2.1. Animals and Diets
All experimental procedures were approved by the University of São Paulo, College of Animal Science and Food Engineering (USP/FZEA), Ethics Committee for the Use of Animals in Experiments (#012606). This study was conducted at the São Paulo Agency for Agribusiness Technology [Agência Paulista de Tecnologia dos Agronegócios (APTA)], in Ribeirão Preto, Brazil, from August to December 2009. This location (47°51′ W and 21°12′ S; altitude 646 m) is considered tropical humid, with average rain precipitation of 1427 mm, an average maximum temperature of 25 °C, and an average minimum temperature of 19 °C.
A total of 24 multiparous lactating Jersey cows (2.4 ± 0.5 lactations; 370.8 ± 35.7 kg of body weight; 62 ± 10 days in milk; and initial milk yield of 12.1 ± 1.8 kg/d, mean ± SD), were used in a completely randomized design trial. The cows were randomly assigned into one of four treatments according to the Microsoft Excel RAND command: (1) Control (CON) cows were fed a basal ration without sunflower oil, an organic mineral source or additional vitamins; (2) Selenium (SEL) cows were fed the basal ration with added organic Se (selenium yeast; Sel-Plex
®, Alltech Inc., Nicholasville, KY, USA) at 2.5 mg kg
−1 dry matter (DM), and 1000 IU of vitamin E, daily; (3) Sunflower oil (SUN) cows received the basal ration with 3% sunflower oil kg
−1 DM; and (4) Selenium + sunflower oil (SEL + SUN) cows were fed the basal ration with added organic Se (selenium yeast; Sel-Plex
®, Alltech Inc.) at 2.5 mg kg
−1 DM, 1000 IU of vitamin E, and 3% of sunflower oil kg
−1 DM (
Table 1). The total mixed ration (TMR) had 50% of roughage (corn silage) and 50% concentrate (DM-basis), according to recommendations described in [
19]. The sunflower oil, Se, and vitamin E were mixed in TMR. The cows were housed in individual pens (17.5 m
2), with sand bedding, feed bunks, and forced ventilation. The adaptation period to the diet was 14 days.
The experimental TMR was provided for 12 weeks after the adaptation period in equal amounts, twice daily (0700 h and 1400 h). Feed intake was determined based on daily feed offered and refusals, in which the latter was kept to 50–100 g/kg of total offered feed. Feed and refusals were collected weekly and stored at −20 °C. The dry matter intake (DMI) and chemical analyses of the experimental diets were performed for each cow in a pooled 4-week sample (weeks 3, 6, 9, and 12). All samples were assessed for dry matter (DM, AOAC 950.15), total nitrogen (AOAC, 984.13) for crude protein (CP) determination (6.25 × total nitrogen), ether extract (EE, AOAC 920.39), and ash (AOAC 942.05), according to the method described in [
20]. The neutral detergent fiber (aNDF) and acid detergent fiber (aADF) content of samples were determined as stated by [
21]. An aNDFanalysis was performed using amylase without sodium sulfide (TE-149 fiber analyzer, Tecnal Equipment for Laboratory Inc., Piracicaba, Brazil). Dry matter intake was quantified as the mean over the entire experimental period.
Table 1.
Ingredients and chemical composition of the experimental diets (values expressed as % of DM, otherwise stated) 1.
Table 1.
Ingredients and chemical composition of the experimental diets (values expressed as % of DM, otherwise stated) 1.
Item | Treatments 1 |
---|
CON | SEL | SUN | SEL + SUN |
---|
Ingredients | | | | |
Corn silage | 50.0 | 50.0 | 50.0 | 50.0 |
Corn meal | 25.0 | 25.0 | 22.0 | 22.0 |
Soybean meal | 18.0 | 18.0 | 18.0 | 18.0 |
Wheat bran | 4.0 | 4.0 | 4.0 | 4.0 |
Urea | 0.9 | 0.9 | 0.9 | 0.9 |
Salt | 0.5 | 0.49 | 0.5 | 0.49 |
Mineral premix 2 | 1.0 | 1.0 | 1.0 | 1.0 |
Ammonia sulfate | 0.04 | 0.04 | 0.04 | 0.04 |
Sodium bicarbonate | 0.53 | 0.53 | 0.53 | 0.53 |
Sunflower oil 3 | - | - | 3 | 3 |
Selenium, mg/kg | - | 2.5 | - | 2.5 |
Vitamin E, IU | - | 1000 | - | 1000 |
Chemical composition | | | | |
DM | 57.7 | 57.8 | 58.3 | 58.2 |
Ash | 6.4 | 6.4 | 6.5 | 6.6 |
CP 4 | 18.1 | 18.6 | 18.4 | 18.4 |
Ether extract | 2.3 | 2.4 | 3.6 | 3.4 |
Nitrogen-free extract | 57.5 | 57.0 | 56.3 | 56.2 |
aADF | 21.1 | 21.3 | 21.6 | 21.3 |
aNDF | 37.9 | 37.8 | 37.7 | 37.1 |
NEL, Mcal/kg DM 5 | 1.70 | 1.69 | 1.82 | 1.81 |
2.2. Milk Production and Composition
Cows were milked twice a day (0600 h and 1600 h), and milk yield was recorded using an electronic device (Alpro
®, DeLaval, Tumba, Sweden). Pooled milk samples for each cow, from two consecutive milkings and keeping a fixed ratio, were collected weekly. All milk analyses were performed in a pooled sample from four experimental weeks (weeks 3, 6, 9, and 12), including milk yield information. Fat-corrected milk (FCM, 3.5%) was calculated according to the method proposed by Sklan et al. [
23], wherein FCM = (0.432 + 0.1625 × percentage of fat) × kg of milk. Plastic bottles containing preservative potassium dichromate tablets were used for milk storage and transport to the laboratory. Milk chemical components were determined by infrared (Bentley 2000
®, Bentley Instruments Inc., Chaska, MN, USA), and SCC by flow cytometry (Somacount 300
®, Bentley Instruments Inc., Chaska, MN, USA). Milk vitamin E concentration was analyzed using high-performance liquid chromatography (HPLC) at the Institute of Food Technology [Instituto de Tecnologia de Alimentos (ITAL)] in Campinas, Brazil, according to [
24]. Briefly, 1 g of whey of milk was weighed into a test tube with 7.3 mL of the saponification solution (11%
v/
v KOH, 45%
v/
v H
2O, 55%
v/
v ETOH, and 0.25 g vitamin C/sample). Four milliliters of isooctane were added to each sample and the tubes were vortexed for 2 min to extract the vitamin E. The tubes were rested to separate the isooctane from the water, which was transferred to a vial for HPLC analysis. Milk Se concentration was analyzed using a wet digestion mixture with nitric-perchloric acid, and a fluorometric reading was subsequently taken, followed by diaminonaphthalene sensitization [
25] at the USP/FZEA Mineral Laboratory in Pirassununga, Brazil.
