Impact of nano zinc supplementation in therapeutic management of sub clinical mastitis in buffaloes

Introduction

The Indian dairy market is amongst the largest and fastest growing markets in the world. India has managed to attain top position in milk production owing to huge bovine population of 302.3 million, of which buffaloes contribute 109.85 million, (Livestock Census, 2019) [4] . In rural India buffaloes are the preferred dairy animal due to fact that buffalo are more efficient converters of low-quality feeds or coarse fodder under harsh climatic conditions and its milk also fetches higher price. Also, they are resistant to many diseases and even genetically resistant to some ailments (Borriello et al., 2006) [2] . Since they are reared mostly by small and landless farmers in unhygienic conditions and unscientific milking practices in hot-humid environmental conditions, the buffaloes are predisposed to diseases like mastitis especially in its hidden form, the subclinical mastitis, the major reason for decreased per animal milk production potential. Livestock farmers being ignorant of sub clinical mastitis, this form is more prevalent and accounts for the majority of losses, yet its early detection and treatment remain a challenge for veterinarians (Singh et al, 2025) [14] . Till date antibiotics are considered the mainstay of mastitis treatment but the growing menace of mastitis has compelled researchers to look for alternatives that can be easily adopted by livestock farmers. A protocol that effectively boosts the udder immunity and strengthens the structural integrity of udder without having any ill effect on animal or human welfare is by far the most suited protocol. Supplementation of zinc sulphate is known to reduce the somatic cell count and lower the prevalence (Shakweer et al., 2010 and El-Nour et al., 2010) [12,5] .

The bioavailability of zinc depends on its source, with organic zinc being more bioavailable than inorganic zinc (Zhao et al., 2014) [19] . Supplementation with zinc through mineral proteinates or mineral amino acid chelates, which are organic sources, effectively had higher retention (Lardy et al., 1992) [7] , more bioavailability (Spears, 1989) [16] , and tissue concentrations (Cao et al., 2000) [3] than inorganic sources. Very limited work has been conducted on mitigating effect of nano zinc supplementation on mastitis in buffaloes. The present study was therefore designed to study the effect of nano zinc supplementation in management of mastitis.

Materials and Methods

Ethical approval

Ethical approval was taken from the institutional ethical committee of the college

Area of study

The study was carried out at university livestock farm complex, Acharya Narendra Deva University of Agriculture & Technology, Ayodhya.

Selection of animals

The animals were examined physically to rule out abnormalities in the udder and teats, followed by screening using the California Mastitis Test (CMT), White Side Test (WST), and Somatic Cell Count (Schalm et al., 1971) [11] . Milk samples were randomly collected aseptically from lactating buffaloes, which were then categorized as unaffected (healthy), clinical mastitis, or sub clinical mastitis cases.

Selection of antibiotic

Antibiotic was selected based on antibiotic sensitivity test. The most sensitive antibiotic, Enrofloxacin, was selected for further studies.

Mineral Analysis by ICP-OES

Serum zinc concentrations were estimated using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Agilent-5800). Prior to analysis, serum samples were digested following standard protocols. Measurements were conducted in triplicate to ensure analytical accuracy.

ZnO Nanoparticles and Zinc Sulphate

Commercially available zinc oxide nanoparticles were procured from Vedayukt India Pvt. Ltd., while zinc sulphate heptahydrate (ZnSO₄•7H₂O) was obtained from Qualigens.

Therapeutic Trials

Thirty buffaloes found positive for sub clinical mastitis were divided into 5 groups, each containing 6 animals (table1) to evaluate the therapeutic efficacy of different protocols. Nano zinc @ 30 ppm basal diet for 30 days II 6 Zinc sulphate @ 10 mg/kg body weight along with basal diet for 30 days III 6 Enrofloxacin @ 5 mg/kg body weight/day for 5 days IV 6 Nano zinc @ 30 ppm basal diet for 30 days with Enrofloxacin @ 5 mg/kg body weight/day for 5 days V 6 Zinc sulphate @ 10 mg/kg body weight along with basal diet for 30 days with Enrofloxacin @ 5 mg/kg body weight/day for 5 days

The milk samples from these animals were collected on Day 0 (before treatment) and Day 30 (after treatment) for evaluating efficacy of different therapeutic protocols based on CMT score and Somatic cell count. The level of zinc was also estimated before and after therapy.

