Early Pregnancy Detection Using Progesterone Hormone and Ultrasonography in Local Iraqi Ewes

Early Pregnancy Detection Using Progesterone Hormone and Ultrasonography in Local Iraqi Ewes | 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 Early Pregnancy Detection Using Progesterone Hormone and Ultrasonography in Local Iraqi Ewes Ahmed Emad Abood, Qusay Muhammad Aboud, Maysam Abdulrahman Ghazi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6986774/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 This study was conducted to evaluate and compare the diagnostic efficacy of transrectal ultrasonography (TRU) and serum progesterone (P4) assay for early pregnancy detection in local Iraqi ewes. A total of fifteen multiparous non-pregnant ewes (2–4 years old) were synchronized using intravaginal sponges containing Medroxyprogesterone Acetate (MPA) and 500 IU equine chorionic gonadotropin (eCG), followed by natural mating. Blood samples were collected on days 0, 14, 21, and 30 post-inseminations (PI) for hormonal analysis. TRU was employed concurrently on the same days to visualize pregnancy indicators. The results revealed that TRU had limited accuracy during the early stages (day 14–21 PI), with accuracy rates ranging from 26.7–60%, while improving substantially by day 30 PI (accuracy: 100%). In contrast, serum P4 levels demonstrated a statistically significant elevation in pregnant ewes compared to non-pregnant and control groups from day 14 onward, achieving full diagnostic accuracy by day 21 PI. Receiver operating characteristic (ROC) analysis confirmed the high predictive value of P4, especially on days 21 and 30 (AUC = 1.000). In conclusion, P4 assay serves as a more sensitive and reliable diagnostic tool for early pregnancy (days 14–21), whereas ultrasonography offers superior accuracy at later gestational stages (day 30 onward), thus supporting a complementary role for both techniques in reproductive management. Iraqi ewes early pregnancy detection progesterone assay transrectal ultrasonography hormonal diagnostics Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Early pregnancy diagnosis constitutes a cornerstone in optimizing reproductive efficiency and reducing economic losses in small ruminant production systems. In the absence of timely detection, numerous ewes are inadvertently culled or slaughtered while gravid, often during the first trimester, as documented in abattoir-based investigations across different contexts, including Iraq (AL-Sariy et al., 2020 ; Pagamici & Stephan, 2022 ). This mismanagement not only compromises animal welfare but also results in substantial fetal wastage and impaired herd productivity. Timely identification of pregnant versus non-pregnant ewes is essential to facilitate strategic decisions such as early nutritional adjustments, rebreeding, or culling of barren females (Kadhem & Al-Thuwaini, 2022 ; Alfathi et al., 2023 ). Moreover, accurate early pregnancy diagnosis is directly linked to improved perinatal outcomes, particularly through better management of twin pregnancies, which are associated with increased risks of dystocia and pregnancy toxemia (Khames et al., 2023 , ). Among the available diagnostic modalities, transrectal ultrasonography (TRU) and serum progesterone (P4) assays represent the most widely employed techniques in clinical and field reproductive practices. Ultrasonography enables visualization of embryonic structures and has been extensively applied in Iraq to assess pregnancy status, fetal number, gestational age, and various reproductive pathologies (Abas et al., 2022; Al-Thuwaini & Al-Hadi, 2022 ; Altememy & Saeed, 2023 ). However, its diagnostic sensitivity during the first three weeks of gestation remains suboptimal due to the small embryonic size and potential for false negatives (Aboud, 2024). Conversely, hormonal evaluation—particularly serum P4 profiling—offers a cost-effective, non-invasive alternative capable of distinguishing pregnant from non-pregnant females as early as day 14 post-mating. The rise in circulating P4 levels reflects corpus luteum (CL) activity and is typically sustained throughout gestation in fertile ewes, with concentrations exceeding 3–4 ng/mL around days 14–16 PI ( Aldeen et al., 2022 ). Nonetheless, the specificity of P4 assays is compromised by potential false positives arising from early embryonic mortality, persistent CLs, or uterine infections (Mohammed et al., 2020 ; Aboud, 2024). Estrus synchronization protocols incorporating Medroxyprogesterone Acetate (MPA) and equine chorionic gonadotropin (eCG) have demonstrated promising fertility outcomes, with conception rates ranging between 74–80% and favorable litter sizes (Yu et al., 2022 ; Santos-Jimenez et al., 2022 ). These protocols are increasingly utilized in Iraqi flocks to enhance reproductive scheduling and facilitate fixed-time insemination (Dhahir et al., 2022, Aziz et al., 2023 ). Despite the availability of these techniques, comparative evaluations of their accuracy in early pregnancy detection—particularly in local Iraqi breeds—remain sparse. Therefore, the present study was undertaken to critically assess and compare the reliability of TRU and serum P4 assay in detecting pregnancy during the early post-insemination period (days 14–30), with a focus on their diagnostic accuracy, sensitivity, and applicability under field conditions. Materials and Methods Animals and Experimental Design This study was conducted at the Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Fallujah, Iraq, between October 2024 and March 2025. Fifteen clinically healthy, multiparous local Iraqi ewes aged 2–4 years and weighing 40–50 kg were included. All animals were cyclic, non-pregnant at the beginning of the study, and were kept in a single flock under semi-intensive management conditions. Four fertile, mature rams were used for estrus detection and natural mating. Ewes were housed in shaded open yards with ad libitum access to water and were fed a balanced ration of alfalfa hay, barley grain, and mineral blocks, in accordance with NRC (2007) guidelines for small ruminants. Prior to the experiment, all ewes underwent a clinical and reproductive examination to exclude any anatomical or pathological abnormalities. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee of the University of Fallujah (Approval No. VET-FAL-2024-35). Estrus Synchronization Protocol Estrus was synchronized using intravaginal sponges (60 mg Medroxyprogesterone Acetate, MPA; Intervet, Netherlands) inserted for 14 days. Upon sponge removal, each ewe received an intramuscular injection of 500 IU equine Chorionic Gonadotropin (eCG; Ceva, France). Estrus was detected 36–48 hours post-treatment by observing behavioral signs and acceptance of mounting by teaser rams. Ewes that exhibited standing estrus were naturally mated by fertile rams, and the day of mating was designated as Day 0 post-insemination (PI). Blood Sample Collection and Progesterone Assay Blood samples (10 mL) were collected aseptically via jugular venipuncture into plain vacuum tubes on Days 0 (baseline), 14, 21, and 30 PI. Samples were immediately centrifuged at 3000 rpm for 15 minutes, and the serum was separated and stored at − 20°C until hormonal analysis. Serum progesterone concentrations were measured using a validated commercial sandwich ELISA kit (Bioassay Technology Laboratory, China), following the manufacturer’s protocol. All samples were run in duplicate, and intra-assay and inter-assay coefficients of variation were recorded to ensure assay precision. Ultrasonographic Examination Transrectal ultrasonography was performed on Days 14, 21, and 30 PI using a real-time B-mode scanner equipped with a 7.5 MHz linear-array probe (ECO1, Chison, China). Ewes were restrained in a standing position, and examinations were conducted with minimal stress and no sedation. The uterus was scanned to detect early pregnancy indicators such as corpus luteum presence, intrauterine fluid, and embryonic vesicles, following diagnostic criteria outlined by Yotov (2007) and Romano & Christians (2008). A ewe was classified as pregnant if an embryonic vesicle or fetal structure was visualized. In ambiguous cases, re-scanning was done within 24 hours. Statistical Analysis All data were analyzed using the Statistical Package for the Social Sciences (SPSS version 26.0; IBM Corp., USA). The data were tested for normality using the Shapiro–Wilk test. Repeated measures ANOVA was applied to analyze P4 concentration changes over time within and between groups. Significant differences between means were determined using the least significant difference (LSD) test at a 95% confidence level (p < 0.05). Chi-square test was used to compare pregnancy detection rates between diagnostic methods. The diagnostic performance of P4 assay was evaluated using Receiver Operating Characteristic (ROC) curve analysis with MedCalc Statistical Software version 16.4.3 (MedCalc Software, Belgium). Area under the curve (AUC), sensitivity, specificity, and Youden’s index were calculated to determine the optimal P4 cut-off points for pregnancy detection on each experimental day. Results Diagnostic Accuracy of Transrectal Ultrasonography (TRU) The Table 1 illustrates the progressive improvement in the diagnostic performance of transrectal ultrasonography (TRU) as gestation advances. On Day 14 post-insemination (PI), TRU exhibited low sensitivity (18.2%) and accuracy (26.7%), which is attributed to the difficulty of detecting early embryonic vesicles or intrauterine fluid in small ruminants at this stage. By Day 21, visualization of embryonic vesicles improved diagnostic sensitivity to 45.5%. By Day 30 PI, TRU achieved optimal diagnostic precision with 100% for sensitivity, specificity, and overall accuracy, confirming its value in mid-gestational confirmation of pregnancy. Table 1 Diagnostic Parameters of TRU at Different Days Post-Insemination Day post-insemination Sensitivity (%) Specificity (%) Accuracy (%) Day 14 18.2 50 26.7 Day 21 45.5 100 60.0 Day 30 100.0 100 100.0 The ROC analysis presented in Table 2 demonstrates the superior diagnostic efficiency of the serum progesterone assay, especially on Days 21 and 30 PI, with an AUC of 1.000 indicating perfect classification of pregnancy status. On Day 14, although the AUC was slightly lower (0.932), it still indicated a high degree of accuracy. The calculated Youden Index values reinforce the reliability of the cutoff thresholds (3.34, 3.95, and 3.67 ng/ml) at each respective time point, with perfect sensitivity and specificity achieved from Day 21 onwards (Table 2 ). Table 2 ROC Parameters for P4 Assay at Different Days Day post-insemination AUC Youden Index Cutoff (ng/ml) Sensitivity (%) Specificity (%) Day 14 0.932 0.75 3.34 100 75 Day 21 1.000 1.00 3.95 100 100 Day 30 1.000 1.00 3.67 100 100 ROC curves illustrating the diagnostic capacity of serum progesterone assay for pregnancy detection at three time points post-insemination (Fig. 1 ). Trend of serum progesterone levels in pregnant and non-pregnant ewes across Days 14 to 30 post-insemination (Fig. 2 ). The Table 3 summarizes the dynamic changes in serum progesterone levels among different ewe groups. Pregnant ewes exhibited a consistent and statistically significant rise in P4 levels from Day 14 (4.75 ng/ml) to Day 30 (7.12 ng/ml), while non-pregnant animals displayed relatively stable but lower values, confirming corpus luteum regression or embryonic loss. Control animals-maintained baseline levels (0.62 ng/ml). These trends support the physiological basis for using P4 as a biomarker of pregnancy establishment and maintenance. Table 3 presents detailed P4 concentrations with corresponding statistical comparisons. Group Day 14 Day 21 Day 30 Control Inseminated/Pregnant 4.75 ± 0.26 5.97 ± 0.23 7.12 ± 0.26 0.62 ± 0.09 Inseminated/Non-pregnant 2.57 ± 0.22 2.59 ± 0.60 2.13 ± 0.54 0.62 ± 0.09 On Day 14 PI, the P4 assay significantly outperformed ultrasonography, demonstrating perfect sensitivity and accuracy (100%), whereas TRU was limited by low sensitivity (18.2%) and poor diagnostic precision (accuracy: 26.7%). These findings validate that hormonal assays are more informative during the early luteal phase, when ultrasonographic visualization of gestational structures remains unreliable (Table 4 ). Table 4 Comparison of Detection Methods on Day 14 PI Comparison of sensitivity, specificity, and accuracy between P4 assay and TRU at Day 14 post-insemination. Method Sensitivity (%) Specificity (%) Accuracy (%) P4 Assay 100.0 75.0 100.0 B-mode Ultrasonography 18.2 50.0 26.7 By Day 21, both methods became diagnostically informative. However, the P4 assay retained a slight advantage with perfect performance across all parameters. Although TRU reached 100% specificity, its lower sensitivity (45.5%) and accuracy (60%) suggest that some conceptuses may still not be reliably visualized, necessitating repeated scanning or parallel hormonal testing (Table 5 ). Table 5 Comparison of Detection Methods on Day 21 PI Comparison of diagnostic performance of P4 assay and TRU on Day 21 post-insemination. Method Sensitivity (%) Specificity (%) Accuracy (%) P4 Assay 100.0 100.0 100.0 B-mode Ultrasonography 45.5 100.0 60.0 On Day 30 post-insemination, both TRU and the P4 assay achieved complete diagnostic performance (100% across sensitivity, specificity, and accuracy). At this stage, embryonic structures are fully developed and distinguishable via ultrasonography, while the sustained elevation in P4 reflects functional luteal support and ongoing pregnancy. These results confirm the diagnostic convergence of both modalities in later gestation (Table 6 ). Table 6 Comparison of Detection Methods on Day 