Evaluation of mineral elements, some antioxidants and biochemicals in follicular fluid of cow ovaries

Evaluation of mineral elements, some antioxidants and biochemicals in follicular fluid of cow ovaries | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of mineral elements, some antioxidants and biochemicals in follicular fluid of cow ovaries Zirak M R Palani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7068159/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Trace elements are essential for follicular development, oocyte maturation and fertility. Nevertheless, as yet, the role of these Trace elements within follicular environment has seldom been investigated in bovines, especially in the presence of contemporary environmental pollution. The objective of this work was to determine the concentrations of toxic elements and trace elements (Cu, Zn, Se, Fe, Co, Mo, Cd, As, Pb, and In) and Malondialdehyde, glutathione, cholesterol, and glucose in bovine follicular fluids (bFFs) in various follicular sizes in different follicular development stages. Important biochemical variables were also measured to correlate them with follicular maturity. Follicles were categorized as small (3–5 mm), medium (6–9 mm), and large (10–22 mm). Follicular fluid (FF) samples were subjected to inductively coupled plasma emission spectrometry (ICPE-9000) trace elements and analyzed spectrophotometrically (glucose, cholesterol, GSH and MDA). A significantly increase (p < 0.05) in the contents of copper, zinc, selenium and cobalt was observed as follicles matured, which may be related to the higher antioxidant and metabolism potential of the mature follicle. Medium follicles showed maximum concentration of iron and molybdenum for follicles of size (6-9 mm). For toxic elements, lead decreased, and arsenic increased in follicle development. Cadmium and indium were found at trace levels, with indium detected for the first time in bovine follicular fluid. Levels of cholesterol and glutathione also increased with the size of the follicles, while MDA decreased, indicating enhanced oxidative status. Glucose was similar in both groups. These findings indicate the stage-dependent regulations of mineral elements in follicle development, which has implications for the maintenance of oocyte quality and follicle health. first-time detection of Inindium (In) raises questions about its potential reproductive effects and environmental implications. Ovaries cows heavy metals antioxidants seminal fluid Introduction Follicular fluid is an avascular compartment within the mammalian ovary, isolated by the follicles wall from the neighboring tissues, which forms the blood–follicle barrier (BBB) (Sorak et al.,2024; Sekulovski et al., 2020; Liu et al., 2022). This fluid, including the filtrate of blood, and local substances secreted by granulosa and theca cells, were indicative of the metabolic and physiological condition of the follicle (Pan et al.,2024). Follicular fluid (FF) acts as an important microenvironment for oocyte growth and maturation, providing nutrition and necessary factors for the requirements of oocytes, and promoting the maturation process to create a favorable local environment there fore as an important mechanism in maintaining female fertility (Sorak et al.,2024; Pan et al.,2024). Minerals, in particular trace elements, are essential for various bodily functions including digestion and bodily functions and are required in biosynthesis. They play a major role in serving structural frames of various organs, tissues, electrolyte, fundamental antigen, and a renewable source in metabolite, catalyst of enzyme and hormone system Sampath et al., 2023; Palomares, 2022). These are fundamental minerals for thegrowth, reproduction and production performance ofthe animals in livestock. Optimal handling of micronutrients also enhances animal products such asmilkandfertility (Palomares et al., 2024; Jyani et al., 2024) .Mineral elements are categorized as major essential minerals ( Ca, P, K, Na, S, Mg) and trace elements (Fe, I, Cu, Mn, Zn, Co, Mo and Se), Trace minerals exert a particularly critical influence on reproduction; however, deficiencies in certain major minerals especially calcium and phosphorus can likewise impair fertilit (Verma & Sweety,2024; Kumar et al.,2025;Al-Hadeedy et al.,2019). The organic factors like mineral chelate and/ or trace elements affect the follicular regrowth which may enhance the fertility rates due to the improvement in Oocyte fertilization rate, decreasing in embryonic mortality, improvement in the uterine environment, and in the increasing of the intensity of mating in an animal (Mion et al., 2023; Morris et al., 2025). The FG composition of follicular fluid changes according to the migration of the oocyte with its surrounding cells because metabolic, hormonal and redox conditions evolve concomitantly to oocytes maturation (Hessock et al., 2023; Pan et al., 2024).Thus, studies on a biochemical characterization of the FF as for trace and bioactive elements (like glucose or cholesterol) as well as on oxidative stress markers (like for example reduced glutathione(GSH), maldondialdehyde (MDA) will be crucial to understanding the etiological grounds of the metabolic and functional balance of the oocyte maturation and follicular health. Trace elements such as Selenium (Se), zinc (Zn), copper (Cu), iron (Fe) Cobalt (Co), son cofactores esenciales para enzimas que protegen a las celulas del estrés oxidativo, como superoxido dismutasa (SOD) glutatión peroxidasa (Spears and Weiss, 2008; Tian et al., 2017). On the other hand, harmful heavy metals such as lead (Pb), arsenic (As) and cadmium (Cd) can exert negative effects on gonadal function by impairing hormone synthesis, damaging cells, and degrading oocyte quality (Zhang et al., 2024; Rzymski et al., 2015). In history, it is a new environmental pollutant, but the reports on its damage to reproductive system are not a lot. It is a leap across the study of contamination using reproductive damage (Liu et al., 2022) to discover indium in the follicular fluid accordingly. The aims of the present study are therefore to assess essential and toxic trace element content of bovine FF and follicles of different sizes (small, medium, and large) and oxidative stress markers, glucose and cholesterol levels in FF. In addition, this work provides the first evidence of indium distribution in environmental and reproductive backgrounds that indium has been slight consideration effect on reproductive efficiency in cows, and, thus, this study which is able to broaden our understanding of how the follicular micro-environment influences fertility in cows. Materials and Methods The study was carried out in winter at the Animal Reproduction Laboratory. Healthy, non-pregnant adult female cattle of unknown genotype were obtained immediately post-mortem from a local abattoir; only reproductive tracts that appeared phenotypically normal were selected. Ovaries were transferred to the laboratory within 2 h of excision in crushed ice (4 °C) placed in a sealed polyethylene bag. Extraction of Follicular-fluid collection Upon arrival, ovaries were rinsed in cold (4 °C) sterile physiological saline (0.9 % NaCl) to remove excess blood and tissue, after delivery in the laboratory. Follicles 3–8 mm in diameter were measured with a Vernier micro-caliper. Follicles were punctured, and their contents aspirated with a sterile 18-gauge needle attached to a syringe. Follicles showing visible blood contamination, pathological fluid, or cystic change were excluded. The FF was centrifuged at 3 000 rpm for 10 min at 4 °C to remove cells and debris; the clear supernatant was aliquoted into polypropylene (Eppendorf) tubes and stored at −20 °C until analysis. Biochemical assays Glucose and cholesterol concentrations in FF were determined colorimetrically with commercial enzymatic kits (spectrophotometer; manufacturer’s instructions. Lipid peroxidation was quantified as malondialdehyde (MDA) using the thiobarbituric acid reactive substances (TBARS) assay (Guidet & Shah, 1989) and expressed as nmol ml⁻¹. Reduced glutathione (GSH) was measured with the modified Ellman reaction (Al-Zamely et al., 2001) and expressed as nmol ml⁻¹. (DTNB) values were expressed in μmol/ml.Mineral Content Trace-element determination, Trace elements were quantified at part-per-billion (µg l⁻¹) levels. Samples (0.50 ml) were microwave-digested, diluted to 10 ml (dilution factor = 20), and analysed on an ICP-OES (ICPE-9000; Shimadzu: RF power 1.2 kW; plasma gas 15 l min⁻¹; nebuliser gas 0.70 l min⁻¹). Calibration curves (0.01–50 µg l⁻¹; six points, r² ≥ 0.9995) were prepared from a multi-element stock solution. Accuracy was checked every ten samples with NIST SRM 1640a and SRM 1643f (recoveries 96–103 %). Procedural blanks were < 0.5 × LOD; LODs (3 σ blanks) ranged from 0.02 to 0.15 µg l⁻¹. Instrument counts were blank-corrected, converted to concentrations (µg l⁻¹ ≈ ppb), and multiplied by the dilution factor. Final follicular-fluid values were obtained by multiplying by the dilution factor. Results are reported to two or three significant figures; instrumental RSD for triplicates was < 4 %. The elements which were analyzed to be nickel (Ni), copper (Cu), iron (Fe), cobalt (Co), selenium (Se), zinc (Zn), cadmium(Cd), molybdenum (Mo), lead (Pb), antimony (Sb) and arsenic (As). : Analysis :Results were analyzed using Addinsoft, (2016). Model Y₍ᵢⱼₖ₎ = μ+ Aᵢ + Bⱼ + (AB)ᵢⱼ + eᵢⱼₖ, means having different lettersin the same column differ significantly at P ≤ 0.05, by Duncan’s, (1955) multiple range tests and are expressed as mean values ± SD. All procedures were approved by the University of Kirkuk ethics committee (Approval No. 03C01L004) and complied with ARRIVE guidelines for ruminant welfare; veterinary assistance was available at all times. Results and Discussion First: Concentration of mineral elements in the follicular fluid Routine findings show that the concentration of trace minerals varies significantly among small, medium and large follicles in the follicular fluid of native cows. Copper (Cu) concentrations increased with follicle size: 0.090 ppb in small follicles, 0.298 ± 0.053 ppb in medium follicles, and 0.428 ± 0.125 ppb in large follicles (P < 0.005Such an increase is consistent with findings in cattle and other ruminants, demonstrating that follicular copper rises with growth as an enzymatic co-factor essential for follicular development (Mion et al., 2023; Zhang et al., 2024; Lou et al., 2025). Zinc (Zn) showed a similar pattern, with concentrations of 0.583 ± 0.180 ppb in small follicles, 0.830 ± 0.347 ppb in medium follicles and 1.813 ± 0.388 ppb in large follicles, a difference that was statistically significant.This accumulation is in agreement with the participation of zinc in cell growth and protein synthesis and in essential activities in the follicle during oocyte maturation (Yao et al., 2023; Liu et al., 2024). As to Se, it significantly increased 23.988 a±0.906 ppb in large follicles, a finding in agreement with its antioxidant role protecting oocytes from oxidative stress, thus making a better follicular environment (Lava Kumar et al., 2024; Ali et al., 2023). The study indicated that adding selenium- or zinc-enriched yeast improved antioxidant status as well as liver and kidney function in local Iraqi goats (Shareef, 2025).The level of Fe increased markedly from 9.373 ± 0.757 ppb in small follicles to 21.368 ± 4.151 ppb in medium follicles and then, decreased slightly to 19.650 ± 1.253 ppb in large follicles, which may suggest that during maturation period, there was more metabolic activity in medium follicles, and the blood perfusion and oxygen transport increased (Hessock et al., 2023; Shahzad et al., 2024;Brantmeier et al., 1987). Also cobalt (Co) content also increased gradually and the contents in small,medium and large follicles were 2.430±0.295,3.255±0.342,5.438±0.377 ppb, respectively. This indicates how essential this element may be in some required biochemical processes in follicular cells, potentially in the process of cell growth as vitamin B ₁₂ (Tang et al., 2024; Ayyat et al., 2025 ;McDowell, 1992). Molybdenum concentrations were 0.390 ± 0.111 ppb in small follicles, 2.125 ± 0.384 ppb in medium follicles (the highest value), and 1.645 ± 0.140 ppb in large follicles, suggesting a possible regulatory interaction between Mo and Cu. For the toxic element Cd, no significant differences were detected among follicles, with values ranging from 0.343 ± 0.098 ppb to 0.488 ± 0.184 ppb. No significant difference was observed also for the In (0.013±0.003 ppb in SF, 0.098±0.048 ppb in MF and 0.093±0.003 ppb in LF). The presence of indium in bovine follicular fluid is a novel observation, and further studies are required to clarify its biological significance. Arsenic (As) also increased with follicle size, reaching 0.318 ± 0.080 ppb in small follicles, 0.675 ± 0.094 ppb in medium follicles, and 1.180 ± 0.234 ppb in large follicles. This progressive accumulation, despite As’s known toxicity, suggests its retention in the follicular environment during growth. The Pb, in turn, reacted inversely reaching 2.188±0.081 ppb in small, 1.645±0.14 ppb in medium and 1.228±0.09 ppb in large follicles, less possibly avoiding its toxic influence when the follicle atresia retards. Taken together, these results indicate that the size of the follicle affects the level of TMs in FF and add in support of the concept that FF is produced by plasma filtration and follicular metabolic activity leading to modifications of the FF (Pan et al., 2024; Hessock et al., 2023). These findings highlight the importance of these elements also in reproduction and the necessity of including it in the rations if fertility rate of cows has to be increased (Mion et al., 2023; Palomares & Ferrer, 2024). Table (1): Concentrations of mineral elements (ppb) in three different size of seminal fluid in bovine ovaries Trace Elements Follicular size Small Follicles (3-5mm) Medium Follicles (6-9mm) Large Follicles (10-22mm) Copper (Cu) 0.090 b±0.033 0.298 ab±0.053 0.428 a±0.125 Zinc (Zn) 0.583 b±0.18 0.830 ab±0.347 1.813 a±0.388 Selenium (Se) 13.730 c±0.638 20.213 b±0.487 23.988 a±0.906 Iron (Fe) 9.363 b±0.757 21.368 a±4.151 19.650 a±1.253 Cobalt (Co) 2.430 b±0.295 3.255 b±0.342 5.438 a±0.377 Molybdenum (Mo) 0.390 c±0.111 2.125 b±0.384 1.645 b±0.14 Cadmium (Cd) 0.343 a±0.098 0.435 a±0.074 0.488 a±0.184 Indium (In) 0.013 a±0.003 0.098 a±0.048 0.093 a±0.003 Arsenic (As) 0.318 b±0.08 0.675 b±0.094 1.180 a±0.234 Lead (Pd) 2.188 a±0.081 1.645 b±0.14 1.228 c±0.09 *Values are expressed as Mean ± Standard Error ) SE ( . *Different superscript letters (a, b, c) within the same column indicate statistically significant differences (P < 0.05) according to Duncan’s multiple range test. In light of these new findings, the elevated levels of copper, zinc, selenium, cobalt, and molybdenum associated with follicular growth suggest that blood serves as the primary reservoir of these elements and that they can readily penetrate follicles— mechanism that weakens the blood–follicle barrier during development. This observation is consistent with earlier studies reporting higher concentrations of these metals in follicular fluid (FF) that correlate with follicle size (Pawliński et al., 2023; Shahzad et al., 2024; Hessock et al., 2023). Iron showed a similar pattern, mirroring previous reports (Pan et al., 2024; Mion et al., 2024).The uniformly high arsenic content and consistently low lead levels in larger follicles suggest a narrow margin between beneficial and detrimental arsenic in follicles and lead occurring at low levels in follicles. Second: Biochemical indicators in the follicle (Table 2) Cholesterol concentration rose with follicle size, reaching 31.073 ± 1.229 mg dL⁻¹ in small follicles, 37.568 ± 1.785 mg dL⁻¹ in medium follicles, and 45.338 ± 2.232 mg dL⁻¹ in large follicles. These differences were statistically significant across all three follicle classes. Glutathione (GSH) levels were 0.185 ± 0.017 nmol ml⁻¹ in small follicles, 0.340 ± 0.039 nmol ml⁻¹ in medium follicles, and 0.348 ± 0.029 nmol ml⁻¹ in large follicles. These values differed significantly among follicle sizes.The larger the follicles. Malondialdehyde (MDA) levels decreased significantly from 0.600 ± 0.077 nmol ml⁻¹ in small follicles to 0.348 ± 0.045 nmol ml⁻¹ in medium follicles and 0.308 ± 0.041 nmol ml⁻¹ in large follicles, confirming marked biochemical differences among follicle sizes. Recent research shows that particular hormonal therapies have markedly enhanced reproductive and haematological parameters in Awassi ewes, underscoring the importance of hormonal regulation in boosting livestock productivity (Alwan, Majid & Ismail, 2018a,b). Table (2): Biochemical levels of follicular fluid in three different sized follicles in cow ovaries Parameter Follicular size Small Follicles (3-5mm) Medium Follicles (6-9mm) Large Follicles 10-22mm) Glucose (mg/dl) 55.718 a±2.522 59.135 a±1.607 57.205 a±2.582 Cholesterol (mg/dl) 31.073 c±1.229 37.568 b±1.785 45.338 a±2.232 GSH (nmol/mL) 0.185 b±0.017 0.340 a±0.039 0.348 a±0.029 MDA (nmol/mL) 0.600 a±0.077 0.348 b±0.045 0.308 b±0.041 *Values are expressed as Mean ± Standard Error )SE(. *Different superscript letters (a, b, c) within the same column indicate statistically significant differences (P < 0.05) according to Duncan’s multiple range test. Biochemically, the nearly constant glucose concentration among small, medium and large bovine follicles suggests tight intrafollicular control of carbohydrate supply (Anderson et al., 2012), a trend that has recently been confirmed in cows of the Holstein-Friesian breed (Pawliński et al., 2023) and in slaughterhouse goats where the slight numerical glucose decline associated with the increase in diameter did not reach statistical significance (Bordoloi et al., 2025). In contrast to dromedaries, however, intrafollicular glucose increases seasonally in Bactrian camels during the breeding season (Abdoon et al., 2025), which emphasizes the species-specific nature of the difference. The greater conversion of cholesterol to steroid hormones in large bovine follicles also corresponds to a Redox shift, involving higher reduced-glutathione and lower oxidative markers such as hydrogen-peroxide and malondialdehyde, providing a more favourable environment for granulosa-cell function (Hill et al. 2014; Lava Kumar et al. 2014) and conflicts with other studies in camels (Huang et al.,2002). The increased cholesterol conversion to steroid hormones is explained by the increased GSH and lower MDA levels in the fluid of larger follicles, indicating reflects less oxidative damage, which benefits cell function and follicle growth in local bovine oocytes. This was supported by the present study and is consistent with the findings of (Rufai et al. 2013; Beg and Ginther, 2006; Zheng et al. 2023) where they describe the conversion of cholesterol into steroid hormones during ontogenesis. With respect to non-enzymatic antioxidants, GSH was showed to be significantly enhanced during follicle development, suggesting an enhanced anti-oxidative force of follicular fluid, a critical factor that protects the oocyte from oxidative stress during maturation (Takahashi et al., 2003; Lava Kumar et al., 2024). There was also a considerable decline in the level of malondialdehyde (MDA), which arises from lipid peroxidation and signifies reduction in oxidative stress with follicles’ maturity, that was accompanied by increased antioxidants, e.g., selenium and glutathione (Duan et al., 2024; Agarwal et al., 2012). Third: Analysis of the correlation relationships between mineral elements in follicular fluid. Thus, the results of this study demonstrate statistically significant pairwise relationships among several mineral elements in bovine follicular fluid, revealing complementary interactions or competition within the oocyte microenvironment. Strong positive correlations for the pairs Se–Cu, Se–Co, and Se–Mo—each biologically complementary—link these elemental interactions to physiological processes of follicle growth and oocyte maturation. It is well known that Se is a trace element required for the enzyme glutathione peroxidase that reduces free radicals and oxidative stress in follicular cells (Lava Kumar et al., 2024; Agarwal et al., 2012), thus improving follicular cell viability and oocyte quality. Furthermore, the study found a high positive correlation between the level of selenium and cobalt (r = 0.875), demonstrating their mutual elevation in the improvement of metabolic processes and protection from oxidative stress. These results are consistent with those of Ingle et al. (2017) finding that elements found in the follicular fluid are directly related to follicular maturation and ovulation quality. Table (3): A table showing the correlation matrix (Pearson): for the mineral elements in the follicular fluid of cows’ ovaries. Variables Cu Copper Zn Zinc Co Cobalt Se Selenium Cd Cadmium As Arsenic Fe Iron Mo Molybdenum Pb Lead indium (In) Cu Copper 1 0.375 0.600 0.706 -0.160 0.661 0.442 0.648 -0.751 0.234 Zn zinc 0.375 1 0.607 0.611 0.193 0.305 0.281 0.515 -0.396 0.120 Co Cobalt 0.600 0.607 1 0.875 0.344 0.693 0.621 0.830 -0.764 0.561 Se Selenium 0.706 0.611 0.875 1 0.173 0.681 0.739 0.793 -0.883 0.623 Cd Cadmium -0.160 0.193 0.344 0.173 1 0.383 0.305 0.339 -0.165 0.287 As arsenic 0.661 0.305 0.693 0.681 0.383 1 0.532 0.925 -0.841 0.481 Fe Iron 0.442 0.281 0.621 0.739 0.305 0.532 1 0.475 -0.592 0.870 Mo Molybdenum 0.648 0.515 0.830 0.793 0.339 0.925 0.475 1 -0.867 0.393 Pb Lead -0.751 -0.396 -0.764 -0.883 -0.165 -0.841 -0.592 -0.867 1 -0.501 indium (In) 0.234 0.120 0.561 0.623 0.287 0.481 0.870 0.393 -0.501 1 Values in bold are different from 0 with a significance level alpha=0.05 By contrast, the research revealed strong negative correlations between Pb with major trace elements particularly Se (r = −0.883), Mo (r = −0.867), and Co (r = −0.764). suggests that lead may be influencing the absorption or stability of these elements in the follicular milieu. The toxic properties of lead, that may interfere with the activities of enzymes and chain of transport of electrons in reproductive cells leading to an increase in intracellular oxidant stress are well known (Flora et al., 2012; El-Boshy et al., 2015). Furthermore, lead deposition in the ovaries was correlated with reduced number of mature follicles and lower oocyte quality, which affect fertility. Iron (Fe) and indium (In) by contrast, showed a strong positive correlation (r = 0.870), although indium is not among the most abundant biological elements. This relationship might reflect similar transport mechanisms or their attachment to carrier proteins, including transferrin, as suggested by Guo et al (2025). Selenium and zinc are considered to be extremely important trace elements for the health of any animal, due to their positive effects on the regulation of hormones and enzymes, enhancement of antioxidant defenses, and support for the activity of metabolic and immunological systems. However, their biological effects are highly dose-dependent, and the two elements can interact synergistically or antagonistically, promoting beneficial actions or increasing adverse effects. Studies by Palani et al. (2019, 2020) and Al-Obaidi et al. (2020) showed that dietary fat proportion significantly alters serum biochemical parameters and antioxidant capacity, producing beneficial effects on animal health and metabolism. Nevertheless, this relationship should be further investigated, taking also into account any impact on either industrial or nutritional contami- nants present in feed sources. Overall, these findings highlight the importance of maintaining mineral equilibrium in follicular fluid due to its direct influence on the microenvironment of oocytes. Additionally, they suggest that the presence of harmful substances like lead in the reproductive environment can disrupt mineral balance, potentially impairing reproductive function in cows. This study reports, for the first time, the presence of indium in bovine ovarian follicular fluid at low concentrations. Further research is warranted to clarify, particularly