Influence of molybdenum and organic sources of copper and sulfur on performance and blood mineral concentration in lambs

doi:10.21203/rs.3.rs-2460108/v1

This study aimed to evaluate the effects of molybdenum (Mo) and organic and inorganic sources of copper (Cu) and sulfur (S) on performance and blood concentration of these minerals in lambs. Forty male non-castrated crossbred Dorper x Santa Inês lambs (20 ± 1.2 kg of body weight and 90 ± 2 d of age) were randomly allocated into one of the 10 following treatments: T0) control, basal diet; T1) Mo; T2) inorganic Cu and inorganic S; T3) inorganic Cu and organic S; T4) organic Cu and inorganic S; T5) organic Cu and organic S; T6) Mo plus inorganic Cu and inorganic S; T7) Mo plus inorganic Cu and organic S; T8) Mo plus organic Cu and inorganic S; T9) Mo plus organic Cu and organic S. The animals were kept in individual pens and received a total mixed ration during 84 days. Body weight and blood sampling was performed every 28 days. All animals were slaughtered after 84 days and carcass traits were evaluated. No effects of treatments were observed on body weight and carcass traits. Dietary molybdenum and sulfur-supplemented animals had reduced serum concentrations of copper and ceruloplasmin. Also, an interaction among Mo x Cu sources x S sources was detected for both copper and ceruloplasmin serum concentrations. Organic sources of Cu and S led to increased serum concentration of S. Further, Mo supplementation influenced positively its blood concentration.

ceruloplasmin activity

mineral antagonism

mineral supplementation

ruminants

Several negative mineral interactions have been reported in ruminants, such as zinc-iron, copper-sulfur, copper-molybdenum, copper-iron, copper-molybdenum-sulfur [1], and copper-sulfur-selenium [2]. Excessive intake of one mineral can modify the requirement of others [3]. For example, Molybdenum (Mo) and sulfur (S) are the main antagonists of copper (Cu), in which high levels of dietary of these minerals increase Cu requirements in ruminant farm animals [4]. Thus, if Cu levels in the body are low, higher amounts of Mo may become a toxic element to the animals [5].

Excessive dietary Mo and S impair Cu absorption through insoluble thiomolybdates and sulfides, which are formed by ruminal microbes. This can lead to reduced liver Cu concentration and eventually reduced blood Cu concentration [6]. Also, thiomolybdate absorption is most likely when its production exceeds the amount that can react with Cu to form insoluble complexes in the rumen. This increases thiomolybdate binding to plasma Cu, reducing the transport of available Cu to the tissues [7]. All these mechanisms, resulting in high plasma Cu concentrations even in ruminants deficient in Cu [4]. Still, this may lead animals to intoxication, promoting red blood cell lysis and, hence, a hemoglobinuric nephrosis state [8].

Besides the negative impact of Mo and S on tissue uptake of Cu, it has also been reported in sheep that dietary Mo plus S affect another critical step of Cu metabolism, its utilization for ceruloplasmin synthesis in the liver [9]. Ceruloplasmin is a copper-protein complex found in blood that is responsible for Cu transport, binding up to 95% of this circulating mineral [10]. As a consequence, this complex may be an indicator of serum Cu concentration in ruminants [11]. Furthermore, ceruloplasmin is an acute phase protein that increases during infection or acute stress, being an important substance for animal survivability [10].

Therefore, this study aimed to evaluate the effects of organic and inorganic sources of Cu and S and their interaction with Mo on performance and blood concentration of these minerals, as well as investigate the serum ceruloplasmin activity in lambs.

Animals, facilities, and treatments

This study was conducted at the experimental facility of the Department of Animal Science of the College of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, SP, Brazil.

Forty male non-castrated crossbred Dorper x Santa Inês lambs (20 ± 1.2 kg of body weight and 90 ± 2 d of age, at the start of the experiment) were used in a completely randomized design to evaluate the effects of Mo and organic and inorganic sources of Cu and S on performance, blood concentration of these minerals, and serum ceruloplasmin activity. Throughout the experiment, animals were housed in individual metabolic cages with 1.0 m² of area/animal, under a sheltered barn containing feed bunks and free access to water. The study lasted 84 days, in which 14 days were allowed for facilities and feeding adaptation.

After the adaptation period, the lambs were allocated in a completely randomized design study consisting of ten treatments (n = 4 per treatment): T0) Control, basal diet without any additional mineral supplementation; T1) Basal diet added 10 mg of Mo; T2) Basal diet added 10 mg of inorganic Cu per kg DM and 0.2% of inorganic S; T3) Basal diet added 10 mg of inorganic Cu and 0.2% organic S; T4) Basal diet added 10 mg of organic Cu and 0.2% of inorganic S; T5) Basal diet added 10 mg of organic Cu and 0.2% of organic S; T6) Basal diet added 10 mg of Mo plus 10 mg of inorganic Cu and 0.2% of inorganic S; T7) Basal diet added 10 mg of Mo plus 10 mg of inorganic Cu and 0.2% of organic S; T8) Basal diet added 10 mg of Mo plus 10 mg of organic Cu and 0.2% of inorganic S; T9) Basal diet added 10 mg of Mo plus 10 mg of organic Cu and 0.2% of organic S. Treatments comprised the same basal diet, which contained 9.96 mg/kg Cu, 1.02 mg/kg Mo, and 2.56 g/kg S on DM basis (Table 1).

