Transrectal Spectral Doppler reveals uterine hyperemia in Tharparkar cows with subclinical endometritis

Transrectal Spectral Doppler reveals uterine hyperemia in Tharparkar cows with subclinical endometritis | 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 Transrectal Spectral Doppler reveals uterine hyperemia in Tharparkar cows with subclinical endometritis Uttam Kumar Sahu, Brijesh Kumar, Meraj Haider Khan, Mayank Singh, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7990665/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 Subclinical endometritis (SCE) is an asymptomatic uterine inflammation that impairs fertility and is hard to detect in field conditions. We serially applied blinded transrectal spectral Doppler to characterize middle uterine artery hemodynamics across the estrous cycle in lactating Tharparkar cows with (n = 10) and without (n = 10) cytology-confirmed SCE. Doppler measures (resistance index [RI], pulsatility index [PI], timed-averaged maximum velocity [TMAX], blood-flow volume [BFV], artery diameter) and serum progesterone (P4) were recorded every 3 days from estrus through the next estrus. Repeated-measures analysis showed that SCE cows had consistently lower RI and PI and higher TMAX, BFV and artery diameter across the cycle (group effect P < 0.05). RI correlated positively with corpus luteum size (r = 0.64) and P4 (r = 0.77), while BFV correlated positively with TMAX (r = 0.82) and diameter (r = 0.78) and negatively with RI (r = − 0.57) and P4 (r = − 0.59) (all P < 0.0001). Limitations include modest sample size (n = 10/group) and absence of concurrent bacteriology or nitric-oxide measurements. With validation in larger cohorts, Doppler metrics could provide a non-invasive adjunct to cytology for detecting uterine inflammation and guiding reproductive management in tropical dairy systems. Doppler indices Nitric oxide Resistance index Subclinical endometritis Tharparkar cattle Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Indigenous Bos indicus cattle underpin rural livelihoods in tropical and subtropical regions, providing milk, draft power and income. Although these breeds exhibit greater thermotolerance than Bos taurus (Cooke et al. 2020 ), tropical climates predispose them to reproductive infections that compromise fertility (Chenoweth 1994 ; Sheldon and Owens 2017 ). Postpartum uterine disorders remain a major cause of subfertility, with metritis (8–40%), clinical endometritis (5–30%) and subclinical endometritis (SCE; 11–70%) reported worldwide (Baithalu et al. 2017 ; Wagener et al. 2017 ). SCE, defined as cytological inflammation of the endometrium without overt clinical signs (Kasimanickam et al. 2004 ), can reduce milk yield, fat and protein content, and increase somatic cell counts, leading to substantial economic losses (Burke et al. 2010 ; Green et al. 2011 ; Ghasemi et al. 2012 ). Detection of SCE is challenging because affected cows appear clinically normal, and diagnosis relies on endometrial cytology, an invasive and labour-intensive procedure poorly suited to field conditions. Conventional B-mode ultrasonography identifies uterine fluid or wall thickening but has limited accuracy for subclinical cases. Doppler ultrasonography, by contrast, provides real-time assessment of uterine blood flow and may serve as a non-invasive indicator of inflammation (Ginther and Utt 2004 ; El-Sayed et al. 2024 ). Inflammatory mediators such as histamine, bradykinin, prostaglandins and nitric oxide (Moncada and Higgs 2006 ) induce vasodilation and tissue hyperaemia, which can be quantified through spectral Doppler indices including the resistance index (RI) and pulsatility index (PI). Reduced RI and PI, reflecting lower vascular impedance, are reported in uterine infections in women (Jaiyeoba and Soper 2011 ), Bos taurus cattle (Sharma et al. 2021 ), buffaloes (El-Sayed et al. 2024 ), mares (Bollwein et al. 2003 ) and ewes (Anderson et al. 1977 ). However, the uterine hemodynamic responses to SCE remain largely unexplored in Bos indicus breeds, which are central to tropical dairy systems. Addressing this gap, the present study employed serial transrectal spectral Doppler ultrasonography across the estrous cycle in Tharparkar cows with and without SCE to characterize alterations in uterine vascular perfusion. We hypothesized that SCE would produce a distinct Doppler signature of uterine hyperaemia, characterized by increased blood flow velocity and vessel diameter with reduced impedance indices. Materials and methods Location and climatic parameters The experiment was conducted at the Cattle and Buffalo Farm, ICAR–IVRI, Izatnagar, India (28° N, 79° E; 564 m above mean sea level). The climate of the region is humid and tropical, with a mean summer temperature of 30.70°C (range 15.10–40.90°C) and mean relative humidity of 53.20%. Animals and feed management Twenty lactating parous Tharparkar cows (parity 2–3; age 4.80 ± 0.81 year; body weight 388.1 ± 9.5 kg; mean milk yield 3.01 ± 0.23 kg day⁻¹) were enrolled. Provide days in milk (DIM) or postpartum interval for each animal (70 ± 3.34). Cows received 1.5–2.5 kg concentrate feed (20% DCP, 70% TDN), ad libitum wheat straw, green fodder twice daily, and free access to water. Animals were managed under semi-intensive conditions with access to open paddocks. Experimental design, inclusion and exclusion criteria of animal Before the start of the experiment, all animals underwent clinical screening to exclude systemic or inflammatory conditions. Reproductive tract evaluation included visual vulvar inspection and vaginal palpation; clear mucus indicated absence of clinical endometritis (Saleem et al. 2021 ). Animals found positive for uterine infections were excluded from the study. Only clinically normal cows with regular estrous cycles, no history of uterine infection, dystocia, or retained placenta, and no abnormal discharge were included. Additionally, transrectal palpation and B-mode ultrasonography confirmed uterine integrity, with no intrauterine fluid or abnormalities. No hormonal synchronization of estrous cycles was performed. Each cow was enrolled on a random day of its cycle, and daily ultrasonographic monitoring was initiated to track the growth of the dominant follicle. Once natural behavioural estrus was detected, confirmed by the presence of a preovulatory follicle and clear mucous discharge, endometrial cytology sampling for SCE diagnosis was performed using the cytobrush technique (Kasimanickam et al. 2004 ). Subsequently, ultrasonographic monitoring continued throughout one complete estrous cycle at regular intervals to assess uterine haemodynamics under natural conditions without exogenous hormonal intervention. Smears were stained with modified Wright’s Giemsa, and 100 cells per slide were counted (Fig. 1 ). Based on established cytological criteria, SCE was defined as the presence of ≥ 5% PMNs at ≥ 47 days postpartum (Kasimanickam et al. 2004 ). Ten cows with SCE formed the treatment group, and ten healthy cows served as controls. Post-cytobrush welfare monitoring was performed for 24 h using a standardized 5-point pain score and observation for abnormal discharge, as per ARRIVE 2.0 guidelines (Sert et al. 2020 ). No adverse reactions were noted. To minimize observer bias, the ultrasonography operator remained blinded to the group allocation (control or SCE-affected). Spectral Doppler acquisition The MUA imaging was performed using a transrectal linear transducer (Exago ECM, France)] with a frequency of 7.5 MHz using color and spectral Doppler. The abdominal aorta was traced caudally to the internal/external iliac bifurcation and the MUA localized within the mesometrium using color Doppler. Spectral Doppler was applied with the sample gate centered within the lumen and gate length set approximately to vessel diameter. The insonation angle was kept ≤ 60° (mean used: 50°), and color gain was standardized (~ 20 dB). Pulse repetition frequency (PRF) was adjusted (typically 3000–5000 Hz; in this study 4000 Hz) to avoid aliasing; when aliasing occurred PRF or scale was adjusted and angle reconfirmed. For each