The milk relative density was determined using a hydrometer at 15 °C. The milk fat content was estimated by the Gerber method. The total dry extract (TDE) was estimated by gravimetry. The non-fat dry extract (NDE) was estimated by the following equation: NDE = %TDE − %fat. These analyses were carried out at the Clínica do Leite Laboratory, University of São Paulo, School of Agriculture Luiz de Queiroz (USP/ESALQ).
For milk fatty acid profile determination, fat extraction was performed by the method described in [
26]. Then, the separated fat (300–400 mg) was methylated and sterols were formed according to the method described in [
27], and fatty acids were quantified by gas chromatography (GC-2010 Plus, with on-column automatized injection, Shiamdzu Co., Kyoto, Japan) using a capillary column (100 m × 25 mm diameter, 0.02 mm thick; Suppelco, Bellfonte, PA, USA). Hydrogen was used as carrier gas at a flow rate of 40 cm/s, and the vaporizer and detector temperatures were 200 and 300 °C, respectively. The oven temperature increased from 70 °C to 230 °C at rates of 13, 4, and 7 °C/min for the three different phases of temperature raising. During the identification procedure, four standards were utilized: C4-C24 standard fatty acid (Supelco
® 37 Component FAME Mix, Sigma-Aldrich Co, St. Louis, MO, USA); C18:1
cis-vaccenic acid (V0384-1G SIGMA, Sigma-Aldrich Co, St. Louis, MO, USA); C18:2 10-
trans, 12-
cis methyl (UC-61-M, Nu-Check Prep, Inc., Elysian, MN, USA); and C18:2 9-
cis, 11-
trans methyl (UC-60-M, Nu-Check Prep, Inc., Elysian, MN, USA).
2.3. Blood Sampling and Analysis
On day 84, blood samples were collected by puncture of the coccygeal vessels. Samples were placed in vacutainer tubes without anticoagulant but with potassium EDTA (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The coagulated and EDTA were analyzed or processed within 1 h after collection. The blood samples from tubes without anticoagulant were centrifuged at 3000×
g for 15 min at 4 °C for serum separation and frozen at −80 °C for further analysis. White cell count was performed using a Neubauer chamber, utilizing a Thoma pipette after dilution to 1:20, using Türk solution. Serum concentration of vitamin E was determined according to the method described in [
24] using liquid chromatography. Serum Se concentration was assessed using a fluorometric method, as described by Olson et al. [
25]. Vitamin E analysis was performed at the Diagnostics and Clinical Analysis Laboratory in Pirassununga, and Se analysis was performed at the USP/FZEA Mineral Laboratory.
Blood serum was analyzed for glucose, total cholesterol, high- (HDL), low- (LDL), and very low-density lipoprotein (VLDL) cholesterol, and triglycerides using commercial kits (Laborlab®; Guarulhos, SP, Brazil) by an enzymatic-colorimetric method.
2.4. Statistical Analysis
All statistical analyses were performed in SAS version 9.4 SAS (SAS Institute Inc., Cary, NC, USA). All variables were analyzed using the MIXED procedure according to the following model:
where Y
ij = represents the observation for animal j in a given treatment i; µ = is the intercept; α
i = fixed effect of ith treatment (i = 1…4); β = linear regression coefficient of initial DMI of each cow; A
j = random effect of the jth animal (j = 1…24) ≈ N (0;σA
2); e
ij = random error associated with each observation ≈ N (0;σ
e2); N = Gaussian distribution; σA
2 = estimated variance associated with animals; and σ
e2 = estimated residual variance. Visual assessment of the distribution plots of the studentized residuals was used to confirm the normality of the distribution. As the values of SCC and white blood cell count did not present a normal distribution, they were transformed to log
e. Means were compared using a Bonferroni adjustment option. Means were obtained by Least Squares Means (LSMEANS). For all analyses, differences detected at
p < 0.05 were considered significant.