Table 1

Ingredients and chemical composition of diets (g/kg DM, otherwise stated).
Item		Basal diet
Ingredients
	Corn meal	553.0
	Wheat middlings	10.0
	Cottonseed hulls	250.0
	Soybean meal	125.0
	Soybean oil	10.0
	Limestone	12.0
	Dicalcium phosphate	20.0
	Urea	10.0
	Vitamin premix^a	05.0
	Mineral premix^b	05.0
Chemical composition
	Dry matter (g/kg OM^c)	906.5
	aNDF	282.0
	aADF	166.4
	Crude protein	157.3
	Ether extract	42.8
	Ash	57.0
	Calcium	11.8
	Phosphorus	5.8
^aContaining (per kg) : 400,000U of vitamin A and 4,000UI of vitamin E.
^bContaining (per kg): 100 mg of I, 4000 mg of Fe, 40 mg of Co, 3000 mg of Mg, 40 mg of Se, 4000 mg of Zn, and 216 g of NaCl.
^cOrganic matter

The basal diet was formulated to meet the nutrient requirements of growing lambs (NRC, 2007). The animals received a total mixed ration (TMR) twice a day, at 0600h and 1700h, in equal amounts in order to maintain refusals at 10%. Ration fed and refusals were daily weighed to control feed intake throughout the experiment. Sulfur was supplemented as elemental S (99.0% S; inorganic source) and sulfur-proteinate (21.5% S; organic source); cooper was supplemented as CuSO₄ (25.0% Cu; inorganic source) and cooper-proteinate (11.0% Cu; organic source). Molybdenum was supplemented as Na₂MoO₄ (39.7% Mo).

Ration samples of each animal were daily taken during the week of d 28 of each period (the study was composed of 3 periods of 28 d each, for 84 total days period of study) to provide a pooled sample. Immediately after collection, samples were stored at − 20°C until further analysis. The chemical composition presented in Table 1 was assessed in a pooled sample from the control diet (mean value of 3 periods). The Mo, Cu, and S content presented in Table 2 were assessed in a pooled sample of each treatment (mean value of 3 periods).

Table 2

Copper, molybdenum and sulfur contents in treatments (mg/kg DM).
Treatment	Molybdenum	Copper	Sulfur
T0	1.02	9.96	2561.88
T1	9.54	8.42	2369.52
T2	1.05	15.70	4340.04
T3	0.97	15.72	5178.19
T4	1.18	17.22	4829.88
T5	0.90	17.61	5209.69
T6	11.60	15.74	4645.46
T7	10.21	19.91	5288.51
T8	11.92	16.67	5190.82
T9	11.72	17.16	4906.29
T0) control, basal diet without any additional mineral supplementation; T1) Basal diet added 10 mg of Mo; T2) Basal diet added 10 mg of inorganic Cu per kg DM and 0.2% of inorganic S; T3) Basal diet added 10 mg of inorganic Cu and 0.2% organic S; T4) Basal diet added 10 mg of organic Cu and 0.2% of inorganic S; T5) Basal diet added 10 mg of organic Cu and 0.2% of organic S; T6) Basal diet added 10 mg of Mo plus 10 mg of inorganic Cu and 0.2% of inorganic S; T7) Basal diet added 10 mg of Mo plus 10 mg of inorganic Cu and 0.2% of organic S; T8) Basal diet added 10 mg of Mo plus 10 mg of organic Cu and 0.2% of inorganic S; T9) Basal diet added 10 mg of Mo plus 10 mg of organic Cu and 0.2% of organic S.

All samples were dried in a forced air oven at 55°C for 72 h and ground 1-mm screen Willey mills (MA340, Marconi, Piracicaba, Brazil) and analyzed for DM (950.15), total N (984.13) and ether extract (920.39) according to AOAC [12] methods. Neutral detergent fiber (aNDF) and acid detergent fiber (aADF) content were assessed according to Van Soest and Mason [13] using α-amylase without sodium sulphide (TE-149, Tecnal Equipment for Laboratory Inc., Piracicaba, Brazil). Molybdenum, Cu, and S were determined quantitatively by atomic absorption spectrophotometry. Briefly, for Cu determination, representative 2-g sample was ashed at 500°C for 5.5 to 6 hours. The ash was taken up in 25 mL of 6 N hydrochloric acid (HCl), and the solution was aspirated directly into the atomic absorption spectrophotometer. For Mo analysis, a 2-g sample was ashed at 550°C for 6–8 hours. The ash was taken up in 25 ml of 6 N HCI, and the solution was aspirated directly into the atomic absorption spectrophotometer. For S determination, samples were weighed into glass tubes, which were digested using nitric acid and perchloric acid at 210°C. Atomic absorption spectrophotometry was used to determine the concentrations of sulfur by monitoring the plasma emission at 181.975 nm.

