Ovarian response in water buffaloes induced to ovulate outside the breeding season

Ovarian response in water buffaloes induced to ovulate outside the breeding season | 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 Ovarian response in water buffaloes induced to ovulate outside the breeding season Yuliana Izquierdo-Camacho, Nadia Ojeda-Robertos, Víctor Hugo Severino-Lendechy, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7048963/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 The study evaluated the effect of two hormonal protocols on the ovarian activity and pregnancy rate of water buffaloes ( Bubalus bubalis ) outside the breeding season. Twenty multiparous Murrah × Mediterranean buffaloes were included. The protocols used were the following: 1) GnRH (n = 10): 100 µg gonadorelin IM (Day 0), 500 µg cloprostenol IM (Day 7), 100 µg gonadorelin IM (Day 9) and fixed time artificial insemination (FTAI) 18 h after the second GnRH; 2) P4 + eCG (n = 10): intravaginal progesterone (P4) device + 2 mg estradiol benzoate (Day 0), device removal + 500 µg cloprostenol + 400 IU eCG (Day 10), 10 µg buserelin acetate (Day 12) and FTAI 54 h after device removal. The numbers of small ( 8.1 mm) follicles (follicular population) were counted through ultrasonography. The diameters of the largest follicle (maximum follicular diameter; MFD) and corpus luteum (CL) were evaluated. Blood samples were obtained on Days 0, 9, 17, 24 and 31 in the GnRH group and on Days 0, 10, 19, 26 and 33 in the P4 + eCG group to determine serum P4 concentrations. No differences were found between groups in follicular population, MFD and CL diameter. The presence of CL and P4 concentrations were greater in the P4 + eCG group. Only females from the P4 + eCG group became pregnant and P4 concentrations were > 3 ng/ml on Days 26 and 33. In conclusion, the P4 + eCG protocol was effective to induce ovulation and achieve pregnancy in buffalo cows outside the breeding season under tropical conditions. corpus luteum reproductive seasonality follicular population Bubalus bubalis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The population of water buffaloes ( Bubalus bubalis ) has increased in the last years due to their ability to adapt to hostile environments (Naveena and Kiran, 2014 ), their resistance to diseases (Grazziotto et al., 2021 ) and their utilization of low-quality forages as food (de Moura Lima et al., 2021 ), allowing them to thrive in environments where cattle cannot. However, it is considered that buffaloes have low reproductive efficiency due to their delayed puberty and manifestation of silent estrus, making it difficult to determine the appropriate moment for artificial insemination (AI) (Qayyum et al., 2018 ). In addition, since buffaloes are short-day breeders (Sagheer et al., 2020 ), they frequently show a prolonged seasonal anestrus (Devkota et al., 2023 ). Therefore, some strategies have been implemented to increase pregnancy rate in buffaloes outside their breeding season, such as the use of hormone treatments to induce ovulation in anestrus females (Carvalho et al., 2017 ), which would allow to produce calves and therefore milk in months when its production is very low or non-existent, thus favoring productivity and economy throughout the year (Frares et al., 2013 ). Some protocols that include progesterone intravaginal devices combined with estradiol (Carvalho et al., 2017 ) and equine chorionic gonadotropin (eCG) (Baruselli et al., 2018 ; Neglia et al., 2020 ) have been used, which, although expensive, have resulted in pregnancy rates after fixed timed AI (FTAI) up to 60.2% (Monteiro et al., 2018 ). Protocols based in GnRH have also been used, resulting in pregnancy rates of 50% during the breeding season (Pottapenjera et al., 2018 ; Du et al., 2021 ) and lower than 24% outside the breeding season (Sharma et al., 2017 ; Rathore et al., 2017 ; Kavita et al., 2018). Therefore, the objective of the study was to determine the ovarian activity and pregnancy rate using two protocols for the induction of ovulation outside the breeding season in water buffaloes under tropical climate conditions. Materials and methods Study location The study was conducted from May to June in a production unit located in the municipality of Centro, in the state of Tabasco, Mexico (17°54'13"N 93°06'06"O), at altitude of 13 m. The climate is warm and wet with summer rains, mean annual temperature of 27°C, mean relative humidity of 80% and annual rainfall of 2550 mm (INEGI, 2024). Management of experimental animals Twenty Murrah × Mediterranean multiparous buffaloes (3.3 ± 1.5 parturitions) were included in the study. The females averaged 3.3 ± 0.4 of body condition score (BCS; 1 to 5 scale; Magsi et al., 2022 ), 117 ± 36 days postpartum and 444 ± 67 kg of live weight. The buffalo cows underwent ultrasonographic evaluation (Mindray® SD-50; 7.5 MHz transducer) to verify the absence of a CL and any uterine or ovarian anomalies. Feeding was based on rotational grazing of native grasses ( Paspalum fasciculatum, P. notatum, P. conjugatum ) plus mineral blocks and 2 kg of commercial concentrate (14% crude protein) during milking, which was once a day at 06:00 h; water was available ad libitum . Hormonal treatments for induction of ovulation and artificial insemination The buffalo cows were randomly assigned to two treatments of 10 females each for induction of ovulation: 1) GnRH and 2) P4 + eCG. In the GnRH group, the females received one intramuscular (IM) injection with 10 µg of a GnRH agonist (buserelin acetate; Sincroforte ® , Ourofino, Mexico) on Day 0; on Day 7 they received 500 µg of a PGF 2α analogue (cloprostenol; Ciclase ® , Lab. Syntex, Argentina) IM; on Day 9 the females were given a second injection with 10 µg of a GnRH agonist and received FTAI 18 h later (Du et al., 2021 b). In the P4 + eCG group, on Day 0 the females received one intravaginal device containing 1 g of natural P4 (Cronipres ® , Lab. Biogénesis Bagó, Mexico) plus 2 mg of estradiol benzoate (Benzoato de Estradiol ® , Lab. Syntex, Argentina) IM; on Day 10 the intravaginal device was removed and the females were given 500 µg of cloprostenol plus 400 IU of eCG (Folligon ® , Intervet, Mexico) IM; on Day 12 one injection with 10 µg of buserelin acetate was administered; finally, FTAI was performed 54 h after the intravaginal device removal (Srirattana et al., 2022 ). In both treatments, FTAI was made with frozen-thawed semen and performed by the same technician. The sequence of hormone administration in both treatments is shown in Fig. 1 . Evaluation of ovarian activity Ovarian activity was evaluated through transrectal ultrasonography (Mindray® SD-50, 7.5 MHz transducer) and all the evaluations were performed by the same technician. The follicle population in the GnRH group was started the day of PGF₂α injection (Day 7) and from then on, every 12 h until FTAI (Day 10). In the P4 + eCG group the follicle population started the day of the intravaginal device removal (Day 10) and from then on, every 12 h until FTAI (Day 12). In both groups the evaluation included both ovaries, counting and measuring the diameter of all the follicles, classifying them as small (< 4 mm), medium (4 − 7.9 mm) and large (≥ 8 mm) (Gaur and Purohit, 2019 ). The presence and diameter of the dominant follicle were recorded until FTAI. The maximum follicular diameter was obtained averaging the horizontal and vertical diameters. The follicle with a diameter ≥ 10.3 ± 0.5 mm was considered as dominant follicle (Abulaiti et al., 2022 ). Evaluation of the corpus luteum (CL) The presence and size of the CL were determined by ultrasonography on Days 17, 24 and 31 in the GnRH group and on Days 19, 26 and 33 in the P4 + eCG group. The diameter of the CL (DCL) was obtained averaging the horizontal and vertical diameters. Measurement of serum progesterone Blood samples were obtained via coccygeal venipuncture using 21 G disposable needles and heparinized tubes (5 ml) on Days 0, 9, 17, 24 and 31 in the GnRH group and on Days 0, 10, 19, 26 and 33 in the P4 + eCG group. The samples were centrifuged at 1500 rpm/15 min at maximum 4 h after being drawn. The serum was separated into aliquots that were frozen at -20°C until hormone determination. Progesterone concentrations were measured through enzyme immunosorbent assay using commercial kits (Progesterone ELISA; DRG, USA). The sensitivity of the assay was 0.045 ng/ml, the coefficient of variation intra and inter assay was 5.4% and 9.9%, respectively. Progesterone concentrations are presented as ng/ml. Pregnancy and embryo loss diagnosis Pregnancy was diagnosed through real-time ultrasonography on Days 30 and 60 after FTAI in both groups. Embryo loss was determined when an embryonic vesicle found on Day 30 was not present on Day 60 (Barile et al., 2021 ). Statistical