Binjharpuri cattle is one of the important indigenous cattle breeds of the state of Odisha serving the farming community as dual-purpose cattle. This breed of cattle is mainly reared by marginal farmers and poorer section of the people for production of milk and agricultural operations like ploughing. In summer season, due to high environmental temperature, the performance of Binjharpuri cattle was reduced. Heat stress reduces feed intake, efficiency of feed conversion and weight gain in cattle (Sultana et al., 2022). The body’s oxidation and antioxidant defense systems become unbalanced as a result of heat stress, which may increase reactive oxygen species (ROS). Selenium (Se) and vitamin E are the antioxidants present in the body reduces free radical damage and inhibit lipid peroxidation, which ultimately helps to save the cells from reactive oxygen species (Salles et al., 2025). Being a constituent of glutathione peroxidase (GPx), Se removes the excess peroxides produced during the summer stress (Jamima et al., 2020). Vit. E and Se also help in lowering the serum interleukin and tumour necrosis factor levels in cattle (Sethy et al., 2015). The requirement of Se may increase during the summer season, which can be fulfilled by nano-Se (Kanana et al., 2019). Nano Se particles (SeNPs) are red in colour, having high availability and low toxicity due to greater surface area, surface activity and catalytic efficiency (Sultana et al., 2022). Since there are few studies on the combined effect of SeNPs and vitamin E in indigenous breeds like Binjharpuri cattle during summer stress, the present study was conducted to assess the growth, blood chemistry, antioxidant and immunity status of the Binjharpuri calves in the summer season.

Location
 
This trial was conducted in summer season in the College of Veterinary Science and Animal Husbandry, Bhubaneswar which was positioned at latitudes between 20.12N and 20.25N and longitudes between 85.44E and 85.55E. Along the experimental timeframe (March to June), the temperatures recorded ranged from a minimum of 26.7°C to a maximum of 43.9°C, with an average humidity level of 63%. A calculated temperature-humidity index (THI) of 95.86 clearly indicated that heat stress conditions were prevalent, particularly during the hottest hours of the day.
 
Experimental Binjharpuri calves and dietary treatments
 
The study was conducted as per the Institutional Animal Ethics Committee (433/GO/Re/S/01CCSEA) guidelines. A total of twenty-four Binjharpuri male calves (Age: 4-6 months, body weight: 64.3±1.95 kg) were randomly distributed into 4 groups of 6 animals each in a randomized block design (RBD). T1 (control) received a basal diet; T2 and T3 received Se @ 0.3 ppm as nano Se and vitamin E (DL alpha-tocopherol acetate@100 IU/day/animal) respectively in addition to basal diet, whereas T4 was supplemented with both (nano Se @ 0.3 ppm and vitamin E @ 100 IU/per day/ animal) in addition to the basal diet. The nano Se and vitamin E was supplemented through the concentrate mixture. The basal diet consists of concentrate mixture and paddy straw. Before the start of the experiment calves were dewormed (Fenbendazole@ 5mg/kg BW) and received vaccinations against Hemorrhagic Septicaemia and foot mouth disease. The feeding practice lasted for 120 days following ICAR, 2024 guidelines to fulfil the nutrient requirements of calves. The animals were given paddy straw once they had finished the concentrate mixture. To meet their vitamin A needs, each calf was given 5 kg of the accessible green fodder once a week. Every animal has access to fresh, clean drinking water thrice a day. The concentrate mixture was composed of 40 maize, 27 soybean, 30 wheat bran, 2 mineral mixture and 1% salt. The proximate composition and P content were estimated as per A.O.A.C. (2000). The calcium content of the feed was estimated as per Talapatra et al. (1940) and Se content by using ICPOES (Inductively coupled plasma optical emission spectroscopy).
 
Preparation of nano selenium
 
25 mM sodium selenite and 25 mM reduced glutathione were mixed with 200 mg of bovine serum albumin to create SeNPs (Zhang et al., 2012). The size and concentration of synthesized nano Se were estimated by SEM (Scanning Electron Microscopy) and ICPOES respectively.
 