30 PI Both methods achieved complete accuracy on Day 30, confirming pregnancy status. Method Sensitivity (%) Specificity (%) Accuracy (%) P4 Assay 100.0 100.0 100.0 B-mode Ultrasonography 100.0 100.0 100.0 Ultrasonographic Images of Pregnancy in Ewes ( Fig. 3 , 4 ). Discussion The current study provides a comparative evaluation of transrectal ultrasonography (TRU) and serum progesterone (P4) assay in detecting early pregnancy in local Iraqi ewes. The diagnostic outcomes obtained in this study highlight the physiological basis and methodological efficiency of both tools, with an emphasis on their performance across different gestational intervals. On Day 14 post-insemination, the markedly low sensitivity and accuracy of TRU (18.2% and 26.7%, respectively) affirm its limitation in visualizing early embryonic structures, likely due to the incomplete formation of gestational vesicles or fluid accumulation at this stage. These findings align with the observations of Ghelardi, ( 2022 ), who noted that the earliest reliable TRU-based detection typically occurs beyond Day 20 post-mating when embryonic vesicles become ultrasonographically distinguishable. Conversely, serum progesterone evaluation at this stage demonstrated a diagnostic sensitivity of 100%, confirming the active function of the corpus luteum and the associated hormonal rise in pregnant animals, consistent with luteal dynamics described by Constantin et al. ( 2024 ). By Day 21, the diagnostic potential of TRU showed noticeable improvement (sensitivity = 45.5%), though still significantly inferior to P4-based diagnosis, which maintained 100% sensitivity and specificity. This phase corresponds to the progressive anatomical delineation of the embryonic vesicle and developing placentomes, which supports improved sonographic identification (Gavanier et al., 2021 , Al-Taee & Saeed, 2022 ). The current ROC analysis further supports the P4 test’s superiority, as indicated by an AUC of 1.000 and a Youden Index of 1.000, thereby confirming its near-perfect classification capability at this stage. At Day 30 PI, both modalities demonstrated identical diagnostic performance (100% for all parameters), suggesting that gestational development at this point reaches a threshold detectable by both endocrine and imaging criteria. Similar outcomes were reported by Lees et al. ( 2022 ), who emphasized that mid-gestation is the optimal window for definitive ultrasonographic confirmation due to prominent anatomical markers such as placentomes, embryonic heartbeat, and fluid delineation. Moreover, the dynamic increase in serum progesterone levels in pregnant ewes observed in this study from Day 14 to Day 30 reinforces its role as a biochemical indicator for pregnancy status. These findings agree with earlier reports by Vatankhah et al., ( 2025 ), who correlated sustained P4 elevation with embryo survival and functional luteal maintenance. The hormonal profiles observed in the inseminated/non-pregnant group, which showed a transient rise followed by a decline in P4 levels, potentially reflect early embryonic loss or luteolysis, as discussed in previous ovine reproductive physiology literature. In addition to hormonal and imaging data, the ROC-derived cut-off values identified in this study—namely 3.34 ng/ml (Day 14), 3.95 ng/ml (Day 21), and 3.67 ng/ml (Day 30)—may serve as useful clinical thresholds for future diagnostic protocols in field conditions. These values mirror those proposed by Araujo-Castro et al. ( 2021 ) and enhance the applicability of P4-based diagnosis within diverse environmental and genetic contexts. Collectively, the comparative analysis indicates that although TRU remains a non-invasive and practical method for pregnancy confirmation, its diagnostic reliability is stage-dependent and suboptimal in early gestation. On the contrary, serum P4 assessment offers robust, highly sensitive screening from Day 14 onward, enabling timely reproductive decisions in sheep husbandry systems. Conclusion The present study highlights the superior diagnostic performance of serum progesterone (P4) assays compared to transrectal ultrasonography (TRU) for early pregnancy detection in local Iraqi ewes. While TRU accuracy improves significantly with gestational advancement—achieving full diagnostic performance by Day 30—its sensitivity during the early luteal phase remains limited. In contrast, P4 assays demonstrated high sensitivity and specificity as early as Day 14 post-insemination, rendering them a reliable biochemical marker for early pregnancy screening. The integration of ROC analysis provided objective cutoff values that could guide field-level reproductive management. These findings suggest that progesterone profiling may serve as a practical, non-invasive, and time-efficient tool for enhancing reproductive efficiency in small ruminant husbandry. Study Limitations Although this study provides robust diagnostic comparisons, certain limitations must be acknowledged. First, the relatively small sample size (n = 15) may restrict the generalizability of the findings. Second, while TRU was performed by an experienced operator, variations in ultrasonographic interpretation can exist under field conditions. Lastly, other physiological or pathological factors influencing progesterone levels—such as pseudopregnancy or early embryonic mortality—were not addressed in this trial. Declarations Acknowledgments The authors wish to express their sincere gratitude to the staff of the College of Veterinary Medicine, University of Mosul, for their technical support during animal handling and laboratory procedures. Special thanks are extended to the farm personnel for their cooperation throughout the study duration. Funding Statement: Self-Funding. Conflict of Interest: None Data Availability Statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. All relevant raw data, histological images, and statistical outputs have been archived and can be shared to ensure transparency and reproducibility in line with journal policies. References AL-Sariy, S.M., AL-Yasiri, E.A., and AL-Hamedawi, T.M. 2020. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6986774","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485259771,"identity":"f2b2b984-9a30-4a9a-8b96-3e43c065861f","order_by":0,"name":"Ahmed Emad Abood","email":"data:image/png;base64,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","orcid":"","institution":"University of Falluja, College of Veterinary Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ahmed","middleName":"Emad","lastName":"Abood","suffix":""},{"id":485259772,"identity":"8382a099-b1e4-459a-97b9-310b92b2be41","order_by":1,"name":"Qusay Muhammad Aboud","email":"","orcid":"","institution":"University of Falluja, College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qusay","middleName":"Muhammad","lastName":"Aboud","suffix":""},{"id":485259773,"identity":"c8a4d944-625e-4a60-a1e6-ef5b36a60e25","order_by":2,"name":"Maysam Abdulrahman Ghazi","email":"","orcid":"","institution":"University of Fallujah, College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Maysam","middleName":"Abdulrahman","lastName":"Ghazi","suffix":""},{"id":485259774,"identity":"3a9ef99a-c85a-46b6-b3fc-293777428089","order_by":3,"name":"Mahmoud Khudair Abbas","email":"","orcid":"","institution":"University of Fallujah, College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"Khudair","lastName":"Abbas","suffix":""}],"badges":[],"createdAt":"2025-06-27 00:06:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6986774/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6986774/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86858810,"identity":"1af6834a-c61d-40ec-bbfa-d42e70a519af","added_by":"auto","created_at":"2025-07-16 11:41:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC Curves of P4 Assay at Days 14, 21, and 30\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6986774/v1/2168a34fd3f666ca9bf939f1.png"},{"id":86857975,"identity":"52f50634-738e-445d-869c-89909e12d443","added_by":"auto","created_at":"2025-07-16 11:33:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum Progesterone levels in pregnant vs. non-pregnant ewes from Day 14 to Day 30 PI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum Progesterone (P4) Profiles in Ewes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6986774/v1/eaf596fa74bb9320b9947efa.png"},{"id":86858813,"identity":"878124bc-9195-4d9e-a245-191fbf2cacd5","added_by":"auto","created_at":"2025-07-16 11:41:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":238293,"visible":true,"origin":"","legend":"\u003cp\u003eTransrectal ultrasonographic image captured on Day 21 post-insemination. The presence of an anechoic embryonic vesicle is visible, indicating early gestational development.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6986774/v1/b9ab25bb8e11f12042475537.png"},{"id":86857978,"identity":"0a383763-60bf-47b0-9e7b-b643f452bd54","added_by":"auto","created_at":"2025-07-16 11:33:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":239360,"visible":true,"origin":"","legend":"\u003cp\u003eTransrectal ultrasonographic image captured on Day 30 post-insemination. A well-defined embryo with measurable gestational sac diameter (GSD = 2.53 cm) is shown, corresponding to approximately 5 weeks of gestation. The anatomical clarity confirms the reliability of TRU in mid-gestation pregnancy diagnosis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6986774/v1/6ac61008201c0b960f2063f9.png"},{"id":86952372,"identity":"e70c1541-7491-4fad-8d80-b68c3b9daacf","added_by":"auto","created_at":"2025-07-17 14:37:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1523946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6986774/v1/cff4c71a-a1c9-4897-a2cf-bb337d188281.pdf"}],"financialInterests":"","formattedTitle":"Early Pregnancy Detection Using Progesterone Hormone and Ultrasonography in Local Iraqi Ewes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEarly pregnancy diagnosis constitutes a cornerstone in optimizing reproductive efficiency and reducing economic losses in small ruminant production systems. In the absence of timely detection, numerous ewes are inadvertently culled or slaughtered while gravid, often during the first trimester, as documented in abattoir-based investigations across different contexts, including Iraq (AL-Sariy et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pagamici \u0026amp; Stephan, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This mismanagement not only compromises animal welfare but also results in substantial fetal wastage and impaired herd productivity.\u003c/p\u003e\u003cp\u003eTimely identification of pregnant versus non-pregnant ewes is essential to facilitate strategic decisions such as early nutritional adjustments, rebreeding, or culling of barren females (Kadhem \u0026amp; Al-Thuwaini, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Alfathi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, accurate early pregnancy diagnosis is directly linked to improved perinatal outcomes, particularly through better management of twin pregnancies, which are associated with increased risks of dystocia and pregnancy toxemia (Khames et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, ).\u003c/p\u003e\u003cp\u003eAmong the available diagnostic modalities, transrectal ultrasonography (TRU) and serum progesterone (P4) assays represent the most widely employed techniques in clinical and field reproductive practices. Ultrasonography enables visualization of embryonic structures and has been extensively applied in Iraq to assess pregnancy status, fetal number, gestational age, and various reproductive pathologies (Abas et al., 2022; Al-Thuwaini \u0026amp; Al-Hadi, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Altememy \u0026amp; Saeed, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, its diagnostic sensitivity during the first three weeks of gestation remains suboptimal due to the small embryonic size and potential for false negatives (Aboud, 2024).\u003c/p\u003e\u003cp\u003eConversely, hormonal evaluation\u0026mdash;particularly serum P4 profiling\u0026mdash;offers a cost-effective, non-invasive alternative capable of distinguishing pregnant from non-pregnant females as early as day 14 post-mating. The rise in circulating P4 levels reflects corpus luteum (CL) activity and is typically sustained throughout gestation in fertile ewes, with concentrations exceeding 3\u0026ndash;4 ng/mL around days 14\u0026ndash;16 PI (\u003cb\u003eAldeen et al., 2022\u003c/b\u003e). Nonetheless, the specificity of P4 assays is compromised by potential false positives arising from early embryonic mortality, persistent CLs, or uterine infections (Mohammed et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Aboud, 2024).