Research Article

Metabolic Dysregulation and Histopathological Insights into Bovine Cystic Ovarian Disease

Eman Mohamed Samy1, Mohammed A. Marzouk2, Amgad Kadah3, Elsaber A.E4, Walaa Raslan3, Zeinab Said5 and Sara Badawy1*

1Department of Pathology, Faculty of Veterinary Medicine, Benha University, Toukh 13736, Egypt; 2Biochemistry and Molecular Biology Department, Faculty of Veterinary Medicine, Benha University, Benha, Egypt; 3Department of Physiology, Faculty of Veterinary Medicine, Benha University, 13736 Qaliobia, Egypt; 4Department of Clinical Pathology, Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh, 13736, QG, Egypt; 5Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Benha University, 13736, Mushtuhur, Toukh, Qalioubia, Egypt.

Received | May 16, 2025; Accepted | June 04, 2025; Published | September 02, 2025

*Correspondence | Sara Badawy, Department of Pathology, Faculty of Veterinary Medicine, Benha University, Toukh 13736, Egypt; Email: [email protected]

Citation | Samy, E.M., M.A. Marzouk, A. Kadah, A.E. Elsaber, W. Raslan, Z. Said and S. Badawy. 2025. Metabolic dysregulation and histopathological insights into bovine cystic ovarian disease. Advanced Analytical Pathology, 1: 102-110.

DOI | https://dx.doi.org/10.17582/journal.aap/2025/1.102.110

Keywords | Cystic ovarian disease, Metabolic dysregulation, Histopathology, Reproductive dysfunction, Oxidative stress, Inflammation

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Cystic ovarian disease (COD) represents a significant reproductive disorder in dairy cattle that substantially impacts both animal welfare and agricultural economics. This condition is characterized by the persistence of large follicular structures that fail to ovulate, affects approximately 10–15% of dairy cows during their lactation period, leading to considerable economic losses in the dairy industry through reduced reproductive efficiency and increased culling rates (BorŞ and BorŞ, 2020). The financial burden of COD extends beyond direct treatment costs, encompassing decreased milk production, extended calving intervals, and increased labor requirements for reproductive management, collectively resulting in estimated losses of $150–200 per cow per year in affected herds (BorŞ and BorŞ, 2020; Pascottini, 2020).

The pathogenesis of COD is complex and multifaceted, involving intricate interactions between metabolic, endocrine, and inflammatory pathways (Fernández-Novo et al., 2020; Pascottini, 2020). While previous research has established various risk factors associated with COD, including genetic predisposition, environmental stressors, and nutritional imbalances, the precise mechanisms underlying the development and progression of ovarian cysts remain incompletely understood (BorŞ and BorŞ, 2020). The condition appears to be particularly prevalent during periods of metabolic stress, such as the transition period and early lactation, suggesting a strong connection between metabolic homeostasis and reproductive function (Hailay et al., 2019).

The endocrine basis of COD involves disruptions in the hypothalamic-pituitary-ovarian axis, particularly in the regulation of gonadotropin-releasing hormone (GnRH) pulses and subsequent luteinizing hormone (LH) secretion patterns (BorŞ and BorŞ, 2020; Pascottini et al., 2020). These hormonal imbalances can lead to failed ovulation and the formation of persistent follicular structures. However, emerging evidence suggests that metabolic factors play a crucial role in modulating these endocrine pathways, potentially serving as initiating or exacerbating factors in the development of ovarian cysts (Hailay et al., 2019; Pascottini et al., 2020).

Recent studies have highlighted the significance of metabolic dysfunction in the etiology of COD through various pathways, including altered steroidogenesis, disrupted follicular development, and compromised endocrine signalling (Abedal-Majed et al., 2019; Tu et al., 2019). The modern dairy cow, bred for high milk production, faces substantial metabolic challenges during early lactation, including negative energy balance, oxidative stress, and inflammatory conditions (Fernández-Novo et al., 2020; Pascottini, 2020). These metabolic perturbations may create an environment conducive to the development of ovarian cysts through direct effects on follicular cells and indirect effects on hormonal signalling pathways (Hailay et al., 2019).