analysis The normality of the data and homogeneity of variances were evaluated. For the variables of follicle population, diameter of the ovulatory follicle, DCL and progesterone levels, a statistical model that included the effects of treatment, time and their interaction was used. Considering the data of time as repeated measures, a GLM analysis was conducted and means were compared through the Tukey’s test. Statistical significance was set at P < 0.05. The variables of pregnancy diagnosis and embryo loss were categorized binomially, and the Chi-square and Fisher’s exact tests were performed using the PROC FREQ procedure. All the tests were from the SAS software (SAS, 2000). Results A lower population of small follicles (< 4mm) was observed in the P4 + eCG group (P 0.05) (Table 1 ). Regarding the maximum follicular diameter, at the time of device removal or PGF₂α administration it averaged 10.8 mm in both groups (P > 0.05); 12 h later the follicular diameter was larger in the GnRH group (12.7 mm) (P 0.05) (Fig. 2 ). Table 1 Population of small (≤ 4 mm), medium (4.1–8 mm) and large (≥ 8.1 mm) follicles in water buffaloes induced to ovulation with GnRH and P4 + eCG outside the breeding season. GnRH group P4 + eCG group Days of hormonal protocol Days of hormonal protocol Follicle size (mm) Day 7 PGF Day 8 Day 9 2nd GnRH Day 10 FTAI Day 10 Device removal PGF + eCG Day11 Day 12 GnRH h 18:00 06:00 18:00 06:00 18:00 12:00 10:00 18:00 06:00 18:00 06:00 FTAI 16:00 < 4 14 ± 4.9 a 12.7 ± 4.5 a 13 ± 4.2 a 11.5 ± 4.9 a 13.1 ± 4 a 12.1 ± 3.2 a 16.9 ± 4.7 b 16.9 ± 4.7 b 15.6 ± 2.0 b 15.6 ± 2.6 b 15.6 ± 2.6 b 15.9 ± 3.4 b 4.1-8 5.2 ± 2.5 a 5.1 ± 2.6 a 6.6 ± 2.1 a 6.9 ± 2.3 a 6.5 ± 2.9 a 6.5 ± 2.9 a 6.2 ± 3.7 a 6.2 ± 3.7 a 7.4 ± 3.1 a 7.4 ± 3.2 a 7.1 ± 3.3 a 7.4 ± 3.5 a < 8.1 1.6 ± 0.7 a 1.6 ± 0.7 a 1.3 ± 0.5 a 1.4 ± 0.5 a 1.2 ± 0.4 a 1.2 ± 0.4 a 1.1 ± 0.3 a 1.1 ± 0.3 a 1.3 ± 0.3 a 1.3 ± 0.7 a 1.3 ± 0.7 a 1.4 ± 0.7 a ab Different literals for each day between groups indicate statistical difference (P 0.05) on Days 17–19, 24–26 and 31–33 (Table 2 ). Likewise, there was no difference in the DCL between groups (Table 3 ). Table 2 Percentage of water buffaloes with a corpus luteum (CL) after fixed-time artificial insemination and early pregnancy diagnosis following induction to ovulation with GnRH or P4 + eCG outside the breeding season. Females with CL % Group Days of hormonal protocol 17 19 24 26 31 33 GnRH 50 NE 40 NE 10 NE P4 + eCG NE 70 NE 60 NE 50 There was no difference between groups (P > 0.05). NE: not evaluated. Table 3 Corpus luteum diameter (mm) after fixed-time artificial insemination (FTAI) in water buffaloes induced to ovulation with GnRH or P 4 + eCG outside the breeding season. Group Days after IATF 7 14 21 n n n GnRH 5 14.8 ± 2.6 4 13.8 ± 7.5 1 12.4 ± 0 P4 + eCG 7 15.8 ± 2.5 6 17.9 ± 4 5 17.2 ± 2.6 There was no difference between groups (P > 0.05). n: number of females. None of the females from the GnRH group became pregnant, contrary to the P4 + eCG group (P < 0.05), in which 50% of the females were pregnant on Days 30 and 60. There was no embryo loss in the P4 + eCG group. Serum P4 concentrations were around 1 ng/ml at the start of both protocols and on Day 9 in the GnRH group and Day 10 in the P4 + eCG group. There was no difference (P > 0.05) between groups in P4 on Days 17 and 19 of the protocol. On Days 26 and 33, P4 was higher (P > 0.05) in the P4 + eCG group (Fig. 3 ). Of all the cows that had a CL after FTAI, the highest serum P4 concentrations were obtained in pregnant females from the P4 + eCG group on Days 19, 26 and 33 of treatment (3.1, 3.8 and 4.4 ng/ml, respectively), in comparison with the GnRH group (P < 0.05), as none of the females became pregnant (Fig. 4 ). Discussion Regarding the follicular population in this study, differences were found in the number of small follicles between treatments, which differs to the findings by Peralta-Torres et al. ( 2020 ), who reported similar number of small, medium and large follicles in Murrah buffaloes synchronized with combinations of gonadorelin, progesterone intravaginal device, estradiol benzoate and eCG in the breeding season. The existence of a larger population of small follicles in the P4 + eCG group suggests that the estradiol benzoate administered at the start of the hormonal treatment induced follicle atresia and in consequence the emergence of a new follicular wave, and that more follicles were recruited in comparison to the GnRH treatment. Regarding the maximum follicular diameter, all the groups had similar means, and an increase was observed in the cows from the P4 + eCG group as the day of FTAI was approaching. This is in agreement with the report by Carvalho et al.(2013), who indicated that buffalo cows treated with P4 + eCG outside the breeding season had the diameter of the ovulatory follicles increased at the end of the induction protocol (Day 9, 7.9 ± 0.4 mm; Day 11, 10.0 ± 0.7 mm; FTAI, 12.6 ± 0.6 mm). Similarly to this, Monteiro et al. ( 2018 ) reported follicle diameters of 13.2 ± 0.2 mm on the day of FTAI in buffalo cows outside the breeding season treated with hormonal protocols that combine P4 and eCG, and they positively correlated it with ovulation and a functional CL on Day 10 after FTAI. Therefore, the maximum diameter of the follicle is of great importance on the functionality of the CL and a higher probability for pregnancy. In this study, there were no differences in the presence and DCL after FTAI between groups; however, serum P4 concentrations were higher in buffalo cows from the P4 + eCG that had a CL, in comparison to the GnRH group. This is coincident with the report by Stouffer ( 2006 ), who mentioned that in seasonal species, luteal regression can occur four to six days after ovulation because of an early release of PGF 2α and low secretion of P4 (< 0.5 ng/ml). As reported by Russo et al. ( 2010 ), luteal function is not influenced by the DCL, as they found similar diameters in pregnant and non-pregnant buffaloes (18.7 mm and 18.9 mm, respectively) on Day 25 after FTAI following the use of protocols based on GnRH; instead, P4 concentration is a more reliable indicator to determine the luteal function (Di Francesco et al., 2012 ; Esposito et al., 2020 ). In this study, the GnRH group likely had a reduced luteal function (short-lived CL) which resulted in lower P4 levels and, in consequence, diminished the probability of pregnancy establishment. In this study, serum P4 concentrations were higher in pregnant females, which concurs with the report by Barile et al. ( 2021 b), who found P4 concentrations > 3 ng/ml in pregnant buffaloes (days 14 and 25) that received a hormonal protocol that included an intravaginal P4 device, whereas non-pregnant buffaloes had P4 concentrations < 2.5 ng/ml. Likewise, Daghash et al. ( 2022 ) found increased P4 concentrations from Day 20 to 40 in pregnant versus non-pregnant buffaloes when they used a GnRH-based protocol. With respect to the pregnancy rate, no female became pregnant in the GnRH group, contrary to the result obtained by Esposito et al. ( 2020 b) of pregnancy rate of 57.7% on Day 27 following synchronization of buffaloes with a functional CL outside the breeding season. In the present study, no embryo loss was registered, which is coincident with the results of Monteiro et al. ( 2018 b), who reported that the season does not interfere with embryo loss. On the contrary, Campanile et al. ( 2007 ) reported embryo loss of 22.9% between Days 25 and 40 after FTAI. Thus, in this study the low pregnancy rate might have resulted from low functionality of the CL (short-lived CL) rather than lack of embryo survival or development. Conclusions The effect of the hormonal protocols on the follicular population, maximum follicular diameter and CL diameter was similar with both induction protocols; however, the protocol based on P4 + eCG resulted in higher prevalence of the CL and higher P4 concentrations, which led to higher pregnancy rate in comparison to the GnRH group. Therefore, the P4 + eCG protocol was effective to induce ovulation and get buffalo cows pregnant outside the breeding season under tropical conditions. Declarations Acknowledgements The authors thank the production unit “La Carolina” for providing the animals, facilities and workers required to conduct this study. Data availability Data are available with the corresponding author at: ( [email protected] ) upon reasonable request. Declaration of animal rights All the authors declare that, during the study, the buffaloes were managed following the experimental procedures approved by the Committee of Animal Care and Ethical Research of the Universidad Juárez Autónoma de Tabasco (approval number: UJAT-CIEI-2023-183). Declaration of conflict of interests The authors declare that they have no conflict of interest regarding the study. References Abulaiti, A., Riaz, U., Naseer, Z., Ahmed, Z., Hua, G., & Yang, L. (2022). Follicular Dynamics during Estrous Cycle of Pubertal, Mature and Postpartum Crossbred (Nili Ravi × Jianghan) Buffaloes. Animals, 12(9), 1208. https://doi.org/10.3390/ani12091208 Barile, V.L., Menchetti, L., Casano, A. B., Brecchia, G., Melo de Sousa, N., Zelli, R., Canali, C., Beckers, J. F., & Barbato, O. (2021). Approaches to Identify Pregnancy Failure in Buffalo Cows. Animals, 11(2), 487. https://doi.org/10.3390/ani11020487 . Baruselli, P. S., Soares, J. G., Bayeux, B. M., Silva, J. C. B., Mingoti, R. D., & Carvalho, N. A. T. (2018). Assisted reproductive technologies (ART) in water buffaloes. Animal reproduction, 15(Suppl 1), 971–983. https://doi.org/10.21451/1984-3143-AR2018-0043 . Campanile, G., Di Palo, R., Neglia, G., Vecchio, D., Gasparrini, B., Prandi, A., Galiero, G., & D'Occhio, M. J. (2007). Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone. Theriogenology, 67(8), 1393–1398. https://doi.org/10.1016/j.theriogenology.2007.03.001 . Carvalho, N. A., Soares, J. G., Porto Filho, R. M., Gimenes, L. U., Souza, D. C., Nichi, M., Sales, J. S., & Baruselli, P. S. (2013). Equine chorionic gonadotropin improves the efficacy of a timed artificial insemination protocol in buffalo during the nonbreeding season. Theriogenology, 79(3), 423–428. https://doi.org/10.1016/j.theriogenology.2012.10.013 Carvalho, N. A. T., Soares, J. G., Souza, D. C., Maio, J. R. G., Sales, J. N. S., Martins Júnior, B., Macari, R. C., D'Occhio, M. J., & Baruselli, P. S. (2017). Ovulation synchronization with estradiol benzoate or GnRH in a timed artificial insemination protocol in buffalo cows and heifers during the nonbreeding season. Theriogenology, 87, 333–338. https://doi.org/10.1016/j.theriogenology.2016.09.006 Daghash, S. M., Yasin, N. A. E., Abdelnaby, E. A., Emam, I. A., Tolba, A., & Abouelela, Y. S. (2022). Histological and hemodynamic characterization of corpus luteum throughout the luteal phase in pregnant and non-pregnant buffalos in relation to nitric oxide levels based on its anatomical determination. Frontiers in veterinary science, 9, 896581. https://doi.org/10.3389/fvets.2022.896581 de Moura Lima, E., Vargas, J. A. C., Gomes, D. I., Maciel, R. P., Alves, K. S., Oliveira, W. F., Aguiar, G. L., de Carvalho Reis, G., Oliveira, L. R. S., & Mezzomo, R. (2021). Intake, digestibility, and milk yield response in dairy buffaloes fed Panicum maximum cv. Mombasa supplemented with seeds of tropical açai palm. Tropical animal health and production, 53(1), 178. https://doi.org/10.1007/s11250-021-02626-1 Devkota, B., Shah, S., & Gautam, G. (2023). Reproduction and Fertility of Buffaloes in Nepal. Animals, 13(1), 70. https://doi.org/10.3390/ani13010070 Di Francesco, S., Neglia, G., Vecchio, D., Rossi, P., Russo, M., Zicarelli, L., D'Occhio, M. J., & Campanile, G. (2012). Influence of season on corpus luteum structure and function and AI outcome in the Italian Mediterranean buffalo (Bubalus bubalis). Theriogenology, 78(8), 1839–1845. https://doi.org/10.1016/j.theriogenology.2012.07.022 Du, C., Nan, L., Sabek, A., Wang, H., Luo, X., Hua, G., & Zhang, S. (2021). Evaluation of Ovsynch versus modified Ovsynch program on pregnancy rate in water buffaloes: a meta-analysis. Tropical animal health and production, 53(3), 397. https://doi.org/10.1007/s11250-021-02828-7 Esposito, L., Salzano, A., Russo, M., de Nicola, D., Prandi, A., Gasparrini, B., Campanile, G., & Neglia, G. (2020). Corpus Luteum Color Doppler Ultrasound and Pregnancy Outcome in Buffalo during the Transitional Period. Animals, 10(7), 1181. https://doi.org/10.3390/ani10071181 . Frares, L., Weiss, R., Kozicki, L., Santangelo, R., Abreu, R., Santos, I., Junior, J. and Breda, J., 2013. Estrus synchronization and fixed time artificial insemination (FTAI) in dairy buffaloes during seasonal anestrus. Brazilian Archives of Biology and Technology, 56, 575–580. Gaur, M. and Purohit, G.N., 2019. Follicular dynamics and colour Doppler vascularity evaluations of follicles and corpus luteum in relation to plasma progesterone during the oestrous cycle of Surti buffaloes. Reproduction in Domestic Animals, 54, 585–594. Grazziotto, N. M., Maidana, S. S., & Romera, S. A. (2021). Susceptibilidad de los búfalos de agua frente a diferentes enfermedades infecciosas. Revista Veterinaria, 31(2), 215–223. https://doi.org/10.30972/vet.3124750 . Kavita, Phogat, J. B., Pandey, A. K., Balhara, A. K., Ghuman, S. S., & Gunwant, P. (2018). Effects of melatonin supplementation prior to Ovsynch protocol on ovarian activity and conception rates in anestrous Murrah buffalo heifers during out of breeding season. Reproductive Biology, 18(2), 161–168. https://doi.org/10.1016/j.repbio.2018.03.001 Magsi, S. H., Ahmad, N., Rashid, M. A., Bah, M., Akhter, M., & Shahid, M. Q. (2022). Validation of a body condition scoring system in Nili Ravi dairy buffaloes (Bubalus bubalis): inter- and intra-assessor variability. The Journal of dairy research, 89(4), 382–385. https://doi.org/10.1017/S0022029922000723 . Monteiro, B. M., Souza, D. C., Vasconcellos, G. S. F. M., Carvalho, N. A. T., & Baruselli, P. S. (2018). Effect of season on dairy buffalo reproductive performance when using P4/E2/eCG-based fixed-time artificial insemination management. Theriogenology, 119, 275–281. https://doi.org/10.1016/j.theriogenology.2018.07.004 Naveena, B.M. and Kiran, M., 2014. Buffalo meat quality, composition, and processing characteristics: Contribution to the global economy and nutritional security. Animal Frontiers, 4, 18–24. https://doi.org/10.2527/af.2014-0029 . Neglia, G., de Nicola, D., Esposito, L., Salzano, A., D'Occhio, M. J., & Fatone, G. (2020). Reproductive management in buffalo by artificial insemination. Theriogenology, 150, 166–172. https://doi.org/10.1016/j.theriogenology.2020.01.016 Peralta-Torres, J.A., Torres-Chablé, O.M., Segura-Correa, J.C., Ojeda-Robertos, N.F., Chay-Canul, A.J., Luna-Palomera, C., Severino-Lendechy, V.H. and Aké-Villanueva, J.R., 2020. Ovarian dynamics of buffalo ( Bubalus bubalis ) synchronized with different hormonal protocols. Tropical Animal Health and Production, 52, 3475–3480. Pottapenjera, V., Rajanala, S. R., Reddy, C., Gangineni, A., Avula, K., Bejjanki, S. K., Sathagopam, S., Kesharwani, S., & Velmurugan, S. (2018). Kisspeptin Modulates Luteinizing Hormone Release and Ovarian Follicular Dynamics in Pre-pubertal and Adult Murrah Buffaloes. Frontiers in veterinary science, 5, 149. https://doi.org/10.3389/fvets.2018.00149 . Qayyum, A., Arshad, U., Yousuf, M. R., & Ahmad, N. (2018). Effect of breeding method and season on pregnancy rate and embryonic and fetal losses in lactating Nili-Ravi buffaloes. Tropical animal health and production, 50(3), 555–560. https://doi.org/10.1007/s11250-017-1468-4 Rathore, R., Sharma, R. K., Phulia, S. K., Mudgal, V., Jerome, A., Ghuman, S. P. S., & Singh, I. (2017). Comparative efficacy of oestrus synchronization protocols in buffalo (Bubalus bubalis). Tropical animal health and production, 49(7), 1377–1382. https://doi.org/10.1007/s11250-017-1337-1 Russo, M., Vecchio, D., Neglia, G., Pacelli, C., Prandi, A., Gasparrini, B., Zicarelli, L., D’Occhio, M. and Campanile, G. (2010). Corpus luteum function and pregnancy outcome in buffaloes during the transition period from breeding to non-breeding season. Reproduction in Domestic Animals, 45: 988–991. https://doi.org/10.1111/j.1439-0531.2009.01472.x Sagheer, M., Ullah, F., Arshad, U., Saleem, M., Nawaz, M., Sarwar, Z., Saad, M., Riaz, A., Naveed Ul Haque, M., Basheer, A., & Ahmad, N. (2020). Effect of photoperiodicity and methods of follicular wave emergence on follicle turn-over, recovery and quality of oocytes, and early in-vitro developmental competence of embryos using ovum pick-up in Nili-Ravi buffaloes: Preliminary evidence. Theriogenology, 157, 508–516. https://doi.org/10.1016/j.theriogenology.2020.07.021 . Sharma, R. K., Phulia, S. K., Jerome, A., & Singh, I. (2017). Ovsynch Plus protocol improves ovarian response in anovular Murrah buffaloes in low-breeding season. Reproduction in domestic animals = Zuchthygiene, 52(6), 1030–1035. https://doi.org/10.1111/rda.13020 Srirattana, K., Hufana-Duran, D., Atabay, E. P., Duran, P. G., Atabay, E. C., Lu, K., Liang, Y., Chaikhun-Marcou, T., Theerakittayakorn, K., & Parnpai, R. (2022). Current status of assisted reproductive technologies in buffaloes. Animal Science Journal, 93(1), e13767. https://doi.org/10.1111/asj.13767 . Stouffer, R. L. (2006). Structure, function, and regulation of the corpus luteum. In Knobil and Neill's Physiology of Reproduction. 3rd Ed. (pp. 475–526). Elsevier Inc. https://doi.org/10.1016/B978-012515400-0/50017-8 . Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7048963","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488610925,"identity":"b605cbaa-97e3-4b66-82b0-23eb6a6ecc0c","order_by":0,"name":"Yuliana Izquierdo-Camacho","email":"","orcid":"","institution":"Universidad Juarez Autonoma de Tabasco Division Academia de Ciencias Agropecuarias","correspondingAuthor":false,"prefix":"","firstName":"Yuliana","middleName":"","lastName":"Izquierdo-Camacho","suffix":""},{"id":488610926,"identity":"dd4dccfd-215d-4f16-b514-f3ef923a9770","order_by":1,"name":"Nadia Ojeda-Robertos","email":"","orcid":"","institution":"Universidad Juarez Autonoma de Tabasco Division Academia de Ciencias Agropecuarias","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Ojeda-Robertos","suffix":""},{"id":488610927,"identity":"8e47bacb-bda3-4276-a511-afbfd0f121ff","order_by":2,"name":"Víctor Hugo Severino-Lendechy","email":"","orcid":"","institution":"Universidad Autonoma de Chiapas","correspondingAuthor":false,"prefix":"","firstName":"Víctor","middleName":"Hugo","lastName":"Severino-Lendechy","suffix":""},{"id":488610928,"identity":"c60814c1-5790-4725-b749-c0ecca1aab99","order_by":3,"name":"Oswaldo M. Torres-Chablé","email":"","orcid":"","institution":"Universidad Juarez Autonoma de Tabasco Division Academia de Ciencias Agropecuarias","correspondingAuthor":false,"prefix":"","firstName":"Oswaldo","middleName":"M.","lastName":"Torres-Chablé","suffix":""},{"id":488610929,"identity":"28ecbb47-fe9f-43cb-9afb-88dc8cceb145","order_by":4,"name":"Alfonso J. Chay-Canul","email":"","orcid":"","institution":"Universidad Juarez Autonoma de Tabasco Division Academia de Ciencias Agropecuarias","correspondingAuthor":false,"prefix":"","firstName":"Alfonso","middleName":"J.","lastName":"Chay-Canul","suffix":""},{"id":488610930,"identity":"21c9b82e-74dc-4373-93c3-94f20bfbef3b","order_by":5,"name":"Concepción Ahuja-Aguirre","email":"","orcid":"","institution":"Universidad Veracruzana","correspondingAuthor":false,"prefix":"","firstName":"Concepción","middleName":"","lastName":"Ahuja-Aguirre","suffix":""},{"id":488610931,"identity":"277378f6-c901-44bf-b679-9c1d4b077178","order_by":6,"name":"JORGE ALONSO PERALTA-TORRES","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBACxgYg8eGHTQKYl1BAnBbGxpk9aQkMbCAtBkRa1MzDdhiihYEYLcztZ48/nMFzPo9fvjvxwwMDBnl+sQMErOjJS2z4YHG7WLKNd7ME0GGGM2cnENDSkGPYOIPnduKGY7wbQFoSDG4T0tL/xhDol3MgLZt/EKdlRg5IywGQlm1E2jLjXeLMmT3JiTPbcrdZJBhIEPaLYX/ugQ8fftgl9jOf3XzzR4WNPL80IS0NPCh8CfzKQUCegYewolEwCkbBKBjhAACAkUgRgLuWgwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-8962-6434","institution":"Universidad Juarez Autonoma de Tabasco Division Academia de Ciencias Agropecuarias","correspondingAuthor":true,"prefix":"","firstName":"JORGE","middleName":"ALONSO","lastName":"PERALTA-TORRES","suffix":""}],"badges":[],"createdAt":"2025-07-04 18:18:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7048963/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7048963/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87430995,"identity":"ab616fa8-588f-46fd-bf81-07d87e1c9829","added_by":"auto","created_at":"2025-07-23 17:31:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23929,"visible":true,"origin":"","legend":"\u003cp\u003eHormonal treatments for induction of ovulation in water buffaloes outside the breeding season. GnRH = 10 µg buserelin acetate IM; PGF\u003csub\u003e2α\u003c/sub\u003e = 500 μg cloprostenol IM; EB = 2 mg estradiol benzoate IM; Intravaginal P4 device = 1 g natural P4; eCG = 400 IU equine chorionic gonadotropin IM; FTAI = fixed-time artificial insemination.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7048963/v1/f9740bbb5f84148c59f31364.png"},{"id":87431657,"identity":"7ce3762e-ab8b-4a58-aa13-40d9d97b0966","added_by":"auto","created_at":"2025-07-23 17:39:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28411,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum follicular diameter (mm) in water buffaloes induced to ovulation with GnRH or P4+eCG outside the breeding season.\u003c/p\u003e\n\u003cp\u003e*Statistical difference P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage22.png","url":"https://assets-eu.researchsquare.com/files/rs-7048963/v1/710d9394719a37307bdcd1fc.png"},{"id":87432167,"identity":"949b10cb-1630-43fb-9a7e-68483ab88a78","added_by":"auto","created_at":"2025-07-23 17:47:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31135,"visible":true,"origin":"","legend":"\u003cp\u003eSerum progesterone concentrations (ng/ml) in water buffaloes induced to ovulation with GnRH or P4+eCG outside the breeding season.\u003c/p\u003e\n\u003cp\u003e*Statistical difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage31.png","url":"https://assets-eu.researchsquare.com/files/rs-7048963/v1/a5249fc7b4fc5312d1a4a23d.png"},{"id":87430996,"identity":"286e0a5f-3925-45b8-8d76-1098014576d2","added_by":"auto","created_at":"2025-07-23 17:31:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30382,"visible":true,"origin":"","legend":"\u003cp\u003eSerum progesterone concentrations (ng/ml) in pregnant and non-pregnant water buffaloes induced to ovulation with GnRH or P4+eCG outside the breeding season.\u003c/p\u003e\n\u003cp\u003e*Statistical difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7048963/v1/352ff50fd39dca27fde253a2.png"},{"id":91185172,"identity":"fbed8a9a-b34c-4f34-81a8-2af25e75532a","added_by":"auto","created_at":"2025-09-12 13:49:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":826689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7048963/v1/0a094b9d-fe1f-4a3d-a543-57da7f00079e.pdf"}],"financialInterests":"","formattedTitle":"Ovarian response in water buffaloes induced to ovulate outside the breeding season","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe population of water buffaloes (\u003cem\u003eBubalus bubalis\u003c/em\u003e) has increased in the last years due to their ability to adapt to hostile environments (Naveena and Kiran, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), their resistance to diseases (Grazziotto et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and their utilization of low-quality forages as food (de Moura Lima et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), allowing them to thrive in environments where cattle cannot.\u003c/p\u003e\u003cp\u003eHowever, it is considered that buffaloes have low reproductive efficiency due to their delayed puberty and manifestation of silent estrus, making it difficult to determine the appropriate moment for artificial insemination (AI) (Qayyum et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, since buffaloes are short-day breeders (Sagheer et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), they frequently show a prolonged seasonal anestrus (Devkota et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, some strategies have been implemented to increase pregnancy rate in buffaloes outside their breeding season, such as the use of hormone treatments to induce ovulation in anestrus females (Carvalho et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which would allow to produce calves and therefore milk in months when its production is very low or non-existent, thus favoring productivity and economy throughout the year (Frares et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSome protocols that include progesterone intravaginal devices combined with estradiol (Carvalho et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and equine chorionic gonadotropin (eCG) (Baruselli et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Neglia et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) have been used, which, although expensive, have resulted in pregnancy rates after fixed timed AI (FTAI) up to 60.2% (Monteiro et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Protocols based in GnRH have also been used, resulting in pregnancy rates of 50% during the breeding season (Pottapenjera et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Du et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and lower than 24% outside the breeding season (Sharma et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rathore et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kavita et al., 2018). Therefore, the objective of the study was to determine the ovarian activity and pregnancy rate using two protocols for the induction of ovulation outside the breeding season in water buffaloes under tropical climate conditions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eStudy location\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study was conducted from May to June in a production unit located in the municipality of Centro, in the state of Tabasco, Mexico (17\u0026deg;54'13\"N 93\u0026deg;06'06\"O), at altitude of 13 m. The climate is warm and wet with summer rains, mean annual temperature of 27\u0026deg;C, mean relative humidity of 80% and annual rainfall of 2550 mm (INEGI, 2024).