Introduction

In dairy cows, major metabolic and physiological changes occur during transition of pregnancy to lactation [17]. Immunological capacity remained lower from 1 month before calving to 1 month after calving [1, 19]. During this period, mostly animals remained in negative energy balance [6] which causes physiological stress of the animals and ultimately leads to immune suppression that adversely affects the animal health and production.

The immune suppression during the transition period increases the susceptibility to diseases of dairy cows [18]. In this period, important neutrophil functions are impaired and lymphocyte proliferation is reduced [12]. The respiratory burst activity of polymorphonuclear leukocytes (PMN) decreased 1 week before parturition and started to increase from 3rd week of lactation [7]. Lymphocyte proliferation in vitro has been used as an indirect measure of immune responsiveness. Concanavalin A (Con A) mitogens are used for stimulating T lymphocyte proliferation in vitro [23] and lipopolysaccharide (LPS) for B lymphocyte proliferation [9].

Among different measures taken to avoid the periparturient immune suppression, supplementation of antioxidant micronutrients considered to be most effective in overcoming this problem. Copper (Cu) is an important part of the superoxide dismutase enzyme. This enzyme safeguards the cells from the deleterious effects of oxygen metabolites produced during phagocytosis [26]. Zinc (Zn) is a structural component of the superoxide dismutase enzyme (SOD) and almost 300 other metabolic enzymes. It is also involved in cell growth, cell replication, cell-mediated immunity and generalized host defense [25].

There is a paucity of information on the effect of in vitro Cu and Zn supplementation on NPA and LPR around peripartum period and it is not known whether there is need for extra Cu and Zn in Cu and Zn fed cows for increasing NPA and LPR around the calving period. Therefore, the present study was carried out to assess the effect of in vitro Cu and Zn supplementation on NPA and LPR in transition dairy cows.

Materials and Methods

Study Site

The present study was carried out at the Cattle Yard of National Dairy Research Institute (NDRI), Karnal, Haryana; located at 29°42′N latitude and 72°02′E longitudes at an altitude of 250 m above the mean sea level. The average annual maximum and minimum ambient temperature ranges between 45 and 1.4 °C in this area. The mean annual relative humidity ranges between 41 and 85 % and the annual rainfall from 760 to 960 mm. The experiment was carried out during August to April.

Animal Selection

The experiment was conducted on Karan-Fries cows, developed for higher milk production at NDRI, Karnal, developed through crossing native Tharparkar and exotic Holstein-Friesian breeds. Eighteen pregnant crossbred (Tharparker × Holstein-Fresian) cows of average body weight around 450 kg were selected from the Institute’s experimental herd during their late gestation 45 days before the expected date of calving.

Feeding

Individual cows were offered with a normal feeding regime i.e., ad libitum green fodder (Trifolium alexandrinum/Zea mays/Avena sativa) and 4 kg concentrate mixture daily. Concentrate mixture composed (90.51 % DM, 19.81 % CP, 8.62 % total ash, 3.7 % EE, 58.97 % NDF, and 13.4 % ADF) of maize, groundnut cake, mustard cake, wheat bran, rice bran, and common salt. After calving, the animals were offered ad libidum green fodder (23.19 % DM, 8.18 % EE, 9.61 % total ash, 1.14 % EE, 49.79 % NDF, and 26.45 % ADF), and 1 kg concentrate mixture was given for every 3 kg milk production.

Experimental Procedures

To assess the effect of in vitro Cu and Zn supplementation on NPA and LPR in transition cows, in the first experiment, six Karan-Fries cows were selected and kept in normal conventional feeding regime. Their blood samples were collected from the jugular vein in 10 ml heparinized vacutainer on 30th, 15th, 7th, 3rd day before calving, on the day of calving and 3rd, 7th, 15th, 30th day after calving. Neutrophil phagocytic activity and LPR were evaluated from the collected blood samples with in vitro supplementation of Cu and Zn in the culture media individually.

In the second experiment, another six Karan-Fries cows were selected and provided with additional 20 ppm Cu/kg DM/day individually from 45 days prepartum to 45 days postpartum. Their blood samples were collected from the jugular vein in 10 ml heparinized vacutainer on 30th, 15th, 7th, 3rd day before calving, on the day of calving and 3rd, 7th, 15th, 30th day after calving. Neutrophil phagocytic activity and LPR were evaluated from the collected blood samples with the addition of Cu in the culture media.

