First Evidence of Phyto-Melatonin as a Protective Bio-Molecule in Capra hircus

Introduction

Melatonin is an evolutionarily conserved molecule found across diverse life forms [1]. Initially known for its presence in animals, it was later identified in plants during the nineteenth century, leading to the concept of phyto-melatonin—the plant-derived form of melatonin [2]. Recent research indicates that phyto-melatonin is not limited to higher plants; it is also present in various macro- and microalgae [3], red algae [4], and metazoans of plant-like origin [5]. However, its occurrence in some plant groups such as bryophytes, pteridophytes, and several gymnosperms remains debated due to inconsistent evidence [1].

The concentration of phyto-melatonin in plants varies widely, influenced by differences in extraction and quantification methods as well as tissue-specific distribution [6]. Studies further suggest that plants can absorb melatonin directly from soil, where it accumulates following the decomposition of microorganisms and fungi [2, 8]. At the same time, plants are also capable of synthesizing melatonin endogenously [3], storing it in edible parts such as fruits [1] and dry seeds [2]. Functionally, phyto-melatonin in plants mirrors many of the roles of melatonin in animals. It regulates circadian rhythms [9], enhances resistance to environmental stressors [10], promotes vegetative growth [11], reduces apoptosis [7], and scavenges free radicals [8]. These functions are particularly crucial under stressful conditions, including exposure to UV radiation [12]. In several European countries, maize (Zea mays) is commonly provided to cattle as a dietary supplement to enhance milk production (www.fao.org). Corn seeds are highly preferred by farmers, breeders, and livestock raisers due to their palatability and ease of incorporation into regular feed. Although maize contains phyto-melatonin, its concentration is relatively low (1.4 ng/g of dry tissue) when compared with phyto-melatonin-rich seeds such as white mustard, which contains approximately 189 ng/g of dry tissue. Despite the established physiological roles of melatonin in animals, the influence of phyto-melatonin derived from plant-based diets on animal physiology remains largely unexplored. Recognizing this gap in existing knowledge, the present study was designed to investigate the effects of a phyto-melatonin-rich diet—specifically Zea mays—on the immune function and general physiology of the Indian goat, Capra hircus.

Materials and methods

Animals and maintenance

Goats of similar age (~1 year) and body weight (20 ± 2 kg) were procured from a commercial goat raiser and housed in a controlled goat shelter under the natural environmental conditions of Varanasi (25°18’ N, 83°01’ E, India). This ensured uniformity in diet, hygiene, and management throughout the study period. At procurement, body weight was measured using a Calf Weighing Sling (Munk’s Livestock, Kansas, USA), and age was determined through dentition following the method described by earlier workers [13]. To avoid any mating-related or pheromonal influences, male and female goats were kept in separate enclosures. The detection of estrus in females was based solely on visual indicators such as increased vocalization, vulvar reddening, and mucorrhea.

All goats received a standard ration of roughage (both dry and green) and concentrate as recommended by the Central Institute for Research on Goats (CIRG), Mathura, Uttar Pradesh, India. A single adult goat typically requires 4–5 kg of fodder per day. Dry roughage consisted of crushed barley (Hordeum vulgare, 1 part), crushed maize (Zea mays, 2 parts), linseed (Linum usitatissimum) or mustard seed cake (Brassica juncea, 2.25 parts), rice bran (Oryza sativa, 2 parts), and small quantities of molasses or salt when required. Green roughage included maize (Zea mays), elephant grass (Pennisetum purpureum), pearl millet (Pennisetum glaucum), sorghum (Sorghum spp.), and oat (Avena sativa). The concentrate mixture contained oilseed cakes and soaked gram (Cicer arietinum), and water was available ad libitum. All goats were allowed approximately 8 hours of outdoor grazing daily and kept indoors for the remaining 16 hours.

Routine health monitoring was performed by authorized veterinary practitioners, including assessment of rectal temperature (normal: 102.5–103°F) and rumen movements. Helminthic treatment was provided twice annually, and acaricidal baths using 0.5% malathion solution were administered following the procedure of Chowdhury et al. (2002). Slaughter of animals, wherever required, was conducted at the city abattoir in accordance with the Slaughter of Animals Act (Central Provinces Gazette, 1915; revised 2002). All procedures adhered strictly to CPCSEA guidelines and were approved under the revised Animal (Specific Procedure) Act, 2007, Government of India.

