Adequate mineral nutrition may be used as a strategy to optimize immune system function by the reduction of metabolic and oxidative stress; and therefore it may have a positive effect on the defense mechanisms of mammary gland against mastitis (Weiss and Wyatt, 2002). Zn, Cu and Se supplementation has been associated with higher antioxidant capacity of superoxide dismutase (CuZnSOD), glutathione peroxidase (GSH-Px) and serum ceruloplasmin (CP) respectively, resulting in reduced somatic cell count (SCC) (Weiss and Wyatt, 2002).
Free radicals are produced as a result of pathogen phagocytosis when mastitis occurs, which may result in lesion of mammary epithelial cell and decreased milk secretion (Barbano et al., 2006).
Organic mineral sources (complexes, proteinates and amino acid chelates) have been reported to have higher bioavailability than inorganic minerals. However, few studies with conflicting results have been published showing their influence on mammary gland health and on milk production (Ashmead and Samford, 2004).
Siciliano-Jones et al. (2008) supplemented dairy cows with organic sources of Zn, Mn, Cu and Co, and reported no changes in milk composition and SCC. Griffiths et al. (2007) studied Zn, Mn and Cu supplementation as an amino acid complex for grazing dairy cows, reported increased milk production, but no differences on SCC and milk composition was observed. In contrast, Kinal et al. (2007) observed a reduction in SCC with organic sources of Zn, Mn and Cu supplementation for dairy cows during 305 days of lactation.
The objectives of this study were to evaluate the effects of dietary organic sources of Zn, Cu and Se for dairy cows on SCC, occurrence of subclinical mastitis, number of clinical mastitis cases and the concentration of CuZnSOD, GSH-Px and CP.
2. Materials and methods
2.1. Experimental design and diets
The experimental design was completely randomized with two treatments and repeated measures during the experimental period. Nineteen clinically healthy dairy cows, six primiparous (614±68 kg of body weight, BW), and 13 multiparous (647±60 kg of BW), pregnant (7 months of gestation), were selected for this study. Cows were paired according to parity, body weight, body condition score and milk yield in the previous lactation for multiparous animals. Cows were housed in individual pens and randomly assigned to one of the two groups, one to receive organic and the other inorganic source of Zn, Cu and Se, during 60 days prior to the expected calving date (-60 days) up to 80 days of lactation.
All animals received the same basal diet, formulated tomeet the recommendations of NRC (2001) (Table 1), according to their stage of pregnancy and lactation: a) after drying off, 60 to 29 days before the expected date of calving; b) pre-partum, 28 days before the expected date of calving until the day of parturition; c) lactation, 1 to 80 days of lactation.
The treatments consisted of providing same amounts of a mixture of micromineral composed of Zn, Cu and Se as carboaminochelates (Tortuga, Companhia Zootecnica Agrária, São Paulo, SP, Brazil) or inorganic (sulfates) form. The total amount of microminerals provided for both groups was administered intraesophageally, twice daily. Cows were fed ad libitum twice daily as total mixed ration (TMR), and consumption was adjusted daily to allow 5% of feed refusal.
After the start of lactation, the animals were milked twice a day and milk production was recorded daily.
2.2. Sampling and laboratory analysis
Blood samples were collected at -60, -21, 1, 21, 40 and 80 days in relation to the expected calving date for the analysis of CuZnSOD, GSH-Px and CP concentration.
GSH-Px was determined by the methodology proposed by Paglia and Valentine (1967), using a commercial kit (Ransel, Randox Laboratories, UK). Similarly, CuZnSOD was determined with a commercial kit (Ransod, Randox Laboratories, UK) by inhibition of a formazan-producing colorimetric reaction (Suttle and McMurray, 1983). As the results were expressed in units per gram of hemoglobin for both CuZnSOD and GSH-Px, hemoglobin concentration was determined by spectrophotometry using a commercial kit for hemoglobin (Labtest Diagnostica, Minas Gerais, Brazil), by the method of hemoglobin cyanide. CP was determined by spectrophotometry according to the methodology proposed by Schoslnsky et al. (1974).
Milk samples were collected weekly after 15 days of the beginning of lactation for SCC determination. The content of somatic cells was determined by the fluoro-opto- lectronic cell counting method (Somacount 300, Bentley, Instrument Inc., Chaska, MN, USA), and the results were converted to log scale (Log SCC) for statistical analysis.
A clinical case of mastitis was defined by the presence of abnormality in the milk suggestive of mammary gland inflammation, such as flakes, clots, andwatery or other unusual appearance. Detection of clinical mastitis was done at every milking.
