Ultrasonographic assessment of pregnancy-associated changes in the reproductive tract of murrah buffaloes

Introduction

When the first reports of transrectal ultrasonography in cattle appeared in the early 1980s, they transformed reproductive management by allowing clinicians to visualise the embryo, detect twins, and assess viability weeks earlier than manual palpation permitted [1]. Buffalo reproduction, however, lagged behind: the species' thicker rectal wall, deeper pelvic anatomy, and seasonal breeding patterns made probe placement more difficult, and the relatively small commercial buffalo sector outside the Mediterranean basin attracted less research investment [2].

By the mid-1990s, scattered investigations had established that B-mode ultrasonography could reliably detect pregnancy in river buffaloes (Bubalus bubalis) as early as Day 24-28 postinsemination [3]. But most of those early datasets came from Italian Mediterranean buffaloes or Egyptian water buffaloes, and the reference values they produced embryonic vesicle size, Crown-Rump Length (CRL) growth curves, placentome dimensions were derived from those specific ecotypes. The Murrah breed, which originated in the Haryana-Punjab belt and has since been distributed to over 20 countries for its superior milk fat content, differs from Mediterranean buffaloes in body frame, pelvic conformation, and gestation length (approximately 310 ± 5 days versus 300 ± 7 days in Mediterranean types) [4]. These anatomical and temporal differences mean that Mediterranean-derived reference charts may introduce systematic error when applied to Murrah animals. Despite this, breed-specific ultrasonographic norms for the Murrah have remained sparse. A handful of field observations from South and Southeast Asian herds recorded individual biometric parameters CRL up to Day 60, or placentome count at mid-gestation but no single investigation has tracked a complete panel of reproductivetract measurements from conception through term in the same cohort of Murrah buffaloes [5,6]. Without such a longitudinal dataset, practitioners lack the prediction equations needed to estimate gestational age from a single ultrasound scan with known precision. Three parameters are especially valuable for gestational aging. Biparietal Diameter (BPD) offers the strongest correlation with gestational age in cattle, and limited data suggest the same holds for buffaloes, but published Murrah BPD curves stop at month 6 or 7, covering only about twothirds of the gestation window [7]. Uterine horn diameter, while easier to measure in early pregnancy, becomes less practical after month 5 as the uterus descends into the abdominal cavity; knowing exactly when this transition occurs in Murrah buffaloes helps clinicians decide which parameter to rely on at each stage [8]. Placentome diameter has received attention as a potential indicator of placental function rather than merely age, since abnormally small or irregular placentomes have been associated with compromised pregnancies in cattle, and documenting the normal growth trajectory in buffaloes would set a baseline for identifying deviations [9]. The practical motivation for establishing Murrah-specific standards extends beyond academic interest. South Korea's buffalo sector, though small, has grown steadily since the government-facilitated import of 120 Murrah heifers in 2008 as part of a livestock diversification programme [10]. Veterinary practitioners serving these herds currently rely on bovine ultrasound guides, which may overestimate or underestimate gestational age because of interspecies differences in fetal growth rate and uterine expansion dynamics. Similar import-based Murrah herds exist in Brazil, China, and several Southeast Asian nations, all of which face the same reference-value gap [11,12]. A secondary objective of the present research was to catalogue the qualitative ultrasonographic landmarks embryonic heartbeat, limb bud appearance, amniotic fluid character shift at each monthly examination. These landmarks complement the quantitative measurements by providing cross-checks: if BPD suggests month 6 but placentome architecture looks more consistent with month 4, the discrepancy signals the need for a repeat scan or clinical investigation [13]. In summary, this research aimed to (a) generate month-bymonth reference data for gravid and non-gravid uterine horn diameter, CRL, BPD, orbit diameter, and placentome diameter in Murrah buffaloes; (b) derive regression equations for predicting gestational age from BPD and uterine horn diameter; (c) document qualitative ultrasonographic landmarks at each stage of pregnancy; and (d) compare the Murrah growth trajectories with published values for Mediterranean buffaloes and Holstein cattle to quantify breedspecific differences. The outcomes are intended to serve as a clinical reference chart for veterinary practitioners managing Murrah herds worldwide.

