The chemicals used in this study were magnesium chloride (Nutricost), insulin (Lantus, 100 IU/mL), ketamine, xylazine, and streptozotocin (STZ), all obtained from standard sources and of analytical grade. Additional materials included ELISA kits for insulin (Cat. No. E0707Ra), IGF-1 (Cat. No. E0709Ra), HER2/neu (Cat. No. E2460Ra), and malondialdehyde (MDA) (Cat. No. E0156Ra), which were purchased from Bioassay Technology Laboratory (BT LAB).

A total of 35 female Wistar rats (Rattus norvegicus) aged 6–8 weeks and weighing 170–200 g were bred and housed at the Laboratory for Research and Animal Testing Unit 4 (LPPT Unit 4), Universitas Gadjah Mada. The rats were kept in ventilated laboratory cages at a controlled room temperature (25–27 °C) and maintained on a 12-hour light–dark cycle with unrestricted access to standard pellet feed and water.

T1DM was induced in 28 rats through a single intraperitoneal injection of STZ at a dose of 65 mg/kg body weight, dissolved in citrate buffer (0.1 M, pH 4.5). Diabetes was confirmed three days after injection by measuring fasting blood glucose (FBG) levels using a photometer (Microlab 300, MERCK). Rats with FBG ≥200 mg/dL were classified as diabetic and included in the experimental groups, while seven rats served as normal controls and received no STZ. Ethical approval for this study was granted by the Ethics Committee of the Faculty of Medicine, Universitas Sebelas Maret (Approval No. 96/UN27.06.11/KEP/EC/2024).

After a seven-day acclimatization period, rats were randomly assigned to five groups (n = 7 per group) using a computer-generated randomization sequence. Treatment allocation was concealed from the investigator responsible for outcome measurements. All treatments were administered by an investigator not involved in histopathological or biochemical assessments. Histological evaluations were performed by a veterinary pathologist blinded to the group assignments to minimize observation bias.

1. Group 1 (P1): Normal control, no STZ induction, placebo (distilled water).
2. Group 2 (P2): STZ-induced T1DM, placebo (distilled water).
3. Group 3 (P3): STZ-induced T1DM, subcutaneous insulin injections (4 IU/200 g body weight/day).
4. Group 4 (P4): STZ-induced T1DM, oral magnesium chloride supplementation (300 mg/kg body weight/day).
5. Group 5 (P5): STZ-induced T1DM, insulin (4 IU/200 g body weight/day), and magnesium chloride (300 mg/kg body weight/day).

Treatments were administered daily for 21 days. Insulin was given subcutaneously, while magnesium chloride was given orally through gavage. At the end of the treatment period, rats were euthanized under general anesthesia induced by ketamine (100 mg/kg) and xylazine (15 mg/kg). Blood and ovarian tissue samples were collected for biochemical and histological analyses.

To collect blood samples, rats were euthanized by cardiac puncture. Approximately 5 mL of blood was drawn from the heart of each rat and collected into sterile specimen bottles. The blood was allowed to clot at room temperature for 30 minutes, then centrifuged at 3,000 g for 20 minutes to separate the serum. The resulting serum was stored at −80 °C until analysis of insulin and IGF-1 levels. Following blood collection, the ovaries were carefully excised. Half of each ovary was rinsed in ice-cold phosphate-buffered saline (PBS, pH 7.4) to remove excess blood, weighed, and homogenized in PBS at a ratio of 1:9 (tissue: PBS) using a glass homogenizer on ice. The homogenate was sonicated to further disrupt the cells and centrifuged at 5,000 g for five minutes. The supernatant was collected and stored at −80 °C for subsequent analysis of HER2/neu and MDA levels. The other half of the ovarian tissue was fixed in 10% formalin for 48 hours, followed by paraffin embedding. Tissue sections of 5–10 μm were cut and stained with hematoxylin and eosin (H&E) for histological evaluation. These sections were examined under a light microscope at 400× magnification to assess folliculogenesis, including the identification and quantification of primary, secondary, and tertiary follicles and corpus luteum.