Oxidative stress and inflammation have emerged as potentially critical factors in the pathophysiology of COD. The intense metabolic demands of lactation can lead to increased production of reactive oxygen species and inflammatory mediators, which may damage ovarian tissue and disrupt normal follicular development (Lu et al., 2018; Pascottini et al., 2020). Furthermore, alterations in lipid metabolism and energy homeostasis may affect the availability of substrates for steroid hormone synthesis, potentially contributing to the hormonal imbalances characteristic of COD (Abedal-Majed et al., 2019; Tu et al., 2019).

The relationship between metabolic status and reproductive function extends beyond direct effects on the ovary. Systemic metabolic alterations can affect multiple organ systems, including the liver and adipose tissue, which play crucial roles in hormone metabolism and energy homeostasis (Hailay et al., 2019; Silva and Mohebbi, 2022). Understanding these complex interactions requires a comprehensive approach that integrates both metabolic profiling and detailed histopathological analyses, yet such studies remain limited in the current literature (BorŞ and BorŞ, 2020).

The present study aims to bridge this knowledge gap by conducting a thorough investigation of the metabolic and histopathological alterations associated with COD in cattle. By employing a multi-parametric approach, we examine various aspects of metabolism, including energy homeostasis, lipid profiles, liver and kidney function, oxidative stress markers, and mineral balance, alongside detailed histopathological evaluations of ovarian and uterine tissues (Hailay et al., 2019; BorŞ and BorŞ, 2020). This integrated analysis provides valuable insights into the complex pathophysiology of COD and its systemic effects on bovine reproductive health.

Our research particularly emphasizes the importance of understanding the bidirectional relationship between metabolic dysregulation and structural tissue alterations in the context of reproductive dysfunction. The comprehensive evaluation of both metabolic parameters and tissue pathology allows for a more nuanced understanding of how systemic metabolic perturbations may manifest in specific cellular and tissue-level changes within the reproductive tract (Abedal-Majed et al., 2019; Domp et al., 2021). This approach may reveal novel therapeutic targets and biomarkers for early detection and intervention in cases of COD.

Furthermore, this investigation has broader implications for comparative reproductive medicine. The pathophysiological features of bovine COD share several similarities with polycystic ovarian syndrome (PCOS) in humans and other reproductive disorders in domestic animals (Hailay et al., 2019; Ryu et al., 2019). Understanding the metabolic and structural alterations associated with COD in cattle may provide valuable insights into the mechanisms underlying similar reproductive disorders across species, potentially contributing to the development of more effective therapeutic strategies in both veterinary and human medicine.

Materials and Methods

Ethical approval and sample collection

All procedures involving animals were conducted in accordance with the institutional guidelines for animal care and use, and the study protocol was approved by the Institutional Animal Ethics Committee (Approval No. BUFVTM 08-04-25), faculty of Veterinary Medicine, Benha University. Samples were collected from cows at various slaughterhouses from Cairo and Giza governorates, such as the El-Basateen slaughterhouse and the El-Warraq slaughterhouse. Immediately post-mortem, blood was drawn via jugular venipuncture into sterile tubes, allowed to coagulate at room temperature, and centrifuged at 3,000 rpm for 20 minutes to separate the serum, which was then allocated and stored at -20°C until further analysis (Abu-El-Hamd et al., 2023). Concurrently, ovarian and uterine tissue specimens were harvested and promptly fixed in 10% neutral buffered formalin for histopathological evaluation.