The study was conducted across three major seasons—summer, monsoon, and winter. The seasonal environmental parameters were as follows:
• Summer (April–June): temperature 43.87 ± 1.02 °C, relative humidity (RH) 36.74 ± 4.28%, day length 13.42 h (light) : 10.18 h (dark)
• Monsoon (July–September): temperature 28.68 ± 2.76 °C, RH 87.04 ± 3.50%, day length 12 h : 12 h
• Winter (November–January): temperature 10.76 ± 3.63 °C, RH 64.12 ± 3.05%, day length 10.35 h (light) : 13.25 h (dark)

All study findings were cross-validated using seasonally collected samples from CIRG to ensure consistency and reliability of results.

Experimental design

A total of 12 goats (six males and six females) were selected from the flock each month during the winter season, and all animals were individually identified using ear tags. Thus, across the three winter months, the total number of goats examined was 36, consisting of 18 males and 18 females. Each goat was supplemented daily with 250 g of maize, providing approximately 350 ng of melatonin per animal per day. The experimental trial was conducted over a period of 40 days. At the end of the supplementation period, blood samples from both male and female goats were collected following the procedures described in the Materials and Methods section. The samples were subsequently processed for hematological, biochemical, hormonal, immunological, and free radical analyses.

Measurement of body weight

The goats were weighed using Calf Weighing Sling of Munk’s Livestock, Kansas, USA.

Hematological parameters

Estimations of AST and ALT activities in plasma

Aspartate aminotransferase (AST), also known as glutamate oxaloacetate transaminase (GOT), is an important transaminase enzyme involved in amino acid metabolism. The estimation of AST activity is based on the following kinetic principle:

L-Aspartate + α-Ketoglutarate → Oxaloacetate + L-Glutamate
Oxaloacetate + NADH + H⁺ → L-Malate + NAD⁺

The decrease in absorbance due to the oxidation of NADH to NAD⁺ is measured spectrophotometrically and is directly proportional to AST activity. Plasma AST levels are commonly used as biochemical indicators of hepatocellular damage and can help assess hepatotoxicity associated with hormonal or drug treatments. Alanine aminotransferase (ALT), also known as glutamate pyruvate transaminase (GPT), is another key transaminase enzyme. The principle behind the ALT assay is as follows:

L-Alanine + α-Ketoglutarate → Pyruvate + L-Glutamate
(Pyruvate is then utilized in an indicator reaction with NADH, allowing spectrophotometric measurement.)

ALT

L-Alanine + α- Ketoglutarate ——–> Pyruvate + LGlutamate

    LDH

Pyruvate + NADH + H⁺  ——–> L-Lactate + NAD⁺

Assessment of Red Blood Cell (RBC) count in blood

The peripheral blood sample was collected and immediately diluted in an RBC counting pipette using Ringer’s solution as the diluting fluid. The mixture was thoroughly homogenized and then charged onto a Neubauer haemocytometer. Red blood cells (RBCs) were counted under the microscope, and the total RBC concentration was calculated accordingly.

Assessment of % haemoglobin (%Hb) blood

Hemoglobin concentration (% Hb) was estimated using Sahli’s hemoglobinometer (Systonic Instruments, India). The method is based on the conversion of hemoglobin to acid hematin by adding 0.1N HCl. The resulting brown-colored acid hematin solution is then diluted gradually with distilled water until its color matches the reference standard in the comparator tube. The hemoglobin concentration is read directly from the calibrated Sahli tube. The percentage of hemoglobin was determined based on the corresponding standard scale provided on the instrument.

Immunological parameters

Total Leukocyte Count (TLC)

            Blood was drawn into a WBC pipette and diluted 20-fold using Natt–Herrick diluent. The diluted sample was then charged onto a Neubauer’s counting chamber (Spencer, USA), and total white blood cells (WBCs) were counted under a microscope. For differential leukocyte count (DLC), thin blood smears were prepared, air-dried, and stained with Leishman’s stain. Leukocyte subpopulations were examined and quantified under an oil-immersion objective of a Nikon microscope (Nikon E200, Japan; Haldar et al., 2004). The lymphocyte count (cells/mm³) was calculated from the total leukocyte count and the percentage of lymphocytes using the following formula:

% Lymphocyte count was performed following the protocol of Haldar et al., (2004) as published elsewhere.

