Effect of Supplemental Chromium, Vitamin E and Selenium on Biochemical and Physiological Parameters of Holstein Friesian Calves under Heat Stress

Livestock rearing is a major continuous income generating activity for the rural households (Mahla et al., 2015). Climatic variables like humidity and temperature have an intense effect on the performance of the animals. The Thermoneutral zone (TNZ) is comfortable for the animal, the core temperature is generally within physiological limits and body temperature regulation is achieved only by sensible heat loss (Charles, 1994; IUPS, 2001). Dairy animal’s TNZ lies in the range of 16°C-25°C, within which a physiological body temperature of 38.4-39.1°C is maintained by them (Yousef, 1985). Animals  are  able  to  adjust  to  adverse  climate  by  means  of  acclimatization  and  adaptation  (Roy  and  Collier,  2012). Extreme climatic conditions that cannot be compensated by thermoregulatory mechanisms result in thermal stress (Roland et al., 2016). Calves and heifers generate less metabolic heat and have greater body surface area relative to body mass, hence efficiently dissipating body heat and are thus considered to be more tolerant of heat stress than mature cattle (West, 2003), still they suffer from heat stress to some degree (Wang et al., 2020). Temperature-Humidity Index (THI) is a parameter widely used to describe heat load on animals and is a good indicator of stressful thermal climatic conditions (Das, 2018; Habeeb et al., 2018).
       
Heat stress brings about changes in immunity and hormonal concentrations. Primary indicators of immune response comprise of protein, glucose, packed cell volume, hemoglobin, red blood cells and white blood cell concentrations in blood which get changed due to thermal stress (Das et al., 2016). The cortisol level is an important indication of heat stress during summer season (Sinha et al., 2019). The heat stress induced changes potentially enhance the formation of reactive oxygen species (El-Kholy et al., 2018). Reactive oxygen species induce oxidative stress and damage the cellular macromolecules causing lipid peroxidation, nucleic acid and protein alterations (Adwas et al., 2019). The animal shows several physiological adaptation mechanisms to cope up the adverse climate condition. Some of the physiological determinants of adaptations to heat stress are respiration rate, rectal temperature, pulse rate, skin temperature and sweating rate (Indu and Pareek, 2015). Respiration rate is one of the ideal biomarkers of heat stress. In homeostasis, PR reflects the circulation and metabolic status (Rashamol et al., 2020).
       
Dietary modifications could help animals to retain their normal physiological and blood biochemical profile thereby maintaining the general wellbeing of the animals. Antioxidants, both non-enzymatic and enzymatic, offer required defense against oxidative stress resulted due to thermal stress (Kumar et al., 2011). Vitamin E and selenium are documented antioxidants (Domoslawska et al., 2018). Chromium is gaining worldwide importance for combating heat stress adverse effects and it is believed as a promising agent. Hence, this study was performed for investigating the chromium propionate effects on physiological as well as the biochemical profile of calves under thermal stress.

Site of study
 
The current study was performed at Kapila Agro Farm, a commercial dairy farm situated at Timmareddypally village of Kondapak Mandal in Siddipet district, Telangana State,  located at 17°58¢25.7² North and 78°51¢56.5² East, at 547 meters altitude above the sea level. Analysis of blood and feed sample was carried out at ICAR-CRIDA (ICAR-Central Research Institute for Dryland Agriculture), Santoshnagar, Hyderabad.
 
Animals and diet
 
The trial was performed for a 90d period during peak summer season (April 15 to July 15) in the year 2017 on 24 H.F calves with an average body weight of 172.96±4.39 kg and aged 7-8 months . They were randomly distributed into four groups of six animals in each group considering their body weights. Calves either received a basal diet (T₀) or were supplemented with vitamin E + selenium (T₁: 500 IU vitamin E + 0.3 mg selenium per kg DM); chromium propionate (T₂: 0.5 mg chromium per kg DM) and chromium propionate + vitamin E + selenium (T₃: 0.5 mg chromium per kg DM+ 500 IU vitamin E + 0.3 mg selenium per kg DM) and fed with a total mixed ration (TMR) as basal diet. All the calves were stall fed, reared under standard management conditions and housed in tie stalls with fans. All the groups were fed total mixed ration (TMR) diet twice daily. Fifty per cent of the daily feed allocation was provided in the morning feeding at 8.00 am and fifty per cent in the evening at 4.00 pm during the trial period. The TMR contained chopped paddy straw, maize fodder (sweet corn with cobs and grains in milk stage) and concentrate mixture (Table 1) with roughage: concentrate ratio of 70:30. The basal diet was formulated to contain all the necessary nutrients to meet the nutrient requirements as recommended by ICAR (2013). Chromium propionate, vitamin E and selenium were supplemented to the individual animal daily in the morning in the form of a 20 g jaggery bolus. The calves had free access to fresh wholesome clean drinking water all the time.
 