Results

Thirty buffaloes with subclinical mastitis were randomly assigned to five treatment groups (n = 6 per group) and evaluated for recovery percentage, serum zinc concentration, California Mastitis Test (CMT) score and somatic cell count (SCC) before and after treatment (Table 2). The therapeutic response differed among the treatment groups. Complete recovery (100%) was observed in Group IV (Nano zinc + Enrofloxacin), where all six animals recovered following treatment. Groups III (Enrofloxacin) and V (Zinc sulphate + Enrofloxacin) each showed a recovery rate of 83.33% (5/6), whereas Groups I (Nano zinc) and II (Zinc sulphate) each recorded a recovery rate of 66.67% (4/6). Serum zinc concentration increased following treatment in all groups except Group III, where only a marginal increase was observed. The highest post-treatment serum zinc concentration was recorded in Group IV (0.88±0.03 ppm), followed by Group V (0.84±0.05 ppm), Group I (0.83±0.04 ppm), Group II (0.81±0.07 ppm) and Group III (0.52±0.07 ppm). The increase in serum zinc concentration was more pronounced in the nano zinc-and zinc sulphatesupplemented groups than in the antibiotic-alone group. The California Mastitis Test (CMT) score decreased in all treatment groups following therapy. Complete normalization of the CMT score (0) was achieved only in Group IV. The post-treatment CMT scores were 0.16±0.40 in Group V, 0.17±0.40 in Group III and 0.33±0.52 in both Groups I and II. Somatic cell count (SCC) decreased after treatment in all groups. The lowest post-treatment SCC was observed in Group IV (0.69±0.10 × 10⁵ cells/mL), followed by Group V (0.74±0.19 × 10⁵ cells/mL), Group III (0.92±0.34 × 10⁵ cells/mL), Group I (0.99±0.32 × 10⁵ cells/mL) and Group II (1.04±0.41 × 10⁵ cells/mL). Compared with pretreatment values, the greatest reduction in SCC was observed in Group IV, followed by Groups V, III, I and II.

Overall, the combination of nano zinc supplementation and enrofloxacin produced the highest recovery rate, the greatest increase in serum zinc concentration and the maximum reduction in CMT score and SCC among the treatment groups (Table 2).

Discussion

The higher recovery rate on nano zinc supplementation and antibiotic treated group can be attributed to the synergistic effect of antibiotics and Nano-Zinc supplementation. Though zinc was supplemented in both Group IV and Group V, the highest efficacy was found in Group IV because the nano form of zinc provides better absorption due to its smaller particle size and higher surface-to-volume ratio, facilitating superior absorption and bioavailability and is also less toxic (Wang et al., 2016, Sahoo et al., 2014) [17,10] , compared to conventional zinc sulphate. Also in Group IV, there was a significant decrease in the somatic cell count (0.69±0.10) as compared to the Group V indicating that the supplementation had helped in boosting the health of the mammary gland. Nano zinc supplementation alone exhibited recovery percent similar to antibiotic group indicating its potent role as an alternate to antibiotic therapy on management of sub clinical mastitis. The results are in close agreement with the findings of Bakhshizadeh et al.

(2019) who reported that nano zinc had significantly (P<0.05) reduced SCC in milk as lactation progressed in Holstein dairy cows. These findings demonstrate that dietary zinc supplementation effectively restored serum zinc concentrations towards normal physiological levels. Rajendran et al., 2013 [9] observed a decline in the somatic cell counts in cows with subclinical mastitis and improved milk production with nano zinc supplementation. Zinc possess phagocytic activity and aid in intracellular killing of mastitis pathogens, protects from oxidative stress and promotes the integrity of teat canals and mammary epithelium (Weng et al., 2018) [18] toughens the epithelial barrier to infection which decreases bacterial invasion and recurrence of mastitis (Kumar et al, 2026) [6] . Zinc helps to establish keratin in the teat canal (Spain et al., 2005) [15] . Padmavathy and Vijayaraghavan, (2008) [8] opined that Zinc oxide nanoparticles (ZnO NP) possess better antibacterial properties than the conventional ZnO. Auffan et al. (2009) [1] reported cell death in bacteria treated with ZnO NP, owing to increased permeability affecting proper transport through the plasma membrane. Shrivastava et al., 2007 [13] also suggested an inversely proportional relation of size to the antibacterial property which means smaller the size of ZnO, better is the antibacterial activity.

Conclusion

The present study demonstrated that nano zinc supplementation is an effective therapeutic intervention for the management of subclinical mastitis in buffaloes. Supplementation with nano zinc significantly improved serum zinc concentration, reduced somatic cell count (SCC) and California Mastitis Test (CMT) scores, indicating improved udder health. The therapeutic protocol combining nano zinc supplementation with enrofloxacin achieved the highest recovery rate (100%), suggesting a synergistic effect between nano zinc and antibiotic therapy. Nano zinc supplementation alone produced therapeutic efficacy comparable to antibiotic treatment, highlighting its potential as an alternative or supportive strategy for reducing antibiotic dependence. The superior bioavailability and biological activity of nano zinc compared with conventional zinc sulphate may contribute to enhanced mammary gland health and immune function. Therefore, nano zinc supplementation can be recommended as a promising adjunctive therapeutic approach for the effective management of subclinical mastitis in buffaloes, while also supporting prudent antimicrobial use in dairy production. Further large-scale field studies are warranted to validate these findings under diverse management and environmental conditions.

Acknowledgement

AcknowledgementThe authors are very grateful to Dean, College of Veterinary Science and Animal Husbandry, for providing the facilities for conducting the research work and for his cooperation and guidance throughout the occupancy.

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