Introduction

Among abiotic stresses, heat stress (HS) is the most challenging hyperthermic condition experienced by small ruminants due to increased surrounding temperature and humidity wherein body fails to regulate the temperature within a normal range, i.e., homeothermy1,2. Heat stress is reported to affect the availability of nutrients3,4,5,6,7, body metabolism, immunity8,9,10,11, performance, and overall production of small ruminants1,2,12. Consequently, the nutrient requirements are increased during HS to support the augmented demands of higher metabolism and to counter or ameliorate the adverse effects of HS-induced oxidative stress4,13. Besides, HS alters the expression of heat shock proteins (Hsp) and acute phase proteins responsible for preventing denaturation, aggregation, and reversion of denatured proteins to native conformation6,12,14. Within this context, several researchers have explored different strategies1,15,16,17 to manage the heat stress in animals. Amongst all, dietary manipulation is cost-effective and simplest method. Several studies have employed supplementation of antioxidant nutrients viz. ascorbic acid, folic acid, zinc, and selenium (Se) in the animals exposed to HS6,15,16,17.

The Se is an ultra-trace element popularly known for its antioxidant function through the glutathione peroxidase (GPx)18. The Se is a cofactor for selenoproteins (SeP) viz. GPx, thioredoxin reductase (TRx), Selenoprotein-P (SePP), iodothyronine deiodinases (ID), and SeP W, that are involved in body metabolism, hormone synthesis, and maintaining other trace minerals concentration in animals13,19. Besides, Se supplementation reduces LDL-cholesterol20 and serum urea due to increased rumen microbial protein synthesis21. Moreover, Se, acting as a cofactor for SeP, is essential for their synthesis. These SePs support immune cells22 and the differentiation and function of T- and B-cells9,23, consequently influencing cytokine synthesis8,10,24,25 and immunocompetence in laboratory animals, poultry18, and livestock26,27. The Se homeostasis is primarily regulated through urinary excretion instead of absorption at the small intestine level28. However, during dietary inadequacy of Se, fecal excretion predominates29. For instance, dietary Se consumed by the animals is primarily used for synthesis of SeP, which plays crucial roles in various physiological functions. Any amount of Se above the required amount and below the toxic limit is excreted primarily through urine. Conversely, when the dietary Se level exceeds the toxic level, the excess Se is metabolized to dimethyl- and trimethyl- selenide to be excreted through respiration, often imparting garlic odor to breath. Furthermore, excess Se may be accumulated in hair, hooves, and other highly keratinous tissues, resulting in selenium toxicity disorders in animals13.

Studies demonstrating the effect of Se supplementation on immune response, antioxidant status, and immunocompetence under healthy conditions are available7,16,25,30,31. Further, few studies in broiler chicken and pigs have reported the positive effects of Se or Se along with vitamin E supplementation on antioxidant status, immune response, and production performance under HS15,16. Likewise, there is a report by De et al.1 studying the effect of Se yeast supplementation on reproduction performance and another report by Alhidary et al.3 demonstrating the effect of Se along with vitamin E supplementation on physiological performance and Se retention in sheep exposed to HS. However, studies on the effects of exclusively Se and its various dietary levels in goats under prolonged heat stress are lacking. Hence, the present study was conducted with the hypothesis that incremental dietary Se levels will positively influence antioxidant status, serum hormones, cytokines levels, Hsp70 mRNA expression, Se metabolism, and immunocompetence of goats under prolonged heat stress, thereby helping to evaluate the optimal dietary Se requirement.

Materials and methods

All the experimental procedures involving feeding, collection of various samples, and housing were in accordance with the institutional animal ethics committee (IAEC; ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, India), which functions under the supervision of the committee for the control and supervision of experiments on animals (CCSEA), India. All the procedures with experimental animals were approved by IAEC (Approval letter no. 25/17/2019-CPCSEA). Also, the experiment was in accordance with the ARRIVE guidelines that are equivalent to CCSEA guidelines of our country. The experimental goats used in the present study were procured from Experimental Animal Farm of Physiology and Climatology Division, ICAR- IVRI, Izatnagar, Bareilly- 243,122 (UP), India. A flow diagram of the methodology followed in the study is depicted in Fig. S1.

Animals, experimental groups, and management

The effect of higher Se concentrations in the diet on the select health attributes under HS vis-à-vis a thermoneutral environment was assessed in goats. Twenty-eight non-growing, non-lactating, and non-descript female goats (a combination of Rohilkhandi breed; 1.5 years age; 21.9 ± 1.32 kg body weight) were randomly assigned based on body weight and age into four groups (n = 7), i.e., CON, CON_HS, T1_HS, and T2_HS (Table 1). The goats were maintained in individual pens with separate provisions for water and feed. The CON group was fed basal diet with 351 ppb Se and maintained under thermo-neutral (TN) condition throughout the experimental period of 60 days, whereas goats in CON_HS, T1_HS, and T2_HS were fed on diet with 351, 1156, and 2018 ppb Se respectively under TN condition for initial 39 days and then exposed to HS during the last 21 days of the experimental period. Selenium was added to the concentrate mixture as sodium selenite (99% Purity, AR grade; CDH, India) through the mineral mixture so that the required Se concentration in the diet of respective groups are achieved.

