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Cuban Journal of Agricultural Science

versión impresa ISSN 0864-0408versión On-line ISSN 2079-3480

Cuban J. Agric. Sci. vol.50 no.3 Mayabeque jul.-set. 2016


Cuban Journal of Agricultural Science, 50(3): 371-380, 2016, ISSN: 2079-3480




Effect of different doses of an injectable compound of Cu, Zn and Mn on bio-productive indicators of milking cows


Efecto de diferentes dosis de un compuesto inyectable de Cu, Zn y Mn en parámetros bioproductivos de la vaca lechera



E. Noval,I J. Ramón,I R. García-López,II J. Jiménez,III

IDepartamento de Medicina Veterinaria y Zootecnia. Facultad de Ciencias Agropecuarias. Universidad Central de las Villas “Marta Abreu”. Carretera a Camajuaní, km 5,5. Santa Clara (54830), Villa Clara, Cuba.
IIInstituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque.
IIIUnidad Básica de Producción Agropecuaria “Desembarco del Granma”. Antón Díaz, Santa Clara. Villa Clara.




Different doses of an injectable compound of Cu, Zn and Mn on bio-productive indicators of milking cows. Four groups were formed, with seven cows each, belonging to Siboney de Cuba (5/8 Holstein 3/8 Zebu) breed genotype, with eight months of gestation. Each group was assigned with the following treatments: A) control; B) 25 mg of Cu, 50 mg of Zn and 25 of Mn; C) 50 mg of Cu, 100 mg of Zn and 50 mg of Mn and D) 75 mg of Cu, 150 mg of Zn and 75 mg of Mn, provided every two months up to completing three applications. Serum concentrations of Cu and Zn and bio-productive indicators were evaluated. Two groups with five cows were formed: one of them received C treatment and the other, used as control, was destined to evaluate hepatic reserves of Cu, Zn and Mn. C and D treatments increased significantly (P< 0.05) serum concentrations of Cu and Zn regarding the rest of them. C treatment increased significantly (P< 0.05) the hepatic levels of Zn and Cu, and Mn (P < 0.001). Reproductive indicators were better (P<0.05) in cows that received C and D treatments, showing the highest levels of milk production (P< 0.05), after 60 d of lactation, regarding A and B groups. It can be concluded that C and D treatment were more effective to reestablish the organic levels of these microelements and favor productive and reproductive indicators of animals.     

Key words: copper, zinc, manganese, doses, injectable, indicator, milking cows.


Se evaluaron diferentes dosis de un compuesto inyectable de Cu, Zn y Mn en los parámetros bioproductivos de la vaca lechera. Se formaron cuatro grupos, de siete vacas cada uno, pertenecientes al genotipo racial Siboney de Cuba (5/8 Holstein 3/8 Cebú), con ocho meses de gestación. A cada grupo se le asignaron los tratamientos siguientes: A) control; B) 25 mg de Cu, 50 mg de Zn y 25 de Mn; C) 50 mg de Cu, 100 mg de Zn y 50 mg de Mn y D) 75 mg de Cu, 150 mg de Zn y 75 mg de Mn, administrados cada dos meses hasta completar tres aplicaciones. Se evaluaron las concentraciones séricas de Cu y Zn y los indicadores bioproductivos. Se conformaron dos grupos de cinco vacas: uno recibió el tratamiento C y otro, utilizado como control, se destinó a evaluar las reservas hepáticas de Cu, Zn y Mn. Los tratamientos C y D aumentaron significativamente (P< 0.05) las concentraciones séricas de Cu y Zn con respectos a los restantes. El tratamiento C incrementó significativamente (P< 0.05) los niveles hepáticos de Zn y Cu y Mn (P < 0.001). Los indicadores reproductivos fueron mejores (P< 0.05) en las vacas que recibieron los tratamientos C y D, que mostraron mayores niveles de producción láctea (P< 0.05), a partir de los 60 d de lactancia, con respecto a los grupos A y B. Se concluye que los tratamientos C y D resultaron más eficaces para restablecer los niveles orgánicos de estos microelementos y favorecer los indicadores reproductivos y productivos de los animales.

Palabras clave: cobre, zinc, manganeso, dosis, inyectable, parámetros, vaca lechera.




Bovine milk production in Cuba is developed under grazing conditions, with climatic and management conditions that are negative and affect the intake of coarse food with low level of yield and quality. During dry period, nutritional deficit is intensified. Mineral supplementation is poor and lacking of trace minerals (García et al. 2010a).

Feeds for bovine cattle not always fulfill the requirements of milking cows for production and, many times, the contribution of trace minerals is unknown (Genther and Hansen 2014). Zn, Cu and Mn are affected due to the antagonism of Mo, Fe, S, and Ca concentrations, among other minerals supplied by feeds (Nayeri et al. 2014). 

