DISCUSSION

Overview of principal findings

The present study evaluated metabolic, inflammatory, biochemical, and hormonal parameters associated with uterine health status, recovery status, and reproductive performance in FH dairy cows. Cows with subclinical endometritis exhibited higher PMN percentages, more pronounced metabolic and inflammatory disturbances, lower recovery rates, and poorer reproductive performance than cows with suspected endometritis. Collectively, these findings indicate that greater uterine inflammation is closely associated with systemic physiological imbalance, delayed uterine recovery, and reduced reproductive efficiency.

Uterine inflammation and recovery status

Endometrial cytology demonstrated that cows with subclinical endometritis had higher PMN percentages than cows with suspected endometritis. Increased PMN infiltration reflects an active inflammatory response within the endometrium and is widely accepted as a reliable indicator of uterine inflammation in dairy cows [20]. Persistent postpartum uterine inflammation may delay uterine involution, alter endometrial function, and reduce the ability of the uterus to support sperm transport, embryo implantation, and early embryonic development [21, 22].

In the present study, cows with suspected endometritis exhibited a higher recovery rate than cows with subclinical endometritis, suggesting that animals with less severe initial endometrial inflammation were more likely to recover during the observation period. Conversely, greater neutrophilic infiltration may indicate persistent inflammatory activity and impaired restoration of normal uterine function. These findings support the value of endometrial cytology not only as a diagnostic method but also as an indicator of recovery potential and the risk of persistent uterine inflammation.

Metabolic imbalance and uterine health

Metabolic status differed markedly between the uterine health groups. Cows with subclinical endometritis had higher NEFAs and BHB concentrations, indicating a greater degree of negative energy balance. During early lactation, high-producing dairy cows commonly experience negative energy balance because the rapid increase in energy demand for milk production exceeds dietary energy intake [23]. This metabolic challenge promotes adipose tissue mobilization and increases circulating NEFAs and BHB concentrations.

Elevated NEFAs and BHB concentrations have been associated with impaired immune competence, reduced neutrophil function, altered inflammatory regulation, and increased susceptibility to postpartum uterine infections [24]. Consequently, cows experiencing greater metabolic stress may have a reduced capacity to eliminate uterine pathogens and resolve endometrial inflammation. The decreases in NEFAs and BHB concentrations from D0 to D30 indicate partial metabolic recovery in both groups. However, the persistently greater metabolic disturbance in cows with subclinical endometritis suggests that prolonged negative energy balance may contribute to persistent uterine inflammation and inferior reproductive performance.

Hp as an indicator of reproductive performance

Among all evaluated physiological parameters, Hp showed the strongest associations with reproductive performance. Although NEFAs and BHB were significantly associated with SC and DO, their correlations were considerably weaker than those observed for Hp. In the present study, Hp showed the strongest negative correlation with estrus intensity and the strongest positive correlations with SC and DO. These findings suggest that systemic inflammatory status may be more closely associated with reproductive impairment than metabolic imbalance alone.

Hp is produced primarily by the liver in response to inflammatory cytokines and tissue injury and is widely recognized as a biomarker of systemic inflammation in cattle [25]. The elevated Hp concentrations observed in cows with subclinical endometritis are consistent with the greater degree of uterine inflammation identified by endometrial cytology. Moreover, the decline in Hp concentrations between D0 and D30 indicates partial resolution of the systemic inflammatory response. Nevertheless, persistently higher Hp concentrations in cows with subclinical endometritis suggest continued inflammatory activity despite partial physiological recovery. These observations highlight the potential utility of Hp in assessing reproductive prognosis in cows with endometritis.

Potential mechanisms linking Hp and fertility

The biological relationship between elevated Hp concentrations and impaired reproductive performance likely involves complex interactions among systemic inflammation, iron homeostasis, ovarian function, and endocrine regulation. Hp binds free hemoglobin released during tissue injury, thereby reducing oxidative damage and restricting iron availability to pathogenic microorganisms. Although this mechanism is physiologically protective, sustained elevation of Hp reflects persistent systemic inflammation.

Prolonged inflammatory activity may disrupt the hypothalamic–pituitary–ovarian axis, impair follicular development and steroidogenesis, compromise luteal function, and reduce embryonic survival [26]. Inflammatory mediators may also interfere with gonadotropin responsiveness and ovarian follicular dynamics, ultimately reducing fertility. Therefore, elevated Hp concentrations may reflect an inflammatory state that contributes to weaker estrus expression, increased insemination requirements, and prolonged DO. This interpretation is consistent with the stronger associations observed for Hp than for NEFAs, BHB, and the other biochemical and hormonal parameters evaluated in this study.

Hormonal alterations and reproductive outcomes

Hormonal parameters were also significantly associated with reproductive performance. PGFM showed positive correlations with SC and DO. Prostaglandins are key mediators of uterine inflammatory responses and play central roles in luteolysis, uterine contractility, and postpartum uterine involution [27]. Elevated PGFM concentrations may therefore reflect persistent uterine inflammation or altered prostaglandin metabolism that adversely affects reproductive function.

