Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recıbıdo: 26/11/2025 Aceptado: 28/01/2026 Publicado: 16/02/2026 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 9 Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Eﬀect of oestrus expression and eCG treatment on reproducve performance in dairy cows subjected to Progesterone plus Ovsynch protocol Efecto de la expresión del estro y del tratamiento con eCG sobre el desempeño reproducvo en vacas lecheras somedas a un protocolo de Progesterona más Ovsynch Baris Guner 1* , Melih Erturk 2 , Buse Ozturk 1 , Aslihan Ayalp-Erkan 1 , Mehmet Dursun 2 , Bekir Aydogan 2 ¹Balıkesir University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology, Balıkesir 10050, Türkiye ²Uluova Dairy Farm, Çanakkale, Türkiye *Corresponding author: baris.guner@balikesir.edu.tr ABSTRACT The objecves were to evaluate the eﬀects of equine chorionic gonadotropin on oestrus expression and ferlity in dairy cows undergoing a med arﬁcial inseminaon protocol. Holstein cows (n = 534) were subjected to a 7-day progesterone-based Ovsynch protocol. The equine chorionic gonadotropin group received 500 IU equine chorionic gonadotropin at progesterone device removal, whereas the control group received no addional treatment. Oestrus expression was monitored using an electronic neck-mounted acvity system and recorded during the voluntary waing period-oestrus expression and during synchronizaon prior to med arﬁcial inseminaon (pre med arﬁcial inseminaon-oestrus expression). The overall pre med arﬁcial inseminaon-oestrus expression rate during synchronizaon was 36.3 %. Administraon of equine chorionic gonadotropin did not (P > 0.05) inﬂuence the pre med arﬁcial inseminaon-oestrus expression (36.0 vs. 36.7 %) and pregnancy per arﬁcial inseminaon at day 30 (36.7 vs. 36.7 %). In addion, pregnancy loss, calving rate, and twinning rate did not diﬀer between equine chorionic gonadotropin and control groups (P > 0.05). During the voluntary waing period, 59.0 % of cows expressed oestrus. Pregnancy per arﬁcial inseminaon was greater (41.3 % vs. 32.4 %) in voluntary waing period-oestrus expression posive cows than voluntary waing period- oestrus expression negave cows (P = 0.04) regardless of group (equine chorionic gonadotropin and control). However, there was no signiﬁcant diﬀerence in pre med arﬁcial inseminaon- oestrus expression between equine chorionic gonadotropin and control groups in voluntary waing period-oestrus expression negave (P = 0.33) and voluntary waing period- oestrus expression posive cows (P = 0.91). Similarly, treatment with equine chorionic gonadotropin did not aﬀect pregnancy per arﬁcial inseminaon, either in voluntary waing period-oestrus expression–negave cows (P = 0.39) or in voluntary waing period-oestrus expression –posive cows (P = 0.69). In conclusion, oestrus expression during the voluntary waing period-oestrus expression and prior to med arﬁcial inseminaon-oestrus expression had a strong associaon with higher ferlity. However, equine chorionic gonadotropin did not enhance ferlity outcomes in high-producing dairy herds using sex-sorted semen within an opmised synchronizaon protocol, regardless of parity or cyclicity status. Key words: Dairy cows; equine chorionic gonadotropin; ferlity; oest- rus expression RESUMEN Los objevos de este estudio fueron evaluar los efectos de la gonadotropina coriónica equina sobre la expresión del estro y la ferlidad en vacas lecheras somedas a un protocolo de inseminación arﬁcial a empo ﬁjo. Vacas Holstein (n = 534) fueron somedas a un protocolo Ovsynch de 7 días basado en progesterona. El grupo gonadotropina coriónica equina recibió 500 UI de gonadotropina coriónica equina al momento de rerar el disposivo de progesterona, mientras que el grupo control no recibió tratamiento adicional. La expresión del estro se monitoreó mediante un sistema electrónico de acvidad montado en el cuello y se registró durante el periodo de espera voluntario (periodo de espera voluntario-expresión del estro) y durante la sincronización antes de la inseminación arﬁcial a empo ﬁjo (Pre-inseminación arﬁcial a empo ﬁjo-expresión del estro). La tasa global de expresión del estro Pre-inseminación arﬁcial a empo ﬁjo-expresión del estro durante la sincronización fue del 36,3 %. La administración de gonadotropina coriónica equina no inﬂuyó (P > 0,05) en la expresión del estro Pre-inseminación arﬁcial a empo ﬁjo-expresión del estro ni en la preñez por inseminación arﬁcial al día 30. Asimismo, la pérdida de gestación, la tasa de parto y