In Cuba, there are severe deficiencies of Cu and Zn in milking cows, in the western (Valera et al. 2011, Figueredo 2016),  central (García et al. 2010a, Noval et al. 2014) and eastern (Fajardo et al. 2011, Galindo et al. 2014) regions.

Mineral supplementation in milking cows have a beneficial effect on the immune condition of the animal, resistance to diseases, feed intake (McDowell and Arthington 2005), energy and oxidative metabolism (Spears and Weiss 2008) and reproduction (Nemec et al. 2012).

Zn, Cu and Mn have an important function on fertility of milking cows (Nayeri et al. 2014). In Cuba, a negative correlation was demonstrated (p< 0.05) between cupremia and reproductive indicators, in a range of serum copper between 9.8 ± 1.0 and 14.0 ± 0.9 μmol/L. It was also confirmed that deficiency of this microelement is a significant risk factor for the appearance of anestrus and repetition of service (García et al. 2010b).

Parental supplementation of minerals reduces the interactions occurring during its ingestion and absorption, avoids mineral antagonism and increases organic reserves of the animal (Pogge et al. 2012). Injectable supply of Cu every two months, up to completing three applications, increased cupremia and hepatic reserve, and benefited reproductive performance of bovine females (García et al. 2012a, b).

The objective of this study was to evaluate different doses of an injectable compound of Cu, Zn and Mn on bio-productive indicators of milking cows.



This research was carried out during dry period (November-May) of 2011, in a milking herd from a Unidad Básica de Producción Cooperativa (UBPC) from the central region of Cuba, with a predominance of typical carbonated brown soils (Hernández et al. 2015).

Animals under study were grazing in a rotational system of natural grasses during the day. At night, they were grazing in areas of Pennisetum purpureum cv. Cuba CT-115, associated with Neonotonia wightii and Teramnus labialis.

Supplementation was accomplished according to DDGs productive level, at a rate of 2.4 kg DM and urea (0.08 kg DM). Cows were manually milked, at double milking and with a restrained suckling system.

Experimental design. The effect of supplementation with Cu, Zn and Mn on blood serum was evaluated, as well as the productive and reproductive response of cows. An amount of 28 crossbred Holstein x Zebu cows, with eight months of gestation, between six and seven years old, in the  third and fourth gestation, was used. Body condition (BC) was between three and four at a scale of five points (Parker 1989), with 11.8 ± 1.2 L cow-1 day-1 as mean of previous milk production.

Four groups of seven animals were established, randomly distributed. Treatments consisted on a control group, which had no parenteral supplementation of the mineral elements of this study. The other three groups received a supplementation of 25, 50, 25; 50, 100, 50 and 75, 150, 75 mg of Cu, Zn and Mn, respectively.

All the animals showed serum deficiency of Cu and Zn (concentrations inferior to 11.77 and 12.62 µmol/L, respectively). From a clinical point of view, they were healthy according to a previous diagnose (Sotolongo 2007). The lack of Mn, diagnosed in herds, was determined by its deficiency on soil and grass (Noval et al. 2014).

The first supply of micro-elements was performed at eight months of gestation and it was repeated every two months up to completing three applications (30 and 90 d after parturition). Every animal underwent a dynamics of cupremia and zinc supplementation, before and every 15 d after the supply of treatments for two months. The maximum serum concentration of Cu and Zn was determined, as well as the time of declination of these values. At 60 d, a final sampling was performed to determine the appearance or not of hypo-cupremia and hypo-zinc supplementation during this period.

From the results of this experiment, two experimental groups were established, with five cows each. One was used as control and the other was parenterally supplemented with C dose. Animal selection was performed according to previously described inclusion criteria, although they were animals destined to sacrifice due to their low milk production. The influence of the supply of these microelements on hepatic reserves was evaluated. Animals were slaughtered at 15 d after the application of the product, which coincided with the peak of cupremia and zinc supplementation, determined during the previous stage.

Sample taking, used procedures and techniques. Through jugular venipuncture, 10 mL of blood were taken, before and every fifteen days after treatment. These samples were located in sterilized and demineralized anticoagulant tubes. Later, they were centrifuged at 3,500 rpm for 10 min., obtaining the blood serum. Liver samples were taken during the 15 min. after the animal slaughter. Samples from the ventral edge of the right lobule (15 cm3) were obtained, with a previous general inspection of this viscera in order to discard any alteration.

Samples of blood serum and hepatic tissue were preserved at -10 ºC up to the analysis. Determinations of Cu, Zn and Mn were performed by atomic absorption spectrophotometry (Miles et al. 2001), with a SP-9 equipment, PYE UNICAM brand, according to procedures of manufacturer.