Performance, blood collection, and analysis

Body weight was assessed at enrollment and every 28 d until the end of the study (84 d after enrollment). These measurements were also used to calculate the ADG during the study period [(final weight – initial weight)/period in days].

For blood sampling handling, the animals were restrained in the pens to minimize movement and stress. Blood samples were collected at enrollment, 28, 56, and 84 d later by jugular venipuncture using a Vacutainer tube without anticoagulant and a 20-gauge × 2.54-cm Vacutainer needle (Becton, Dickinson and Co.). After collection, tubes were immediately placed in a cooler containing iced water and transported to the laboratory for processing. Samples were analyzed or processed within 2 h after collection. Blood samples collected without anticoagulant were centrifuged at 2,000 × g for 15 min at 4°C for serum separation (SL 16R Centrifuge, ThermoFisher Scientific Inc.)

Serum samples were used to determine the trace-mineral concentration and ceruloplasmin activity. For serum Mo, Cu, and S concentration, the stored serum samples were thawed and diluted at a 1:1 ratio with destilled water. Afterward, serum Mo, Cu, and S were determined by an atomic absorption spectrophotometer. For ceruloplasmin determination, the methodology proposed by Schosinsky et al. (1974) was followed. Briefly, O-Dianisidine dihydrochloride was used as substrate, which was converted to a yellow product on ceruloplasmin and oxygen presence. After adding sulfur acid, this enzymatic essay stop, producing a pinkish stable solution. Subsequently, the absorbance of this solution was measured at 450 nm on a spectrophotometer reader (Perkin Elmer^™ Lambda 35 UV/Vis, Waltham, MA, USA). All these analyses were performed at the Laboratory of Minerals of the College of Animal Science and Food Engineering of the University of Sao Paulo (FZEA/USP).

Slaughter and carcass evaluation

On day 84 of the study, all animals were weighted and slaughtered at the abattoir of the College of Animal Science and Food Engineering of the University of Sao Paulo (Pirassununga, Brazil). All procedures were performed according to the Sanitary and Industrial Inspection Regulation for Animal Origin Products of Humanitarian Slaugther Guidelines as required by Brazilian law [15]. After slaughter, the carcasses were skinned, eviscerated, washed, and weighed for the determination of hot carcass weight (HCW). After, the pH 0h was obtained with a digital pH meter (model HI8314, Hanna Instruments), measured in the longissimus muscle at the height of the 12th rib equipped using a penetration probe. The carcasses were then stored in a cold room (0–2°C) for 24 h. After this period, the carcasses were again weighed for the determination of cold carcass weight (CCW), and pH 24h was measured at the same site.

Statistical analysis

All statistical analyses were performed in SAS software, version 9.4 (SAS Institute Inc.). The normality of the residuals was verified by the Shapiro-Wilk test using the UNIVARIATE procedure. The experimental unit was the lamb. The parameters were analyzed considering a 2 x 2 x 2 factorial arrangement (with and without molybdenum, organic and inorganic Cu, organic and inorganic S, in addition to a basal and a basal diet with molybdenum).

To evaluate the effect of treatments on performance, serum Cu, S, and Mo concentration, and ceruloplasmin activity throughout the 4 sampling points (enrollment day, 28, 56, and 84 d later), repeat measures models were fitted by multiple mixed linear models using the MIXED procedure. For these repeated-measure models, a first-order autoregressive covariance structure [(AR(1)] was applied to account appropriately for within-lamb residuals correlation among times evaluated. This variance-covariance structure is indicated for equally spaced data collection and assumes correlation decline as a function of time. the variables treatment, time, and their interaction were forced into all statistical models even in the absence of statistical significance. Carcass traits, which did not include the time factor, were analyzed using the MIXED effect procedure with a model that included the fixed effects of treatment.

The orthogonal contrasts studied were: Control vs. others = T0 vs. (T1 + T2 + T3 + T4 + T5 + T6 + T7 + T8 + T9); Mo vs. others = T1 vs. (T0 + T2 + T3 + T4 + T5 + T6 + T7 + T8 + T9); Control vs. Mo = T0 vs. T1; Cu source = (T3 + T4) vs. (T5 + T6); S source = (T3 + T5) vs. (T4 + T6); Mo vs. Cu source interaction = (T2 + T4 + T7 + T9) vs. (T3 + T5 + T6 + T8); Mo vs. S source interaction = (T2 + T3 + T8 + T9) vs. (T4 + T5 + T6 + T7); Cu source vs. S source = (T3 + T4 + T7 + T8) vs. (T2 + T5 + T6 + T9) ; and Mo vs. Cu source vs. S source interaction = (T3 + T4 + T6 + T9) vs. (T2 + T5 + T7 + T8). Means were adjusted using Least Square Means (LSMeans) procedure of SAS and differences were determined by T-Test using the PDIFF command. Statistical significance was declared at P ≤ 0.05.