regarding its associations with other trace metals, characterise potential synergistic or antagonistic interactions, and assess its impact on reproductive efficiency. Conclusion This trial revealed substantial changes in the concentrations of essential trace elements: copper, zinc, selenium, and cobalt, in the bovine follicular fluid alongside the growth and maturation of follicles.These findings indicate that these endogenous elements promote metabolic and antioxidant activities within the follicular micro-environment conducive to oocyte quality. In contrast, the toxic metal lead showed strong negative associations with these elements. suggesting that mineral imbalance and excess oxidative stress can impair follicular function and interference with vital enzymes. Of particular interest is the first-time identification of indium in follicular fluid, where a number of elements showed positive correlations with iron. Further research is needed to unearth additional knowledge about the potential role of in in the reproductive system and the underlying mineral interactions. Overall, the results highlight the importance of a delicate mineral balance in follicular fluid for healthy follicles and efficient oocyte maturation and the demand to curb exposure to toxic heavy metals in the farm setting to enhance cows’ fertility. References Abdoon, A. S. S., Soliman, S. S., Hussein, N. S., Haggag, S. H. I., El-Sanea, A. M., & Abdel-Hamid, A. Z. (2025). Metabolomic profile of dromedary camel follicular fluid during the breeding and non-breeding seasons. Scientific Reports, 15, 8923. https://doi.org/10.1038/s41598-025-91710-9 Addinsoft. (2016). XLSTAT statistical and data analysis solution (Version 2016.1) [Computer software]. Addinsoft. https://www.xlstat.com Agarwal, A., Aponte-Mellado, A., Premkumar, B. J., Shaman, A., & Gupta, S. (2012). The effects of oxidative stress on female reproduction: A review. Reproductive Biology and Endocrinology, 10, 49. https://doi.org/10.1186/1477-7827-10-49 Al-Hadeedy, I. Y., Al-Nuaimi, M. I. A., & Ali, A. R. (2019). Effect of adding local minerals clay (meqdadia) in the female japanese quail diets upon production performance, egg quality and carcass traits. Indian Journal of Public Health Research and Development, 10(8). https://doi.org/10.5958/0976-5506.2019.02020.5 Ali, M. E., Abd-Elkariem, M. F. A., Fahmy, S., Hussein, H. A., Abdelrahman, M., & Ahmed, A. E. (2023). Dietary supplementation with different nano and organic selenium doses improves ovarian activity, fertility rate, and progesterone level in Ossimi ewes. Italian Journal of Animal Science, 22, 651–659. https://doi.org/10.1080/1828051X.2023.2231946 Al-Obaidy, M.H.A., Palani, Z.M.R., Sirwan, K., Hameed, K. EFFECT OF ADDING DIFFERENT PERCENTAGES OF GLYCERIN IN RATIONS ON SOME BLOOD SERUM BIOCHEMICAL AND ANTIOXIDANTS TRAITS IN MALE LAMBS AWASSI BREED SHEEP. Biochemical and Cellular Archives, 2020, 20(1), pp. 2253–2257. Alwan, A. S., Majid, A. F., & Ismail, H. I. (2018a). The effect of using some hormonal treatments on some reproductive performance traits in Awassi ewes. Kirkuk University Journal for Agricultural Sciences, 9(4), 17–23. Alwan, A. S., Majid, A. F., & Ismail, H. I. (2018b). The effect of a hormonal program on some physiological and biochemical blood traits in Awassi ewes. Kirkuk University Journal for Agricultural Sciences, 9(4), 52–59. Al-Zamely; Mizil, Y. O. and Al-Nimer, M. S.(2001). Detection the level of peroxynitrite and related with antioxidants status in the serum of patients with acute myocardial infraction national. J. of Chemistry . 4 : 625-637. Ayyat, M. S., Abdel Monem, U. M., Mostafa, T. H., Thabet, R. M., Abd El-Latif, K. M., & Al-Sagheer, A. A. (2025). Influence of long-term dietary cobalt supplementation on lactation performance and reproductive efficiency in Maghrabi she-camels. Biological Trace Element Research. https://doi.org/10.1007/s12011-025-04551-y Bordoloi PK, Sarmah BC, Dutta DJ, Deka BC. Follicular fluid cholesterol in goat ovary. Indian Vet J. 2000;77:638–639. Bordoloi, P. K., Sarmah, B. C., Dutta, D. J., & Deka, B. C. (2025). Follicular fluid glucose concentration of goat ovary. Indian Journal of Animal Research, 35(1), 81–82. Brantmeier, S.A., Grummer, R.R., Ax, R.L., 1987. ConcentraƟons of high density lipoproteins vary among follicular sizes in the bovine. J. Dairy. Sci. 70, 2145–2149. Chen, Y., Yang, J., & Zhang, L. (2023). The impact of follicular-fluid oxidative stress levels on the outcomes of assisted reproductive therapy. Antioxidants, 12(12), 2117. https://doi.org/10.3390/antiox12122117 Duan, H., Wang, F., Wang, K., Yang, S., Zhang, R., Xue, C., … Xiao, L. (2024). Quercetin ameliorates oxidative-stress-induced apoptosis of granulosa cells in dairy-cow follicular cysts by activating autophagy via the SIRT1/ROS/AMPK signaling pathway. Journal of Animal Science and Biotechnology, 15, 119. https://doi.org/10.1186/s40104-024-01078-5 Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11(1), 1–42. https://doi.org/10.2307/3001478 El-Boshy, M. E., Risha, E. F., Abdelhamid, F. M., Mubarak, M. S., & Hadda, T. B. (2015). Protective effects of selenium against cadmium-induced hematological disturbances, immunosuppressive oxidative stress and hepatorenal damage in rats. Journal of Trace Elements in Medicine and Biology, 29, 104–110. https://doi.org/10.1016/j.jtemb.2014.05.009 Flora, G., Gupta, D., & Tiwari, A. (2012). Toxicity of lead: A review with recent updates. Interdisciplinary Toxicology, 5(2), 47–58. https://doi.org/10.2478/v10102-012-0009-2 Fortune, J.E., Hansel, W., 1985. ConcentraƟons of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulaƟon. Biol. Reprod. 32, 1069-1079. Guidet, B., & Shah, S. V. (1989). Enhanced in vivo H₂O₂ generation by rat kidney in glycerol-induced renal failure. American Journal of Physiology: Renal Physiology, 257(3 Pt 2), F440–F445. https://doi.org/10.1152/ajprenal.1989.257.3.F440 Guo, Q., Qian, C., Wang, X., & Qian, Z.-M. (2025). Transferrin receptors. Experimental & Molecular Medicine, 57, 724–732. https://doi.org/10.1038/s12276-025-01436-x Harmon RJ. Trace minerals and dairy cattle: importance for udder health. In: Biotechnology in the Feed Industry. 1988;Proc Alltech’s 14 th Annual Symp:485-495. Hessock, E. A., Edwards, J. L., Schrick, F. N., Payton, R. R., Campagna, S. R., Pollock, A. B., Clark, H. M., Stokes, A. E., Klabnik, J. L., Hill, K. S., Roberts, S. R., Hinson, M. G., & Moorey, S. E. (2023). Metabolite abundance in bovine preovulatory follicular fluid is influenced by follicle developmental progression post-estrous onset in cattle. Frontiers in Cell and Developmental Biology, 11, 1156060. https://doi.org/10.3389/fcell.2023.1156060 Hidiroglou, M. 1979. Manganese in ruminant nutriƟon. Can. J. Anim. Sci. 59:217-236. Hill, K. S., Edwards, J. L., Payton, R. R., Schrick, F. N., Campagna, S. R., Hessock, E. A., & Moorey, S. E. (2024). Relationships of circulating and pre-ovulatory follicular fluid hydrogen peroxide levels with body condition score and metabolome profiles of lactating beef cows. Agriculture, 14(8), 1406. https://doi.org/10.3390/agriculture14081406 Huang WT, Lu SG, Tang PC, Wu SC, Cheng SP, Ju, JC. Biochemical compositions of follicular fluid and the effects of culture conditions on the in vitro development of pig oocytes. Asian Aust J Anim Sci. 2002;15:1403–1411. Ingle, M. E., Bloom, M. S., Parsons, P. J., Steuerwald, A. J., Kruger, P., & Fujimoto, V. Y. (2017). Associations between IVF outcomes and essential trace elements measured in follicular fluid and urine: A pilot study. Journal of Assisted Reproduction and Genetics, 34(2), 253 – 261. https://doi.org/10.1007/s10815-016-0853-7 Jyani, V., Mudgal, V., Gupta, M., & Bharadwaj, A. (2024). Supplementation of critical micro-nutrients in peri-parturient dairy buffaloes improves lactation performance. Journal of Trace Elements and Minerals, Article 100136.https://doi.org/10.1016/j.jtemin.2024.100136 Kumar, R., Singh, K. D., Chauhan, S. S., Verma, M. K., Verma, A. K., … Srivastva, S. (2025). Trace minerals in growth, production and reproduction in farm animals (Review). The Indian Journal of Animal Reproduction, 46(1), 1–15. https://doi.org/10.48165/ijar.2025.46.01.1 Lava Kumar, S., Kushawaha, B., Mohanty, A., Kumari, A., Kumar, A., Beniwal, R., … Prasada Rao, H. B. D. (2024). Glutathione peroxidase (GPX1)—Selenocysteine metabolism preserves the follicular fluid’s redox homeostasis via IGF-1-NMD cascade in follicular ovarian cysts (FOCs). Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease, 1870(6), 167235. https://doi.org/10.1016/j.bbadis.2024.167235 Leroy, J.L.M.R., Vanholder, T., Delanghane, J.R., Opsomer, G., Van-Soom, A., Bols, P.E.J., de Kruif, A., 2004. Metabolite and ionic composition of follicular fluid from different-sized follicles and their relaƟonship to serum concentraƟons in dairy cows. Anim. Reprod. Sci. 80, 201–211. Liu, N., et al. (2022). Pulmonary effects of exposure to indium and its compounds: cross-sectional survey of exposed workers and experimental findings in rodents. Particle and Fibre Toxicology, 19, 59. https://doi.org/10.1186/s12989-022-00510-w Liu, T., Qu, J., Tian, M., Yang, R., Song, X., Li, R., … Qiao, J. (2022). Lipid metabolic process involved in oocyte maturation during folliculogenesis. Frontiers in Cell and Developmental Biology, 10, 806890. https://doi.org/10.3389/fcell.2022.806890 Liu, W.-J., Li, L.-S., Lan, M.-F., Shang, J.-Z., Zhang, J.-X., Xiong, W.-J., Lai, X.-L., & Duan, X. (2024). Zinc deficiency deteriorates ovarian follicle development and function by inhibiting mitochondrial function. Journal of Ovarian Research, 17, Article 115. https://doi.org/10.1186/s13048-024-01442-z Lyons, T.P. 1993. Bioscience Centres: Forging links between industry and academia. In Biotechnology in the Feed Industry", Proc. Alltech's 9th Sym. pp. 1-25. McDowell, L. R. 1992. Minerals in Animal and Human Nutrition. Academic Press Inc. Harcourt Brace Jovanovich Publishers, San Diego, CA. Mion, B., Madureira, G., Spricigo, J. F. W., King, K., Van Winters, B., LaMarre, J., LeBlanc, S. J., Steele, M. A., & Ribeiro, E. S. (2023). Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows. Journal of Dairy Science, 106(7), 5074–5095. https://doi.org/10.3168/jds.2022-22784 Mion, B., Madureira, G., Sprícigo, J. F. W., Van Winters, B., LaMarre, J., LeBlanc, S. J., Steele, M. A., & Ribeiro, E. S. (2023). Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows. Journal of Dairy Science, 106, 5074–5095. https://doi.org/10.3168/jds.2022-22784 Morris, C. A., Neves, D., Carroll, M., Moon, J., Orellana, L., Burin, R., Morris, C., Jasek, A., & Macklin, K. S. (2025). Impact of trace mineral supplementation and translucency score on eggshell quality, hatchability, fertility, and chick quality. Poultry Science, 104(1), 104400. https://doi.org/10.1016/j.psj.2024.104400 Palani, Z.M.R., Al-Obaidy, M.H.A., Hameed, K., Sirwan, K. Effect of selenium and zinc supplements on each individual or mixture on some carcass characteristics and glutathione activity in male lambs kurdi breed sheep. Plant Archives, 2020, 20, pp. 231–234. Palani, Z.M.R.K., Al-Jaf, H.I., Raheem, S.M. Effect of addition of selenium to Kurdi sheep and its interactions with some necessary and toxic elements on health and the environment. Plant Archives, 2019, 19(2), pp. 3963–3970. Palomares, R. A. (2022). Trace mineral supplementation with great impact on beef cattle immunity and health. Animals, 12, 2839. https://doi.org/10.3390/ani12202839 Palomares, R., Ferrer, M., & Jones, L. (2024). Role of trace minerals in cow’s reproductive function and performance: A clinical theriogenology perspective. Clinical Theriogenology, 16, Article 10529. https://doi.org/10.58292/CT.v16.10529 Pan, Y., Pan, C., & Zhang, C. (2024). Unraveling the complexity of follicular fluid: Insights into its composition, function, and clinical implications. Journal of Ovarian Research, 17, 237. https://doi.org/10.1186/s13048-024-01551-9 Pawliński, B., Petrajtis-Gołobów, M., Trela, M., & Witkowska-Piłaszewicz, O. (2023). Acid-base, gas, ions, and glucose analysis in follicular fluid in Holstein-Friesian dairy cows is associated with the follicle size in Poland. Animals, 13(10), 1636. https://doi.org/10.3390/ani13101636 Rufai, N., Razzaque, W. A. A., & Shah, A. (2013). Biochemical parameters of follicular fluid in cyclic and acyclic sheep. VETSCAN, 7(2), 15–20. Rzymski, P., Tomczyk, K., Rzymski, P., Poniedziałek, B., & Opala, T. (2015). Impact of heavy metals on the female reproductive system. Annals of Agricultural and Environmental Medicine, 22(2), 259–264. Sampath, V., Sureshkumar, S., Seok, W. J., & Kim, I. H. (2023). Role and functions of micro and macro-minerals in swine nutrition: A short review. Journal of Animal Science and Technology, 65(3), 479–489. https://doi.org/10.5187/jast.2023.e9 Sekulovski, N., Whorton, A. E., Shi, M., Hayashi, K., and MacLean, J. A. (2020). Periovulatory Insulin Signaling Is Essential for Ovulation, Granulosa Cell Differentiation, and Female Fertility. FASEB j. 34 (2), 2376–2391. doi:10.1096/fj.201901791R Shahzad, M., Cao, J., Kolachi, H. A., et al. (2024). Unravelling the signature follicular fluid metabolites in dairy cattle follicles growing under negative energy balance: An in-vitro approach. International Journal of Molecular Sciences, 25(23), 12629. https://doi.org/10.3390/ijms252312629 Shareef, M. A. (2025). Effect of yeast supplemented with selenium or zinc on some antioxidant indices and liver and kidney functions in local Iraqi goats. Kirkuk University Journal for Agricultural Sciences, 16(1), 191–200. Sorak, M. P., Nikolov, A. B., Sazdanovic, P. S., Arsenijevic, N. S., Milicic, V. M., Cekovic, J. M., Parandilovic, A. Z., & Gavrilovic, A. Z. (2024). Activity of enzymes in the follicular fluid and outcome of in vitro fertilization. Medicine, 103(4), e36851. https://doi.org/10.1097/MD.0000000000036851 Spears, J. W., & Weiss, W. P. (2008). Role of antioxidants and trace elements in health and immunity of transition dairy cows . The Veterinary Journal, 176(1), 70–76. https://doi.org/10.1016/j.tvjl.2007.12.015 Takahashi, T., Takahashi, E., Igarashi, H., Tezuka, N., & Kurachi, H. (2003). Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization. Molecular Reproduction and Development, 66(2), 143–152. https://doi.org/10.1002/mrd.10341 Tang, W., Zhu, X., Chen, Y., Yang, S., Wu, C., … Wang, S. (2024). Towards prolonging ovarian reproductive life: Insights into trace-element homeostasis. Ageing Research Reviews, 97, 102311. https://doi.org/10.1016/j.arr.2024.102311 Tian, T., Wang, Z., & Zhang, J. (2017). Pathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies . Oxidative Medicine and Cellular Longevity, 2017, Article ID 4535194. https://doi.org/10.1155/2017/4535194 Verma, A., & Sweety. (2024). Important minerals affect the fertility of dairy animals: A review. International Journal of Veterinary Sciences and Animal Husbandry, 9(2), 290–292. Yao, Y., Tang, Y., Qin, H., Meng, R., Zhang, C., Zhang, Y., Yang, Y., Qiao, P., Liu, J., & Su, J. (2023). Zinc supplementation promotes oocyte maturation and subsequent embryonic development in sheep. Theriogenology, 206, 161–169. https://doi.org/10.1016/j.theriogenology.2023.04.025 Zhang, P.-P., Ding, G.-C., Tao, C.-Y., Zhang, L., Wang, Y.-X., Yuan, Q.-Y., Zhang, S.-M., & Wang, L.-P. (2024). Levels of trace metals and their impact on oocyte: A review. Taiwanese Journal of Obstetrics and Gynecology, 63(3), 307–311. https://doi.org/10.1016/j.tjog.2024.02.003 Zheng, M., Andersen, C. Y., Rasmussen, F. R., Cadenas, J., Christensen, S. T., & Mamsen, L. S. (2023). Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development. Frontiers in Endocrinology, 14, Article 1268248. https://doi.org/10.3389/fendo.2023.1268248 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7068159","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487572936,"identity":"77e0bd14-9b9c-40a9-9736-3e78a2e875c4","order_by":0,"name":"Zirak M R Palani","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0006-3502-803X","institution":"University of Kirkuk","correspondingAuthor":true,"prefix":"","firstName":"Zirak","middleName":"M R","lastName":"Palani","suffix":""}],"badges":[],"createdAt":"2025-07-07 18:38:40","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7068159/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7068159/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90426118,"identity":"b81e0098-98a1-4524-b92b-9768ff1e5834","added_by":"auto","created_at":"2025-09-02 14:45:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":601545,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7068159/v1/8b7131ff-4ce3-4662-9cb2-d061b5a7cbde.pdf"}],"financialInterests":"","formattedTitle":"Evaluation of mineral elements, some antioxidants and biochemicals in follicular fluid of cow ovaries","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFollicular fluid is an avascular compartment\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003ewithin the mammalian ovary, isolated by the follicles wall from the neighboring tissues, which forms the blood\u0026ndash;follicle barrier (BBB) (Sorak\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eet al.,2024; Sekulovski et al., 2020; Liu et al., 2022). This fluid, including the filtrate of blood, and local substances secreted by granulosa and\u0026ensp;theca cells, were indicative of the metabolic and physiological condition of the follicle (Pan et al.,2024). Follicular fluid (FF) acts as an important microenvironment for oocyte growth and maturation, providing nutrition and necessary factors for the requirements of oocytes, and promoting the maturation process to create a favorable local environment there fore as an important mechanism in\u0026ensp;maintaining female fertility (Sorak et al.,2024; Pan et al.,2024). Minerals, in particular trace elements, are essential for various bodily functions including digestion and bodily functions and are\u0026ensp;required in biosynthesis. They play a major role in serving structural frames of various organs,\u0026ensp;tissues, electrolyte, fundamental antigen, and a renewable source in metabolite, catalyst of enzyme and hormone system Sampath et al., 2023; Palomares, 2022). These are fundamental minerals for thegrowth, reproduction and\u0026ensp;production performance ofthe animals in livestock. Optimal handling of micronutrients also enhances animal products such\u0026ensp;asmilkandfertility (Palomares et al., 2024; Jyani et al., 2024) .Mineral elements are categorized as major essential minerals ( Ca, P, K, Na, S, Mg) and trace elements (Fe, I, Cu, Mn,\u0026ensp;Zn, Co, Mo and Se), Trace minerals exert a particularly critical influence on reproduction; however, deficiencies in certain major minerals especially calcium and phosphorus can likewise impair fertilit (Verma \u0026amp; Sweety,2024; Kumar et al.,2025;Al-Hadeedy et al.,2019).\u003c/p\u003e\n\u003cp\u003eThe organic factors like mineral chelate and/ or trace elements affect\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003ethe follicular regrowth\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003ewhich may enhance the fertility rates due to the improvement in Oocyte fertilization rate, decreasing in embryonic mortality, improvement in the\u0026ensp;uterine environment, and in the increasing of the intensity of mating in an animal (Mion et al., 2023; Morris et al., 2025). The FG composition of follicular fluid changes according to the migration of\u0026ensp;the oocyte with its surrounding cells because metabolic, hormonal and redox conditions evolve concomitantly to oocytes maturation (Hessock et al., 2023; Pan et al., 2024).Thus, studies on a biochemical characterization of the FF as for trace and bioactive elements (like glucose or cholesterol) as well as on oxidative stress markers (like for\u0026ensp;example reduced glutathione(GSH), maldondialdehyde (MDA) will be crucial to understanding the etiological grounds of the metabolic and functional balance of the oocyte maturation and follicular health. Trace elements such as Selenium (Se), zinc (Zn), copper (Cu), iron (Fe) \u0026nbsp;Cobalt (Co), son cofactores esenciales para enzimas que protegen a las celulas\u0026ensp;del estr\u0026eacute;s oxidativo, como superoxido dismutasa (SOD) \u0026nbsp;glutati\u0026oacute;n peroxidasa (Spears and Weiss, 2008; Tian et al., 2017). On the other hand, harmful\u0026ensp;heavy metals such as \u0026nbsp;lead (Pb), arsenic (As) and cadmium (Cd) can exert negative effects on gonadal function by impairing hormone synthesis, damaging cells, and degrading oocyte quality (Zhang et al., 2024; Rzymski et al., 2015). In history, it is a new environmental pollutant, but the reports on\u0026ensp;its damage to reproductive system are not a lot. It is a leap across the study of contamination using reproductive\u0026ensp;damage (Liu et al., 2022) to discover indium in the follicular fluid accordingly.\u003c/p\u003e\n\u003cp\u003eThe aims of the present study are therefore to assess essential and toxic trace element content of bovine FF and follicles of different sizes (small, medium, and large) and oxidative stress markers,\u0026ensp;glucose and cholesterol levels in FF. In addition, this work provides the first evidence of indium distribution in environmental and reproductive backgrounds that indium has been slight consideration effect on reproductive efficiency in cows, and, thus, this study which is able to broaden our understanding of how the follicular micro-environment influences fertility in cows.\u003c/p\u003e"},{"header":"Materials and Methods ","content":"\u003cp\u003eThe study was carried out in winter at the Animal Reproduction Laboratory. Healthy, non-pregnant adult female cattle of unknown genotype were obtained immediately post-mortem from a local abattoir; only reproductive tracts that appeared phenotypically normal were selected. Ovaries were transferred to the laboratory within 2 h of excision in crushed ice (4 \u0026deg;C) placed in a sealed polyethylene bag. Extraction of Follicular-fluid collection\u0026emsp;Upon arrival, ovaries were rinsed in cold (4 \u0026deg;C) sterile physiological saline (0.9 % NaCl) to remove excess blood and tissue, after delivery in the laboratory. Follicles 3\u0026ndash;8 mm in diameter were measured with a Vernier micro-caliper. Follicles were punctured, and their contents aspirated with a sterile 18-gauge needle attached to a syringe. Follicles showing visible blood contamination, pathological fluid, or cystic change were excluded. The FF was centrifuged at 3 000 rpm for 10 min at 4 \u0026deg;C to remove cells and debris; the clear supernatant was aliquoted into polypropylene (Eppendorf) tubes and stored at \u0026minus;20 \u0026deg;C until analysis. Biochemical assays\u0026emsp;Glucose and cholesterol concentrations in FF were determined colorimetrically with commercial enzymatic kits (spectrophotometer; manufacturer\u0026rsquo;s instructions. Lipid peroxidation was quantified as malondialdehyde (MDA) using the thiobarbituric acid reactive substances (TBARS) assay (Guidet \u0026amp; Shah, 1989) and expressed as nmol ml⁻\u0026sup1;. Reduced glutathione (GSH) was measured with the modified Ellman reaction (Al-Zamely et al., 2001) and expressed as nmol ml⁻\u0026sup1;. (DTNB) values were expressed in \u0026mu;mol/ml.Mineral Content Trace-element determination, Trace elements were quantified at part-per-billion (\u0026micro;g l⁻\u0026sup1;) levels. Samples (0.50 ml) were microwave-digested, diluted to 10 ml (dilution factor = 20), and analysed on an ICP-OES (ICPE-9000; Shimadzu: RF power 1.2 kW; plasma gas 15 l min⁻\u0026sup1;; nebuliser gas 0.70 l min⁻\u0026sup1;). Calibration curves (0.01\u0026ndash;50 \u0026micro;g l⁻\u0026sup1;; six points, r\u0026sup2; \u0026ge; 0.9995) were prepared from a multi-element stock solution. Accuracy was checked every ten samples with NIST SRM 1640a and SRM 1643f (recoveries 96\u0026ndash;103 %). Procedural blanks were \u0026lt; 0.5 \u0026times; LOD; LODs (3 \u0026sigma; blanks) ranged from 0.02 to 0.15 \u0026micro;g l⁻\u0026sup1;. Instrument counts were blank-corrected, converted to concentrations (\u0026micro;g l⁻\u0026sup1; \u0026asymp; ppb), and multiplied by the dilution factor. Final follicular-fluid values were obtained by multiplying by the dilution factor. Results are reported to two or three significant figures; instrumental RSD for triplicates was \u0026lt; 4 %. The elements which were analyzed to be nickel (Ni), copper (Cu), iron (Fe), cobalt (Co), selenium (Se), zinc (Zn), cadmium(Cd), molybdenum (Mo), lead (Pb), antimony\u0026ensp;(Sb) and arsenic (As). : Analysis :Results were analyzed\u0026ensp;using Addinsoft, (2016). Model Y₍ᵢⱼₖ₎ = \u0026mu;+ Aᵢ + Bⱼ + (AB)ᵢⱼ + eᵢⱼₖ, means having different lettersin the same column differ\u0026ensp;significantly at P \u0026le; 0.05, by Duncan\u0026rsquo;s, (1955) multiple range tests and are expressed as mean values \u0026plusmn; SD. All procedures were approved by the University of Kirkuk ethics committee (Approval No. 03C01L004) and complied with ARRIVE guidelines for ruminant welfare; veterinary assistance was available at all times.