%SR of Peripheral Blood Mononuclear Cells (PBMCs)

Separation of Peripheral Blood Mononuclear Cells (PBMCs)

Peripheral blood was diluted with phosphate-buffered saline (PBS, room temperature) in a 1:1 ratio. Three milliliters of Ficoll (HiSep, Cat. No. LSM 1084) was transferred into a sterile 15 mL centrifuge tube, and the diluted blood (6 mL) was carefully layered over it to avoid mixing of the phases. The tubes were centrifuged at 400 × g for 30 minutes at room temperature without brake, as centrifugation at lower temperatures often leads to cell clumping and reduced mononuclear cell yield. Following centrifugation, erythrocytes and polymorphonuclear leukocytes formed a pellet at the bottom, while a distinct opaque interface containing peripheral blood mononuclear cells (PBMCs) appeared above the Ficoll layer.

The plasma layer containing platelets was gently aspirated without disturbing the PBMC interface. The mononuclear cell band was carefully collected using a sterile glass Pasteur pipette and transferred to a fresh 15 mL centrifuge tube. Approximately 10 mL of PBS or appropriate cell culture medium was added, and the tube was gently inverted to ensure proper mixing without causing cell damage. The suspension was centrifuged at 250 × g for 10 minutes, and the supernatant was discarded. This washing step was repeated three times to remove residual Ficoll and platelets. Finally, PBMCs were resuspended, counted, and assessed for viability using the trypan blue exclusion method, ensuring a cell viability of greater than 95%.

Cell harvesting and MTT assay

Cell harvesting and the MTT assay were performed following the protocol of [15], with minor modifications as described by others [16]. Thymocyte and splenocyte cultures were incubated at 37 °C in a humidified CO₂ incubator (Heracell, Germany) with 5% CO₂ for 48 hours. The blastogenic response was assessed using a standard colorimetric assay based on the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SRL, Mumbai, India), following the method outlined by [17].

After 48 hours of incubation, 200 µL of acidified propanol (0.04 M HCl in isopropanol) was added to each well to dissolve the formazan crystals. The optical density (OD) was measured at 570 nm using a microplate reader (ELx-800, BioTek Instruments, Winooski, VT, USA). Mean OD values from triplicate wells were used for statistical analysis. The blastogenic response was expressed as percent stimulation ratio (%SR), calculated as the ratio of absorbance of mitogen-stimulated cultures (Con A-treated) to that of basal cultures (without Con A) for each experimental group.

Metabolic parameters

Estimation of plasma glucose

Plasma glucose levels were estimated using a commercially available glucose assay kit (Beacon India Pvt. Ltd., Mumbai), following the manufacturer’s instructions. Plasma samples were used directly for the analysis without any prior treatment.

Estimation of plasma cholesterol

Cholesterol levels were estimated following the method described by [18]. A stock solution of cholesterol (1 mg/mL) was prepared, and serial dilutions ranging from 0–200 µg/mL in chloroform were used to generate the standard curve. To each standard, a mixture of acetic anhydride and sulfuric acid (20:1) was added, followed by incubation in the dark for 30 minutes. The optical density (OD) was then recorded at 640 nm. For plasma samples, cholesterol was extracted using an ether:ethanol mixture (3:1), followed by centrifugation at 3000 rpm for 10 minutes. The resulting supernatant was evaporated to dryness in a boiling water bath and reconstituted in 5 mL of chloroform. One milliliter of the acetic anhydride–sulfuric acid mixture (20:1) was added, and samples were incubated in the dark for 30 minutes. The OD was measured at 640 nm using a microplate reader (ELx-800, Biotek Instruments, Winooski, VT, USA).

Estimation of Plasma Protein

Plasma protein concentration was estimated using commercially available Bradford reagent following the method of Bradford (1976). Plasma samples were used directly for protein quantification.

Estimation of Plasma HDL, LDL Levels and HDL:LDL Ratio

Plasma HDL and LDL concentrations were determined using a commercial assay kit (Sigma Aldrich, USA; Cat. No. MAK045) as per the manufacturer’s instructions. The assay sensitivity ranged from 2 mg/dL to 300 mg/dL. The HDL:LDL ratio was calculated from the obtained values.