Table 1 Description of experimental setup.
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Before initiation of the experiment, all goats were dewormed and vaccinated against Peste des Petits Ruminants (PPR) disease. The goats were offered the respective concentrate mixtures and wheat straw as a roughage source daily at 09:30 h. The potable water was accessible to goats twice (10:00 and 16:00 h) daily, and the water offered was found to be devoid of detectable Se. The concentrate mixture was formulated to satisfy the nutrient requirement given by ICAR32. The concentrate mixture consisted of crushed maize grains, 28%; wheat bran, 29%; soybean meal, 40%; salt, 1%; and mineral mixture, 2%. The specific mineral mixture was formulated using inorganic salts (ACS grade) procured from CDH chemicals, India, to suffice all the mineral requirements (Table S2). The proximate principles, mineral composition (except Se) and fibre fractions of the composite diet were as per the ICAR32 requirements. All goats were administered with 2 mL/10 kg BW multivitamin injection, HIVIT Plus (Zydus AH, India), once a month to meet the requirement of vitamins A and E. Each mL of injection contained Vitamin A- 1000 IU, Vitamin D3- 500 IU, Niacinamide- 10 mg, Thaimine HCL- 10 mg, Pyridoxine HCL- 5 mg, Riboflavin Sodium Phosphate- 5 mg, Cyanocobalamine- 10 mcg, D-Biotin- 10 mcg, D-Panthenol- 1 mg, Choline Chloride- 5 mg, Alpha Tocopheryl Acetate- 5 mg, and Sodium Acid Phosphate- 10 mg.

Environmental variables of animal houses

The dry and wet bulb temperatures in the animal sheds were recorded at every two-hour interval during the HS exposure period. The relative humidity (RH) of the animal house was calculated from the psychrometric chart, and the THI was calculated from the equation given by NRC33 as depicted below:

$${\text{THI}} = 0.72 \times \left\{ {\left( {{\text{dry bulb temprature}}\left( {^\circ {\text{C}}} \right) + {\text{wet bublb temperature }}\left( {^\circ {\text{C}}} \right)} \right)} \right\} + 40.6$$

The higher ambient temperature conditions were created and maintained by using halogen lamps of 500 Watts each (to simulate the radiation effect of the sun during summer) at a height of six feet from the floor of the animal shed. Simultaneously, the goats of the TN groups were housed in an air-conditioned animal house. The average temperature(°C), RH (%) and THI of the animal house for the 39-day pre-exposure period were 31.2 ± 0.083, 76.6 ± 0.1.07, and 81.3 ± 0.0.085, respectively. The animal house with higher ambient temperature, where goats in CON_HS, T1_HS and T2_HS were kept, had an average 36.6 ± 0.079 °C temperature, 80.7 ± 0.295 % RH, and 90.0 ± 0.088 THI for six hours and 35.0 ± 0.117 °C temperature, 78.1 ± 0.282 % RH, and 87.5 ± 0.132 THI for the remaining 18 h daily. The air-conditioned animal house where goats of TN groups were housed had 26.7 ± 0.067, 77.4 ± 0.752, and 75.0 ± 0.165 average temperature (°C), RH (%) and THI, respectively (Table S1).

Physiological indices of goats

The physiological indices, namely rectal temperature (RT) and respiration rate (RR) of all goats were recorded daily at 12:00 h during the last 21 days of the experimental period. These physiological indices were repeated three times to record an average. The RR was measured by counting flank movement for one minute. The RT was measured using a clinical digital rectal thermometer.

Analysis of proximate constituents and minerals

The concentrate mixture and wheat straw were analyzed for proximate principles, namely dry matter (DM; 930.15), total ash (942.05), crude protein (CP; 984.13), and ether extract (EE; 945.16) following AOAC34. The neutral detergent fibre (NDF) and acid detergent fibre (ADF) were analyzed as per the method described by Van Soest et al.35. The feed and feces samples were subjected to triple-acid (AR grade nitric acid, perchloric acid, and sulphuric acid in a 4:2:1 proportion) digestion as per the method of Sahrawat et al.36 with slight modifications as a preparatory step for mineral analysis. Briefly, the weighed quantity of sample (≈1000 mg) was transferred to a 200 mL Kjeldahl flask and to the sample, 20 mL of the triple acid mixture was added. Following this, the flasks with the sample were heated at 70 °C till the brown fumes emission stopped. Subsequently, after cooling, the digested samples were transferred to a 100 mL volumetric flask to make up the volume with Millipore water. The macro-minerals calcium and phosphorus were analyzed as per the gravimetric method described by Talpatra et al.37 and the calorimetric method of AOAC34, respectively. The macro-mineral magnesium and trace minerals, copper (Cu), iron (Fe), and manganese (Mn) content of feed, urine, feces, and serum were analyzed by inductively coupled plasma-optical emission spectroscopy (ICP-OES; 5800 ICP-OES, Agilent, CA, USA) after calibrating with ICP Multi-element standard (Merck Chemicals, Germany). The multimode sample introduction system (Agilent Technologies, USA) was used for the analysis of selenium by ICP-OES. The wavelengths (nm) used for analyzing minerals were 196.0 for Se, 279.5 for Mg, 238.2 for Fe, 257.6 for Mn, 324.7 for Cu, and 213.8 for Zn. The instrument conditions were 12 L/min plasma gas flow, 0.7 L/min nebulizer gas flow, and the viewing mode was axial at 8 mm height. All the samples were run in triplicate.

Dry matter intake and body weight changes

Weighted quantity of feed was offered to goats at 09:30 h daily, and the orts left were recorded after 24 h. The orts and feed offered were dried in a hot air oven to calculate dry matter content. The dry matter intake (DMI) was calculated by subtracting dry matter refused by goats from dry matter offered. The weekly body weight (BW) changes of all goats were recorded to observe the effect of higher dietary Se levels under HS and TN conditions.

