Blood mineral response and field performance of a nano-calcium-phosphorus-selenium oral supplement in dairy cattle: comparative evidence from Bangladesh and India

Introduction

Calcium homeostasis is challenged sharply at the onset of lactation. Even in the absence of overt milk fever, subclinical hypocalcaemia can be common in early-lactation cows and has been associated with impaired immune and metabolic function and with postpartum disease risk (Martinez et al., 2012) [1] . Oral calcium supplementation is therefore widely used; however, the response depends on product chemistry, dose, timing, parity and baseline nutritional management. A 2024 meta-analysis of 22 studies found that oral calcium supplementation increased blood calcium and reduced the risk of clinical and subclinical hypocalcaemia, while effects on milk yield and several other outcomes were inconsistent (Ma et al., 2024) [2] . Nanotechnology has been proposed as a means of altering mineral dispersion, surface area and dissolution behaviour. Reviews of nano-minerals in livestock nutrition describe potential for biological effects at lower inclusion rates, although the evidence base varies substantially among minerals and formulations (Swain et al., 2015) [3] . Selenium is especially relevant because it is incorporated into glutathione peroxidases and other selenoproteins. Controlled studies in dairy cows have reported that nano-selenium can increase plasma or milk selenium and glutathione-peroxidase activity compared with sodium selenite, while effects on milk yield have differed among studies (Han et al., 2021; Xiao et al., 2024) [4,5] . NanoCal Se is a liquid oral supplement developed by Invati Creations Private Limited. The formulation combines nano-calcium and nano-phosphorus with selenium, vitamin D3, vitamin B12 and biotin. The present manuscript integrates a 42-day Bangladesh comparative field evaluation with an independent set of Indian field observations, and reports a transparent statistical re-analysis of the available summary data. A secondary objective was to relate biological findings to available physicochemical characterization, while explicitly separating analytical evidence from claims of in-vivo superiority.

Materials and Methods

Test formulation and daily oral dose

The evaluated formulation was NanoCal Se, supplied as an aqueous colloidal liquid. The label/formulation specification used in the supplied technical dossier is shown in Table 1. The cattle dose used in the Bangladesh evaluation was 50 mL orally once daily.

Physicochemical characterization

The supplied analytical dossier included a TEM micrograph with a 500 nm scale bar showing a dispersed population of nanoscale/submicron particles. A Malvern Panalytical zeta-potential measurement recorded a mean of -40.33 mV and a principal peak mean of -40.41 mV at 25 °C. An absolute zeta-potential magnitude around 40 mV is consistent with strong electrostatic stabilization under the measurement conditions. These measurements support nanoparticulate morphology and colloidal stability; they do not, by themselves, demonstrate gastrointestinal absorption or clinical superiority. Representative analytical evidence is shown in Figures 567. The current source package included a TEM micrograph and a Malvern zeta-potential report; however, a full batch-wise DLS particle-size distribution printout was not supplied. Accordingly, Figure 7 summarizes the declared nanoscale particle-size specification (< 100 nm) of the three mineral phases from the product dossier rather than a measured DLS distribution curve. Note: Total dietary selenium exposure from the basal ration and all supplements should be checked against applicable species-specific feed limits.

Bangladesh comparative field evaluation

The Bangladesh field report described a 42-day evaluation conducted in Dhaka from 12 April 2025 [6] to 24 May 2025 [6] . One hundred cattle were enrolled and allocated to two groups of 50. Group 1 received NanoCal Se at 50 mL orally once daily. Group 2 received a regular calcium-phosphorus supplement at the label-recommended daily dose. Both groups remained on the normal farm ration. Serum samples were collected at baseline (Day 0) and Day 42 for total calcium and inorganic phosphorus. The analysis in this manuscript uses the signed summary report values supplied in the company study file.

Supporting Indian field observations

A February-April 2025 [7] field feedback dataset from West Bengal contained 75 recipient-level records, 25 each from Rakab, Maanbazar and Mahadevpur. Each record reported a percentage "development in milk production" after use of the nano-calcium product. The dataset did not include a contemporaneous untreated control, standardized baseline milk yield, duration for every animal, parity, stage of lactation or ration covariates. Therefore, these records were analysed as observational field-support data rather than as a controlled efficacy trial.

