Status and correlation of progesterone, 17β-estradiol and minerals on treatment with conventional Ovysynch protocol and medicinal herbs in postpartum anestrum buffaloes

Introduction

Microminerals play a critical role in the reproductive performance of buffaloes. They significantly influence the synthesis, secretion and systemic regulation of progesterone and 17-β estradiol in buffaloes (Abhijit B et al., 2015) [1] . For optimal reproductive performance there should essentially a specific balance between reproductive hormones and microminerals. Anestrus buffaloes have been reported to exhibit low concentrations of estradiol, thyroid hormones, inorganic phosphorus, iron and copper. (Ankita et al., 2018; Sharma et al., 2012) [3,9] . Reproductive disorders such as anestrum and repeat-breeding in buffaloes consistently associated with suppressed phosphorus and progesterone levels compared to normal cycling animals. An optimum concentration of Calcium and Phosphorus is essential for reproductive performance. Calcium influences the ability of an animal to utilize other trace elements while inorganic phosphorus shows a positive correlation with cyclic ovarian activity. An imbalance in the Calcium to Phosphorus ratio can impair ovarian activity. Altered Ca:P ratio affects ovarian function through its blocking action on pituitary gland which results in prolongation of first oestrus, ovulation and prolonged uterine International Journal of Advanced Biochemistry Research https://www.biochemjournal.com involution. Ca:P ratio of under 1.5:1 or >3:1 could potentially lead to reproductive problems. (Pashudhan Praharee, 2024; Chaurasia et al., 2010) [6,4] . Trace minerals such as zinc (Zn), copper (Cu), manganese (Mn), selenium (Se) and cobalt (Co) serve as cofactors for critical enzymes that regulate steroidogenesis, corpus luteum (CL) lifespan and follicular development. Deficiency of these microminerals leads to altered enzymatic profiles, diminished circulating steroid levels, silent estrus and reproductive pathologies such as postpartum anestrus and repeat breeding (Wang et al., 2026; Hafez et.al., 2019; Yatoo et al., 2016) [12,5,13] . Feeding of certain herbal plants ameliorates anestrus and repeat breeding in postpartum buffaloes by providing phytochemicals along with mineral supplementation. Saraca asoca leaves contain high concentrations of minerals such as calcium, phosphorus, sodium, iodine, iron and manganese (Suresh et al., 2024) [ 11] . [8,2] . These herbs provide mineral supplementation and also aid in exhibiting estrus in postpartum anestrus buffaloes by stimulating the production of progesterone and 17β-estradiol. The concentration of minerals estimated in Saraca asoca leaves in recent ICP-OES studies showed total ash 7.28%, Calcium [7] .

The present study was undertaken with the objective of feeding herbal plants Saraca asoca leaves and Trigonella foenum-graecum seeds, which are rich in microminerals, to postpartum anestrus buffaloes to induce estrus and to assess the correlation of sex hormones (progesterone and 17βestradiol) with trace minerals.

Materials and Methods

Postpartum anestrus buffaloes (>90 days postpartum) were identified and selected for the study. A total of thirty-two animals were randomly divided into four groups, with eight buffaloes in each group. Group I served as the control group and was treated with the Ovsynch protocol, consisting of GnRH on day 0, PGF2α on day 7, and GnRH on day 9.

Group II was supplemented orally with 130 g per day of Saraca asoca leaves powder for 24 days. Group III received 200 g per day of Trigonella foenum-graecum seed powder orally for 24 days. Group IV was administered a combination of 65 g of Saraca asoca leaves powder and 100 g of Trigonella foenum-graecum seed powder orally per day for 24 days. Blood samples were collected on days 0, 4, 8, 12, 16 and 24 for estimation of serum progesterone and 17βestradiol by UV-Vis spectrophotometry. For mineral estimation, blood samples collected on days 0, 8, 16 and 24 were analyzed for calcium, phosphorus, zinc, selenium, iron and copper by ICP-OES. Data obtained was subjected to statistical analysis by using Analysis of variance (ANOVA) as per the procedure of Snedecor and Cochran (1994) [10] by using statistical software IBM SPSS Ver.23.0 for windows.

Results

The overall mean value of serum progesterone and 17 βestradiol were given in Table 1. The serum progesterone value in group II is higher compared to Group I, III, and Group IV.

Minerals

The serum calcium levels (Table 2 andFig 1) were significantly (p < 0.01) were higher in order of group III followed by group II and group IV. Group I values were significant at p < 0.05 which showed the lower value in comparison with other groups. The serum phosphorus levels (Table 3 andFig

Correlation between Hormones (Progestrone and 17 β-Estradiol) and minerals

Correlation co-efficient between serum mineral levels and serum hormone (Progesterone and 17β-estradiol) levels in four experimental groups is given in table 8.

Macrominerals

In Group I, serum calcium and phosphorus showed a positive correlation with progesterone concentration, with phosphorus exhibiting a positive and significant correlation (p < 0.01). In contrast, both calcium and phosphorus were negatively correlated with 17β-estradiol.In Group II, serum calcium and phosphorus showed a positive and significant correlation (p < 0.01) with progesterone concentration. The correlation of phosphorus with progesterone was more significant in Group II compared to Group I. Calcium was negatively correlated with 17β-estradiol, while phosphorus showed no significant correlation.In Group III, serum calcium and phosphorus exhibited a positive and significant correlation (p < 0.01) with progesterone concentration. The correlation of calcium with progesterone was higher in Group III compared to Groups II and I. Neither calcium nor phosphorus showed a significant correlation with 17βestradiol. In Group IV, serum calcium and phosphorus showed a positive and significant correlation (p < 0.01) with progesterone concentration. The correlation of calcium with progesterone was significantly higher (p < 0.01) in Group IV compared to Groups III, I, and II. Furthermore, calcium was positively and significantly correlated (p < 0.05), while phosphorus was positively and significantly correlated (p < 0.01) with 17β-estradiol. Notably, both calcium and phosphorus exhibited a significant correlation with 17βestradiol in Group IV compared to Groups I, II, and III.