\u003c/p\u003e\u003cp\u003eEstrus synchronization protocols incorporating Medroxyprogesterone Acetate (MPA) and equine chorionic gonadotropin (eCG) have demonstrated promising fertility outcomes, with conception rates ranging between 74\u0026ndash;80% and favorable litter sizes (Yu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Santos-Jimenez et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These protocols are increasingly utilized in Iraqi flocks to enhance reproductive scheduling and facilitate fixed-time insemination (Dhahir et al., 2022, Aziz et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the availability of these techniques, comparative evaluations of their accuracy in early pregnancy detection\u0026mdash;particularly in local Iraqi breeds\u0026mdash;remain sparse. Therefore, the present study was undertaken to critically assess and compare the reliability of TRU and serum P4 assay in detecting pregnancy during the early post-insemination period (days 14\u0026ndash;30), with a focus on their diagnostic accuracy, sensitivity, and applicability under field conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals and Experimental Design\u003c/h2\u003e\u003cp\u003eThis study was conducted at the Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Fallujah, Iraq, between October 2024 and March 2025. Fifteen clinically healthy, multiparous local Iraqi ewes aged 2\u0026ndash;4 years and weighing 40\u0026ndash;50 kg were included. All animals were cyclic, non-pregnant at the beginning of the study, and were kept in a single flock under semi-intensive management conditions. Four fertile, mature rams were used for estrus detection and natural mating. Ewes were housed in shaded open yards with ad libitum access to water and were fed a balanced ration of alfalfa hay, barley grain, and mineral blocks, in accordance with NRC (2007) guidelines for small ruminants.\u003c/p\u003e\u003cp\u003ePrior to the experiment, all ewes underwent a clinical and reproductive examination to exclude any anatomical or pathological abnormalities. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee of the University of Fallujah (Approval No. VET-FAL-2024-35).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEstrus Synchronization Protocol\u003c/h3\u003e\n\u003cp\u003eEstrus was synchronized using intravaginal sponges (60 mg Medroxyprogesterone Acetate, MPA; Intervet, Netherlands) inserted for 14 days. Upon sponge removal, each ewe received an intramuscular injection of 500 IU equine Chorionic Gonadotropin (eCG; Ceva, France). Estrus was detected 36\u0026ndash;48 hours post-treatment by observing behavioral signs and acceptance of mounting by teaser rams. Ewes that exhibited standing estrus were naturally mated by fertile rams, and the day of mating was designated as Day 0 post-insemination (PI).\u003c/p\u003e\n\u003ch3\u003eBlood Sample Collection and Progesterone Assay\u003c/h3\u003e\n\u003cp\u003eBlood samples (10 mL) were collected aseptically via jugular venipuncture into plain vacuum tubes on Days 0 (baseline), 14, 21, and 30 PI. Samples were immediately centrifuged at 3000 rpm for 15 minutes, and the serum was separated and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until hormonal analysis. Serum progesterone concentrations were measured using a validated commercial sandwich ELISA kit (Bioassay Technology Laboratory, China), following the manufacturer\u0026rsquo;s protocol. All samples were run in duplicate, and intra-assay and inter-assay coefficients of variation were recorded to ensure assay precision.\u003c/p\u003e\n\u003ch3\u003eUltrasonographic Examination\u003c/h3\u003e\n\u003cp\u003eTransrectal ultrasonography was performed on Days 14, 21, and 30 PI using a real-time B-mode scanner equipped with a 7.5 MHz linear-array probe (ECO1, Chison, China). Ewes were restrained in a standing position, and examinations were conducted with minimal stress and no sedation. The uterus was scanned to detect early pregnancy indicators such as corpus luteum presence, intrauterine fluid, and embryonic vesicles, following diagnostic criteria outlined by Yotov (2007) and Romano \u0026amp; Christians (2008). A ewe was classified as pregnant if an embryonic vesicle or fetal structure was visualized. In ambiguous cases, re-scanning was done within 24 hours.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using the Statistical Package for the Social Sciences (SPSS version 26.0; IBM Corp., USA). The data were tested for normality using the Shapiro\u0026ndash;Wilk test. Repeated measures ANOVA was applied to analyze P4 concentration changes over time within and between groups. Significant differences between means were determined using the least significant difference (LSD) test at a 95% confidence level (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Chi-square test was used to compare pregnancy detection rates between diagnostic methods.\u003c/p\u003e\u003cp\u003eThe diagnostic performance of P4 assay was evaluated using Receiver Operating Characteristic (ROC) curve analysis with MedCalc Statistical Software version 16.4.3 (MedCalc Software, Belgium). Area under the curve (AUC), sensitivity, specificity, and Youden\u0026rsquo;s index were calculated to determine the optimal P4 cut-off points for pregnancy detection on each experimental day.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eDiagnostic Accuracy of Transrectal Ultrasonography (TRU)\u003c/h2\u003e\u003cp\u003eThe Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the progressive improvement in the diagnostic performance of transrectal ultrasonography (TRU) as gestation advances. On Day 14 post-insemination (PI), TRU exhibited low sensitivity (18.2%) and accuracy (26.7%), which is attributed to the difficulty of detecting early embryonic vesicles or intrauterine fluid in small ruminants at this stage. By Day 21, visualization of embryonic vesicles improved diagnostic sensitivity to 45.5%. By Day 30 PI, TRU achieved optimal diagnostic precision with 100% for sensitivity, specificity, and overall accuracy, confirming its value in mid-gestational confirmation of pregnancy.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDiagnostic Parameters of TRU at Different Days Post-Insemination\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay post-insemination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSensitivity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpecificity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccuracy (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e45.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e60.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe ROC analysis presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrates the superior diagnostic efficiency of the serum progesterone assay, especially on Days 21 and 30 PI, with an AUC of 1.000 indicating perfect classification of pregnancy status. On Day 14, although the AUC was slightly lower (0.932), it still indicated a high degree of accuracy. The calculated Youden Index values reinforce the reliability of the cutoff thresholds (3.34, 3.95, and 3.67 ng/ml) at each respective time point, with perfect sensitivity and specificity achieved from Day 21 onwards (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eROC Parameters for P4 Assay at Different Days\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay post-insemination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAUC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYouden Index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCutoff (ng/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSensitivity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSpecificity (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eROC curves illustrating the diagnostic capacity of serum progesterone assay for pregnancy detection at three time points post-insemination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTrend of serum progesterone levels in pregnant and non-pregnant ewes across Days 14 to 30 post-insemination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e summarizes the dynamic changes in serum progesterone levels among different ewe groups. Pregnant ewes exhibited a consistent and statistically significant rise in P4 levels from Day 14 (4.75 ng/ml) to Day 30 (7.12 ng/ml), while non-pregnant animals displayed relatively stable but lower values, confirming corpus luteum regression or embryonic loss. Control animals-maintained baseline levels (0.62 ng/ml). These trends support the physiological basis for using P4 as a biomarker of pregnancy establishment and maintenance.