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The effect of injectable copper, selenium, zinc, and manganese on cattle vaccine response and conception in Australia

Matthew Ball (Virbac Australia), Luc Durel (Virbac France), and Jerry Liu (Virbac Australia)

Posted Flock & Herd December 2019


Trace mineral supplementation has traditionally been used only for animals in areas of known deficiency, especially after the manifestation of clinical signs. However, more recent research has found production responsive benefits as a result of aqueous trace mineral injections, administered at strategic times of the production cycle, even in the absence of apparent deficiency. Very few of these studies have been replicated in field conditions in Australia. Ruminants are less able to rapidly utilise oral trace minerals compared to monogastrics, so injectable administration provides a mechanism to reliably increase levels around periods of high demand such as weaning, pre-joining and pre-calving. Administration at these times may improve immunity and/or fertility.



To assess the effect of a single dose of different commercially available trace mineral injections on the conception of beef cattle to first round artificial insemination in Australian field conditions.


To assess the effect of an injectable trace mineral injection (ITM)—containing copper, zinc, selenium and manganese—on the leptospirosis antibody response when administered concurrently with a 7 in 1 vaccination to cattle in Australian field conditions.



Female Angus cattle of mixed ages (n=1093) were enrolled in the study. Blood samples were taken at day -7 to assess trace mineral status and product safety from a representative sample. On day 0 animals were blocked by age group and randomly assigned to one of four treatment groups; 1. Negative control, 2. Cobalife VB12 Plus Selenium (Bayer Australia, containing 4 mg/mL sodium selenite and 2 mg/mL hydroxocobalamin) and Coppernate (Troy Laboratories, containing 60 mg/mL copper as copper glycinate), 3. Multimin Injection for Cattle (Virbac Australia, containing 40 mg/mL zinc as disodium zinc EDTA, 15 mg/mL copper as disodium copper EDTA, 10 mg/mL manganese as disodium manganese EDTA, and 5 mg/mL selenium as sodium selenite) and HyB12 (Virbac Australia, containing 1.8 mg hydroxocobalamin and 0.2 mg cyanocobalamin/mL, or 4. Multimin Copper Free Injection for Sheep & Cattle (Virbac Australia, containing 40 mg/mL zinc as disodium zinc EDTA, 10 mg/mL manganese as disodium manganese EDTA, and 5 mg/mL selenium as sodium selenite) and HyB12. All treatments were applied as per label guidelines. Between day 28 and 35, all females underwent fixed time artificial insemination (AI). Up to three AI sessions were performed, and all cattle were pregnancy tested 35 to 49 days after the final AI session. This study was approved by the Virbac Animal Ethics Committee under protocol no. 578-15.


Female Droughtmaster cattle 18 months of age (n=20) were enrolled in this study. Blood samples were taken from ten animals at day -21 to assess trace mineral status and product safety for selenium and copper. Zinc levels were not measured in this study. On day 0 animals were randomly allocated to one of two treatment groups; 1. Control with no treatment and 2. Multimin Injection for Cattle. All cattle in both groups were vaccinated with a commercially available 7 in 1 vaccine. This was the first time the heifers had received Leptospirosis vaccine antigens. A 5 in 1 vaccination had been given at marking and weaning. On Day 0 and Day 28 blood samples were collected and submitted to the NSW State Diagnostic Laboratory for Leptospirosis pomona and L. hardjo MAT titres. Statistical analysis of the results was undertaken using a standard t-test after Box-Cox normalisation. This study was approved by the Virbac Animal Ethics Committee under protocol no. 594-18-QLD.



Blood mineral analysis showed that the herd was not considered to be deficient in copper, selenium, zinc, or vitamin B12 as per the laboratory reference ranges. Although injectable trace mineral (ITM) supplementation did not have a significant effect on pregnancy failure compared to control, treatment did have a significant effect (p=0.036) on the number of cattle conceiving to first round AI compared to control. The proportion of cows pregnant after first round AI was significantly greater for cows receiving treatment 3 than control animals (56% pregnant vs 48% pregnant, p=0.04). A similar effect was observed with cows receiving treatment 4 compared to control (58% vs 48%, p=0.012). Treatments 3 and 4 were not significantly different from each other (p=0.629).


Blood mineral analysis showed that the cattle were not considered to be deficient in copper or selenium as per the laboratory reference ranges. Day 0 results indicated all cattle naïve to leptospirosis with negative titres. Cattle responded to primary vaccination with 70% having positive L. hardjo MAT titres ranging from 0 - 200 (raw data) and 100% having positive MAT L. pomona titres ranging from 100 - 3200 (raw data). Once data was normalised by Box-Cox transformation mean titre levels were not significantly different for L. hardjo (p=0.13). However, there was a significant difference in favour of the ITM treatment group for L. pomona (p=0.03). Mean normalised titre levels for L. pomona in the control group was 5.99 and in the ITM treatment group was 7.74.