la tasa de gemelaridad no diﬁrieron entre los grupos gonadotropina coriónica equina y control (P > 0,05). Durante el periodo de espera voluntario, el 59,0 % de las vacas expresó estro. La preñez por inseminación arﬁcial fue mayor en las vacas posivas a periodo de espera voluntario-expresión del estro en comparación con las vacas negavas a periodo de espera voluntario-expresión del estro (41,3 vs. 32,4 %; P = 0,04), independientemente del grupo de tratamiento. Sin embargo, no se observaron diferencias signiﬁcavas en la expresión del estro Preinseminación arﬁcial a empo ﬁjo-expresión del estro entre los grupos gonadotropina coriónica equina y control tanto en vacas periodo de espera voluntario-expresión del estro negavas (P = 0,33) como en vacas periodo de espera voluntario-expresión del estro posivas (P = 0,91). De manera similar, el tratamiento con gonadotropina coriónica equina no afectó la preñez por inseminación arﬁcial ni en vacas periodo de espera voluntario-expresión del estro negavas (P = 0,39) ni en vacas periodo de espera voluntario- expresión del estro posivas (P = 0,69). En conclusión, la expresión del estro durante el periodo de espera voluntario (periodo de espera voluntario-expresión del estro) y antes de la inseminación arﬁcial a empo ﬁjo (Pre-inseminación arﬁcial a empo ﬁjo-expresión del estro) se asoció fuertemente con una mayor ferlidad. Sin embargo, la gonadotropina coriónica equina no mejoró los resultados reproducvos en hatos lecheros de alta producción que ulizan semen sexado dentro de un protocolo de sincronización opmizado, independientemente de la paridad o del estatus cíclico. Palabras clave: Vacas lecheras; gonadotropina coriónica equina; fer- lidad; expresión del estro https://doi.org/10.52973/rcfcv-e361870

eCG and oestrus expression in synchronised dairy cows / Guner et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Intensive genec selecon of modern dairy cows for higher milk yield makes it diﬃcult to maintain postpartum health and subsequent ferlity [1]. As dairy herd producvity increases, new approaches are needed to improve herd management and ensure the eﬃcient use of available resources [2]. The increasing scale of herds and milk yields has rendered tradional oestrous detecon more challenging. Previous studies reported that monitoring the oestrus expression of cows (Bos taurus) during the voluntary waing period (VWP- OE, before the me of inseminaon has yet arrived) will be a strategic predictor for postpartum health and higher ferlity [1 ,[3]. However, med arﬁcial inseminaon (TAI) protocols have sll been in the dairy industry, eliminang the need for oestrous detecon [4]. Recent studies in dairy cows emphasize the crical role of oestrous expression (OE) at the near me of med arﬁcial inseminaon (TAI) in determining pregnancy per AI [4 , 5 ,6,7, 8 ,9]. Increased estradiol concentraons not only impact oestrous expression [10 , 11] but also inﬂuence sperm transport, ferlizaon, and create an uterine environment aﬀecng embryo development and pregnancy establishment [12 , 13 , 14]. Although increasing circulang estrogen levels by using addional oestradiol preparaon in synchronizaon protocols [10 , 15 , 16 , 17] seems to be the most logical opon, oestradiol preparaons in cale are restricted in the European countries [18]. Equine chorionic gonadotropin (eCG), a glycoprotein hormone with approximately a 40-hour half-life, is secreted by the endometrial cups of pregnant mares (Equus caballus) and exhibits both FSH and LH-like acvies in cale [19]. Treatment with eCG during late dioestrous/prooestrous stages accelerates the development of the dominant follicle, increases ovulatory follicle size, and elevates P4 concentraons in the subsequent cycle [20]. In progesterone-based TAI protocols, eCG is oﬅen co- administered with the removal of a progesterone-releasing intravaginal device (PRID) due to its aﬃnity for FSH and LH receptors [21 , 22]. The administraon of eCG concurrent with removing the progesterone implant has demonstrated enhancements in preovulatory follicle development, consequently leading to an augmented pregnancy per AI in beef cale [23], 24 , 25 , 26]. However, previous reports demonstrated that using eCG in TAI protocols increased [27 , 28 , 29] or did not change [30 , 31 , 32 , 33 , 34] the pregnancy rate in dairy cale. The primary objecve of this study was to invesgate the eﬀect of oestrus expression at the near me of AI on pregnancy per AI in dairy cows undergoing progesterone plus Ovsynch protocol. The secondary objecve of this