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Influence of Temperature Humidity Index and Dietary Energy on Certain Stress Marker Profile of Murrah Buffalo Heifers

Papori Talukdar Shivlal Singh Kundu Gautom Mondal
Vol 7(4), 195-204

The present study was undertaken to assess the influence of temperature humidity index (THI) and dietary energy (DE) on certain metabolic and endocrine profile of Murrah buffalo heifers. Thirty six heifers were distributed into three groups (T1, T2, and T3) comprising six animals in each group. The ration was formulated as control group (T1) having ME content according to ICAR and T2 and T3 having 115% and 85% than control respectively, in total mixed based ration. The effect of rations was observed in high THI, 78-85 (summer) and low THI, 50-61 (winter) separately for a period of 120days. The Blood samples were collected aseptically at monthly interval. The respiratory rate, pulse rate and rectal temperature were higher during high THI (summer season) in compression to low THI (winter season). Though there was no significant variation on plasma thyroid (T3, T4) and IGF-1 level among the groups but in high THI period, the level were significantly (P<0.01) decrease in comparison to low THI period. Plasma cortisol level showed significantly (P<0.01) higher in T3 group in both the season in comparisons to T1 and T2 which indicated stress to the animals. In conclusion, increasing ME content could be helpful in alleviating stress during both the seasons and moreover, during summer period, where the animals were at higher stress, increasing ME content will support to climatic resilient livestock production.

Keywords : Energy Level Season Metabolic Hormone Stress Hormone Murrah Buffalo Heifer


Environmental stress has a major concern in animal productivity especially in tropical condition. Temperature, humidity, sunshine, and wind velocity may increase or decrease energy demand depending upon region which will be reflected on various metabolic indices in the body. Their levels in the blood may vary in response to various dietary, physiological and environmental stimuli (Bova et al., 2014). Understanding of the system physiology and effects of dietary and environmental stressors that interfere with the metabolic and endocrine system may be helpful to reduce the negative impacts and/or to acclimatizing animal to these stressors to the greatest possible degree for proper health and enhanced product quality. Exposure to stressful environment produces reduction in the rates of metabolism and alteration of various indicators of stress. Also acclimation to thermal stress imposes physiological and metabolic adjustments associated with reduction of performance and compromising of health (Bernabucci et al., 2010). Dietary energy levels play important role on reducing environmental stress by a shift toward carbohydrate use and minimize heat-induced damage. Dietary energy levels have influence on maintenance of thermal balance as a portion of the metabolizable energy typically used for production must be diverted to regulate thermal balance. Naturally, a reduction in nutrient intake during stress combined with increased energy expenditure for maintenance lowers energy balance, and the animal lose significant amounts of body weight during climatic stress. Glucose becomes the favoured fuel of heat-stressed animals as chronic heat stress decreases circulating glucose levels and the increased glucose pool entry coupled with the decreased blood glucose may suggest a higher rate of glucose leaving the circulating blood pool. Also due to the increased reduction of fatty acid oxidation under chronic heat stress, heat-stressed animals become increasingly dependent on glucose for their energy needs (Slimen et al., 2016). Thus adequate level of additional energy beyond maintenance will be required for metabolic adaptation to stress. Moreover, Murrah buffalo (Bubalus bubalis) is an important breed in northern tropical part of India for its contribution to its superior quality of milk, better ability to utilize low quality roughage. But the buffaloes are more susceptible to extreme climatic conditions due to their scarcely distributed sweat glands, dark body color and sparse hair on body surface. The increased susceptibility of buffaloes to climatic stress is primarily due to their high metabolic rate, compared with that of other ruminants. Impact of varying dietary energy levels at different season on endocrine stress marker profile in tropical countries for Murrah buffalo heifers is limited. Therefore, the present study was undertaken to reduce stress by adjustment in feeding system energy requirements at different season besides climate resilient livestock production

Material and Method

The experiment was conducted at the Livestock Research Centre, ICAR-National Dairy Research Institute (NDRI), Karnal (India) situated on an altitude of 250 meter above the sea level, latitude and longitude position being 29o42”N and 79o54”E respectively.

Recording of Climatic and Physiological Variables

Microclimatic data viz., dry bulb temperature, wet bulb temperature, maximum and minimum temperature and relative humidity was recorded at 7:30 h and 14:30 h using Zeal (UK) dry bulb and wet bulb thermometer and relative humidity by automatic hygrometer every day during experimental period. Temperature Humidity Index (THI) was calculated using the formula of Johnson et al., 1963.

THI= 0.72 (Tdb + Twb) + 40.6

Where, Tdb = Dry bulb temperature (ºC), Twb = Wet bulb temperature (ºC)

Physiological parameters viz. Respiration Rate (RR), Pulse Rate (PR), Rectal Temperature (RT) was recorded daily between 8.00 – 9.00AM using standard method.

