Effect of Parenteral Supplementation with a Mineral Compound of Cu, Zn, Mn on Bovine Mastitis

The objective of this investigation was to evaluate the effect of supplementing an injectable compound of Cu, Zn and Mn on bovine mastitis. The investigation was carried out in the dairy farms of San Quintín (Lat. 22.4070, Long. 80.0448) and Tres Caminos (Lat. 22.4019, Long. 80.0563) of “Desembarco de Granma”, Plant and Livestock Company, Yabu of Santa Clara during the dry period. The animals used were crossbreeds of Holstein and zebu, these animals were milked two times daily and fed with CT-115 and natural grasses. 5 mL of the injectable compound (50 mg Cu, 100 mg of Zn and 50 mg Mn) was administered parentally to the cows in production and the cows in the last trimester of gestation, with repetitions every two months until three applications were completed. In the animals treated the prevalence and indice of subclinical mastitis was reduced significantly (P< 0.05) from 67.9% to 40.3% y 1.48 to 0.81; 62% to 33.3% y 1.27 to 0.55, in San Quintín and in Tres Caminos respectively. The risk index was estimated and it demonstrated that in the group of cows which was given the mineral supplement was affected 0.34 times less with mastitis subclinical with each cow that was not supplemented. The incidence rate was evaluated and it was discovered that the group of animals exposed to the mineral supplement was 6.3%, meanwhile the animals not exposed to the mineral supplement was 18.5%. It can concluded that the supplementation of Cu, Zn and Mn via parenteral reduced the presentation of mastitis subclinical, demonstrating a protective effect to reduce the relative risk for this pathology.


Introduction
In milk production, the most important element is the cow. According Barlow, the term "mastitis" is a non-specific descriptor of the inflammatory changes in the mammary gland, for which there are numerous etiology. From a practical perspective, mastitis is the response of the gland to infection commonly acquired by invasion of pathogenic bacteria through the orifice and teat canal. This disease is an inflammatory process of the mammary gland that causes changes in the composition of milk, the secretion of the epithelial cells and reduced functional capacity of the udder [1]. It is the disease with the most prevalence in dairy herds [2], with a great economic impact on milk production [3]. Overall, between 15 and 40% of the milking cows have one or more mammary quarters with subclinical mastitis; while between 1 to 8% may be going through the stage of clinical mastitis [4].
For infection (mastitis) to occur in the udder many factors are involved. In some dairy herds in the incidence of mastitis may be greater due to certain nutritional deficiencies. If cows are not consuming or absorbing enough minerals and vitamins needed to have a functional immune system may have a higher incidence of mastitis. This could be due to a weakened immune system that cannot successfully fight bacteria that invade the udder [5]. O'Rourke, stated that there is a relationship between nutrition and mastitis in dairy cows and nutritional deficiency is linked to the quality of soil [6], in the tropics 66% of soils are found between very little productive and unproductive, with the following constraints: salinity and sodium 14.9%, 43.3% erosion, poor drainage 40.3%, 44.8% low fertility and low organic matter content of 69.6% [7].
Recent studies on brown soils with carbonates in animal units of the southern province of Villa Clara showed Cu deficiency in 100% of samples of soil and grass, 75% of serum samples and 72% of liver tissue [8]; deficiencies in this ecosystem of Zn and Mn were detected in the soil plant animal axis [9].
The main impact of nutrition on udder health is by suppressing the immune system. Cows with negative energy balance are at increased risk of developing ketosis, and clinical ketosis is associated with a twofold increase in the risk of clinical mastitis. The trace minerals and vitamins that can affect the health of the udder are selenium and vitamin E, copper, zinc, manganese and vitamin A and β-carotene.
Mastitis is a constant challenge for all dairy farmers. There is a positive genetic correlation between mastitis and milk production, as milk production increases, susceptibility to mastitis also increases. Mineral deficiency is rarely a primary cause of mastitis. Certain trace elements (Cu, Se, Zn, Co) and vitamins (E and A) are known to influence the immune response and its deficiency may be associated with a high incidence of clinical or subclinical infection.

General objective
To assess the effect of an injectable composite Cu, Zn and Mn, on mastitis in dairy cows.

Specific objectives
To evaluate the effect of supplementation of a mineral compound on the epizootic bovine mastitis structure before and during the administration of the compound.
To determine the prevalence of subclinical mastitis and infectious pathogens involved as causes of the disease, before and during the administration of the compound: Determining the level of subclinical mastitis before and during the administration of the compound.
Determine cumulative incidence and incidence rate of subclinical mastitis in the course of the investigation.
Evaluate the economic feasibility of the results obtained in the investigation.