Selenium metabolism

To study the selenium metabolism of goats fed different dietary Se levels under respective environmental conditions, goats of all groups were shifted to metabolic cages on the 51st day of the experimental period for conducting a metabolic trial. The metabolic trial was conducted for nine days, comprising the initial three days of the adaptation period and the remaining six days of the collection period. The urine excreted by goats during the 24 h period was collected and stored in airtight amber colored glass bottles till further processing. The feces excreted during the last six days were recorded daily, and the dried fecal sample was used for Se analysis. The feed offered and residue left by goats were recorded and the dried samples were pooled for further analysis of Se. The sample preparation and Se analysis were conducted by the method described in “Analysis of proximate constituents and minerals” section of material and methods.

Collection of blood samples and serum separation

About 10 mL of blood was collected from goats at the end of the experiment by jugular venipuncture. Of 10 mL of blood, 1 mL was transferred to a vacutainer containing ethylenediaminetetraacetic acid (EDTA; an anticoagulant) under the ice for hemoglobin (Hb) estimation, and the rest was transferred to a tube containing a gel clot activator (Krupa Labequi, India) for serum separation. The serum was separated by centrifugation at 2000 g for 10 min. The harvested serum samples were aliquoted into five places and stored at − 20 °C till further analysis.

Estimation of hemato-biochemical constituents and hormones

The blood Hb concentration was analyzed following the method of Drabkin and Austin38. The serum glucose, total protein, albumin, globulin, albumin: Globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, urea, and total cholesterol were analyzed using Tulip Diagnostics (Coral Clinical Systems, India) biochemical metabolite assay kits following the manufacturer’s instructions. The levels of serum hormones, viz., cortisol, insulin, and thyroid hormones triiodothyronine (T3) and tetraiodothyronine (T4) were analyzed by ELISA kits (LDN, Germany) as per the manufacturer’s instructions.

Estimation of serum minerals

The macro-minerals, Ca and P in the serum were analyzed by using biochemical metabolite assay kits supplied by Coral Clinical Systems, Goa, India. For micromineral analysis, a measured quantity (~ 500 µL) of serum samples was digested with 10 mL nitric acid and 100 µL hydrogen peroxide in a microwave digester (Anton Paar Multiwave 5000, India). Subsequently, the digested samples were transferred to a volumetric flask and the volume was made to 100 mL with Millipore water. The mineral extract was then used for Se, Mg, Cu, Zn, Mn, and Fe analysis by ICP-OES.

Serum oxidative stress indices and cytokines

The activity of antioxidant enzymes, namely GPx, SOD, and catalase, and non-enzymatic antioxidants, i.e., reduced glutathione (GSH) and MDA levels in serum samples, were analyzed using ELISA kits supplied by Cayman Chemical Company (Michigan, USA) as per the manufacturer’s instructions. Various serum cytokines viz. Tumour Necrosis Factor-α (TNF-α) and IL-1, IL-6 and IL-10 levels in serum samples were analyzed in duplicate by ELISA kits supplied by Bioassay Technology Laboratory (unit of Shanghai Korain Biotech Co. Ltd., China).

Immune response

Humoral immune response

The humoral immune response against chicken red blood cells (CRBC) was assessed by the Hemagglutination (HA) test39,40. For this, the blood was collected in a heparinized vial from the healthy broiler chickens. Subsequently, CRBC was obtained by centrifugation and collected in sterile phosphate-buffered saline (PBS). One mL of 20% CRBC was intravenously administered to all goats on day 1 of the HS exposure period. Subsequently, blood samples of goats were collected on days 7, 14, and 21 post-CRBC inoculation, and serum samples were separated and stored at − 20 °C for the HA test. For this, initially, serum samples were incubated at 56 °C for 15 min for the inactivation of fibrin. The normal saline (100 μL) was added to all the wells of the round (U) bottom HA plate. To the first column of the plate, 100 μL of respective serum samples were added, and serial dilution was made for every sample in subsequent rows, and finally, 100 μL was discarded from the penultimate wells. The last column was kept as a control without serum. Subsequently, 100 μL of 1% CRBC was added to all wells. Following incubation at 37 °C for 2 h, a clear presence of button shape agglutination was examined in the sample. The HA plate well with the lowest or no agglutination, or a well just before the clear settlement of RBCs was taken as the maximum HA titer.

Cell-mediated immune (CMI) response

For assessing the CMI response, around three mL of blood was collected from all the goats in sterile heparinized syringes at the termination of the experiment. The CMI response was analyzed by employing a lymphocyte transformation test (LTT)41 with slight modifications as follows: Following blood collection, the peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Histopaque (1.077 g/mL; Sigma-Aldrich, UK) density gradient centrifugation. Following centrifugation, the cells were suspended in RPMI-1640 complete medium (Sigma-Aldrich, USA) containing 10% fetal calf serum as a nutrient source and 100 IU/mL penicillin and 50 µg/mL streptomycin as antibiotics. After determining cell viability by the trypan blue dye exclusion method, the cells (100 µL) were plated in 96-well cell culture plates. The RPMI 1640 medium (100 µL) was added to the wells in triplicate, and similarly, RPMI 1640 medium with concanavalin A (Con-A (10 µg/mL)) was added to the wells in triplicate @ 100 µL/well. Subsequently, the plates were incubated in a humidified CO2 incubator having 5% CO2 at 37°C for 72 h. At the end of incubation, 20 µL of 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich, USA) was added, and the plates were again incubated under the same conditions for a further four hours. From each well, a definite amount of 100 µL culture supernatant was removed. Herein, the formazan crystals formed were dissolved by dimethyl sulfoxide (100 µL; Amresco, USA) in each well and optical density (OD) was read in an ELISA reader (Multiskan Go, Thermo Scientific, Finland) at a wavelength of 570 nm. The LTT was assessed in terms of blastogenic response, which was calculated by dividing the mean OD of the ConA-stimulated cultures by the mean OD of unstimulated control cultures and expressed as a stimulation index (S.I.).