The results of this trial are in agreement with previously published papers. Mundel et al. (2012) demonstrated that there was a 12.5% increase (p=0.01) in the number of calves born in the first 20 days of calving from dams treated with a trace mineral injection similar to treatment 3, compared to control animals. A similar improvement was also reported by Sales et al. (2011) using a timed embryo transfer protocol in Bos indicus x Bos taurus animals. However, this study was unable to detect differences in overall conception (or pregnancy failure), which has been previously reported (Hawkins, 2007). This may be due to the power of analysis, in which Hawkins (2007) had enrolled approximately 2000 animals, compared to the ~1000 enrolled in this study. The mechanism of action has been hypothesised to be a result of improved early embryo survival from trace mineral supplementation (Hawkins, 2007). In humans, there is evidence to suggest that reactive oxygen species (ROS) may be responsible for some instances of infertility, and supplementation with antioxidants may be beneficial (Agarwal & Gupta, 2006). Providing bioavailable sources of copper, selenium, zinc, and manganese is effective in increasing in vivo synthesis of superoxide dismutase and glutathione peroxidase enzymes (Machado et al., 2014 & Teixeira et al., 2014), which are both necessary for neutralisation of ROS.


The increased L. pomona antibody titres in this study are consistent with other published trials where ITM at time of vaccination also increased antibody titres and proportion of protected animals. Arthington and Havenga (2012) found that concurrent use of ITM increased Bovine Herpes virus titres at days 10, 30 and 60 after vaccination. Arthington et al. (2014) subsequently found that ITM also increased response to a novel antigen challenge (Porcine RBC). Palomares et al. (2014) found BVDV titres were also higher with concurrent use of ITM and vaccine. The same authors established that without the use of ITM only 53% of cattle had a 4-fold seroconversion to BVDV when compared to concurrent ITM and vaccine use which led to 80% seroconversion rate by day 28. Ball (2016) found a higher percentage of cattle reaching a protective threshold at days 10, 20, 28 and 42 days against Clostridium botulinum when ITM was given at the same time as a commercial botulism vaccine.

The mechanism of action for increased antibody response with concurrent injectable trace minerals and vaccines is not fully understood but it has been established by Arthington and Havenga (2012) that the body stores of trace minerals such as zinc and selenium are reduced following vaccination.

The lack of a significant difference in the L. hardjo titres may be explained by the decreased number of positive animals to this antigen and also the lower overall antibody levels. This is typically seen with L. hardjo testing.

While the results in this trial do not necessarily suggest the treated animals are any better protected against leptospirosis they do provide measurable evidence that injectable trace minerals enhance the immune response. Other immune research with injectable trace minerals has demonstrated improved acute phase response (Arthington et al., 2014), elevated antioxidant levels (Machado et al., 2014 & Teixeira et al., 2014) and reduced morbidity (Teixeira et al., 2014). This suggests that ITM may be a useful tool in animal health management.


The results from both trials have shown that in spite of no apparent trace mineral deficiency, providing trace minerals through aqueous injections at strategic times of the production cycle can be beneficial for various productivity outcomes. The commercially available formulation containing a combination of copper, selenium, zinc, and manganese was effective at increasing conception to first round AI and improving markers of immune function in Australian field conditions. Both these benefits may prove to provide measurable financial benefits for Australian cattle producers.


  1. Agarwal, A. & Gupta, S. (2006). The role of free radicals and antioxidants in female infertility and assisted reproduction. US Genito-Urinary Disease, pp. 60–65
  2. Arthington, J. & Havenga, L. (2012). Effect of injectable trace minerals on the humoral immune response to multivalent vaccine administration in beef calves. Journal of Animal Science 2012, 90, 1966-71
  3. Arthington, J. et al. (2014). Effects of trace mineral injections on measures of performance and trace mineral status of pre and post weaned beef calves, J. Anim. Sci., 92:2630-2640
  4. Ball, M. (2016). Trace mineral injection enhances antibody response to botulism vaccination. AVA Proceedings, Adelaide
  5. Hawkins, D. (2007). The effect of injectable trace elements (Multimin) on health and reproduction parameters in NZ dairy herds, New Zealand Dairy Cattle Veterinarians Newsletter, 24(3):12-16. 9
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  7. Mundell, L. et al. (2012). Effects of prepartum and postpartum bolus injections of trace minerals on performance of beef cows and calves grazing native range. The Professional Animal Scientist, 28:82-88
  8. Palomares, R. et al. (2016). Effects of injectable trace minerals on humoral and cell-mediated immune responses to Bovine Viral Diarrhea Virus, Bovine Herpes Virus 1 and Bovine Respiratory Syncytial Virus following administration of a modified-live virus vaccine in dairy calves. Vet. Immunol. Immunopathol., 178:88-98
  9. Sales, J. et al. (2011). Effect of injectable copper, selenium, zinc and manganese on the pregnancy rate of crossbred heifers (Bos indicus x Bos taurus) synchronised for timed embryo transfer. Livestock Science, 142:59-62
  10. Teixeira, A. et al. (2014). Effect of an injectable trace mineral supplement containing selenium, copper, zinc, and manganese on immunity, health, and growth of dairy calves. J. Dairy Sci., 97:4216–4226


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