MUA, recordings were accepted only when at least three consecutive, high-quality, consistent cardiac cycles were visualized; three such waveforms were frozen and used to calculate indices, and the mean of these three measurements per side was used for analysis (Bollwein et al. 2000 ; Hassan et al. 2017 ). Each session required ~ 30–45 min per cow and was performed every 3 days from estrus (D0) through the next estrus (D21). Operator training and quality control A single, experienced operator performed all Doppler scans using a standardized protocol and machine presets to minimize observer-related variation. Weekly reliability tests demonstrated excellent reproducibility (intra-observer ICC = 0.92, inter-observer ICC = 0.88). All waveforms were quality-checked, and those with artifacts (e.g., motion, aliasing) were excluded and reacquired. Measurement of spectral Doppler attributes of the middle uterine artery The RI and PI were calculated automatically by the ultrasound system using standard formulas: RI = (PSV − EDV) / PSV (Gosling & King, 1974 ) PI = (PSV − EDV) / mean velocity (Gosling & King, 1974 ) The author Gosling & King, ( 1974 ) commonly used convention; see Ginther & Utt, ( 2004 ) for Doppler principles. Timed-averaged maximum velocity (TMAX; units cm s⁻¹) was taken as the system’s time-averaged peak velocity across the cardiac cycle. Blood flow volume (BFV, mL min⁻¹) was estimated using: BFV = TMAX × π × (D/2)² × 60 (Varughese et al. 2013 ), where D is the internal vessel diameter (cm) measured intima-to-intima, and the factor 60 converts seconds to minutes. Reported BFV thus represents an approximation assuming laminar flow and circular cross-section; limitations of this calculation are acknowledged. Measurement of diameter of MUA Internal vessel diameter (intima-to-intima) was measured on three cardiac-matched frozen B-mode frames at the Doppler sampling site; the mean of three measurements per side was used (Hassan et al. 2017 ). Blood sampling and Progesterone (P4) estimation After each Doppler exam, 4 mL blood was collected by jugular venipuncture into serum clot-activator tubes, allowed to clot for 30 min at room temperature, and centrifuged at 3000 rpm for 10 min. Serum was aliquoted and stored at − 20°C. Progesterone (P4) was measured by DRG Progesterone ELISA (EIA-1561); assay sensitivity = 0.045 ng mL⁻¹, intra-assay CV = 6.4%, inter-assay CV = 6.6%. Samples were analyzed in duplicate. Statistical analysis Data were analyzed using GraphPad Prism 10.6.0 (GraphPad Software, LLC, San Diego, CA, USA) following standard methods. Normality and distribution of data were determined using Shapiro-wilk and Kolmogorov-Smirnov normality test. The mean values of serum P4 concentration, Doppler attributes of MUA viz., Doppler indices (RI, PI), TMAX, vessel diameter and BFV were tested for significance using independent t-test. The RI, PI, TMAX, diameter of MUA and BFV were subjected for analysis of variance of repeated measurement considering the variance between animals as well as between days of estrous cycle, using GLM procedure to determine the principal effects and their interaction. The statistical model was yij = µ + αi + τj + (ατ)ij + \(\:\:\text{ϵ}\) ij, where αi is the day effect and τj is the animal effect and (ατ)ij is their interaction effect. The significance of the differences was tested using post hoc Sidak test. The association among Doppler indices, TMAX, BFV, Cl size, and serum P4 concentration were calculated using Pearson’s correlation coefficient. All the data presented are as mean ± standard error of mean. The difference in mean values for all data analyzed with P < 0.05 was considered significant. Result PMNs count in endometrial cytology screening Ten cows classified as SCE-positive had a mean endometrial PMN% of 6.80 ± 0.24%, whereas the control group had mean PMN% = 3.30 ± 0.21% (group difference P < 0.0001). Spectral Doppler attributes of MUA Mean RI (average of left and right MUA) was significantly lower (group effect P < 0.05) in cows with SCE compared with controls at ovulation (day 0), early luteal (days 3, 6), mid-luteal (days 9, 12), late luteal (days 15, 18) and at subsequent ovulation (day 21), consistent with increased uterine perfusion in SCE cows (Figs. 2 , 3 a). Further, a significant ( P 0.05). Additionally, a strong positive correlation between the size of the CL and mean RI values (r = 0.64; P < 0.0001), as well as between RI values and serum P4 concentrations (r = 0.77; P < 0.0001) (Fig. 4a). Additionally, the RI exhibited a coefficient of variation of 25.54% among the cows. Pulsatility index reflects the relative difference between PSV and EDV across the cardiac cycle (Figs. 2 , 3 b). PI was significantly lower in cows with SCE at ovulation (day 0), mid-luteal days 9 and 12, and at the subsequent ovulation (day 21) ( P < 0.05). Additionally, there was a strong effect of both animal status and days of the estrous cycle on PI values ( P 0.05). A strong positive correlation between RI and PI values was observed (r = 0.77; P < 0.0001) (Fig. 4b) and a strong negative correlation between PI and serum P4 (r = 0.64; P < 0.0001) was observed (Fig. 4c). In addition to that, PI of MUA exhibited a coefficient of variation of 50.37% among the cows. Mean TMAX (average of right and left MUA) was higher in cows with SCE ( P < 0.05), indicating increased flow velocity in the inflamed uterus. Furthermore, there was a significant effect of both animal status and days of the estrous cycle on TMAX values ( P 0.05) interaction was observed (Fig. 3 c). Further, a strong negative correlation (r = -0.67; P < 0.0001) between TMAX and RI was observed (Fig. 4d). The quantitative mean value of BFV in mL min − 1 revealed that uterine BFV was markedly higher ( P < 0.05) in SCE-affected cows compared to healthy animals on days 0, 6, 9, 12, 15, and 18 of the estrous cycle. Additionally, there was a significant effect of animal status and days of the estrous cycle on BFV ( P 0.05) (Fig. 3 d). In addition to that BFV was significantly positively correlated with TMAX (r = 0.82; P < 0.0001) (Fig. 4e) and MUA (r = 0.78; P < 0.0001) (Fig. 4f), while it was significantly negative in correlation with RI (r = -0.57; P < 0.0001) (Fig. 4g) and serum P4 (r = -0.59; P < 0.0001) (Fig. 4h), which is well explanation of a flaccid quiescent uterus during diestrus associated with high RI and P4 but low BFV and TMAX. The diameter of the MUA is a key determinant of uterine blood perfusion, and changes in its diameter can be used to predict UBF across various reproductive events. In this study, the diameter of both MUA (in cm) was monitored to assess the impact of SCE. Results indicated that the average diameter of the MUA in the cows with SCE was consistently larger throughout the estrous cycle compared to healthy cows, with significant ( P < 0.05) differences observed on days 6, 9, 12, 15, and 18 of the cycle. Additionally, the diameter of the MUA was significantly influenced by both the animal and the day of the estrous cycle ( P 0.05) (Fig. 3 e). An inverse relationship was also observed between the MUA diameter and the RI value, with a correlation coefficient of r = − 0.24 ( P < 0.0001) indicative of higher resistance in those blood vessels with lesser diameter and thus, lower blood perfusion to the associated organ (Fig. 4i). Effect of production traits on blood flow indices This study enrolled all cows were of similar physiological and management status, maintained under uniform feeding, housing, and milking schedules. Thus, effect of production traits (milk yield, parity, or days in milk were non-significant ( P > 0.05) uterine haemodynamics indices (RI and PI) in either group, indicating physiological uniformity among experimental animals. All cows were maintained under similar nutritional and management conditions, minimizing the likelihood of production-related variation. Hence, the observed alterations in UBF can be primarily attributed to subclinical endometritic status rather than differences in production performance. Discussion This study provides clear evidence that subclinical endometritis (SCE) in