In the third experiment, another group of six Karan-Fries cows were selected and provided with additional 80 ppm/kg DM/day Zn individually. Their blood samples were collected from the jugular vein in 10 ml heparinized vacutainer on 30th, 15th, 7th, 3rd day before calving, on the day of calving and 3rd, 7th, 15th, 30th day after calving. Neutrophil phagocytic activity and LPR were evaluated from the collected blood samples with the addition of Zn in the culture media.

Estimation of In Vitro Phagocytic Activity of Blood Neutrophils

Neutrophils were isolated from blood samples by hypotonic lysis of erythrocytes and adjusted to 5 × 106 live cells/ml in the culture media (RPMI 1640) containing 10 % fetal calf serum (FCS). 200 μl of the cell suspension was placed per well in triplicate in a 96-well flat-bottomed tissue culture plate. Zymosan (650 µg/ml) and nitroblue tetrazolium (NBT) (250 µg/ml) were added to the cell suspension and incubated at 37 °C in a humidified CO2 incubator (95 % air and 5 % CO2) for 2 h. NBT is yellow in color, but it gets changed to blue after phagocytosis, which can be measured spectrophotometrically [2].

Estimation of In Vitro Lymphocyte Proliferation Response

The blood samples were centrifuged at 3000 rpm for 30 min, then buffy coat was collected. 1:1 v/v Dulbecco’s phosphate saline (DBPS) was added to re-suspend the buffy coat. The suspended buffy coat was carefully layered on lymphocyte separation medium (Histopaque 1077) at concentration of 4:1 v/v in sterile 15 ml polypropylene centrifuge tube and centrifuged at 2500 rpm for 30 min at room temperature to separate the lymphocytes. After separation of the lymphocytes, culture media (RPMI 1640) with 10 % FCS was added to the lymphocyte suspension to adjust at 5 × 106 live lymphocytes/ml. 200 μl of the cell suspension was placed per well in triplicate in a 96-well flat-bottomed tissue culture plate. The mitogen Concanavalin A (5 µg/ml) and Lipopolysaccharide (50 ug/ml) were used to stimulate T and B lymphocytes, respectively. The cells were allowed to proliferate with and without mitogen, for determining the difference between cell proliferations. All cultures were incubated at 37 °C in a humidified CO2 incubator (95 % air and 5 % CO2) for 24 h. The proliferative response of lymphocyte was estimated using the colorimetric MTT (tetrazolium) according to the procedure given by Mosmann [14].

Supplementation of Cu and Zn in the Culture Media

Normal plasma level of Cu and Zn was 12.234 μM and 9.442 μM, respectively. Before conducting this experiment, different doses of Cu and Zn were supplemented in the culture medium to see their influence on the NPA for standardization of the best suitable supplementation dose. Micronutrients were added in seven different doses, i.e., 5, 10, 25, 50, 75, 100, and 150 % of the plasma level in the culture media where 100 % is equal to the level found in plasma of normal lactating Karan-Fries cows. Culture media, which were without any extra micronutrient supplementation served as control. The maximum effect was found with in vitro supplementation of 25 % (2.360 μM) of Cu of normal plasma level and 75 % (9.175 μM) of Zn of normal plasma level [4]. Therefore, we supplemented 2.360 μM Cu and 9.175 μM Zn in the culture media of NPA and LPR of transition cows during the peripartum period.

Statistical Analysis

The data were analyzed using the GLM procedure by analysis of variance which includes the effect of in vitro Cu and Zn supplementation on NPA and LPR. A post hoc analysis was performed using Tukey’s tests for pairwise comparisons. Data were presented as mean ± SEM, and statistical analysis was carried out using SPSS software, version 14.0. The level of statistical significance was set at P < 0.05.

Results and Discussion

Table 1 describes the effect of in vitro micronutrient supplementation in the culture media on NPA. It was found that in vitro supplementation of micronutrient had significant (P < 0.05) effect on the NPA. Addition of Zn in the culture media increased the NPA significantly (P < 0.05) as compared to non-supplemented neutrophils whereas addition of Cu increased the NPA (10 %). Neutrophil phagocytic activity increased by 38.95 % with the addition of Zn in the culture media on day of the calving. There was increase in NPA from calving to 30 days postpartum with the addition of Cu and Zn. Peripartum days had significant (P < 0.001) effect on the NPA while interaction between peripartum days and micronutrients had no significant effect on the in vitro NPA.

