MATERIALS AND METHODS

Ethical approval

All experimental procedures involving animals were reviewed and approved by the Ethics Committee of the Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia (Approval No. 015/UN4.12/EC/VI/2025). The study was conducted in accordance with the institutional guidelines for the care and use of animals in research, the applicable national regulations governing animal experimentation in Indonesia, and the internationally accepted principles for the ethical use of animals in scientific research.

Written permission to conduct the study was obtained from the participating commercial dairy farm before animal enrolment. All procedures, including clinical examination, endometrial cytobrush sampling, and jugular venous blood collection, were performed by licensed veterinarians or trained personnel using standardized aseptic veterinary techniques to minimize stress, pain, and the risk of infection. Animals were gently restrained for the shortest possible duration, and no invasive surgical procedures were performed.

Throughout the study, all cows remained under routine commercial herd management and had unrestricted access to feed and water in accordance with normal farm practice. Animal health and welfare were continuously monitored during the sampling period, and any cow requiring veterinary treatment received appropriate clinical care immediately. No animal was deliberately harmed, euthanized, or excluded from receiving treatment for research purposes, and participation in the study did not interfere with routine herd health management or reproductive management practices.

The observational nature of the study involved procedures commonly performed during routine reproductive health examinations, and every effort was made to minimize animal discomfort while maintaining the highest standards of animal welfare throughout the investigation.

Study period and location

The study was conducted from August 2024 to April 2025 under commercial tropical dairy production conditions in Indonesia. The study location was characterized by high ambient temperature and humidity, environmental conditions known to influence postpartum metabolic status, inflammatory responses, and reproductive performance in dairy cattle. All laboratory analyses were performed using standardized analytical procedures under controlled laboratory conditions.

Study design

A longitudinal observational study was conducted to evaluate metabolic, inflammatory, biochemical, and hormonal parameters associated with uterine health status, uterine recovery, and reproductive performance in Friesian-Holstein (FH) dairy cows. Sixty multiparous FH dairy cows were enrolled based on postpartum uterine health status and their availability for longitudinal follow-up.

The inclusion criteria comprised multiparous FH cows with parity ≥2, clinically stable, available for sampling on day 0 (D0) and day 30 (D30), and complete reproductive records. Cows presenting severe systemic illness, concurrent clinical diseases that could markedly influence metabolic or inflammatory status, incomplete biological sampling, or incomplete reproductive records were excluded. Throughout the study, all cows were maintained under comparable housing, nutritional, and reproductive management conditions.

Based on the initial endometrial cytological findings, cows were allocated into two experimental groups: suspected endometritis (n = 30) and subclinical endometritis (n = 30). Physiological assessments and sample collections were performed at two predetermined time points, D0 and D30. D0 represented the initial postpartum uterine cytological diagnosis and baseline physiological evaluation, whereas D30 was selected to assess short-term changes in physiological status and uterine recovery.

The principal strength of the study design was the paired longitudinal assessment of metabolic, inflammatory, biochemical, and hormonal biomarkers within the same animals, allowing comprehensive evaluation of temporal physiological changes and their associations with uterine recovery and reproductive performance.

At the completion of the observation period, cows were further classified according to uterine recovery status as recovered or not recovered. Reproductive performance variables, including estrus intensity, SC, and DO, were subsequently recorded for all animals. No animals were withdrawn from the study during the experimental period.

Diagnosis and group classification

Uterine health status was determined by endometrial cytology, evaluating the proportion of PMNs in endometrial epithelial samples, a widely accepted diagnostic method for identifying uterine inflammation in dairy cows [12]. Endometrial samples were collected using a sterile cytobrush (Minitube GmbH, Tiefenbach, Germany). The cytobrush was carefully introduced through the cervix into the uterine lumen using a protective sheath to minimize contamination during sample collection. Cellular material was immediately transferred onto clean microscope slides (Thermo Fisher Scientific, Waltham, MA, USA), air-dried, and stained using a standard cytological staining procedure as previously described [13].

Stained slides were examined using a light microscope (Olympus BX43; Olympus Corporation, Tokyo, Japan). A minimum of 200 endometrial cells were counted per sample to determine the PMN percentage, in accordance with established cytological evaluation procedures [14]. To minimize observer bias, cytological evaluations were performed by trained personnel blinded to the animals' reproductive performance records.

Based on the PMN percentage, cows were classified into two uterine health categories. Cows with PMN percentages between 5% and <10% were classified as having suspected endometritis, whereas cows with PMN percentages ≥10% were classified as having subclinical endometritis. This classification differentiated cows with mild cytological evidence of uterine inflammation from those exhibiting more severe endometrial neutrophil infiltration.