study was to invesgate the eﬀect of eCG on oestrus expression and ferlity the ﬁrst and subsequent services in dairy cows undergoing progesterone plus Ovsynch protocol. MATERIALS AND METHODS All procedures for the maintenance and use of animals were approved by the Balıkesir University Animal Care and Use Commiee (Approval No: 2022/1-4). Herd management The study was conducted on a commercial dairy farm located in the northwestern region of Türkiye, with an average herd size of 1,900 lactang cows. The annual replacement rate of the herd was approximately 30 %. The lactaon number (mean ± Standard Error of Mean [SEM]) of mulparous cows was 2.2 ± 0.06. The voluntary waing period was ~90 d for primiparous cows and ~60 d for mulparous cows. Milking was carried out three mes per day at 8-hour (h) intervals, and mean daily milk yield per cow ranged between 40 and 43 kg throughout the lactaon period. Milk yield on the day of inseminaon and the corresponding week (± 3 d) was recorded via the herd management program. According to milk yield and days in milk (DIM), cows were grouped in free-stall barns and fed a total mixed raon based on NRC recommendaons [35], with ad libitum access to water. The farm had an automated acvity monitoring system (Dataﬂow II™, Allﬂex, Israel) necessary for data collecon. All cows were ﬁed with a neck collar containing an electronic idenﬁcaon tag. The system ulized a wireless receiver box to record average acvity every 2 h and calculate an acvity index (0–100). Oestrus was deﬁned as an acvity index > 35, consistent with previously validated thresholds [36]. Oestrus expression was deﬁned in two diﬀerent stage in this study. First, spontaneous oestrus expression was evaluated from calving unl the iniaon of the synchronizaon protocol (VWP- OE). Cows exhibit oestrus during this period were classiﬁed as oestrus-expressing, and those not showing oestrus were classiﬁed as anestrous. Second, protocol-induced oestrus expression was assessed from PRID removal unl the me of med arﬁcial inseminaon (preTAI-OE) during synchronizaon. These two deﬁnions were analyzed independently according to the objecves of the study. Experiment Experiment was designed as a randomized controlled trial to assess the eﬀect of eCG supplementaon on oestrus expression and ferlity. 534 Holstein-Friesian cows (primiparous, n = 153; mulparous, n = 381) were enrolled between April 2022 and December 2023. DIM was 82.8 ± 0.3 in primiparous cows and 64.3 ± 0.5 in mulparous cows. Cows were randomly allocated by odd versus even ear tag numbers into a control group (n = 259) and an eCG group (n = 275). Cows in the eCG group received 500 IU eCG (Folligon®, MSD Animal Health) intramuscularly at the me of progesterone device removal (d 7), while the control group did not. Synchronizaon protocol In both experiments, cows were synchronized using a 7-day progesterone-based Ovsynch protocol prior to the ﬁrst TAI. At the start of the protocol (day 0), an intramuscular injecon of gonadotropin releasing hormone (GnRH,0.1 mg gonadorelin acetate; Gestavet GnRH®, Hipra, Spain) was administered together with the inseron of a PRID containing 1.55 g P4 2 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico (PRID-DELTA®, Ceva, France). On day 7, the intravaginal device was withdrawn and cows were treated intramuscularly with cloprostenol (0.15 mg d-cloprostenol; Gestavet Prost®, Hipra, Spain). A second GnRH dose (0.1 mg) was given 56 h aﬅer device removal, followed by TAI using sexed semen 16 h later. Sexed semen was also used for the second and third inseminaons, whereas convenonal semen was applied from the fourth to the eighth services. Ultrasonographic examinaon A subsample of cows (n = 75) was examined to determine preovulatory follicle size at TAI. Ovarian structures were evaluated by transrectal ultrasonography using a 7.5 MHz linear- array probe (Ibex Pro®, E.I. Medical Imaging, Loveland, CO). The largest follicle was measured in two perpendicular diameters, and the mean was recorded as follicle size. All examinaons were performed by the same experienced operator to minimize variaon. Pregnancy diagnoses were performed with the same system on days 31 ± 3 and 60 ± 3 post-AI. Pregnancy per arﬁcial inseminaon (P/AI) was deﬁned as the number of pregnant cows divided by the number of inseminated cows at each diagnosis. Pregnancy loss was calculated from the inial diagnosis of pregnancy (d 31 ± 3) to conﬁrmaon of pregnancy (d 60 ± 3). Pregnancy losses were reported for d 30–60 and d 30–calving. Calving rate was