Table 1 In vitro phagocytic activity of blood neutrophils after in vitro Cu and Zn supplementation during different peripartum days in crossbred cows
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Table 2 describes the effect of in vitro micronutrient supplementation in the culture media on T lymphocyte LPR. No significant difference was found with in vitro micronutrient supplementation in the culture media in T lymphocyte blastogenic response with mitogen (Con A). T lymphocyte LPR increased (9.22 %) with Zn supplementation in the media as compared to non-supplemented media. Cu supplementation increased the LPR of T lymphocyte by 2 %. It was found that the days around peripartum period had significant (P < 0.001) effect on LPR of T lymphocyte.

Table 2 In vitro lymphocyte stimulation index of T lymphocytes after in vitro Cu and Zn supplementation during different peripartum days in crossbred cows
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Table 3 describes the effect of in vitro micronutrient supplementation in the culture media on B lymphocyte LPR. There was significant (P < 0.05) influence of in vitro micronutrient supplementation on mitogen (LPS)-induced B lymphocyte balstogenic response. The result showed that in vitro Zn supplementation in the culture media significantly (P < 0.05) increased the B lymphocyte LPR as compared to control groups. The increase in LPR of B lymphocyte was 16.92 % in in vitro Zn supplemented group as compared to the control group. On 30th day postpartum, mitogen (LPS)-induced lymphocyte blastogenic response was significantly (P < 0.001) higher in Zn supplemented group as compared to the other groups. It was found that different days of peripartum period had a significant (P < 0.001) effect on the B lymphocyte LPR.

Table 3 In vitro lymphocyte stimulation index of B lymphocytes after in vitro Cu and Zn supplementation during different peripartum days in crossbred cows
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In dairy cattle, a few weeks before calving dry matter intake starts reducing [13] and at the same time there is a demand for nutrient to initiate milk synthesis. Therefore, a marked decline in serum concentrations of Cu and Zn at calving occurred. Drop in the serum concentrations of these nutrients is associated with impaired immune functions. The drop in serum concentrations of Cu and Zn is largely due to colostrum formation, changes in dry matter intake and ruminal metabolism [10].

Spears [20] observed that phagocytic activity is reduced in Cu deficiency and it can be changed with Cu supplementation. Similarly, in the present study, NPA was increased by 10 % with in vitro supplementation of Cu in the culture media of neutrophils (Table 1) around the periparturient period. Our results are also in agreement with the findings of Dang et al. [3] who also reported increase in NPA during the prepartum and postpartum period with Cu supplementation. A significant increase in NPA was found with in vitro Zn supplementation in the culture media (Table 1). De et al. [5] reported increase in NPA in dairy cattle with extra Zn feeding during the peripartum period. In the present study, the LPR of B lymphocyte increased significantly (P < 0.05) with in vitro supplementation of Zn in the culture media as compared to non-supplemented lymphocytes (Table 3). Weiss and Spears [22] reported that Zn affects the cell proliferation, replication and immunity. Zinc is essential for lymphocyte replication [21] as indicated by higher B lymphocyte LPR observed with in vitro Zn supplementation in the lymphocyte culture of dairy cows without extra Zn supplementation (experiment 1).

In vitro Cu supplementation in the culture media on NPA and LPR of neutrophils and lymphocytes isolated from Cu fed cows showed no increase in NPA (Table 4) of the Cu fed cows. However, on the day of calving, NPA increased by 15.38 % as compared to control only Cu fed cows. Lymphocyte proliferation response of T lymphocyte increased significantly (P < 0.05) on 3 days prepartum, on the day of calving and 7 days postpartum with in vitro supplementation of Cu in the culture media of lymphocyte from Cu supplemented animals. On the day of calving the increase was 21.64 %. In vitro Cu supplementation in the lymphocyte culture media of Cu fed cows, increased the B lymphocyte LPR significantly (P < 0.05). The increase was 21.40 %. Almost all the peripartum days showed higher LPR of B lymphocyte with in vitro supplementation of Cu in the culture media of lymphocyte of Cu fed animals. On the day of calving the increase was 38.58 %. Our study showed that, B lymphocyte LPR significantly increased with in vitro Cu supplementation in the culture media of lymphocyte of Cu fed animals (Table 4). Senthilkumar et al. [15] also reported higher LPR in Cu supplemented lambs. Solaiman et al. [16] also reported higher lymphocyte proliferation (P < 0.05) in kids supplemented with 100 mg Cu/day.