Grouping and Feeding of Animals during Growth Experiment

Thirty six growing Murrah buffalo heifers of 9-13 months of age (body weight 158.51 ± 16.5 kgwere selected and divided into 3 groups in a randomized block design (RBD), comprising six animals viz. control group (T1) and experimental groups (T2 and T3). The animals were fed on three total mixed rations (TMR) along with concentrate (Table 1 and 2).

Table 1: Ingredients and their proportions (% parts) in the concentrate mixture (on % DM basis)



T1 T2 T3
ME (ICAR, 2013) (+15%) ME (-15%) ME
Maize grain 30 30 10
Mustard cake-deoiled 10 10 30
Soyabean meal 21 21 12
Wheat bran 16 16 30
Rice bran 20 20 15
Mineral mix. 2 2 2
Salt 1 1 1
Total 100 100 100
CP (%) 20.3 20.3 22.3
ME(Mcal/ Kg) 2.82 2.82 2.58

The control ration (T1) having ME content according to ICAR (2013) and T2 and T3 were having 115% and 85% ME than control respectively, in an isonitrogenous diet. The feeding trial was conducted 120 days each in high and low THI period i.e. THI, 78-85 (summer) and THI, 50-61 (winter). Maize green (Zea mays) fodder was used during summer season while during winter season oat fodder (Avena sativa) was used as green in TMR preparation. The offered feed and the remaining residue were weighed daily in each animal. Fresh and clean drinking water was provided ad libitum.

Table 2: The ratio and chemical composition (%DM) of rations fed to different experimental groups during high THI (summer) and low THI (winter) conditions

Particular TMR-1 TMR-2 TMR-3
ME levels ME as per ICAR, 2013 ME (>15%) than ICAR, 2013 ME(<15%) than ICAR, 2013
Concentrate 0.35 0.45 0.3
Green(Maize/Oat) 0.3 0.35 0.25
Wheat straw 0.35 0.2 0.45
Particular TMR-1 TMR-2 TMR-3
DM 61.22 ± 0.71 58.11 ± 0.84 67.73 ± 0.62
OM 91.39 ± 0.05 91.44 ± 0.09 89.69 ± 0.06
CP 11.61 ± 2.06 13.59 ± 1.52 11.78 ± 1.71
EE 2.39 ± 0.08 2.61 ± 0.11 2.10 ± 0.07
NDF 55.45 ± 0.33 50.02 ± 0.29 58.08 ± 0.18
ADF 27.50 ± 0.22 24.08 ± 0.18 29.02 ± 0.20
Lignin 6.25 ± 0.23 5.69 ± 0.19 4.16 ± 0.10
NDICP 3.16 ± 0.07 3.42 ± 0.07 2.18 ± 0.11
ADICP 1.50 ± 0.03 1.54 ± 0.06 0.65 ± 0.04
Ash 7.65 ± 0.12 7.36 ± 0.23 10.31 ± 0.18
NFC 21.94 ± 0.10 25.23 ± 0.18 17.73 ± 0.14
TDN (kg) 58.49 ± 0.43 62.85 ± 0.34 55.37 ± 0.27
DE (Mcal/kg) 2.46 ± 0.07 2.69 ± 0.06 2.11 ± 0.07
DE (MJ/kg) 10.30 ± 0.17 11.52 ± 0.20 8.81 ± 0.23
ME (Mcal/kg) 2.07 ± 0.16 2.35 ± 0.24 1.95 ± 0.22
ME (MJ/kg) 8.67 ± 0.09 9.84 ± 0.10 8.15 ± 0.08

* Conc.= concentrate ration; DM= dry matter; OM= organic matter; CP= crude protein; EE= ether extract; NDF= neutral detergent fiber; ADF= acid detergent fiber; NFC= nonfibrous carbohydrate; TDN= total digestible nutrient; DE= digestible energy; ME= metabolizable energy

Collection of Samples

Blood samples (10 ml) were collected from each animal in a sterile heparinized vacutainer tubes from jugular vein with a minimum stress on day 0, 30, 60, 90, and 120 in both the season. Immediately after collection, plasma were separated and stored in -20°C for further estimation of plasma cortisol, T3, T4 and IGF-1.

Assay for Plasma Hormones

Plasma levels of thyroxine (T4), triiodothyronine (T3) were estimated by radioimmuno assay method using RIA kits as per standard procedure specified along with the kits (supplied by Board of Radiation and Isotope Technology, Mumbai, India). The hormone cortisol and IGF1 was estimated in heparinized plasma samples using ELISA kit (supplied by Cayman Chemical Co., USA).