Animals and experimental diet

No health disorder occurred with the lambs throughout the experiment. Experimental diets had an identical nutrient composition, only differing in Mo, Cu, and S content (Table 2). The experimental diet had 57.0 g/kg DM of ashes, 42.8 g/kg DM of ether extract, 282.0 g/kg DM of neutral detergent fiber, 166.4 g/kg DM of acid detergent fiber, 157.3 g/kg DM of crude protein, 11.8g/kg DM of calcium and 5.8 g/kg DM of phosphorus.

Performance and carcass traits

No treatment effects were observed on body weight measurements (P > 0.05; Table 3); however, ADG was affected by Mo, Cu source, and S source interaction (P = 0.0283; Table 4 depicted in Fig. 1). Also, body weight and ADG were affected by time (P = 0.0001 and P = 0.0014, respectively; Table 3 and Table 4). Mineral supplementation did not affect carcass traits (P > 0.05; Table 5).

Table 3

Body weight (kg) of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Time (days)
Mo	Cu	S	0	28	56	84	Mean
Comparisons
Without	Without	Without	23.87	27.71	32.91	37.21	30.42
With	Without	Without	21.53	24.31	29.54	34.28	27.41
Without	Inorganic	Inorganic	21.98	24.68	30.28	33.85	27.69
	Inorganic	Organic	22.38	25.65	31.89	35.85	28.94
	Organic	Inorganic	22.61	25.83	31.23	36.01	28.92
	Organic	Organic	22.64	25.88	30.94	35.25	28.68
With	Inorganic	Inorganic	22.31	25.63	30.56	35.4	28.48
	Inorganic	Organic	21.66	25.45	30.11	34.8	28.01
	Organic	Inorganic	20.91	23.98	28.79	32.86	26.63
	Organic	Organic	21.6	25.26	31.45	36.75	28.77
Principal effects
Without			22.4	25.51	31.08	35.24	28.56
With			21.62	25.08	30.23	34.95	27.97
	Inorganic		22.08	25.35	30.71	34.98	28.28
	Organic		21.94	25.23	30.6	35.22	28.25
		Inorganic	21.95	25.03	30.21	34.53	27.93
		Organic	22.07	25.56	31.1	35.67	28.6
Average data
Mean			22.23	25.34	30.87	35.32	28.49
SEM			0.67	0.83	0.96	1.09	0.59
Statistical Probabilities of Contrasts
Control vs Others			NS
Mo vs Others			NS
Mo presence			NS
Cu source			NS
S source			NS
Mo*Cu			NS
Mo*S			NS
Cu*S			NS
MoCuS			NS
P value for treatment: 0.9986
P value fot time: 0.0001
Treatment*time: 0.7909
SEM = Standard error of the mean; NS = not significant

Table 4

Average daily gain (ADG; kg/d) of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Time (days)
Mo	Cu	S	0	28	56	84	Mean
Comparisons
Without	Without	Without	.	0.14	0.19	0.15	0.16
With	Without	Without	.	0.1	0.19	0.17	0.15
Without	Inorganic	Inorganic	.	0.1	0.2	0.14	0.15
	Inorganic	Organic	.	0.12	0.22	0.14	0.16
	Organic	Inorganic	.	0.11	0.19	0.17	0.16
	Organic	Organic	.	0.12	0.18	0.15	0.15
With	Inorganic	Inorganic	.	0.12	0.18	0.17	0.16
	Inorganic	Organic	.	0.12	0.17	0.17	0.15
	Organic	Inorganic	.	0.1	0.17	0.15	0.14
	Organic	Organic	.	0.13	0.22	0.19	0.18
Principal effects
Without				0.11	0.2	0.15	0.15
With				0.12	0.18	0.17	0.16
	Inorganic			0.11	0.19	0.16	0.15
	Organic			0.11	0.19	0.16	0.16
		Inorganic		0.11	0.19	0.16	0.15
		Organic		0.12	0.2	0.16	0.16
Average data
Mean				0.12	0.19	0.16	0.16
SEM				0.007	0.007	0.007	0.005
Statistical Probabilities of Contrasts
Control vs others			NS
Mo vs Others			NS
Mo presence			NS
Cu source			NS
S source			NS
Mo*Cu			NS
Mo*S			NS
Cu*S			NS
MoCuS			0.0283
P value for treatment: 0.2912
P value fot time: 0.0014
Treatment*time: 0.7918
SEM = Standard error of the mean; NS = not significant