Tharparkar cows induces pronounced alterations in uterine hemodynamics, which can be reliably quantified by transrectal spectral Doppler ultrasonography across the estrous cycle. The consistent reduction in resistance index (RI) and pulsatility index (PI) observed in SCE-affected cows reflects a sustained decrease in uterine vascular impedance and, consequently, a state of localized hyperaemia. Such hemodynamic shifts are characteristic of inflammatory responses and signify an adaptive attempt to enhance blood flow and immune surveillance within the endometrium. The pattern observed here parallels those reported in buffaloes (El-Sayed et al. 2024 ), Bos taurus cattle with uterine infections (Debertolis et al. 2016 ; Sharma et al. 2019 , 2021 ), and women with pelvic inflammatory disease (Jaiyeoba and Soper 2011 ), suggesting that uterine inflammation across species elicits a conserved vascular response involving increased perfusion and reduced vascular resistance. This conserved hemodynamic signature supports the notion that inflammation, even when subclinical, reorganizes uterine vascular function to accommodate increased metabolic demands and immune cell infiltration. The lower RI and PI values during the follicular phase in both healthy and SCE-affected cows further reflect the normal cyclic modulation of uterine blood flow (UBF), driven by the endocrine milieu. Estrogen is known to upregulate endothelial nitric oxide synthase (eNOS) and modulate calcium flux in smooth muscle cells, promoting vasodilation and tissue perfusion (Acosta et al. 2003 ; Stice et al. 1987 ; Chen et al. 2004 ; Sumiyoshi et al. 2014 ). During estrus, this estrogen-mediated vascular relaxation supports follicular maturation, uterine receptivity, and endometrial oedema (Ford and Christenson 1979 ; Bollwein et al. 2000 ). The heightened vascular conductance observed in SCE cows, however, extends beyond physiological vasodilation and likely represents an inflammation-driven amplification of the same mechanisms. The strong correlations between Doppler indices and endocrine variables in this study reinforce the intricate coupling between uterine perfusion and hormonal regulation. The positive relationship between RI, PI, corpus luteum (CL) size, and serum progesterone (P4) indicates that progesterone-mediated vasoconstriction dominates during the luteal phase, while lower impedance indices during follicular phases coincide with estrogenic vasorelaxation. These relationships are consistent with previous bovine Doppler studies (Hassan et al. 2017 ; Sharma et al. 2021 ), confirming that uterine blood flow is dynamically tuned to hormonal fluctuations. In SCE cows, however, these cyclic vascular oscillations appear blunted, suggesting that persistent low-grade inflammation overrides the normal endocrine control of vascular tone. The significant elevations in time-averaged maximum velocity (TMAX) and blood-flow volume (BFV) in SCE-positive cows further confirm the presence of uterine hyperaemia. Similar findings have been reported in buffaloes (El-Sayed et al. 2024 ), Bos taurus cows (Sharma et al. 2019 , 2021 ), mares (Bollwein et al. 2003 ), and ewes with experimentally induced uterine inflammation (Still and Greiss 1978 ). Mechanistically, this hyperaemia can be attributed to the action of inflammatory mediators such as prostaglandins, histamine, bradykinin, and especially nitric oxide (NO), which exerts potent vasodilatory effects on the uterine microcirculation (Moncada and Higgs 2006 ). Elevated NO levels in uterine secretions of SCE- and endometritis-affected cattle (Li et al. 2010 ) provide direct biochemical evidence for this pathway. While NO was not measured in the present study, the hemodynamic pattern strongly implies enhanced endothelial activation and NO bioavailability in SCE cows. The relationships among the Doppler parameters themselves are physiologically coherent. The negative correlations between BFV and RI, and between BFV and P4, coupled with positive correlations between BFV, TMAX, and MUA diameter, indicate that increased flow is accompanied by both structural vessel dilation and decreased impedance. This reflects the fundamental hemodynamic principle that vessel expansion lowers resistance and promotes higher volumetric perfusion (Bollwein et al. 2000 ; Varughese et al. 2013 ; Hassan et al. 2017 ). The enlarged middle uterine artery (MUA) diameter in SCE cows observed here supports this interpretation and indicates vascular remodelling consistent with chronic low-grade inflammation. Similar arterial dilation has been documented in cattle (Sharma et al. 2019 , 2021 ) and buffaloes (El-Sayed et al. 2024 ) with endometritis. However, some induced infection models report inconsistent associations between vessel diameter and flow (Debertolis et al. 2016 ), suggesting that the chronicity and intensity of inflammation influence vascular adaptation. The inverse relationship between MUA diameter and RI observed in this study further strengthens the conclusion that subclinical inflammation drives functional and structural vascular changes that facilitate hyper perfusion of the uterus. From a physiological perspective, these findings reveal that SCE modifies uterine vascular dynamics not merely as a passive consequence of inflammation but as an integrated response aimed at maintaining tissue oxygenation, immune cell access, and metabolic exchange within the endometrium. While adaptive in the short term, chronic hyperaemia could also perpetuate tissue stress and disrupt endometrial receptivity, contributing to the subfertility associated with SCE. The recognition of this vascular phenotype opens possibilities for using Doppler indices as objective indicators of uterine health in field conditions, offering a more practical alternative to cytology, particularly in indigenous breeds where handling constraints often limit invasive testing. The modest sample size (n = 10 per group) limits the detection of subtle inter-animal variations and interaction effects. Moreover, the absence of bacteriological culture, systemic inflammatory markers (such as serum amyloid A or haptoglobin), and direct NO quantification restricts the ability to link vascular changes to specific pathogen loads or molecular pathways. Nevertheless, all animals were of comparable parity, milk yield, and management status, minimizing confounding factors. These data, therefore, provide reliable physiological insight specific to lactating Tharparkar cows and form a foundation for broader, multi-breed studies integrating biochemical and microbial profiling. In conclusion, SCE in Tharparkar cows elicits a consistent Doppler-defined hemodynamic pattern of uterine hyperaemia elevated blood-flow velocity and volume, enlarged arterial diameter, and reduced impedance indices. These vascular adaptations represent measurable manifestations of endometrial inflammation and offer potential diagnostic markers for early, non-invasive detection of uterine disease. Establishing such Doppler reference values for Bos indicus breeds not only enhances our understanding of uterine physiology in tropical livestock but also lays the groundwork for field-applicable diagnostic frameworks. Future research should combine Doppler assessments with quantification of inflammatory mediators and metabolic markers to refine diagnostic accuracy and support timely therapeutic interventions, ultimately improving reproductive efficiency and animal welfare in tropical dairy systems. Declarations Acknowledgement The authors extend their gratitude to the Director, ICAR- Indian Veterinary Research Institute, Izatnagar for providing the necessary facilities and institutional support for carrying out this research. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Institutional facilities of ICAR- Indian Veterinary Research Institute (ICAR-IVRI), Izatnagar were utilized. Ethical approval All experimental procedures were approved by the Institute Animal Ethics Committee of ICAR-IVRI, Izatnagar, U.P. India (26-3/2020-21/JD(R)/IAEC). Data availability The datasets generated and/or analyzed during the current study, including Excel spreadsheets, ultrasound images, and related data files, are available from the corresponding author on reasonable request. Conflict of Interest The authors declare that there is no conflict of interest in publishing the article. Consent of publication All authors have reviewed and consented to the submission of this manuscript for publication. They confirm that the work is original and has not been published elsewhere. Author Contribution Statement Conceptualization: Uttam Kumar Sahu, Brijesh Kumar Methodology: Uttam Kumar Sahu, Brijesh Kumar, Meraj Haider Khan Investigation: Uttam Kumar Sahu, Mayank Singh, Chinmay Warghat Formal analysis: Athidi Lokavya Reddy, Laxmi Sahu, Nitish Singh Kharayat Data curation: Uttam Kumar Sahu, Amit Kumar, Brijesh Kumar Resources: Meraj Haider Khan, Sanjay Kumar Singh Writing – original draft preparation: Uttam Kumar Sahu, Amit Kumar, Brijesh Kumar Writing – review and editing: All authors Supervision: Brijesh Kumar, Sanjay Kumar Singh Project administration: Meraj Haider Khan, Sanjay Kumar Singh Funding acquisition: Not applicable ORCIDs Uttam Kumar Sahu: 0009-0003-7680-4996 Brijesh Kumar: 0000-0001-6153-0791 Meraj Haider Khan: 0000-0002-9934-4772 Mayank Singh Baghel: 0000-0002-9951-1788 Chinmay Warghat: 0009-0002-7924-2055 Athidi Lokavya Reddy: 0000-0003-2295-9303 Laxmi Sahu: 0009-0006-3099-4327 Nitish Singh Kharayat: 0000-0001-7341-1925 Amit Kumar: 0000-0001-7272-898x Sanjay Kumar Singh: Not Available References Acosta TJ, Hayashi KG, Ohtani M, Miyamoto A (2003) Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows. 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Am J Obstet Gynecol 130(1):1–8 https://doi.org/10.1016/0002-9378(78)90430-1 Sumiyoshi T, Tanaka T, Kamomae H (2014) Relationships between the appearances and changes of estrous signs and the estradiol-17β peak, luteinizing hormone surge and ovulation during the periovulatory period in lactating dairy cows kept in tie-stalls. J Reprod Dev 60(2):106–114 https://doi.org/10.1262/jrd.2013-119 Tepper R, Aviram R, Cohen N, Cohen I, Holtzinger M, Beyth Y (1998) Doppler flow characteristics in patients with pelvic inflammatory disease: Responders versus nonresponders to therapy. J Clin Ultrasound 26(5):247–249 https://doi.org/10.1002/(SICI)1097-0096(199806)26:5 Thurmond RL, Gelfand EW, Dunford PJ (2008) The role of histamine H1 and H4 receptors in allergic inflammation: The search for new antihistamines. Nat Rev Drug Discov 7(1):41–53 https://doi.org/10.1038/nrd2465 Tinkanen H, Kujansuu E (1992) Doppler ultrasound studies in pelvic inflammatory disease. Gynecol Obstet Invest 34(4):240–242 https://doi.org/10.1159/000292770 Varughese EE, Brar PS, Dhindsa SS (2013) Uterine blood flow during various stages of pregnancy in dairy buffaloes using transrectal Doppler ultrasonography. Anim Reprod Sci 140(1–2):34–39 https://doi.org/10.1016/j.anireprosci.2013.05.011 Wagener K, Gabler C, Drillich M (2017) A review of the ongoing discussion about definition, diagnosis and pathomechanism of subclinical endometritis in dairy cows. Theriogenology 94:21–30 https://doi.org/10.1016/j.theriogenology.2017.02.005 Supplementary Files Highlights.docx Graphicalabstract.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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09:16:32","extension":"html","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134303,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/3aa2a3275793eba810d4a0fe.html"},{"id":97863657,"identity":"191759a5-32b1-45e7-8415-30da05a7e97f","added_by":"auto","created_at":"2025-12-10 09:16:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148782,"visible":true,"origin":"","legend":"\u003cp\u003eEndometrial cytology stained with Wright’s Giemsa Stain (40X)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/da2dd1268819fa61d60a9094.png"},{"id":97899390,"identity":"6cdcbafb-de56-4015-a03e-c894ffc0cc5b","added_by":"auto","created_at":"2025-12-10 15:43:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114754,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative spectral Doppler sonograms showing the variation of blood flow measured from the middle uterine artery on (a) Follicular phase (b) Luteal Phase in SCE affected Tharparkar cows\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/7e89fdee6bc9eb665b6522a2.png"},{"id":97863648,"identity":"4e3db196-bad9-4136-ba52-a074704b5c03","added_by":"auto","created_at":"2025-12-10 09:16:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127337,"visible":true,"origin":"","legend":"\u003cp\u003eSpectral Doppler attributes (a) Resistance index (RI), (b) Pulsatility index (PI), (c) Timed-averaged maximum velocity (TMAX), (d) Blood flow volume (BFV) and (e) diameter of middle uterine artery (MUA) of healthy and subclinical endometritis affected Tharparkar cows. Value with asterisks (*) mark the significant differences.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/8aee3c64a75fb13e12835423.png"},{"id":97863651,"identity":"1483e9da-cea3-4cee-867b-21b0a1bf63ab","added_by":"auto","created_at":"2025-12-10 09:16:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":185622,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of correlation among Doppler indices, Timed-averaged maximum velocity (TMAX), Blood flow volume (BFV), diameter of middle uterine artery (MUA\u003c/p\u003e\n\u003cp\u003eand serum progesterone (P4) concentration in Tharparkar cow with and without subclinical endometritis\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/c8703df399d4580546c093a7.png"},{"id":105565945,"identity":"5af5d9a1-ba0f-4e64-b69c-468cf398c8e3","added_by":"auto","created_at":"2026-03-27 12:54:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1326360,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/bbb388a6-59be-41da-8c2e-3b9a74f57ab3.pdf"},{"id":97863650,"identity":"394f690a-a365-4a9e-b5c3-eb6cdb6440df","added_by":"auto","created_at":"2025-12-10 09:16:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15875,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/6199f8e53952f693cca9940d.docx"},{"id":97900031,"identity":"fdaf38d2-ac39-4d4a-ae6f-cb27a9d8b679","added_by":"auto","created_at":"2025-12-10 15:45:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":654246,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7990665/v1/10e5698f4b62f59cd36196ad.docx"}],"financialInterests":"","formattedTitle":"Transrectal Spectral Doppler reveals uterine hyperemia in Tharparkar cows with subclinical endometritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIndigenous \u003cem\u003eBos indicus\u003c/em\u003e cattle underpin rural livelihoods in tropical and subtropical regions, providing milk, draft power and income. Although these breeds exhibit greater thermotolerance than \u003cem\u003eBos taurus\u003c/em\u003e (Cooke et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), tropical climates predispose them to reproductive infections that compromise fertility (Chenoweth \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Sheldon and Owens \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Postpartum uterine disorders remain a major cause of subfertility, with metritis (8\u0026ndash;40%), clinical endometritis (5\u0026ndash;30%) and subclinical endometritis (SCE; 11\u0026ndash;70%) reported worldwide (Baithalu et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wagener et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). SCE, defined as cytological inflammation of the endometrium without overt clinical signs (Kasimanickam et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), can reduce milk yield, fat and protein content, and increase somatic cell counts, leading to substantial economic losses (Burke et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Green et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ghasemi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Detection of SCE is challenging because affected cows appear clinically normal, and diagnosis relies on endometrial cytology, an invasive and labour-intensive procedure poorly suited to field conditions. Conventional B-mode