Sampling and Analysis of Feeds and Fodders

Representative sample of feed, fodder and residue were collected daily and dried in hot air oven at 70ºC for 48 h to estimate their DM content. The feed samples were analyzed for proximate constituents (AOAC, 2005). The TDN, DE and ME of the feeds were estimated from their chemical composition (NRC, 2001).

Statistical Analysis

The data of the growth trials were compared by analysis of variance as per two way analysis method using the general linear model (GLM) procedure of the statistical analysis system (SAS, 1996), version 9.3 using the model Yijk = μ + Ei + Sj + (E*S) jk + Ɛijk. Where, Yijk represented individual observations of the variable, μ was the overall mean, Ei and Sj was the fixed effect of the ith and jth variable, (E*S) jk was interaction effect of jth and ith variable and Ɛijk was the random error associated with Yijk, i.e., not accounted in the fixed effect. Significant differences of the treatments and season were considered at P<0.05 level and at P<0.01 for highly significant.

Results and Discussion

The average minimum and maximum temperature inside the experimental shed, throughout the hot dry, hot humid summer and winter experimental period are depicted in Table 3. The temperature humidity index (THI) varied from 78.35 to 85.11 during summer, 80 during mid-summer period and 50 to 60 during winter period. The animals were in stress during mid-summer period as per previous report (LPHSI, 1990) who quantified heat stress, as, THI up to 72 no heat stress, 72-78, moderate heat stress, 78-85, severe heat stress.

Table 3: Climatological variables recorded during experimental periods

Season Temperature (ºC)
Av. Maximum Av. minimum Average Relative humidity (%) Temperature humidity index (THI)
Summer 41.77 22.0 31.86 51-74%, 78.35 – 85.11
Winter 19.0 3.0 11.0 56-83% 50 – 61

Effect of Energy Levels at Different Season on Plasma Hormonal Profile

The mean values of plasma tri-iodothyronine (T3) and thyroxin (T4), cortisol and insulin like growth factor-1 (IGF-1) in different experimental groups during high and low THI period are presented in Table 4.

Table 4: Effect of different energy levels on physiological and plasma metabolic and stress hormone levels of Murrah buffalo heifers during high THI summer and low THI winter season

Season High THI (Summer) Low THI (Winter) P value
Treatment T1 T2 T3 T1 T2 T3 S T S*T
Hormonal and Metabolic variables
T3(ng/ml) 0.99± 0.07 1.03± 0.07 0.96± 0.05 2.04± 0.06 2.05± 0.08 2.0± 0.09 <0.001 ns ns
T4(ng/ml) 19.70B± 1.12 20.80± 1.48 19.40± 1.11 40.61± 1.07 38.02± 1.25 42.25± 1.29 0.002 ns ns
Cortisol (ng/ml) 4.43Ab± 0.10 4.23Ab ± 0.09 4.98Aa ± 0.11 3.21Bb ± 0.13 3.18Bb ± 0.09 3.46Ba ± 0.07 <0.001 0.003 0.03
IGF-1(ng/ml) 108.61B± 5.12 113.76B± 3.88 105.06B± 5.51 122.02A± 7.87 129.64A± 7.45 118.14A± 4.94 0.03 ns ns
Physiological variables
Rectal temperature(ºC ) 38.49Ab± 0.16 38.53 Ab± 0.20 39.46 Aa± 0.24 37.25Bb± 0.02 37.14 Bb± 0.12 37.33Ba± 0.04 0.05 0.05 ns
Respiration rate(breath/min) 36.35Ab± 0.23 32.77Ab± 0.34 47.86Aa± 0.21 22.15Bb± 0.24 22.37Bb± 0.10 32.44Ba± 0.21 0.01 0.05 ns
Pulse rate (pulse/min) 77.67A± 0.09 75.45 A± 0.21 78.39 A± 0.31 61.37 B± 0.27 67.2 B ± 0.32 64.41 B ± 0.30 0.05 ns ns

A, B and a, b means bearing different superscripts in the same row, differ at P<0.05 by least squares means for season and treatment level effect respectively (*ns= not significant; S= season; T= treatment; S*T= interaction effect between season and treatment)

Plasma Thyroid Hormones

There was no significant effect of energy levels on T3 and T4 hormone levels among all groups. As in the present study energy content was not limiting or excess in high or low ME group to alter the body homeostasis and major changes in the hormone concentration. Although T3concentrations were indicative of energy balance and T4 appears to be positively associated with energy consumption (Silva et al., 2014). Nowak et al., 2013 reported that serum thyroxin concentration was not affected by medium (0.69 UFL/kg DM) to low (0.61 UFL/kg DM) level energy density diet of dry Holstein cows (UFL is French energy system, used as a unit of net energy, which is equivalent to 1 kg standard air-dried barley). Oler and Glowinska (2013) reported no significant changes in blood serum thyroxin (T4) concentrations in bulls fed with ration having energy limited to 80% of the maintenance requirement but there was decrease in (P<0.05) the triiodothyronine (T3) concentration.