Biomarker levels in serum and ovarian tissue were assessed using commercially available ELISA kits. The following biomarkers were measured:

• Insulin: Serum insulin levels were quantified using the Rat Insulin ELISA Kit (Bioassay Technology Laboratory, Cat. No. E0707Ra).
• IGF-1: Serum IGF-1 levels were measured using the Rat IGF-1 ELISA Kit (Bioassay Technology Laboratory, Cat. No. E0709Ra).
• HER2/Neu: Ovarian HER2/neu levels were quantified using the Rat HER2/neu ELISA Kit (Bioassay Technology Laboratory, Cat. No. E2460Ra).
• MDA: Ovarian malondialdehyde (MDA) levels, a marker of oxidative stress, were measured using the Rat MDA ELISA Kit (Bioassay Technology Laboratory, Cat. No. E0156Ra).

The ELISA assays were conducted according to the manufacturer’s instructions. Briefly, samples and standards were prepared, and the plates were incubated with the appropriate antibodies for 1 hour at 37 °C. After washing, substrate solutions were added, and the plates were incubated for an additional 10 minutes. Absorbance was measured at 450 nm using a microplate reader.

The ovarian tissue sections were examined using light microscopy at 400× magnification. The number of ovarian follicles at different developmental stages (primary, secondary, tertiary, and Graafian follicles) was counted. Additionally, the presence of primary, secondary, and Graafian follicles was recorded. The histological evaluation was carried out at the Pathology Laboratory, Faculty of Veterinary Medicine, Universitas Gadjah Mada.

The data were expressed as mean ± standard deviation (SD). The normality of the data was assessed using the Shapiro–Wilk test. Statistical comparisons between groups were made using one-way analysis of variance (ANOVA), followed by the post-hoc Duncan’s multiple range test for pairwise comparisons. Statistical significance was considered at p < 0.05. All data analyses were performed using SPSS version 26.0.

The FBG levels of all experimental groups were measured three days after STZ induction to confirm the development of T1DM. As shown in Table 1, the FBG levels in all STZ-induced groups (P2–P5) were significantly higher than in the normal control (P1) (p < 0.0001). Specifically, the FBG levels in the STZ-induced groups exceeded 400 mg/dL, a value well above the diabetic threshold of 200 mg/dL, confirming successful induction of T1DM. The FBG values among the STZ-induced groups (P2–P5) were comparable, with no significant differences observed between them (p > 0.05). These results validated the use of these rats as diabetic models for subsequent biomarker and histological analysis.

The effects of magnesium supplementation and insulin therapy on key biomarkers, including serum insulin, IGF-1, HER2/neu, and MDA, are summarized in Table 1 and visualized in Fig. 1. These biomarkers reflect metabolic status, oxidative stress, and ovarian health in STZ-induced diabetic rats.

Figure 1. 

Effects of treatments on biomarkers in STZ-induced T1DM rats. a. Serum insulin levels (mIU/L), b. Serum IGF-1 levels (ng/mL), c. Ovarian HER2/neu levels (µg/mL), and d. Ovarian MDA levels (nmol/mL) across treatment groups. Data are expressed as mean ± SD. Different superscripts indicate significant differences between groups (p < 0.05, one-way ANOVA with Duncan’s post-hoc test). Treatment groups: P1 (normal control), P2 (STZ-induced untreated), P3 (STZ-induced + insulin), P4 (STZ-induced + magnesium), and P5 (STZ-induced + insulin + magnesium).

Serum insulin levels showed significant differences across the groups (p < 0.0001). Diabetic rats in the untreated group (P2) had significantly lower insulin levels (2.41 ± 0.15 mIU/L) compared to the normal control group (P1: 4.05 ± 0.37 mIU/L). Treatment with insulin alone (P3) or magnesium supplementation (P4) partially restored insulin levels, but the combination therapy (P5) proved most effective. Insulin levels in P5 (4.19 ± 0.29 mIU/L) were comparable to those in the normal control group (P1), with no significant differences between these groups (p > 0.05). This underscores the synergistic effect of insulin and magnesium in regulating blood glucose and improving pancreatic function. For IGF-1, the same sort of trend was observed, with significant differences between groups (p < 0.0001). IGF-1 was lowest in the untreated diabetic rats (P2: 40.95 ± 2.96 ng/mL). Insulin (P3) and magnesium (P4) caused improvement, and P5 (combination therapy) restored IGF-1 levels almost back to normal (54.51 ± 4.45 ng/mL), close to the control group (P1: 55.20 ± 9.69 ng/mL). This shows that magnesium, with insulin, supports IGF-1, which is important for follicle growth and overall health.

