Seasonal variation in haemato-biochemical profile during growth phase in indigenous Red Kandhari (RK) calves

Introduction

Indigenous livestock species have always been an important contributor to the rural economy, and especially, form the backbone of the small and marginal agri-livestockecosystems (Singh and Rao, 2021) [43] . These animals are well adapted to the regional environmental conditions and can sustainably produce, as compared to exotic or crossbreeds (Naseer et al., 2020) [35] . In the last 2 decades as the earth's temperature are rising, and the impact of heat stress and decrease in production is more evident in the colder regions, with the tropics (IPCC, 2018) [14] . When the animals are not able to dissipate the excess heat, effectively, the heat load increases and immediately various physio-metabolic mechanisms are activated to preserve homeothermy (Collier et al., 2019; Wankar et al., 2019) [5,51] . In dairy cattle THI values over, 68, resulted a decline in milk production and its components, while for the beef cattle the THI threshold is marginally higher, but nevertheless, the production is compromised (Bunning and Wall, 2020; WANKAR et al., 2024) [4,49] . With the changing climate, rapid urbanization and increased demand for animal products, the preservation of native cattle becomes even more pertinent, for sustained food supply to humans (Wankar et al., 2024; Gokirmakli and Bayram, 2017; Wankar et al., 2021; World Consumption of Meat, 2023) [49,11,50,52] . The Indian subcontinent is bestowed with rich livestock bio-diversity. According to the National Bureau of Animal Genetic Resources (NBAGR), there are 53 registered Indigenous cattle breeds (NBAGR, 2022) [36] , while the State of World's Animal Genetic Resources (SoW-AnGR), has recorded 60 Indian cattle breeds (Sharma et al., 2015) [42] . The Red Kandhari [RK] cattle breed is one of the oldest recognised [Red Kandhari, Maharashtra, NDIA_CATTLE_1100_REDKANDHARI_03024] from Maharashtra. The animals are of medium build and dark reddish to brownish in color (Ingle et al., 2016) [13] .

Bulls are used for farming operations, while the cows are mainly reared for their milk, which is marginal, 500-600 l of milk/lactation/cow (Ingle et al., 2016; Kauthale et al., 2019) [13, 21] . As a drought breed, it possesses extraordinary resilience and adaptation to extreme climatic events, droughts, and thrives optimally under hydritic and nutritional stress (Dzavo et al., 2020; Jadhav et al., 2025) [9,16] . Also, many workers have reported that native cow's milk is more nutritious, more palatable, easily digestible, with better proteins, casein, lacto-globulin and fats as compared to exotic breeds, which make it an important dietary nutrient for humans (Kauthale et al., 2019) [21] . However, in the past decades, the farmers, dairy owners, and breeders have incorporated high yielding dairy cross breeds for higher production and profits (Livestock Census, 2012) [28] . Also, there is cross-breeding with the exotic cattle, which led to depletion of indigenous germplasm (Sarang et al., 2024) [40] . The 20 th livestock census has reported just 11% of this breed as pure graded, which forms a meagre proportion in comparison to that of native Khillar cattle breed, accounting for 50% in Maharashtra (Livestock Census, 2019) [29] [Fig. 1]. Hence, proper planning, awareness and favourable government policies are essential for the breed conservation. [6] . A recent phylogenetic research work on 13 indigenous breeds, using singlenucleotide polymorphism (SNP), confirmed minimum genetic variation and a common ancestry for the Bos indicus cattle as compared to Bos taurus breeds (Strucken et al., 2021) [46] , indicating minimum divergence and higher regional and climatic adaptability (Decker et al., 2014) [7] . The Red Kandhari is an ancient cattle breed, regionally called "Lakhalbunda". The breed is distributed across Nanded, Latur, Parbhani, Ahmadangar, and Hingoli districts of the Marathwada region in Maharashtra (Yadav et al., 2018) [53] . The region-wise distribution for adult RK males and females is presented in Fig. 2. This region exhibits extremes of climatic conditions, and the summer temperatures range from 44-46℃, while the rainfall here is scanty (1150 mm/yr), and droughts are very common (dcmsme.gov.in). The animals are dark red to brownish colored and medium-sized. This breed is mainly reared as draught animals, and the bullocks are used for farming (Ingle et al., 2016) [13] , while the cows yield 500-600 L/lactation (Kauthale et al., 2019; Yadav et al., 2018) [21,53] . The Red Kandhari are maintained extensively, in small herds, by small and marginal farmers, thriving mainly, either on community pastures and agricultural waste (Kauthale et al., 2019) [21] . This ancient breed of cattle is threatened, as a result of the adoption of cross-breed cows for milk, dilution of the indigenous germplasm, and mechanization of the farming operations (Jadhav et al., 2025) [16] . In response to the conservation of native animals, recently in 2024, the government of Maharashtra has passed a resolution to "Establishment of cattle breeding farm for conservation and multiplication of Red Kandhari and Deoni cattle at Beed District of Maharashtra" (archive.org/details/in.gov.maharashtra). (Verma et al., 2020) [48] . Similarly, study of hematological and biochemical components also gives animals nutritional, metabolic, and disease status, essential for early treatment of culling (Radostits et al., 2006) [38] .