Table 5

Carcass traits of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Variables
Mo	Cu	S	pH 0h	pH 24h	Hot carcass weigth (kg)	Cold carcass weigth (kg)
Comparisons
Without	Without	Without	6.17	5.62	19.65	19.04
With	Without	Without	6.15	5.63	17.5	16.8
Without	Inorganic	Inorganic	6.48	5.77	17.3	16.7
	Inorganic	Organic	6.46	5.6	18.5	17.9
	Organic	Inorganic	6.36	5.68	19.03	18.33
	Organic	Organic	6.16	5.66	19.05	18.43
With	Inorganic	Inorganic	6.6	5.71	18.15	17.5
	Inorganic	Organic	6.45	5.85	18	17.43
	Organic	Inorganic	6.51	5.7	16.8	16.18
	Organic	Organic	6.34	5.64	18.93	18.23
Principal effects
Without			6.36	5.68	18.47	17.84
With			6.47	5.72	17.97	17.33
	Inorganic		6.5	5.73	17.99	17.38
	Organic		6.34	5.67	18.45	17.79
		Inorganic	6.49	5.72	18.82	17.18
		Organic	6.35	5.69	18.62	17.99
Average data
Mean			6.36	5.68	18.35	17.72
SEM			0.052	0.024	0.608	0.598
Statistical Probabilities of Contrasts
Control vs others			NS	NS	NS	NS
Mo vs Others			NS	NS	NS	NS
Mo presence			NS	NS	NS	NS
Cu source			NS	NS	NS	NS
S source			NS	NS	NS	NS
Mo*Cu			NS	NS	NS	NS
Mo*S			NS	NS	NS	NS
Cu*S			NS	NS	NS	NS
MoCuS			NS	NS	NS	NS
SEM = Standard error of the mean; NS = not significant

Blood mineral concentration

The data regarding serum Cu concentration was presented in Table 6. At 56 and 84 days after enrollment, serum Cu concentration was higher for Control group (T0) compared to other groups (P = 0.0212 and P = 0.0088, respectively). On the other hand, serum Cu concentration was lower for the Mo group (T1) when compared to other treatments (P < 0.0024). In addition, at 84 days of study, the interaction among Mo, Cu, and S, affected serum Cu concentration (P = 0.0022; Fig. 2).

Table 6

Serum copper concentration (mg/L) of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Time (days)
Mo	Cu	S	0	28	56	84	Mean
Comparisons
Without	Without	Without	0.45	0.43	0.46	0.45	0.45
With	Without	Without	0.35	0.36	0.35	0.25	0.33
Without	Inorganic	Inorganic	0.43	0.39	0.37	0.4	0.4
	Inorganic	Organic	0.44	0.33	0.3	0.34	0.35
	Organic	Inorganic	0.44	0.33	0.3	0.32	0.35
	Organic	Organic	0.35	0.4	0.38	0.43	0.39
With	Inorganic	Inorganic	0.3	0.33	0.32	0.31	0.31
	Inorganic	Organic	0.45	0.36	0.41	0.43	0.41
	Organic	Inorganic	0.41	0.36	0.37	0.39	0.39
	Organic	Organic	0.3	0.35	0.34	0.35	0.33
Principal effects
Without				0.36	0.34	0.37	0.37
With				0.35	0.36	0.37	0.36
	Inorganic			0.35	0.35	0.37	0.37
	Organic			0.36	0.35	0.37	0.36
		inorganic		0.35	0.34	0.36	0.36
		Organic		0.36	0.36	0.39	0.37
Average data
Mean			0.4	0.37	0.36	0.37	0.37
SEM			0.015	0.013	0.016	0.013	0.007
Statistical Probabilities of Contrasts
Control vs. others			NS	NS	0.0212	0.0088	NS
Mo vs. Others			NS	NS	NS	0.0024	NS
Mo presence			NS	NS	NS	NS	NS
Cu source			NS	NS	NS	NS	NS
S source			NS	NS	NS	NS	NS
Mo*Cu			NS	NS	NS	NS	NS
Mo*S			NS	NS	NS	NS	NS
Cu*S			NS	NS	NS	NS	NS
MoCuS			NS	NS	NS	0.0022	NS
P value for treatment: 0.2413
P value fot time: 0.0057
Treatment*time: 0.0001
SEM = Standard error of the mean; NS = not significant

Regardless of time, organic source of Cu and S interaction increased serum S concentration (P = 0.0355; Table 7). At 84 days after enrollment, serum Mo concentration was lower for Control group (T0) compared to others (P = 0.0021; Table 8). Otherwise, Mo group (T1) increased serum Mo concentration at 28, 56, and 84 days after enrollment (P = 0.0001; P = 0.0001; and P = 0.0001, respectively; Table 8). Also, Mo group (T1) increased serum Mo concentration at 28 and 84 days after enrollment, when compared to the Control group (T0; P = 0.0182 and P = 0.0054, respectively; Table 8).