ultrasonography identifies uterine fluid or wall thickening but has limited accuracy for subclinical cases. Doppler ultrasonography, by contrast, provides real-time assessment of uterine blood flow and may serve as a non-invasive indicator of inflammation (Ginther and Utt \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; El-Sayed et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Inflammatory mediators such as histamine, bradykinin, prostaglandins and nitric oxide (Moncada and Higgs \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) induce vasodilation and tissue hyperaemia, which can be quantified through spectral Doppler indices including the resistance index (RI) and pulsatility index (PI). Reduced RI and PI, reflecting lower vascular impedance, are reported in uterine infections in women (Jaiyeoba and Soper \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), \u003cem\u003eBos taurus\u003c/em\u003e cattle (Sharma et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), buffaloes (El-Sayed et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), mares (Bollwein et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and ewes (Anderson et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). However, the uterine hemodynamic responses to SCE remain largely unexplored in \u003cem\u003eBos indicus\u003c/em\u003e breeds, which are central to tropical dairy systems. Addressing this gap, the present study employed serial transrectal spectral Doppler ultrasonography across the estrous cycle in Tharparkar cows with and without SCE to characterize alterations in uterine vascular perfusion. We hypothesized that SCE would produce a distinct Doppler signature of uterine hyperaemia, characterized by increased blood flow velocity and vessel diameter with reduced impedance indices.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eLocation and climatic parameters\u003c/h2\u003e\u003cp\u003eThe experiment was conducted at the Cattle and Buffalo Farm, ICAR–IVRI, Izatnagar, India (28° N, 79° E; 564 m above mean sea level). The climate of the region is humid and tropical, with a mean summer temperature of 30.70°C (range 15.10–40.90°C) and mean relative humidity of 53.20%.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals and feed management\u003c/h3\u003e\n\u003cp\u003eTwenty lactating parous Tharparkar cows (parity 2–3; age 4.80 ± 0.81\u0026nbsp;year; body weight 388.1 ± 9.5 kg; mean milk yield 3.01 ± 0.23 kg day⁻¹) were enrolled. Provide days in milk (DIM) or postpartum interval for each animal (70 ± 3.34). Cows received 1.5–2.5 kg concentrate feed (20% DCP, 70% TDN), ad libitum wheat straw, green fodder twice daily, and free access to water. Animals were managed under semi-intensive conditions with access to open paddocks.\u003c/p\u003e\n\u003ch3\u003eExperimental design, inclusion and exclusion criteria of animal\u003c/h3\u003e\n\u003cp\u003eBefore the start of the experiment, all animals underwent clinical screening to exclude systemic or inflammatory conditions. Reproductive tract evaluation included visual vulvar inspection and vaginal palpation; clear mucus indicated absence of clinical endometritis (Saleem et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Animals found positive for uterine infections were excluded from the study. Only clinically normal cows with regular estrous cycles, no history of uterine infection, dystocia, or retained placenta, and no abnormal discharge were included. Additionally, transrectal palpation and B-mode ultrasonography confirmed uterine integrity, with no intrauterine fluid or abnormalities. No hormonal synchronization of estrous cycles was performed. Each cow was enrolled on a random day of its cycle, and daily ultrasonographic monitoring was initiated to track the growth of the dominant follicle. Once natural behavioural estrus was detected, confirmed by the presence of a preovulatory follicle and clear mucous discharge, endometrial cytology sampling for SCE diagnosis was performed using the cytobrush technique (Kasimanickam et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Subsequently, ultrasonographic monitoring continued throughout one complete estrous cycle at regular intervals to assess uterine haemodynamics under natural conditions without exogenous hormonal intervention. Smears were stained with modified Wright’s Giemsa, and 100 cells per slide were counted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on established cytological criteria, SCE was defined as the presence of ≥ 5% PMNs at ≥ 47 days postpartum (Kasimanickam et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Ten cows with SCE formed the treatment group, and ten healthy cows served as controls. Post-cytobrush welfare monitoring was performed for 24 h using a standardized 5-point pain score and observation for abnormal discharge, as per ARRIVE 2.0 guidelines (Sert et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). No adverse reactions were noted. To minimize observer bias, the ultrasonography operator remained blinded to the group allocation (control or SCE-affected).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eSpectral Doppler acquisition\u003c/h3\u003e\n\u003cp\u003eThe MUA imaging was performed using a transrectal linear transducer (Exago ECM, France)] with a frequency of 7.5 MHz using color and spectral Doppler. The abdominal aorta was traced caudally to the internal/external iliac bifurcation and the MUA localized within the mesometrium using color Doppler. Spectral Doppler was applied with the sample gate centered within the lumen and gate length set approximately to vessel diameter. The insonation angle was kept ≤ 60° (mean used: 50°), and color gain was standardized (~ 20 dB). Pulse repetition frequency (PRF) was adjusted (typically 3000–5000 Hz; in this study 4000 Hz) to avoid aliasing; when aliasing occurred PRF or scale was adjusted and angle reconfirmed. For each MUA, recordings were accepted only when at least three consecutive, high-quality, consistent cardiac cycles were visualized; three such waveforms were frozen and used to calculate indices, and the mean of these three measurements per side was used for analysis (Bollwein et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hassan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Each session required ~ 30–45 min per cow and was performed every 3 days from estrus (D0) through the next estrus (D21).\u003c/p\u003e\n\u003ch3\u003eOperator training and quality control\u003c/h3\u003e\n\u003cp\u003eA single, experienced operator performed all Doppler scans using a standardized protocol and machine presets to minimize observer-related variation. Weekly reliability tests demonstrated excellent reproducibility (intra-observer ICC = 0.92, inter-observer ICC = 0.88). All waveforms were quality-checked, and those with artifacts (e.g., motion, aliasing) were excluded and reacquired.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of spectral Doppler attributes of the middle uterine artery\u003c/h2\u003e\u003cp\u003eThe RI and PI were calculated automatically by the ultrasound system using standard formulas:\u003c/p\u003e\u003cp\u003eRI = (PSV − EDV) / PSV (Gosling \u0026amp; King, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e)\u003c/p\u003e\u003cp\u003ePI = (PSV − EDV) / mean velocity (Gosling \u0026amp; King, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe author Gosling \u0026amp; King, (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) commonly used convention; see Ginther \u0026amp; Utt, (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) for Doppler principles. Timed-averaged maximum velocity (TMAX; units cm s⁻¹) was taken as the system’s time-averaged peak velocity across the cardiac cycle. Blood flow volume (BFV, mL min⁻¹) was estimated using:\u003c/p\u003e\u003cp\u003eBFV = TMAX × π × (D/2)² × 60 (Varughese et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e),\u003c/p\u003e\u003cp\u003ewhere D is the internal vessel diameter (cm) measured intima-to-intima, and the factor 60 converts seconds to minutes. Reported BFV thus represents an approximation assuming laminar flow and circular cross-section; limitations of this calculation are acknowledged.