\u003c/p\u003e\u003cp\u003e\u003cb\u003eManagement of experimental animals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwenty Murrah \u0026times; Mediterranean multiparous buffaloes (3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 parturitions) were included in the study. The females averaged 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 of body condition score (BCS; 1 to 5 scale; Magsi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), 117\u0026thinsp;\u0026plusmn;\u0026thinsp;36 days postpartum and 444\u0026thinsp;\u0026plusmn;\u0026thinsp;67 kg of live weight. The buffalo cows underwent ultrasonographic evaluation (Mindray\u0026reg; SD-50; 7.5 MHz transducer) to verify the absence of a CL and any uterine or ovarian anomalies. Feeding was based on rotational grazing of native grasses (\u003cem\u003ePaspalum fasciculatum, P. notatum, P. conjugatum\u003c/em\u003e) plus mineral blocks and 2 kg of commercial concentrate (14% crude protein) during milking, which was once a day at 06:00 h; water was available \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHormonal treatments for induction of ovulation and artificial insemination\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe buffalo cows were randomly assigned to two treatments of 10 females each for induction of ovulation: 1) GnRH and 2) P4\u0026thinsp;+\u0026thinsp;eCG. In the GnRH group, the females received one intramuscular (IM) injection with 10 \u0026micro;g of a GnRH agonist (buserelin acetate; Sincroforte\u003csup\u003e\u0026reg;\u003c/sup\u003e, Ourofino, Mexico) on Day 0; on Day 7 they received 500 \u0026micro;g of a PGF\u003csub\u003e2α\u003c/sub\u003e analogue (cloprostenol; Ciclase\u003csup\u003e\u0026reg;\u003c/sup\u003e, Lab. Syntex, Argentina) IM; on Day 9 the females were given a second injection with 10 \u0026micro;g of a GnRH agonist and received FTAI 18 h later (Du et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb). In the P4\u0026thinsp;+\u0026thinsp;eCG group, on Day 0 the females received one intravaginal device containing 1 g of natural P4 (Cronipres\u003csup\u003e\u0026reg;\u003c/sup\u003e, Lab. Biog\u0026eacute;nesis Bag\u0026oacute;, Mexico) plus 2 mg of estradiol benzoate (Benzoato de Estradiol\u003csup\u003e\u0026reg;\u003c/sup\u003e, Lab. Syntex, Argentina) IM; on Day 10 the intravaginal device was removed and the females were given 500 \u0026micro;g of cloprostenol plus 400 IU of eCG (Folligon\u003csup\u003e\u0026reg;\u003c/sup\u003e, Intervet, Mexico) IM; on Day 12 one injection with 10 \u0026micro;g of buserelin acetate was administered; finally, FTAI was performed 54 h after the intravaginal device removal (Srirattana et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In both treatments, FTAI was made with frozen-thawed semen and performed by the same technician. The sequence of hormone administration in both treatments is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of ovarian activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOvarian activity was evaluated through transrectal ultrasonography (Mindray\u0026reg; SD-50, 7.5 MHz transducer) and all the evaluations were performed by the same technician. The follicle population in the GnRH group was started the day of PGF₂α injection (Day 7) and from then on, every 12 h until FTAI (Day 10). In the P4\u0026thinsp;+\u0026thinsp;eCG group the follicle population started the day of the intravaginal device removal (Day 10) and from then on, every 12 h until FTAI (Day 12). In both groups the evaluation included both ovaries, counting and measuring the diameter of all the follicles, classifying them as small (\u0026lt;\u0026thinsp;4 mm), medium (4 \u0026minus;\u0026thinsp;7.9 mm) and large (\u0026ge;\u0026thinsp;8 mm) (Gaur and Purohit, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The presence and diameter of the dominant follicle were recorded until FTAI. The maximum follicular diameter was obtained averaging the horizontal and vertical diameters. The follicle with a diameter\u0026thinsp;\u0026ge;\u0026thinsp;10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm was considered as dominant follicle (Abulaiti et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of the corpus luteum (CL)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe presence and size of the CL were determined by ultrasonography on Days 17, 24 and 31 in the GnRH group and on Days 19, 26 and 33 in the P4\u0026thinsp;+\u0026thinsp;eCG group. The diameter of the CL (DCL) was obtained averaging the horizontal and vertical diameters.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of serum progesterone\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBlood samples were obtained via coccygeal venipuncture using 21 G disposable needles and heparinized tubes (5 ml) on Days 0, 9, 17, 24 and 31 in the GnRH group and on Days 0, 10, 19, 26 and 33 in the P4\u0026thinsp;+\u0026thinsp;eCG group. The samples were centrifuged at 1500 rpm/15 min at maximum 4 h after being drawn. The serum was separated into aliquots that were frozen at -20\u0026deg;C until hormone determination. Progesterone concentrations were measured through enzyme immunosorbent assay using commercial kits (Progesterone ELISA; DRG, USA). The sensitivity of the assay was 0.045 ng/ml, the coefficient of variation intra and inter assay was 5.4% and 9.9%, respectively. Progesterone concentrations are presented as ng/ml.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePregnancy and embryo loss diagnosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePregnancy was diagnosed through real-time ultrasonography on Days 30 and 60 after FTAI in both groups. Embryo loss was determined when an embryonic vesicle found on Day 30 was not present on Day 60 (Barile et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe normality of the data and homogeneity of variances were evaluated. For the variables of follicle population, diameter of the ovulatory follicle, DCL and progesterone levels, a statistical model that included the effects of treatment, time and their interaction was used. Considering the data of time as repeated measures, a GLM analysis was conducted and means were compared through the Tukey\u0026rsquo;s test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The variables of pregnancy diagnosis and embryo loss were categorized binomially, and the Chi-square and Fisher\u0026rsquo;s exact tests were performed using the PROC FREQ procedure. All the tests were from the SAS software (SAS, 2000).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA lower population of small follicles (\u0026lt;\u0026thinsp;4mm) was observed in the P4\u0026thinsp;+\u0026thinsp;eCG group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the populations of medium and large follicles were similar in both groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Regarding the maximum follicular diameter, at the time of device removal or PGF₂α administration it averaged 10.8 mm in both groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); 12 h later the follicular diameter was larger in the GnRH group (12.7 mm) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and from then on until FTAI the mean follicular diameter was similar (13.1 mm) in both groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\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\u003ePopulation of small (\u0026le;\u0026thinsp;4 mm), medium (4.1\u0026ndash;8 mm) and large (\u0026ge;\u0026thinsp;8.1 mm) follicles in water buffaloes induced to ovulation with GnRH and P4\u0026thinsp;+\u0026thinsp;eCG outside the breeding season.