Livestock Research for Rural Development 24 (12) 2012 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Effect of supplementation of vitamin E, copper and zinc around peripartum on udder health, milk yield and composition of Sahiwal cows

G Mutoni, Shiv Prasad, Kalyan De, Shashi Pal, J Mukherjee, S Kapila, R Kapila, Harjit Kaur, A K Mohanty and A K Dan

National Dairy Research Institute, Karnal 132 001, Haryana, India


To study the effect of vitamin E (VE), Copper (Cu), Zinc (Zn) supplementation on the udder health, milk production and composition, 30 pregnant Sahiwal cows were taken and divided into 5 groups. One group was supplemented with VE, other with Cu, next with Zn and also one group with a combination of VE, Cu and Zn from 30 days before to 45 days after calving. Unsupplemented group served as control. Milk samples were collected weekly from cows.

Significantly higher (P<0.01) somatic cell counts (SCC), was found in colostrums of control group followed by VE, Cu and Zn group. Significantly higher (P<0.01) milk SCC was found in control group followed by VE, combination, Zn and Cu group. Milk neutrophils percentage were significantly lower (P<0.01) in combination group followed by Cu, Zn, VE and control group. Total immunoglobulins in colostrum were highest (P<0.01) in combination group. Supplemented cows had significantly (P<0.05) higher fat and protein content. Micronutrient supplementation lowered milk SCC and neutrophils, leading to better udder health and milk production. Supplementation increased the total colostral immunoglobulins and thus may improve calf health.

Keywords: immunoglobulins, micronutrients, milk yield, neutrophils, SCC


Immune and innate host defence mechanism of the dairy cows remains suppressed at 3 weeks before calving, is maximal at parturition and remains low until 3 weeks after calving (Dobbelaar et al  2010). These impaired immune responses may relate to the stresses of late pregnancy and parturition stimulating release of stress hormones such as corticosteroids (Sordillo  2005). Major metabolic changes around this period are also associated with the initiation and rapid increase in milk production (Dobbelaar et al  2010). This abruptly increased metabolic rate associated with late pregnancy, parturition, and initiation of lactation results in a greater release of reactive oxygen species (ROS). An imbalance between metabolic entry and disposal of ROS may also contribute to periparturient disorders in cows (Miller et al 1993; Sordillo and Aitken 2009). A reduction in dry matter intake has been found about seven to ten days before calving, but the nutrient demand for the growing foetus and initiation of milk production is increasing at the same time (Grummer 1995). Therefore, a gap in nutrient demand occurs which affects the nutrient metabolism resulting in metabolic disorders and decreased productivity. Provision of adequate mineral and vitamin nutrition during the transition period may be used as a strategy to not only enhance the cow’s immunity against disease (Dang et al  2012) but also maintain milk quality and production (Weiss and Wyatt  2002; Cortinhas et al  2010). Therefore, the present study was initiated to see the effect of supplementing Vitamin E, Copper and Zinc and their combination during the periparturient period and study its subsequent effect on milk yield, quality and milk composition of indigenous Sahiwal cows. 

Material and methods

Thirty Sahiwal pregnant cows in their late gestation (i.e. 30 days before the expected date of calving) were selected from the experimental herd. All the cows were apparently free from any physiological, anatomical and infectious disorders. Only animals which were expected to calve from November, 2010 to February, 2011 were considered. Based on their milk yield and parity, the animals within the same range of yield and at the same parity were blocked and randomly allocated to one of the five different treatments; each group composed by six animals.  Group 1, without any supplementation acted as control. The experimental cows were supplemented individually with Vitamin E (Group 2), Copper (Group 3), Zinc (Group 4), and also with a combination of Vitamin E, Copper and Zinc (Group 5), to study the cumulative effect of all micronutrients.  