deﬁned as calved cows divided by enrolled cows, and twinning rate as twin calvings divided by total calvings. Stascal analysis Data were analyzed using SPSS 27.0 (IBM, Chicago, IL, USA). The primary outcomes included two diﬀerent measures of oestrus expression: (1) VWP-OE (Voluntary Waing Period Oestrus Expression), deﬁned as oestrus observed from calving unl the iniaon of the synchronizaon protocol, and (2) preTAI-OE, deﬁned as oestrus observed from PRID removal unl med arﬁcial inseminaon (TAI). Addional outcomes were P/ AI, calving rate, twinning rate, pregnancy loss (d 30–60 and d 60– calving), total pregnancy loss, return to oestrus, second-service P/AI, days open, number of AIs per pregnancy, and overall P/AI. Categorical variables (e.g., VWP-OE, preTAI-OE, P/AI, calving rate) were compared using chi-square or Fisher’s exact test. Connuous variables (DIM, milk yield etc.) were analyzed by Student’s t-test or Mann–Whitney U-test depending on distribuon. Logisc regression was used to evaluate associaons between oestrus expression, eCG treatment, and P/AI, with parity included as a ﬁxed eﬀect. Survival analysis (Kaplan–Meier with log-rank test) was used for d open. Fixed eﬀects were considered signiﬁcant at P ≤ 0.05. Results are presented as mean ± S.E.M. for connuous outcomes and as percentages for categorical outcomes. RESULTS AND DISCUSSION In this study, milk yield (mean ± S.E.M.) at the d of TAI was 44.2 ± 1.5 kg and 46.2 ± 1.1 kg for primiparous cows in the eCG and control groups, respecvely, and 53.4 ± 1.3 kg and 54.1 ± 1.1 kg for mulparous cows (TABLE I). Similarly, average weekly milk yield during the two weeks preceding TAI did not diﬀer between treatments in primiparous cows (45.1 ± 1.4 vs. 46.5 ± 1.2 kg/day) or mulparous cows (52.7 ± 1.3 vs. 53.3 ± 1.2 kg/d). Mean milk yield during the ﬁrst 100 DIM was also comparable between the eCG and control groups in primiparous (41.6 ± 1.2 vs. 42.3 ± 1.0 kg/d) and mulparous cows (49.8 ± 1.1 vs. 50.5 ± 1.0 kg/d). Likewise, 305-d mature-equivalent milk yield did not diﬀer between treatments, with primiparous cows producing 10,420 ± 215 kg and 10,570 ± 208 kg, and mulparous cows producing 11,960 ± 240 kg and 12,030 ± 225 kg in the eCG and control groups, respecvely. Overall, milk producon parameters at all evaluated me points were similar between treatments. TABLE I Comparison of oestrus expression, pregnancy per arﬁcial inseminaon, pregnancy loss, calving rate and twinning rate between equine chorionic gonadotropin and control groups according to parity in dairy cows subjected to Progesterone plus Ovsynch protocol Primiparous Mulparous p value Variables eCG Control eCG Control Parity Treatment Parity × Treatment Oestrus expression at preTAI; % (n/n) 40.3 (31/77) 46.1 (35/76) 34.3 (68/198) 32.8 (60/183) 0.04 0.64 0.42 P/AI on d 30; % (n/n) 41.6 (32/77) 44.7 (34/76) 34.8 (69/198) 33.3 (61/183) 0.05 0.86 0.61 Pregnancy loss 30 to 60 d; % (n/n) nd (0/32) nd (0/34) 2.9% (1/69) 8.2 (1/61) 0.31 0.95 0.95 P/AI on d 60; % (n/n) 41.6 (32/77) 44.7 (34/76) 34.3 (68/198) 32.8 (60/183) 0.04 0.86 0.61 Pregnancy loss 30 d to calving; % (n/n) 3.1 (1/32) nd (0/34) 7.3 (5/68) 6.6 (4/60) 0.11 0.56 0.73 Total Pregnancy loss; % (n/n) 3.1 (1/32) nd (0/34) 8.7 (6/69) 8.1 (5/61) 0.04 0.50 0.88 Calving rate; % (n/n) 40.3 (31/77) 44.7 (34/76) 32.3 (64/198) 31.1 (57/183) 0.02 0.53 0.72 Twinning rate; % (n/n) nd (0/31) nd (0/34) 4.7 (3/64) 5.3 (3/57) 0.07 0.92 0.92 Oestrus expression at preTAI: Pre–Timed Arﬁcial Inseminaon Oestrus Expression (preTAI-OE), deﬁned as oestrus detected by the automated acvity monitoring system from PRID removal unl TAI during synchronizaon. P/AI: Pregnancy per arﬁcial inseminaon. eCG: Cows received 500 IU eCG on days 7 in TAI protocol. Control: Cows did not addional hormone in TAI protocol. nd: not detected. Calving rate calculated by divided the number of calving cows to enrolled cows to study. Twinning rate was also calculated by divided the calving cows that had twin calves to calving cows. 3 of 9