Biochemical analysis

Serum biochemical parameters including glucose, total protein, triglycerides, cholesterol, urea, creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were quantified using spectrophotometric assays on an automated clinical chemistry analyzer following standardized protocols (Ali et al., 2020). Serum total antioxidant capacity (TAC) was determined calorimetrically using commercially available kits (Biodiagnostic, Egypt) in accordance with the method described by Koracevic et al. (2001). The concentration of C-reactive protein (CRP) was evaluated using a rapid latex slide test kit (Spectrum, Egypt). Additionally, the serum concentrations of calcium and phosphorus were measured using established colorimetric methods as described by Tietz (2006).

Histopathological examination and special stains

Ovarian and uterine tissue specimens were concurrently harvested and fixed in 10% neutral buffered formalin for histopathological evaluation. After fixation, tissues were processed through graded ethanol, cleared in xylene, and embedded in paraffin. Serial sections (4–5 µm thick) were then cut and mounted on glass slides. For histological assessment, sections were stained with hematoxylin and eosin (H&E) to evaluate general tissue architecture. In a continuous staining protocol, additional special stains were applied to further characterize specific tissue components: Masson’s Trichrome was used to assess collagen deposition and fibrosis; Sirius Red facilitated detailed visualization of collagen fiber organization; periodic acid-Schiff (PAS) staining highlighted basement membranes, glycogen, and mucopolysaccharides; and methyl violet was employed to delineate nuclear structures and components of the extracellular matrix (Bancroft and Layton, 2019). Microscopic evaluation of the stained sections was performed by an experienced pathologist to elucidate the structural and cellular alterations associated with cystic ovarian disease.

Statistical analysis

Data were analyzed using statistical software (SPSS). Descriptive statistics were calculated as mean ± standard deviation (SD). Comparisons between cows with cystic ovarian disease and cyclic controls were performed using Student’s t-test. Statistical significance was set at p < 0.05 (*), and p < 0.001 (**) was considered highly significant.

Results

In this study, thirty cows (15 with cystic ovarian disease and 15 cyclic controls) were evaluated to compare their metabolic profiles, which are presented as mean ± standard deviation (SD) in Table 1. Statistical analyses were conducted using Student’s t-test, with significance defined at p < 0.05 (*) and p < 0.001 (**).

Cyclic cows demonstrated significantly higher serum glucose levels (4.8 ± 1.3 mmol/L) compared with their cystic counterparts (3.5 ± 0.4 mmol/L, **p < 0.001), suggesting a potential disruption in carbohydrate metabolism associated with cystic ovarian disease. Although cyclic cows exhibited a tendency toward higher insulin concentrations (2.5 ± 1.6 µU/mL) than cystic cows (2.1 ± 1.4 µU/mL), this difference did not reach statistical significance (p > 0.05).

In terms of lipid metabolism, triglyceride levels were comparable between groups (0.21 ± 0.09 mmol/L in cystic cows versus 0.18 ± 0.11 mmol/L in cyclic cows, p > 0.05). However, total cholesterol was significantly elevated in cystic cows (3.7 ± 0.9 mmol/L) relative to cyclic cows (2.5 ± 0.6 mmol/L, p < 0.05), indicating an alteration in lipid metabolism linked to the disease state.

Evaluations of liver function revealed that serum aspartate aminotransferase (AST) levels were significantly higher in cystic cows (105 ± 25 U/L) than in cyclic animals (85 ± 30 U/L, p < 0.05), possibly reflecting hepatic stress. Conversely, alanine aminotransferase (ALT) activity remained similar between the two groups (23 ± 5 U/L in cystic versus 22 ± 6 U/L in cyclic cows, p > 0.05).

Renal function assessments showed that serum urea concentrations were significantly lower in cystic cows (1.5 ± 0.3 mmol/L) compared to cyclic cows (4.1 ± 3.0 mmol/L, **p < 0.001), which may indicate alterations in protein catabolism or renal filtration. In contrast, serum creatinine levels were similar between groups (110 ± 30 µmol/L in cystic cows versus 115 ± 28 µmol/L in cyclic cows, p > 0.05), suggesting comparable glomerular filtration rates.