Calculation of reproductive indicators. The main reproductive indicators (parturition-first insemination period (PFIP), service period (SP), parturition-parturition period (PPP), insemination index (II) and percentage of gestation at first service) were determined according to procedures recommended by Brito (2010). In addition, milk production of each animal was determined during the first 240 d of lactation.

Milk weighing. Weighing of individual milk production per month was carried out by means of a certified scale, dynamometer type and graduated in kilograms.

Statistical procedure. In order to compare concentrations of Cu and Zn in blood serum among the different groups in each sampling, reproductive indicators (PFIP, SP, PPP and II) and milk production in each weighing, an analysis of variance of simple classification (ANOVA) was performed. Duncan (1955) test was used to compare means in the cases with significant differences, with a previous verification of variance homogeneity. Comparison of hepatic reserves of microelements was conducted through t-Student test for independent samples. Statgraphics Centurion (StatPoint Technologies 2010) program was used for each procedure.



Table 1 shows results of parenteral supplementation of the different studied doses on Cu levels in blood. At the beginning of this research (day 0), there were no significant differences among the four groups. However, levels of cupremia were significantly superior (P< 0.001) in treatments with 50, 100, 50 and 75, 150, 75 mg of Cu, Zn and Mn respectively, at 15, 30 and 45 d, regarding the control group and the treatment with 25, 50 and 25 mg of Cu, Zn and Mn, respectively. At 60 d, there were no differences (P > 0.05) in cupremia among the groups supplemented with 25, 50, 25; 50, 100 and 50; 75, 150 and 75 mg of and Mn, respectively. The same happened between control and treatment with 25, 50 and 25 mg de Cu, Zn and Mn.

Cu blood levels, for C and D treatments, reached standard physiological patterns for bovine species (McDowell and Arthington 2005) and they maintained for 60 d, moment in which they began to decrease under the critical limit of deficiency, suggested by McDowell and Arthington (2005). 

These results are similar to those obtained by García et al. (2012a), who increased cupremia and reached its peak at five days after treatment, when it is stabilized up to 15 d, and began to have a significant decrease and hypo-cupremia appears (< 11.77 µmol/L) at 60 d.

García et al. (2012a) conducted determinations of serum Cu every five days, which may be the cause for the non-coincidence of cupremia peak regarding the results of this study. Another aspect that could have influenced is that those authors provided Cu alone, and this study supplemented it combined with Zn and Mn.

Table 2 shows blood levels of Zn at different moments, before and after the parenteral application of mineral compounds. Before the supplementation (day 0) and at 60 d, there were no differences (P>0.05) among treatments. However, treatments with 50, 100, 50 and 75, 150, 75 mg of Cu, Zn and Mn, respectively, were superior to control group and to the one of lowest concentration of minerals under study at 15, 30 and 45 d. Values of zinc supplementation were superior to standard (12.62 µmol/L) physiological values (McDowell and Arthington 2005).

 In Angus and Simental cattle, with parenteral supplementation of Cu, Zn, and Mn, cupremia tended to increase. Serum concentrations of Zn and Mn had a significant increase at 15 d after treatment regarding control, while Mn tended to be superior (Pogge et al. 2012).

Concentration of microelements on the hepatic tissue (table 3) had statistical differences on Zn (P<0.05), Cu and Mn (P<0.001) levels, favoring the animals treated with the injectable compound of these microelements. In all cases, their hepatic concentrations reached superior values to those of the physiological limits for bovine species, reported by García et al. (2012a), after studying Cu supplementation on milking cows.

Liver Zn and Cu concentrations of this research are similar to other publications, pointing out that hepatic Zn ranges between 100 and 400 mg/kg on dry basis, and Cu varies from 200 to 300 mg/kg. Concentrations become toxic when their levels on this organ are superior to 900 mg/kg on dry basis (McDowell and Arthington 2005).

In this study, hepatic levels of Mn are similar to those reported by NRC (2001), and range between 10 and 24 mg/kg on dry basis. In addition, liver Mn concentrations are directly proportional to those of this mineral on the consumed feed.

C treatment (50 mg of Cu, 100 mg of Zn and 50 mg of Mn) was used for evaluating concentration of those mineral on hepatic tissue because serum concentrations of Cu and Zn of animals supplemented with this treatment differed significantly from the group of cows treated with D dose. In addition, in C treatment, a lower concentration of microelements under study was used, so it was more economic.

Determination of hepatic concentrations is the most precise method for evaluating organic reserves of these minerals, especially Cu and Zn, and also for determining the efficiency of the supplementation because the liver is the reserve organ and it is the accurate indicator of the state of these minerals in the organism (Rosa et al. 2008).     

Reproductive performance of these animals was favored by treatments with 50, 100, 50; 75, 150, 75 mg of Cu, Zn and Mn, respectively (table 4). In these treatments, there was a significant (P < 0.001) reduction of the PFIP, SP, PPP and II, regarding the group control and that of lowest concentration.