mRNA expression of Hsp70

Isolation and quantification of RNA: The isolated PBMCs were stored in Triazol at − 20 °C till further use for mRNA expression. The total RNA was extracted from the PBMC (n = 6) following the Trizol method of Chomczynski and Mackey,42. The nanodrop spectrophotometer (QIAxpert, QIAGEN, Germany) was used for determining the purity and quantity of the RNA and the samples with an A260/A280 ratio of more than 1.5 and an A260/A230 ratio of more than 1.3 were used for first-strand cDNA synthesis.

First-strand cDNA synthesis: The first-strand cDNA synthesis was done by employing M-MLV Reverse transcriptase RNAse H minus kit (GCC Biotech, Kolkata, India) according to the manufacturer’s instructions in a thermal cycler. The contents of the kit included: 5X Reaction buffer, 10 mM dNTP Mix, 100 pM/μL Oligo (dT)18 Primer, 100 pM/μL Random Hexamer Primer, Reverse Transcriptase (RT) enzyme, RNase Inhibitor and nuclease-free water (NFW). The reactions were carried out in 0.2 ml RNase-free PCR tubes. The following thermal cycler conditions were used: 5 min at 65 °C, followed by snap chill on ice, 5 min at 25 °C, 60 min at 42 °C, and 10 min at 70 °C. The resulting cDNA was stored at − 20 °C until further use.

Real-time PCR: Real-time PCR was carried out in triplicate by using 2X qPCR master mix, SYBR (GCC Biotech, India), gene-specific primers (Table S3), and a real-time PCR system (Stratagene Mx3000P, Agilent, Germany). Primers were designed by primer3 input (v. 0.4.0), an online primer designing software tool43 having an annealing temperature of 56 °C. β-actin was used as a housekeeping gene. Thermal cycling conditions employed in the present study were 95 °C, 3 min, followed by 40 cycles of 95 °C, 30 s; 56 °C, 30 s; and 72 °C, 30 s. The melting curve analysis of each sample was performed to determine the specificity of the PCR product. Intra-assay and inter-assay variability were determined for each target gene. After normalizing with β-actin, the relative expression of mRNA was assessed by using log2 fold change (2−ΔΔCT), as described by Livak and Schmittgen44. The log2 fold change values were transformed for a graphical presentation of relative gene expression. However, statistical analysis was done on dCTs employing ANOVA to find the significant differences between groups.

Statistical analysis

The data collected during the experiment were analyzed using the statistical software package of SPSS version 2045. One-way analysis of variance was used to compare the treatment means. A fixed effect model was used to perform all the analysis (except weekly BW changes and DMI):

$$y_{ij} = \mu + \alpha_{i} + e_{ij}$$

where, yij = findings on a parameter of the i th treatment, μ = overall mean effect, αi = i th fixed treatment effect, eij = random error associated with the j th goat assigned to the i th treatment.

The repeated-measure analysis (with treatment and time as fixed effects, and individual animals nested under treatment as a random effect) was employed for assessing weekly BW changes and DMI of goats in different treatment groups by using an in house custom Python pipeline (https://github.com/ganaderao/Repeated-measure-analysis_). The treatment means were decided to be significantly different at p < 0.05. The Tukey’s Honestly Significant Difference test was used as a post hoc test for pairwise comparison of treatment means. The trend towards significance was decided at 0.05 < p < 0.10.

Results

Physiological indices of goats

The RT and RR of goats fed different dietary Se levels exposed to HS conditions are shown in Fig. 1 and Fig. 2, respectively. The average RT (°F) and RR (counts per minute) of goats fed different dietary Se levels during the last 21 days of the experiment were higher (p ≤ 0.001) in CON_HS, followed by T1_HS and T2_HS, and lowest in CON.

Fig. 1
figure 1

Effect of dietary Se levels on rectal temperature of goats under heat stress and thermo-neutral conditions. (CON group: 351 ppb Se (Na2SeO3) under TN condition; CON_HS, T1_HS and T2_HS groups: 351, 1156 and 2018 ppb Se (Na2SeO3), respectively with heat stress for 6 h/d during last three weeks). (Created using custom python script: https://github.com/ganaderao/Violin-plot/tree/main). ns-P > 0.05; *- P ≤ 0.05; **-P ≤ 0.01; ***-P ≤ 0.001; ****-P ≤ 0.0001 The rectal temperature of all goats was recorded daily at 12:00 h during the last 21 days of the experimental period using a clinical digital rectal thermometer.

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Fig. 2
figure 2

Effect of dietary Se levels on respiration rate of goats under heat stress and thermo-neutral conditions. (CON group: 351 ppb Se (Na2SeO3) under TN condition; CON_HS, T1_HS and T2_HS groups: 351, 1156 and 2018 ppb Se (Na2SeO3), respectively with heat stress for 6 h/d during last three weeks). (Created using custom python script: https://github.com/ganaderao/Violin-plot/tree/main). ns-P > 0.05; *- P ≤ 0.05; **-P ≤ 0.01; ***-P ≤ 0.001; ****-P ≤ 0.0001

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Chemical composition of feeds

The total diet contained (g/kg DM) 923 g organic matter (OM); 187 g crude protein (CP); 18.6 g ether extract (EE); 469 g neutral detergent fibre (NDF); 231 g acid detergent fibre (ADF); 76.3 g total ash; 6.37 g Ca; 7.47 g P and 1.15 g Mg (Table 2). The content of trace minerals (kg−1 DM) were, Cu- 9.08 mg; Zn- 33.5 mg; Fe- 445 mg and Mn- 37.0 mg. The Se content in the diet of CON and CON_HS groups was 351 ppb, and in the T1_HS and T2_HS diets, it was 1156 ppb and 2018 ppb, respectively.