 
Temperature humidity index (THI)
 
Ambient air temperature and humidity were measured for calculation of THI. Dry bulb and wet bulb temperatures were recorded twice a day at 10.00 am and 3.00 pm using dry and wet bulb thermometers placed about 1.5 m above the ground level. The degree of heat stress was determined according to mean THI values measured. THI was estimated for the entire study period as per Bianca (1962).
 
 
 
Where
Tdb:   Represents dry bulb temperature.
Twb:   Represents Wet bulb temperature (°C).
 
Blood collection
 
Blood samples were collected fortnightly from the jugular vein of all the experimental animals, prior to the morning feeding with the help of sterilized needles. Each time two blood samples were collected from each animal; one sample of 5 ml in heparinized vacutainer, for hematology and the other 10 ml in the vacutainer coated with clot activator for serum separation. The samples were refrigerated at 8°C and transported immediately to the laboratory for analysis. Within an hour, the blood samples were centrifuged for 10 minutes at 3500 rpm and blood serum was collected and stored -20°C for further use.
       
Whole blood samples were analyzed using automatic haematology analyzer (ABX Micros 60, USA). Thyroid profile and cortisol were estimated using chemi luminescent immuno assay method with FUJI DRI-CHEM NX 500V (Japan). Alkaline phosphatase, aspartate amino aransferase, alanine amino transferase, total protein, albumin, cholesterol, triglycerides, glucose, urea, creatinine, were analyzed by kits (Bio systems, Spain) using blood biochemical semi auto analyzer (ichem~168, Spain).

Physiological responses measurement
 
Physiological responses including rectal temperature (RT), respiration rate (RR) and pulse rate (PR) of all the experimental animals were recorded twice daily at 10.00 am and 3.00 pm during the entire length of the experimental period of three months. The rectal temperature was measured by inserting clinical thermometer per rectum and observation was recorded after one min. The respiration rate was recorded by observing the flank movements for one minute in which each inward and outward movement of the flank was counted as one complete respiration. The pulse rate of the animals was recorded by observing the pulsation of middle coccygeal artery at the base of the tail.
 
Chemical and statistical analysis
 
Samples of TMR were collected, dried overnight in a hot air oven at 100±5°C and then ground in laboratory Willey mill and preserved in airtight containers for further chemical analysis. The proximate analysis and fiber fractions of feeds was performed as per the procedures described by AOAC (2005) and Van Soest et al., (1991) methods respectively.
       
Statistical analysis of the data was carried out according to the procedures suggested by Snedecor and Cochran (1994). Least-square Analysis of variance was used to test the significance of various treatments and the difference between treatments means was tested for significance by Duncan’s new multiple range and F Test (Duncan, 1955).

Chemical composition of the feed
 
The chemical composition of experimental rations fed to the H.F calves is presented in Table 2. The per cent of crude protein in the TMR, maize fodder and paddy straw fed to the growing calves were 13.90, 8.68 and 3.40 per cent on a dry matter basis, respectively.
 

 
Environmental conditions and microenvironment
 
Temperature humidity index (THI) at the start of experiment (1^st week of April) averaged 75.83±0.22 in the morning (10.00 am) and 80.44±0.85 in the afternoon (3.00 pm) and the THI decreased with the decrease in environmental temperature in July to 72.25±0.31 and 76.69±0.48 in the morning and afternoon, respectively. The mean THI during 90 days of experimental period was 75.10±0.42 in the morning and 80.01±0.64 in the afternoon indicating the animals were under mild to moderate stress. High THI was found in the May month with 83.48±1.41 THI value in the afternoon (Fig 1).
 

 
Biochemical profile
 
Hematological profile has been depicted in Table 3. Overall  mean values for the hematological profile were comparable among calves fed experimental rations. No significant difference was found in groups T₀, T₁, T₂ and T₃. Similar findings were reported by Kumar et al., (2016) and Shinde et al., (2009) who did not find any effect of supplementation of chromium and vitamin E with selenium respectively, on calves.
 