Materials and Methods
The research was conducted in the San Quintín (Lat 22.4070, Long. 80.0448) and Tres Caminos (Lat 22.4019, Long. 80.0563) dairy farms of Plant and Animal Production company "EL YABU" of the "Desembarco de Granma" branch in Santa Clara, Villa Clara, Cuba during the dry period.
The animals are crossbred Holstein and Zebu undergoing double milking which takes place in the early morning hours (2:30 am) and evening (2.30 pm) and a feeding system using CT-115, natural pasture grasses, supplementation of cane, commercial feed (north gold), common salt and urea. A parenteral doses of 5 mL of injectable compound (50 mg Cu, 100 mg Zn and 50 mg Mn) was administered to the cows in production and those in the last trimester of pregnancy in the farms under study, repeating the injection every two months to complete three applications, monitoring and evaluation of variables to investigate (before an during) the application of the compound in the cows are and those which were incorporated.
Total prevalence of intra-mammary infection.

Subclinical Mastitis index (IMSC).
Economic analysis of losses by the prevalence of subclinical bovine mastitis.

Mathematical design and statistical analysis
Prevalence: An observational study with a transversal design was done for processing of electronic data tab by using Microsoft -Excel to determine the prevalence of different clinical forms of mastitis and infections caused by microorganisms that produces the disease, such epziootiological indicators are generally determined by the following expression: Prevalence=Total of positive quarters/Total of quarters exposed to the risk of infection × 100

Subclinical Mastitis Index (SCMI)
To calculate the SCMI: which considers all cows that are lactating and excludes the teats having clinical mastitis during the time of evaluation and the blind or dysfunctional teats, to determine the subclinical mastitis index (SCMI), values are assigned following, according to the results: To calculate the index of subclinical mastitis the following formula was used:

Statistical analysis
Relative risk (RR) was estimated by forming contingency tables 2 × 2 [14], applying a prospective observational cohort study of the analytical type, to determine cumulative incidence and incidence rate, with the following scheme (Table 1): Further analysis of comparing proportions between the different samples with different forms of presentations of the disease was done, using the statistical package STAGRAPHICS ® -Centurion XV, version 6.0 for Windows.
The processing of the data was done using the program for epidemiological analysis of tabulated data EPIDAT. Version 3.1. (Contingency test, type of study: Cohort) to assess the cumulative incidence and incidence rate.

Economic analysis
To determine the loss of production the relationship between the degree of positive reaction and production losses was taken into consideration; Trace (8%); Weak positive (9 to 18%); Positive (19 to 25%) and Strong positive (over 25%), where losses due to grade of reaction to the detection of the test (CMT) was calculated, which was estimated the losses from the percent that is not produced from the actual production from the beginning of the investigation [15]. Tables 2 and 3 shows the results of the epizootic characterization of the dairy farm Tres Caminos, where a prevalence of 62% bovine clinical mastitis was found before the application of the injectable compound and a prevalence of detecting pathogens 90%, while for the dairy farm San Quintín was 67.9% and 45% respectively (Figure 1), in      A study of the tendency in relation to the percent of the prevalence and the period of application of the injectable compound showed that the cows that were exposed to the mineral compound for a longer period, the prevalence of subclinical mastitis decreased (Figures 2 and  3).    [16] reported index values of 1.42 and 1.62 respectively as results in other studies, then 1.27 to 1.48 before application of the mineral compound, achieving a reduction in the indices to 0.81 and 0.55, respectively, considering them as desirable indices according to Castle et al. [17]. These results may be influenced by the prevalence of pathogens isolated Staphylococcus aureus and Staphylococcus coagulase negative (25 and 27%) respectively in the unit of San Quíntin, unlike Tres Caminos where the overall prevalence is greater than 100% in the isolates, with 37% and 47% (Streptococcus agalactiae and others). When we estimated the risk index (hazard ratio) in the animals evaluated according to the presence or absence of the parenteral supplementation with the mineral compound (Table 4) and taking into account the odds ratio, it was demonstrated, that in the group of cows where the mineral compound was applied, cows were affected 0.34 times less with subclinical mastitis per cow that was not supplemented with the mineral compound; this association is significant (CI. 95% [0.27, 0.23] and statistically significant (p ≤ 0.05). According to Novoa et al. undernourishment is a risk factor in the health of the udder; with decreased intake of natural pasture grasses in quantity and quality, characteristic dunging the dry season rain, causes a low intake of Cu, Zn, Mn, Se and Vitamin E to body, compounded by the absence of supplementation of these minerals, which act on the immune function, so that its administration is associated with a better activation of circulating white blood cells in response to microbial invasion, destruction of bacteria and maintaining a low rate of colony forming units, short duration of clinical signs and reducing the incidence of environmental mastitis, thus faster removal of udder infections. The prevalence of subclinical mastitis episodes in cows in the control group was 65%, while in cows supplemented with the mineral compound were affected only by 38%. As a result of this association, it was determined that the attributable fraction in cows exposed is 0.40, i.e., if these cows had been supplemented with mineral complex could have prevented the disease in 40% of the cows in this group and 35% in the entire population (attributable fraction in population of 0.35).  Table 4: Study of the relative risk regarding the supplementation or not of the mineral compound and prevalence of subclinical bovine mastitis. AFE=attributable fraction in exposed AFP=attributable fraction in the population.