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eGnRH group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c14\" namest=\"c9\"\u003e\u003cp\u003eP4\u0026thinsp;+\u0026thinsp;eCG group\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eDays of hormonal protocol\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c14\" namest=\"c9\"\u003e\u003cp\u003eDays of hormonal protocol\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFollicle size (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDay 7 PGF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eDay 8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eDay 9\u003c/p\u003e\u003cp\u003e2nd GnRH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDay 10 FTAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eDay 10\u003c/p\u003e\u003cp\u003eDevice removal\u003c/p\u003e\u003cp\u003ePGF\u0026thinsp;+\u0026thinsp;eCG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eDay11\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003eDay 12\u003c/p\u003e\u003cp\u003eGnRH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eh\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e18:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e06:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e18:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e06:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e18:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e12:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e10:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e18:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e06:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e18:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003e06:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cb\u003eFTAI\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e16:00\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4.1-8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;8.1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003e\u003csup\u003eab\u003c/sup\u003eDifferent literals for each day between groups indicate statistical difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWith respect to the presence of a CL after FTAI, there was no difference between groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) on Days 17\u0026ndash;19, 24\u0026ndash;26 and 31\u0026ndash;33 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Likewise, there was no difference in the DCL between groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\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\u003ePercentage of water buffaloes with a corpus luteum (CL) after fixed-time artificial insemination and early pregnancy diagnosis following induction to ovulation with GnRH or P4\u0026thinsp;+\u0026thinsp;eCG outside the breeding season.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eFemales with CL %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eDays of hormonal protocol\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGnRH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNE\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eP4\u0026thinsp;+\u0026thinsp;eCG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eThere was no difference between groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). NE: not evaluated.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\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\u003eCorpus luteum diameter (mm) after fixed-time artificial insemination (FTAI) in water buffaloes induced to ovulation with GnRH or P\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;eCG outside the breeding season.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\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=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" 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=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\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\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003eDays after IATF\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGnRH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eP4\u0026thinsp;+\u0026thinsp;eCG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e17.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eThere was no difference between groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). n: number of females.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNone of the females from the GnRH group became pregnant, contrary to the P4\u0026thinsp;+\u0026thinsp;eCG group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), in which 50% of the females were pregnant on Days 30 and 60. There was no embryo loss in the P4\u0026thinsp;+\u0026thinsp;eCG group.\u003c/p\u003e\u003cp\u003eSerum P4 concentrations were around 1 ng/ml at the start of both protocols and on Day 9 in the GnRH group and Day 10 in the P4\u0026thinsp;+\u0026thinsp;eCG group. There was no difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between groups in P4 on Days 17 and 19 of the protocol. On Days 26 and 33, P4 was higher (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the P4\u0026thinsp;+\u0026thinsp;eCG group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOf all the cows that had a CL after FTAI, the highest serum P4 concentrations were obtained in pregnant females from the P4\u0026thinsp;+\u0026thinsp;eCG group on Days 19, 26 and 33 of treatment (3.1, 3.8 and 4.4 ng/ml, respectively), in comparison with the GnRH group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as none of the females became pregnant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRegarding the follicular population in this study, differences were found in the number of small follicles between treatments, which differs to the findings by Peralta-Torres et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who reported similar number of small, medium and large follicles in Murrah buffaloes synchronized with combinations of gonadorelin, progesterone intravaginal device, estradiol benzoate and eCG in the breeding season. The existence of a larger population of small follicles in the P4\u0026thinsp;+\u0026thinsp;eCG group suggests that the estradiol benzoate administered at the start of the hormonal treatment induced follicle atresia and in consequence the emergence of a new follicular wave, and that more follicles were recruited in comparison to the GnRH treatment.\u003c/p\u003e\u003cp\u003eRegarding the maximum follicular diameter, all the groups had similar means, and an increase was observed in the cows from the P4\u0026thinsp;+\u0026thinsp;eCG group as the day of FTAI was approaching. This is in agreement with the report by Carvalho et al.(2013), who indicated that buffalo cows treated with P4\u0026thinsp;+\u0026thinsp;eCG outside the breeding season had the diameter of the ovulatory follicles increased at the end of the induction protocol (Day 9, 7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mm; Day 11, 10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mm; FTAI, 12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm). Similarly to this, Monteiro et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported follicle diameters of 13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mm on the day of FTAI in buffalo cows outside the breeding season treated with hormonal protocols that combine P4 and eCG, and they positively correlated it with ovulation and a functional CL on Day 10 after FTAI. Therefore, the maximum diameter of the follicle is of great importance on the functionality of the CL and a higher probability for pregnancy.\u003c/p\u003e\u003cp\u003eIn this study, there were no differences in the presence and DCL after FTAI between groups; however, serum P4 concentrations were higher in buffalo cows from the P4\u0026thinsp;+\u0026thinsp;eCG that had a CL, in comparison to the GnRH group. This is coincident with the report by Stouffer (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), who mentioned that in seasonal species, luteal regression can occur four to six days after ovulation because of an early release of PGF\u003csub\u003e2α\u003c/sub\u003e and low secretion of P4 (\u0026lt;\u0026thinsp;0.5 ng/ml). As reported by Russo et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), luteal function is not influenced by the DCL, as they found similar diameters in pregnant and non-pregnant buffaloes (18.7 mm and 18.9 mm, respectively) on Day 25 after FTAI following the use of protocols based on GnRH; instead, P4 concentration is a more reliable indicator to determine the luteal function (Di Francesco et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Esposito et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the GnRH group likely had a reduced luteal function (short-lived CL) which resulted in lower P4 levels and, in consequence, diminished the probability of pregnancy establishment.\u003c/p\u003e\u003cp\u003eIn this study, serum P4 concentrations were higher in pregnant females, which concurs with the report by Barile et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb), who found P4 concentrations\u0026thinsp;\u0026gt;\u0026thinsp;3 ng/ml in pregnant buffaloes (days 14 and 25) that received a hormonal protocol that included an intravaginal P4 device, whereas non-pregnant buffaloes had P4 concentrations\u0026thinsp;\u0026lt;\u0026thinsp;2.5 ng/ml. Likewise, Daghash et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found increased P4 concentrations from Day 20 to 40 in pregnant versus non-pregnant buffaloes when they used a GnRH-based protocol.