Material and Methods

Experimental animals

Present work was conducted at the Red Kandhari Unit, located at, Instructional Livestock Farm Complex, College of Veterinary and Animal Sciences, Parbhani, from July-December, 2023. A total of 32 animals were selected for the study and equally divided into 4 groups, based on their age. Each group consisted of a total of 8 calves, four male and four female, respectively. RK1 consisted of calves between 0-3 m, similarly, RK2, RK3, and RK4 had claves aged 3-6 m, 6-9 m, and 9-12 m, respectively. All the experimental procedures were approved by Scientific Research and Animal Ethics Board of the institute (PHY/34 th BOS/02, 14/06/2023).

Animal management and housing

All the treatment groups were reared under a semi-extensive rearing system and went to grazing from 0900-300 hrs. Only the RK1 animals were completely stall-fed. After grazing the animals from RK2-RK4, treatments were also stall-fed as per their nutritional requirements, as per the ICAR standards (Udeybir and Mandal, 2014) [47] . The calves had free access to drinking water in the calf shed throughout the experimental period.

Sampling

For all treatments, blood was collected from all the animals, in the morning hours in the mid of the treatment period (on 45 th days), before the grazing. 10 ml of blood was drawn by jugular venipuncture, and immediately, 2-3 ml was poured into sterile tubes coated with anticoagulants. This portion was used for haematological estimations, and, analysed within 2 hrs of blood collection. Remaining, 7 ml of blood was poured into glass tubes, kept at a 45°, angle and allowed to clot naturally for 30 min. After that, this clot was centrifuged at 3000 RPM for 10 min for harvesting serum, and stored at -20℃ for biochemical estimations. The weather data for the experimental duration was collected form World-Weather.info website (World-Weather.info) and minimum, maximum, average temperatures, relative humidity, average precipitation, cloud cover, sunny days were recorded. While, the temperature humidity index [THI], was calculated by formula given by Mader et al.

(2006) [30] , THI = (0.8 × Tdb) + [(RH/100) × (Tdb -14.4)] + 46.4 Where, Tdb = dry bulb temperature and RH = relative humidity

Sample analysis

In all the blood samples, haematological parameters like, haemoglobin (Hb), total erythrocyte count (TEC), total leukocyte count (TLC), differential leukocyte count (DLC), packed cell volume (PCV), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), were estimated on a fully automatic haematology analyser. Similarly, total protein, albumin, globulin, A:G ratio, triglycerides, Alkaline phosphatase, glucose, uric acid, creatinine, calcium, and phosphorus, were estimated by using commercial kits, on a fully automatic biochemical analyser (UNICORN 120).

Statistical analysis

Statistical analysis was done by using one-way ANOVA, IBM SPSS Statistics software (SPSS 26.0). Level of significance was fixed at P<0.05, and differences were indicated by Tukey's test ( a,b,c,d ).

Results

Haematology variables

The variation for the haematological components in the different RK groups is presented in Table 1. There was no significant difference in hemoglobin concentration within groups, but RK2 had the lowest levels. For RBC concentrations, there was no variation at RK1 and RK3, but RBC was significantly highest at RK2 as compared to RK4 (p ≤ 0.05), group, respectively. The white blood cell concentration didn't vary within groups (p ≥ 0.05), but RK4 had the lowest levels within groups. The haematocrit values didn't differ significantly from RK1-RK4, but RK2 had the lowest levels of PCV. Likewise, the mean corpuscular volume (MCV) was also lowest for the RK2 groups as compared to other treatments (p ≥ 0.05). Similarly, the mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration didn't vary significantly, amongst groups but, lower MCH and MCHC were recorded for RK2 and RK1 than the other groups. There was no significant variation for the white blood cells (p ≥ 0.05), like lymphocytes, monocytes, neutrophils, and eosinophils (Table 2). However, the RK3 group had concurrently higher levels of monocytes and eosinophils and lower levels of neutrophils and lymphocytes than all other groups.