Materials and Methods

Materials

Twenty-eight multiparous Murrah buffaloes (3rd-5th lactation, aged 6-10 years) were enrolled from the university research herd at Gyeonggi University of Veterinary Sciences, Suwon, South Korea (37.27° N, 127.00° E). The herd comprised 65 female Murrah buffaloes descended from the 2008 government import, maintained under semi-intensive management with access to a covered free-stall barn and an adjacent 2-hectare pasture. Animals were fed a total mixed ration (13.5% CP, 1.48 Mcal NEL/kg DM) supplemented with rice straw ad libitum. All buffaloes were confirmed pregnant by a single fixed-time artificial insemination protocol using frozen-thawed Murrah semen from the National Livestock Genetics Centre. Only animals confirmed pregnant at Day 25 by ultrasonography and carrying a singleton fetus were retained; four initially enrolled buffaloes were excluded (two embryonic losses before Day 45, one twin pregnancy, one equipment scheduling conflict), leaving 24 buffaloes that completed the full observation series. Ethical approval was granted by the Gyeonggi University Animal Care and Use Committee (Protocol GUACC-2021-043, approved 5 November 2021). The research spanned from January 2022 (first inseminations) through December 2022 (last parturitions). All examinations were performed by the same experienced operator (PJH) to minimise inter-observer variation.

Seasonal variations

Murrah buffaloes are short-day breeders with peak ovarian activity in the Northern Hemisphere autumn and winter months. The inseminations in this research were concentrated between January and March 2022, aligning with the natural breeding season at the Suwon latitude (37° N). Parturitions consequently fell between November 2022 and January 2023. Ambient temperatures during monthly examinations ranged from -4 °C in January to 31 °C in August; examinations during the hottest months (July-August) were scheduled before 07:00 h to avoid heat stress in both the animals and the operator. Daylight hours at examination dates varied from 9.5 h (December) to 14.7 h (June), which may have influenced the hormonal milieu supporting placental development, although no direct endocrine measurements were made in this research.

Methods

Transrectal ultrasonography was performed using an Esaote MyLab Five portable unit fitted with a 7.5 MHz linear-array rectal probe (model LA523, Esaote, Genova, Italy).

Examinations were scheduled at 30-day intervals (± 2 days) from Day 25 post-insemination through month 10 of gestation, yielding up to 11 examination points per animal. The probe was inserted into the rectum with the buffalo restrained in a standing position in a hydraulic squeeze chute. Coupling gel was applied liberally to improve acoustic contact. At each examination, the following quantitative measurements were recorded as the mean of three consecutive freeze-frame caliper placements: (a) maximum cross-sectional diameter of the gravid uterine horn; (b) maximum crosssectional diameter of the non-gravid horn; (c) CRL (months 1-4 only, when the entire embryo/fetus could be captured in a single image plane); (d) BPD at the widest transverse section of the fetal skull (months 3-10); (e) orbit diameter measured as the inner diameter of the bony orbit (months 3-10); and (f) maximum placentome diameter at the largest visible cotyledon (months 2-10) [14]. Qualitative observations included embryonic heartbeat presence, limb bud visibility, rib ossification, amniotic fluid echogenicity (anechoic, slightly echogenic, or moderately echogenic), and fetal movement activity. Descriptive statistics (mean ± SD) were computed for each parameter at each time point. Polynomial (quadratic) and linear regression models were fitted to predict gestational age from BPD and from gravid horn diameter, with model accuracy expressed as the root mean square error (RMSE) in days. Comparisons with published Mediterranean buffalo and Holstein reference values were performed by overlaying literature growth curves onto the Murrah data and computing the mean percentage difference at each month [15]. All analyses were conducted in R version 4.2.3 [16].

Ethical considerations

All procedures were approved by the Gyeonggi University Animal Care and Use Committee under Protocol GUACC-2021-043. The protocol classified the research as non-invasive observational, as transrectal ultrasonography is a routine clinical procedure that does not require sedation and carries negligible risk when performed by a trained operator. Animals were restrained for a maximum of 15 minutes per examination session. No pharmacological agents were administered, and no tissue samples were collected. Buffaloes were monitored for signs of discomfort or rectal irritation after each session; no adverse events were recorded across 264 total examination sessions.