\u003c/p\u003e\u003cp\u003eWith respect to the pregnancy rate, no female became pregnant in the GnRH group, contrary to the result obtained by Esposito et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003eb) of pregnancy rate of 57.7% on Day 27 following synchronization of buffaloes with a functional CL outside the breeding season. In the present study, no embryo loss was registered, which is coincident with the results of Monteiro et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003eb), who reported that the season does not interfere with embryo loss. On the contrary, Campanile et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) reported embryo loss of 22.9% between Days 25 and 40 after FTAI. Thus, in this study the low pregnancy rate might have resulted from low functionality of the CL (short-lived CL) rather than lack of embryo survival or development.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe effect of the hormonal protocols on the follicular population, maximum follicular diameter and CL diameter was similar with both induction protocols; however, the protocol based on P4\u0026thinsp;+\u0026thinsp;eCG resulted in higher prevalence of the CL and higher P4 concentrations, which led to higher pregnancy rate in comparison to the GnRH group. Therefore, the P4\u0026thinsp;+\u0026thinsp;eCG protocol was effective to induce ovulation and get buffalo cows pregnant outside the breeding season under tropical conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors thank the production unit \u0026ldquo;La Carolina\u0026rdquo; for providing the animals, facilities and workers required to conduct this study.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eData are available with the corresponding author at: ([email protected]) upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eDeclaration of animal rights\u003c/h2\u003e\n\u003cp\u003eAll the authors declare that, during the study, the buffaloes were managed following the experimental procedures approved by the Committee of Animal Care and Ethical Research of the Universidad Ju\u0026aacute;rez Aut\u0026oacute;noma de Tabasco (approval number: UJAT-CIEI-2023-183).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDeclaration of conflict of interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest regarding the study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbulaiti, A., Riaz, U., Naseer, Z., Ahmed, Z., Hua, G., \u0026amp; Yang, L. (2022). Follicular Dynamics during Estrous Cycle of Pubertal, Mature and Postpartum Crossbred (Nili Ravi \u0026times; Jianghan) Buffaloes. Animals, 12(9), 1208. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani12091208\u003c/span\u003e\u003cspan address=\"10.3390/ani12091208\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarile, V.L., Menchetti, L., Casano, A. B., Brecchia, G., Melo de Sousa, N., Zelli, R., Canali, C., Beckers, J. F., \u0026amp; Barbato, O. (2021). Approaches to Identify Pregnancy Failure in Buffalo Cows. Animals, 11(2), 487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani11020487\u003c/span\u003e\u003cspan address=\"10.3390/ani11020487\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaruselli, P. S., Soares, J. G., Bayeux, B. M., Silva, J. C. B., Mingoti, R. D., \u0026amp; Carvalho, N. A. T. (2018). Assisted reproductive technologies (ART) in water buffaloes. Animal reproduction, 15(Suppl 1), 971\u0026ndash;983. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21451/1984-3143-AR2018-0043\u003c/span\u003e\u003cspan address=\"10.21451/1984-3143-AR2018-0043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampanile, G., Di Palo, R., Neglia, G., Vecchio, D., Gasparrini, B., Prandi, A., Galiero, G., \u0026amp; D'Occhio, M. J. (2007). Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone. Theriogenology, 67(8), 1393\u0026ndash;1398. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2007.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2007.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarvalho, N. A., Soares, J. G., Porto Filho, R. M., Gimenes, L. U., Souza, D. C., Nichi, M., Sales, J. S., \u0026amp; Baruselli, P. S. (2013). Equine chorionic gonadotropin improves the efficacy of a timed artificial insemination protocol in buffalo during the nonbreeding season. Theriogenology, 79(3), 423\u0026ndash;428. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2012.10.013\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2012.10.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarvalho, N. A. T., Soares, J. G., Souza, D. C., Maio, J. R. G., Sales, J. N. S., Martins J\u0026uacute;nior, B., Macari, R. C., D'Occhio, M. J., \u0026amp; Baruselli, P. S. (2017). Ovulation synchronization with estradiol benzoate or GnRH in a timed artificial insemination protocol in buffalo cows and heifers during the nonbreeding season. Theriogenology, 87, 333\u0026ndash;338. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2016.09.006\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2016.09.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDaghash, S. M., Yasin, N. A. E., Abdelnaby, E. A., Emam, I. A., Tolba, A., \u0026amp; Abouelela, Y. S. (2022). Histological and hemodynamic characterization of corpus luteum throughout the luteal phase in pregnant and non-pregnant buffalos in relation to nitric oxide levels based on its anatomical determination. Frontiers in veterinary science, 9, 896581. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2022.896581\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2022.896581\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Moura Lima, E., Vargas, J. A. C., Gomes, D. I., Maciel, R. P., Alves, K. S., Oliveira, W. F., Aguiar, G. L., de Carvalho Reis, G., Oliveira, L. R. S., \u0026amp; Mezzomo, R. (2021). Intake, digestibility, and milk yield response in dairy buffaloes fed Panicum maximum cv. Mombasa supplemented with seeds of tropical a\u0026ccedil;ai palm. Tropical animal health and production, 53(1), 178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11250-021-02626-1\u003c/span\u003e\u003cspan address=\"10.1007/s11250-021-02626-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevkota, B., Shah, S., \u0026amp; Gautam, G. (2023). Reproduction and Fertility of Buffaloes in Nepal. Animals, 13(1), 70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani13010070\u003c/span\u003e\u003cspan address=\"10.3390/ani13010070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDi Francesco, S., Neglia, G., Vecchio, D., Rossi, P., Russo, M., Zicarelli, L., D'Occhio, M. J., \u0026amp; Campanile, G. (2012). Influence of season on corpus luteum structure and function and AI outcome in the Italian Mediterranean buffalo (Bubalus bubalis). Theriogenology, 78(8), 1839\u0026ndash;1845. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2012.07.022\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2012.07.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDu, C., Nan, L., Sabek, A., Wang, H., Luo, X., Hua, G., \u0026amp; Zhang, S. (2021). Evaluation of Ovsynch versus modified Ovsynch program on pregnancy rate in water buffaloes: a meta-analysis. Tropical animal health and production, 53(3), 397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11250-021-02828-7\u003c/span\u003e\u003cspan address=\"10.1007/s11250-021-02828-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEsposito, L., Salzano, A., Russo, M., de Nicola, D., Prandi, A., Gasparrini, B., Campanile, G., \u0026amp; Neglia, G. (2020). Corpus Luteum Color Doppler Ultrasound and Pregnancy Outcome in Buffalo during the Transitional Period. Animals, 10(7), 1181. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani10071181\u003c/span\u003e\u003cspan address=\"10.3390/ani10071181\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrares, L., Weiss, R., Kozicki, L., Santangelo, R., Abreu, R., Santos, I., Junior, J. and Breda, J., 2013. Estrus synchronization and fixed time artificial insemination (FTAI) in dairy buffaloes during seasonal anestrus. Brazilian Archives of Biology and Technology, 56, 575\u0026ndash;580.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGaur, M. and Purohit, G.N., 2019. Follicular dynamics and colour Doppler vascularity evaluations of follicles and corpus luteum in relation to plasma progesterone during the oestrous cycle of Surti buffaloes. Reproduction in Domestic Animals, 54, 585\u0026ndash;594.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrazziotto, N. M., Maidana, S. S., \u0026amp; Romera, S. A. (2021). Susceptibilidad de los b\u0026uacute;falos de agua frente a diferentes enfermedades infecciosas. Revista Veterinaria, 31(2), 215\u0026ndash;223. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.30972/vet.3124750\u003c/span\u003e\u003cspan address=\"10.30972/vet.3124750\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKavita, Phogat, J. B., Pandey, A. K., Balhara, A. K., Ghuman, S. S., \u0026amp; Gunwant, P. (2018). Effects of melatonin supplementation prior to Ovsynch protocol on ovarian activity and conception rates in anestrous Murrah buffalo heifers during out of breeding season. Reproductive Biology, 18(2), 161\u0026ndash;168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.repbio.2018.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.repbio.2018.