Statistical analysis

For the Bangladesh evaluation, group means and standard deviations were available at Day 0 and Day 42 (n = 50/group). Between-group differences at each time point were assessed post hoc from summary statistics using Welch independent-samples t tests. Mean differences are presented with 95% confidence intervals, and standardized effects as Hedges g. Because animal-level paired values were not available for this re-analysis, within-animal paired tests and a formal treatment-by-time interaction were not calculated. Percentage changes reported in the source field report are presented descriptively.

For the Indian field observations, mean, standard deviation, median, range and 95% confidence intervals were calculated by site and overall. Site heterogeneity was explored using one-way ANOVA, with Kruskal-Wallis as a non-parametric sensitivity analysis. P < 0.05 was considered statistically significant. The site test should not be interpreted as evidence that geography itself caused the differences, because allocation and management factors were uncontrolled.

Results

Serum total calcium

At baseline, mean serum calcium was 8.12 ± 0.41 mg/dL in the NanoCal Se group and 8.10 ± 0.43 mg/dL in the regular-supplement group; the 0.02 mg/dL difference was not significant (P = 0.812). At Day 42, means were 9.38 ± 0.39 and 8.55 ± 0.46 mg/dL, respectively. The between-group difference was 0.83 mg/dL (95% CI 0.66-1.00; P < 0.001), with Hedges g = 1.93. Relative to baseline, the source report recorded increases of 15.52% for NanoCal Se and 5.56% for the regular supplement.

Serum inorganic phosphorus

Baseline serum inorganic phosphorus was also similar between groups (4.68 ± 0.45 vs 4.70 ± 0.47 mg/dL; P = 0.828). At Day 42, the NanoCal Se group had a mean of 5.64 ± 0.42 mg/dL compared with 5.01 ± 0.49 mg/dL in the regular-supplement group. The betweengroup difference was 0.63 mg/dL (95% CI 0.45-0.81; P < 0.001), with Hedges g = 1.37. The reported baseline-to-Day-42 increases were 20.51% and 6.60%, respectively.

Indian field observations on milk production

Physicochemical evidence

The supplied TEM image directly demonstrated a population of small particulate structures at the nanoscale/submicron scale. The measured mean zeta potential was -40.33 mV (principal peak -40.41 mV), supporting a strongly charged colloidal dispersion under the stated measurement conditions. These findings provide a physicochemical basis for examining altered dispersion and dissolution, but a direct link to systemic mineral uptake requires controlled in-vivo pharmacokinetic or balance studies. The representative TEM image (Figure 5) and the zeta-potential report (Figure 6) are reproduced below. In the absence of a supplied DLS printout, Figure 7 summarizes the declared < 100 nm specification for the nano-calcium, nano-phosphorus and nano-selenium phases.

Discussion

The principal finding was a larger Day-42 serum calcium and inorganic phosphorus concentration in cattle receiving NanoCal Se than in cattle receiving a regular mineral supplement, with very small baseline differences and large standardized between-group effects at Day 42. The direction of the calcium finding is biologically consistent with the broader oral-calcium literature: a recent meta-analysis reported a modest overall increase in blood calcium and a reduced risk of postpartum hypocalcaemia after oral calcium supplementation (Ma et al., 2024) [2] . However, direct comparison of effect size is inappropriate because the present field evaluation used a different formulation, a 42-day schedule and a conventional active comparator rather than no supplementation.