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003epresents detailed P4 concentrations with corresponding statistical comparisons.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay 14\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDay 21\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDay 30\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInseminated/Pregnant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInseminated/Non-pregnant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOn Day 14 PI, the P4 assay significantly outperformed ultrasonography, demonstrating perfect sensitivity and accuracy (100%), whereas TRU was limited by low sensitivity (18.2%) and poor diagnostic precision (accuracy: 26.7%). These findings validate that hormonal assays are more informative during the early luteal phase, when ultrasonographic visualization of gestational structures remains unreliable (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eComparison of Detection Methods on Day 14 PI\u003c/b\u003e Comparison of sensitivity, specificity, and accuracy between P4 assay and TRU at Day 14 post-insemination.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSensitivity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpecificity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccuracy (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP4 Assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e75.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB-mode Ultrasonography\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eBy Day 21, both methods became diagnostically informative. However, the P4 assay retained a slight advantage with perfect performance across all parameters. Although TRU reached 100% specificity, its lower sensitivity (45.5%) and accuracy (60%) suggest that some conceptuses may still not be reliably visualized, necessitating repeated scanning or parallel hormonal testing (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eComparison of Detection Methods on Day 21 PI\u003c/b\u003e Comparison of diagnostic performance of P4 assay and TRU on Day 21 post-insemination.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSensitivity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpecificity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccuracy (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP4 Assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB-mode Ultrasonography\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e45.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e60.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOn Day 30 post-insemination, both TRU and the P4 assay achieved complete diagnostic performance (100% across sensitivity, specificity, and accuracy). At this stage, embryonic structures are fully developed and distinguishable via ultrasonography, while the sustained elevation in P4 reflects functional luteal support and ongoing pregnancy. These results confirm the diagnostic convergence of both modalities in later gestation (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eComparison of Detection Methods on Day 30 PI\u003c/b\u003e Both methods achieved complete accuracy on Day 30, confirming pregnancy status.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSensitivity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpecificity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccuracy (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP4 Assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB-mode Ultrasonography\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eUltrasonographic Images of Pregnancy in Ewes (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study provides a comparative evaluation of transrectal ultrasonography (TRU) and serum progesterone (P4) assay in detecting early pregnancy in local Iraqi ewes. The diagnostic outcomes obtained in this study highlight the physiological basis and methodological efficiency of both tools, with an emphasis on their performance across different gestational intervals. On Day 14 post-insemination, the markedly low sensitivity and accuracy of TRU (18.2% and 26.7%, respectively) affirm its limitation in visualizing early embryonic structures, likely due to the incomplete formation of gestational vesicles or fluid accumulation at this stage. These findings align with the observations of Ghelardi, (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who noted that the earliest reliable TRU-based detection typically occurs beyond Day 20 post-mating when embryonic vesicles become ultrasonographically distinguishable. Conversely, serum progesterone evaluation at this stage demonstrated a diagnostic sensitivity of 100%, confirming the active function of the corpus luteum and the associated hormonal rise in pregnant animals, consistent with luteal dynamics described by Constantin et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBy Day 21, the diagnostic potential of TRU showed noticeable improvement (sensitivity\u0026thinsp;=\u0026thinsp;45.5%), though still significantly inferior to P4-based diagnosis, which maintained 100% sensitivity and specificity. This phase corresponds to the progressive anatomical delineation of the embryonic vesicle and developing placentomes, which supports improved sonographic identification (Gavanier et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Al-Taee \u0026amp; Saeed, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The current ROC analysis further supports the P4 test\u0026rsquo;s superiority, as indicated by an AUC of 1.000 and a Youden Index of 1.000, thereby confirming its near-perfect classification capability at this stage.