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMagsi, S. H., Ahmad, N., Rashid, M. A., Bah, M., Akhter, M., \u0026amp; Shahid, M. Q. (2022). Validation of a body condition scoring system in Nili Ravi dairy buffaloes (Bubalus bubalis): inter- and intra-assessor variability. The Journal of dairy research, 89(4), 382\u0026ndash;385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0022029922000723\u003c/span\u003e\u003cspan address=\"10.1017/S0022029922000723\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonteiro, B. M., Souza, D. C., Vasconcellos, G. S. F. M., Carvalho, N. A. T., \u0026amp; Baruselli, P. S. (2018). Effect of season on dairy buffalo reproductive performance when using P4/E2/eCG-based fixed-time artificial insemination management. Theriogenology, 119, 275\u0026ndash;281. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2018.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2018.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaveena, B.M. and Kiran, M., 2014. Buffalo meat quality, composition, and processing characteristics: Contribution to the global economy and nutritional security. Animal Frontiers, 4, 18\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2527/af.2014-0029\u003c/span\u003e\u003cspan address=\"10.2527/af.2014-0029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNeglia, G., de Nicola, D., Esposito, L., Salzano, A., D'Occhio, M. J., \u0026amp; Fatone, G. (2020). Reproductive management in buffalo by artificial insemination. Theriogenology, 150, 166\u0026ndash;172. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2020.01.016\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2020.01.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeralta-Torres, J.A., Torres-Chabl\u0026eacute;, O.M., Segura-Correa, J.C., Ojeda-Robertos, N.F., Chay-Canul, A.J., Luna-Palomera, C., Severino-Lendechy, V.H. and Ak\u0026eacute;-Villanueva, J.R., 2020. Ovarian dynamics of buffalo (\u003cem\u003eBubalus bubalis\u003c/em\u003e) synchronized with different hormonal protocols. Tropical Animal Health and Production, 52, 3475\u0026ndash;3480.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePottapenjera, V., Rajanala, S. R., Reddy, C., Gangineni, A., Avula, K., Bejjanki, S. K., Sathagopam, S., Kesharwani, S., \u0026amp; Velmurugan, S. (2018). Kisspeptin Modulates Luteinizing Hormone Release and Ovarian Follicular Dynamics in Pre-pubertal and Adult Murrah Buffaloes. Frontiers in veterinary science, 5, 149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2018.00149\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2018.00149\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQayyum, A., Arshad, U., Yousuf, M. R., \u0026amp; Ahmad, N. (2018). Effect of breeding method and season on pregnancy rate and embryonic and fetal losses in lactating Nili-Ravi buffaloes. Tropical animal health and production, 50(3), 555\u0026ndash;560. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11250-017-1468-4\u003c/span\u003e\u003cspan address=\"10.1007/s11250-017-1468-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRathore, R., Sharma, R. K., Phulia, S. K., Mudgal, V., Jerome, A., Ghuman, S. P. S., \u0026amp; Singh, I. (2017). Comparative efficacy of oestrus synchronization protocols in buffalo (Bubalus bubalis). Tropical animal health and production, 49(7), 1377\u0026ndash;1382. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11250-017-1337-1\u003c/span\u003e\u003cspan address=\"10.1007/s11250-017-1337-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRusso, M., Vecchio, D., Neglia, G., Pacelli, C., Prandi, A., Gasparrini, B., Zicarelli, L., D\u0026rsquo;Occhio, M. and Campanile, G. (2010). Corpus luteum function and pregnancy outcome in buffaloes during the transition period from breeding to non-breeding season. Reproduction in Domestic Animals, 45: 988\u0026ndash;991. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1439-0531.2009.01472.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1439-0531.2009.01472.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSagheer, M., Ullah, F., Arshad, U., Saleem, M., Nawaz, M., Sarwar, Z., Saad, M., Riaz, A., Naveed Ul Haque, M., Basheer, A., \u0026amp; Ahmad, N. (2020). Effect of photoperiodicity and methods of follicular wave emergence on follicle turn-over, recovery and quality of oocytes, and early in-vitro developmental competence of embryos using ovum pick-up in Nili-Ravi buffaloes: Preliminary evidence. Theriogenology, 157, 508\u0026ndash;516. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theriogenology.2020.07.021\u003c/span\u003e\u003cspan address=\"10.1016/j.theriogenology.2020.07.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharma, R. K., Phulia, S. K., Jerome, A., \u0026amp; Singh, I. (2017). Ovsynch Plus protocol improves ovarian response in anovular Murrah buffaloes in low-breeding season. Reproduction in domestic animals\u0026thinsp;=\u0026thinsp;Zuchthygiene, 52(6), 1030\u0026ndash;1035. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/rda.13020\u003c/span\u003e\u003cspan address=\"10.1111/rda.13020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSrirattana, K., Hufana-Duran, D., Atabay, E. P., Duran, P. G., Atabay, E. C., Lu, K., Liang, Y., Chaikhun-Marcou, T., Theerakittayakorn, K., \u0026amp; Parnpai, R. (2022). Current status of assisted reproductive technologies in buffaloes. Animal Science Journal, 93(1), e13767. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/asj.13767\u003c/span\u003e\u003cspan address=\"10.1111/asj.13767\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStouffer, R. L. (2006). Structure, function, and regulation of the corpus luteum. In Knobil and Neill's Physiology of Reproduction. 3rd Ed. (pp. 475\u0026ndash;526). Elsevier Inc. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B978-012515400-0/50017-8\u003c/span\u003e\u003cspan address=\"10.1016/B978-012515400-0/50017-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\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":"corpus luteum, reproductive seasonality, follicular population, Bubalus bubalis","lastPublishedDoi":"10.21203/rs.3.rs-7048963/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7048963/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study evaluated the effect of two hormonal protocols on the ovarian activity and pregnancy rate of water buffaloes (\u003cem\u003eBubalus bubalis\u003c/em\u003e) outside the breeding season. Twenty multiparous Murrah \u0026times; Mediterranean buffaloes were included. The protocols used were the following: 1) GnRH (n\u0026thinsp;=\u0026thinsp;10): 100 \u0026micro;g gonadorelin IM (Day 0), 500 \u0026micro;g cloprostenol IM (Day 7), 100 \u0026micro;g gonadorelin IM (Day 9) and fixed time artificial insemination (FTAI) 18 h after the second GnRH; 2) P4\u0026thinsp;+\u0026thinsp;eCG (n\u0026thinsp;=\u0026thinsp;10): intravaginal progesterone (P4) device\u0026thinsp;+\u0026thinsp;2 mg estradiol benzoate (Day 0), device removal\u0026thinsp;+\u0026thinsp;500 \u0026micro;g cloprostenol\u0026thinsp;+\u0026thinsp;400 IU eCG (Day 10), 10 \u0026micro;g buserelin acetate (Day 12) and FTAI 54 h after device removal. The numbers of small (\u0026lt;\u0026thinsp;4 mm), medium (4.1-8 mm) and large (\u0026gt;\u0026thinsp;8.1 mm) follicles (follicular population) were counted through ultrasonography. The diameters of the largest follicle (maximum follicular diameter; MFD) and corpus luteum (CL) were evaluated. Blood samples were obtained on Days 0, 9, 17, 24 and 31 in the GnRH group and on Days 0, 10, 19, 26 and 33 in the P4\u0026thinsp;+\u0026thinsp;eCG group to determine serum P4 concentrations. No differences were found between groups in follicular population, MFD and CL diameter. The presence of CL and P4 concentrations were greater in the P4\u0026thinsp;+\u0026thinsp;eCG group. Only females from the P4\u0026thinsp;+\u0026thinsp;eCG group became pregnant and P4 concentrations were \u0026gt;\u0026thinsp;3 ng/ml on Days 26 and 33. In conclusion, the P4\u0026thinsp;+\u0026thinsp;eCG protocol was effective to induce ovulation and achieve pregnancy in buffalo cows outside the breeding season under tropical conditions.\u003c/p\u003e","manuscriptTitle":"Ovarian response in water buffaloes induced to ovulate outside the breeding season","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 17:31:41","doi":"10.21203/rs.3.rs-7048963/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":"e14607f0-d1c7-47e2-a3bd-bad38e7d451c","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-12T13:41:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 17:31:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7048963","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7048963","identity":"rs-7048963","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}