Biochemical variables

The biochemical components at different growth periods are shown in Table 3. There was no significant variation (p ≥ 0.05) for serum total protein, albumin, and globulin, within different groups, but numerically, the highest values were found in the RK3, than other groups. Similar to the serum proteins, the A/G ratio also did not differ significantly within groups. Glucose levels decreased linearly from RK1 to RK4 (p ≤ 0.05). The highest levels for serum glucose were noted at RK1 (115.39 ± 4.43 mg/dl), which decreased gradually in RK2-RK3, and the lowest levels were noted at RK4 (51.29 ± 3.12 mg/dl). For serum creatinine and uric acid, we noted no significant variation, but notably higher levels were at RK3 and RK4 as compared to that at RK2 and RK1, respectively. In Table 4, the lipid, enzyme, and mineral variation for RK calves is presented. Serum concentration of triglycerides was non significantly higher in the RK1 and RK2 groups as compared to RK3 and RK4, respectively. A similar trend was also noted for alkaline phosphatase enzyme, which was higher initially in RK1 and RK2, and then decreased at RK3 and RK4 (p ≥ 0.05). Serum phosphorus also increased non significantly with age in the treatment groups, while for calcium, no peculiar trend was observed within treatments.

Weather parameters

The variation in the meteorological variables and THI during experimental period is presented in Table 5, Higher temperatures were recorded from July-November, October being the hottest month. While lower temperatures were seen in November and December. The average humidity was higher from July-September, as compared to October-December period. Similarly, highest THI was noted for August, followed by, July and September, while, during October to December THI was low. The average rainfall and cloudy days, were maximum from July-September, while clear sunny days, were highest from October to December.

Discussion

For most of the hematological variables, the RK calves showed no significant age wise variations. The slightly higher values for the hemoglobin in the RK1 group might be due to elevated hemoglobin synthesis during the initial 3 months after birth. Present findings are corroborated by Dzavo et al. (2020) [9] who reported no notable impact of age or seasons on the hemoglobin content in Mashona cattle. Variation in the blood-biochemical components in accordance with age is one of the most notable signs of adaptability to the environment and growth, and many researchers consider the indigenous breeds, to be more prolific than the cross breeds or the exotic cattle (Mazzullo et al., 2014) [32] . Similarly, Kalyani et al. (2018) [19] , who studied seven native cattle breeds, viz, Gir, Sahiwal, Deoni (Milch); Kangayam (Draught), Ongole, Rathi, and Tharparkar (Dual purpose), reported optimum hemoglobin concentration in these breeds, according to sex and climatic conditions, indicating the adaptability and resilience (Kalyani et al., 2018) [19] . The hemoglobin range of the RK calves in this study was comparable to Gir and Hanwoo claves (Jacob et al., 2019; Kim et al., 2021) [15,23] and Holstein Friesian and crossbred calves, respectively (Jezek et al., 2011; Bouda and Jagos, 2014) [17,3] . In the RK1 calves, the RBC levels were higher than the RK4 group (9-12 m), which indicate higher erythropoiesis during early life, which gradually decreases with puberty. Also, Bouda and Jagos, (2014) [3] in crossbred calves (0-6 m); Karumalaisamy et al. (2022) [20] in indigenous Kangayam calves, and Kumar et al. (2019) [26] in Hariana cattle, respectively have observed an age-related decline in RBC levels, which confirms the present findings. This adaptability of the erythrocytic indices also reflects the breed resilience and robustness, environmental adaptation and hemopoietic homeostasis with age, in the RK cattle. (Dutta and Bora, 2020) [8] . Marginally higher leukocytes till the 6 months of age (RK2) in our study, indicate higher susceptibility environmental stressors or growth-related increase during this phase, which later decreased (Karumalaisamy et al., 2022) [20] . Similarly, a higher trend for leukocyte concentrations has been reported by Dzavo et al. (2020) [9] and Sripad et al. (2018) [45] in drought and Malnadgidda cattle breeds. The white blood cells, neutrophils, lymphocytes, monocytes, and eosinophils, all mediate the immune response against different pathogens, infections, and parasites. In this study, there was no notable variation or trend, which indicates a lack of infections and immune response, and our results are corroborated by other workers, reporting non-significant variation for leukocytes, with respect to age in different cattle breeds (Kumar et al., 2019; Kumar et al., 2017) [26,27] . However, other studies show a significant age wise variation in the leukocytic concentration in growing calves (Kumar et al., 2019; Dzavo et al., 2020; Park et al., 2019) [26,9,37] .

