Set-up of a multivariate approach based on serum biomarkers as an alternative strategy for the screening evaluation of the potential abuse of growth promoters in veal calves

Figures & data

Table 1. Investigated analytes in serum and urines from veal calves (n = 24) of the ‘unknown’ group.

Substance	Matrix	LOD (µg l^−1)	LOQ (µg l^−1)
Sexual steroids
17β-Oestradiol	Serum	0.03	0.03
17β-Testosterone	Serum	1	2
Progesterone	Serum	0.06	0.15
Ethinyloestradiol	Urine	0.30	1.5
Fluossimesterone	Urine	0.55	1.5
16β-Hydroxystanozolol	Urine	0.15	0.55
Methylboldenone	Urine	0.30	1.5
Methyltestosterone	Urine	0.30	1.5
17α-Nortestosterone	Urine	0.30	1
17β-Nortestosterone	Urine	0.30	1
Stanozolol	Urine	0.30	0.55
17α-Trenbolone	Urine	0.25	1.5
17β-Trenbolone	Urine	0.55	1.5
17α-Boldenone	Urine	0.30	0.55
17β-Boldenone	Urine	0.30	0.55
Corticosteroids
Beclomethasone	Urine	0.2	1.5
Betamethasone	Urine	0.2	1.5
Dexamethasone	Urine	0.2	1.5
Flumethasone	Urine	0.2	1.5
Fluorometholone	Urine	0.2	1.5
Methylprednisolone	Urine	0.2	1.5
Prednisolone	Urine	0.2	1.5
Prednisone	Urine	0.2	1.5
Triamcinolone acetonide	Urine	0.2	1.5

Table 2. Serum biomarker values from veal calves in the control group (n = 50).

Biomarker	Mean ± SD	Minimum	Median	Maximum
Osteocalcin (ng ml^−1)	13.73 ± 3.29	6.70	14.15	21.60
Cortisol (ng ml^−1)	22.82 ± 7.1	8.81	22.03	48.53
Inhibin (ng ml^−1)	3.32 ± 1.86	0.60	3.03	10.24
Urea (mg dl^−1)	10.28 ± 3.03	5.00	10.00	20.00
SAC (µmol HClO ml^−1)	352.63 ± 60.01	250.27	354.61	488.66

Note: SAC, serum antioxidant capacity.

Table 3. Serum biomarker values from veal calves in the unknown group (n = 421).

Biomarker	Mean ± SD	Minimum	Median	Maximum
Osteocalcin (ng ml^−1)	9.62 ± 2.17	0.10	9.42	9.83
Cortisol (ng ml^−1)	24.83 ± 17.01	3.13	19.45	106.41
Inhibin (ng ml^−1)	1.13 ± 0.63	0.04	1.04	6.36
Urea (mg dl^−1)	14.21 ± 4.89	0.23	13.75	14.67
SAC (µmol HClO ml^−1)	348.01 ± 72.12	3.47	342.21	661.04

Note: SAC, serum antioxidant capacity.

Figure 1. Distribution of the Mahalanobis distance (log values) for Group C and Group U animals. Blue figures = Group C; green figures = Group U, compliant animals; and red figures = Group U, non-compliant animals.

Figure 2. (colour online) Flow chart of the study.

Table 4. Results of the histological evaluation (n = 52) and chemical analysis (n = 24) of selected samples from the ‘unknown veal calves’ group classified as ‘non-compliant’ with respect to the UNEQ class model based on the levels of five serum biomarkers.