Growth performance
 
Fortnightly body weight of individual calves was estimated by using electronic weighing balance and body weight gain and average daily gain (ADG) were calculated during the experimental period of 120 days.
 
Blood collection and processing
 
At the end of the experiment, 4 ml of blood was drawn from each animal through jugular vein puncture in a centrifuge tube and placed at an angle for forty-five minutes, followed by centrifugation at 1500 × g for 10 minutes. The extracted serum samples were stored at -40°C and used for estimation of blood biochemicals (glucose, total protein, albumin, globulin, triglycerides and total cholesterol) using kit purchased from Coral Clinical Systems (Goa, India). In order to assess the erythrocytic antioxidant status, two millilitres of blood were placed in a collecting vial containing acid citrate dextrose as an anticoagulant. After centrifuging for ten minutes at 3000 × g for 10 minutes, the plasma and buffy coats were separated and the resultant RBC pellets were washed thrice with normal saline solution. The RBC pellets were diluted in distilled water at a ratio of 1:10 to create 10% RBC hemolysates.
 
Erythrocytic antioxidant analysis
 
Lipid peroxidation value was quantified as per Placer et al., (1966), by calculating the amount of malondialdehyde (MDA) produced, whereas Bergmeyer (1983) method was used to know catalase activity in erythrocytes (10% RBC hemolysates). The method described by Paglia and Valentine (1967) was used to evaluate the activity of glutathione peroxidase (GSH-Px).
 
Cellular immune response
 
At the conclusion of the 120-day trial, Phytohaemaglutinin-P (100 µg in 0.1 ml normal saline) was injected intradermally in skin fold at the neck of calves. A digital vernier calliper was used to measure the thickness of the neck skin fold, before and 24 hours after injection. The CBH (Cutaneous Basophilic Hypersensitivity) response was calculated as:

 
Humoral immune response
 
Antibody production against foot and mouth disease vaccine injected in experimental animals was estimated by using indirect hemagglutination (IHA) techniques (Abdallah et al., 2009).
 
Statistical analysis
 
Data collected by the above methods were analyzed using a analysis of variance with Statistical Packages for the Social Science (SPSS, version 16.0) and Tukey Least Significant Difference test (Snedecor and Cochran, 1994).

Experimental diets and synthesized nano selenium
 
The crude protein content in concentrate mixture and paddy straw was 18.60 and 2.50 %, respectively (Table 1).


       
Low Se content (0.002 ppm) was observed in the basal diet compared to the requirement (0.3 ppm) as suggested by ICAR, 2024. Hence, Se supplementation is highly recommended for calves in the summer season. Likewise, the synthesis of red colour powder denotes the formation of nano Se (Vajpeyee et al., 2022). The average size of SeNPs was 55 nm as observed in SEM and the shape of the nano Se particle was mostly spherical (Fig 1 and Fig 2). The concentration of Se in synthesized SeNPs was 7300 ppm. Similarly, Zhang et al., (2012) observed red colour spherical nanoparticles of Se and size varied from 40 to 60 nm.




 
Effect on production parameters
 
The initial body weights of calves were statistically similar (P>0.05) among all treatment groups, indicating uniformity at the start of the experiment. However, significant (P<0.05) differences were observed in final BW, BW gain and average daily gain (ADG) among the treatments. Combined supplementation showed the highest body weight gain compared to all other supplementation (Table 2). Similarly, individual supplementation of SeNPs and vitamin E improved body weight gain compared to control. Our findings closely match those of Kalmath and Swamy (2020), who found that supplementing Hallikar cattle with vitamin E and Se during summer stress increased their dry matter intake and body weight gain. According to Kassim et al., (2021), Awassi lambs administered with vitamin E and nano Se supplements showed higher BW and ADG than the control. Additionally, Choonkham et al., (2021) also found that dairy cows given vitamin E and nano Se had improved feed consumption and faster growth rates in the summer season.


       
The improvement in body weight gain in the combined supplementation group indicates the synergistic role of these antioxidants under summer stress conditions. The significantly higher (P<0.01) weight gain and ADG in supplemented groups could be attributed to improved antioxidant defense and better nutrient utilization (Sultana et al., 2022). Se, especially in nano form, possesses higher bioavailability and efficient cellular uptake, which aids in protecting tissues from oxidative damage and maintaining metabolic homeostasis during summer stress conditions. Vitamin E, a potent lipid-soluble antioxidant, complements Se by preventing peroxidation of cell membranes and regenerating other antioxidants such as glutathione for better growth.
 
