Abstract: The objective of this study was to investigate the effect of supplemental recombinant bovine somatotropin and cooling management on changes in oxidative stress index in the plasma of crossbred Holstein cattle at different stages of lactation. Ten primiparous crossbred 87.5% Holstein cattle were randomly divided into 2 groups of five animals each which were housed in Normal Shaded (NS) as non-cooled cows and in shaded with Misters and Fans (MF) as cooled cows. The cows in each group were supplemented of recombinant bovine Somatotropin (rbST) in each stage of lactation with three consecutive subcutaneous injections with 500 mg of rbST in every 14 days. The ambient temperature at 13:00 h in the MF barn was significantly lower while relative humidity was higher than that of the NS barn. Cows under NS barn showed high rectal temperatures and respiration rate as compared with cows under the MF barn. Milk yield significantly increased in both cooled and non-cooled cows treated with rbST in each stage of lactation. The plasma concentrations of Sulfhydryl (SH) residue and Thiobarbituric Acid Reactive Substance (TBARS) did not alter in both groups of cows with or without rbST supplementation but the ascorbic acid concentrations in the plasma and in milk of cows under NS barn were significantly lower than those of cows under MF barn during early and mid lactation. The present study, demonstrates that changes in antioxidant reserve of ascorbic acid in both plasma and milk of dairy cows would be an appropriate and sensitive indicator of oxidative stress in crossbred dairy cows during exposure to high temperatures.

INTRODUCTION

Low milk yield is one of the most problems for dairy animal production in the tropic. Thermal effect with heat stress is probably, the most important factor and is widely recognized as one of the leading causes on the performance of dairy cows. It affects their dry matter intake, feed efficiency and milk production (Fuquay, 1981; Shibata and Mukai, 1979) including disruption their homeostasis and metabolism. The effect of heat stress is known to induce oxidative stress which induces production of Reactive Oxygen Species (ROS) (Mitchell and Russo, 1983; Loven, 1988). The high production of reactive oxygen species and a decrease in antioxidant defense, leads to cause of many diseases (Halliwell and Gutteridge, 1990) and leading to the onset of health disorders in cattle (Miller et al., 1993).

Oxidative stress markers can be divided into nonenzymatic antioxidants and antioxidant enzymes such as Superoxide Dismutase (SOD), catalase and glutathione peroxidase (Zalba et al., 2001). The plasma concentration of vitamin C in ruminants is an oxidative stress indicator which is affected by heat stress. A negative correlation between rectal temperatures and ascorbic acid concentrations of Bos taurus cattle in the hot season was reported (Tanaka et al., 2007). The different responses of radical scavengers for antioxidant mechanisms may be due to interaction with factors that involve in the activation of oxidative stress. Different breeds of dairy cows may show different responses of the level of oxidative stress marker during heat stress. Temperate breeds (Bos taurus) have higher milk production but they also have inherent disadvantageous traits particular low heat tolerance as compared with crossbred cows containing Bos indicus gene which have high heat tolerance in the tropical environment. Few data are available for studies of the oxidative status in crossbred cows under high ambient temperatures although, a number of studies have been reported in heat stressed exotic Holstein dairy cows (Bernabucci et al., 2002; Lakritz et al., 2002; Tanaka et al., 2007).

The previous studies in crossbred Holstein cattle (Chaiyabutr et al., 2007) have shown that growth hormone act as a regulator of fluid homeostasis with an increase in total body water after rbST administration. Increment of body temperature during bST treatment has been noted (Johnson et al., 1991). The restoring body fluids involving heat dissipation mechanism during rbST administration in crossbred Holstein cattle under high ambient temperatures led to raise the question of whether rbST treated cows show a novel mechanism to defend heat stress which need for further investigation.

As described previous, there are many reports examining the effect of heat stress alone on oxidative stress index in many species animals including poultry (Altan et al., 2003; Mujahid et al., 2005). Oxidative stress can be monitored with several biomarkers (antioxidants and pro-oxidants) in the plasma. However, little information of oxidative status regarding crossbred Holstein cattle given exogenous rbST under high temperatures is available. Therefore, the aim of the present study was to examine, the effect of cooling and supplemental rbST on the responses of oxidant defenses in crossbred Holstein cows in the tropic.