The feed grade dl-α-tocopheryl acetate dry powder of 50% purity was taken as a source of vitamin E, Copper (II) sulphate pentahydrate GR (cupric sulphate) of 99% purity and the Zinc sulfate heptahydrate purified (99% purity) were taken as a source of Copper and Zinc.  All the above micro-nutrients were accurately weighed daily and mixed with a small amount of concentrate and directly fed to the animals from 30 days before calving to 45 days after calving.

Doses of Vitamin E, Copper and Zinc supplemented daily have been depicted in Table 1.

Table 1. Supplemented doses of micronutrients to Sahiwal cows


No. of cows





Vitamin E


1000 IU



20 ppm



80 ppm

Vitamin E + Zn + Cu


Combination of the above

Milk samples from all the four quarters of cows were collected at weekly intervals and pooled. These samples were brought to the laboratory immediately after collection for cell enumeration (SCC, Differential leucocyte counts (DLC)) and milk composition. For SCC and DLC the slides were prepared within one hour of collection of milk samples (Dang et al 2008). Milk composition i.e. fat, protein, lactose and SNF was estimated by Automatic milk analyzer, Funke Gerber, Berlin, Germany. Total immunoglobulin of colostrums was estimated by using Zinc sulphate turbidity method as described by Mc Ewan et al (1970). Milking was done three times daily and milk yield records was maintained throughout the experimental period (75 days after the onset of lactation). 

Statistical analysis

The statistical analysis was performed to compare Milk SCC, DLC, milk composition of different experimental groups, using the least squares method using Sigma Plot 11.0. Detailed Comparison of the least squares means of the different experimental groups was done by using Tukey’s test. 

Results and discussion

Milk SCC


The results of the changes occurring in the colostrum and milk SCC in control and micronutrient supplemented Sahiwal cows have been presented group wise in Figure 1.

Figure 1.  Change in the colostrum and milk SCC (x 105) in control and micronutrient supplemented Sahiwal cows during different days of lactation


Somatic cells counts were significantly higher (P<0.001) in day-1 colostrum of all the cows. These values decreased significantly (P<0.001) on day-3 and day-5 respectively. Overall milk SCC was higher in control groups followed by vitamin E, zinc, copper and supplemented group. Combine feeding of all the micronutrients resulted in significantly (P<0.001) low milk SCC as compared to control group. During the various days of lactation, milk SCC also decreased significantly (P<0.05) in the supplemented groups as compared to the control ones. 


Colostrum SCC which was higher became normal in subsequent milkings. The SCC was maximum in control group followed by Vitamin E and combination, Zinc and copper group.  Similar results have also been reported by Chawla and Kaur (2004) and Brozos et al. (2009) after supplementation of both Vitamin E and selenium.  Like Cortinhas et al (2010), we also found that the supplementation of combination of micronutrients reduced the milk somatic cell counts significantly (P<0.001) as compared to control group.


Milk DLC


On estimating the milk DLC, neutrophils percentage were found to be significantly higher (P<0.001) and lymphocytes percentage were significantly lower (P<0.01) in day-1 colostrum of all the cows as reported earlier (Dang et al 2008). Result of milk DLC has been presented in Figures 2, 3 and 4 respectively.

Figure 2.  Milk neutrophil % in control and micronutrient supplemented cows


Figure 3. Milk lymphocyte % in control and micronutrient supplemented cows


Figure 4. Milk macrophages % in control and micronutrient supplemented cows


The overall mean of milk neutrophils percentage were significantly lower (P<0.01) in combination group (19.34 0.31) followed by copper (19.80 0.29) , zinc (20.26 0.21), vitamin E (20.59 0.21) and control group (24.61 0.33) (Figure 2). The overall mean of milk lymphocytes percentage were significantly higher (P<0.001) in combination group (60.31 0.26) followed by zinc (60.03 0.32), copper (59.96 0.21), vitamin E (59.17 0.24) and control group (59.02 0.15) (Figure 3). The maximum percentage of milk macrophages were found in combination group (20.36 0.43) followed by vitamin E (20.26 0.31), copper (20.24 0.30), zinc (19.77 0.37), and control group (16.43 0.38) (Figure 4). However, the data could not be compared with cows, as there is no literature available on milk DLC after micronutrient supplementation.