Table 4 Effect of in vitro Cu supplementation in the culture media on PA and LPR of Cu fed cows
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Table 5 gives the result of the effect of in vitro Zn supplementation in the culture media on NPA and LPR isolated from Zn fed cows. There was no significant variation in NPA with in vitro supplementation of Zn in the culture of neutrophils of Zn fed cows. There was also no significant change in mitogen (Con A) induced T lymphocyte LPR with in vitro supplementation of Zn in the culture media of Zn fed cows. However, a positive response in T lymphocyte LPR was found from 3 day prepartum to the 7 day postpartum with in vitro supplementation of Zn in the culture media of lymphocyte of Zn fed cows. Our result showed a significant (P < 0.001) increase in the LPR of T lymphocyte on 3rd day prepartum with in vitro supplementation of Zn in the culture media of lymphocytes. The increase was 25.78 %. On the day of calving and 3rd day after calving the increase in mitogen (Con A) induced LPR of T lymphocyte was 7.86 and 11.18 %, respectively, with in vitro addition of Zn in the culture media of lymphocyte of Zn fed cow. A similar trend was also found in B lymphocyte LPR. A positive response in B lymphocyte proliferation was observed from 7th day prepartum to the 15th day postpartum with in vitro supplementation of Zn in the culture media of Zn fed cows. There was a significant (P < 0.001) increase in LPR of B lymphocyte on 3 days before calving with in vitro supplementation of Zn in the culture media of lymphocyte of Zn fed animal. The increase was 14.52 %. However, peripartum days had significant (P < 0.001) influence on the changes of NPA and LPR.

Table 5 Effect of in vitro Cu supplementation in the culture media on PA and LPR of Zn fed cows
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In the present study, there was no overall significant difference in NPA and LPR with in vitro Zn supplementation in culture media of neutrophils and lymphocyte of Zn fed cows (experiment 3). It may be attributed to the fact that the cows have got a sufficient amount of Zn with extra Zn feeding during their transition period to maintain NPA and LPR.

It was observed that the NPA and LPR changes significantly during the transition period in all the three experiments. It is gradually decreased toward parturition and again increased gradually as postpartum days progressed. Similar finding was previously reported by Dang et al. [3]. It may be due to diversion of micronutrients towards fetal growth and colostrum formation [3].

In the present study, it was found that the effect of in vitro supplementation of Cu and Zn on NPA of neutrophils and LPR was more pronounced in close to calving periods in all the experiments. Earlier it was indicated that the feed intake was reduced and most of the micronutrients were diverted towards fetal growth, milk and colostrum formation [3], which may lead to insufficiency of micronutrients for immune activity of blood cells. The plasma Cu level was decreased before calving because of drainage to the fetal liver [24]. That causes inadequate availability of Cu for NPA and LPR during very close to calving. Therefore, when in vitro supplementation of Cu was done in the culture media, it increased the NPA and T lymphocyte LPR of Cu non-fed cows (experiment 1) and, NPA and LPR of both T and B lymphocyte of Cu fed cows (experiment 2) on the day of calving and post-calving days. Similarly, the level of Zn was reduced significantly around calving and post-calving periods [11] that also leads to inadequacy of Zn for NPA and LPR around calving period. As a consequence, in vitro Zn supplementation in the culture media increased the NPA, B lymphocyte LPR of Zn non-fed cows (experiment 1) and T lymphocyte LPR of Zn fed cows on the calving day and post-calving period (experiment 3). Ibs and Rink [8] had already reported that Zn is important in B cell activation. It also affects the phagocytic activity of neutrophils.

Conclusions

We found an increase in NPA and LPR on the day of calving and early postpartum (7 days postpartum) period with in vitro supplementation of Cu and Zn in the culture media of Cu and Zn fed and non-fed cows. Keeping this view, it is concluded that extra micronutrient supplementation (Cu and Zn) can improve immunity on the day of calving and postpartum period by increasing NPA and LPR.