The project was approved by the Animal Use Ethics Committee of the Animal Science Institute, with protocol number CEUA/IZ 268-18. We confirm that this study was carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and all methods were performed following the relevant guidelines and regulations.
2.1. Location and experimental design
The experiment was carried out on the experimental farm of the Animal Science Institute of Ribeirão Preto/SP using 42 male Holstein calves (initial weight 38.33 ± 4.09 kg) coming from a commercial farm and acquired at ages between 2 and 6 days. All animals were properly fed with colostrum immediately after birth with the provision of 4 liters of quality colostrum (Brix > 21%) per animal within four hours after birth and then another 2 liters of milk from the second milking. Calves received second milking milk (6L per day) on the second and third day. After the fourth day of life, they received 6 liters of milk a day until they were transported to the farm where the experiment was carried out.
On the day of arrival at the experimental farm, calves remained for 12 hours in a collective pen, with wood shavings bedding and heating with infrared light lamp, and received 2 liters of serum (Calf Care®, De Laval) per animal via bottle. After this period, animals were allocated to individual tropical huts, with sand bedding. Treatments began one day after calves arrived at the experimental farm, considered the first day of the experiment, and all received treatments for 60 days.
The design was completely randomized, with calves allocated to the following treatments: C (control milk replacer, N = 14); SeVitE (milk replacer supplemented with 0.6 mg organic selenium/kg + 100 IU vitamin E, N = 14); SeVitEFe (milk replacer supplemented with 0.6 mg organic selenium/kg + 100 IU vitamin E + 200 mg Fe chelate/kg, N = 14).
Supplementation was administered through capsules previously manipulated for each treatment and added to the milk replacer individually at the time of its daily preparation in the bucket with the respective bottle for each calf. The sources of supplemented nutrients were amino acid selenium (YES- Mineral Selenium), ferrous glycine hydrate chelate (BASF Iron Glycinate), and alpha tocopherol acetate (LUTAVIT E 50, BASF). The calculated amount for capsules was to ensure that the animals received at least 0.6 mg of Se; 200 mg of Fe and 100 IU of vitamin E (149 mg) per kg of dry matter, a value calculated taking into account the amount of the respective nutrients from the intake of concentrate and milk replacer according to the dietary program adopted.
2.2. Management and experimental parameters
Calves were fed for 60 days, receiving 6 liters of milk replacer/day until 30 days from the beginning of the experiment and 4 liters of milk/day, from 31 to 60 days of experiment, and then weaned. Commercial milk replacer was used (Sprayfo® Violeta, Trouw Nutrition), which was diluted in drinking water preheated to 39ºC to reach dry matter concentration of 14%. The grounded concentrate was offered ad libitum, always in small amounts, being gradually increased according to the animal's consumption. The milk replacer and concentrate composition is shown in Table 1.
Table 1. Chemical composition of the calf starter and milk replacer offered to calves during the experiment.
|
Composition (%)
|
Starter
|
Milk Replacer
|
|
Ground corn
|
36.0
|
-
|
|
Wheat bran
|
20.0
|
-
|
|
Soybean meal
|
40.0
|
-
|
|
Salt
|
1.0
|
-
|
|
Premix¹
|
3.0
|
-
|
|
Nutrients (%)
|
|
|
|
Dry matter
|
90.30
|
89.93
|
|
Crude protein
|
25.67
|
20.53
|
|
Ether extract
|
3.88
|
8.81
|
|
Neutral detergent fiber
|
17.81
|
2.56
|
|
Acid detergent fiber
|
7.20
|
2.23
|
|
Lignin
|
1.05
|
1.34
|
|
Non-fibrous carbohydrates
|
45.94
|
60.42
|
|
Total digestible nutrients2
|
70.55
|
89.09
|
|
Selenium (mg/kg)
|
0.48
|
0.17
|
|
Iron (mg/kg)
|
115.03
|
191.96
|
|
Alpha tocopherol (mg/kg)
|
37.54
|
82.38
|
|
¹ Maximum level - calcium: 270 g; cobalt: 25 mg; copper: 840 mg; fluoride: 500 mg; phosphorus: 50 g; iodine: 50 mg; magnesium: 15 g; manganese: 2,500 mg; selenium: 25 mg; sodium: 55 g; virginiamycin: 800 mg; vitamin A 333,330 IU; vitamin D3: 33,330 IU; vitamin E: 1,210 IU; Zinc: 3,360 mg.