Table 2 Chemical composition of concentrate mixture, wheat straw and composite diet.
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Dry-matter intake and body weight changes

The weekly DMI and BW changes observed throughout the experimental period are given in Table 3. The DMI was similar (p > 0.05) among the treatment groups throughout the experimental period. However, the DMI was different (p = 0.029) throughout the weeks among different groups with no interaction (p = 0.192) of groups and DMI. Similarly, the BW changes were not influenced (p > 0.05) by either environmental variables or levels of Se. However, the BW was significantly increased with weeks (p ≤ 0.001), and there was an interaction (p ≤ 0.001) between groups and weeks for BW changes.

Table 3 Effect of dietary Se levels on weekly dry matter intake and body weight changes of goats under heat stress and thermo-neutral conditions.
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Selenium metabolism of goats

The Se metabolism of goats fed different Se levels under HS conditions is depicted in Table 4. The Se intake (μg/d) was found to be significantly higher (p ≤ 0.001) in T2_HS, followed by T1_HS, as compared to CON and CON_HS groups. The fecal Se excretion (μg/d) was significantly higher (p ≤ 0.001) in T2_HS as compared to CON, CON_HS and T1_HS groups. Further, significantly higher urinary (p = 0.026) and fecal Se excretion and consequently higher total (p = 0.001) Se excretion (μg/d) were observed in T2_HS and T1_HS groups as compared to CON_HS and CON groups. The Se absorption was significantly higher (p ≤ 0.001) in T2_HS, followed by T1_HS, compared to CON and CON_HS groups. Further, it was observed that the Se retention (μg/d) of goats was significantly higher (p = 0.003) in T2_HS and T1_HS groups as compared to CON and CON_HS groups.

Table 4 Effect of dietary Se levels on selenium metabolism of goats under heat stress and thermo-neutral conditions.
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Blood biochemical attributes

The blood Hb was significantly higher (p = 0.019) in CON, T1_HS, and T2_HS as compared to CON_HS (Table 5). The serum glucose, total protein, albumin, globulin and albumin: globulin ratio were comparable (p > 0.05) among the treatments (Table 5). The serum urea level was significantly higher (p = 0.022) in CON_HS as compared to T2_HS, whereas the CON and T1_HS groups had an intermediate value. The serum cholesterol was found to be significantly higher (p = 0.006) in CON_HS as compared to others. The serum AST activity (p = 0.003) and creatinine concentrations (p = 0.004) were significantly higher in CON_HS as compared to other groups. Further, the serum ALT activity was significantly lower (p = 0.046) in T1_HS and T2_HS as compared to CON_HS, wherein CON had an intermediate ALT activity.

Table 5 Effect of dietary Se levels on blood biochemical attributes of goats under heat stress and thermo-neutral conditions.
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Serum hormones

The concentrations of serum hormones are depicted in Table 6. The serum cortisol level was lower (p ≤ 0.001) in CON, T1_HS, and T2_HS as compared to CON_HS. The serum insulin was lower (p ≤ 0.001) in CON_HS compared to other groups. Also, it was highest in T2_HS with a significant difference (p ≤ 0.001) compared to T1_HS, which in turn was higher compared to CON. The serum T3 level was higher (p = 0.040) in T2_HS as compared to CON and CON_HS, whereas the T1_HS had an intermediate T3 level. Serum T4 level was higher (p ≤ 0.001) in T1_HS and T2_HS as compared to CON and CON_HS.

Table 6 Effect of dietary Se levels on circulating hormone levels of goats under heat stress and thermo-neutral conditions.
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Serum minerals

There was a trend (p = 0.055) towards higher serum Ca concentration in T1_HS and T2_HS compared to CON and CON_HS; however, the P concentration was similar among groups (Table 7). The Se and Fe concentrations were significantly higher (p ≤ 0.001) in T1_HS and T2_HS in comparison with both CON and CON_HS groups. The Mn concentration was significantly higher (p ≤ 0.001) in T1_HS, followed by T2_HS, as compared to CON, whereas CON_HS had an intermediate value. The Cu concentration was higher (p = 0.018) in CON_HS compared to other groups. Also, the Cu concentration was found to be higher (p = 0.018) in T1_HS and T2_HS compared to CON. The Zn concentration was higher (p = 0.025) in CON_HS, T1_HS, and T2_HS compared to CON.

Table 7 Effect of dietary Se levels on serum mineral concentration, oxidative stress indices, cytokine levels, and cell mediated immune response of goats under heat stress and thermo-neutral conditions.
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Oxidative stress indices

The serum MDA level was lower (p ≤ 0.001) in CON as compared to CON_HS, T1_HS, and T2_HS groups (Table 7). Further, it was observed that the serum MDA was lower (p ≤ 0.001) in T1_HS and T2_HS compared to CON_HS. The GSH level was higher (p ≤ 0.001) in T2_HS as compared to T1_HS, CON, and CON_HS. Further, the serum GSH level was found to be lower (p ≤ 0.001) in CON_HS compared to other treatment groups. The serum GPx activity was higher (p ≤ 0.001) in T1_HS and T2_HS as compared to CON and CON_HS. Further, the GPx activity was significantly lower in CON_HS as compared to CON. The serum catalase and SOD activity were higher (p = 0.002) in T2_HS, T1_HS, and CON as compared to CON_HS.