Combination therapy in P5 restored IGF-1 levels to near-normal values (54.51 ± 4.45 ng/mL), closely approximating those in the control group (P1: 55.20 ± 9.69 ng/mL), suggesting magnesium, particularly in combination with insulin, plays a crucial role in regulating IGF-1. It is a key factor in ovarian folliculogenesis and metabolic health. The treatments also significantly affected ovarian HER2/neu levels (p < 0.0001). The untreated diabetic group (P2) had substantially lower HER2/neu levels (4.47 ± 0.76 µg/mL) compared to the normal control group (P1: 9.45 ± 2.13 µg/mL).

Magnesium supplementation (P4: 7.58 ± 1.56 µg/mL) and combination therapy (P5: 7.46 ± 2.48 µg/mL) significantly improved HER2/neu levels compared to P2. However, the HER2/neu levels in P4 and P5 were almost the same as in P1. This means magnesium might help ovarian receptors work better and ameliorate ovarian problems in diabetes. Malondialdehyde (MDA), which indicated oxidative stress, was higher in the untreated diabetic group (P2: 1.96 ± 0.54 nmol/mL) than in the control group (P1: 0.94 ± 0.30 nmol/mL).

Treatments with insulin alone (P3: 1.01 ± 0.21 nmol/mL), magnesium (P4: 0.94 ± 0.16 nmol/mL), or combination therapy (P5: 0.97 ± 0.14 nmol/mL) significantly reduced MDA levels (p < 0.0001). The single magnesium treatment (P4) and the combination (P5) restored MDA levels to the normal control (P1). These results confirm the antioxidant potential of magnesium in combating oxidative stress caused by diabetes.

The effects of treatments on ovarian follicle counts are summarized in Table 2 and visualized in Fig. 2. Ovarian follicle count serves as a key indicator of reproductive health and reflects the degree of restoration of ovarian function in diabetic rats.

Figure 2. 

Effects of treatments on ovarian follicle counts in STZ-induced T1DM rats. Ovarian follicle counts across treatment groups: P1 (normal control), P2 (STZ-induced untreated), P3 (STZ-induced + insulin), P4 (STZ-induced + magnesium), and P5 (STZ-induced + insulin + magnesium). Data are expressed as mean ± SD. Different superscripts indicate significant differences (p < 0.05). Combination therapy (P5) showed the highest restoration among STZ-induced groups.

Fig. 2 shows that the untreated diabetic group (P2) had a significantly lower ovarian follicle count (9.14 ± 2.04) than the normal control group (P1: 22.14 ± 1.35) (p < 0.0001). Insulin therapy (P3: 14.00 ± 1.41) and magnesium supplementation (P4: 14.43 ± 2.07) both significantly improved follicle counts compared to P2 (p < 0.05). However, combination therapy (P5: 18.57 ± 2.37) was the most effective intervention, resulting in follicle counts that closely approached those of the normal control group. These results demonstrate the synergistic effects of magnesium and insulin in promoting folliculogenesis and mitigating reproductive dysfunction in diabetic rats.

The histopathological analysis of ovarian tissues showed significant differences in follicular development across the treatment groups (Fig. 3). In the normal control group (P1), ovarian tissues demonstrated well-organized structures with a balanced presence of primary, secondary, and Graafian follicles. In contrast, the untreated diabetic group (P2) had disorganized ovarian architecture, with a marked reduction in the number of secondary and Graafian follicles as well as an increase in atretic follicles. These results confirm the detrimental effects of diabetes on ovarian histology. In the group that received insulin (P3), the ovaries showed partial improvement, with more secondary and Graafian follicles compared to P2. Magnesium supplementation (P4) also provided benefits, with healthier-looking follicles and reduced atresia. The combined treatment group (P5) had the best results, with ovarian structures resembling those of the normal control group (P1). This treatment markedly improved follicle development, with more secondary and Graafian follicles than in the insulin-only group (P3) or the magnesium-only group (P4).

Figure 3. 

Histopathological analysis of ovarian tissues across the treatment groups revealed distinct follicular stages. Representative ovarian tissue sections highlighted primary follicles (green arrows), secondary follicles (blue arrows), and Graafian follicles (red arrows) in the following groups: (A) P1 (normal control), (B) P2 (untreated diabetic), (C) P3 (diabetic + insulin), (D) P4 (diabetic + magnesium), and (E) P5 (diabetic + insulin + magnesium). The combination therapy group (P5) demonstrated the most significant restoration of follicular development.