\u003c/p\u003e\u003cp\u003eAt Day 30 PI, both modalities demonstrated identical diagnostic performance (100% for all parameters), suggesting that gestational development at this point reaches a threshold detectable by both endocrine and imaging criteria. Similar outcomes were reported by Lees et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who emphasized that mid-gestation is the optimal window for definitive ultrasonographic confirmation due to prominent anatomical markers such as placentomes, embryonic heartbeat, and fluid delineation.\u003c/p\u003e\u003cp\u003eMoreover, the dynamic increase in serum progesterone levels in pregnant ewes observed in this study from Day 14 to Day 30 reinforces its role as a biochemical indicator for pregnancy status. These findings agree with earlier reports by Vatankhah et al., (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), who correlated sustained P4 elevation with embryo survival and functional luteal maintenance. The hormonal profiles observed in the inseminated/non-pregnant group, which showed a transient rise followed by a decline in P4 levels, potentially reflect early embryonic loss or luteolysis, as discussed in previous ovine reproductive physiology literature. In addition to hormonal and imaging data, the ROC-derived cut-off values identified in this study\u0026mdash;namely 3.34 ng/ml (Day 14), 3.95 ng/ml (Day 21), and 3.67 ng/ml (Day 30)\u0026mdash;may serve as useful clinical thresholds for future diagnostic protocols in field conditions. These values mirror those proposed by Araujo-Castro et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and enhance the applicability of P4-based diagnosis within diverse environmental and genetic contexts.\u003c/p\u003e\u003cp\u003eCollectively, the comparative analysis indicates that although TRU remains a non-invasive and practical method for pregnancy confirmation, its diagnostic reliability is stage-dependent and suboptimal in early gestation. On the contrary, serum P4 assessment offers robust, highly sensitive screening from Day 14 onward, enabling timely reproductive decisions in sheep husbandry systems.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study highlights the superior diagnostic performance of serum progesterone (P4) assays compared to transrectal ultrasonography (TRU) for early pregnancy detection in local Iraqi ewes. While TRU accuracy improves significantly with gestational advancement\u0026mdash;achieving full diagnostic performance by Day 30\u0026mdash;its sensitivity during the early luteal phase remains limited. In contrast, P4 assays demonstrated high sensitivity and specificity as early as Day 14 post-insemination, rendering them a reliable biochemical marker for early pregnancy screening. The integration of ROC analysis provided objective cutoff values that could guide field-level reproductive management. These findings suggest that progesterone profiling may serve as a practical, non-invasive, and time-efficient tool for enhancing reproductive efficiency in small ruminant husbandry.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStudy Limitations\u003c/h2\u003e\u003cp\u003eAlthough this study provides robust diagnostic comparisons, certain limitations must be acknowledged. First, the relatively small sample size (n\u0026thinsp;=\u0026thinsp;15) may restrict the generalizability of the findings. Second, while TRU was performed by an experienced operator, variations in ultrasonographic interpretation can exist under field conditions. Lastly, other physiological or pathological factors influencing progesterone levels\u0026mdash;such as pseudopregnancy or early embryonic mortality\u0026mdash;were not addressed in this trial.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to express their sincere gratitude to the staff of the College of Veterinary Medicine, University of Mosul, for their technical support during animal handling and laboratory procedures. Special thanks are extended to the farm personnel for their cooperation throughout the study duration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement:\u0026nbsp;\u003c/strong\u003eSelf-Funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request. All relevant raw data, histological images, and statistical outputs have been archived and can be shared to ensure transparency and reproducibility in line with journal policies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAL-Sariy, S.M., AL-Yasiri, E.A., and AL-Hamedawi, T.M. 2020. Limitation of Gestational Age in Local Iraqi Ewes and Does by Using Number and Measurement of Placentomes Diameter in Abattoirs. \u003cem\u003eBiochemical and Cellular Archives\u003c/em\u003e, 20(1), 1089\u0026ndash;1191. DOI : 10.35124/bca.2020.20.1.1089 \u003c/li\u003e\n\u003cli\u003ePagamici, C., \u0026amp; Stephan, R. 2022. Pregnancy in Slaughtered Lambs and Sheep\u0026mdash;A Cross-Sectional Study in Three Abattoirs in Switzerland. \u003cem\u003eAnimals\u003c/em\u003e, 12(10), 1328. https://doi.org/10.3390/ani12101328MDPI\u003c/li\u003e\n\u003cli\u003eKadhem, A.F., \u0026amp; Al-Thuwaini, T.M. 2022.Influence of litter size on the hematologic profile of Awassi ewes during gestation and lactation. \u003cem\u003eVeterinary Integrative Sciences\u003c/em\u003e, 20(3), 625\u0026ndash;633. DOI: 10.12982/VIS.2022.047ThaiJo2.1: Thai Journal Online\u003c/li\u003e\n\u003cli\u003eKhames, M.M., Shareef, S.O., \u0026amp; Abdulealah, I.M. 2023. Clinical and Biochemical Study of Pregnancy Toxemia in Iraqi Ewes. \u003cem\u003eArchives of Razi Institute\u003c/em\u003e, 78(3), 1131\u0026ndash;1139. https://doi.org/10.22092/ari.2022.359420.2029\u003c/li\u003e\n\u003cli\u003eAlfathi, M., Alabdaly, Y. and Al-Hayyali, F., 2023. Ameliorative Effect of Spirulina against gentamicin toxicity in liver and kidney tissues of male rat. Egyptian Journal of Histology, 46(4), pp.1666-1675.\u003c/li\u003e\n\u003cli\u003eAbas, H.K., EA, A., Al-Hamedawi, T.M., and Ajeel, H.M. 2022. Pregnancy Rate in Synchronized Iraqi Awassi Ewes Inseminated Artificially and Naturally. \u003cem\u003eIndian Journal of Forensic Medicine \u0026amp; Toxicology\u003c/em\u003e, 16(1), 1\u0026ndash;6. DOI: 10.37506/ijfmt.v16i1.17808Medico Publication\u003c/li\u003e\n\u003cli\u003eAl-Thuwaini, T.M., and Al-Hadi, A.B. 2022. Association of Lamb Sex with Body Measurements in Single and Twin on the Awassi Ewes. \u003cem\u003eAdvances in Animal and Veterinary Sciences\u003c/em\u003e, 10(8), 1849\u0026ndash;1853. DOI | https://dx.doi.org/10.17582/journal.aavs/2022/10.8.1849.1853\u003c/li\u003e\n\u003cli\u003eAltememy, M. H., \u0026amp; Saeed, M. G. (2023). Prevalence and pathological study of trichomoniasis in the racing pigeons in Mosul city, Iraq. Iraqi Journal of Veterinary Sciences, 37(5).