Serum cytokines

The IL-1 levels in T1_HS and T2_HS were at par with each other and lower (p ≤ 0.001) in comparison with CON and CON_HS (Table 7). Moreover, the CON_HS had a significantly higher serum IL-1 concentration compared to CON. Serum IL-6 and IL-10 levels were higher (p ≤ 0.001) in T2_HS, T1_HS, and CON as compared to CON_HS. The serum TNF-α level was significantly higher (p ≤ 0.001) in CON_HS as compared to CON, T1_HS, and T2_HS. Moreover, serum TNF-α level was found to be similar among T1_HS and T2_HS, and T1_HS and CON groups.

Immune response of goats

The CMI response (SI) was similar (p > 0.05) among the treatments (Table 7). The humoral immune (HI) response in terms of HA titer of goats is shown in Fig. 3. The HI response of goats against CRBC (log2 of HA titer) was similar (p > 0.05) on day 7 post-CRBC administration among the groups. However, the HI response was significantly higher (p = 0.001) in T1_HS and T2_HS as compared to CON and CON_HS on day 14 and day 21 post-CRBC administration. Further, there was an increase (p ≤ 0.001) in HA titer with an increase in the period. Nevertheless, no treatment and period interaction were found in the HI response of goats against CRBC.

Fig. 3
figure 3

Effect of dietary Se levels on humoral immune response of goats under heat stress condition (CON group: 351 ppb Se (Na2SeO3) under TN condition; CON_HS, T1_HS and T2_HS groups: 351, 1156 and 2018 ppb Se (Na2SeO3), respectively with heat stress for 6 h/d during last three weeks). The humoral immune response against chicken red blood cells (CRBC) was assessed by administering CRBC intravenously to all goats on the day 1 of the TN/HS exposure period and performing the Haemagglutination (HA) test using serum from the blood samples collected on days 7, 14, and 21 post-CRBC inoculation. ns-P > 0.05; *- P ≤ 0.05; **-P ≤ 0.01; ***-P ≤ 0.001; ****-P ≤ 0.0001.

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mRNA expression of Hsp70

The relative mRNA expression of Hsp70 in goats fed various Se levels and exposed to HS is depicted in Fig. 4. The level of Hsp70 mRNA expression was significantly down regulated in CON_HS as compared to CON. Further, the Hsp70 mRNA expression in T1_HS and T2_HS was equivalent to CON group.

Fig. 4
figure 4

Relative mRNA expression of Hsp70 in goats under heat stress condition. (CON group: 351 ppb Se (Na2SeO3) under TN condition; CON_HS, T1_HS and T2_HS groups: 351, 1156 and 2018 ppb Se (Na2SeO3), respectively with heat stress for 6 h/d during last three weeks). ns-P > 0.05; *- P ≤ 0.05; **-P ≤ 0.01; ***-P ≤ 0.001; ****-P ≤ 0.0001

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Discussion

The HS is reported to impair health, immunocompetence, and production performance of livestock17. The HS in animals can be assessed using various techniques, of which THI is the simplest and easiest. A THI value above 80 is reported to induce HS in goats46. In the present experiment, all groups were initially reared for a 39-day pre-exposure period under similar environmental conditions, during which the THI remained within the TN range33, whereas, during the HS exposure period, the THI at 90.0 ± 0.088 was higher than the TN CON group environmental conditions (75.0 ± 0.165) inflicting moderate to severe HS in goats2,12. Moreover, physiological indices, such as RR and RT, which are commonly used to assess the actual impact of HS47, confirmed that CON_HS, T1_HS, and T2_HS experienced stress. At the same time, it was observed that goats in higher dietary Se fed groups T1_HS and T2_HS experienced milder HS than the CON_HS group, reflecting lower RR and RT values. These findings corroborated the reports of Zhou et al.46 and Srikandakumar et al.47. The findings of the present study indicate the potential beneficial effects of higher dietary Se at 1154 and 2018 ppb against 351 ppb concentrations in reducing HS, resulting in regulating the respiration rates in goats exposed to HS.

HS is widely known to affect the hypothalamic-pituitary axis2 and thereby influence the DMI to maintain homeothermy. Lately, it is known that the reduction in DMI during HS is primarily due to the adverse effects of excess ROS on the feeding center located in the hypothalamus5,12. Consequently, similar DMI of goats in the HS groups compared to the CON group in the present study may be attributed to the adequate nutrient composition and chiefly adequate Se content of the diet. In the present study, the nutrient composition was found to be adequate for the goats, as there were no apparent deficiency symptoms or digestive and health disturbances during the experimental period. It is reported that adequate Se and nutrient content32 reduced the oxidative stress and ROS production in goats exposed to HS through various SeP e.g. GPX, SePP etc. Also, it may be possible that reduced DMI during the heat stress period in goats was compensated during the cooler hours of the night. Thus, it can be deduced that the recommended dietary Se level is adequate for maintaining DMI and BW changes in goats under HS.