The investigation of oxidative stress and inflammation revealed that cystic cows had a significantly reduced total antioxidant capacity (0.48 ± 0.03 mM/L) relative to cyclic cows (0.75 ± 0.04 mM/L, **p < 0.001). Furthermore, C-reactive protein (CRP) concentrations were markedly higher in cystic cows (5.5 ± 0.5 mg/L) compared to cyclic cows (2.2 ± 0.3 mg/L, **p < 0.001), reflecting an enhanced inflammatory state.

While serum calcium levels were consistent between cystic (10.5 ± 1.5 mg/dL) and cyclic cows (10.8 ± 1.4 mg/dL, p > 0.05), serum phosphorus levels were significantly lower in cystic cows (6.2 ± 0.9 mg/dL) than in cyclic cows (7.8 ± 0.7 mg/dL, p < 0.05). These results suggest that cystic ovarian disease is accompanied by a range of metabolic disturbances, including impairments in carbohydrate and lipid metabolism, hepatic and renal function, and antioxidant defenses, as well as an elevated inflammatory response.

 

Table 1: Metabolic profile of cystic vs. cyclic cows.

Parameter	Cystic cows (n=15)	Cyclic cows (n=15)	Significance
Energy metabolism
Glucose (mmol/L)	3.5 ± 0.4	4.8 ± 1.3	***
Insulin (µU/mL)	2.1 ± 1.4	2.5 ± 1.6	ns
Lipid profile
TG (mmol/L)	0.21 ± 0.09	0.18 ± 0.11	ns
Chol (mmol/L)	3.7 ± 0.9	2.5 ± 0.6	*
Liver function
AST (U/L)	105 ± 25	85 ± 30	*
ALT (U/L)	23 ± 5	22 ± 6	ns
Kidney function
Urea (mmol/L)	1.5 ± 0.3	4.1 ± 3.0	***
Creatinine (µmol/L)	110 ± 30	115 ± 28	ns
Oxidative stress/inflammation
Total antioxidant capacity (mM/L)	0.48 ± 0.03	0.75 ± 0.04	***
C-Reactive protein (mg/L)	5.5 ± 0.5	2.2 ± 0.3	***
Minerals
Ca (mg/dl)	10.5 ± 1.5	10.8 ± 1.4	ns
P (mg/dl)	6.2 ± 0.9	7.8 ± 0.7	*

 

Histopathological findings

Examination of cystic ovarian tissue revealed a complex array of pathological changes. The ovarian parenchyma was largely replaced by multiple, variably sized cystic structures. These cysts were characterized by thin, delicate walls lined predominantly by a single layer of cuboidal to low columnar granulosa cells. In several sections, the granulosa cell lining appeared attenuated or focally disrupted, with evidence of cellular degeneration and apoptosis. The adjacent theca interna was notably thickened and exhibited hyperplasia, with clusters of steroidogenic cells displaying abundant eosinophilic cytoplasm and hyperchromatic nuclei. In addition, areas of follicular atresia were apparent, intermingled with fibrous connective tissue deposition and accumulations of hemosiderin-laden macrophages, indicative of previous hemorrhagic events. Focal inflammatory infiltrates primarily lymphocytes with occasional neutrophils were observed around the cystic lesions, suggesting a chronic inflammatory response. Concomitant vascular congestion and limited zones of necrosis further underscored the compromised microenvironment within the ovarian stroma, collectively supporting a diagnosis of cystic ovarian degeneration likely related to hormonal imbalance and impaired follicular maturation (Figure 1).