Plasma Cortisol

Plasma cortisol was significantly (P<0.01) lower in both the season in high ME (T1, T2) groups. Among the treatment groups reduced cortisol values in T1 and T2 group indicated that these animals had less stress compared to T3 group in both high and low THI period. Reduced (P<0.01) serum cortisol with increased energy availability was also reported by Singh et al., 2013 in Muzaffarnagari lambs when fed diets with 100, 80 and 70 per cent of their metabolizable energy requirement during 180 days study.

Plasma Insulin like Growth Factor-1 (IGF-1)

The IGF-1 concentration (ng/ml) increased with increasing ME levels in different treatment groups during both the seasons. Campanile et al. (2010) observed no difference in plasma IGF-1 concentrations on low or high energy diets and the level ranged from 79.1 (low energy) to 95.5 ng/ml (high energy) in buffalo heifers. The IGF-1 levels varied within a wide range in 5 month old buffalo calves (115-470 ng/ml) and IGF-1 level increased with increasing age (months) of the buffalo calves (Bova et al., 2014). Wu et al. (2010) also observed that cows fed concentrates with high energy have greater magnitude of the GH-induced increase in serum IGF1 concentration than low energy hay feeding.

Effect of Season on Plasma Hormone Profile

The level of plasma T3, T4 and IGF-1 was significantly (P<0.01) lower during high THI summer season but among the treatment groups it did not show any significant difference. But plasma cortisol showed significant (P<0.01) difference in between the season and among the treatment groups.

Plasma Thyroid Hormones

In the present study the thyroid hormone levels in the summer months were less as compared to winter months. Thyroid hormones decreased in the present study with increase in the thermal temperature might be an adaptive mechanism followed by animals to reduced metabolic rate and heat production. Rasooli et al. (2004) reported significantly lower levels of T3 and T4 in Holstein heifers during hot summer compared to the winter season. Similarly, Mayahi et al. (2014) reported that thyroid hormone level was maximum in winter, decreased in spring and reached to the lowest value in summer in both buffaloes and Friesian cows. It has been reported that thyroid hormones are the primary determinants of basal metabolic rate and have a positive correlation to weight gain/tissue production, feed intake and energy and nitrogen balance (Caldeira et al., 2007) which might be the reason for more circulatory thyroid hormones in the present study among the treatment groups during winter as compared to the summer season.

Plasma Cortisol

In between seasons there was significantly (P<0.001) higher plasma cortisol level was observed during summer season compared to the winter season. It has been reported that cortisol level was affected by the thermal stress. It indicates that cortisol level was affected by the thermal temperature variation. Yousef et al. (1997) observed that plasma cortisol concentration increased from 11 to 29 ng/ml when exposed to direct solar radiation of the hot summer in Friesian calves. Marai and Haeeb (2010) reported that the cortisol values during February and July, were 9.07 and 12.53 ng/ml respectively, in Egyptian buffaloes and also stated that concentration of cortisol is altered by acute and chronic heat exposure along with changes in photoperiod. In the present experiment, reduced cortisol values in T1 and T2 group indicated that these animals had less stress compared to T3 group.

Plasma Insulin like Growth Factor-1 (IGF-1)

The significantly (P<0.05) lower IGF-1 level was found during summer season in all treatment groups than winter season. Somal and Aggarwal (2014) reported significantly (P<0.001) lower plasma IGF-1 levels during summer as compared to those in thermo neutral conditions in both dry Sahiwal and Karan Fries cows.

Effect of Energy Levels and Season on Physiological Parameters

The increase (P<0.05) RT and RR in low ME (T3) group might be due to more fiber in diet leads to more heat increment generation. The significantly (P<0.05) higher RR, PR and RT indicates that the animals are under stress and was higher during summer season. Among the physiological parameters respiratory rate is the best predictor of heat stress in dairy cattle (Dalcin et al., 2016). The increase in respiration rate with the increasing temperature may be due to the more demand of oxygen by the tissues in stressful condition. The results of physiological responses are in agreement with the observations of Haque et al., 2012.


In the present study it was observed that there was impact of season on plasma metabolic and stress hormone profile in growing Murrah buffalo heifers. But dietary energy levels have significant impact on cortisol hormone levels and were significantly lower in high energy groups in both the season. Thus it can be concluded that increasing dietary energy levels can be a strategy to overcome the adverse impact of temperature variability for sustained productivity and climatic resilient livestock production.


The authors wish to express their thanks to Director, ICAR-National Dairy Research Institute, Karnal (India) and Dairy Cattle Nutrition Division, for their collaboration and support and provide funds for conducting the research work.

Conflict of Interest

There is no conflict of interest.


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