A second plausible component is selenium-mediated antioxidant support. (Han et al.) [4] reported greater plasma selenium and glutathione-peroxidase activity with nano-selenium than sodium selenite in lactating cows, although milk yield was unchanged in that small study. In contrast, (Xiao et al.) [5] found higher milk yield and feed efficiency with nanoselenium during peak lactation. These differing results reinforce the need to separate mineral status, antioxidant biomarkers and productivity outcomes rather than assuming that improvement in one necessarily translates into another. The Indian feedback dataset showed an overall mean reported milkproduction improvement of approximately one-third, but the wide SD and marked site heterogeneity are important. Without a concurrent control, standardized pre-treatment milk records, parity and stage-oflactation adjustment, ration data and blinded outcome capture, regression to the mean and management changes cannot be excluded. Accordingly, these data are best treated as hypothesis-generating field surveillance. This interpretation is also consistent with the 2024 metaanalysis, which did not find a pooled milk-yield benefit from oral calcium supplementation despite clearer effects on blood calcium and hypocalcaemia risk [2] . The physicochemical evidence strengthens the formulation description but should not be overextended. TEM is direct evidence of particulate morphology, and a zeta potential of approximately -40 mV indicates substantial electrostatic stabilization in the tested medium. Neither measurement proves intestinal uptake, systemic bioavailability or prevention of milk fever. Future studies should characterize each test batch by DLS size distribution/PDI, TEM, zeta potential, elemental assay and phase identity, and should pair these measurements with serial serum ionized and total calcium, phosphorus, magnesium, selenium status, glutathione-peroxidase activity and clinical outcomes.

Strengths and limitations

The Bangladesh evaluation included an active comparator and equal group sizes (50/group), with baseline and Day-42 bloodmineral measurements.

The manuscript provides confidence intervals and standardized effect sizes rather than relying only on percentage change.

The statistical re-analysis is constrained by summary-level data; raw paired values were unavailable, so a treatment-by-time interaction could not be tested. Randomization, blinding, detailed eligibility criteria, parity distribution, lactation stage, ration composition, compliance and adverse-event capture were not fully documented in the source report.

The Indian milk-response dataset was uncontrolled and should not be used as confirmatory evidence of a milk-yield claim.

TEM and zeta potential establish physicochemical characteristics, not in-vivo bioavailability or clinical efficacy.

Recommended confirmatory trial

A publication-grade confirmatory study should be prospective, randomized and blinded at outcome assessment, with predefined samplesize calculation. A pragmatic design could include NanoCal Se, a conventional oral calcium-phosphorus supplement and standard-of-care control where ethically acceptable. Primary endpoints should include serial serum ionized calcium or total calcium area under the curve and incidence of predefined subclinical hypocalcaemia. Secondary endpoints may include phosphorus, magnesium, selenium status, GPx activity, milk yield, feed intake, retained placenta/metritis, recurrence of hypocalcaemia and tolerability. Animal-level data should be retained to permit mixed-effects repeated-measures analysis.

Conclusion

In the available 42-day Bangladesh field evaluation, NanoCal Se was associated with higher Day-42 serum total calcium and inorganic phosphorus than a regular mineral supplement, while baseline values were similar. The magnitude of the between-group differences was large in the post-hoc summary-statistic analysis. Supporting Indian field observations showed increased reported milk production in many recipients, but the absence of a concurrent control and the strong site heterogeneity mean that these milk data should remain exploratory. The combined evidence supports further randomized, animal-level validation of this nano-mineral platform, with rigorous safety and batch characterization. Commercially, NanoCal-Se is already available in Bangladesh under Renata PLC (Animal Health). According to Invati's commercial declaration, the Bangladesh white-label/marketing arrangement is handled through Nanovet; the product formulation and underlying nanoscaling technology remain exclusively owned by Invati Creations Private Limited, and Nanovet is required under the applicable commercial arrangement to procure the corresponding white-label formulation from Invati. This ownership and supply statement is a commercial/IP disclosure and is not part of the efficacy analysis.

Declarations

Author contributions: Nilanjan Deb -conceptualization, formulation development, scientific interpretation and manuscript drafting; Parijat Bhattacharya -scientific interpretation and manuscript drafting; Biresh Koley -formal analysis; Ritam Santra -formal analysis Conflict of interest and commercial disclosure: The test formulation and nano-scaling technology are proprietary to Invati Creations Private Limited. The author is affiliated with Invati. NanoCal-Se is commercially listed in Bangladesh under Renata PLC (Animal Health). The white-label/marketing and supply relationship involving Nanovet is reported here as a company-declared commercial arrangement. These interests should be disclosed to the target journal in full. Data availability: Summary datasets used for the analyses are contained in the company field reports. De-identified animal-level laboratory data should be made available to editors/reviewers or deposited as supplementary material before/at submission where permitted.

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