Table 7

Serum S concentration (mg/L) of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Time (days)
Mo	Cu	S	0	28	56	84	Mean
Comparisons
Without	Without	Without	1227.11	1021.41	1159.69	1170.3	1144.63
With	Without	Without	1437.58	1245.49	1559.48	1479.99	1430.63
Without	Inorganic	Inorganic	1318.22	1144.77	1488.13	1481.75	1358.22
	Inorganic	Organic	1324.11	1121.26	1330.45	1351.19	1281.75
	Organic	Inorganic	1311.83	1127.96	1200.98	1220.72	1215.37
	Organic	Organic	1401.44	1255.69	1503.38	1687.64	1462.03
With	Inorganic	Inorganic	1400.63	1224.19	1490.01	1506.15	1405.24
	Inorganic	Organic	1348.67	1200.14	1422.88	1458.74	1357.6
	Organic	Inorganic	1241.01	1021.26	1101.2	1067.52	1107.75
	Organic	Organic	1393.11	1234.41	1451.48	1517.24	1399.06
Principal effects
Without				1162.42	1380.74	1435.32	1329.34
With				1170	1366.39	1387.41	1317.41
	Inorganic			1172.59	1432.87	1449.46	1350.7
	Organic			1159.83	1314.26	1373.28	1296.05
		inorganic		1129.54	1320.08	1319.04	1271.64
		Organic		1202.87	1427.05	1503.7	1375.11
Average data
Mean			1334.98	1153.07	1360.72	1383.46	1308.06
SEM			23.36	20.67	49.25	53.39	20.81
Statistical Probabilities of Contrasts
Control vs. others			NS	NS	NS	NS	NS
Mo vs. Others			NS	NS	NS	NS	NS
Mo presence			NS	NS	NS	NS	NS
Cu source			NS	NS	NS	NS	NS
S source			NS	NS	NS	NS	NS
Mo*Cu			NS	NS	NS	NS	NS
Mo*S			NS	NS	NS	NS	NS
Cu*S			NS	NS	NS	NS	0.0355
MoCuS			NS	NS	NS	NS	NS
P value for treatment: 0.1992
P value fot time: 0.0001
Treatment*time: 0.4175
SEM = Standard error of the mean; NS = not significant

Table 8

Serum molybdenum concentration (mg/L) of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Time (days)
Mo	Cu	S	0	28	56	84	Mean
Comparisons
Without	Without	Without	0.036	0.014	0.055	0.054	0.07
With	Without	Without	0.022	0.89	0.715	0.991	0.654
Without	Inorganic	Inorganic	0.032	0.075	0.212	0.032	0.087
	Inorganic	Organic	0.028	0.072	0.027	0.027	0.039
	Organic	Inorganic	0.024	0.068	0.031	0.027	0.037
	Organic	Organic	0.027	0.087	0.024	0.026	0.041
With	Inorganic	Inorganic	0.029	0.141	0.097	0.113	0.095
	Inorganic	Organic	0.033	0.142	0.128	0.103	0.102
	Organic	Inorganic	0.042	0.146	0.147	0.139	0.118
	Organic	Organic	0.027	0.134	0.064	0.097	0.081
Principal effects
Without				0.075	0.073	0.028	0.051
With				0.141	0.109	0.113	0.099
	Inorganic			0.108	0.116	0.069	0.081
	Organic			0.109	0.067	0.072	0.069
		inorganic		0.107	0.122	0.078	0.085
		Organic		0.109	0.06	0.063	0.066
Average data
Mean			0.03	0.187	0.145	0.156	0.129
SEM			0.0029	0.0372	0.0372	0.0441	0.0176
Statistical Probabilities of Contrasts
Control vs. others			NS	NS	NS	0.0021	NS
Mo vs. others			NS	0.0001	0.0001	0.0001	NS
Mo presence			NS	0.0182	NS	0.0054	NS
Cu source			NS	NS	NS	NS	NS
S source			NS	NS	NS	NS	NS
Mo*Cu			NS	NS	NS	NS	NS
Mo*S			NS	NS	NS	NS	NS
Cu*S			NS	NS	NS	NS	NS
MoCuS			NS	NS	NS	NS	NS
P value for treatment: 0.0001
P value fot time: 0.0001
Treatment*time: 0.0001
SEM = Standard error of the mean; NS = not significant