 

The uterine histopathology provided further insights into the reproductive pathology associated with ovarian dysfunction. The endometrium exhibited heterogeneity in glandular architecture; focal regions demonstrated marked endometrial hyperplasia, with elongated, crowded glands lined by tall, columnar epithelium. However, these hyperplastic areas were interspersed with regions where glandular atrophy and focal epithelial degeneration were evident. The underlying stroma was infiltrated by a mixed inflammatory cell population predominantly lymphocytes with sporadic plasma cells and neutrophils alongside areas of mild edema and early fibrotic changes. These inflammatory changes were most pronounced in regions adjacent to the hyperplastic glands, potentially reflecting a reactive process to abnormal hormonal stimulation or secondary insult. The myometrium retained a largely normal architecture, with well-organized bundles of smooth muscle fibers; nevertheless, focal areas of mild inflammatory cell infiltration and occasional microhemorrhages were noted, suggesting that the uterine changes might represent a secondary response to the altered ovarian hormonal milieu (Figure 2).

 

Special stain results

The application of a series of special stains provided an in-depth characterization of the extracellular matrix alterations and cellular changes in ovarian and uterine tissues affected by cystic ovarian disease (COD). Masson’s Trichrome staining revealed a marked increase in collagen deposition within the stromal compartments of cystic ovaries. Specifically, extensive blue-stained collagen fibers were observed surrounding cystic follicles and within fibrotic areas, suggesting a chronic reparative response secondary to repeated follicular degeneration (Figure 3).

 

Sirius red staining, examined under polarized light, further corroborated these findings by highlighting densely packed collagen fibers with intense red birefringence. This observation indicates not only an increased collagen content but also a reorganization of type I collagen within the cystic ovarian stroma, which is consistent with a fibrotic remodeling process (Figure 4).

 

In addition, PAS staining demonstrated an abnormal accumulation of glycoproteins and mucopolysaccharides. In ovarian sections, PAS-positive material was notably present along the basement membranes of follicles and within the granulosa cell cytoplasm, reflecting altered metabolic activity and extracellular matrix composition. Uterine sections similarly showed well-defined PAS-positive basement membranes in the endometrial glands, though regions of glandular atrophy exhibited diminished staining intensity, indicative of degenerative changes.

Methyl Violet staining provided enhanced visualization of nuclear morphology and extracellular matrix components. In the ovaries, this stain accentuated hyperchromatic nuclei and an increased nuclear-to-cytoplasmic ratio in granulosa cells lining the cysts, suggesting cellular stress and degeneration. In uterine tissue, methyl violet highlighted focal inflammatory infiltrates, particularly in proximity to hyperplastic endometrial glands, thus confirming a reactive inflammatory process (Figure 5).

 

Discussion

The present study elucidates the intricate metabolic alterations associated with cystic ovarian disease (COD) in cows. Our findings indicate that cows with COD exhibit significant disturbances in several metabolic domains compared to their cyclic counterparts, which may have profound implications for reproductive efficiency and overall health (BorŞ and BorŞ, 2020).

A notable finding is the diminished serum glucose concentration in cystic cows, suggesting an impairment in carbohydrate metabolism. Lower glucose levels may reflect a reduced availability of energy substrates necessary for optimal ovarian function and follicular development (Abedal-Majed, 2019; Xue, 2019). Although insulin levels were not significantly different between groups, the observed trend toward lower insulin in the COD group raises the possibility of altered insulin sensitivity or secretion dynamics an important consideration given insulin’s role in both glucose homeostasis and reproductive function (Abedal-Majed, 2019; Ryu, 2019).

The lipid profile further underscores metabolic disturbances in cystic cows. Elevated total cholesterol in the COD group, in the absence of significant changes in triglyceride levels, implies selective alterations in lipid metabolism. Such dyslipidemia could be a consequence of disrupted steroidogenesis, since cholesterol serves as the precursor for steroid hormones (Xue, 2019; Pavithran et al., 2020). Thus, elevated cholesterol levels might not only be a marker of metabolic imbalance but could also directly impact the endocrine milieu, further exacerbating reproductive dysfunction (Tu et al., 2019).

Hepatic function, as assessed by the activity of aspartate aminotransferase (AST), was compromised in cystic cows, whereas alanine aminotransferase (ALT) remained unchanged. The selective increase in AST could indicate hepatic stress or subclinical liver dysfunction, potentially linked to the heightened metabolic demands imposed by COD (Chastant and Saint-Dizier, 2019; Silva and Mohebbi, 2022). These observations suggest that the liver a central organ in metabolic regulation may be adversely affected in cows with ovarian cysts, contributing to the overall metabolic profile observed.