Supplementation of a mineral compound of Zn, Cu, Mn and Co glucoheptonate reduced parturition-first insemination periods and parturition-gestation in 12 and 62 d, respectively, in milking cows, compared with the group that received no treatment. In the supplemented animals, conception services were 1.80 and, in control group, 2.60, as reported by Uchida et al. (2001).

 According to reports of Campbell et al. (1999), in an experiment in which inorganic and organic minerals were supplemented to cows with voluntary waiting periods of 60 d, there were no differences in days at the first service, parturition-conception periods and first service to gestation, and services per gestation among animals, according to the mineral source.

In Cuba, injectable Cu supplementation favored reproductive performance of animals with hypo-cupremic and normal-cupremic animals, with cupremia next to 14 µmol/L / L (Garcia et al. 2012b). Parenteral supply of 50 mg Cu in female bovines at eight months of gestation, without knowing the state of serum Cu, favored postpartum reproductive performance (Figueredo 2016).

Milk production means of groups supplemented with 50, 100, 50; 75, 150, 75 mg  of Cu, Zn and Mn, respectively, had no significant differences. However, there were differences regarding control group and that of lower concentration of Cu, Zn and Mn (figure 1).

Significant increase of milk production of groups supplemented with 50,  100, 50; 75, 150, 75 mg of Cu, Zn and Mn, respectively, regarding control group and that of the lowest applied doses,  could be favored by the significant increase provoked by supplementation to blood and hepatic concentrations of Cu (table 1) and Zn (table 2).

Rises of milk production increased mineral requirements and marginal deficiencies of microelements, decreased productive levels and affected the functioning of many enzymes and proteins, involving different physiological, biochemical and metabolic processes, as well as the functioning of immune system, contributing to decrease productive potential and defenses of the organism (McDowell & Arthington 2005).

Cu requirements during lactation are 0.15 mg/kg of milk produced during early lactation (< 100 days). Levels of 0.5 mg of Cu are introduced to fetus, placenta and uterine tissues every day, increasing the requirements from 1.5 to 2 mg/day during the last month of gestation. Mn requirements during gestation grow up to 0.3 mg/day at the end of it, and its concentrations are 0.16 mg/kg in colostrum and 0.03 mg/kg in milk. Those of Zn, 12 mg/day are retained during gestation up to 190 days and at the end of it are duplicated (ARC & CAB 1980). Zn content in milk is 4 mg/kg, ranging from 3.4 to 5.8 mg/kg (NRC 2001).

Therefore, transition period increases Cu, Zn and Mn requirements in milking cows and it is the most adequate moment for its supplementation. Similar results to those of this experiment were obtained in the supply of 50 mg of Cu, parenterally, in female bovines at eight months of gestation. It produced an increase of cupremia, immunoglobulin, decreases mastitis and favored postpartum reproductive performance (Figueredo 2016).

Oral mineral supplementation to milking cows, three weeks before parturition, increase milk production in the moment of lactation peak (Kincaid & Socha 2004), between 2.9 and 3.2 % in the thirteenth week (Siciliano-Jones et al. 2008) and in the fourteenth week (Hackbart et al. 2010).

Supplementing an amino acid complex including Zn, Cu and Mn favors dry matter intake and the functioning of many metalloenzymes like Cu-Zn- superoxide dismutase, carbonic anhydrase, alcohol dehydrogenase, carboxypeptidase, alkaline phosphatase and RNA polymerase, which act on metabolism of carbohydrates, proteins, lipids and nucleic acids, improving the performance of milking cows (NRC 2001).

Benefits of organic supplements of Cu, Zn and Mn include prevention of mastitis, improvement of fertility and reduction of incidences of podal injuries (Uchida et al. 2001, Kellogg et al. 2003, 2004), digestibility of nutrients and udder health (Machado et al. 2014).

Zn-deficient animals have a low intake and deficient use of nutrients (McDowell & Arthington 2005). In this sense, Rosa et al. (2008) state that even with initial symptoms of Zn deficiency, there is a reduction of food efficiency and low production and secretion of hormones.

It can be concluded that parenteral supplementation of 50 mg of Cu, 100 mg of Zn and 50 mg of Mn in milking cows from 8 months of gestation was the most efficient to reestablish organic levels of these microelements and favors reproductive and productive performance of treated animals.



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Received: 13/3/2015
Accepted: 7/6/2016



E. Noval, Departamento de Medicina Veterinaria y Zootecnia. Facultad de Ciencias Agropecuarias. Universidad Central de las Villas “Marta Abreu”. Carretera a Camajuaní, km 5,5. Santa Clara (54830), Villa Clara, Cuba. Email:

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