Free Radical Parameters

Superoxide Dismutase (SOD) Activity in Plasma

Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined following the method described by [19]. Briefly, 0.5 mL of plasma was added to 1.4 mL of reaction mixture containing 50 mM phosphate buffer (pH 7.4), 20 mM L-methionine, 1% (v/v) Triton X-100, 10 mM hydroxylamine hydrochloride, and 50 mM EDTA. The reaction mixture was pre-incubated at 37 °C for 5 minutes. Subsequently, 0.8 mL of riboflavin was added to all tubes, including a control tube (buffer in place of plasma).

The samples were exposed to illumination using two parallel 20 W fluorescent lamps inside an aluminium-foil–lined wooden chamber. After 10 minutes of exposure, 1 mL of Griess reagent was added to each tube. The absorbance of the resulting chromophore was measured at 543 nm using a microplate reader (ELx-800, Biotek Instruments, Winooski, VT, USA). One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of nitrite formation under the assay conditions.

Catalase Activity in Plasma

Catalase (CAT; EC 1.11.1.6) activity was measured following the method described by [20]. The assay is based on the reduction of dichromate in acetic acid to chromic acetate upon heating in the presence of residual hydrogen peroxide (H₂O₂), forming an unstable intermediate, perchromic acid. The amount of chromic acetate formed, which develops a characteristic green coloration, is quantified colorimetrically.

A 10% tissue homogenate was prepared in PBS (10 mM; pH 7.0) and centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was used for enzyme estimation. For the reaction, 5 mL of PBS was mixed with 4 mL of H₂O₂ (200 mM), followed by the addition of 1 mL of plasma. After 1 minute, 1 mL of this reaction mixture was transferred into a clean tube, and 2 mL of 5% potassium dichromate (K₂Cr₂O₇) in acetic acid was added to terminate the reaction. The tubes were then boiled for 10 minutes, and absorbance was recorded at 570 nm using a microplate reader (ELx-800, Biotek Instruments, Winooski, VT, USA). Catalase activity was expressed as the amount of H₂O₂ degraded per minute under assay conditions.

GPx Activity in Plasma

Glutathione peroxidase (GPx; EC 1.11.1.9) activity was measured following the protocol described by [21]. The 1 mL reaction mixture consisted of:

• 50 μL plasma
• 398 μL of 50 mM phosphate buffer (pH 7.0)
• 2 μL of 1 mM EDTA
• 10 μL of 1 mM sodium azide
• 500 μL of 0.5 mM NADPH
• 40 μL of 0.2 mM reduced glutathione (GSH)
• 1 U glutathione reductase

The reaction mixture was equilibrated for 1 minute at room temperature, and the reaction was initiated by adding H₂O₂ (final concentration: 100 mM). The decrease in absorbance was monitored kinetically at 340 nm for 3 minutes using a microplate reader (ELx-800, Biotek Instruments, Winooski, VT, USA). GPx activity was expressed as nmol of NADPH oxidized to NADP⁺ per minute per mg protein, using the extinction coefficient for NADPH (ε = 6.22 mM⁻¹ cm⁻¹).

MDA Level in Plasma

Lipid peroxidation in plasma was quantified by estimating malondialdehyde (MDA) using the thiobarbituric acid (TBA) assay as described by Ohkawa et al. (1978) [22]. The plasma supernatant was mixed with 8.1% sodium dodecyl sulfate (SDS), 20% acetic acid, and 0.8% TBA, and the reaction mixture was digested for 1 hour at 95 °C. After incubation, the tubes were cooled under running tap water and vigorously shaken with 2.5 mL of n-butanol:pyridine (15:1). The mixture was centrifuged at 1500 × g for 10 minutes, and the absorbance of the resulting upper organic layer was recorded at 534 nm using a microplate reader (ELx-800, Biotek Instruments, Winooski, VT, USA). Total thiobarbituric acid–reactive substances (TBARS) were expressed as malondialdehyde (MDA; nmol/g tissue weight) using 1,1,3,3-tetraethoxypropane (TEP) as the standard. A standard curve was generated using serial dilutions of 10 nM TEP. For plasma assays, equal volumes of plasma were used in place of tissue homogenate.

Hormonal Parameters

Plasma Testosterone

Peripheral testosterone levels were measured using a commercial ELISA kit (DiaMetra, Italy; Lot No. DKO 002) following the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were <9% and <15%, respectively. All samples were assayed in triplicate.