Changes in antioxidative stress markers in plasma for concentrations of Sulfhydryl (SH) residue and Thiobarbituric Acid Reactive Substance (TBARS) including concentrations of ascorbic acid in both plasma and milk were studied.

MATERIALS AND METHODS

Animals and management: Animal managements in the present study were similar to those in the previous study (Chaiyabutr et al., 2011). Ten primiparous, non pregnant lactating crossbred cattle, containing 87.5% Holstein (HF) genes were used and divided into 2 groups of five animals each. Cows in the 1st group were housed in open-sided barn with a tiled roof in Normal Shaded house (NS) as the non-cooled cows. Cows in the 2nd group were housed in open-sided barn of normal shaded house with two sets of Misty Fan cooling (MF) as cooled cows. Each system of misty fan cooling consisted of a 65 cm. Diameter blade fan circulating 81 m³ min^-1 of air with oscillation coverage of 180°. The amount of water discharged from 4 splay heads was 7.5 L h^-1 and size of mist droplet 0.01 mm. Animals were exposed to MF for 45 min at 15th min intervals from 06:00-18:00 h. At night, animals were exposed to MF for 15 min at 45th min intervals from 18:00- 06:00 h.

Cows in both groups were offered, the same ration of Total Mixed Ration (TMR) ad libitum twice a day around 06:00 and 17:00 h when the animals were milking. Ingredient and chemical compositions of feed are shown in Table 1. Meteorological data of both barns were performed using dry bulb thermometer for measurement of the ambient temperature. The relative humidity at NS and MF barns were read by psychrometric chart depending on wet and dry bulb temperature.

Ambient temperatures, humidity, rectal temperature and respiratory rate were measured weekly at 09:00 and 13:00 h on specified day. A Temperature-humidity Index (THI) was calculated from the average ambient temperature of dry and wet bulb temperatures according to McDowell (1972) as follow: THI = 0.72 (wb+db)+40.6. Where; wb = wet bulb temperature and db = dry bulb temperature expressed in °C. Rectal temperature and respiratory rates were measured at different parts of the day with electronic thermometers and observing flank movements, respectively.

The procedures used in the present study were performed in accordance with national animal care guidelines of National Research Council of Thailand and were pre-approved by the faculty ethics committee.

Experimental design: The design of experimental protocol in cooled and non-cooled cows was also similar as that of the previously described (Chaiyabutr et al., 2011). Briefly, the experiments were carried out throughout lactating period in each group.

Table 1:	Feed ingredients and chemical compositions of the diet

The experiment in each group was divided into 3 phases, namely early (day 60 postpartum), mid (day 120 postpartum) and late lactating periods (Day 180 postpartum).

The pretreatment study was conducted on the starting day of each phase. At the end of the pretreatment within the same day, the animal was injected with the 1st dose subcutaneous injection of 500 mg of recombinant bovine Somatotropin (rbST) (POSILAC, Monsanto, USA). Subsequently, the animal was injected with two consecutive doses injections of rbST every 2 weeks. Thereafter within 2 days after the 3rd injection, the treatment study was conducted. The pretreatment, 3 doses of injections and the treatment periods were performed during the 1st 30 days and the same procedures were followed for each phase. During the last 30 days of each phase, no experiments were conducted in order to allow the milk yield from the effect of rbST treatment to return to the control level.

Determinations of plasma concentrations of SH residue, TBARS, albumin and concentration of ascorbic acid in both plasma and milk: On each specified day after the morning milking at around 10 h, blood sample from each cow was collected from the coccygeal vessel by venopuncture into the tube containing heparin 25 i.u. mL^-1 blood. Plasma was separated by centrifugation (3500 x g, 15 min) immediately after sampling and kept at -40°C until analysed.

The concentration of Sulfhydryl residue (SH), the Thiobarbituric Acid-Reactive Substances (TBARS) concentration and the total ascorbate concentration in plasma were measured by spectrophotometry as described in details by Tanaka et al. (2007). In brief, the concentration of plasma Sulfhydryl (SH) residue was measured via the method of Motchnik et al. (1994) using Dithionitrobenzene (DTNB) in the reaction. The TBARS concentration in the plasma, a breakdown product of lipid peroxidation was carried out with an extraction solution contained pyridine and 1-buthanol using the concentration of malondialdehyde as the standard (Ohkawa et al., 1979; Chitra et al., 2003). The milk sample was collected from each animal at the morning milking on the day of study of each period.