Table 9

Serum ceruloplasmin activity (U/L) of lambs receiving a control diet or supplemented with molybdenum (Mo) or sources of copper (Cu) and sulfur (S).
Treatments			Time (days)
Mo	Cu	S	0	28	56	84	Mean
Comparisons
Without	Without	Without	21.58	17.28	23.37	20.15	20.59
With	Without	Without	13.32	14.82	17.06	11.44	14.16
Without	Inorganic	Inorganic	22.96	17.12	17.98	15.65	18.3
	Inorganic	Organic	20.7	15.85	18.26	21.41	19.05
	Organic	Inorganic	18.85	15.99	13.02	11.43	14.82
	Organic	Organic	17.33	15.76	16.62	16.88	16.65
With	Inorganic	Inorganic	13.04	14.47	12.73	8.02	12.06
	Inorganic	Organic	19.63	15.24	16.59	18.26	17.43
	Organic	Inorganic	17.73	17.04	17.86	15.92	17.14
	Organic	Organic	14.38	16.34	13.12	11.03	13.71
Principal effects
Without				16.18	16.47	16.34	17.21
With				15.77	15.07	13.31	15.09
	Inorganic			15.67	16.39	15.84	16.71
	Organic			16.28	15.15	13.81	15.58
		inorganic		16.15	15.4	12.75	15.58
		Organic		15.8	16.14	16.89	16.71
Average data
Mean			18.08	16.05	16.98	15.26	16.59
SEM			0.76	0.64	0.97	0.82	0.41
Statistical Probabilities of Contrasts
Control vs. others			0.0345	NS	NS	0.0013	0.0053
Mo vs. others			0.043	NS	NS	NS	NS
Mo presence			0.0196	NS	NS	0.0254	NS
Cu source			NS	NS	NS	NS	NS
S source			NS	NS	NS	0.031	NS
Mo*Cu			NS	NS	NS	NS	NS
Mo*S			NS	NS	NS	NS	NS
Cu*S			NS	NS	NS	0.0055	NS
MoCuS			NS	NS	NS	0.0074	NS
P value for treatment: 0.0246
P value fot time: 0.0021
Treatment*time: 0.0349
SEM = Standard error of the mean; NS = not significant

Ceruloplasmin activity

The control diet had the highest value of serum ceruloplasmin activity compared to other treatments (P = 0.0053). At 84 days after enrollment, ceruloplasmin had reduced activity when Mo (P = 0.0254) and inorganic S (P = 0.0310) were supplemented to the lambs. There was an interaction between Cu and S sources (P = 0.0055), in which serum ceruloplasmin level was higher when Cu supplementation, regardless of source, was associated with organic S. In addition, there was interaction among Mo x Cu source x S source (P = 0.0074; Fig. 3).

Animals and experimental diet

No health problems were diagnosed in all animals during the experimental period. It ensures that all effects herein detected were exclusively through mineral addition into diets. Furthermore, as shown, only mineral contents were different among treatments. Thus, there was no difference in the experimental basal diet, which might interfere with the results herein presented.

Performance and carcass traits

The time influenced ADG, which ranged from 140 to 180 g/day. It was expected once we were studying growing lambs. When contrasts were applied, the inclusion of organic Cu and S added with Mo, increased ADG (0.180 kg/d) when compared to the same sources without Mo addition (0.150 kg/d; Fig. 1). Molybdenum is an essential trace-mineral required for animals’ growth; however, its excessive dietary inclusion may impair Cu absorption. Further, dietary S may reduce Cu absorption [16]. Insoluble thiomolybdates and sulfides formed by ruminal microbes bind dietary Cu, decreasing its bioavailability [3]. Dick et al. [17] reported that the limiting effect of Mo on Cu nutrition is dependent on S level in the feed; further, Suttle [23] reported that organic Mo may be more effective Cu antagonist than inorganic source. Thus, Mo, Cu, and S supply balancing and sources (inorganic and organic) may affect ruminant growth. It is of great importance for lambs, which are more susceptible to Cu toxicity than either cattle or goats [16]. Dezfoulian et al. [19] studied doses (10 and 20 mg/kg DM) and Cu sources (CuSO₄ and Cu-proteinate) in sheep diets and did not observe any effect on ADG; however, Cu supplementation, regardless of source, had effect on feed conversion ratio. Therefore, our findings suggest that organic minerals interaction can influence the performance of growing lambs being less sensitive to impaired interactions.

Although effect was detected on ADG, it did not reflect on heavier body weight in the organic Cu and S added with Mo group. Likewise, HCW and CCW also did not differ, ranging from 16.7 to 19.65 kg. These values were similar to those observed by Garrine et al. [20], who supplemented crossbreed Merino x Texel lambs with different doses and sources of Cu. In this study, no influence of treatments was observed on pH values, which showed normal values for initial (0h) and final (24h) measurements, indicating a normal rate of muscle pH fall due to postmortem glycolysis [22].