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eFirst:\u003c/strong\u003e Concentration of mineral elements in the follicular fluid\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Routine findings show that the concentration of trace minerals varies significantly among small, medium and large follicles in the follicular fluid of native cows. Copper (Cu) concentrations increased with follicle size: 0.090 ppb in small follicles, 0.298 \u0026plusmn; 0.053 ppb in medium follicles, and 0.428 \u0026plusmn; 0.125 ppb in large follicles (P \u0026lt; 0.005Such an increase is consistent with findings in cattle and other ruminants, demonstrating that follicular copper rises with growth as an enzymatic co-factor essential for follicular development (Mion et al., 2023; Zhang et al., 2024; Lou et al., 2025). Zinc (Zn) showed a similar pattern, with concentrations of 0.583 \u0026plusmn; 0.180 ppb in small follicles, 0.830 \u0026plusmn; 0.347 ppb in medium follicles and 1.813 \u0026plusmn; 0.388 ppb in large follicles, a difference that was statistically significant.This accumulation is in agreement with the participation of zinc in cell growth and protein synthesis and in essential activities in the follicle during oocyte maturation (Yao et al., 2023; Liu et al., 2024). As to Se, it significantly increased 23.988 a\u0026plusmn;0.906\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eppb in large follicles, a finding in agreement with its antioxidant role protecting oocytes from oxidative stress, thus making a better follicular environment (Lava Kumar et al., 2024; Ali et al., 2023). The study indicated that adding selenium- or zinc-enriched yeast improved antioxidant status as well as liver and kidney function in local Iraqi goats (Shareef, 2025).The level of Fe increased markedly from 9.373 \u0026plusmn; 0.757 ppb in small follicles\u0026ensp;to 21.368 \u0026plusmn; 4.151 ppb in medium follicles and then, decreased slightly to 19.650 \u0026plusmn; 1.253 ppb in large follicles, which may suggest that during maturation period, there was more metabolic activity in medium follicles, and the blood perfusion and oxygen transport increased (Hessock et al., 2023; Shahzad et al., 2024;Brantmeier et al., 1987). Also cobalt (Co) content also increased gradually and the contents\u0026ensp;in small,medium and large follicles were 2.430\u0026plusmn;0.295,3.255\u0026plusmn;0.342,5.438\u0026plusmn;0.377 ppb, respectively. This indicates how essential this element may be in some required biochemical processes in follicular cells, potentially in the\u0026ensp;process of cell growth as vitamin B ₁₂ (Tang et al., 2024; Ayyat et al., 2025 ;McDowell, 1992). Molybdenum concentrations were 0.390 \u0026plusmn; 0.111 ppb in small follicles, 2.125 \u0026plusmn; 0.384 ppb in medium follicles (the highest value), and 1.645 \u0026plusmn; 0.140 ppb in large follicles, suggesting a possible regulatory interaction between Mo and Cu. For the toxic element Cd, no significant differences were detected among follicles, with values ranging from 0.343 \u0026plusmn; 0.098 ppb to 0.488 \u0026plusmn; 0.184 ppb. No significant difference was observed also for the In (0.013\u0026plusmn;0.003 ppb in SF, 0.098\u0026plusmn;0.048 ppb in\u0026ensp;MF and 0.093\u0026plusmn;0.003 ppb in LF). The presence of indium in bovine follicular fluid is a novel observation, and further studies are required to clarify its biological significance. Arsenic (As) also increased with follicle size, reaching 0.318 \u0026plusmn; 0.080 ppb in small follicles, 0.675 \u0026plusmn; 0.094 ppb in medium follicles, and 1.180 \u0026plusmn; 0.234 ppb in large follicles. This progressive accumulation, despite As\u0026rsquo;s known toxicity, suggests its retention in the follicular environment during growth. The Pb, in turn, reacted inversely reaching 2.188\u0026plusmn;0.081 ppb in small, 1.645\u0026plusmn;0.14 ppb in medium and\u0026ensp;1.228\u0026plusmn;0.09 ppb in large follicles, less possibly avoiding its toxic influence when the follicle atresia retards. Taken together, these results indicate that the size of the follicle affects the level of TMs in FF and add in support of the concept that FF\u0026ensp;is produced by plasma filtration and follicular metabolic activity leading to modifications of the FF (Pan et al., 2024; Hessock et al., 2023). These findings highlight the importance of these elements also in reproduction and the necessity of including it in the rations if fertility rate of cows has\u0026ensp;to be increased (Mion et al., 2023; Palomares \u0026amp; Ferrer, 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (1):\u003c/strong\u003e Concentrations of mineral elements (ppb) in three different size of seminal fluid in bovine ovaries\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"520\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTrace Elements\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 378px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFollicular size\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmall Follicles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(3-5mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedium Follicles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(6-9mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLarge Follicles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(10-22mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCopper (Cu)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.090 b\u0026plusmn;0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.298 ab\u0026plusmn;0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.428 a\u0026plusmn;0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eZinc (Zn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.583 b\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.830 ab\u0026plusmn;0.347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e1.813 a\u0026plusmn;0.388\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eSelenium (Se)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e13.730 c\u0026plusmn;0.638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e20.213 b\u0026plusmn;0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e23.988 a\u0026plusmn;0.906\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIron (Fe)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e9.363 b\u0026plusmn;0.757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e21.368 a\u0026plusmn;4.151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e19.650 a\u0026plusmn;1.253\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCobalt (Co)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2.430 b\u0026plusmn;0.295\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e3.255 b\u0026plusmn;0.342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e5.438 a\u0026plusmn;0.377\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eMolybdenum (Mo)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.390 c\u0026plusmn;0.111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e2.125 b\u0026plusmn;0.384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e1.645 b\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCadmium (Cd)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.343 a\u0026plusmn;0.098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.435 a\u0026plusmn;0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.488 a\u0026plusmn;0.184\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIndium (In)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.013 a\u0026plusmn;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.098 a\u0026plusmn;0.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.093 a\u0026plusmn;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eArsenic (As)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.318 b\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.675 b\u0026plusmn;0.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e1.180 a\u0026plusmn;0.234\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eLead (Pd)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2.188 a\u0026plusmn;0.081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1.645 b\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e1.228 c\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Values are expressed as Mean \u0026plusmn; Standard Error \u003cspan dir=\"RTL\"\u003e)\u003c/span\u003eSE\u003cspan dir=\"RTL\"\u003e(\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*Different superscript letters (a, b, c) within the same column indicate statistically significant differences (P \u0026lt; 0.05) according to Duncan\u0026rsquo;s multiple range test.\u003c/p\u003e\n\u003cp\u003eIn light of these new findings, the elevated levels of copper, zinc, selenium, cobalt, and molybdenum associated with follicular growth suggest that blood serves as the primary reservoir of these elements and that they can readily penetrate follicles\u0026mdash; mechanism that weakens the blood\u0026ndash;follicle barrier during development. This observation is consistent with earlier studies reporting higher concentrations of these metals in follicular fluid (FF) that correlate with follicle size (Pawliński et al., 2023; Shahzad et al., 2024; Hessock et al., 2023). Iron showed a similar pattern, mirroring previous reports (Pan et al., 2024; Mion et al., 2024).The uniformly high arsenic content and consistently low lead levels in larger follicles suggest a narrow margin between beneficial and detrimental arsenic in follicles and lead occurring at low levels in follicles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecond:\u003c/strong\u003e Biochemical indicators in the follicle (Table 2)\u003c/p\u003e\n\u003cp\u003eCholesterol concentration rose with follicle size, reaching 31.073 \u0026plusmn; 1.229 mg dL⁻\u0026sup1; in small follicles, 37.568 \u0026plusmn; 1.785 mg dL⁻\u0026sup1; in medium follicles, and 45.338 \u0026plusmn; 2.232 mg dL⁻\u0026sup1; in large follicles. These differences were statistically significant across all three follicle classes. Glutathione (GSH) levels were 0.185 \u0026plusmn; 0.017 nmol ml⁻\u0026sup1; in small follicles, 0.340 \u0026plusmn; 0.039 nmol ml⁻\u0026sup1; in medium follicles, and 0.348 \u0026plusmn; 0.029 nmol ml⁻\u0026sup1; in large follicles. These values differed significantly among follicle sizes.The larger the follicles. Malondialdehyde (MDA) levels decreased significantly from 0.600 \u0026plusmn; 0.077 nmol ml⁻\u0026sup1; in small follicles to 0.348 \u0026plusmn; 0.045 nmol ml⁻\u0026sup1; in medium follicles and 0.308 \u0026plusmn; 0.041 nmol ml⁻\u0026sup1; in large follicles, confirming marked biochemical differences among follicle sizes. Recent research shows that particular hormonal therapies have markedly enhanced reproductive and haematological parameters in Awassi ewes, underscoring the importance of hormonal regulation in boosting livestock productivity (Alwan, Majid \u0026amp; Ismail, 2018a,b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (2):\u003c/strong\u003e Biochemical levels of follicular fluid in three different sized follicles in cow ovaries\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"518\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 373px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFollicular size\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmall Follicles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(3-5mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedium Follicles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(6-9mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLarge Follicles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;10-22mm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eGlucose (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e55.718 a\u0026plusmn;2.522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e59.135 a\u0026plusmn;1.607\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e57.205 a\u0026plusmn;2.582\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eCholesterol (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e31.073 c\u0026plusmn;1.229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e37.568 b\u0026plusmn;1.785\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e45.338 a\u0026plusmn;2.232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eGSH (nmol/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.185 b\u0026plusmn;0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e0.340 a\u0026plusmn;0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.348 a\u0026plusmn;0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003eMDA (nmol/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.600 a\u0026plusmn;0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e0.348 b\u0026plusmn;0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.308 b\u0026plusmn;0.041\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Values are expressed as Mean \u0026plusmn; Standard Error )SE(.