Milk aliquots were kept at -40°C until the day of analysis of ascorbic acid in milk. Briefly, the milk samples were centrifuged at 12,000 rpm for 15 min at 4°C. The aqueous phase below the solidified fat layer was removed for the measurement of ascorbic acid concentration. The total ascorbate concentration in both plasma and milk was measured by the oxidation of ascorbic acid with copper ions to form dehydroascorbic acid and diketogulonic acid which react with 2, 4-dinitrophenylhydrazine to form the derivative bis-2, 4dinitrophenylhydrazone (Omaye et al., 1979). The total protein and albumin concentrations in the plasma were determined by an automatic analyzer (Operator Manual BT 2000 Plus, Biotecnica Instruments S.P.A Via Licenza, Rome, Italy).

Statistical analyses: The statistic analyses were performed using General Linear Model (GLM) procedures of statistical software package SPSS (SPSS for windows, V14.0; SPSS Inc., Chicago, IL, USA). The model used for each analysis was:

Where:

Means values were used to evaluate the effect for all variables. Statistical significance was declared at p<0.05 and trends were declared at 0.05<p≤0.10. Duncan’s new multiple range tests were used to detect the statistical significance between different treatment groups. The unpaired t-test was also done for comparison of average meteorological data between cooling and non-cooling barns.

RESULTS

Ambient temperature, relative humidity, temperature humidity index, rectal temperature and respiration rate in animals treated with rbST under Normal Shade (NS) and Misters and Fans cooling (MF) at different stages of lactation: Mean values of environmental parameters in NS and MF barns at morning (09:00 h) and after- noon (13:00 h) are shown in Fig. 1. Ambient temperatures at 09:00 h were not different between NS and MF barns while ambient temperatures in the afternoon of NS barn were significantly higher than those of MF barn (average 37.7 vs. 31.6°C) throughout experimental periods.

Fig. 1:	Ambient temperature, relative humidity and temperature humidity index at morning (09:00 h) and afternoon (13:00 h) in Normal Shade (NS) and Misters and Fans cooling (MF) barns at different stages of location. Comparisons were made between mean values of measurement at the same time of both barns: values marked with significant differences using unpaired t-test, *p<0.05, **p<0.01

The values of relative humidity of NS barn were significantly lower than those of MF barn throughout experimental periods. Values of THI in both barns ranged from 77.9-85.5 which the THI of MF barn in the afternoon were significantly lower than those of NS barn during mid and late stage of lactation.

In afternoon, cows without rbST housed under MF barn showed significantly lower rectal temperature and the respiration rate than those of cows housed under NS barn (Fig. 2). Rectal temperature and the respiration rate were increased after rbST supplementation in both cooled and non-cooled cows when compared with pre-treatment in each stage of lactation.

Effects of misty fan cooling and supplementation with rbST on milk yield, plasma concentrations of sulfhydryl residue, lipid peroxidation-end product (TBARS), plasma albumin and plasma and milk concentrations of ascorbate of crossbred Holstein cows: Milk yield significantly increased after supplemental rbST as compared with the pretreatment period in each stage of lactation in both cooled and non-cooled cows. The decline in milk yield were apparent as lactation advanced in both cooled and non-cooled cows whether supplemental rbST or not.

The concentrations of ascorbic acid in both plasma and milk of non-cooled cows were lower than those of cooled cows. These changes were significantly apparent in early and mid-lactation while it was not affected by rbST supplementation (Table 2).