eCG and oestrus expression in synchronised dairy cows / Guner et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The overall preTAI-OE rate during synchronizaon was 36.3 % (194/534) in cows. Primiparous cows had a higher preTAI- OE rate than mulparous cows (43.1 % vs. 33.6 %, P = 0.04) regardless of treatment group (eCG and control). In primiparous cows, preTAI-OE was 40.3 % in the eCG group and 46.1 % in the control group, while in mulparous cows the corresponding rates were 34.3 % and 32.8 %, respecvely. Administraon of eCG did not aﬀect preTAI-OE P = 0.64), and no signiﬁcant parity × treatment interacon was detected for preTAI-OE (P = 0.42, TABLE I). The ﬁrst objecve of this study was to determine whether the expression of oestrus preTAI-OE was associated with improved P/AI in lactang dairy cows synchronised with a progesterone- based Ovsynch protocol. In this study, preTAI-OE was 36.3 % during the synchronizaon, a proporon that is greater than the approximately 20 % reported in earlier studies using convenonal Ovsynch [37 , 38], and consistent with more recent studies reporng oestrus expression rates ranging between 30 % and 40 % [4 ,7,[39]. Diﬀerences among studies appear to be strongly inﬂuenced by the type of inseminaon protocol used. Protocols incorporang presynchronizaon, such as Double- Ovsynch, typically yield higher oestrus expression than protocols that rely solely on Ovsynch. For example, Laplacee et al. [4] and Lauber and Fricke [39] observed oestrus expression rates of 36.1 and 29.1 %, respecvely, in cows enrolled in a Double- Ovsynch protocol. These ﬁndings indicate that milk yield was not a confounding factor aﬀecng reproducve outcomes. Although high- producing cows typically exhibit enhanced metabolic clearance of ovarian steroids, leading to reduced circulang estradiol and progesterone [11], the hormonal synchronizaon protocol used (PRID + Ovsynch) may have minimised such metabolic eﬀects by providing suﬃcient endocrine control during the periovulatory period. Addionally, supplementaon of progesterone device in this study may also have contributed to the preTAI-OE. Progesterone supplementaon provides a controlled luteal-phase environment that supports the emergence of a new follicular wave and enhances the uniformity of follicular development [5]. Higher circulang progesterone prior to luteolysis, parcularly 3–4 d before TAI, has been associated with enhanced preovulatory estradiol (E2) producon and stronger behavioural expression of oestrus [40]. Despite this, the reproducve physiology of high-producing dairy cows presents unique metabolic challenges that can modulate the hormonal responsiveness expected during synchronizaon. Elevated hepac blood ﬂow and intensiﬁed metabolic acvity in high-yielding cows accelerate the clearance of ovarian steroids, reducing circulang concentraons of both progesterone and estradiol [11]. This increased metabolic clearance rate may aenuate the ancipated smulatory eﬀects of progesterone on follicular E2 synthesis, thereby reducing the manifestaon of oestrus despite adequate follicular development. Addionally, the altered endocrine environment may limit the potenal for exogenous gonadotropins, such as eCG, to augment follicular development or increase E2 producon [22]. In this study, preovulatory follicle size at inseminaon was not aﬀected (P = 0.25) by eCG treatment. In primiparous cows, follicle size was 14.50 ± 0.44 mm in the eCG group and 14.88 ± 0.55 mm in the control group. In mulparous cows, follicle size was 15.41 ± 0.33 mm in the eCG group and 14.68 ± 0.34 mm in the control group.These metabolic constraints likely contributed to the absence of an eCG eﬀect on preovulatory follicle size in this study. Oestrus expression at preTAI remains an important determinant of ferlity in TAI programs. High-producing dairy cows oﬅen exhibit diminished LH pulsality and reduced circulang estradiol, both of which weaken the behavioural signs of oestrus [11]. Reduced intensity or duraon of oestrus is parcularly consequenal because robust oestrus expression is ghtly linked to improved P/AI [7 , 8]. Adequate preovulatory E2 promotes the onset of behavioural oestrus and opmises the uterine environment, sperm transport, and early embryonic development [12 , 14]. In agreement with previous ﬁndings, cows that expressed oestrus before TAI had higher P/AI than those that did not in this study. Several recent studies corroborate this relaonship, reporng similar associaons between oestrus expression and improved ferlity under synchronised breeding programs [7 , 8 ,9], 39 , 40 , 41]. Reduced detecon of oestrus by automated acvity monitoring systems has been associated with lower ferlity in TAI programs [40]. Although TAI protocols such as Ovsynch were designed to reduce dependence on heat detecon [42], the presence of pre-TAI oestrus expression connues to serve as a reliable indicator of opmal endocrine status and improved reproducve potenal. Regarding the ﬁrst TAI, P/AI was higher in primiparous cows compared with mulparous cows both on d 30 (43.1 % vs. 34.1 %; P = 0.05) and d 60 (43.1 % vs. 33.6 %; P = 0.04). However, P/ AI did not diﬀer between eCG and control groups on either d 30 (P = 0.86) or d 60 (P = 0.86). The second major objecve of this study was to assess whether administraon of 500 IU eCG at the me of progesterone device removal inﬂuenced reproducve outcomes in cows synchronised with a progesterone-based Ovsynch protocol. The administraon of eCG did not inﬂuence preTAI-OE or P/AI in this study. Previous studies have produced inconsistent results regarding the beneﬁts of eCG in dairy cows. While some studies have reported no signiﬁcant eﬀect of eCG on oestrus expression or ferlity [34 , 43], others have noted improvements in anoestrous cows or in speciﬁc subgroups [27 , 29]. For example, Bryan et al. [27] observed improved ferlity only in anoestrous cows older than ﬁve years, and Bryan et al. [28] reported enhanced P/AI in anovulatory cows treated with eCG during progesterone-based synchronizaon. Garcia-Ispierto et al. [29] found increased ferlity at 120 d in milk in cows without luteal acvity prior to treatment, suggesng a potenal beneﬁt of eCG in cows with compromised ovarian cyclicity. The inconsistent response to eCG between dairy and beef cale has been widely discussed. Many studies have documented posive eﬀects of eCG in beef cows, parcularly in postpartum anoestrous animals [23], 24 , 25 , 26], 44 , 45 , 46]. Several mechanisc diﬀerences appear to underpin this disparity. Compared with dairy cows, beef cale have lower metabolic demands, more stable endocrine proﬁles, and slower metabolic clearance of ovarian steroids. These physiological condions support higher circulang estradiol levels following eCG-induced follicular smulaon and allow for a more pronounced LH surge [23], 24]. In contrast, in high-producing dairy cows, negave energy balance, reduced 4 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico IGF-1 concentraons, and impaired LH pulsality limit follicular responsiveness to gonadotropins [47]. These factors likely explain why eCG commonly enhances follicular acvity and ferlity in beef cale but yields limited beneﬁts in high-yielding dairy herds. Pregnancy loss between d 30 and 60 was not observed in primiparous cows in either group, while mulparous cows exhibited rates of 2.9 % in the eCG group and 8.2 % in the control group. Pregnancy loss from day 30 to calving was 3.1 % in primiparous cows in the eCG group, whereas no losses were recorded in the control group. In mulparous cows, the corresponding values were 7.3 % in the eCG group and 6.6 % in the control group. None of these diﬀerences were stascally signiﬁcant (P > 0.05). Overall, pregnancy loss from days 30–60 (P = 0.95), days 30–calving (P = 0.56), and total pregnancy loss (P = 0.50) was not inﬂuenced by eCG treatment (TABLE I). Consistent with previous studies, eCG treatment did not aﬀect pregnancy loss in this study. Pregnancy loss between d 30 and 60 of gestaon was low, and no diﬀerences were noted between the treatment groups, consistent with reports indicang that eCG does not inﬂuence pregnancy loss during this stage [43 , 48]. Since pregnancy losses are relavely uncommon in this window [21], a signiﬁcant treatment eﬀect would not be expected. The lack of diﬀerences in pregnancy loss from day 60 to calving further supports the conclusion that supplementaon with eCG in this protocol does not inﬂuence late gestaonal outcomes. The role of eCG is generally more prominent in the early post-ovulatory period, when its acons on follicular development and luteal funcon may be more biologically relevant. No twinning cases were observed in primiparous cows. In mulparous cows, the twinning rate was 4.7% in the eCG group and 5.3 % in the control group, with no signiﬁcant diﬀerence between groups (P = 0.92). Therefore, parity had a signiﬁcant eﬀect on P/AI, calving rate, and twinning rate, while no treatment × parity interacon was observed (TABLE I). Finally, the absence of a treatment eﬀect on twinning rate is noteworthy. Twinning is an undesirable outcome associated with increased pregnancy loss [49], postpartum disorders, and reduced longevity in dairy cows [50], 51 , 52 , 53]. Although doses exceeding 1000 IU of eCG can induce mulple ovulaons [22], the 500 IU dose used in this study is generally considered insuﬃcient to increase twinning risk. This aligns with previous ﬁndings in dairy cale [54], and our results further demonstrate that the administraon of eCG at this dose does not increase the likelihood of twin pregnancies, even when ovulaon