On comparing the result of milk DLC, it was found that PMN and macrophage percentage was lower and lymphocyte percentages were higher in milk samples which are in agreement to the result of Gargouri et al (2008). They reported that lymphocytes were the predominant cell types, but their proportion declined with total bulk milk SCC. However, Paape et al (2002), reported that the dominant cell types in milk samples in healthy quarters were macrophages, followed by lymphocytes and PMN in cow milk. In the normal healthy mammary gland, macrophages predominate and act as sentinels to invading mastitis-causing pathogens. 


Total Immunoglobulins

Results of the total immunoglobulin secreted in colostrums on days 1, 3 and 5 respectively have been presented in Figure 5.


Figure 5. Total immunoglobulins in control and micronutrient supplemented cows

Colostrum immunoglobulins were significantly (P<0.01) higher in the supplemented group, which may increase the survivability of calves born to these cows as it is largely responsible for providing protective antibody and possibly lymphocytes to the calf (Caspari and Schmidt 1991). Also for the transport of antibody and cells from the blood across the endothelium into epithelial tissue and then into mammary gland secretions, the status of the immune system of the peripartum cow plays an important influence on calf health (Mallard et al 1998). Increased synthesis of Ig in the mammary gland of cows supplemented with Vitamin E and selenium during dry period has also been observed by Nicola et al (1996), which resulted in increased concentration of Ig in colostrum. The reason for this is because Vitamin E enhances the ability of the immune cells of the mammary gland to produce more Ig (Nicola et al 1996).  

Milk Yield

Results of the milk yield obtained in control and vitamin supplemented group of cows have been presented in Figure 6.


Figure 6. Milk yield in control and micronutrient supplemented cows

Milk yield increased in all the supplemented group of cows as compared to the control ones. The beneficial effect of supplementation of Vitamin E and Beta carotene on milk production has been reported by Chawla and Kaur (2004). Possible reason for this may be that various micronutrients supplemented to the transition cows reduce the milk SCC and thus the animal has fewer udder infections, which subsequently increases its milk production. However, Brozos et al (2009) reported that daily administration of a blend containing 60 g ammonium chloride, 1000 IU Vitamin E and 0.05 ppm Se throughout the dry period seemed to be safe, but without any effect on milk yield at 30 and 60 days postpartum. Cows which were given intramuscular injections of 2100 mg of Vitamin E (and 7 g of sodium selenite) 2 weeks before calving and on the day of calving showed no effect on milk yield (Bourne et al 2008).  Whitaker et al (1997) compared the effects of providing supplemental Zn from a mixture of Zn proteinate and inorganic Zn and found that source of Zn had no effect on infection rate, new infections, clinical mastitis and SCC.  

Beneficial effect was observed after the supplementation of copper in this study. A deficiency of Cu has been associated with a decreased ability of these cells to multiply. Failure to multiply quickly may result in a competitive advantage by mastitis-causing bacteria (Costello, 1998). Xin et al (1991), however, did not reported any difference among treatments where the average daily actual milk yields in the first 2 months were 32.9, 33.2, and 33.1 kg/d for the control, +10 Cu, and +20 Cu treatment groups, respectively. Nocek et al (2006) reported that first lactation cows had no differences in SCC when fed Zn, Mn, Cu and Co in complex or inorganic form at 75 or 100% of NRC above the basal diets, but a small significant milk production response was noted between the organic and the inorganic minerals, even when the inorganic minerals were fed at 75% of NRC. Cope et al (2009) evaluated the effects of the level and form of dietary Zn on milk performance, and found that cows supplemented with organically chelated Zn at the recommended level had a higher milk yield (37.6 kg/day) than those fed inorganic Zn at the recommended level (35.2 kg/day), or organically chelated Zn at low level (35.2 kg/day), but there was no difference from those fed inorganic Zn at the low level (36.0 kg/day).   