|
2 NDT = (0.98 × CNF) + (0.93 × PB) + 2.25 × (EE-1) + 0.75 × (NDLPb – Lig) × [1 – (Lig/NDFlpb)0.667] – 7 (NRC, 2001).
|
Water was offered ad libitum to calves throughout the experimental period and had selenium concentration of 0.008 mg/L and iron concentration of 0.010 mg/L.
2.3. Performance
The intake of milk replacer and concentrate was daily monitored, recording the volume of the liquid diet consumed and weighting the concentrate offered and the following day's leftovers.
All animals were weighed on a digital scale, measuring height at withers, chest circumference, croup width and body length, at the beginning of the experiment, and every 15 days until the end of 60 days. Height measurements were taken using appropriate ruler, fixed to a right-angled support, measuring the vertical distance between the ground and the withers. Chest circumference, croup length and width measurements were taken using a measuring tape. Body length was measured by measuring the distance from the scapula to the beginning of the tail insertion. Croup width was measured by measuring the distance between ischia.
2.4. Immunological challenge and oxidative metabolism parameters
Immunological challenge was performed on all animals on day 40 of the experiment and inoculation was carried out by applying 1 mL of solution containing 1x107 red blood cells infected with Anaplasma marginale (CEPA UFMG1) per mL of blood via jugular puncture. Blood samples inoculated with the pathogen were kept in liquid nitrogen with dimethyl sulfoxide cryoprotectant until the date of inoculation. Blood samples were collected from the jugular vein at 40 days of experiment, before inoculation and after 20 days of inoculation, that is, at 60 days of experiment, to quantify the activity of the glutathione peroxidase enzyme (GPx), total plasma antioxidant activity, reduced glutathione quantification (GSH) and cytokine IFN-γ measurement.
From the date of the immunological challenge, daily monitoring of rectal temperature was performed at 7:30 am and 4:30 pm, as well as observation of the eye score and hematocrit measurement.
To certify the inoculation and its effectiveness in the animals' bodies, blood smears were collected at 46, 53 and 60 days of experiment for Anaplasma marginale quantification and blood samples were collected 20 days after inoculation (60 days of experiment) to perform serology (IFAT) and PCR for Anaplasma marginale. As the period of clinical manifestation of the disease generally occurs on average 20 to 38 days after the animal has contact with the pathogen, blood was collected on a fixed date of 20 days after inoculation (60 days of experiment) to maintain equal inoculation days among animals. Animals showed clinical signs of the disease after 60 days of experimentation and within 68 days. Blood smears were collected to quantify the number of pathogens. Clinical signs of the disease were considered when animals had rectal temperature above 40 °C and hematocrit below 60% of the basal value of each calf. Treatment was carried out with enrofloxacin (Knetomax, Bayer Animal Health, Brazil) intramuscularly with a single dose equivalent to 3 ml/40 kg of body weight.
2.5. Blood metabolism parameters and blood count
Blood samples were collected from calves by jugular puncture on the first day, 40 and 60 days of the experiment, two hours after offering a milk replacer, for analysis of selenium, iron, blood count, ferritin, and the following biochemical parameters: ß-hydroxybutyrate, glucose, total protein, urea, creatinine, creatinine kinase, lactate dehydrogenase (LHD), lactate, albumin, and IgG. For vitamin E analysis, blood samples were collected from animals on the first day and at 60 days of experiment.