\u003c/li\u003e\n\u003cli\u003eMohsin, M.K., \u0026amp; Hussein, K.A. 2022. Monitoring Influence of PGF2\u0026alpha;, Estrogen and Oxytocin on Postpartum Uterine Involution by Ultrasonography in Ewes. \u003cem\u003eIndian Journal of Ecology\u003c/em\u003e, 49(2), 651\u0026ndash;654. DOI : 10.55362/IJE/2022/3575\u003c/li\u003e\n\u003cli\u003eMohammed, T.R., Majeed, A.F., \u0026amp; Alawiy, I.K. 2020. Pregnancy diagnosis in local Iraqi ewes: a comparative study. \u003cem\u003eAnnals of Tropical Medicine and Public Health\u003c/em\u003e, 23(7), 1119\u0026ndash;1125. http://doi.org/10.36295/ASRO.2020.23736\u003c/li\u003e\n\u003cli\u003eAldeen, E., Albayati, O.A. and Ismaeel, M.A. 2022. Study of Some Biochemical and Vitamins Changes in the Occurrence of Repeated Fertilization Failure in Sheep. \u003cem\u003eAnnals of the Romanian Society for Cell Biology\u003c/em\u003e, 26(01), 3262\u0026ndash;3269. DOI: Not available.DOI+4PubMed+4annalsofrscb.ro+4\u003c/li\u003e\n\u003cli\u003eAboud, Q. M. 2024. Individual Variation in Semen Attributes and Number of Straws Produced by Holstein Bulls Born in Iraq During Different Ages and Months. \u003cem\u003eEgyptian Journal of Veterinary Sciences\u003c/em\u003e, 55(7), 1857\u0026ndash;1867. DOI: 10.21608/ejvs.2024.263952.1790SCiNiTO\u003c/li\u003e\n\u003cli\u003eYu, X., Bai, Y., Yang, J., Zhao, X., Zhang, L., \u0026amp; Wang, J. 2022. Comparison of five protocols of estrous synchronization on reproductive performance of Hu sheep. \u003cem\u003eFrontiers in Veterinary Science\u003c/em\u003e, 9, 843514. https://doi.org/10.3389/fvets.2022.843514\u003c/li\u003e\n\u003cli\u003eSantos-Jimenez, Z., Mart\u0026iacute;nez-Ros, P., Encinas, T., Morales-Cruz, J.L., Guerrero-Gallegos, H.Z., Gonzalez-Avalos, R., Gonzalez-Bulnes, A., \u0026amp; Guillen-Mu\u0026ntilde;oz, J.M. 2022. Ovarian Response and Fertility after Short-Term Progestagen/eCG Treatments Are Compromised in Nulliparous Sheep during Non-Breeding Season. \u003cem\u003eVeterinary Sciences\u003c/em\u003e, 9(12), 663. https://doi.org/10.3390/vetsci9120663\u003c/li\u003e\n\u003cli\u003eDhahir, N.N., Ismaeel, M.A., \u0026amp; Aldoori, Z.T. 2022. Effect of adding carrots as feed supplementation on reproductive performance in Awassi ewes. \u003cem\u003eIraqi Journal of Veterinary Sciences\u003c/em\u003e, 36(2), 413\u0026ndash;417. DOI: 10.33899/ijvs.2022.132924.2159\u003c/li\u003e\n\u003cli\u003eAziz, Z. W., Saeed, M. G., \u0026amp; Tawfeeq, K. T. (2023). Formalin Versus Bouin Solution for Rat Testicular Tissue Fixation: A Histochemical and Immunohistochemical Evaluation. International Journal of Medical Toxicology and Forensic Medicine, 13(2), 40267-40267.\u003c/li\u003e\n\u003cli\u003eGhelardi, C., 2022. Non-invasive biomarkers of embryo quality: how they can contribute to the success of Assisted Reproduction Techniques.\u003c/li\u003e\n\u003cli\u003eConstantin, N.T., Posastiuc, F.P. and Andrei, C.R., 2024. Progesterone: An Essential Diagnostic Resource in Veterinary Medicine.\u003c/li\u003e\n\u003cli\u003eGavanier, D., Berthet, G., Hajri, T., Allias, F., Atallah, A., Massoud, M., Golfier, F., Bolze, P.A. and Massardier, J., 2021. Vesicules or placental lakes in ultrasonography, determining the correct etiology. \u003cem\u003eJournal of Gynecology Obstetrics and Human Reproduction\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(6), p.101738.\u003c/li\u003e\n\u003cli\u003eAl-Taee, Z., \u0026amp; Saeed, M. G. (2022). Molecular Diagnosis of Adeno Virus Associated with Hydropericardium Hepatitis Syndrome of the Broiler Chickens in Nineveh Province, Iraq. Egyptian Journal of Veterinary Sciences, 53(4), 583-589.\u003c/li\u003e\n\u003cli\u003eLees, C.C., Romero, R., Stampalija, T., Dall\u0026rsquo;Asta, A., DeVore, G.R., Prefumo, F., Frusca, T., Visser, G.H., Hobbins, J.C., Baschat, A.A. and Bilardo, C.M., 2022. The diagnosis and management of suspected fetal growth restriction: an evidence-based approach. \u003cem\u003eAmerican journal of obstetrics and gynecology\u003c/em\u003e, \u003cem\u003e226\u003c/em\u003e(3), pp.366-378.\u003c/li\u003e\n\u003cli\u003eVatankhah, S., Ebrahimi, M., Taghizadeh, A. and Asadpour, R., 2025. Impacts of Trace Minerals Supplementation on Reproductive Performance and Blood Parameters of Ghezel Ewes. \u003cem\u003eIranian Journal of Applied Animal Science\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(1).\u003c/li\u003e\n\u003cli\u003eAraujo-Castro, M., Garc\u0026iacute;a Cano, A., Jim\u0026eacute;nez Mendiguch\u0026iacute;a, L., Escobar-Morreale, H.F. and Valderr\u0026aacute;bano, P., 2021. Diagnostic accuracy of the different hormonal tests used for the diagnosis of autonomous cortisol secretion. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(1), p.20539.\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":"Iraqi ewes, early pregnancy detection, progesterone assay, transrectal ultrasonography, hormonal diagnostics","lastPublishedDoi":"10.21203/rs.3.rs-6986774/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6986774/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study was conducted to evaluate and compare the diagnostic efficacy of transrectal ultrasonography (TRU) and serum progesterone (P4) assay for early pregnancy detection in local Iraqi ewes. A total of fifteen multiparous non-pregnant ewes (2\u0026ndash;4 years old) were synchronized using intravaginal sponges containing Medroxyprogesterone Acetate (MPA) and 500 IU equine chorionic gonadotropin (eCG), followed by natural mating. Blood samples were collected on days 0, 14, 21, and 30 post-inseminations (PI) for hormonal analysis. TRU was employed concurrently on the same days to visualize pregnancy indicators. The results revealed that TRU had limited accuracy during the early stages (day 14\u0026ndash;21 PI), with accuracy rates ranging from 26.7\u0026ndash;60%, while improving substantially by day 30 PI (accuracy: 100%). In contrast, serum P4 levels demonstrated a statistically significant elevation in pregnant ewes compared to non-pregnant and control groups from day 14 onward, achieving full diagnostic accuracy by day 21 PI. Receiver operating characteristic (ROC) analysis confirmed the high predictive value of P4, especially on days 21 and 30 (AUC\u0026thinsp;=\u0026thinsp;1.000). In conclusion, P4 assay serves as a more sensitive and reliable diagnostic tool for early pregnancy (days 14\u0026ndash;21), whereas ultrasonography offers superior accuracy at later gestational stages (day 30 onward), thus supporting a complementary role for both techniques in reproductive management.\u003c/p\u003e","manuscriptTitle":"Early Pregnancy Detection Using Progesterone Hormone and Ultrasonography in Local Iraqi Ewes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 11:33:05","doi":"10.21203/rs.3.rs-6986774/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":"387beb75-7365-4c20-b7c2-aeb5259f128f","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-17T14:29:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-16 11:33:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6986774","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6986774","identity":"rs-6986774","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}