	Histological evaluation			Chemical analysis
Sample	Thymus	Prostate	Bulbourethral glands	Serum	Urine^a
V01	Suspect	Compliant	Compliant	Compliant	Compliant
V02	Compliant	Compliant	Compliant	Compliant	17β-BOLD detected < LOQ
V03	Compliant	Compliant	Compliant	Compliant	Compliant
V04	Uncertain	Compliant	Compliant	n.a.	n.a.
V05	Uncertain	Compliant	Uncertain	n.a.	n.a. n.a. Compliant
V06	Uncertain	Compliant	Compliant	n.a.
V07	Uncertain	Compliant	Compliant	Compliant
V08	Uncertain	Uncertain	Compliant	n.a.	n.a.
V09	Compliant	Compliant	Compliant	n.a.	n.a.
V10	Compliant	Compliant	Compliant	n.a.	n.a.
V11	Compliant	Compliant	Compliant	Compliant	17β-BOLD detected < LOQ
					PRED detected < LOQ
V12	Uncertain	Uncertain	Compliant	Compliant	Compliant
V13	Compliant	Compliant	Compliant	Compliant	Compliant
V14	Uncertain	Compliant	Compliant	n.a.	n.a.
V15	Suspect	Uncertain	Compliant	n.a.	n.a.
V16	Compliant	Uncertain	Compliant	Compliant	Compliant
V17	Uncertain	Compliant	Compliant	Compliant	Compliant
V18	Uncertain	Compliant	Compliant	n.a.	n.a.
V19	Compliant	Compliant	Compliant	n.a.	n.a.
V20	Compliant	Compliant	Compliant	Compliant	17β-BOLD detected < LOQ
V21	Uncertain	Compliant	Compliant	n.a.	n.a.
V22	Uncertain	Compliant	Compliant	Compliant	Compliant
V23	Uncertain	Compliant	Compliant	n.a.	n.a.
V24	Compliant	Compliant	Compliant	n.a.	n.a.
V25	Compliant	Compliant	Compliant	Compliant	Compliant
V26	Uncertain	Uncertain	Compliant	n.a.	n.a.
V27	Compliant	Uncertain	Compliant	n.a.	n.a.
V28	Uncertain	Compliant	Compliant	Compliant	Compliant
V29	Uncertain	Compliant	Compliant	Compliant	Compliant
V30	Suspect	Compliant	Compliant	n.a.	n.a.
V31	Uncertain	Compliant	Compliant	Compliant	Compliant
V32	Compliant	Compliant	Compliant	n.a.	n.a.
V33	Compliant	Compliant	Compliant	n.a.	n.a.
V34	Compliant	Uncertain	Compliant	Compliant	Compliant
V35	Compliant	Compliant	Compliant	Compliant	Compliant
V36	Compliant	Compliant	Compliant	n.a.	n.a.
V37	Compliant	Compliant	Compliant	n.a.	n.a.
V38	Compliant	Compliant	Compliant	Compliant	Compliant
V39	Uncertain	Compliant	Compliant	n.a.	n.a.
V40	Compliant	Compliant	Compliant	n.a.	n.a.
V41	Compliant	Compliant	Compliant	n.a.	n.a.
V42	Compliant	Compliant	Compliant	Compliant	17β-BOLD detected < LOQ
V43	Compliant	Compliant	Compliant	Compliant	17β-BOLD detected < LOQ
V44	Suspect	Compliant	Compliant	n.a.	n.a.
V45	Compliant	Compliant	Compliant	n.a.	n.a.
V46	Compliant	Compliant	Compliant	Compliant	Compliant
V47	Compliant	Compliant	Compliant	Compliant	Compliant
V48	Compliant	Compliant	Compliant	Compliant	PRED detected < LOQ
V49	Compliant	Compliant	Compliant	Compliant	Compliant
V50	Compliant	Compliant	Compliant	n.a.	n.a.
V51	Compliant	Compliant	Compliant	n.a.	n.a.
V52	Compliant	Compliant	Compliant	n.a.	n.a.

Note: Serum samples were assayed for the presence of natural sexual steroids; urine specimens were analysed for the presence of synthetic sexual steroids and of corticosteroids. The full list of the searched analytes and the corresponding LOD and LOQ values is depicted in Table 1.

17β-BOLD, 17β-boldenone; PRED, prednisolone; n.a., not analysed.

The concentration of the identified analytes was in all cases higher than the LOD but lower than the LOQ.