Recovery status was reassessed at D30 using repeat endometrial cytology. Cows with PMN percentages below the diagnostic threshold for subclinical endometritis were classified as recovered, whereas those with persistent PMN percentages ≥10% were classified as not recovered.

Sample collection

Blood samples were collected from each cow at D0 and D30 to evaluate longitudinal changes in metabolic, inflammatory, biochemical, and hormonal biomarkers. To minimize physiological variation associated with circadian rhythm and feed intake, blood sampling was consistently performed during the morning before the primary feeding period.

Approximately 10 mL of blood was collected from the jugular vein using sterile disposable needles (Terumo Corporation, Tokyo, Japan) and vacuum blood collection tubes without anticoagulant (Vacutainer®, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), following standard blood collection procedures for dairy cattle.

After collection, blood samples were allowed to clot at room temperature for approximately 30 min, then centrifuged at 3,000 × g for 15 min using a refrigerated centrifuge (Eppendorf 5810 R, Eppendorf AG, Hamburg, Germany) to obtain serum [15]. Serum was carefully transferred into sterile microcentrifuge tubes (Eppendorf AG). Samples exhibiting visible hemolysis were excluded from subsequent analyses. All serum samples were stored at −20°C until biochemical analyses were performed.

Metabolic, biochemical, inflammatory, and hormonal analyses

Serum samples were analyzed to determine concentrations of metabolic, biochemical, inflammatory, and hormonal biomarkers associated with uterine health status. The evaluated biomarkers included NEFAs, BHB, calcium (Ca), albumin, globulin, total protein (TP), Hp, PGFM, and cortisol.

Serum NEFAs and BHB concentrations were determined using commercial enzymatic assay kits (Randox Laboratories Ltd., Crumlin, United Kingdom) according to the manufacturer's instructions and previously described methods [16]. Absorbance values were measured using a microplate reader (BioTek ELx800, BioTek Instruments Inc., Winooski, VT, USA).

Serum Ca, albumin, and TP concentrations were measured using an automated biochemical analyzer (Mindray BS-120, Mindray Bio-Medical Electronics Co., Shenzhen, China) based on standardized colorimetric methods [17]. Globulin concentration was calculated by subtracting albumin concentration from TP concentration.

Serum Hp concentrations were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource Inc., San Diego, CA, USA) according to the manufacturer's protocol. Optical density measurements were obtained using the same microplate reader, and concentrations were calculated using standard calibration curves [18].

Serum PGFM and cortisol concentrations were determined using commercial ELISA kits (MyBioSource Inc.) according to the manufacturer's instructions. Hormone concentrations were calculated from the respective standard curves generated from absorbance measurements.

All enzymatic, biochemical, and ELISA assays were performed strictly according to the manufacturers' protocols. Blank controls, calibration standards, and kit-provided quality controls were included where applicable to ensure analytical reliability. Assay sensitivity and intra- and inter-assay coefficients of variation complied with the manufacturers' specifications.

Reproductive performance assessment

Reproductive performance was evaluated using estrus intensity, SC, and DO. Estrus intensity was assessed by visual observation of behavioral indicators, including standing heat, mounting, restlessness, increased physical activity, and vulvar mucus discharge. Estrus detection was routinely performed by trained farm personnel and independently verified by the research team to minimize observer variation. Estrus expression was scored on a three-point ordinal scale, where score 1 represented weak estrus expression, score 2 represented moderate estrus expression, and score 3 represented strong estrus expression according to previously published scoring systems [19].

Artificial insemination was performed in cows exhibiting estrus, in accordance with the farm's routine reproductive management program. The voluntary waiting period and insemination schedule followed the farm's standard reproductive protocol. Frozen semen was administered by trained artificial insemination technicians.

SC was defined as the number of inseminations required to achieve successful conception. Pregnancy diagnosis was performed by a veterinarian using the farm's routine pregnancy diagnostic procedures. DO was calculated as the interval between parturition and confirmed conception.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Data normality was evaluated using the Shapiro–Wilk test. Descriptive data are presented as mean ± standard deviation (SD).

Comparisons between cows with suspected endometritis and subclinical endometritis were performed using the independent-samples t-test. Changes between D0 and D30 within each experimental group were analyzed using the paired-samples t-test. Comparisons between recovered and not recovered cows were performed using the independent-samples t-test where appropriate.

Associations between physiological biomarkers and reproductive performance variables, including estrus intensity, SC, and DO, were evaluated using Pearson correlation analysis.

Data were screened for completeness and potential outliers before statistical analysis. An a priori sample size calculation was not performed because the study was based on an available longitudinal field dataset. Adjustment for multiple comparisons was not applied; therefore, the findings were interpreted based on statistical significance, biological plausibility, and the consistency of observed responses. Statistical significance was established at p < 0.05.