Renal function parameters provided additional insight into the metabolic alterations in COD. The significantly lower urea levels in cystic cows may reflect changes in protein catabolism or altered renal processing of nitrogenous waste, while the absence of significant differences in creatinine levels indicates that glomerular filtration remains largely intact (Marczuk et al., 2018; Abedal-Majed et al., 2019). These findings highlight a possible shift in nitrogen metabolism that may be secondary to or even contribute to the metabolic disturbances associated with ovarian dysfunction.

Oxidative stress and inflammation emerged as critical factors in the pathophysiology of COD. The marked reduction in total antioxidant capacity, coupled with elevated C-reactive protein levels in cystic cows, underscores an environment of heightened oxidative stress and systemic inflammation (Lu, 2018; Liang, 2021). Oxidative stress can impair cellular function and may exacerbate reproductive abnormalities by disrupting follicular development and steroid hormone synthesis, while chronic inflammation may further impair ovarian function, creating a vicious cycle of metabolic and endocrine disruptions (Abedal-Majed, 2019).

Mineral homeostasis was also affected, as evidenced by the significantly lower phosphorus levels despite comparable calcium concentrations between groups were normal. Given phosphorus’s vital role in energy metabolism, its reduction could further compromise cellular energy balance and overall metabolic function (Marczuk et al., 2018; Xue et al., 2019). This selective mineral imbalance may reflect the broader metabolic derangements present in COD and warrants further investigation.

In addition to the metabolic disturbances described above, histopathological evaluation of the ovary and uterus offers critical insights into the structural and cellular alterations underlying COD. Ovarian tissue in cystic cows exhibited multiple, variably sized cystic structures that largely replaced normal parenchyma, with cyst walls lined by attenuated granulosa cells and a thickened, hyperplastic theca interna findings that are indicative of aberrant folliculogenesis likely resulting from persistent gonadotropin stimulation and disrupted steroidogenesis (Tu, 2019; Domp, 2021). Similarly, uterine histopathology revealed heterogeneous changes, including focal endometrial hyperplasia with elongated, crowded glands interspersed with areas of glandular atrophy and epithelial degeneration, as well as inflammatory infiltrates composed primarily of lymphocytes, occasional plasma cells, and neutrophils (Sheldon, 2018). These alterations likely reflect abnormal hormonal stimulation and a reactive process secondary to systemic metabolic stress.

The interplay between these histopathological findings and the metabolic data is striking. Ovarian cystic lesions and uterine inflammatory responses likely represent morphological manifestations of metabolic imbalances such as impaired carbohydrate and lipid metabolism, hepatic stress, and oxidative stress that contribute to ovarian dysfunction (Lu et al., 2018; Pavithran et al., 2020). Collectively, these findings suggest that COD in cows is characterized by a complex interplay of metabolic dysregulation, chronic inflammation, and tissue remodelling. This integrated perspective is critical for elucidating the pathogenesis of COD and for identifying potential targets for metabolic, nutritional, and hormonal interventions (Abedal-Majed, 2019; Xue et al., 2019). Future research should focus on unraveling the mechanistic links between these metabolic disturbances and ovarian dysfunction through longitudinal studies and comprehensive molecular and histopathological analyses.

Collectively, these findings suggest that cystic ovarian disease in cows is characterized by a complex interplay of metabolic dysregulation, which spans disturbances in energy metabolism, lipid and protein processing, liver function, and oxidative stress, and is compounded by chronic inflammation and tissue remodeling. The convergence of these metabolic and histopathological alterations provides a comprehensive framework for understanding the multifactorial physiological challenges imposed by COD. Specifically, the observed reductions in serum glucose and antioxidant capacity, along with elevated cholesterol and markers of inflammation, underscore a systemic endocrine disruption that likely contributes to impaired ovarian and uterine function.