For selenium, iron, ferritin and vitamin E measurements, blood samples were collected in vacutainer tubes without anticoagulant and centrifuged at 3,000 RPM to obtain serum, being stored frozen in plastic tubes. The tubes used for vitamin E analysis were protected with aluminum foil and the serum was stored in amber plastic tubes.
2.6. Stool consistency score and incidence of diarrhea
The stool consistency score (0 = normal, well-formed stools; 1 = abnormal, but not yet showing diarrhea, little pasty stools; 2 = moderate diarrhea, pasty stools; 3 = severe diarrhea; very pasty or liquid stools) was daily assessed. Cases of diarrhea were considered when calves presented fecal scores > 2. When presenting score of 2, animals received 4 L of daily oral serum, divided into two meals, two hours after offering a milk replacer and probiotic based on lactobacilli. In the case of score 3 and clinical signs of dehydration, calves received probiotic based on lactobacilli, intravenous serum until body rehydration occurred and could be medicated with antibiotic based on sulfamethoxazole + trimethoprim, an anti-inflammatory based on flunixin meglumine.
Health and presence of clinical cases of other diseases were daily observed, and daily environmental temperature and relative humidity data were recorded.
2.7. Laboratory analysis methodologies
Grounded concentrate and milk replacer samples were weekly collected and frozen to prepare a monthly pool of each food for bromatological analysis, carried out at the Laboratory of Bromatology - FMVZ/USP. Samples were ground in mill with 1 mm orifice sieve, for bromatological analyses of dry matter (DM, method 930.15), crude protein (CP, method 984.13), ether extract (EE, method 920.39), mineral matter (MM, method 942.05), acid detergent lignin (method 973.18), according to AOAC8. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) analyses were also carried out, according to Van Soest9 and Goering & Van Soest10, respectively.
To calculate non-fibrous carbohydrates (NFC) and total digestible nutrients (TDN), the following NRC formulas2 were used:
NFC = 100 – (crude protein - ether extract - ash – neutral detergent fiber)
TDN = (0.98 × NFC) + (DC × CP) + 2.25 × (EE-1) + 0.75 × (NDFcp – lignin) × [1 – (lignin/NDFlpb)0.667] – 7, where CD = digestibility coefficient (93% for milk replacer, 86.45% concentrate), CP = crude protein, NDFlpb = neutral detergent fiber free from crude protein.
Water samples were stored in 300-mL plastic bottles and frozen at –6ºC for later sending to the laboratory for selenium and iron analyses11.
Selenium and iron analyses of feed, milk replacer, and serum samples were carried out in the Laboratory of Minerals of the Faculty of Animal Science and Food Engineering at USP/Pirassununga. Concentrate and milk replacer samples underwent wet digestion with nitric-perchloric acid and to detect selenium in food and serum, using an adapted fluorimetric method12. Serum iron concentration was analyzed using the colorimetric method (Fe liquiform, Labtest kit).
Analyses of alpha tocopherol in the concentrate, milk replacer and vitamin supplement were carried out at the CBO Laboratory (Valinhos, SP) using the methodology with HPLC (Isocratic, fluorescence detector) and reverse phase column separation technique, detection at 326 nm of emission and 294 nm of excitation and quantification by external standard13. Samples were saponified to hydrolyze and solubilize fats and proteins and extracted with hexane. Serum alpha tocopherol analyses were performed using high-performance liquid chromatography with fluorescence detector14.
Serum ferritin was analyzed by two-site enzyme immunoassay (sandwich), the chemiluminescent substrate (Lumi-Phos* 530) was added to the container and the light generated by the reaction was measured with luminometer.
To perform blood count, blood samples were collected using vacutainer tubes containing EDTA as an anticoagulant. Immediately after collection, samples were refrigerated and transported to the veterinary laboratory for analysis. Blood samples were analyzed in automated pochi-100ivdiff® cell counter, which uses impedance and spectrophotometry techniques. The following parameters were analyzed: global erythrocyte, hematocrit and hemoglobin count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), global leukocyte count and global platelet count.