Plasma Estradiol

Estradiol levels were estimated using a commercial ELISA kit (Biotron Diagnostics Inc., Palm Ave Hemet, CA, USA) following the manufacturer’s protocol. The analytic sensitivity of the assay was 10 pg/mL, with intra-assay variation <5% and inter-assay variation <14%. All measurements were performed in triplicate.

Plasma Melatonin

Melatonin levels were assessed in night-time blood samples using a commercial ELISA kit (Biosource, Nivelles, Belgium; Cat. No. KIPL3300). The limit of detection was 2 pg/mL, and intra- and inter-assay variations ranged from 9% to 15%. All measurements were performed in triplicate.

Cytokine Parameters

Plasma IL-6

Plasma IL-6 concentration was quantified using a sandwich ELISA kit (KomaBiotech, Seoul, Korea; Cat. No. K0331230) according to the manufacturer’s instructions. The analytic sensitivity ranged from 16 pg/mL to 1000 pg/mL. Assays were performed in triplicate.

Plasma TNF-α

Plasma TNF-α level was measured using a sandwich ELISA kit (KomaBiotech; Cat. No. K0331186). The analytic sensitivity ranged from 16 pg/mL to 2000 pg/mL. All samples were run in triplicate.

Statistical analyses

Data were expressed as mean ± standard error of the mean (SEM). Hematological, biochemical, hormonal, immunological, and free radical parameters were analyzed using one-way ANOVA followed by Student’s unpaired t-test for pairwise comparisons. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), IBM, version 17.0, following the guidelines described by Bruning and Kintz (1977).

Results

Since the study is ongoing (unpublished data), representative findings along with relevant p-values are summarized below.

Effect on Body Weight

Phyto-melatonin supplementation resulted in a significant increase in body weight in both male and female goats (p < 0.05).

Effect on Hematological Parameters

Plasma AST and ALT levels—markers of hepatotoxicity—remained unchanged following phyto-melatonin supplementation. However, a significant increase in RBC count and %Hb was observed exclusively in female goats compared to males (p < 0.05).

Effect on Immunological Parameters

The percent stimulation ratio (%SR) of PBMCs increased significantly in both sexes after phyto-melatonin treatment (p < 0.05). Total leukocyte count (TLC) also increased significantly in both sexes (p < 0.01), with males showing a significantly higher TLC than females (p < 0.05). Additionally, the percentage of lymphocytes (%LC) was significantly elevated in both males and females upon supplementation (p < 0.05).

Effect on Metabolic Parameters

Plasma glucose levels increased significantly in both sexes following phyto-melatonin supplementation (p < 0.01), with females showing a comparatively higher increase than males (p < 0.05). Plasma cholesterol levels were also significantly elevated in both sexes (p < 0.05), while plasma protein levels remained unchanged. Plasma HDL concentrations increased significantly in both males and females (p < 0.05), with females showing a greater elevation (p < 0.05). In contrast, plasma LDL concentrations decreased significantly in males (p < 0.05) and more strongly in females (p < 0.01). As a result, the HDL:LDL ratio increased significantly (p < 0.01), particularly in females (p < 0.01).

Effect on Free Radical Parameters

SOD activity increased significantly in both sexes following phyto-melatonin supplementation (p < 0.05). Catalase and GPx activities also increased significantly—catalase (p < 0.05 in males; p < 0.01 in females*) and GPx (p < 0.01 in both sexes*).

Conversely, plasma MDA levels showed a significant decline in both male and female goats (p < 0.01), indicating a reduction in lipid peroxidation.

Effect on Hormonal Parameters

Phyto-melatonin supplementation resulted in a significant increase in plasma melatonin levels in male goats (p < 0.01), whereas females showed no measurable change. Plasma estradiol concentrations increased significantly in both sexes (p < 0.05), while plasma testosterone levels remained unaffected by the supplementation.

Effect on Cytokine Parameters

Plasma IL-6 levels increased significantly in both males (p < 0.05) and females (p < 0.01), with females exhibiting a comparatively higher IL-6 concentration (p < 0.05). Similarly, plasma TNF-α levels were significantly elevated in both sexes (p < 0.01) following phyto-melatonin supplementation.

