Indigenous cattle breeds play a vital role in sustaining rural livelihoods in developing regions by contributing to milk production, draft power, employment, and household income security. These breeds display superior thermotolerance and adaptation to tropical climates (Cooke et al., 2020; Asmarasari et al., 2023), however, they generally produce lower milk yields than Bos taurus breeds (Al Kalaldeh et al., 2023). The sustained genetic selection and extensive use of artificial insemination have substantially enhanced productivity in Bos taurus (Miglior et al., 2017). However, these gains in Bos taurus have often come with diminished fertility due to physiological trade-offs (Lucy, 2001; Walsh et al., 2011). Understanding follicular dynamics, corpus luteum (CL) function, and endocrine changes across the estrous cycle is fundamental to improving fertility. Such understanding provides the basis for developing synchronization and fixed-time ovulation protocols, that rely on hormonal interventions. Furthermore, pre-synchronization and resynchronization strategies have demonstrated satisfactory fertility outcomes in Bos taurus (Stevenson et al., 1999; Wiltbank and Pursley, 2014). However, their effectiveness in indigenous cattle remains inconsistent, largely due to differences in follicular wave patterns, CL development kinetics, and endocrine sensitivity (Pinheiro et al., 1998; Sartori et al., 2016; Jiamjariyatam et al., 2024; Widyastuti et al., 2025). Therefore, systematic characterization of estrous cycle physiology in indigenous breeds is essential for the development of breed-specific reproductive management protocols.

Doppler ultrasonography enables non-invasive assessment of reproductive hemodynamics and functional vascular changes, complementing anatomical imaging (Herzog and Bollwein, 2007). Earlier invasive methods showed rhythmic uterine and ovarian blood flow changes across the cycle (Ford and Christenson, 1979). In addition to that, Doppler assessment of reproductive hemodynamics, including vascular adaptations during pregnancy (Honnens et al., 2008), placental separation (Mo and Rogers, 2008), puerperium (Heppelmann et al., 2013), incomplete cervical dilatation (Chaudhari et al., 2023) and endometritis (Sharma et al., 2021) has been carried out. Doppler ultrasonography has facilitated assessment of vascular perfusion in the follicle (Acosta et al., 2003), super ovulatory response (Chandra et al., 2023), CL function (Lüttgenau and Bollwein, 2014), ovulatory structures (Aslan et al., 2011) and more recently has been applied to predict pregnancy at embryo transfer (Kanazawa et al., 2016). Despite these advances, breed-specific uterine artery hemodynamic patterns across the estrous cycle in indigenous cattle remain poorly defined.

To date, no study has systematically established Doppler reference values for middle uterine artery perfusion throughout the estrous cycle in Tharparkar (Bos indicus) cows. Generating such baseline data is critical for developing reproductive management protocols that align with the physiological characteristics of indigenous breeds. This is particularly relevant for the Tharparkar cow, a resilient dual-purpose breed from the South Asian region, globally recognized for its remarkable heat tolerance, drought resilience, and consistent milk production under challenging climatic conditions (Patel et al., 2021). Their genetic traits make them a vital resource for improving dairy productivity and enhancing climate adaptability in tropical and arid regions worldwide (Patel et al., 2022), although they differ in economic importance and physiological characteristics from Bos taurus breeds (Patel et al., 2022). Therefore, this study aimed to characterize cyclic changes in uterine blood perfusion in Tharparkar cows and examine their relationship with luteal function using spectral Doppler ultrasonography.