The erythrocytic indices like PCV, MCV, MCH, and MCHC didn't vary significantly, but a peculiar trend was noted for all the indices. All the indices were lower at age 3-6 m (RK2), and then increased marginally till 12 m of age (RK3-RK4), which indicates a transition period for the haemopoietic system, with approaching puberty. Contrastingly, Karumalaisamy et al. (2022) [20] [15,23] . The protein metabolism also showed an interesting trend; lower at 3-6 m of age (RK2), and thereafter increased non statistically at RK3 and RK4 (p ≥ 0.05). Serum protein carries many important functions, and gradually the levels stabilize, indicating a balance between protein anabolism and catabolism with the growth phase. Protein levels are also known to fluctuate with an animal's health status, reproductive stage, and environmental conditions. These protein stores (serum total protein, protein ratios, albumins, and globulins) and their metabolites also reflect the critical nitrogen balance in an animal (Sejian et al., 2012) [41] . The marginally higher levels of protein, albumin, globulin, creatinine, and uric acid RK3 and RK4, claves clearly indicate higher hepatic production and anabolism, common during the growth phase (Mamun et al., 2013) [31] . Also, the animal's nutritional status doesn't entirely affect the creatine levels, and it mainly depends on the anabolic muscle metabolism (Klinkon and Jezek, 2012) [24] . The calves are primarily monogastric and dependant entirely on cow's milk during the 1 st 3-4 m after birth. The highest levels for serum glucose (p ≤ 0.05), in RK1, indicate monogastric digestive function and dependency on glucose for energy. Further decline in glucose levels in RK2 and RK3, indicates a transition from monogastric to ruminant digestion, which coincides at weaning, evident by higher consumption of roughages or starter feed (Khan et al., 2007; Rashid et al., 2013) [22,39] . Similar decreasing levels for glucose in growing calves have been earlier reported by many workers, which corroborate the present findings (Kumar et [26,27,54] .

Triglyceride levels started declining at RK2 and thereafter declined, which might be due to higher utilization or lipid storage with age, in RK calves. This corresponds with the decrease in glucose levels, indicating a metabolic shift from glucose to lipids or VFA's in the claves (Bagade et al., 2024) [1] . Similar decrease in serum triglyceride and lipids have been reported in neonatal calves (Knowles et al., 2000) [25] , in Sahiwal cattle (Kumar et al., 2017) [27] , and in unweaned calves and piglets (Yu et al., 2019) [54] , which validate present findings. While others have reported an increase in triglyceride levels with age in beef and Bonsmara calves, respectively (Jill et al., 2022) [18] . Alkaline phosphatase is a tissue nonspecific enzyme, extensively produced in the liver, kidneys, and bones and is indispensable for bone metabolism, its mineralization, and phosphorus metabolism. Its primary functions are bone metabolism, mineralization, and calcium and phosphorus turnover, and the levels are quite high in growing animals and decrease by puberty (Narisawa et al., 2013; Gaikwad et al., 2007) [34,10] . The low levels of the enzyme in our study, correspond to higher concentrations of calcium and phosphorus (RK3-RK4), which is indicative of maximum growth during the phase. Similarly, Knowles et al. (2000) [25] and Mohri et al. (2007) [33] , have reported low enzyme levels in growing calves, while higher calcium and phosphorus levels were observed by Mamun et al. (2013) [31] and Sreedhar et al. (2013) [44] , which confirms the present findings and the interrelationship for ALP, calcium, and phosphorus turnover in growing calves.

In the present study, the RK calves were reared on a semiextensive system, exposing them to all the environmental variables, like, fluctuations of temperature, humidity, rainfall, solar intensity and THI etc. The temperatures ranges, of 86-89℉, humidity between 79-82% and its combination i.e., THI's of 78-85, are considered severe enough to subject the animals to moderate-severe thermal stress (Bohmanova et al., 2007) [2] . Despite these challenging environmental conditions, the RK calves didn't showed any adverse hemato-biochemical alterations, during their growth period, which is indicative of superior acclimatization and adaptation of the indigenous cattle, as compared to exotic breeds (Hansen, 2004) [12] . Also, we recorded a solid, transition trend for most of the biochemical components, after 6 m till 12 m, in the RK claves, which strongly imply, optimized growth, in accordance to the environmental conditions.

Conclusion

The results of the present study indicate that the RK claves are acclimatized to the environmental conditions under the semi-extensive management. The breed shows natural genetic adaptation to the climatic conditions, like, high temperatures, humidity and THI, with minimum or no impact on the metabolism during the growth phase. Our observations for the hematological and biochemical components confirms that, for this breed, the major transition period starts beyond the 3-6 m period and continues till the 9-12 m. The red kandhari is an important native, robust, cattle breed of Maharashtra, well adapted to local climatic stressors and heat stress. Comprehensive future research and rigorous conservation is pertinent, to preserve indigenous germplasm, in the climate-changing scenario.

Ethics approval

All the experimental procedures were approved by the Animal ethics committee and were followed. We declare that no animals were harmed during the present research.

Disclaimer (Artificial Intelligence)

Authors declare that, no generative AI tools were used for preparation or editing this manuscript.

Declaration of competing interest

The author declares no conflict of interest.

Acknowledgement

The authors humbly acknowledge the Associate Dean of the College of Veterinary and Animal Sciences, Parbhani, and the In-charge Red Kandhari Unit, Instructional Livestock Farm Complex, for providing the funding, support and other necessary facilities for the present work.

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