To estimate the concentration of plasma proteins, Megabrix® portable manual refractometer (IONLab Barigui, Brazil) was used. Blood samples were centrifuged in a laboratory centrifuge model CEOO1-B1® - Kacil (Recife, Brazil), at speed of 2,500 rpm for 5 minutes.
For the analysis of biochemical parameters, blood samples were collected with the aid of a vacuum tube containing sodium fluoride to obtain plasma and tubes without anticoagulant to obtain blood serum. Samples were centrifuged at 3,000 rpm for 15 minutes. Serum and plasma were collected, placed in 1.5-mL tubes and stored at -20 ˚C for later analysis. The determination of metabolites and enzymes to describe the metabolic profile of animals through commercial kits was performed using the Automatic System for Biochemistry – Model SBA – 200 kit (CELM, Barueri, SP, Brazil). Labtest Diagnóstica S.A. kits (Lagoa Santa, MG, Brazil.) were used to determine glucose (Ref. 85), total protein (Ref. 92-250), urea (Ref. 104), albumin (Ref. 19), creatinine (Ref. 35), creatinine kinase (Ref. 96), lactate dehydrogenase (Ref. 86), lactate (Ref. 138). For BHBA determinations, kit from Randox Laboratories – Life Sciences Ltda (Crumlin, Uk - Ranbut – Ref. RB1007) was used.
IFN-γ, IgG, GPx, GSH and AST concentrations were determined at the FMVZ/USP laboratory. IFN-γ concentrations were determined using kit (IFN-γ DuoSet, RD system®), according to manufacturer's instructions.
IgG concentration in calf serum was measured using the sandwich ELISA test, in which rabbit anti-bovine IgG antibody (capture antibody, B5645; Sigma, St. Louis, MO), diluted 1:400 in sodium carbonate buffer, was used to coat Immulon 4HBX plates (Thermo Corp., Milford, MA) at 4-8°C, and the analysis methodology was followed according to manufacturer's recommendation.
Vacuum total blood samples were collected in two 8.5-mL tubes containing lytic heparin as an anticoagulant for GPx and AST analyses. Samples were centrifuged at 3,000 rpm for 10 min in centrifuge refrigerated at 4°C with slow deceleration, with plasma and buffy coat being collected. After the first centrifugation, an aliquot of plasma was removed, stored in amber Eppendorf tubes and kept frozen at -80 ºC. The total antioxidant activity (AST) was determined using commercial kit (RANSEL® Laboratories, Randox, Crumlin, UK) according to protocol described by the manufacturer. Centrifugation was repeated two more times with phosphate buffer solution (PBS 10%), until the supernatant was completely clear. The red blood cell mash obtained was packaged in amber microtubes and frozen in freezer at -80°C for subsequent GPx analysis. Serum GPx activity was determined on a Randox automatic biochemical analyzer (model RX Daytona) using Randox® commercial kits (RANSOD and RANSEL) to determine the values and corrected by the hemoglobin concentration measured by spectrophotometry.
To determine GSH, whole blood collected in vacuum tubes containing EDTA was used. In a test tube, 200 μl of whole blood was hemolyzed with 1,800 μl of distilled water, then, 3 mL of precipitating solution (3.34 g of metaphosphoric acid, 0.44 g of EDTA Na2H20, 60 g of NaCl and distilled water q.s.p 200 ml) were added to the hemolysate. The solution remained at rest for 5 minutes and was then centrifuged for 5 minutes at 3,000 rpm. The intermediate content was collected, stored in amber Eppendorf tubes and kept in freezer at -80 °C until analysis. To determine the GSH concentration, a 200-μl aliquot of the supernatant was added to 800 μl of Na2HPO4 solution (300 mmol/l). After homogenization, 100 μl of 0.05% DTNB (2-dinitrobenzoic acid) solution was added to the system, with reading being performed at 412 nm within 30 seconds after the addition of DTNB. From the standard curve values, the straight line equation was calculated and the absorbance values of analyzed samples were converted into mg/dL.