Milk Composition

Change in the fat, protein and lactose percentage in the colostrums and milk samples of various groups of cows have been presented in Table 2, 3 and 4 respectively.


Table 2.  The average of fat % in colostrum and milk of control and micronutrient supplemented cows



Vitamin E






















Values within a row bearing different letters are significantly different


Table 3.  The average of protein % in colostrum and milk of control and micronutrient supplemented cows



Vitamin E






















Values within a row bearing different letters are significantly different


Table 4. The average of lactose  % in control and colostrum and milk of micronutrient supplemented cows



Vitamin E






















Values within a row bearing different letters are significantly different

Supplementation of micronutrients to peripartum cows not only increased the milk yield but also significantly (P<0.01) increased the fat and protein percentage. Maximum beneficial effect was seen in the combination group which showed a significant (P<0.01) increase in the fat, protein and lactose percentage as compared to the control groups. Kellogg et al (2004) indicated that Zn methionine increased lactation performance (produced more (P < 0.01) milk, energy-corrected milk and fat-corrected milk) and improved udder health (as a 33.3% reduction in SCC), but milk composition did not change (P > 0.15). Popovic (2004) evaluated replacing 33% of the supplemental inorganic Zn sulphate with organic Zn for 45 days pre-calving until 100 days post-calving and found that organic Zn had significantly (P < 0.05) lower SCC by day 10 of lactation (158,840/mL vs. 193,530/mL) and at the end of the trial (62,670/mL vs. 116,440/mL), the average milk yield was numerically greater (27.75 kg/day vs. 26.22 kg/day).  However, according to Cope et al (2009), milk composition was unaffected by dietary treatment, but animals that received the low level of Zn had higher SCC. 

Ashworth et al (1967) observed that milk fat percentage falls as a result of udder infection and this could be due to impaired synthetic and secretory activity of the udder epithelial cells (Schultz  1977). The fall is less in copper supplemented group (Engle et al (2001)), who reported that copper supplementation alters lipid metabolism in high-producing dairy cows. The mechanism by which copper affects the profile of fatty acids is not clear but it might be due to its effect on desaturation, esterification and mobilization from triacylglycerols. In addition, Kinal et al (2005) reported that replacing 30% of the inorganic Cu, Zn and manganese (Mn) for 6 weeks pre-calving until 305 days of lactation in dairy cows resulted in a 6.5% increase in milk yield (22.35 vs. 21.20 kg/day, P < 0.05). However, Uchida et al (2001) reported that feeding a combination of Zn amino acid (AA), Mn AA and Cu AA complexes, and Co glucoheptonate to early lactation Holstein cows had no effect on milk production, milk fat and protein content, and linear SCC. A significant increase in SNF was seen in copper supplemented group and decrease in unsupplemented group was seen at 3–7 days post calving. This decrease in fat percentage in unsupplemented group could be due to the presence of subclinical infections (Gakhar et al 2010). 


In a study by Griffiths et al (2007), supplementing cows with CTM (providing daily 360 mg Zn, 200 mg Mn, 125 mg Cu as amino acid complexes and 12 mg cobalt (Co) from Co glucoheptonate) resulted in (P ≤ 0.05) a 6.3% increase in milk production, 5.6% increase in milk energy, 6.4% improvement in fat yield, 6.5% improvement in crude protein yield, 5.8% improvement in production of milk solids, and a trend for a reduction in mastitis cases (P ≤ 0.10). Milk composition was not affected by treatment, although fat, crude protein and the solids content of milk produced by CTM supplemented cows were numerically (P ≥  0.10) higher than that produced by the control cows. 


Although this study was initiated in a limited number of cows by taking a few important micronutrients, it clearly indicates that supplementation of micronutrients around the peripartum period reduces mammary stress in cows.  This reduction in mammary stress may help to achieve higher peak lactation for which studies need to be carried out in larger group of cows. 



The authors are thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India for providing financial assistance for carrying out this research work.   


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Received 24 March 2012; Accepted 12 November 2012; Published 2 December 2012

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