A cow was considered to be subclinically infected when clinical signs were not present and SCC level was greater than the threshold value of 200,000 cells/ml, with or without positive isolation of the udder pathogen. The number of infected cows was then summed to obtain the number of subclinical mastitis cases for each cow during the experimental period. A variable called subclinical mastitis case was created to describe the udder health of cows during each week over the test period. A new infection case was defined as a change in state from a SCC<200,000 cells/ml to a SCC>200,000 cells/ml. A minimum of one SCC-negative test was needed to a new infection case to be considered.
For microbiological culture, milk samples were collected at day 1 and 7 after parturition, when a clinical case was diagnosed, and when a mammary quarter was positive on CMT (California mastitis test), which was performed weekly during lactation. Bacteriological examination of milk samples was performed as recommended by National Mastitis Council (NMC, 1999). Samples yielding more than one species of bacteria were excluded from data analysis.
2.3. Statistical analysis
Statistical analysis of SCC and CuZnSOD, GSH-Px, and CP concentration was performed considering the main effect of treatment, time and interaction between treatment and time in a completely randomized design, with repeated measurements over time, by PROC MIXED of SAS software (SAS, 1999). A logarithmic (log 10) transformation was used on SCC, CuZnSOD and GSH-Px to normalize the distribution of these data. The association among the number of subclinical mastitis cases, new infection cases, and treatment was analyzed by PROC GENMOD of SAS software (SAS, 1999).
3. Results and discussion
3.1. Udder health
The number of new and total subclinical mastitis cases was lower in animals fed with organic sources of Zn, Cu and Se in comparison to the group fed with inorganic sources of these microminerals (Table 2). These results may indicate an increase in immune response capacity that resulted in improved mammary gland health, for cows fed with organic sources of Zn, Cu and Se. Few studies evaluated the effect of different sources of microminerals supplementation on the number of clinical and subclinical cases, levels of infection and causative agents of mastitis. Whitaker et al. (1997) studied organic Zn supplied to Holstein cows, from 3 weeks pre-partum to 100 days of lactation, observed no significant difference in the number of clinical and subclinical mastitis cases, new infections, and type of isolated microorganism and SCC.
Average SCC during the first 80 days of lactation had a tendency (P=0.056) to be lower for the group fed with organic Zn, Cu and Se (Table 3). Average SCC for cows in the inorganic group (237,370 cells/ml) was 4.2 times higher than the average in the organic group (55,579 cells/ml). Studies regarding the effect of supplemental organic microminerals sources on SCC, milk yield and composition have shown variable results. Some authors (Griffiths et al., 2007; Ballantine et al., 2002; Siciliano- ones et al., 2008; Campbell et al., 1999; Uchida et al., 2001; Nocek et al., 2006) reported no effect of different organic microminerals sources supplementation for dairy cows on somatic cell count. Other authors (Kinal et al., 2007; Cunha Filho et al., 2007; Pechova et al., 2006) reported a decrease in SCC with different sources of microminerals. Kinal et al. (2007) associated the reduction of SCC with quick formation of keratin in teat canal provided by the supplementation of organic Zn.
3.2. Concentration of GSH-Px, CuZnSOD and CP
No effects of Zn, Cu and Se supplementation, as organic source, were observed on CuZnSOD, GSH-Px and CP concentrations (Table 3). Similarly to results were found in the present study. Gunter et al. (2003) did not observe an effect of organic Se supplied to beef cows on GSH-Px. Du et al. (1996) also reported no effect of the micromineral source on plasma concentration of CP in dairy cows supplemented with organic Cu (proteinate) for 60 days. Knowles et al. (1999) observed an increase in total serum glutathione concentration in dairy cows with Se supplementation compared to animals without supplementation, but no effect of organic source of Se was observed on glutathione and SCC.
No significant interaction was found between time and treatment on CuZnSOD, GSH-Px and CP, and no effect of time was observed on GSH-Px and CP. However, a positive linear effect of time on CuZnSOD concentration was observed from 60 days pre-partum up to 80 days of lactation.
Under the conditions of this study, the supplementation of organic microminerals (Zn, Cu and Se) had no effect on antioxidant enzyme system as measured by the concentration of CuZnSOD, GSH-Px and CP. But, it was effective on reducing the occurrence of subclinical mastitis cases and SCC. The organic source of microminerals may be implicated to other mammary gland defense mechanisms, in addition to antioxidant mechanism.
Based on the results of this study it may be concluded that feeding organic source of Zn, Cu and Se had a tendency to reduce SCC during the first 80 days of lactation, and a reduction on the number of subclinical mastitis cases, but did not alter the concentration of serum superoxide dismutase, glutathione peroxidase and ceruloplasmin.
The authors are grateful to Antonio Carlos da Silva Bueno, Gilmar Edson Botteon, José Garcia Moreno Franchini, Lucinéia Mestieri, and Clara Satsuki Mori for technical assistance in accomplishing this research project.
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This article was originally published in a journal published by Elsevier in September 2009.