Understanding the Se metabolism of goats under higher ambient environmental conditions is essential to determine the Se requirements during HS. In the current experiment, the higher (p ≤ 0.001) Se intake in T1_HS and T2_HS as compared to CON and CON_HS goats is obviously attributed to the varying levels of Se in the respective groups’ composite diet. Although being a minor route of Se excretion, the faecal route contributes to a significant Se loss from the body. However, the comparable faecal Se excretion observed in CON, CON_HS, and T1_HS groups suggests an increased requirement of Se under HS, as already reported by Wang et al.29 in Qinghai-Tibetian sheep. The urinary Se excretion is the principal route of Se excretion in animals fed higher dietary Se levels, whereas the faecal route predominates when the diet is low in Se29. The increased urinary Se excretion indicates excess dietary Se or a rise in catabolic changes13. In the present study, the increased urinary Se excretion (p = 0.026) with increased dietary Se level in goats under HS may be attributed to excess dietary Se concentration rather than the Se requirement13,29 and the heat stress-induced excretion of trace minerals48. The significantly higher (p = 0.003) Se retention in T1_HS and T2_HS groups compared to CON and CON_HS may be attributed to the higher dietary Se level26,29. However, on careful observation, it can be seen that Se retention in CON_HS was decreased (p > 0.05) by two-fold as compared to CON, suggesting an increase in catabolism of antioxidant selenoproteins during HS13. However, no such reduction was observed in goats fed with a higher dietary Se level under HS conditions. This suggests that feeding higher dietary Se helps retain more Se, expecting more beneficial functional outcomes under HS conditions. The Se level used in the present study was lower than the toxic threshold (3000 ppb) reported for prolonged dietary exposure i.e. > 6 months13. Consequently, no adverse effects were observed in goats even at the highest Se level (2018 ppb), indicating the safety of this dietary concentration.

The MDA is used as a direct indicator of lipid peroxidation and an indirect marker of oxidative damage to the body49. In the present study, increased serum MDA levels in goats exposed to HS are in accordance with the findings of Sejian et al.2, indicating increased oxidative stress. However, higher dietary Se levels significantly reduced MDA concentrations in T1_HS and T2_HS goats, which is corroborated by the observations of Kumbhar et al.16 in broiler chickens, and Sheiha et al.25 and Bashar et al.7 in rabbits fed nano form of Se. This reduction in MDA aligns with increased activities of GPx, other antioxidant enzymes, and antioxidant metabolite (i.e. GSH) in the serum of goats in the present study, wherein we found that the concentration of GSH and activities of GPx, SOD, and catalase were lower (p ≤ 0.001) in the CON_HS than in T1_HS and T2_HS. The higher GSH concentrations and GPx activity in goats fed higher dietary Se in T1_HS and T2_HS groups are in line with findings of Malyar et al.50 and Zheng et al.31. Also, the higher GPx activity corresponded with significantly increased serum Se concentrations (p ≤ 0.001), which may be due to greater Se absorption (p ≤ 0.001), and retention (p = 0.003) in T1_HS and T2_HS groups. The increase in catalase activity of T1_HS and T2_HS goats was reflected in higher serum Fe concentrations46, whereas increased SOD activity correlated with higher serum Zn, Cu, and Mn levels, which are essential for SOD activity13,40. At the same time, SOD activity declined in CON_HS despite adequate trace mineral levels, likely due to enzyme catabolism during HS, wherein dietary Se levels were lower. Similar observations were reported by Kumar et al.27 in lambs and Mudgal et al.19 in buffaloes supplemented with supra-nutritional Se and Cu levels under TN conditions. Surprisingly, serum Cu and Zn levels increased in goats exposed to HS, indicating an adaptive mechanism to enhance SOD production. It is revealed that the higher dietary Se at 1154 and 2018 ppb improved the antioxidant indices and reduced oxidative stress in goats under HS conditions.

The cytokines are essential for regulating immunity, tissue repair and managing inflammatory responses. The cytokines are primarily produced by T-helper cells. Selenium is important for maintaining the T-helper (Th) cell 1/Th2 balance of dendritic cells, and its deficiency causes a change in the Th1/Th2 balance towards Th1. The synthesis of pro-inflammatory cytokines like IL-1 and TNF-α increases, and anti-inflammatory cytokines like IL-6 and IL-10 diminish during HS51,52,53. In the present experiment, the CON_HS goats had higher levels of pro-inflammatory cytokines (IL-1 and TNF-α), and lower levels of anti-inflammatory cytokines (IL-6 and IL-10) compared to CON, which is in accordance with the findings of Chen et al.53 in dairy cows supplemented with Se. Corroboratively, Ansar30 reported increased anti-inflammatory cytokines and reduced pro-inflammatory cytokines in Se-supplemented rats. Malyar et al.50 also observed a reduction in the IL-10 level by Se/Zn-enriched probiotic supplementation in Wistar rats exposed to HS. Thus, the findings of the present investigation suggest that feeding higher concentrations of Se in the diet positively modulates the cytokine response in goats under heat stress conditions.

The blood metabolic indices are responsive to varied dietary interventions and indicate health or diseased conditions. In the present study, the Hb levels were lower (p = 0.019) in the CON_HS group compared to T1_HS and T2_HS groups, which may be attributed to HS-induced oxidative stress and Se-influenced antioxidant property of GPx54. Other than this, we observed no alterations in serum glucose, total protein, albumin, globulin, or albumin: globulin ratio across groups, suggesting that Se level in CON_HS was adequate for maintaining these attributes during HS, as also reported by Kumar et al.27. It is reported that HS-induced oxidative stress adversely affects liver and kidney function indices1,6,11. In the present investigation, the reduced activity of ALT and AST enzymes and creatinine levels in T1_HS and T2_HS groups may be attributed to the enhanced antioxidant enzyme activity (GPx, SOD, catalase) and lower MDA levels in these groups7,11. Another metabolite, serum urea, which indicates protein utilization efficiency in ruminants, showed higher levels in CON_HS, indicating reduced rumen microbial protein synthesis. In contrast, lower urea in the T2_HS group suggested that the dietary Se concentration (2018 ppb) was adequate to maintain the equilibrium of rumen microbes21. The HS increased serum cholesterol levels in the goats in the present experiment55; however, higher dietary Se mitigated this rise, which is consistent with the cholesterol-lowering effects of antioxidant nutrients20.