                 
In the present study, hormonal profile revealed (Table 4) that in T₁ group supplemented with vitamin E and selenium the cortisol values were numerically lower than control. This lower cortisol levels could be due to reduced production of reactive oxygen metabolites by supplemental vitamin E and selenium (Gupta et al., 2005). Chromium supplementation reduced the cortisol levels in groups T₂ (P>0.05) and T₃ groups (P<0.05). Obtained results are consistent with the research studies of Chang and Mowat (1992) who suggested that chromium potentiates the insulin action that may reciprocally prevent cortisol. The present results agreed with the findings of Soltan et al., (2012) and Kumar et al., (2016) in buffalo calves with chromium supplementation. There was no significant difference among the groups in Triiodothyronine (T₃), Thyroxin (T₄) activity and T₄: T₃ in calves fed ration T₀, T₁, T₂ and T₃.
       
The mean values for the liver enzymes activity ALP, AST and ALT didn’t vary significantly (p> 0.05) between the experimental group which was in accordance with the results of Nejad et al., (2016), Patil et al., (2017) with chromium supplementation and Shinde et al., (2009), Hala et al., (2014) with vitamin E and selenium in calves and kids, respectively. The T₀ group fed only on basal diet showed higher values for the liver enzymes activity indicating more leakage of these enzymes into the extracellular fluids which might be due to heat stress-induced oxidative damage to the liver cells.
       
Overall mean total serum protein was significantly (P<0.05) high in the T₃ group followed by T₂, T₁ and T₀ indicating the critical role of chromium. This could be due to the decrease of serum cortisol concentration or an increase of sensitivity tissue to insulin (Roginski and Mertz, 1969). The role of insulin is proved at increase of synthesis of proteins (Roginski and Mertz, 1969). The findings of the current research corroborated with the results of Hala et al., (2014) and Pechova et al., (2002). Perusal of the table indicates that the albumin values were greater in chromium supplemented groups compared to T₀ and T₁. Khalili et al., (2011) suggested an increase in the synthesis of amino acid in the liver probably through insulin due to chromium supplementation. The results are in agreement with Nejad et al., (2016) who found relatively higher albumin levels in Holstein steers supplemented with chromium. The mean value for serum globulin and albumin: globulin ratio didn’t vary significantly among the groups.
       
No difference (p>0.05) was observed among the groups in triglyceride and cholesterol concentration in the current study. Nejad et al., (2016) also found no deviation in cholesterol and triglyceride values in Holstein steers supplemented chromium from that of the control group.  Similarly, Shinde et al., (2009) also revealed that total cholesterol and triglycerides concentration was statistically similar in vitamin E and selenium supplemented group and the group devoid of supplementation. Overall mean values of the serum glucose, urea as well as creatinine concentration were similar across the treatments (P>0.05). No effect of chromium (Pechova et al., 2002; Nejad et al., 2016) and vitamin E and selenium (Tahmasbi et al., 2012; Alhidary et al., 2015) was observed on glucose in previous studies during summer. Likewise, no effect on blood urea nitrogen and creatinine was observed in trials conducted by Zhang et al., (2014) with chromium supplementation and Hala et al., (2014) with vitamin E and selenium supplementation.
 
Feed intake and physiological measures
 
The results indicated that the DMI did not differ significantly (p>0.05) among four groups of calves fed experimental rations but relatively higher DMI was found in the supplemented groups compared to control group (Fig 2). Increased DMI may be attributed to the lower stress levels as reflected by lower serum cortisol level in the supplemented calves. The RT, PR as well as RR did not vary significantly (P>0.05) in the morning among the four experimental groups (Table 5). However, in the afternoon calves in the control group had higher RT, PR and RR compared to supplemented groups with a significant (P<0.05) variation in T3 group. Lower serum cortisol levels in T₁, T₂ and T₃ group calves indicate heat stress mitigation potential of dietary supplemented chromium and vitamin E with selenium by lowering the oxidative stress at the cellular level and thus resulting in better thermoregulation and lower RT, PR and RR in calves in T₁, T₂ and T₃ groups. Chauhan et al., (2016) also reported a reduction in RT (P=0.08) and RR in heat-stressed lambs fed supra nutritional selenium and vitamin E levels. Yari et al., (2010) also concluded that chromium supplementation lowered the respiration rate in calves.
 

Dietary supplementation of chromium reduced the thermal stress impacts in Holstein Friesian calves more effectively than vitamin E and selenium. Further, supplementation of vitamin E and selenium along with chromium had significant additional benefit which is reflected in the physiological variables and the cortisol levels in the present study.  Hence, supplementation of chromium along with vitamin E and selenium could be more beneficial to mitigate negative impacts of heat stress and improving the performance of H.F. calves.