The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Nuclear Institute for Agriculture and Biology (NIAB), Pakistan (Approval No. NIAB/ASD/11-2025). (Approval No. SAU-FAHVS-00125). All procedures complied with international guidelines for the ethical care and use of animals in research.

The experiment was conducted from December 2019 to February 2020 at Chak Jhumra experimental goat farm, Faisalabad, Pakistan. Forty healthy, multiparous crossbred Beetal goats (average body weight [BW]: 40.0 ± 0.5 kg) in third parity with a body condition score above 3.5 were selected. No previous reproductive failures were recorded. Goats were individually housed in well-ventilated pens (1.5–2.5 m² per animal) bedded with straw (10–15 cm depth), with bedding replaced every 2–3 days.

Environmental conditions, including temperature (10°C–24°C), relative humidity (50%–70%), and air quality (ammonia <10 ppm; CO₂ <3,000 ppm), were monitored daily. A natural photoperiod of 11–13 h of light was maintained. All animals had free access to fresh water and mineral blocks, and pasture grazing was offered twice daily.

Goats were allocated to two physiological categories: mid-pregnancy (n = 20) and late pregnancy to early lactation (n = 20). Each category was subdivided into a control group and a treatment group antioxidant supplementation (Loxidan at the rate of 120 mg/kg BW per day, Kaesler Nutrition GmbH, Germany). Randomization was based on parity and BW to ensure uniform distribution across groups.

All animals received a basal diet formulated according to NRC (2007) [16] recommendations, consisting of mung bean straw, wheat bran, rice bran, corn, linseed, vitamins, and a mineral premix. The antioxidant supplement (Loxidan, Kaesler Nutrition GmbH) contained a mixture of tocopherols (E-306) and rosemary extract. The supplement was mixed daily with the concentrate portion and fed for 90 days at a dosage of 120 mg/kg BW. Daily feed intake and refusals were recorded. The compositions of the antioxidant-enriched and basal diets are provided in Tables 1 and 2. The Schematic diagram of the experiment is illustrated in Figure 1.

Estrus synchronization was performed with two prostaglandin injections (Cloprostinol; Synchromate®, 125 μg; Bremer, Germany) administered 12 days apart. Does were mated with fertile bucks, and pregnancy was confirmed on day 30 post-breeding using ultrasonography (ExaGO, Anshili, China) with a 7.5 MHz linear probe. Antioxidant supplementation was administered from gestational days 60–105 for mid-pregnant goats and from days 120–165 for late pregnancy to early lactation goats.

Blood samples (5 mL) were collected via jugular venipuncture at gestational days 60, 75, 90, and 105 for mid-pregnancy goats, and days 120, 135, 150, and 165 for late pregnancy to early lactation goats. Samples were centrifuged (1000 × g, 10 min, 4°C), and serum was stored at −20°C until analysis.

Colostrum and milk samples were collected on day 0 (colostrum), day 15, and day 30 postpartum using aseptic hand-milking. Samples were defatted by centrifugation (2,500 × g, 15 min) and stored at −20°C. Freeze–thaw cycles were minimized, and all samples were analyzed within three months. Sequential sampling enabled the tracking of enzymatic changes and oxidative trends throughout gestation and lactation, an approach rarely documented in small ruminants.

Data normality was assessed using the Shapiro–Wilk test. A mixed-model analysis of variance with repeated-measures evaluated treatment, time, and treatment × time interactions, with individual goats as random effects (SPSS v.20.0; IBM Corp., NY, USA). Fisher’s least significant difference test was used for post hoc comparisons. Results are presented as mean ± Standard error of the mean, and differences were considered significant at p < 0.05. Chi-square tests analyzed categorical variables (litter size and survival rate). Figures were generated using GraphPad Prism v.9 (GraphPad software, USA).