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMeasurement of diameter of MUA\u003c/h3\u003e\n\u003cp\u003eInternal vessel diameter (intima-to-intima) was measured on three cardiac-matched frozen B-mode frames at the Doppler sampling site; the mean of three measurements per side was used (Hassan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBlood sampling and Progesterone (P4) estimation\u003c/h3\u003e\n\u003cp\u003eAfter each Doppler exam, 4 mL blood was collected by jugular venipuncture into serum clot-activator tubes, allowed to clot for 30 min at room temperature, and centrifuged at 3000 rpm for 10 min. Serum was aliquoted and stored at − 20°C. Progesterone (P4) was measured by DRG Progesterone ELISA (EIA-1561); assay sensitivity = 0.045 ng mL⁻¹, intra-assay CV = 6.4%, inter-assay CV = 6.6%. Samples were analyzed in duplicate.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using GraphPad Prism 10.6.0 (GraphPad Software, LLC, San Diego, CA, USA) following standard methods. Normality and distribution of data were determined using Shapiro-wilk and Kolmogorov-Smirnov normality test. The mean values of serum P4 concentration, Doppler attributes of MUA viz., Doppler indices (RI, PI), TMAX, vessel diameter and BFV were tested for significance using independent t-test. The RI, PI, TMAX, diameter of MUA and BFV were subjected for analysis of variance of repeated measurement considering the variance between animals as well as between days of estrous cycle, using GLM procedure to determine the principal effects and their interaction. The statistical model was yij = µ + αi + τj + (ατ)ij +\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\text{ϵ}\\)\u003c/span\u003e\u003c/span\u003eij, where αi is the day effect and τj is the animal effect and (ατ)ij is their interaction effect. The significance of the differences was tested using post hoc Sidak test. The association among Doppler indices, TMAX, BFV, Cl size, and serum P4 concentration were calculated using Pearson’s correlation coefficient. All the data presented are as mean ± standard error of mean. The difference in mean values for all data analyzed with \u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e was considered significant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003ch2\u003ePMNs count in endometrial cytology screening\u003c/h2\u003e\u003cp\u003eTen cows classified as SCE-positive had a mean endometrial PMN% of 6.80 ± 0.24%, whereas the control group had mean PMN% = 3.30 ± 0.21% (group difference \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001).\u003c/p\u003e\u003ch2\u003eSpectral Doppler attributes of MUA\u003c/h2\u003e\u003cp\u003eMean RI (average of left and right MUA) was significantly lower (group effect \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05) in cows with SCE compared with controls at ovulation (day 0), early luteal (days 3, 6), mid-luteal (days 9, 12), late luteal (days 15, 18) and at subsequent ovulation (day 21), consistent with increased uterine perfusion in SCE cows (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Further, a significant (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05) effect of both animal status and days of the estrous cycle on RI values could be noted, although their interaction was non-significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). Additionally, a strong positive correlation between the size of the CL and mean RI values (r = 0.64; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001), as well as between RI values and serum P4 concentrations (r = 0.77; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;4a). Additionally, the RI exhibited a coefficient of variation of 25.54% among the cows. Pulsatility index reflects the relative difference between PSV and EDV across the cardiac cycle (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). PI was significantly lower in cows with SCE at ovulation (day 0), mid-luteal days 9 and 12, and at the subsequent ovulation (day 21) (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05). Additionally, there was a strong effect of both animal status and days of the estrous cycle on PI values (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05), while their interaction remained non-significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). A strong positive correlation between RI and PI values was observed (r = 0.77; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;4b) and a strong negative correlation between PI and serum P4 (r = 0.64; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) was observed (Fig.\u0026nbsp;4c). In addition to that, PI of MUA exhibited a coefficient of variation of 50.37% among the cows.\u003c/p\u003e\u003cp\u003eMean TMAX (average of right and left MUA) was higher in cows with SCE (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05), indicating increased flow velocity in the inflamed uterus. Furthermore, there was a significant effect of both animal status and days of the estrous cycle on TMAX values (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05), while a non-significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) interaction was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Further, a strong negative correlation (r = -0.67; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) between TMAX and RI was observed (Fig.\u0026nbsp;4d). The quantitative mean value of BFV in mL min\u003csup\u003e− 1\u003c/sup\u003e revealed that uterine BFV was markedly higher (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05) in SCE-affected cows compared to healthy animals on days 0, 6, 9, 12, 15, and 18 of the estrous cycle. Additionally, there was a significant effect of animal status and days of the estrous cycle on BFV (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05), while their interaction remained non-significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In addition to that BFV was significantly positively correlated with TMAX (r = 0.82; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;4e) and MUA (r = 0.78; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;4f), while it was significantly negative in correlation with RI (r = -0.57; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;4g) and serum P4 (r = -0.59; \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) (Fig.\u0026nbsp;4h), which is well explanation of a flaccid quiescent uterus during diestrus associated with high RI and P4 but low BFV and TMAX. The diameter of the MUA is a key determinant of uterine blood perfusion, and changes in its diameter can be used to predict UBF across various reproductive events. In this study, the diameter of both MUA (in cm) was monitored to assess the impact of SCE. Results indicated that the average diameter of the MUA in the cows with SCE was consistently larger throughout the estrous cycle compared to healthy cows, with significant (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05) differences observed on days 6, 9, 12, 15, and 18 of the cycle. Additionally, the diameter of the MUA was significantly influenced by both the animal and the day of the estrous cycle (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05), though their interaction was not statistically significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). An inverse relationship was also observed between the MUA diameter and the RI value, with a correlation coefficient of r = − 0.24 (\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0001) indicative of higher resistance in those blood vessels with lesser diameter and thus, lower blood perfusion to the associated organ (Fig.\u0026nbsp;4i).