For blood collected for indirect fluorescent antibody test (IFAT), tube containing EDTA as an anticoagulant and frozen at -20°C was used. For PCR analysis, blood was collected in tube without anticoagulant, centrifuged at 3.00 rpm for 15 minutes and the serum was frozen at -20°C until sent for analysis. Both analyses were carried out at the Laboratory of Veterinary Protozoology / ICB/UFMG. The presence of anti-Anaplasma marginale antibodies was detected using IFAT and anti-bovine monoclonal antibody (AbD Serotec) conjugated to fluorescein isothiocyanate (FITC). A. marginale antigen (UFMG1strain)15. Samples were considered positive if they showed fluorescence at dilution of 1:40. For each reaction, positive and negative control serum was used16.
Polymerase chain reaction (PCR) was used to diagnose Anaplasma marginale infection using the following primers Am1(F) [5'-CAA TCG TGA GGG ATA GCC TTG TAC-3'] and Am1(R) [5' -TGG TAT CAC GGT CAA AAT CTT TGC T-3'], which amplify a 300-bp fragment specific to the msp1a A. marginale gene17. All reactions were carried out under the same reagent conditions and thermal cycles. To this end, each reaction tube had final total volume of 25µL, with mixture containing 12.6µL of ultrapure water, 2.5µL of buffer, 2.5µL of tick DNA sample, 4.0µL of dNTPs, 0. 75µL of MgCl2, 1.25µL of each primer and 0.15µL of Taq-polymerase enzyme (Taq-Platinum, Invitrogen). PCR conditions were 95 oC for 5 min, followed by 40 cycles of 95 oC for 15 seconds, 58 oC for 30 seconds, 72 oC for 30 seconds; and final extension at 72 oC for 5 minutes. Samples were amplified in thermocycler apparatus (Biocycler®) and the products amplified by PCR were submitted to electrophoresis in a 1.5% Agarose gel, under 100 V (≅ 1 hour) stained with Ethidium Bromide (30 minutes). DNA bands separated by electrophoresis were visualized using ultraviolet light transilluminator. Samples with amplified product corresponding to the same migration pattern as the band generated by the corresponding positive control were considered positive. Blood from all calves were sent for analysis, but there were failures in the transport and delivery system of these samples between the experiment site and the laboratory (600 km) and in the end, 12 samples were reliable for carrying out the analyses.
The blood smear technique to obtain the number of Anaplasma marginale was performed with 5 μL of blood and using the GIEMSA dye. There were problems in producing and storing these smear slides and it was decided to keep only slides in excellent reading conditions. Due to this fact, readings were taken from 14, 18, 13 and 14 slides from 46, 53, 60 experimental days and 14 from the day of clinical diagnosis of the disease (peak), respectively.
2.8. Statistical analysis
Results were submitted to statistical analysis according to a completely randomized design using PROC MIXED from SAS 9.418 according to the model below:
Yij = µ + Di + A(Di)j + Ɛij,
Where Y ijklm = dependent variable; µ = overall average; Di = diet effect (i = 1 to 3); A(Di)j = random effect of animal within treatment (j =1 to 3); Ɛijklm = assumed random error NID ~ (0,σ2).
Blood variables were collected over time according to the previous design considering the effect of time as a repeated measurement, using PROC MIXED from SAS, according to the following model:
Yijklmn = µ + Di + A(Di)j + Ɛij,+ Tn + D×Tln + ωijln
Where Yijklmn is the dependent variable value, μ is the overall mean, Ai, A(Di)j, Tl, Am, T×Dlm + Ɛijklm were previously described, and ωijlmn is the random error associated with the assumed effect of time NID ~ (0, σ2); Tn is the fixed effect of sampling time (l = 1 to 3); D×Tln is the fixed effect of the interaction between diet and time; D×Tmn is the fixed effect of the interaction between diet and time. All means were obtained using LSMEANS.
Treatment responses were tested and significance level of 0.05 was adopted.