Microminerals

In Group I, serum zinc and copper were found to be positively and significantly (p < 0.01) correlated with progesterone concentration, while selenium was positively correlated but non-significant. The correlation of zinc, copper, and selenium with 17β-estradiol was positive but non-significant. In Group II, serum zinc and copper were positively and significantly (p < 0.01) correlated with progesterone, while selenium was positively and significantly (p < 0.05) correlated. The correlation of zinc, copper, and selenium with 17β-estradiol remained positive but non-significant. In Group III, serum zinc, copper, and selenium were positively and significantly (p < 0.01) correlated with progesterone. The correlation of these minerals with 17β-estradiol was positive. In Group IV, serum zinc, copper, and selenium were positively and significantly (p < 0.01) correlated with progesterone. The correlation of zinc and copper with 17β-estradiol was positive and significant (p < 0.01), while selenium showed a positive and significant (p < 0.05) correlation with 17βestradiol. Regarding iron, in Group I it was negatively correlated with progesterone and positively correlated with 17β-estradiol, but both correlations were non-significant. In Groups II and III, iron was positively correlated with both progesterone and 17β-estradiol, but the correlations were non-significant. In Group IV, iron was positively correlated with progesterone and showed a positive and significant (p < 0.05) correlation with 17β-estradiol. Overall, the correlation of minerals with progesterone was found to be positive and significant in all groups, except for iron. In contrast, the correlation of minerals with 17β-estradiol was non-significant in all groups except Group IV, which showed significant correlations.

Discussion

In the present study, serum macrominerals (Ca, P) and trace minerals (Zn, Cu, Se, Fe) showed a predominantly positive correlation with progesterone in all treatment groups, while their correlation with 17β-estradiol was significant only in the combination group (Group IV). This differential relationship explains the reproductive physiology of buffaloes and the role of herbal mineral supplementation.

Calcium and Phosphorus:

The positive and significant correlation of Ca and P with progesterone observed in all groups supports the findings of Chaurasia et al. (2010) [4] who reported that optimum Ca and P are essential for cyclic ovarian function. Calcium influences steroidogenesis as a second messenger in gonadotropin-mediated LH action on luteal cells, and regulates the utilization of other trace elements (Sharma et al., 2012) [9] . The improved significance in Group II (Saraca asoca leaves) and Group III (Trigonella foenum-graecum seeds) compared to control is attributable to high mineral content reported in these herbs (Suresh et al., 2024 [11] ; Rabia Praveen et al., 2024 [8] ). Saraca asoca leaves are rich in Ca, P, Na, I, Fe and Mn, while fenugreek seeds are rich in Ca, Mg, Na, K, Fe, Zn and Cu (Ahmad et al., 2015) [2] . The negative correlation of Ca and P with 17β-estradiol in Group I (Ovsynch) can be explained by hormonal antagonism during follicular phase. In contrast, the positive and significant correlation observed in Group IV indicates restoration of Ca:P ratio, which is critical. An imbalanced Ca:P ratio blocks pituitary function and prolongs ovulation (Chaurasia et al., 2010) [4] . The combination of herbs at reduced doses (65g + 100g) provided a more balanced mineral supply, resulting in significant correlation of both Ca (p < 0.05) and P (p < 0.01) with estradiol, unlike individual herb groups.

Zinc, Copper and Selenium: Zn and Cu were positively and significantly correlated (p < 0.01) with progesterone in all groups, confirming their role as cofactors for enzymes governing steroidogenesis, corpus luteum lifespan and antioxidant defense (Wang B et al., 2026 [12] ; Hafez et.al., 2019 [5] ). Copper is essential for cytochrome oxidase and lysyl oxidase involved in steroid synthesis, while zinc regulates progesterone synthesis via Zn-finger transcription factors. Selenium, as a component of glutathione peroxidase, protects luteal cells from oxidative damage, thereby maintaining progesterone secretion (Yatoo et al., 2016) [13] . The non-significant correlation of these trace elements with 17β-estradiol in Groups I, II and III and significant correlation in Group IV suggests that estradiol synthesis is more sensitive to combined mineral deficiency. Abhijit et al. (2015) [1] also emphasized that microminerals influence synthesis and systemic regulation of both progesterone and estradiol. The synergistic phytochemicals (β-sitosterol and flavonoids in Saraca asoca leaves and phytoestrogens. diosgenin in Trigonella foenum graceum seeds) along with Zn and Se supplementation in Group IV may have stimulated aromatase activity, enhancing conversion of androgens to estradiol in developing follicles.

Iron: Iron showed a negative to non-significant correlation with progesterone in Group I, which corroborates reports of low serum iron in anestrus buffaloes (Ankita et al., 2018 [3] ; Sharma et al., 2012 [9] ). Iron is required for heme-containing enzymes like cytochrome P 450 , which catalyzes cholesterol side-chain cleavage for steroidogenesis. The shift to positive correlation in Groups II, III and significant correlation with estradiol (p < 0.05) in Group IV indicates that herbal supplementation corrected the iron deficiency-induced anestrus.

In conclusion, the correlation pattern indicates that progesterone is highly mineral-dependent, while 17βestradiol requires a synergistic balance of minerals. The combination of Saraca asoca leaves and Trigonella foenumgraecum seeds was superior in establishing significant correlations for both hormones, suggesting its potential as a cost-effective herbal strategy to ameliorate postpartum anestrus by improving mineral-hormonal cross-talk and exhibiting estrus in buffaloes.

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