Blood biochemicals of calves
 
The findings show that nano-Se and vitamin E did not affect blood chemistry of calves (Table 3). Chen et al. (2023) also found that supplementing dairy cows with vitamin E and Se did not change their blood metabolic profile (protein fractions, glucose and cholesterol). Asadi et al. (2024) showed that calves born from cows injected with vitamin E and Se did not exhibit significant (P<0.05) variations in albumin, glucose and total protein concentration during the hot season, which is consistent with our findings. Additionally, Gavidel et al., (2025) confirmed the negligible impact on biochemical indicators by showing that supplementing suckling calves with various types of Se had no effects on glucose, protein fractions, or lipid profiles. Contrary to these, Kassim et al., (2021) found increased albumin and total protein levels in the serum of Awassi lambs supplemented with vitamin E and nano Se. These differences may be due to species variation and the form of Se used.


 
Antioxidant enzyme activity
 
Nano-Se and vitamin E markedly enhanced the antioxidant status of calves, as evidenced by higher GPx and catalase activities and lower LPO values (Table 4).


       
Similar findings were reported by Shams et al., (2020) in Friesian calves, where nano-Se supplementation significantly increased GPx activity in the summer season. Salles et al., (2025) also demonstrated enhanced stress-preventing status in Se and vitamin E-supplemented calves during the summer season. The marked reduction in MDA concentration in the supplemented groups agrees with the observations of Asadi et al., (2024) and Gavidel et al., (2025), who reported decreased lipid peroxidation in calves after nano-Se and vitamin E administration. Furthermore, increased catalase activity recorded in the current study corroborates the results of Raghunandan et al., (2022) and Salles et al., (2025), who reported improved oxidative defense in nano-Se-supplemented cattle in the summer season. These improvements indicate better redox balance and protection against oxidative damage under summer stress. The superior bioavailability of nano-Se and the membrane-stabilizing effect of vitamin E act synergistically to strengthen the cellular antioxidant defense system (Ali et al., 2025). Thus, combined supplementation of nano-Se and vitamin E effectively mitigates oxidative stress and enhances physiological resilience in calves under high temperature-humidity conditions.
 
Effect on immunity
 
The CBH response against PHA-P and indirect HA titre (log2) against the FMD vaccination was significantly (P<0.05) better in all the supplemented calves as compared to the control during the summer season (Fig 3 and Fig 4). Combined supplementation (T4) showed highest titre, followed by vitamin E (T3) and nano-Se (T2). The control group (T1) had the lowest titre.




         
Similarly, Prince et al., (2017) found that vitamin E and Se supplementation significantly increased antibody titers against the HS vaccine in buffalo calves, consistent with our findings.  Similarly, simultaneous supplementation has been shown to uplift the humoral and cell-mediated immunity in calves, as shown by Mohamed et al., (2023) and Salles et al., (2025). Additionally, Se plays a crucial role in the regulation of immune function through its incorporation into selenoproteins such as glutathione peroxidase, which protect immune cells from oxidative damage and support lymphocyte proliferation. The nano form of Se further improves this effect owing to its higher bioavailability and efficient cellular uptake. Vitamin E acts as a lipid-soluble antioxidant that protects cell membranes from peroxidative damage, maintains structural integrity of immune cells and enhances antibody production. The synergistic interaction of Se and vitamin E therefore provides comprehensive immune protection by reducing oxidative stress and promoting better functional activity of lymphocytes and macrophages.

From this experiment, it may be concluded that dietary supplementation of nano Se @ 0.3 ppm along with 100 IU of vitamin E in the summer season enhanced the growth rate by 33% increasing immunity and stress response without any negative effect.

The present study was supported by “All India coordinated project on “Nutritional and Physiological Approaches for Enhancing Reproductive Performance in Animal”, OUAT, Bhubaneswar, Odisha, India.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.