number is not directly assessed. Overall, during the voluntary waing period, the proporon of anestrous (VWP-OE negave) cows was found to be 41 % (219/534). The presence or absence of oestrus expression during VWP did not aﬀect (P = 0.17) preTAI-OE during the TAI protocol (33.9 vs. 39.7 %). However, VWP-OE posive cows had greater (41.3 vs. 32.4 %) P/AI aﬅer the ﬁrst AI compared with VWP-OE negave cows (P = 0.04, TABLE II). TABLE II Oestrus expression near med arﬁcial inseminaon and pregnancy per arﬁcial inseminaon relave to oestrus expression during voluntary waing period in dairy cows Variables VWP-OE Posive VWP-OE Negave P value Number of animals; (n/n) 59.0 a (315/534) 41.0 b (219/534) < 0.01 Oestrus expression at preTAI; % (n/n) 33.9 (107/315) 39.7 (87/219) 0.17 P/AI on day 30; % (n/n) 41.3 a (125/303) 32.4 b (71/219) 0.04 a,b: Percentages with diﬀerent superscripts within a row are diﬀerent (P < 0.05). VWP-OE posive: Cows expressed oestrus during voluntary waing period. VWP-OE negave: Cows did not expressed oestrus during voluntary waing period. WP-OE: Voluntary Waing Peri- od Oestrus Expression, deﬁned as oestrus detected by the automated acvity monitoring system from calving unl iniaon of the synchronizaon protocol Oestrus expression at preTAI (preTAI-OE): deﬁned as oestrus detected by the automated acvity monitoring sys- tem from PRID removal unl TAI during synchronizaon P/AI: Pregnancy per arﬁcial inseminaon During the voluntary waing period, 59.0 % of cows expressed oestrus (VWP-OE posive) in this study (TABLE II). Higher percentage of anoestrous cows may reﬂect the physiological demands imposed by early lactaon. High- producing cows undergo substanal metabolic stress during this period, requiring large amounts of energy to support milk synthesis. Failure to adapt to this metabolic load may delay the resumpon of ovarian cyclicity and prolong the period of anoestrus. Low circulang concentraons of estradiol resulng from accelerated steroid metabolism [11] may further limit the expression of oestrus and subsequent ferlity [12 , 14]. Nevertheless, cows that expressed oestrus during the VWP exhibited approximately 7 % higher P/AI aﬅer ﬁrst service, consistent with previous studies reporng oestrus expression rates of 50–55 % within 60 d in milk [3 , 55] and even higher rates of 65–70 % in more recent cohorts [1 , 56]. Diﬀerences in parity distribuon and the shorter duraon of monitoring in mulparous cows may partly explain the lower oestrus expression observed in some animals [3]. Variability in environmental condions, management pracces, and metabolic adaptaon could also contribute to divergent ﬁndings across studies [40]. Increasing evidence suggests that automated oestrus detecon during the VWP can serve as a useful predictor of reproducve potenal, further highlighng the value of early oestrus expression [2 , 3]. There was no signiﬁcant diﬀerence in preTAI-OE between eCG and control groups in VWP-OE negave (P = 0.33) and VWP-OE posive cows (P = 0.91). Similarly, treatment of eCG did not aﬀect P/AI, either in VWP-OE–negave cows (P = 0.39) or in VWP-OE–posive cows (P = 0.69, TABLE III). It is possible that the opmised synchronizaon protocol used here provided adequate endocrine control to migate the negave eﬀects of high metabolic load on ferlity. While eCG can support follicular growth and enhance luteal funcon, its eﬀects are typically short-lived [22] and may not extend into subsequent cycles. Moreover, sex-sorted semen inherently has lower ferlisaon potenal [35], which may diminish any subtle beneﬁt of eCG. 5 of 9

eCG and oestrus expression in synchronised dairy cows / Guner et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE III Distribuon of anoestrus, oestrus expression, oestrus frequency during the voluntary waing period, and pregnancy per arﬁcial inseminaon by parity and equine chorionic gonadotropin treatment Primiparous Mulparous P value eCG (n = 77) Control (n = 76) eCG (n = 198) Control (n = 183) Parity eCG eCG x Parity Oestrus expression at pre-TAI in anestrus cows; % (n/n) 40.0 (14/35) 53.3 (16/30) 36.7 (33/90) 37.5 (24/64) 0.19 0.33 0.39 P/AI in anestrus cows % (n/n) 40.0 (14/35) 33.3 (10/30) 30.3 (27/90) 31.2 (20/64) 0.70 0.39 0.57 Oestrus expression at preTAI in cows expressed oestrus during VWP % (n/n) 40.5 (17/42) 41.3 (19/46) 32.4 (35/108) 30.2 (36/119) 0.11 0.91 0.80 P/AI in cows expressed oestrus during VWP % (n/n) 42.9 (18/42) 50.0 (23/46) 38.9 (42/108) 34.5 (41/119) 0.08 0.69 0.26 Oestrus expression at preTAI (preTAI-OE): deﬁned as oestrus