It is reported that the stress hormone cortisol increases during HS in small ruminants1,2. In the present study, serum cortisol level was higher in CON_HS, but it reduced in goats fed higher dietary Se under HS. This reduction may be attributed to the antioxidant properties of SeP, which were higher in these groups7,16. Further, we observed increased blood glucose levels in the CON_HS group compared to the higher Se fed groups, with no significant difference in blood glucose levels between CON and CON_HS. No change in blood glucose level despite heat stress in the present study could not be explained. However, low blood glucose levels in T1_HS and T2_HS compared to CON_HS may be ascribed to reduced HS-induced oxidative stress, responsible for increasing blood glucose in response to increased cortisol levels and prevention of damage to pancreatic β cells, responsible for synthesis and secretion of insulin56. Further, HS lowers thyroid hormones (T3 and T4) synthesis by affecting the hypothalamic-pituitary axis12. However, higher T3 and T4 levels in higher Se-fed goats under HS (T1_HS, T2_HS) suggest enhanced synthesis due to increased SeP and ID-1 activity13. Nevertheless, similar T3 and T4 levels in CON and CON_HS indicate the HS adaptive potential of goats.

The HS is known to alter immunocompetence of animals against foreign antigens2,17,53. However, higher dietary Se is reported to improve immunocompetence by modulating the synthesis of inflammatory mediators8,10, supporting T- and B-cell development23, and maintaining the Th1/Th2 balance. In the present study, the humoral immune response, as indicated by antibody titers on day 14 and 21 post-CRBC injection, was significantly higher in T1_HS and T2_HS compared to CON and CON_HS groups (Fig. 3). This higher antibody titer may be attributed to higher dietary Se that is reported to have role in alteration of B-cell development, diversion of Th1/Th2 balance towards Th2,7,9,23, ability to reduce oxidative stress, enhancement of antioxidant defenses, and promotion of anti-inflammatory cytokine production24,55. In the present study, under HS, the recommended dietary Se concentration was inadequate to elicit an optimal immune response (as evident from antibody titer) in the HS groups compared to the CON group, indicating the requirement for a higher dietary Se concentration. Although the CMI response, measured as SI, was similar across groups, it was apparently improved by higher dietary Se, indicating that the lowest dietary Se level employed in the present study was sufficient to maintain CMI under TN conditions.

During HS, cells increase the production of Hsp to prevent the denaturation and aggregation of proteins and revert the denatured proteins to native conformation2,12. The HS-induced oxidative damage is known to induce Hsp production in livestock and poultry15,16,17. However, Se through GPx and SePP reduced oxidative stress13,18,19. Consequently, in the present study, reduced mRNA expression of Hsp70 in CON_HS may be ascribed to inadequate dietary Se level to cope with the HS-induced oxidative damage, and the similar Hsp70 expression in T1_HS and T2_HS compared to CON may be ascribed to the higher serum Se and resultant GPx activity.

Limitations and future prospects

The present study provides valuable insights into the effects of higher dietary Se levels under HS conditions; however, we report some limitations and prospects for addressing them. In the present study, among the incremental dietary Se levels, only the lowest Se level was evaluated under TN conditions, limiting the ability to determine if higher Se levels may also benefit goats under TN environments. Besides, in the present study, we did not find a clear plateau in Se retention at higher dietary Se concentrations, suggesting the inadequacy of the tested Se levels to estimate the accurate Se requirements under HS conditions. Moreover, the sparing effect of Se and vitamin E on the antioxidant status of animals is widely known. In the present study, evaluating higher dietary Se levels at a fixed vitamin E level poses the question of whether these benefits of higher dietary Se levels are indicative of an absolute increase in dietary Se demands and/or compensatory effect of a dietary vitamin E deficiency under HS conditions in goats. Iodine is a crucial factor in thyroid hormone metabolism and synthesis, and Se also plays an important role in thyroid hormone metabolism. In view of this, future studies may be conducted with HS with varied dietary levels of iodine and Se for a clear understanding of their interaction. The Se biomarkers, namely SePP and Se content of different tissues, may be studied in future for a holistic and comprehensive understanding of Se metabolism and requirement under HS conditions. Sodium selenite, being the widespread, predominant and cost-effective source of Se used in practical livestock feeding, was employed in the present study to understand the Se (inorganic) requirements in goats under HS conditions. However, future studies may be conducted using different organic Se sources having higher bioavailability to reduce the need for high dietary inorganic Se.

Conclusion

From the present study, it can be concluded that 351 ppb dietary Se is not sufficient for goats under heat stress conditions. Higher dietary Se (i.e., 2018 ppb) is beneficial for improving Se retention, hepatic health, antioxidant status, serum cytokine and mineral concentrations, humoral immune response, and Hsp70 mRNA expression in goats under heat stress conditions. Further studies may be conducted to evaluate the effects of higher dietary Se levels beyond the present supplementation to address the Se metabolism, safety of its long-term use, the impact of higher dietary Se levels under thermoneutral conditions, and the sparing effect of vitamin E and Se in animals under heat stress conditions.