Fig. 2:

Mean values of rectal temperature (a) and respiration rate (b) in cows treated with rbST under Normal Shade (NS) and Misters and Fans cooling (MF) at morning (09:00 h) and (13:00 h) in different stages of location. Duncan’s new multiple range tests were used to detect the statistical significance between different treatments. Means with the same letter in each time of measurement are not significantly different (p<0.05)

Table 2:	Effects of misty fan cooling and supplementation with rbST on milk yield, plasma and milk concentrations of ascorbic acid of crossbred Holstein cows
SEM = Standard Error of the Mean;1p values for the effects; MF = Misty Fan cooling effect, rbST = rbST effect, MF x rbST = Interaction effect of MF and rbST

Table 3:	Effects of misty fan cooling and supplementation with rbST on plasma concentrations of lipid peroxidation end product (TBARS), sulfhydryl residue, and plasma albumin concentration of crossbred Holstein cows
SEM = Standard error of the mean; 1p values for the effects ; MF = Misty Fan cooling effect, rbST = rbST effect, MF x rbST = interaction effect of MF and rbST

Another antioxidative component, plasma concentration of the SH residue and the concentration of TBARS, oxidation products of polyunsaturated lipid did not significantly differ between cooled and non-cooled cows with or without rbST supplementation.

The plasma albumin concentration did not differ between cooled and non-cooled cows with or without rbST supplementation (Table 3).

DISCUSSION

The values of ambient temperature in NS barn recording at 13:00 h were significantly higher than those of MF barn throughout the experimental periods. Values of the daytime THI in both barns ranged from 77.9-85.5 which were higher than the threshold level (THI 72) of comfortable zone (Armstrong, 1994). It indicates that cows in both groups were subjected to moderate heat stress (Fuquay, 1981). However both respiratory rate and rectal temperature of cows under MF were affected by the cooling effect in comparison with those of non-cooled cows which indicate a partial alleviation of heat stress by MF system especially in the afternoon.Milk yield of cows under NS barn in each stage of lactation were smaller than those of cows in MF barn. The reduction in milk production in a hot environment is usually attributed to a fall in feed intake. However, cows in both groups in the present study were fed similar diet throughout studies. Therefore, the low milk yields of cows under NS barn as compared with those of cows under MF barn would attribute to direct effect of high temperatures even cows were fed with fixed level of feed intake (Wayman et al., 1962).

The previous studies in 87.5% crossbred Holstein cattle in the tropic showed that the decline in milk yield as lactation progressed to mid and late lactation would concomitant to the decrease in the concentration of plasma bST (Chaiyabutr et al., 2000). Cows under NS and MF barns receiving rbST, significantly increased milk yields after treatment in each stage of lactation and decreased as lactation advanced to mid and late lactation. These results confirm that an increase in milk yield in response to rbST administration will not be sustained indefinitely (Bauman, 1992) and it is influenced by the stage of lactation (Phipps et al., 1991). However, rbST is one of the factors capable of stimulating milk production efficiency in crossbred lactating animals in the tropical environment.

It is known that heat stress, induces oxidative stress resulting increase in formation of ROS and/or decreased antioxidant reserve. Oxidative stress affecting to cells by the oxidizing of macromolecules resulting from increased formation of ROS have been studied in heat-stressed cells (Flanagan et al., 1998) and in cultured tissue model (Skibba et al., 1990). In the present study in different stages of lactation, clarifications of the oxidative stress of crossbred cows under moderate heat stress and supplemental rbST with determinations of the alteration of plasma biomarkers were performed. Under high ambient temperatures, a marked increase in the respiratory rates of cows under NS barn might increase oxygen pressure of blood which might be the cause of alteration of oxidative status. Crossbred Holstein cows under moderate heat stress (high THI) in NS barn seemed to induce oxidative stress leading to reduction of the plasma ascorbic acid concentration in early and mid lactation. Reduction of the plasma ascorbic acid concentration in late lactation was less pronounced.

Ascorbic acid is known as an antioxidative vitamin serving as a water-soluble reductant to donate a free molecule of hydrogen that detoxifies the harmful ROS generated by the body to a less toxic form during heat stress (Frei et al., 1989).

Changes in the antioxidative activity of ascorbic acid during heat stressed is also observed in Bos Taurus dairy cattle (Tanaka et al., 2007) when the body ascorbic acid is exhausted (Altan et al., 2003; Tauler et al., 2003). Cows have not been considered to need dietary ascorbic acid because they are able to synthesize a high enough level of ascorbic acid in themselves. The significant reduction of ascorbic acid concentrations in plasma of non-cooled cows during early lactation and mid lactation were apparent independent on the effect of rbST. A reduction of antioxidant activity in plasma during mid lactation has also been observed in heat-stressed Holstein cows (Harmon et al., 1997). The reductions of the concentration of ascorbic acid in plasma have also been observed in calves under heat stressful conditions of cowshed environments (Cummins and Brunner, 1991). High ambient temperatures in NS barn increased the rectal temperature of cows up to 39.9°C in mid lactation. Negative relationship of ascorbic acid concentration in plasma and rectal temperature of dairy cows has also been reported (Tanaka et al., 2007). It is possible in the present study that high milk yield in early lactation have more metabolic activity and produce more body heat production (West, 1994).