Antioxidant supplementation markedly influenced the serum enzymatic antioxidant profile of goats. CAT activity increased significantly in the treated group on gestational days 75, 90, and 105 during mid-pregnancy (p < 0.05; Figure 2A). During late pregnancy and early lactation, CAT activity remained consistently higher than in the control group across all sampling days (p < 0.05; Figure 3A). GPx and SOD activities followed a similar pattern. The highest activities were observed on days 90 and 105 in the mid-pregnancy–treated group (Figures 2B and 2C), whereas in late pregnancy to early lactation, GPx and SOD values increased on days 135, 150, and 165 (p < 0.05; Figures 3D–3F). In colostrum and milk, CAT and SOD concentrations were significantly higher in the supplemented animals than in the controls (p < 0.05; Figure 3A and 3C). However, GPx activity in milk did not differ significantly between the two groups (p > 0.05; Figure 3B).

Supplementation resulted in significant increases in serum concentrations of total phenolics, lycopene, carotenoids, and total flavonoids. These effects were observed consistently across mid-pregnancy on days 75, 90, and 105 (Figures 4A–D) and during late pregnancy to early lactation on days 135, 150, and 165 (p < 0.05; Figures 5A–D). These findings demonstrate that antioxidant supplementation enhanced the availability of multiple non-enzymatic antioxidant compounds throughout the transition period.

Total protein concentration and TAC were significantly higher in supplemented goats during both mid-pregnancy (days 75, 90, 105; Figures 6A and 6B) and late pregnancy to early lactation (days 135, 150, 165; Figures 6E and 6F) (p < 0.05). Conversely, TOS and MDA levels were significantly reduced in the treated groups across both physiological stages (p < 0.05; Figures 6C, 6D, 5G, and 6H), indicating improved oxidative balance. Milk biochemical parameters followed the same trend. Milk protein content and TAC were significantly higher in the supplemented groups (p < 0.05; Figures 7A and 7B), whereas milk TOS and MDA concentrations were significantly lower (p < 0.05; Figures 7C and 7D), reflecting enhanced milk oxidative stability.

Growth performance indicators are shown in Figure 8. Antioxidant supplementation did not affect litter size (Figure 8A), initial birth weight of female kids (Figure 8C), total weight gain of male (Figure 8D) or female kids (Figure 8E), or postnatal survival (Figure 8F). However, a significant improvement was observed in the initial birth weight of male kids born to supplemented dams (p < 0.05; Figure 8B). No additional growth advantages were detected.

Antioxidant supplementation supports sustainable goat production by mitigating climate-induced metabolic stress, reducing oxidative burden, and improving neonatal viability, consistent with the One Health framework for animal productivity and welfare [26]. Oxidative species generated during gestation can damage fetal tissues, underscoring the importance of prenatal nutrition for offspring developmental resilience. Factors such as oxidative stress, micronutrient deficiencies, and environmental stressors influence fetal susceptibility and health outcomes [27]. The present study demonstrates that antioxidant supplementation enhances enzymatic defenses (CAT, SOD, and GPx) and reduces MDA, thereby improving maternal redox homeostasis during the transition period [28].

During advanced pregnancy and early lactation, animals experience heightened metabolic demands associated with fetal growth and milk synthesis, leading to increased oxygen consumption [29]. Previous research in goats shows elevated ROS biomarkers during gestation, indicating oxidative stress and the need for a robust antioxidant defense [30, 31]. Our findings confirm that prenatal antioxidant supplementation modulates oxidative stress indicators in maternal blood and milk, supporting enzymatic systems that counteract ROS. CAT, GPx, and SOD are primary enzymatic defenses, neutralizing ROS and preventing cellular damage [32]. The observed increase in CAT activity suggests enhanced free radical breakdown.

Unlike studies conducted in controlled environments, this work evaluates a commercial antioxidant blend in naturally grazing Beetal goats, integrating both biochemical and productive outcomes. Under metabolic stress, antioxidants upregulate catalytic enzyme activity [33]. GPx and SOD are particularly important in detoxifying hydrogen peroxide and preventing oxidative damage to proteins, nucleic acids, and lipids [34]. Their activity depends on glutathione availability [35]. Although initially non-significant, GPx and SOD levels later increased in supplemented goats, consistent with findings in buffaloes showing elevated antioxidant enzyme activity following dietary supplementation [36].