Diabetes mellitus is characterized by chronic hyperglycemia, which results from impaired insulin secretion or action (Merovci et al. 2021). In this study, untreated diabetic rats (P2) displayed significantly elevated fasting blood glucose (FBG) levels and reduced insulin levels, indicating β-cell destruction due to STZ-induced oxidative stress. These results match an earlier study that linked hyperglycemia in T1DM to oxidative stress and inflammation, which aggravates insulin resistance and disrupts β-cell function (Davies et al. 2022).

Insulin therapy (P3) helped increase serum insulin levels, but magnesium (P4) improved them further by enhancing insulin sensitivity and reducing oxidative stress (Massaro et al. 2016). When both therapies were combined (P5), insulin levels were nearly normalized, showing a stronger effect (Qi et al. 2023). Magnesium works by increasing insulin receptor sensitivity, modulating tyrosine kinase activity, and improving glucose transport, and this study shows that it could be a useful treatment for T1DM (Islam and Nyholt 2022). In both magnesium-treated (P4) and combined therapy (P5) groups, IGF-1 levels rose significantly (Nagao et al. 2021). IGF-1 helps regulate glucose metabolism and supports follicle growth in the ovaries (Zhang et al. 2023). The fact that magnesium restored IGF-1 indicates that it could help regulate endocrine signaling, probably through its antioxidant effects and modulation of the insulin/IGF-1 pathway (Abozaid et al. 2023).

Oxidative stress is a critical pathogenic factor in diabetes, contributing to structural and functional impairment of ovarian tissue (Yang et al. 2021). Untreated diabetic rats (P2) had high MDA levels, indicating significant lipid and cellular damage. However, magnesium (P4) and combination therapy (P5) reduced MDA levels (Zhang et al. 2023). This is probably because magnesium can mitigate oxidative stress by scavenging reactive oxygen species (ROS) and preventing lipid peroxidation (Parcheta et al. 2021).

Magnesium also improved HER2/neu levels in P4 and P5, suggesting that it supports receptor function and cellular signaling (Slezak et al. 2024). HER2/neu is important for cell survival and growth, particularly in the ovaries, where it contributes to follicular development (Abdel Hamid et al. 2022). In untreated diabetic rats, receptor expression was lower, showing damage from oxidative stress. However, magnesium treatment countered this effect and reduced cell death (Bhatti et al. 2022). By lowering MDA levels and restoring HER2/neu expression, magnesium alleviated oxidative stress and supported ovarian cell viability. These findings suggest that magnesium may mitigate diabetes-induced cellular damage by attenuating oxidative stress and preserving ovarian function (Bhatti et al. 2022).

Diabetes causes oxidative stress, which disrupts ovarian function. In rats, this resulted in fewer follicles and more follicular atresia in the untreated group (P2) (Kanak et al. 2024). This study looked at tissue samples and found that magnesium (P4) and insulin (P3) helped improve follicular development and restored follicle counts, including primary, secondary, and Graafian follicles (Wang 2020). When both treatments were used together (P5), the effect was strongest, almost comparable to the control group (P1) (Wang 2020). This shows that magnesium and insulin together help restore ovarian structure and function (Sengupta et al. 2024). Magnesium may promote follicular growth by preventing granulosa cell death and reducing oxidative stress in the ovaries (Kanafchian et al. 2020). Insulin also promotes follicular growth by enhancing gonadotropin signaling and supporting steroidogenesis (Ipsa et al. 2019). When both are used together, the effects are amplified, as magnesium enhances insulin’s action by increasing ovarian steroid production and improving signaling.

The results highlight the importance of addressing oxidative stress and insulin resistance in managing diabetes and its effects on reproduction. Insulin therapy is a mainstay in diabetes treatment, but adding magnesium may further improve insulin sensitivity, reduce oxidative stress, and support ovarian health. Therefore, combined use of insulin and magnesium could be a promising strategy for addressing reproductive complications in people with T1DM. This study examined how magnesium influences factors such as HER2/neu, IGF-1, and MDA, which are directly implicated in oxidative stress and ovarian health. More clinical studies are needed to establish effective dosing, treatment duration, and long-term outcomes of using magnesium with insulin in patients with diabetes.

Although this study provides valuable evidence of magnesium’s benefits, there are limitations to consider. The short-term design may not capture long-term effects on ovarian function, and the use of an animal model may not fully replicate human pathophysiology. Future studies should investigate the molecular pathways underlying the protective effects of magnesium, particularly its interaction with insulin signaling and oxidative stress regulation. Long-term studies focusing on fertility outcomes, hormonal regulation, and pregnancy success rates are also needed.