detected by the automated acvity monitoring system from PRID removal unl TAI during synchronizaon. Anestrus cows: Cows did not expressed oestrus during voluntary waing period (VWP-OE negave). Expressed oestrus during VWP: Cows expressed oestrus during voluntary waing period (VWP-OE posive). P/AI: Pregnancy per arﬁcial inseminaon The rate of cows returning to oestrus aﬅer the ﬁrst TAI was 47.3 % (160/338). This parameter did not diﬀer between groups (P = 0.92) or paries (P = 0.96). The P/AI aﬅer the second service was 27.5 %, with no diﬀerences detected between treatment groups (P = 0.58) or paries (P = 0.43). The number of AI per pregnancy ranged from 2.04 to 2.49. Overall P/AI did not diﬀer between treatment groups (P = 0.15). The addion of eCG to the ﬁrst TAI protocol did not inﬂuence return-to-oestrus rates, P/AI at the second service, number of AI per pregnancy, or days open compared with the control group (P > 0.05, TABLE IV). Despite expectaons that high milk yield and the use of sex-sorted semen could negavely impact ferlity [35], no adverse eﬀect was detected on days open or AI per pregnancy. TABLE IV Return rate to oestrus aﬅer the ﬁrst TAI, pregnancy per arﬁcial inseminaon aﬅer the second service, the number of arﬁcial inseminaon per pregnancy, and open days between equine chorionic gonadotropin and control groups in cows Primiparous Mulparous P value eCG Control eCG Control Parity Treatment Parity × Treatment Return rate to oestrus; % (n/n) 46.7 (21/45) 47.6 (20/42) 47.3 (61/129) 47.5 (58/122) 0.96 0.92 0.96 Second service P/AI; % (n/n) 37.8 (17/45) 23.8 (10/42) 22.5 (29/129) 30.3 (37/122) 0.43 0.58 0.05 Open days 128.97 ± 5.69 139.64 ± 6.54 125.76 ± 4.87 121.88 ± 4.43 0.07 0.55 0.21 Number of AI per pregnancy 2.04 ± 0.15 2.42 ± 0.19 2.49 ± 0.13 2.46 ± 0.13 0.12 0.28 0.21 Overall P/AI; % (n/n) 87.0 (67/77) 97.4 (74/76) 81.8 (162/198) 81.4 (149/183) <0.01 0.15 0.12 Return rate to oestrus refers to cows expressed oestrus aﬅer the ﬁrst inseminaon. Open days refers to the interval between calving and the establishment of a conﬁrmed pregnancy The opmised progesterone-based synchronizaon may have provided adequate endocrine support to standardise responses, thereby masking any potenal long-term beneﬁts of eCG. Since eCG’s luteotropic eﬀects are short-lived and typically do not extend beyond the immediate post-ovulatory period [22], its inability to improve second-service ferlity or days open is consistent with physiological expectaons. Finally, in the present study, neither P/AI nor other ferlity parameters such as d open, the number of AI per pregnancy, or responses at the second service diﬀered between the eCG and control groups. 6 of 9

Revista Cienﬁca, FCV-LUZ / Vol. XXXVI UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico CONCLUSION The present study demonstrates that oestrus expression during the voluntary waing period (VWP-OE) and during synchronizaon prior to med arﬁcial inseminaon (preTAI- OE) remains a strong indicator of enhanced ferlity in high- producing dairy cows, even within synchronised breeding programs. Although eCG oﬀers well-documented beneﬁts in beef cale and certain subsets of dairy cows, its administraon at 500 IU did not improve preTAI-OE, P/AI, or other reproducve outcomes in this high-yielding herd managed under an opmised progesterone-based Ovsynch protocol. The standardisaon of management pracces, consistent monitoring, and robust hormonal control provided by the protocol may have stabilised reproducve outcomes suﬃciently to eliminate measurable diﬀerences between groups. Finally, these ﬁndings suggest that the usefulness of eCG in dairy cows may be limited value in well-managed herds characterised by high producon levels and eﬃcient reproducve management. Conﬂict of Interest All authors declare that they have no conﬁct of interest. Funding This study was supported by the Scienﬁc Research Projects Coordinaon Unit of Balıkesir University (BAP project no. 2022/008). BIBLIOGRAPHIC REFERENCES [1] Bruinjé TC, Morrison EI, Ribeiro ES, Renaud DL, Couto- Serrenho R, LeBlanc SJ. Postpartum health is associated with detecon of estrus by acvity monitors and reproducve performance in dairy cows. J. Dairy Sci. [Internet]. 2023; 106(12):9451–9473. doi: hps://doi. org/qqzs [2] Rial C, Giordano JO. Combining reproducve outcomes predictors and automated estrus alerts recorded during the voluntary waing period idenﬁed subgroups of cows with diﬀerent reproducve performance potenal. J. Dairy Sci. 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