Cortisol also showed positive correlations with SC and DO, although these relationships were weaker than those observed for Hp. Cortisol is a principal mediator of physiological stress responses, and prolonged increases in cortisol may suppress gonadotropin secretion, impair ovarian follicular development, and reduce estrus expression [28]. The higher cortisol concentrations observed in cows with subclinical endometritis suggest that more severe uterine inflammation was accompanied by greater physiological stress. The decreases in PGFM and cortisol between D0 and D30 indicate partial normalization of endocrine responses during recovery.

Integrated relationships among physiological parameters

The correlation analysis emphasized the close interaction among metabolic stress, systemic inflammation, endocrine regulation, and reproductive performance. NEFAs and BHB were negatively associated with estrus intensity and positively associated with SC and DO. These findings agree with previous reports demonstrating that postpartum metabolic stress impairs follicular growth, oocyte quality, steroidogenesis, and endocrine signaling, thereby reducing reproductive efficiency in dairy cattle [29].

Albumin was positively associated with estrus intensity and negatively associated with SC and DO, whereas globulin and TP showed positive associations with poorer reproductive outcomes. These relationships likely reflect the combined effects of inflammation, hepatic protein metabolism, and nutritional status. Nevertheless, the substantially stronger correlations observed for Hp suggest that persistent systemic inflammation may be a dominant physiological factor underlying impaired reproductive performance in cows with endometritis.

Relevance under tropical dairy production conditions

The present findings are particularly relevant to dairy production under tropical Indonesian conditions. High ambient temperature and humidity may reduce dry matter intake, exacerbate negative energy balance, increase physiological stress, and compromise immune and reproductive function [9, 10]. Consequently, FH cows maintained in tropical environments may experience greater physiological challenges than those raised in temperate conditions.

Heat stress may interact with postpartum uterine inflammation by intensifying metabolic imbalance, endocrine disturbances, and systemic inflammatory responses, thereby delaying uterine recovery and impairing fertility. The present study provides contextual novelty by integrating endometrial cytology with longitudinal assessment of metabolic, inflammatory, biochemical, and hormonal parameters under tropical dairy production conditions. This comprehensive approach improves understanding of the physiological mechanisms linking uterine inflammation with reproductive performance in tropical dairy herds.

Practical implications

From a practical perspective, these findings have important implications for precision dairy herd management. Routine monitoring of Hp may facilitate early identification of cows at increased risk of weak estrus expression, repeated insemination, delayed conception, and prolonged DO. Compared with NEFAs and BHB, Hp showed stronger associations with reproductive outcomes, suggesting that monitoring systemic inflammation may add value to reproductive management.

Furthermore, combining endometrial cytology with circulating Hp assessment may improve the identification of cows requiring intensified reproductive monitoring, nutritional intervention, or veterinary treatment. However, the present study did not establish diagnostic cutoff values or validate predictive models for Hp. Consequently, additional validation is required before its routine implementation as a cow-side decision-support biomarker.

Strengths and limitations

A major strength of this study was the longitudinal evaluation of metabolic, inflammatory, biochemical, and hormonal parameters within the same animals at D0 and D30. Integrating endometrial cytology, uterine recovery status, and reproductive performance enabled a comprehensive assessment of the relationships between local uterine inflammation and systemic physiological responses. Conducting the study under tropical field conditions further enhanced its practical relevance to dairy production systems in Indonesia and similar environments.

Nevertheless, several limitations should be considered. The observational design precludes definitive conclusions regarding causality between physiological parameters and reproductive outcomes. Environmental, nutritional, parity-related, and management factors may also have influenced metabolic, inflammatory, hormonal, and reproductive responses. In addition, an a priori sample size calculation and adjustment for multiple comparisons were not performed.

Formal predictive modeling, regression-based effect-size estimation, interaction analyses between metabolic and inflammatory parameters, and machine-learning approaches were beyond the scope of the available dataset. Detailed economic analyses of reproductive losses associated with elevated Hp concentrations were also not performed because comprehensive farm-level economic data were unavailable. Furthermore, an independent validation cohort was not included, and uterine microbiome profiling, oxidative stress markers, cytokines, microRNA expression, and adipokine measurements were not investigated.

Future research

Future investigations should include larger and more diverse dairy populations, additional longitudinal sampling points, controlled experimental designs, and independent validation cohorts. Multivariable analyses are needed to determine whether Hp independently predicts reproductive outcomes after adjustment for parity, milk yield, body condition score, nutritional status, and environmental variables.

Future studies should also establish clinically relevant Hp cutoff values, evaluate rapid field-based Hp assays, and develop predictive models integrating Hp with NEFAs, BHB, PGFM, cortisol, cytokines, oxidative stress markers, and uterine microbiome profiles. Economic analyses are additionally warranted to determine whether Hp-guided reproductive monitoring can improve herd fertility and profitability under commercial dairy production conditions.

Summary of the discussion

The present study demonstrates that metabolic imbalance, systemic inflammation, and hormonal alterations are closely associated with uterine health status, recovery, and reproductive performance in FH dairy cows. Cows with subclinical endometritis exhibited more severe physiological disturbances, lower recovery rates, weaker estrus expression, greater SC, and prolonged DO than cows with suspected endometritis. Among all evaluated physiological parameters, Hp showed the strongest associations with estrus intensity, SC, and DO, indicating that systemic inflammation is a major physiological feature associated with reduced reproductive efficiency in cows with endometritis. These findings support further investigation of Hp as a biomarker for reproductive monitoring and management, particularly under tropical dairy production systems.