The combination of body heat increment either consequence of rbST treatment (Cole and Hansen, 1993) and high environmental temperatures would lead to sensitive to oxidative stress. In the present results, there was dissociation among plasma biomarkers of oxidative status.

The components of SH residue, Thiobarbituric Acid Reactive Substance (TBARS) as an index of lipid peroxidation and the plasma albumin concentration were not affected in cows under NS barn as compared with cows under MF barn whether supplemental rbST or not. The study of Calamari et al. (1999) in Bos tuarus cows showed that a reduction of plasma lipid soluble antioxidants (vitamin E and β-carotene) and an increase of plasma TBARS under moderately heat-stressed during summer were apparent particularly in mid-lactation.

In the present results, the concentration of SH residues in plasma of both cooled and non-cooled cows supplemental rbST were closed to the plasma albumin concentrations indicating that most protein SH groups are found on plasma albumin (Radi et al., 1991).

Albumin is thought to be one of resources of SH residue in the plasma. The reversible oxidations of sulfur residues are common and fundamentally important in the control of cell functions (Moran et al., 2001). SH residues are abundant both inside and outside the cell as non-protein and protein SH groups which predominate over the oxidized form. Albumin possess only one SH residue in a molecule (Carter and Ho, 1994). It was observed that no alterations in the ratio of SH residues to albumin concentrations were apparent in plasma of both cooled and non-cooled cows supplemental rbST. It is possible that crossbred cows using in the present study containing indigenous genes have heat tolerance.

Cows did not expose to severe heat stress which a marked increase in plasma total solids including plasma protein concentrations causing of alteration of plasma albumin concentration have been noted in both cattle and buffalo under severe heat exposure (Bianca, 1957; Chaiyabutr et al., 1987). In the present results, there was dissociation among plasma biomarkers of oxidative status. The plasma SH concentration did not show pattern as the plasma ascorbic acid concentration although, oxidized ascorbic acid by oxidative stress has been reported to be reduced using the reducing equivalent of SH residues in rat plasma (Vethanayagam et al., 1999). It is possible that the oxidation of the SH residues by oxidative stress did not utilize the same mechanism as that of ascorbic acid oxidation. An ascorbic acid concentration in the plasma of dairy cows seems to be important in reducing its equivalent in body fluid and is sensitive to oxidative stress. These results suggest that ascorbic acid concentrations in cow plasma are sensitive to high environmental temperature.

The TBARS concentrations in plasma of crossbred cows in both groups were not derived from oxidative stress under moderately heat stress although, the concentration of a breakdown product of lipid peroxidation, TBARS is known to be one of the oxidative stress markers in the plasma (Flemming et al., 1997). However, Armstrong and Browne (1994) reported that the thiobarbituric acid test would be considered to be a good general indicator of oxidative stress rather than a marker of lipid peroxidation. Weak negative effects of moderate heat stress on the plasma SH and TBARS levels for oxidative status were apparent in crossbred cows.

It is possible that the primiparous cows in both groups using in the present study may be less affected than multiparous cows during exposure to high environmental temperatures (Folman et al., 1979). Other findings (Miller et al., 1993) have also shown that an imbalance of the oxidative status during different stages of lactation was observed in Holstein cows.

CONCLUSION

From the present results, different plasma biomarkers using for determination of the effect of heat stress on oxidative status suggest that measurement of plasma level of TBARS is not sensitive to the oxidant-antioxidant balance in crossbred dairy cows exposure to high environmental temperatures. The study of antioxidant reserve of ascorbic acid concentration in plasma is an appropriate and sensitive model to study the oxidative status of crossbred dairy cows during exposure to high temperatures.

ACKNOWLEDGEMENTS

This study has been partly supported by The Thailand Research Fund (BRG498004).