This study provides the first detailed characterization of uterine artery hemodynamics across the estrous cycle in Tharparkar cows using spectral Doppler ultrasonography. Distinct, cyclical changes were observed in resistance (RI, PI), velocity (TMAX), volume (BFV), and vessel diameter, demonstrating that uterine perfusion is dynamically regulated throughout the cycle. These findings align with previous studies in Bos taurus and Sahiwal cows (Hassan et al., 2017; Abdelnaby et al., 2018), but importantly extend existing knowledge by establishing breed-specific perfusion patterns in an indigenous Bos indicus breed that differs physiologically from intensively selected dairy cattle. In our study, the RI and PI were lowest at estrus, increased progressively during the early luteal phase, peaked mid-luteal, and declined before the subsequent estrus. This pattern corresponds with earlier invasive and Doppler-based studies, which demonstrated maximal uterine blood flow around estrus in cows and ewes (Anderson et al., 1977; Ford and Christenson, 1979; Abdelnaby et al., 2020). The resistance indices peaked at D12 of the estrous cycle in the present study, suggesting that Tharparkar cows may exhibit a relatively pronounced luteal-phase uterine vascular resistance compared with Sahiwal cow, in which peak resistance is often observed slightly earlier on D10 (Hassan et al., 2017). This pattern likely reflects sustained progesterone dominance during mid-luteal development, leading to vasoconstriction and reduced uterine perfusion. The positive correlations of RI and PI with P4 and CL size further support the role of luteal activity in regulating uterine blood flow, consistent with prior reports (Lüttgenau and Bollwein, 2014; Hassan et al., 2017; Sharma et al., 2021).

In contrast, time-averaged maximum velocity (TMAX), blood flow volume (BFV), and middle uterine artery diameter exhibited reciprocal cyclic patterns, with maximal uterine perfusion at estrus, a pronounced decline during the luteal phase, and recovery toward the subsequent estrus. This estrus-associated increase in uterine perfusion is consistent with estradiol-mediated vasodilation and hyperemia of the reproductive tract during follicular dominance, a phenomenon well documented across the bovine estrous cycle (Ando et al., 2007; Ford and Christenson, 1979). Elevated uterine blood flow during estrus coincides with prominent behavioral and anatomical signs of estrus, including opening of the external uterine orifice and changes in cervical mucus viscosity that facilitate sperm transport and fertilization (Chen et al., 2004; Sumiyoshi et al., 2014). At the cellular level, estradiol is known to induce rapid vasodilatory responses in uterine arteries through non-genomic mechanisms involving G-protein–coupled estrogen receptor (GPER) signaling, activation of calcium- and ERK1/2-dependent pathways, and enhanced endothelial nitric oxide synthase (eNOS) activity, collectively promoting smooth muscle relaxation and increased uterine perfusion (Stice et al., 1987; Acosta et al., 2003; Li et al., 2022; Tropea et al., 2022). Consistent with these mechanisms, Doppler studies have repeatedly demonstrated that uterine blood flow velocity indices peak around estrus when circulating estradiol concentrations are high and decline during progesterone-dominated luteal phases (Ando et al., 2007; Abdelnaby et al., 2023, 2024). Importantly, deviations from these normal cyclic perfusion patterns have been associated with compromised luteal function, embryonic loss, and reduced fertility, underscoring that uterine hemodynamic changes are not merely physiological phenomena but are directly linked to reproductive efficiency and management outcomes in cattle (Abdelnaby et al., 2024; Reddy et al., 2025; Sahu et al., 2025). The strong correlations of BFV with TMAX and vessel diameter, and its inverse association with RI and P4, indicate that uterine perfusion is jointly regulated by functional vascular tone and blood flow velocity rather than large cyclic changes in vessel caliber alone. The relatively low variability observed in uterine artery diameter compared with velocity and resistance indices suggests that dynamic modulation of vascular resistance is the primary driver of cyclic perfusion changes. Similar observations have been reported in both veterinary and human reproductive hemodynamic studies, where Doppler velocity indices are more sensitive indicators of functional blood flow regulation than arterial diameter measurements (Bernstein, 2002; Browne et al., 2015).