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e*Different superscript letters (a, b, c) within the same column indicate statistically significant differences (P \u0026lt; 0.05) according to Duncan\u0026rsquo;s multiple range test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBiochemically, the nearly constant glucose concentration among small, medium and large bovine follicles suggests tight intrafollicular control of carbohydrate supply (Anderson et al., 2012), a trend that has recently been confirmed in cows of the Holstein-Friesian breed (Pawliński et al., 2023) and\u0026ensp;in slaughterhouse goats where the slight numerical glucose decline associated with the increase in diameter did not reach statistical significance (Bordoloi et al., 2025). In contrast to dromedaries, however, intrafollicular glucose increases seasonally in Bactrian\u0026ensp;camels during the breeding season (Abdoon et al., 2025), which emphasizes the species-specific nature of the difference. The greater conversion of cholesterol to steroid hormones in large bovine follicles also corresponds to a Redox shift, involving higher reduced-glutathione and lower oxidative\u0026ensp;markers such as hydrogen-peroxide and malondialdehyde, providing a more favourable environment for granulosa-cell function (Hill et al. 2014; Lava Kumar et al. 2014) and conflicts with other studies in camels (Huang et al.,2002). The increased cholesterol conversion to steroid hormones is explained by the increased GSH and lower MDA levels in the fluid of larger follicles, indicating reflects less oxidative damage, which benefits cell function and follicle growth in local bovine oocytes.\u003c/p\u003e\n\u003cp\u003eThis was\u0026ensp;supported by the present study and is consistent with the findings of (Rufai et al. 2013; Beg and Ginther, 2006; Zheng et al. 2023) where they describe the conversion of cholesterol into\u0026ensp;steroid hormones during ontogenesis. With respect to non-enzymatic antioxidants, GSH was showed\u0026ensp;to be significantly enhanced during follicle development, suggesting an enhanced anti-oxidative force of follicular fluid, a critical factor that protects the oocyte from oxidative stress during maturation (Takahashi et al., 2003; Lava Kumar et al., 2024). There was also a considerable decline in the level of malondialdehyde (MDA), which arises from lipid peroxidation and signifies reduction in\u0026ensp;oxidative stress with follicles\u0026rsquo; maturity, that was accompanied by increased antioxidants, e.g., selenium and glutathione (Duan et al., 2024; Agarwal et al., 2012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThird:\u003c/strong\u003e Analysis of the correlation relationships between mineral elements in follicular fluid.\u003c/p\u003e\n\u003cp\u003eThus, the results of this study demonstrate statistically significant pairwise relationships among several mineral elements in bovine follicular fluid, revealing complementary interactions or competition within the oocyte microenvironment. Strong positive correlations for the pairs Se\u0026ndash;Cu, Se\u0026ndash;Co, and Se\u0026ndash;Mo\u0026mdash;each biologically complementary\u0026mdash;link these elemental interactions to physiological processes of follicle growth and oocyte maturation.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eIt is well known that Se is a trace element required for the enzyme glutathione peroxidase that reduces free radicals and oxidative stress in follicular cells (Lava Kumar\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e et al., 2024; Agarwal et al., 2012), thus improving follicular cell viability and oocyte\u0026ensp;quality. Furthermore, the study found a high positive correlation between the level of selenium and cobalt (r = 0.875),\u0026ensp;demonstrating their mutual elevation in the improvement of metabolic processes and protection from oxidative stress. These results are consistent with those of\u0026ensp; Ingle et al. (2017) finding that elements found in the follicular fluid are directly related to\u0026ensp;follicular maturation and ovulation quality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (3):\u003c/strong\u003e A table showing the correlation matrix (Pearson): for the mineral elements in the follicular fluid of cows\u0026rsquo; ovaries.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"813\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003eCu Copper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003eZn Zinc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003eCo Cobalt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003eSe Selenium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003eCd Cadmium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003eAs Arsenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003eFe Iron\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eMo Molybdenum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003ePb Lead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003eindium (In)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eCu Copper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.600\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.706\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e-0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.661\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.648\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.751\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e0.234\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eZn zinc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.607\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.611\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e0.515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e-0.396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e0.120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eCo Cobalt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.600\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.607\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.875\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.693\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.621\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.830\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.764\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eSe Selenium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.706\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.611\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.875\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.681\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.739\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.793\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.883\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.623\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eCd Cadmium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e-0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e0.344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e0.173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e0.383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e0.339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e-0.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e0.287\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eAs arsenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.661\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.693\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.681\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.925\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.841\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e0.481\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eFe Iron\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e0.442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.621\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.739\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e0.532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e0.475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.592\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.870\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eMo Molybdenum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.648\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.830\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.793\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.925\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.867\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e0.393\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003ePb Lead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.751\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e-0.396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.764\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.883\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e-0.