This integrated perspective is critical not only for elucidating the underlying pathogenesis of COD but also for identifying potential targets for metabolic, nutritional, and hormonal interventions. Future research should endeavor to unravel the mechanistic links between these metabolic disturbances and ovarian dysfunction through longitudinal studies and larger sample cohorts. Incorporating molecular and hormonal analyses alongside detailed histopathological assessments will be essential to confirm these associations, identify early biomarkers of disease onset, and ultimately guide the development of more effective diagnostic and therapeutic strategies.

Acknowledgement

Faculty of veterinary medicine, Banha university, Egypt.

Novelty Statement

This study uniquely combines metabolic profiling with detailed histopathological analysis to uncover the underlying mechanisms of bovine cystic ovarian disease, offering new insights into its pathogenesis and potential diagnostic biomarkers

Author’s Contribution

Eman Samy and Sara Badawy conceptualized the study, developed the research design, and wrote the original draft. Mohammed A. Marzouk, Amgad Kadah and Walaa Raslan contributed to data analysis and interpretation. Zeinab Said and Elsaber A.E provided supervision, critical revisions, and project guidance. All authors read and approved the final manuscript.

Generative AI or AI-assisted Technology Statement

The author(s) declare that no Genrative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Abedal-Majed, M.A., Romereim, S.M., Davis, J.S. and Cupp, A.S., 2019. Perturbations in lineage specification of granulosa and theca cells may alter corpus luteum formation and function. Front. Endocrinol., pp. 10. https://doi.org/10.3389/fendo.2019.00832

Abu El-Hamd, M.A., Gabr, S., Soliman, S., Sayah, M., Badr, E. and Ouf, G., 2023. Effect of different zinc sources on milk production and reproductive performance of Friesian cows. Egypt. J. Vet. Sci.,  54(7): 45–56. https://doi.org/10.21608/ejvs.2023.232883.1587

Ali, S.H., Al-Obaidi, Q.T., Aliraqi, O.M.M. and Alhamo, M.A., 2020. Clinical, hematological and some serum biochemical alterations in local cows affected with milk fever in Gogjalee Region, Mosul, Iraq. Egypt. J. Vet. Sci., 51(2): 143–151. https://doi.org/10.21608/ejvs.2020.19294.1120

Bancroft, J.D. and Layton, C., 2019. The hematoxylin and eosin, In: (eds. S.K. Suvarna, C. Layton and J.D. Bancroft), Bancroft’s theory and practice of histological techniques, 8th ed. Elsevier, Philadelphia, pp. 126–138. https://doi.org/10.1016/B978-0-7020-6864-5.00010-4

BorŞ, S.I. and BorŞ, A., 2020. Ovarian cysts, an anovulatory condition in dairy cattle. J. Vet. Med. Sci., 82(10): 1515-1522. https://doi.org/10.1292/jvms.20-0381

Chastant, S. and Saint-Dizier, M., 2019. Inflammation friend or foe of bovine reproduction. Anim. Reprod., 16: 539-547. https://doi.org/10.21451/1984-3143-AR2019-0057

Dompe, C., Kulus, M., Stefańska, K., Kranc, W., Chermuła, B., Bryl, R., Pieńkowski, W., Nawrocki, M.J., Petitte, J.N., Stelmach, B., Mozdziak, P., Jeseta, M., Pawelczyk, L., Jaśkowski, J.M., Piotrowska-Kempisty, H., Spaczyński, R.Z., Nowicki, M. and Kempisty, B., 2021. Human granulosa cells-stemness properties, molecular cross-talk and follicular angiogenesis. Cells, 10(6): 1396. https://doi.org/10.3390/cells10061396

Fernández-Novo, A., Pérez-Garnelo, S.S., Villagrá, A., Pérez-Villalobos, N. and Astiz, S., 2020. The effect of stress on reproduction and reproductive technologies in beef cattle. A review. Animals, 10(11): 2096. https://doi.org/10.3390/ani10112096