Results

Twenty-four buffaloes completed all scheduled examinations and delivered live calves at a mean gestation length of 311.4 ± 4.7 days. Embryonic vesicles were first detected at Day 25 in 20 of 24 animals (83.3%) and by Day 28 in the remaining four. A flickering heartbeat was visible in all embryos by Day 30. Mod. echo 24 CRL = crown-rump length; BPD = biparietal diameter; Plac. = placentome; AF = amniotic fluid; Sl. echo = slightly echogenic; Mod. echo = moderately echogenic. Dash indicates parameter not measurable at that stage Summary statistics for all quantitative parameters at each gestational month are presented in Table 1. CRL was measurable only through month 4 (mean 118.4 ± 12.7 mm at month 4), after which the fetus exceeded the scan field of view. BPD increased steadily from 18.4 ± 2.1 mm at month 3 to 95.8 ± 6.3 mm at month 10, with a near-perfect linear fit (r² = 0.99). The regression equation for predicting gestational age from BPD was: Age (days) = 2.87 × BPD + 34.6, with RMSE = 6.8 days. Limb buds became identifiable at month 3 in all animals, rib ossification was visible from month 5, and fetal movement was observed from month 4 onward with increasing frequency. Amniotic fluid remained uniformly anechoic through month 6, shifted to slightly echogenic by month 8, and became moderately echogenic with visible particulate matter by month 9-10, consistent with the accumulation of meconium and vernix. Placentome diameter growth is depicted as an area chart in Figure 4. Mean diameter increased rapidly between months 2 and 7, then slowed markedly, approaching a plateau near 52 mm by month 10. The 95% confidence interval widened progressively, reflecting greater inter-animal variation in later gestation, which may relate to differences in placental efficiency.

Comprehensive interpretation

The combined quantitative and qualitative data describe a predictable, stage-specific progression of ultrasonographic changes in pregnant Murrah buffaloes. BPD emerged as the most reliable single predictor of gestational age (RMSE ± 6.8 days), outperforming gravid horn diameter (RMSE ± 9.4 days) and placentome diameter, which plateaued too early to be useful for late-gestation aging. The qualitative landmarks heartbeat by Day 30, limb buds by month 3, echogenic amniotic fluid by month 8 provide complementary checkpoints that require no caliper measurement and can be assessed even when image quality is suboptimal. Compared with published Mediterranean buffalo reference values, Murrah gravid horn diameters were 8-14% larger at equivalent months, consistent with the breed's heavier body frame and longer gestation, while BPD values differed by only 3-5%, suggesting that fetal skull growth is more conserved across buffalo ecotypes than maternal tract changes.

Discussion

The main findings of this research are a complete set of month-by-month ultrasonographic reference values for the reproductive tract of Murrah buffaloes and regression equations that allow gestational age estimation from a single BPD or horn diameter measurement. The BPD-based equation achieved a prediction accuracy of ± 6.8 days, comparable to the ± 5.7 days reported for Holstein cattle and the ± 7.4 days for Mediterranean buffaloes [7,15]. Several points of agreement with prior work deserve mention. The sigmoid pattern of gravid horn expansion mirrors the trajectory described by Zambrano-Varon et al. [5] in Colombian river buffaloes, suggesting that uterine growth dynamics are relatively conserved across buffalo populations despite breed and management differences. Similarly, the earliest detection of the embryonic vesicle at Day 25 aligns with the Day 24-28 window reported consistently in Italian, Egyptian, and Brazilian studies [3,6,17]. The heartbeat visualisation window of Day 28-30 also matches published data for both buffaloes and cattle. Where the Murrah data diverge from Mediterranean references is informative. Gravid horn diameters in the present cohort exceeded Italian values by 8-14% at equivalent months, a gap that widened as gestation progressed. This likely reflects the Murrah's larger adult body weight (600-700 kg vs. 500-600 kg for Mediterranean females) and deeper body cavity, which permits greater uterine expansion before the horn descends past the pelvic brim [4]. The practical consequence is that horn-diameter reference charts derived from Mediterranean animals would systematically underestimate gestational age if applied to Murrah buffaloes a clinically relevant error of approximately 8-12 days at midgestation.