Discussions

The present study is the first systematic attempt to explore the role of phyto-melatonin in regulating health and immunity in goats, and more broadly, in any ruminant species. Since maize is not a routine component of goat feed in India, it was offered as a dietary supplement rather than a replacement for the regular diet. Before drawing any conclusions on its physiological benefits, it was essential to ensure that corn supplementation did not exert adverse effects on the digestive or metabolic organs, particularly the liver, which plays a central role in maintaining body homeostasis. For this reason, plasma levels of AST and ALT—two major enzymes used as biomarkers of hepatic function—were evaluated.

AST is widely distributed in tissues such as the liver, heart, skeletal muscle, and kidney, whereas ALT is more specific to the liver. Elevated levels of these enzymes typically indicate hepatocellular injury. In the current study, neither AST nor ALT exhibited significant variation between the treated and control groups, nor were any sex-dependent patterns observed. These findings strongly suggest that corn—when used as a source of phyto-melatonin—does not disrupt hepatic physiology and is safe as a supplementary feed for goats. One of the most striking observations was the significant increase in body mass in phyto-melatonin-supplemented goats. This increase may be attributed to enhanced basal metabolic activity, particularly anabolic processes. To investigate this possibility, key metabolic indicators were examined. Plasma glucose levels, representing readily available energy, increased significantly in both sexes, with females showing comparatively higher values. Circulating cholesterol—representing longer-term, stored energy—was also significantly elevated in treated goats, while total plasma protein remained unchanged between sexes and treatment groups. Since protein is generally mobilized as an energy source only under starvation or disease conditions, its stable level indicates that animals remained physiologically normal.

The rise in glucose and cholesterol suggests an overall enhancement of metabolic turnover, likely supporting increased tissue deposition and, ultimately, greater body weight. An additional noteworthy observation was the favorable modulation of lipid profile. HDL, often termed “good cholesterol” due to its cardioprotective role, was significantly elevated in treated groups, whereas LDL, or “bad cholesterol,” was significantly reduced. Consequently, the HDL:LDL ratio improved markedly in supplemented animals, reinforcing the beneficial metabolic effects of phyto-melatonin. Collectively, these findings indicate that phyto-melatonin supplementation does not disrupt liver function, enhances metabolic efficiency, and leads to a healthier lipid profile, thereby contributing to improved body condition in goats.

Higher circulating cholesterol in the phyto-melatonin–supplemented goats appears to be physiologically advantageous. Elevated plasma glucose indicates enhanced metabolic activity, while increased peripheral cholesterol suggests that animals may have been storing additional energy reserves. This stored energy is likely mobilized during energy-intensive physiological events. In goats, winter coincides with two major energy-demanding processes—reproduction and immune modulation—occurring simultaneously; thus, the elevated metabolic substrates may play a critical adaptive role. To explore these interactions, hematological, immunological, and hormonal parameters were assessed. Total RBC count and %Hb were significantly higher in the supplemented groups, particularly in females. These enhanced hematological values suggest improved oxygen-carrying capacity, supporting the higher metabolic rates observed. Consequently, females appear to achieve better physiological fitness during the stressful winter months.

Cell-mediated immune parameters—including Total Leukocyte Count (TLC), % Lymphocyte Count (%LC), and the Stimulation Ratio of Peripheral Blood Mononuclear Cells (%SR of PBMCs)—were also significantly higher in phyto-melatonin–treated goats. This may reflect an increase in peripheral melatonin concentration following supplementation, which in turn could augment cell-mediated immune activity. These findings are in agreement with earlier reports demonstrating the immunostimulatory role of melatonin [24–26]. A similar trend was obtained for cytokines: both TNF-α and IL-6 levels increased significantly in males and females, indicating that cytokine responses were aligned with elevated melatonin levels and the heightened inflammatory readiness of the animals.