ROS are natural by-products of metabolism; excess ROS oxidize cellular components, impairing structure and function [37]. In untreated animals, elevated TOS reflects an inability to neutralize free radicals during gestation [38]. Increased metabolic rate in late pregnancy further elevates oxidant production, contributing to disrupted cellular physiology [39]. MDA, a key indicator of lipid peroxidation, increases under oxidative stress due to hydrogen peroxide accumulation [40]. In this study, supplemented goats exhibited significantly reduced MDA levels, consistent with a previous study showing that flavonoid supplementation decreases pregnancy-induced oxidative damage [41].

Protein depletion is common during the peripartum period due to fetal amino acid demands [42]. The significantly lower total protein levels in the control group suggest greater oxidative and metabolic strain. The higher protein concentrations in supplemented goats may reflect enhanced enzyme synthesis and antioxidant status.

Biological systems possess multiple protective strategies to neutralize harmful ROS [43], yet antioxidant capacity naturally declines during pregnancy [44]. Phenolic compounds and flavonoids, often deficient in grazing diets, suppress ROS formation, enhance enzymatic reactivity, and strengthen antioxidant defense pathways [45]. Vitamins and plant-derived antioxidants work synergistically to support fetal development, reproductive performance, and maternal oxidative balance [46]. The present findings confirm elevated phenolic and flavonoid levels following supplementation, demonstrating attenuation of pregnancy-induced oxidative stress.

Lycopene and carotenoids, potent plant-derived antioxidants, protect tissues from oxidative damage through complementary mechanisms [47]. Previous studies report reduced levels under pathological conditions [48]. In our study, supplementation significantly increased lycopene and carotenoid concentrations, indicating enhanced antioxidant capacity from combined phenolic and pigment sources.

The placenta serves as the critical interface for maternal–fetal nutrient exchange, while colostrum and milk are major postnatal sources of immune and antioxidant protection [49, 50]. Although colostrum contains ROS-generating mechanisms involved in microbial defense, the macromolecules it delivers are highly susceptible to oxidative injury [51]. Maternal diet markedly influences colostrum quality, particularly protein composition and antioxidant potential, which are essential for neonatal immunity and gastrointestinal development [52]. Improved maternal oxidative balance enhances the transfer of immunoglobulins and bioactive compounds to offspring [53].

This study demonstrated that antioxidant supplementation increased milk protein levels, consistent with earlier findings linking antioxidant intake to improved milk macronutrient composition [54]. High-yielding animals often exhibit reduced milk antioxidant potential due to oxidative stress in mammary somatic cells, leading to increased MDA [55]. Dietary antioxidants reduce oxidative damage, reduce the incidence of intramammary infections, and enhance milk TAC [56]. Our results align with studies showing improved antioxidant capacity following Moringa supplementation [55].

Newborns face significant oxidative challenges when transitioning from a hypoxic intrauterine environment to ambient oxygen conditions [56]. Adequate antioxidant support is therefore critical. Mammary-derived enzymes such as SOD and GPx contribute significantly to milk’s antioxidant defense [57, 58]. In this study, antioxidant supplementation strengthened SOD activity in colostrum and milk, consistent with reports showing that these enzymes synergistically improve milk oxidative stability [58]. GPx and CAT activities were also enhanced, supporting nutrient preservation and milk quality, in agreement with Paraskevakis’s observations [60–62].

Supplementation increased birth weight in male offspring during late pregnancy and early lactation, supporting the role of antioxidants in reducing oxidative stress and promoting fetal growth [63]. However, litter size, survival rate, and postnatal growth were not significantly affected, likely due to genetic variability and environmental influences that are difficult to modify through dietary interventions [64].