BPD differences between breeds were smaller (3-5%), supporting the idea that fetal skull growth is under tighter genetic control and less influenced by maternal frame size. This makes BPD a more portable parameter across buffalo ecotypes, and practitioners serving mixed herds can probably use Murrah BPD equations for other river buffalo types with only modest loss of accuracy. That said, the orbit-to-BPD ratio of 0.44-0.45 observed here is slightly higher than the 0.41-0.42 ratio in Holsteins, hinting at subtle species-level differences in craniofacial proportions that warrant further investigation [18]. Placentome diameter followed a growth-then-plateau curve, with the inflection near month 7. A similar pattern has been reported in cattle, where placentome growth ceases around day 220 of a 280-day gestation proportionally equivalent to month 8 of a 310-day buffalo pregnancy [9]. The progressive widening of the 95% confidence interval in later months reflects genuine biological variation rather than measurement error, as intra-observer repeatability remained below 5% throughout. Buffaloes with the largest placentomes at month 10 tended to produce heavier calves (data not shown), a relationship that merits formal analysis in a larger sample. From a clinical standpoint, the integrated reference chart combining quantitative cutoffs with qualitative landmarks gives practitioners a decision-support tool for pregnancy evaluation. If a scan at month 5 shows BPD below the 5th percentile or anechoic fluid at a stage when slight echogenicity is expected, these flags prompt closer surveillance. The dataset also enables retrospective pregnancy dating when breeding records are incomplete a common scenario in imported or rescue herds where insemination dates may not be recorded.

Conclusion

This research set out to fill a long-standing gap in buffalo reproductive ultrasonography by generating breed-specific reference data for the Murrah across the full gestation period. Previous work had supplied fragmentary measurements CRL through early pregnancy, or placentome dimensions at midterm but no single longitudinal dataset had tracked all major parameters from Day 25 through month 10 in the same cohort of Murrah buffaloes.

The key findings can be condensed into four points. First, gravid uterine horn diameter follows a sigmoid growth curve, expanding from roughly 38 mm at month 1 to 362 mm at month 10, with the steepest increase between months 4 and 8. Second, BPD offers the strongest linear correlation with gestational age (r² = 0.99) and predicts parturition date within ± 6.8 days accurate enough for most clinical and management purposes. Third, placentome diameter grows rapidly until month 7 and then plateaus near 52 mm, making it unreliable for aging beyond that point but potentially useful as an indicator of placental adequacy. Fourth, qualitative landmarks such as heartbeat detection (Day 28-30), limb bud visibility (month 3), and amniotic fluid echogenicity shifts (months 8-9) provide low-technology cross-checks that any practitioner with a basic ultrasound unit can use. The practical message for veterinary practitioners and herd managers is clear. When managing Murrah buffaloes whether in South Korea, South America, or Southeast Asia breedspecific reference charts should be used instead of generic bovine or Mediterranean buffalo guides. The BPD regression equation presented here (Age = 2.87 × BPD + 34.6) is simple to apply in the field: measure BPD in millimetres, multiply by 2.87, add 34.6, and the result is gestational age in days. For early pregnancy, the gravid horn diameter equation (Age = 0.84 × Horn diameter + 2.1) provides an alternative when fetal structures are not yet large enough for BPD measurement. Printing these equations on a laminated field card, alongside the qualitative landmark checklist, would give practitioners a pocket reference that costs almost nothing to distribute. Several directions for future work emerge from these results. Extending the sample size and including primiparous buffaloes would clarify whether parity influences fetal growth rates or uterine expansion dynamics. Doppler ultrasonography of the middle uterine artery could complement the morphometric data by adding a functional assessment of uteroplacental blood flow, which has proven valuable for detecting compromised pregnancies in cattle. Threedimensional ultrasound, now available in portable veterinary units, could improve placentome volume estimation and fetal weight prediction beyond what two-dimensional crosssections allow. Lastly, applying the same monthly examination protocol to Nili-Ravi or Jaffarabadi buffaloes would determine how far the Murrah equations can be extrapolated to other Bubalus bubalis breeds or whether each ecotype needs its own reference set. In sum, the present dataset offers a practical, clinically validated reference standard for Murrah buffalo pregnancy ultrasonography that covers the entire gestational span and integrates both measurement-based and landmark-based approaches.

Acknowledgement

AcknowledgementsInstitutional support: The authors thank the Large Animal Teaching Hospital at Gyeonggi University of Veterinary Sciences for providing ultrasound equipment and animal handling facilities throughout the research period.Contributions not qualifying for authorship: Mr. Yoon Tae-sung assisted with animal restraint and data recording during monthly examination sessions.Funding sources: This research was supported by internal departmental funds from the Department of Veterinary Obstetrics and Reproduction, Gyeonggi University of Veterinary Sciences.

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