Hormonal analysis revealed a significant rise in plasma melatonin particularly in males, while testosterone remained unchanged. In females, estradiol levels increased significantly. These findings suggest sex-specific physiological pathways: in males, elevated melatonin alone may have enhanced immune responses, which would increase energy demand and therefore be consistent with the elevated metabolic parameters. In contrast, females exhibited elevated levels of both melatonin and estrogen, the latter of which is known to upregulate inflammatory cytokines in other species [27]. The combined influence of these hormones likely contributed to the heightened cytokine responses and the superior immune status observed in females, supported by their elevated metabolic and hematological parameters. In males, melatonin alone was sufficient to maintain enhanced immune function, especially since testosterone is recognized as an immunosuppressive hormone [28]. The elevated metabolic activity observed in the phyto-melatonin–supplemented goats is expected to generate higher levels of reactive oxygen species (ROS). ROS production can be indirectly assessed by measuring the activities of endogenous antioxidant enzymes. The primary enzymatic antioxidants responsible for scavenging free radicals include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidases (GPx). Lipid peroxidation, a downstream consequence of oxidative stress, was assessed by measuring malondialdehyde (MDA), a widely accepted biomarker of membrane lipid peroxidation and cellular damage [29]. Interestingly, MDA levels were significantly lower in both sexes of the phyto-melatonin–treated groups, with females showing comparatively lower values. Assessment of antioxidant enzyme activities revealed that SOD, CAT, and GPx were significantly elevated in the supplemented groups compared to controls, although no significant sex-dependent variation was observed. These findings indicate that phyto-melatonin supplementation effectively upregulated the enzymatic antioxidant defense system in both males and females. Thus, as metabolic activity increased, the corresponding rise in circulating melatonin levels appears to have maintained proportional enhancement of free radical scavenging mechanisms. Despite the expected rise in ROS due to heightened metabolism, the significantly reduced MDA levels suggest minimal lipid peroxidation. This indicates that the increased ROS generation was likely a direct consequence of elevated metabolic activity, but adequate antioxidant defense—supported by phyto-melatonin—prevented oxidative damage to cellular membranes.

            For the first time, the present study proposes a protective and health-enhancing role of phyto-melatonin in the Indian goat Capra hircus. The findings indicate that phyto-melatonin supplementation can restore multiple physiological parameters toward an improved or normalized state, suggesting that dietary phyto-melatonin may act through mechanisms similar to those of synthetic melatonin. Importantly, numerous inexpensive, naturally abundant plant sources contain substantial levels of phyto-melatonin. With appropriate scientific understanding and systematic utilization, these sources hold significant potential for improving the health, immunity, and overall productivity of livestock—and may even offer translational benefits for human health in the future.

Acknowledgement:

Authors are grateful to Alexander von Humboldt Foundation, Germany as instrumental gift to Prof. Chandana Haldar. Financial support to SG by Council of Scientific and Industrial Research (CSIR, New Delhi) as CSIR-NET –SRF is gratefully acknowledged.

Conflict of interest:

There is no conflict of interest between the authors including financial.