The cyclic uterine perfusion patterns identified in this study have strong applied relevance for reproductive management in indigenous Bos indicus cattle, particularly under tropical and low-input production systems. Inefficiency of estrus detection remains a major constraint in Bos indicus breeds due to attenuated behavioral signs, shortened estrus duration, heat stress, and nutritional variability, which together contribute to mistimed artificial insemination and reduced conception rates (Pinheiro et al., 1998; Lucy, 2001; Walsh et al., 2011; Sartori et al., 2016; Cooke et al., 2020). Under such conditions, reliance on behavioral estrus expression or endocrine measurements alone often fails to provide sufficient resolution for precise reproductive decision-making. In this context, Doppler ultrasonography offers a functional, real-time assessment of uterine and luteal perfusion that can complement conventional reproductive examinations, particularly for estrus confirmation in cows with weak or ambiguous estrous signs. Doppler ultrasonography has therefore been increasingly investigated as an applied tool that provides functional information on uterine and luteal blood flow, directly reflecting reproductive status. Multiple studies have demonstrated that cyclic changes in uterine and ovarian perfusion correspond closely with estrous stage, luteal competence, and early pregnancy establishment (Lüttgenau and Bollwein, 2014; Hassan et al., 2017; Chandra et al., 2023). In applied herd settings, deviations from the expected luteal-phase resistance and perfusion patterns may assist in early identification of luteal insufficiency or abnormal luteal regression, enabling timely resynchronization or therapeutic intervention. Moreover, Doppler-based assessment of uterine and luteal blood flow has been shown to improve early identification of non-pregnant cows, support earlier resynchronization, and refine the timing of artificial insemination and embryo transfer programs (Kanazawa et al., 2016; Pugliesi et al., 2018; Fontes and Oosthuizen, 2022; Gawai et al., 2024).

Functionally, elevated uterine perfusion during estrus may support follicular growth, estrus expression, and gamete transport, whereas reduced perfusion during the luteal phase may facilitate endometrial receptivity and luteal maintenance (Ruiz et al., 2022; Sangkate et al., 2025). Accordingly, the reference values generated in the present study may also support donor selection and prediction of super-ovulatory response, as adequate uterine and luteal vascularization is closely linked with follicular recruitment, corpus luteum functionality, and embryo yield. Importantly, most Doppler reference values and reproductive management strategies have been developed in Bos taurus cattle, despite well-documented physiological and endocrine differences from Bos indicus breeds. Comparative studies consistently report differences in follicular dynamics, luteal function, metabolic status, and reproductive hormone sensitivity that influence vascular regulation and fertility outcomes (Sartori et al., 2016; Cooke et al., 2020; Chandra et al., 2023; Gawai et al., 2024). The breed-specific uterine perfusion reference patterns generated in the present study therefore fill a critical applied knowledge gap and provide a practical framework for adapting Doppler-guided estrus confirmation, luteal assessment, donor selection, and fertility monitoring strategies to Tharparkar and other indigenous Bos indicus cattle.

A limitation of the present study is the relatively small sample size (n = 10), which reflects the intensive, longitudinal nature of the experimental design but may restrict extrapolation of the derived Doppler reference values to larger or more heterogeneous cattle populations. Accordingly, validation of these findings in broader herds and under diverse management conditions is warranted. In addition, the absence of concurrent estradiol measurements precluded direct correlation of uterine perfusion parameters with circulating estrogen concentrations, despite the well-established role of estradiol in regulating uterine vascular tone. While cyclic hemodynamic changes were interpreted in the context of existing endocrine knowledge, future studies integrating estradiol profiling would allow more direct confirmation of hormone–perfusion relationships. Doppler examinations and blood sampling involve animal handling, which may introduce mild, transient stress with the potential to influence endocrine measurements; however, standardized handling procedures and consistent examination timing were employed to minimize this effect. Although repeated-measures ANOVA was appropriate for the balanced longitudinal design of the present study, future investigations with larger sample sizes may benefit from the use of linear mixed-effects models to further account for individual variability and enhance statistical robustness. Further studies incorporating larger populations, steroid hormone profiling, assessment of local vasoactive mediators (e.g., nitric oxide), seasonal influences, and longitudinal fertility outcomes will strengthen mechanistic understanding and enhance the translational applicability of uterine Doppler ultrasonography in herd-level reproductive management.

This study demonstrates that Tharparkar cows display clear and repeatable uterine perfusion patterns across the estrous cycle, corresponding with changes in luteal function and vascular regulation. The Doppler hemodynamic values established here provide useful breed-specific reference standards and support the incorporation of transrectal spectral Doppler ultrasonography into reproductive evaluation and herd fertility management in Bos indicus cattle. Compared with Bos taurus breeds, the observed perfusion dynamics suggest distinctive vascular regulation that should be considered when applying reproductive monitoring and synchronization protocols to indigenous cattle. Future work should focus on characterizing these vascular signatures in cows with reproductive disorders, evaluating seasonal and herd-level variability, and integrating Doppler indices with endocrine profiling to advance Doppler-guided diagnostic and decision-making tools for indigenous breeds.