\u003c/p\u003e\u003ch2\u003eEffect of production traits on blood flow indices\u003c/h2\u003e\u003cp\u003eThis study enrolled all cows were of similar physiological and management status, maintained under uniform feeding, housing, and milking schedules. Thus, effect of production traits (milk yield, parity, or days in milk were non-significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) uterine haemodynamics indices (RI and PI) in either group, indicating physiological uniformity among experimental animals. All cows were maintained under similar nutritional and management conditions, minimizing the likelihood of production-related variation. Hence, the observed alterations in UBF can be primarily attributed to subclinical endometritic status rather than differences in production performance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides clear evidence that subclinical endometritis (SCE) in Tharparkar cows induces pronounced alterations in uterine hemodynamics, which can be reliably quantified by transrectal spectral Doppler ultrasonography across the estrous cycle. The consistent reduction in resistance index (RI) and pulsatility index (PI) observed in SCE-affected cows reflects a sustained decrease in uterine vascular impedance and, consequently, a state of localized hyperaemia. Such hemodynamic shifts are characteristic of inflammatory responses and signify an adaptive attempt to enhance blood flow and immune surveillance within the endometrium. The pattern observed here parallels those reported in buffaloes (El-Sayed et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), Bos taurus cattle with uterine infections (Debertolis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and women with pelvic inflammatory disease (Jaiyeoba and Soper \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), suggesting that uterine inflammation across species elicits a conserved vascular response involving increased perfusion and reduced vascular resistance. This conserved hemodynamic signature supports the notion that inflammation, even when subclinical, reorganizes uterine vascular function to accommodate increased metabolic demands and immune cell infiltration. The lower RI and PI values during the follicular phase in both healthy and SCE-affected cows further reflect the normal cyclic modulation of uterine blood flow (UBF), driven by the endocrine milieu. Estrogen is known to upregulate endothelial nitric oxide synthase (eNOS) and modulate calcium flux in smooth muscle cells, promoting vasodilation and tissue perfusion (Acosta et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Stice et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sumiyoshi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). During estrus, this estrogen-mediated vascular relaxation supports follicular maturation, uterine receptivity, and endometrial oedema (Ford and Christenson \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Bollwein et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The heightened vascular conductance observed in SCE cows, however, extends beyond physiological vasodilation and likely represents an inflammation-driven amplification of the same mechanisms. The strong correlations between Doppler indices and endocrine variables in this study reinforce the intricate coupling between uterine perfusion and hormonal regulation. The positive relationship between RI, PI, corpus luteum (CL) size, and serum progesterone (P4) indicates that progesterone-mediated vasoconstriction dominates during the luteal phase, while lower impedance indices during follicular phases coincide with estrogenic vasorelaxation. These relationships are consistent with previous bovine Doppler studies (Hassan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), confirming that uterine blood flow is dynamically tuned to hormonal fluctuations. In SCE cows, however, these cyclic vascular oscillations appear blunted, suggesting that persistent low-grade inflammation overrides the normal endocrine control of vascular tone.\u003c/p\u003e\u003cp\u003eThe significant elevations in time-averaged maximum velocity (TMAX) and blood-flow volume (BFV) in SCE-positive cows further confirm the presence of uterine hyperaemia. Similar findings have been reported in buffaloes (El-Sayed et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), Bos taurus cows (Sharma et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), mares (Bollwein et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and ewes with experimentally induced uterine inflammation (Still and Greiss \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Mechanistically, this hyperaemia can be attributed to the action of inflammatory mediators such as prostaglandins, histamine, bradykinin, and especially nitric oxide (NO), which exerts potent vasodilatory effects on the uterine microcirculation (Moncada and Higgs \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Elevated NO levels in uterine secretions of SCE- and endometritis-affected cattle (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) provide direct biochemical evidence for this pathway. While NO was not measured in the present study, the hemodynamic pattern strongly implies enhanced endothelial activation and NO bioavailability in SCE cows. The relationships among the Doppler parameters themselves are physiologically coherent. The negative correlations between BFV and RI, and between BFV and P4, coupled with positive correlations between BFV, TMAX, and MUA diameter, indicate that increased flow is accompanied by both structural vessel dilation and decreased impedance. This reflects the fundamental hemodynamic principle that vessel expansion lowers resistance and promotes higher volumetric perfusion (Bollwein et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Varughese et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hassan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The enlarged middle uterine artery (MUA) diameter in SCE cows observed here supports this interpretation and indicates vascular remodelling consistent with chronic low-grade inflammation. Similar arterial dilation has been documented in cattle (Sharma et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and buffaloes (El-Sayed et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) with endometritis. However, some induced infection models report inconsistent associations between vessel diameter and flow (Debertolis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), suggesting that the chronicity and intensity of inflammation influence vascular adaptation. The inverse relationship between MUA diameter and RI observed in this study further strengthens the conclusion that subclinical inflammation drives functional and structural vascular changes that facilitate hyper perfusion of the uterus.\u003c/p\u003e\u003cp\u003eFrom a physiological perspective, these findings reveal that SCE modifies uterine vascular dynamics not merely as a passive consequence of inflammation but as an integrated response aimed at maintaining tissue oxygenation, immune cell access, and metabolic exchange within the endometrium. While adaptive in the short term, chronic hyperaemia could also perpetuate tissue stress and disrupt endometrial receptivity, contributing to the subfertility associated with SCE. The recognition of this vascular phenotype opens possibilities for using Doppler indices as objective indicators of uterine health in field conditions, offering a more practical alternative to cytology, particularly in indigenous breeds where handling constraints often limit invasive testing. The modest sample size (n\u0026thinsp;=\u0026thinsp;10 per group) limits the detection of subtle inter-animal variations and interaction effects. Moreover, the absence of bacteriological culture, systemic inflammatory markers (such as serum amyloid A or haptoglobin), and direct NO quantification restricts the ability to link vascular changes to specific pathogen loads or molecular pathways. Nevertheless, all animals were of comparable parity, milk yield, and management status, minimizing confounding factors. These data, therefore, provide reliable physiological insight specific to lactating Tharparkar cows and form a foundation for broader, multi-breed studies integrating biochemical and microbial profiling.