Hailay, T., Hoelker, M., Poirier, M., Gebremedhn, S., Rings, F., Saeed-Zidane, M., Salilew-Wondim, D., Dauben, C., Tholen, E., Neuhoff, C., Schellander, K. and Tesfaye, D., 2019. Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status. Sci. Rep., 9(1): 12851. https://doi.org/10.1038/s41598-019-49029-9

Koracevic, D., Koracevic, G., Djordjevic, V., Andrejevic, S. and Cosic, V., 2001. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol., 54(5): 356–361. https://doi.org/10.1136/jcp.54.5.356

Liang, C., Zhang, X., Qi, C., Hu, H., Zhang, Q., Zhu, X. and Fu, Y., 2021. UHPLC-MS-MS analysis of oxylipins metabolomics components of follicular fluid in infertile individuals with diminished ovarian reserve. BioMed. Centr., 19: 143. https://doi.org/10.1186/s12958-021-00825-x

Lu, J., Wang, Z., Cao, J., Chen, Y. and Dong, Y., 2018. A novel and compact review on the role of oxidative stress in female reproduction. Reprod. Biol. Endocrinol., 16(1): 80. https://doi.org/10.1186/s12958-018-0391-5

Marczuk, J., Brodzki, P., Brodzki, A. and Kurek, Ł., 2018. The concentration of free amino acids in blood serum of dairy cows with primary ketosis. Polish J. Vet. Sci., 21(1): 149-156. https://doi.org/10.24425/119033

Pascottini, O.B., Leroy, J. and Opsomer, G., 2020. Metabolic stress in the transition period of dairy cows: Focusing on the prepartum period. Animals, 10(8): 1419. https://doi.org/10.3390/ani10081419

Pavithran, P.V., Ladjouze, A., Sauter, K.S., Pulickal, A., Katharopoulos, E., Trippel, M., Perren, A., Pandey, A.V. and Flck, C.E., 2020. Novel CYP19A1 mutations extend the genotype-phenotype correlation and reveal the impact on ovarian function. J. Endocrine Soc., 4(4). https://doi.org/10.1210/jendso/bvaa030

Ryu, Y., Kim, S.W., Kim, Y.Y. and Ku, S.Y., 2019. Animal models for human polycystic ovary syndrome (PCOS) focused on the use of indirect hormonal perturbations: A review of the literature. Int. J. Mol. Sci., 20(11): 2720. https://doi.org/10.3390/ijms20112720

Sheldon, I.M., Cronin, J. and Bromfield, J.J., 2018. Tolerance and innate immunity shape the development of postpartum uterine disease and the impact of endometritis in dairy cattle. Ann. Rev., 7: 361-384. https://doi.org/10.1146/annurev-animal-020518-115227

Silva, P.H.I. and Mohebbi, N., 2022. Kidney metabolism and acid-base control: Back to the basics. Pflügers Arch. Eur. J. Physiol., 474(7): 919–934. https://doi.org/10.1007/s00424-022-02696-6

Tietz, N.W., 2006. Fundamentals of clinical chemistry (4th ed.). Sunders, Philadelphia, United States.

Tu, J., Cheung, H.H., Chan, C.L.K. and Chan, W.Y., 2019. The role of microRNAs in ovarian granulosa cells in health and disease. Front. Endocrinol., 10. https://doi.org/10.3389/fendo.2019.00174

Xue, J., Li, X., Liu, P., Li, K., Sha, L., Yang, X., Zhu, L., Wang, Z., Dong, Y., Zhang, L., Lei, H., Zhang, X., Dong, X. and Wang, H., 2019. Inulin and metformin ameliorate polycystic ovary syndrome via anti-inflammation and modulating gut microbiota in mice. Japan Endocrine Soc., https://doi.org/10.1507/endocrj.EJ18-0567