References

1. Ahmad, R and Haldar, C, Melatonin and androgen receptor expression interplay modulates cell-mediated immunity in tropical rodent Funambulus pennanti: an in-vivo and in-vitro study, Scand J Immunol, 71, 420-430, 2010.
2. Badria, FA, Melatonin, serotonin, and tryptamine in some Egyptian food and medicinal plants, J Med Food, 5, 153–157, 2002.
3. Balzer, I, Bartolomaeus, B and Ho¨cker, B, Circadian rhythm of melatonin content in Chlorophyceae, Proc Conference News Plant Chronobiol Res Markgrafenheide,  55–56, 1998.
4. Bruning, JL and Knitz, BL, Computational handbook of statistics, Eds. Scott, F and Company, Illinois, 1977.
5. Calippe, B, Douin-Echinard, V and Laffargue, M, Chronic estradiol administration in vivopromotes the proinûammatory response of macrophages to TLR4 activation: involvement of the phosphatidylinositol 3-kinase pathway, J Immunol, 180, 7980-7988, 2008.
6. Carrillo-Vico, A, Patricia, JL, Álvarez-Sánchez, N, Rodríguez-Rodríguez, A, and Guerrero JM, Melatonin: Buffering the Immune System, Int J Mol Sci, 14, 8638-8683, 2013.
7. Chowdhury, SA, Bhuiyan, MSA and Faruk, S, Rearing Black Bengal goat under semi-intensive management 1. Physiological and reproductive performances, Asian-Aust J Anim Sci, 15, 477-484, 2002.
8. Das, K, Samanta, L and Chainy, GBN, A modified spectrophotometric assay of superoxide dismutase using nitrite formation by superoxide radicals, Ind J Biochem Biophys, 37, 201–204, 2000.
9. Dubbels, R, Reiter, RJ, Klenke, E, Goebel, A, Schnakenberg, E and Ehlers, C, Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry, J Pineal Res, 18, 28–31, 1995.
10. Fandos P, Orueta JF and Olanda A, Tooth wear and its relation to kind of food: the repercussion on age criteria in Capra pyrenaica, Acta Theriol, 38, 93-102, 1993.
11. Ghosh, S, Singh, AK, Haldar, C, Seasonal modulation of immunity by melatonin and gonadal steroids in a short day breeder goat Capra hircus, Theriogenol, 82, 1121-1130.
12. Haldar, C, Rai, S and Singh, R,Melatonin blocks dexamethasone-induced immunosuppression in a seasonally breeding rodent Indian palm squirrel, Funambulus pennanti, Steroids, 69, 367-377, 2004.
13. Hardeland, R and Poeggeler, B, Non-vertebrate melatonin, J Pineal Res, 34, 233–324, 2003.
14. Herna´ndez-Ruiz, J, Cano, A and Arnao, MB, Melatonin: a growth-stimulating compound present in lupin tissues, Planta, 220, 140–144, 2004.
15. Kola´r, J and Macha´ckova´ I, Occurrence and possible function of melatonin in plants, A review, Endocytobio Cell Res, 14, 75–84, 2001.
16. Lei, XY, Zhu, RY, Zhang, GY and Dai, YR, Attenuation of cold induced apoptosis by exogenous melatonin in carrot suspension cells: the possible involvement of polyamines, J Pineal Res, 36, 126–131, 2004.
17. Lorenz, M and Lu¨ning, K, Detection of endogenous melatonin in the marine red macroalgae Porphyra umbilicalis and Palmaria palmata by enzyme-linked immunoassay (ELISA) and effects of melatonin administration on algal growth, Proc Conf Plant Chronobiol Res, Markgrafenheide, 42–43, 1998.
18. Mantha, SV, Prasad, M, Kalra, J and Prasad, K, Anti oxidant enzymes in hypercholesteromia and effect of vitamin E in rabbits, Atherosclerosis, 101, 135–144, 1993.
19. Mueller, U and Hardeland, R, Transient accumulations of exogenous melatonin indicate binding sites in the dinoflagellate Gonyaulax polyedra, In: Studies on antioxidants and their metabolites, ed. R. Hardeland, 140–147, Gottingen: Cuvilier, 1999.
20. Ohkawa, H, Ohishi, N and Yagi, K, Reaction of linoleic acid hydroperoxide with thiobarbituric acid, J Lipid Res, 19, 1053–1057, 1978.
21. Pauly, JL, Sokal, JE and Han, T, Whole-blood culture technique for functional studies of lymphocyte reactivity to mitogens, antigens, and homologous lymphocytes, J Lab Clin Med, 82, 500–512, 1973.
22. Posmyk, MM, Kuran, H, Marciniak, K and Janas, KM, Pre-sowing seed treatment with melatonin protects red cabbage seedlings against toxic copper ion concentrations, J Pineal Res, 45, 24–31, 2008.
23. Reiter, RJ, Tan, DX, Manchester, LC, Simopoulos, AP, Maldonado, MD and Flores, LJ, Melatonin in edible plants (phyto-melatonin): identification, concentrations, bioavailability and proposed functions, World Rev Nutr Diet, 97, 211–230, 2007.
24. Sackett, DL, Clinical epidemiology, Am J Epidemiol, 89, 125- 128, 1969.
25. Sharma, S, Haldar, C and Chaube SK, Effect of exogenous melatonin on X-ray induced cellular toxicity in lymphatic tissue of Indian tropical male squirrel, Funambulus pennanti, Int J Radiat Biol, 84, 363-374, 2008.
26. Sinha, AK, Colorimetric assay of catalase, Anal Biochem, 47, 389–394, 1972.
27. Tan, DX, Manchester, LC, Di Mascio, P, Martinez, GR, Prado, FM and Reiter, RJ, Novel rhythms of N1-acetyl-N2- formyl-5-methoxykynuramine and its precursor melatonin in water hyacinth: importance for phyto-remediation, The FASEB J, 21, 1724–1729, 2007.
28. Tettamanti, C, Cerabolini, B, Gerola, P and Conti, A, Melatonin identification in medicinal plants, Acta Phytotherap, 3, 137–144, 2000.
29. Wong-ekkabut, J, Xu,Z, Triampo, W, Tang, I-M, Tieleman, DP and Monticelli, L, Effect of Lipid Peroxidation on the Properties of Lipid Bilayers: A Molecular Dynamics Study, Biophy J,93,4225–4236, 2007.