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.841\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.592\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.867\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e-0.501\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003eindium (In)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.9531%;\"\u003e\n \u003cp\u003e0.234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e0.120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.3807%;\"\u003e\n \u003cp\u003e0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.5558%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.623\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.8993%;\"\u003e\n \u003cp\u003e0.287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6.182%;\"\u003e\n \u003cp\u003e0.481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.870\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\n \u003cp\u003e0.393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\n \u003cp\u003e-0.501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"bottom\" style=\"width: 48.8838%;\"\u003e\n \u003cp\u003e\u003cem\u003eValues in bold are different from 0 with a significance level alpha=0.05\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.7613%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.1517%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 5.6096%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBy contrast, the research revealed strong negative correlations between\u0026nbsp;Pb\u0026nbsp;with\u0026ensp;major trace elements particularly Se (r = \u0026minus;0.883), Mo (r = \u0026minus;0.867), and Co (r = \u0026minus;0.764). suggests that\u0026ensp;lead may be influencing the absorption or stability of these elements in the follicular milieu. The toxic properties of lead, that may interfere with the activities of enzymes and chain of transport\u0026ensp;of electrons in reproductive cells leading to an increase in intracellular oxidant stress are well known (Flora et al., 2012; El-Boshy et al., 2015). Furthermore, lead deposition in\u0026ensp;the ovaries was correlated with reduced number of mature follicles and lower oocyte quality, which affect fertility. Iron (Fe) and indium (In) by contrast, showed a strong positive correlation (r = 0.870), although indium is not\u0026ensp;among the most abundant biological elements. This relationship\u0026ensp;might reflect similar transport mechanisms or their attachment to carrier proteins, including transferrin, as suggested by Guo et al (2025). Selenium and zinc are considered to be extremely important trace elements for the health of any animal, due to their positive effects on the regulation of hormones and enzymes, enhancement of antioxidant defenses, and support for the activity of metabolic and immunological systems. However, their biological effects are highly dose-dependent, and the two elements can interact synergistically or antagonistically, promoting beneficial actions or increasing adverse effects. Studies by Palani et al. (2019, 2020) and Al-Obaidi et al. (2020) showed that dietary fat proportion significantly alters serum biochemical parameters and antioxidant capacity, producing beneficial effects on animal health and metabolism. Nevertheless, this\u0026ensp;relationship should be further investigated, taking also into account any impact on either industrial or nutritional contami- nants present in feed sources.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eOverall, these findings highlight the importance of maintaining mineral equilibrium in follicular fluid due to its direct influence on the microenvironment of oocytes. Additionally, they suggest that the presence of harmful substances like lead in the reproductive environment can disrupt mineral balance, potentially impairing reproductive function in cows.\u003c/p\u003e\n\u003cp\u003eThis study reports, for the first time, the presence of indium in bovine ovarian follicular fluid at low concentrations. Further research is warranted to clarify, particularly regarding its associations with other trace metals, characterise potential synergistic or antagonistic interactions, and assess its impact on reproductive efficiency.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis trial revealed substantial changes in the concentrations of essential trace elements: copper, zinc, selenium, and cobalt, in the bovine follicular fluid alongside the growth and maturation of follicles.These findings indicate that these endogenous elements promote metabolic and antioxidant activities within the follicular micro-environment conducive to oocyte quality. In contrast, the toxic metal lead showed strong negative associations with these elements. suggesting that mineral imbalance and excess oxidative stress can impair follicular function and interference with vital enzymes. Of particular interest is the first-time identification of indium in follicular fluid, where a number of elements showed positive correlations with iron. Further research is needed to unearth additional knowledge about the potential role of in in the reproductive system and the underlying mineral interactions. Overall, the results highlight the importance of a delicate mineral balance in follicular fluid for healthy follicles and efficient oocyte maturation and the demand to curb exposure to toxic heavy metals in the farm setting to enhance cows\u0026rsquo; fertility.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdoon, A. S. S., Soliman, S. S., Hussein, N. S., Haggag, S. H. I., El-Sanea, A. M., \u0026amp; Abdel-Hamid, A. Z. (2025). Metabolomic profile of dromedary camel follicular fluid during the breeding and non-breeding seasons. Scientific Reports, 15, 8923. https://doi.org/10.1038/s41598-025-91710-9 \u003c/li\u003e\n\u003cli\u003eAddinsoft. (2016). XLSTAT statistical and data analysis solution (Version 2016.1) [Computer software]. Addinsoft. https://www.xlstat.com \u003c/li\u003e\n\u003cli\u003eAgarwal, A., Aponte-Mellado, A., Premkumar, B. J., Shaman, A., \u0026amp; Gupta, S. (2012). The effects of oxidative stress on female reproduction: A review. Reproductive Biology and Endocrinology, 10, 49. https://doi.org/10.1186/1477-7827-10-49 \u003c/li\u003e\n\u003cli\u003eAl-Hadeedy, I. Y., Al-Nuaimi, M. I. A., \u0026amp; Ali, A. R. (2019). Effect of adding local minerals clay (meqdadia) in the female japanese quail diets upon production performance, egg quality and carcass traits. Indian Journal of Public Health Research and Development, 10(8). https://doi.org/10.5958/0976-5506.2019.02020.5 \u003c/li\u003e\n\u003cli\u003eAli, M. E., Abd-Elkariem, M. F. A., Fahmy, S., Hussein, H. A., Abdelrahman, M., \u0026amp; Ahmed, A. E. (2023). Dietary supplementation with different nano and organic selenium doses improves ovarian activity, fertility rate, and progesterone level in Ossimi ewes. Italian Journal of Animal Science, 22, 651\u0026ndash;659. https://doi.org/10.1080/1828051X.2023.2231946 \u003c/li\u003e\n\u003cli\u003eAl-Obaidy, M.H.A., Palani, Z.M.R., Sirwan, K., Hameed, K. EFFECT OF ADDING DIFFERENT PERCENTAGES OF GLYCERIN IN RATIONS ON SOME BLOOD SERUM BIOCHEMICAL AND ANTIOXIDANTS TRAITS IN MALE LAMBS AWASSI BREED SHEEP. Biochemical and Cellular Archives, 2020, 20(1), pp. 2253\u0026ndash;2257.\u003c/li\u003e\n\u003cli\u003eAlwan, A. S., Majid, A. F., \u0026amp; Ismail, H. I. (2018a). The effect of using some hormonal treatments on some reproductive performance traits in Awassi ewes. Kirkuk University Journal for Agricultural Sciences, 9(4), 17\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eAlwan, A. S., Majid, A. F., \u0026amp; Ismail, H. I. (2018b). The effect of a hormonal program on some physiological and biochemical blood traits in Awassi ewes. Kirkuk University Journal for Agricultural Sciences, 9(4), 52\u0026ndash;59.\u003c/li\u003e\n\u003cli\u003eAl-Zamely; Mizil, Y. O. and Al-Nimer, M. S.(2001). Detection the level of peroxynitrite and related with antioxidants status in the serum of patients with acute myocardial infraction national. J. of Chemistry . 4 : 625-637.\u003c/li\u003e\n\u003cli\u003eAyyat, M. S., Abdel Monem, U. M., Mostafa, T. H., Thabet, R. M., Abd El-Latif, K. M., \u0026amp; Al-Sagheer, A. A. (2025). Influence of long-term dietary cobalt supplementation on lactation performance and reproductive efficiency in Maghrabi she-camels. Biological Trace Element Research. https://doi.org/10.1007/s12011-025-04551-y \u003c/li\u003e\n\u003cli\u003eBordoloi PK, Sarmah BC, Dutta DJ, Deka BC. Follicular fluid cholesterol in goat ovary. Indian Vet J. 2000;77:638\u0026ndash;639. \u003c/li\u003e\n\u003cli\u003eBordoloi, P. K., Sarmah, B. C., Dutta, D. J., \u0026amp; Deka, B. C. (2025). Follicular fluid glucose concentration of goat ovary. Indian Journal of Animal Research, 35(1), 81\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eBrantmeier, S.A., Grummer, R.R., Ax, R.L., 1987. ConcentraƟons of high density lipoproteins vary among follicular sizes in the bovine. J. Dairy. Sci. 70, 2145\u0026ndash;2149.\u003c/li\u003e\n\u003cli\u003eChen, Y., Yang, J., \u0026amp; Zhang, L. (2023). The impact of follicular-fluid oxidative stress levels on the outcomes of assisted reproductive therapy. Antioxidants, 12(12), 2117. https://doi.org/10.3390/antiox12122117\u003c/li\u003e\n\u003cli\u003eDuan, H., Wang, F., Wang, K., Yang, S., Zhang, R., Xue, C., \u0026hellip; Xiao, L. (2024). Quercetin ameliorates oxidative-stress-induced apoptosis of granulosa cells in dairy-cow follicular cysts by activating autophagy via the SIRT1/ROS/AMPK signaling pathway. Journal of Animal Science and Biotechnology, 15, 119. https://doi.org/10.1186/s40104-024-01078-5\u003c/li\u003e\n\u003cli\u003eDuncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11(1), 1\u0026ndash;42. https://doi.org/10.2307/3001478\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eEl-Boshy, M. E., Risha, E. F., Abdelhamid, F. M., Mubarak, M. S., \u0026amp; Hadda, T. B. (2015). Protective effects of selenium against cadmium-induced hematological disturbances, immunosuppressive oxidative stress and hepatorenal damage in rats. Journal of Trace Elements in Medicine and Biology, 29, 104\u0026ndash;110. https://doi.org/10.1016/j.jtemb.2014.05.009 \u003c/li\u003e\n\u003cli\u003eFlora, G., Gupta, D., \u0026amp; Tiwari, A. (2012). Toxicity of lead: A review with recent updates. Interdisciplinary Toxicology, 5(2), 47\u0026ndash;58. https://doi.org/10.2478/v10102-012-0009-2 \u003c/li\u003e\n\u003cli\u003eFortune, J.E., Hansel, W., 1985. ConcentraƟons of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulaƟon. Biol. Reprod. 32, 1069-1079.\u003c/li\u003e\n\u003cli\u003eGuidet, B., \u0026amp; Shah, S. V. (1989). Enhanced in vivo H₂O₂ generation by rat kidney in glycerol-induced renal failure. American Journal of Physiology: Renal Physiology, 257(3 Pt 2), F440\u0026ndash;F445. https://doi.org/10.1152/ajprenal.1989.257.3.F440 \u003c/li\u003e\n\u003cli\u003eGuo, Q., Qian, C., Wang, X., \u0026amp; Qian, Z.-M. (2025). Transferrin receptors. Experimental \u0026amp; Molecular Medicine, 57, 724\u0026ndash;732. https://doi.org/10.1038/s12276-025-01436-x\u003c/li\u003e\n\u003cli\u003eHarmon RJ. Trace minerals and dairy cattle: importance for udder health. In: Biotechnology in the Feed Industry. 1988;Proc Alltech\u0026rsquo;s 14 th Annual Symp:485-495.\u003c/li\u003e\n\u003cli\u003eHessock, E. A., Edwards, J. L., Schrick, F. N., Payton, R. R., Campagna, S. R., Pollock, A. B., Clark, H. M., Stokes, A. E., Klabnik, J. L., Hill, K. S., Roberts, S. R., Hinson, M. G., \u0026amp; Moorey, S. E. (2023). Metabolite abundance in bovine preovulatory follicular fluid is influenced by follicle developmental progression post-estrous onset in cattle. Frontiers in Cell and Developmental Biology, 11, 1156060. https://doi.org/10.3389/fcell.2023.1156060\u003c/li\u003e\n\u003cli\u003eHidiroglou, M. 1979. Manganese in ruminant nutriƟon. Can. J. Anim. Sci. 59:217-236.\u003c/li\u003e\n\u003cli\u003eHill, K. S., Edwards, J. L., Payton, R. R., Schrick, F. N., Campagna, S. R., Hessock, E. A., \u0026amp; Moorey, S. E. (2024). Relationships of circulating and pre-ovulatory follicular fluid hydrogen peroxide levels with body condition score and metabolome profiles of lactating beef cows. Agriculture, 14(8), 1406. https://doi.org/10.3390/agriculture14081406 \u003c/li\u003e\n\u003cli\u003eHuang WT, Lu SG, Tang PC, Wu SC, Cheng SP, Ju, JC. Biochemical compositions of follicular fluid and the effects of culture conditions on the in vitro development of pig oocytes. Asian Aust J Anim Sci. 2002;15:1403\u0026ndash;1411. \u003c/li\u003e\n\u003cli\u003eIngle, M. E., Bloom, M. S., Parsons, P. J., Steuerwald, A. J., Kruger, P., \u0026amp; Fujimoto, V. Y. (2017). Associations between IVF outcomes and essential trace elements measured in follicular fluid and urine: A pilot study. Journal of Assisted Reproduction and Genetics, 34(2), 253 \u0026ndash; 261. https://doi.org/10.1007/s10815-016-0853-7 \u003c/li\u003e\n\u003cli\u003eJyani, V., Mudgal, V., Gupta, M., \u0026amp; Bharadwaj, A. (2024). Supplementation of critical micro-nutrients in peri-parturient dairy buffaloes improves lactation performance. Journal of Trace Elements and Minerals, Article 100136.https://doi.org/10.1016/j.jtemin.2024.100136 \u003c/li\u003e\n\u003cli\u003eKumar, R., Singh, K. D., Chauhan, S. S., Verma, M. K., Verma, A. K., \u0026hellip; Srivastva, S. (2025). Trace minerals in growth, production and reproduction in farm animals (Review). The Indian Journal of Animal Reproduction, 46(1), 1\u0026ndash;15. https://doi.org/10.48165/ijar.2025.46.01.1 \u003c/li\u003e\n\u003cli\u003eLava Kumar, S., Kushawaha, B., Mohanty, A., Kumari, A., Kumar, A., Beniwal, R., \u0026hellip; Prasada Rao, H. B. D. (2024). Glutathione peroxidase (GPX1)\u0026mdash;Selenocysteine metabolism preserves the follicular fluid\u0026rsquo;s redox homeostasis via IGF-1-NMD cascade in follicular ovarian cysts (FOCs). Biochimica et Biophysica Acta (BBA) \u0026ndash; Molecular Basis of Disease, 1870(6), 167235. https://doi.org/10.1016/j.bbadis.2024.167235 \u003c/li\u003e\n\u003cli\u003eLeroy, J.L.M.R., Vanholder, T., Delanghane, J.R., Opsomer, G., Van-Soom, A., Bols, P.E.J., de Kruif, A., 2004. Metabolite and ionic composition of follicular fluid from different-sized follicles and their relaƟonship to serum concentraƟons in dairy cows. Anim. Reprod. Sci. 80, 201\u0026ndash;211.