\u003c/p\u003e\u003cp\u003eIn conclusion, SCE in Tharparkar cows elicits a consistent Doppler-defined hemodynamic pattern of uterine hyperaemia elevated blood-flow velocity and volume, enlarged arterial diameter, and reduced impedance indices. These vascular adaptations represent measurable manifestations of endometrial inflammation and offer potential diagnostic markers for early, non-invasive detection of uterine disease. Establishing such Doppler reference values for Bos indicus breeds not only enhances our understanding of uterine physiology in tropical livestock but also lays the groundwork for field-applicable diagnostic frameworks. Future research should combine Doppler assessments with quantification of inflammatory mediators and metabolic markers to refine diagnostic accuracy and support timely therapeutic interventions, ultimately improving reproductive efficiency and animal welfare in tropical dairy systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their gratitude to the Director, ICAR- Indian Veterinary Research Institute, Izatnagar for providing the necessary facilities and institutional support for carrying out this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Institutional facilities of ICAR- Indian Veterinary Research Institute (ICAR-IVRI), Izatnagar were utilized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Institute Animal Ethics Committee of ICAR-IVRI, Izatnagar, U.P. India (26-3/2020-21/JD(R)/IAEC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study, including Excel spreadsheets, ultrasound images, and related data files, are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest in publishing the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent of publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed and consented to the submission of this manuscript for publication. They confirm that the work is original and has not been published elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e Uttam Kumar Sahu, Brijesh Kumar \u003cstrong\u003eMethodology:\u003c/strong\u003e Uttam Kumar Sahu, Brijesh Kumar, Meraj Haider Khan \u003cstrong\u003eInvestigation:\u003c/strong\u003e Uttam Kumar Sahu, Mayank Singh, Chinmay Warghat \u003cstrong\u003eFormal analysis: \u003c/strong\u003eAthidi Lokavya Reddy, Laxmi Sahu, Nitish Singh Kharayat\u003cstrong\u003e Data curation:\u003c/strong\u003e Uttam Kumar Sahu, Amit Kumar, Brijesh Kumar \u003cstrong\u003eResources:\u003c/strong\u003e Meraj Haider Khan, Sanjay Kumar Singh \u003cstrong\u003eWriting – original draft preparation:\u003c/strong\u003e Uttam Kumar Sahu, Amit Kumar, Brijesh Kumar \u003cstrong\u003eWriting – review and editing:\u003c/strong\u003e All authors \u003cstrong\u003eSupervision:\u003c/strong\u003e Brijesh Kumar, Sanjay Kumar Singh \u003cstrong\u003eProject administration:\u003c/strong\u003e Meraj Haider Khan, Sanjay Kumar Singh \u003cstrong\u003eFunding acquisition:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCIDs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUttam Kumar Sahu: 0009-0003-7680-4996\u003c/p\u003e\n\u003cp\u003eBrijesh Kumar: 0000-0001-6153-0791 \u003c/p\u003e\n\u003cp\u003eMeraj Haider Khan: 0000-0002-9934-4772\u003c/p\u003e\n\u003cp\u003eMayank Singh Baghel: 0000-0002-9951-1788\u003c/p\u003e\n\u003cp\u003eChinmay Warghat: 0009-0002-7924-2055\u003c/p\u003e\n\u003cp\u003eAthidi Lokavya Reddy: 0000-0003-2295-9303 \u003c/p\u003e\n\u003cp\u003eLaxmi Sahu: 0009-0006-3099-4327\u003c/p\u003e\n\u003cp\u003eNitish Singh Kharayat: 0000-0001-7341-1925\u003c/p\u003e\n\u003cp\u003eAmit Kumar: 0000-0001-7272-898x\u003c/p\u003e\n\u003cp\u003eSanjay Kumar Singh: Not Available\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcosta TJ, Hayashi KG, Ohtani M, Miyamoto A (2003) Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows. 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Biol Reprod 21(3):617\u0026ndash;624 https://doi.org/10.1095/biolreprod21.3.617\u003c/li\u003e\n\u003cli\u003eGhasemi F, Gonzalez-Cano P, Griebel PJ, Palmer C (2012) Proinflammatory cytokine gene expression in endometrial cytobrush samples harvested from cows with and without subclinical endometritis. Theriogenology 78(7):1538\u0026ndash;1547 https://doi.org/10.1016/j.theriogenology.2012.06.022\u003c/li\u003e\n\u003cli\u003eGinther OJ, Utt MD (2004) Doppler ultrasound in equine reproduction: Principles, techniques, and potential. 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J Clin Ultrasound 26(5):247\u0026ndash;249 https://doi.org/10.1002/(SICI)1097-0096(199806)26:5\u003c/li\u003e\n\u003cli\u003eThurmond RL, Gelfand EW, Dunford PJ (2008) The role of histamine H1 and H4 receptors in allergic inflammation: The search for new antihistamines. Nat Rev Drug Discov 7(1):41\u0026ndash;53\u003cbr\u003e https://doi.org/10.1038/nrd2465\u003c/li\u003e\n\u003cli\u003eTinkanen H, Kujansuu E (1992) Doppler ultrasound studies in pelvic inflammatory disease.\u003cbr\u003e Gynecol Obstet Invest 34(4):240\u0026ndash;242 https://doi.org/10.1159/000292770\u003c/li\u003e\n\u003cli\u003eVarughese EE, Brar PS, Dhindsa SS (2013) Uterine blood flow during various stages of pregnancy in dairy buffaloes using transrectal Doppler ultrasonography. Anim Reprod Sci 140(1\u0026ndash;2):34\u0026ndash;39 https://doi.org/10.1016/j.anireprosci.2013.05.011\u003c/li\u003e\n\u003cli\u003eWagener K, Gabler C, Drillich M (2017) A review of the ongoing discussion about definition, diagnosis and pathomechanism of subclinical endometritis in dairy cows.\u003cbr\u003e Theriogenology 94:21\u0026ndash;30 https://doi.org/10.1016/j.theriogenology.2017.02.005\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":false,"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":"Doppler indices, Nitric oxide, Resistance index, Subclinical endometritis, Tharparkar cattle","lastPublishedDoi":"10.21203/rs.3.rs-7990665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7990665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Subclinical endometritis (SCE) is an asymptomatic uterine inflammation that impairs fertility and is hard to detect in field conditions. We serially applied blinded transrectal spectral Doppler to characterize middle uterine artery hemodynamics across the estrous cycle in lactating Tharparkar cows with (n\u0026thinsp;=\u0026thinsp;10) and without (n\u0026thinsp;=\u0026thinsp;10) cytology-confirmed SCE. Doppler measures (resistance index [RI], pulsatility index [PI], timed-averaged maximum velocity [TMAX], blood-flow volume [BFV], artery diameter) and serum progesterone (P4) were recorded every 3 days from estrus through the next estrus. Repeated-measures analysis showed that SCE cows had consistently lower RI and PI and higher TMAX, BFV and artery diameter across the cycle (group effect P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RI correlated positively with corpus luteum size (r\u0026thinsp;=\u0026thinsp;0.64) and P4 (r\u0026thinsp;=\u0026thinsp;0.77), while BFV correlated positively with TMAX (r\u0026thinsp;=\u0026thinsp;0.82) and diameter (r\u0026thinsp;=\u0026thinsp;0.78) and negatively with RI (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.57) and P4 (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.59) (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Limitations include modest sample size (n\u0026thinsp;=\u0026thinsp;10/group) and absence of concurrent bacteriology or nitric-oxide measurements. With validation in larger cohorts, Doppler metrics could provide a non-invasive adjunct to cytology for detecting uterine inflammation and guiding reproductive management in tropical dairy systems.","manuscriptTitle":"Transrectal Spectral Doppler reveals uterine hyperemia in Tharparkar cows with subclinical endometritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 09:16:23","doi":"10.21203/rs.3.rs-7990665/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":"2bc811d1-2631-482a-9655-49b70d01ac04","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T18:02:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 09:16:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7990665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7990665","identity":"rs-7990665","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}