Blood mineral concentration

Serum Cu concentration was higher in the control group compared to other treatments. Otherwise, serum Cu levels decreased when supplementing Mo and through Mo x Cu x S interaction. As earlier mentioned, excessive dietary Mo and S may impair Cu absorption through insoluble thiomolybdates and sulfides, formed by ruminal microbes [6]. This can lead to reduced Cu in the liver and eventually reduced plasma Cu concentration [4]. Lower serum Cu concentration was detected in this present study in Mo supplemented group compared to others, confirming the negative effect of Mo on Cu absorption in bloodstream. Corroborating, Dias et al. (2013) in their meta-analytical study, found reduced plasmatic Cu concentration as increasing levels of Mo and S in the diet of growing-finishing cattle. Further, Suttle (1974) observed reduced serum Cu concentrations by increasing S levels in sheep diets, both in organic and inorganic form. A similar reduction was found in sheep when including high S levels, which increases ruminal sulfide concentration [24]. In this study, all treatments were composed of either Mo or S inclusion, except the control group, which afford, in this study, higher serum Cu concentration. Thus, Cu absorption, even due to its higher level of supplementation, may be jeopardized by Mo and S interaction, reflecting in lower serum Cu concentration.

Otherwise, at 84 days after enrollment, higher serum Cu concentration was detected in animals receiving organic Cu plus organic S and in animals receiving Mo + inorganic Cu + organic S compared to other treatments. One possible explanation is that thiomolybdate absorption is most likely when the production of thiomolybdates exceeds the amount that can react with Cu to form insoluble complexes in the rumen. One of the systemic effects is thiomolybdates binding strongly to plasma Cu bound to albumin, which results in a reduced transport of available Cu for biochemical processes [7]. This can result in high plasma Cu concentrations even in ruminants deficient in Cu [4]. Noteworthy that total serum Cu is of little significance in Mo and S presence, because Cu concentration may be increased in plasma, but not available for tissue absorption due to its insoluble bonds.

In this study, lambs fed with organic Cu and S sources had increased serum S concentration compared to inorganic sources or interaction between inorganic and organic sources. It may be explained due to strong bond between Cu and S in the bloodstream, which increases serum S retention by decreasing its tissue uptake [7].

Higher serum Mo concentrations found in this study for Mo fed animals, agree with those found by Pott et al. [25] and Galbraith et al. [26] who observed an increase in plasma Mo concentration when 7.5 mg Mo/kg DM or 15 mg Mo/kg DM were added to the sheep and goat diets, respectively. Interestingly, the control diet had higher serum Mo levels than the treatments that received Cu and S supplementation with no Mo addition, regardless of sources. It may be through the negative effect of S, which has been reported to reduce plasma Mo concentration, regardless of its source [18].

Ceruloplasmin activity

Regarding serum ceruloplasmin activity, the reduced values found in the presence of Mo, inorganic S, or their interaction corroborate with McDowell [27], who indicated that as lower Cu availability as lower ceruloplasmin activity. It is due to the copper-molybdenum-sulfur interaction, once the increase of Mo and S in the diet, alone or in combination, can reduce Cu use [4, 6, 17, 23], as it reduces the Cu absorption [7, 24].

Molybdenum and inorganic and organic sources of Cu and S did not improve performance, nor did they have an impact on carcass traits in this study. However, a clear negative effect of Mo and S was detected on Cu bioavailability and metabolism either by serum Cu concentrations or by ceruloplasmin activity. In addition, organic sources of Cu and S interacted by themselves with no defined pattern for each variable herein studied.

Acknowledgements

The authors express appreciation to the College of Animal Science and Food Engineering, University of Sao Paulo for providing staff and facilities. In addition, the authors thanks the National Council for Scientific and Technological Development (CNPq) and the Coordination for the Improvement of Higher Education Personnel (CAPES) for providing fellowships.

Author Contributions

R.M.C. Conti: Conceptualization, Investigation, Methodology, Writing – Original draft preparation T.H. Silva: Writing – Original draft preparation, Writing – review & editing I.C.S.B. Guimarães: Writing – review & editing H.V.A. Bezerra: Investigation A. Saran Netto: Investigation, Methodology P.H.M. Rodrigues: Formal analysis J.C.G. Pacheco: Methodology M.A. Zanetti: Funding acquisition, Visualization, Supervision.

Funding Information

This work was supported by the Sao Paulo Research Foundation (FAPESP) [grant number 2011/21377–0]

Data Availability

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics Approval

The experimental procedures were reviewed and approved by the Bioethics Committee for Animal Experimentation of the College of Animal Science and Food Engineering (FZEA/USP, #14.1.1410.74.7).

Competing interest

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

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No competing interests reported.

Influence of molybdenum and organic sources of copper and sulfur on performance and blood mineral concentration in lambs

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Animals, facilities, and treatments

Performance, blood collection, and analysis

Slaughter and carcass evaluation

Statistical analysis

Results

Animals and experimental diet

Performance and carcass traits

Blood mineral concentration

Ceruloplasmin activity

Discussion

Animals and experimental diet

Performance and carcass traits

Blood mineral concentration

Ceruloplasmin activity

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1