\u003c/li\u003e\n\u003cli\u003eLiu, N., et al. (2022). Pulmonary effects of exposure to indium and its compounds: cross-sectional survey of exposed workers and experimental findings in rodents. Particle and Fibre Toxicology, 19, 59. https://doi.org/10.1186/s12989-022-00510-w\u003c/li\u003e\n\u003cli\u003eLiu, T., Qu, J., Tian, M., Yang, R., Song, X., Li, R., \u0026hellip; Qiao, J. (2022). Lipid metabolic process involved in oocyte maturation during folliculogenesis. Frontiers in Cell and Developmental Biology, 10, 806890. https://doi.org/10.3389/fcell.2022.806890\u003c/li\u003e\n\u003cli\u003eLiu, W.-J., Li, L.-S., Lan, M.-F., Shang, J.-Z., Zhang, J.-X., Xiong, W.-J., Lai, X.-L., \u0026amp; Duan, X. (2024). Zinc deficiency deteriorates ovarian follicle development and function by inhibiting mitochondrial function. Journal of Ovarian Research, 17, Article 115. https://doi.org/10.1186/s13048-024-01442-z \u003c/li\u003e\n\u003cli\u003eLyons, T.P. 1993. Bioscience Centres: Forging links between industry and academia. In Biotechnology in the Feed Industry\u0026quot;, Proc. Alltech\u0026apos;s 9th Sym. pp. 1-25.\u003c/li\u003e\n\u003cli\u003eMcDowell, L. R. 1992. Minerals in Animal and Human Nutrition. Academic Press Inc. Harcourt Brace Jovanovich Publishers, San Diego, CA.\u003c/li\u003e\n\u003cli\u003eMion, B., Madureira, G., Spricigo, J. F. W., King, K., Van Winters, B., LaMarre, J., LeBlanc, S. J., Steele, M. A., \u0026amp; Ribeiro, E. S. (2023). Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows. Journal of Dairy Science, 106(7), 5074\u0026ndash;5095. https://doi.org/10.3168/jds.2022-22784\u003c/li\u003e\n\u003cli\u003eMion, B., Madureira, G., Spr\u0026iacute;cigo, J. F. W., Van Winters, B., LaMarre, J., LeBlanc, S. J., Steele, M. A., \u0026amp; Ribeiro, E. S. (2023). Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows. Journal of Dairy Science, 106, 5074\u0026ndash;5095. https://doi.org/10.3168/jds.2022-22784 \u003c/li\u003e\n\u003cli\u003eMorris, C. A., Neves, D., Carroll, M., Moon, J., Orellana, L., Burin, R., Morris, C., Jasek, A., \u0026amp; Macklin, K. S. (2025). Impact of trace mineral supplementation and translucency score on eggshell quality, hatchability, fertility, and chick quality. Poultry Science, 104(1), 104400. https://doi.org/10.1016/j.psj.2024.104400\u003c/li\u003e\n\u003cli\u003ePalani, Z.M.R., Al-Obaidy, M.H.A., Hameed, K., Sirwan, K. Effect of selenium and zinc supplements on each individual or mixture on some carcass characteristics and glutathione activity in male lambs kurdi breed sheep. Plant Archives, 2020, 20, pp. 231\u0026ndash;234.\u003c/li\u003e\n\u003cli\u003ePalani, Z.M.R.K., Al-Jaf, H.I., Raheem, S.M. Effect of addition of selenium to Kurdi sheep and its interactions with some necessary and toxic elements on health and the environment. Plant Archives, 2019, 19(2), pp. 3963\u0026ndash;3970.\u003c/li\u003e\n\u003cli\u003ePalomares, R. A. (2022). Trace mineral supplementation with great impact on beef cattle immunity and health. Animals, 12, 2839. https://doi.org/10.3390/ani12202839\u003c/li\u003e\n\u003cli\u003ePalomares, R., Ferrer, M., \u0026amp; Jones, L. (2024). Role of trace minerals in cow\u0026rsquo;s reproductive function and performance: A clinical theriogenology perspective. Clinical Theriogenology, 16, Article 10529. https://doi.org/10.58292/CT.v16.10529\u003c/li\u003e\n\u003cli\u003ePan, Y., Pan, C., \u0026amp; Zhang, C. (2024). Unraveling the complexity of follicular fluid: Insights into its composition, function, and clinical implications. Journal of Ovarian Research, 17, 237. https://doi.org/10.1186/s13048-024-01551-9 \u003c/li\u003e\n\u003cli\u003ePawliński, B., Petrajtis-Gołob\u0026oacute;w, M., Trela, M., \u0026amp; Witkowska-Piłaszewicz, O. (2023). Acid-base, gas, ions, and glucose analysis in follicular fluid in Holstein-Friesian dairy cows is associated with the follicle size in Poland. Animals, 13(10), 1636. https://doi.org/10.3390/ani13101636 \u003c/li\u003e\n\u003cli\u003eRufai, N., Razzaque, W. A. A., \u0026amp; Shah, A. (2013). Biochemical parameters of follicular fluid in cyclic and acyclic sheep. VETSCAN, 7(2), 15\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eRzymski, P., Tomczyk, K., Rzymski, P., Poniedziałek, B., \u0026amp; Opala, T. (2015). Impact of heavy metals on the female reproductive system. Annals of Agricultural and Environmental Medicine, 22(2), 259\u0026ndash;264.\u003c/li\u003e\n\u003cli\u003eSampath, V., Sureshkumar, S., Seok, W. J., \u0026amp; Kim, I. H. (2023). Role and functions of micro and macro-minerals in swine nutrition: A short review. Journal of Animal Science and Technology, 65(3), 479\u0026ndash;489. https://doi.org/10.5187/jast.2023.e9\u003c/li\u003e\n\u003cli\u003eSekulovski, N., Whorton, A. E., Shi, M., Hayashi, K., and MacLean, J. A. (2020). Periovulatory Insulin Signaling Is Essential for Ovulation, Granulosa Cell Differentiation, and Female Fertility. FASEB j. 34 (2), 2376\u0026ndash;2391. doi:10.1096/fj.201901791R\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e \u003cspan dir=\"RTL\"\u003e \u003c/span\u003e \u003c/li\u003e\n\u003cli\u003eShahzad, M., Cao, J., Kolachi, H. A., et al. (2024). Unravelling the signature follicular fluid metabolites in dairy cattle follicles growing under negative energy balance: An in-vitro approach. International Journal of Molecular Sciences, 25(23), 12629. https://doi.org/10.3390/ijms252312629 \u003c/li\u003e\n\u003cli\u003eShareef, M. A. (2025). Effect of yeast supplemented with selenium or zinc on some antioxidant indices and liver and kidney functions in local Iraqi goats. Kirkuk University Journal for Agricultural Sciences, 16(1), 191\u0026ndash;200. \u003c/li\u003e\n\u003cli\u003eSorak, M. P., Nikolov, A. B., Sazdanovic, P. S., Arsenijevic, N. S., Milicic, V. M., Cekovic, J. M., Parandilovic, A. Z., \u0026amp; Gavrilovic, A. Z. (2024). Activity of enzymes in the follicular fluid and outcome of in vitro fertilization. Medicine, 103(4), e36851. https://doi.org/10.1097/MD.0000000000036851 \u003c/li\u003e\n\u003cli\u003eSpears, J. W., \u0026amp; Weiss, W. P. (2008). \u003cem\u003eRole of antioxidants and trace elements in health and immunity of transition dairy cows\u003c/em\u003e. The Veterinary Journal, 176(1), 70\u0026ndash;76. https://doi.org/10.1016/j.tvjl.2007.12.015 \u003c/li\u003e\n\u003cli\u003eTakahashi, T., Takahashi, E., Igarashi, H., Tezuka, N., \u0026amp; Kurachi, H. (2003). Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization. Molecular Reproduction and Development, 66(2), 143\u0026ndash;152. https://doi.org/10.1002/mrd.10341 \u003c/li\u003e\n\u003cli\u003eTang, W., Zhu, X., Chen, Y., Yang, S., Wu, C., \u0026hellip; Wang, S. (2024). Towards prolonging ovarian reproductive life: Insights into trace-element homeostasis. Ageing Research Reviews, 97, 102311. https://doi.org/10.1016/j.arr.2024.102311 \u003c/li\u003e\n\u003cli\u003eTian, T., Wang, Z., \u0026amp; Zhang, J. (2017). \u003cem\u003ePathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies\u003c/em\u003e. Oxidative Medicine and Cellular Longevity, 2017, Article ID 4535194. https://doi.org/10.1155/2017/4535194 \u003c/li\u003e\n\u003cli\u003eVerma, A., \u0026amp; Sweety. (2024). Important minerals affect the fertility of dairy animals: A review. International Journal of Veterinary Sciences and Animal Husbandry, 9(2), 290\u0026ndash;292. \u003c/li\u003e\n\u003cli\u003eYao, Y., Tang, Y., Qin, H., Meng, R., Zhang, C., Zhang, Y., Yang, Y., Qiao, P., Liu, J., \u0026amp; Su, J. (2023). Zinc supplementation promotes oocyte maturation and subsequent embryonic development in sheep. Theriogenology, 206, 161\u0026ndash;169. https://doi.org/10.1016/j.theriogenology.2023.04.025\u003c/li\u003e\n\u003cli\u003eZhang, P.-P., Ding, G.-C., Tao, C.-Y., Zhang, L., Wang, Y.-X., Yuan, Q.-Y., Zhang, S.-M., \u0026amp; Wang, L.-P. (2024). Levels of trace metals and their impact on oocyte: A review. Taiwanese Journal of Obstetrics and Gynecology, 63(3), 307\u0026ndash;311. https://doi.org/10.1016/j.tjog.2024.02.003\u003c/li\u003e\n\u003cli\u003eZheng, M., Andersen, C. Y., Rasmussen, F. R., Cadenas, J., Christensen, S. T., \u0026amp; Mamsen, L. S. (2023). Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development. Frontiers in Endocrinology, 14, Article 1268248. https://doi.org/10.3389/fendo.2023.1268248\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ovaries, cows, heavy metals, antioxidants, seminal fluid","lastPublishedDoi":"10.21203/rs.3.rs-7068159/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7068159/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Trace elements are essential for follicular development, oocyte maturation and fertility. Nevertheless, as yet, the role of these Trace elements within follicular environment has seldom been investigated in bovines, especially in the presence of contemporary environmental pollution. The objective of this work was to determine the concentrations of toxic elements and trace elements (Cu, Zn, Se, Fe, Co, Mo, Cd, As, Pb, and In) and Malondialdehyde, glutathione, cholesterol, and glucose in bovine follicular fluids (bFFs) in various follicular sizes in different follicular development stages. Important biochemical variables were also measured to correlate them with follicular maturity. Follicles were categorized as small (3–5 mm), medium (6–9 mm), and large (10–22 mm). Follicular fluid (FF) samples were subjected to inductively coupled plasma emission spectrometry (ICPE-9000) trace elements and analyzed spectrophotometrically (glucose, cholesterol, GSH and MDA). A significantly increase (p \u0026lt; 0.05) in the contents of copper, zinc, selenium and cobalt was observed as follicles matured, which may be related to the higher antioxidant and metabolism potential of the mature follicle. Medium follicles showed maximum concentration of iron and molybdenum for follicles of size (6-9 mm). For toxic elements, lead decreased, and arsenic increased in follicle development. Cadmium and indium were found at trace levels, with indium detected for the first time in bovine follicular fluid. Levels of cholesterol and glutathione also increased with the size of the follicles, while MDA decreased, indicating enhanced oxidative status. Glucose was similar in both groups. These findings indicate the stage-dependent regulations of mineral elements in follicle development, which has implications for the maintenance of oocyte quality and follicle health. first-time detection of Inindium (In) raises questions about its potential reproductive effects and environmental implications.","manuscriptTitle":"Evaluation of mineral elements, some antioxidants and biochemicals in follicular fluid of cow ovaries","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 08:53:05","doi":"10.21203/rs.3.rs-7068159/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"313e38f2-7482-42a2-be6e-46566b3c43c9","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-17T23:38:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 08:53:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7068159","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7068159","identity":"rs-7068159","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}