Z,14Z-^eicosatetraenoic ^acid	1.38	2.58	up	ko04270
	3’-Aenylic Acid	1.53	0.1	down	ko00230
	Adenosine	1.88	0	down	ko00230, ko0407, ko04270

VIP: Variable importance projection, FC: Multiple of difference. A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.

Figure 4. The correlation between metabolomics and nutrient digestibility during control A and treatment B (Figure 4a), C (Figure 4b), D (Figure 4c), and E (Figure 4d).

Figure 5. The correlation between metabolomics and blood biochemical index during control A and treatment B (Figure 5a), C (Figure 5b), D (Figure 5c), and E (Figure 5d).

Figure 6. The correlation between metabolomics and blood antioxidant indicators during control A and treatment B (Figure 6a), C (Figure 6b), D (Figure 6c), and E (Figure 6d). A: dietary supplements 0% hemp oil in teddy dog (as control), B: dietary supplements 0.5% hemp oil in teddy dog, C: dietary supplements 1% hemp oil in teddy dog, D: dietary supplements 2% hemp oil in teddy dog and E: dietary supplements 4% hemp oil in teddy dog.