Results and Discussion
For the assessment of the study of the incidence rate ( Figure 5), we can say that the group of animals exposed the incidence was 6.3%, and for the unexposed 18.5%. In all cases the confidence intervals (CI) is less than (1), of which there was association, thus existing statistically significant differences (p ≤ 0.05), we can infer that the application of the mineral compound exerts a protective effect on incidence of bovine mastitis, considering the application of injectable compound, a factor for the prevention of this disease. Note that the incidence of bovine mastitis, affects not only paying for quality, but a considerable amount is lost in individual production, such as the high prevalence in cows affected with subclinical bovine mastitis in our dairy and causes greater economic losses in dairy herds specialized herds, as is the case of our research, where according to Hernández et al. [15] there is a relationship between positive degrees of infection and losses in production (Trace (8%), weak positive (9 to 18%), positive (19 to 25%) and strong positive (over 25%), where losses were calculated for the degrees of reaction to the detection of the test (CMT), which stresses that losses from the percent that is left to produce in a day before the application of mineral compound is 73.22 and 46.78 liters of milk, which is making a conservative estimate; would be losing 2197 liters in San Quíntin and 1403 liters of milk in Tres Caminos monthly ( Figure 6). This would represent a refund for our economy $ 2,040, knowing the price of powder milk in the international market 5100 USD/MT according to FAO, for the purchase of 400 Kg milk powder to restore the alleged losses, the concept of reduced production, if we apply the mineral compound it can reduce losses by more than 50% of the initial losses [18]. The study has enabled the production of a product in quantity and best quality for obtained for every peso invested 446 pesos was gained (1:446), giving the measure of efficiency for the 6 months of research. According to Ponce et al. mammary gland can resist most infections if they are provided with the nutrients necessary to maintain this resistance to new infections by acting as a prophylactic measure against bovine mastitis [19]. The important minerals and vitamins are Selenium, Copper, Zinc, Manganese, Vitamins A and E. When these are not provided in adequate amounts, the rate of new infections may increase. According to Garcia in animal units in the central province of Villa Clara deficiencies Cu, Zn and Mn were detected in the soil -plant -animal axis, therefore the mineral supplementation is necessary to decrease the incidence rate mastitis [8,9].
According to the results, it is evident that the mineral complex has a positive effect on bovine subclinical mastitis in the two farms, saying there was a significant decrease in the prevalence of bovine mastitis when prevalence is compared before and during mineral supplementation. Before supplementation the prevalence of subclinical mastitis was 67.9% in San Quíntin and 62% in Tres Caminos and in the last application of the compound (sample IV), the prevalence decreased to 40.3% in San Quíntin and 33.3% in Tres Caminos. It is shown that these trace minerals are beneficial in increasing the immune response in dairy cows (Berger) and according to Scaletti et al. at 34 days of lactation decreases the severity of intramammary infection and cell counting somatic (CCS) supplementing the diet with copper.
The strong positive relationship between the state of trace minerals and the immune response is well known. Research showed a correlation between the Cu, Zn, Mn, and is in the blood and reduced the incidence of bovine mastitis. Recent research from the University of Missouri showed that organic zinc supplementation increases the synthesis of keratin in the teat, which provides a physical barrier to bacteria. This physical barrier of keratin is the first line of defense before the entry of bacteria into the udder, thus acting as a bactericide in the teat canal, thus decreasing the incidence of bovine mastitis and CCS. Copper and manganese also play a key role in immune function. The increase in trace elements in dairy cows with the application of an injectable product could increase immune function, right after birth and therefore reduce mastitis infections [20]. However keep in mind that in addition to inadequate nutrition are other factors that increase the incidence of bovine mastitis, the main ones are; correct milking procedure, proper hygiene and injury to the mammary gland.

Conclusion
According to the results obtained and the literature consulted: In the treated animals the prevalence rate of subclinical mastitis was significantly decreased in the farms under study.
It was shown that the risk index in the group of cows that the mineral compound was applied, were less affected with subclinical mastitis.
Supplementation of trace minerals complex had a protective effect in reducing the relative risk for this disease.
Reducing the prevalence and incidence of bovine mastitis also reduced economic and production losses.
For every Cuban Pesos invested 446 Cuban Pesos was gained.

Recommendations
Continue strategic studies with supplementation of these minerals. Include in the mineral compound selenium and vitamin E.