Follicular status, estrus behavior, and recombinant bovine somatotropin improve the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol

Follicular status, estrus behavior, and recombinant bovine somatotropin improve the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol | 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 Follicular status, estrus behavior, and recombinant bovine somatotropin improve the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol Cyndi G. Hernández-Coronado, Rene C. Calderón-Robles, Ana María Rosales-Torres, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7745904/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 To evaluate the effect of rbST on insulin-like growth factor 1 (IGF-1) serum concentration, ovarian dynamics, and the reproductive response of beef cows subjected to an estradiol-based fixed-time artificial insemination (FTAI) protocol, 107 Bos indicus × Bos taurus multiparous, postpartum beef cows on two commercial herds were used. The animals were randomly divided into two experimental groups balanced by body condition score. Females in the control group did not receive any additional treatment, whereas cows in the treatment group received a subcutaneous injection of 500 mg of rbST. The ovarian dynamics and pregnancy were determined by transrectal ultrasonography. Ovarian dynamics were recorded at the beginning of the hormonal treatment (day 0) and when the intravaginal device was removed (day 8). IGF-1 serum concentrations were measured by enzyme-linked immunosorbent assay on days 0 and 8. The diameter of the dominant follicle and the IGF-1 serum concentrations on day 8 were higher (P < 0.05) in rbST-treated cows than in control cows. Logistic regression model indicated that cows with a dominant follicle on days 0 and 8 and in estrus had greater probability of pregnancy (P < 0.05), whereas use of rbST tended (P < 0.10) to increase the rate of pregnancy per artificial insemination (P/AI). The presence of corpus luteum on day 0 positively affected (P < 0.05) the P/AI rate. Our research indicated that beef cows subjected to the estradiol-based FTAI protocol with rbST injected on the first day had improved P/AI rate likely because the increased IGF-1 serum concentrations induced by rbST enlarge the diameter of the preovulatory follicle. beef cows recombinant bovine somatotropin IGF-1 ovarian dynamics estrus synchronization pregnancy Figures Figure 1 Figure 2 Introduction The use of fixed-time artificial insemination (FTAI) protocols in beef cows worldwide has increased recently because they facilitate the use of artificial insemination and improve reproductive performance in cow-calf production systems (Bó and Menchaca 2023). Even when use of these protocols have acceptable rates of pregnancy per artificial insemination (P/AI), the results are highly variable (Baruselli et al. 2018; Hernández-Coronado et al. 2023). For example, Bó et al. (2016) mentioned that P/AI ranged 6–100%, whereas females treated with a CO-Synch protocol had very low P/AI (17.5%), which was improved to 35.3% by an estradiol-progesterone-based protocol (Vázquez-López et al. 2024). However, these P/AI rates are lower than the expected 50–60% P/AI reported by Hernández-Coronado et al. (2023). For this reason, it is important to develop strategies that may improve the P/AI rate in animals subjected to FTAI protocols. Several reports on beef cows subjected to different protocols of FTAI have shown that the increased diameter of the dominant follicle (DF) after administration of prostaglandin F2α (PGF2α) or the diameter of the preovulatory follicle increased the rate of P/AI (Bishop et al. 2017; Pessoa et al. 2016). These results support the idea that the diameter of the preovulatory follicle is one of the most important factors that determines P/AI, which could be associated with a better oocyte quality and with the formation of a large corpus luteum (CL) capable of producing a large amount of progesterone (Vázquez-López et al. 2024). Therefore, treatments that promote preovulatory follicle growth may favor success of FTAI protocols. Follicular growth is regulated by several signals, including hormones and growth factors (Guzman et al. 2023). Among these molecules, insulin-like growth factor-I (IGF-1) and gonadotropins are the most important promoters of antral follicular growth (Rosales-Torres et al. 2012). IGF-1 is secreted by almost all body cells, but the main source of IGF-1 is the liver, which secretes large amounts of the growth factor in response to growth hormone (Hernández-Cerón and Gutierrez-Aguilar 2013). IGF-1 coming from the liver or synthesized locally stimulates proliferation of granulosa and theca cells and increases aromatase expression and estradiol secretion (Liu et al. 2021). Following its development in the 1980s, recombinant bovine somatotropin (rbST) has been widely used in several countries to stimulate milk production in dairy animals (Raux et al. 2022). However, because of the effect somatotropin has on IGF-1 secretion by the liver, it is likely that the use of rbST during the development of the preovulatory follicle may promote its growth and increase the P/AI rate in cows in a FTAI protocol. In non-lactating, multiparous cows in a FTAI protocol, the use of rbST increased the diameter of the preovulatory follicle and the CL diameter (Kaminski et al. 2019). Whereas an increased P/AI rate was seen in Bos taurus , but not Bos indicus , treated with rbST on the first day of a FTAI protocol (Gasseferth et al. 2023). Therefore, the objective of the present study was to evaluate the effect of rbST on IGF-1 serum concentration, diameter of preovulatory follicle, and the reproductive response of beef cows subject to an estradiol/progesterone FTAI protocol. Material and methods Location and animals All animal handling procedures were approved by the Research Ethics Committee of the División de Ciencias Biológicas y de la Salud at the Universidad Autónoma Metropolitana-Xochimilco under protocol number CEI. 2022.014 in accordance with Section 2, Article 20, 3rd Title of the Federal Law on Animal Health (2018), Mexico. The study was performed on two commercial ranches. One ranch was located between 19°59´39” and 20°08´39” north latitude and 97°21´39” and 97°27´39” west longitude in Ayotoxco de Guerrero, Puebla, Mexico, and the other was located between 20°08’ and 20°18’ north latitude and 96°46’ and 97° 02’ west longitude in San Rafael, Veracruz, Mexico. A total of 107 B. indicus × B. taurus multiparous, postpartum beef cows were allocated in pastureland and fed common Star of Africa grass ( Cynodon plectostachyus (K. Schum.) Pilg.) mixed with native grasses ( Axonopus spp. and Paspalum spp.) as a basic diet supplemented with mineral salts (Ganafos 12%; Mexico) containing calcium (12%), phophorus (12%), and manganese (12,000 ppm) offered ad libitum . Experimental design All the cows were subjected to an estradiol-based FTAI protocol (Fig. 1 ). On the first day of the protocol (day 0), the animals received an injection of 2 mg of estradiol benzoate (EB; Sincrodiol®, Ourofino Animal Health, Brazil), and an intravaginal progesterone device (Sincrogest®, Ourofino Animal Health, Brazil). The intravaginal device was removed eight days later, and 0.5 mg of cloprostenol (Sincrocio®, Ourofino Animal Health, Brazil) was injected. Between 48 and 56 hours after intravaginal device removal, all the cows were inseminated, and an injection of 150 mg of buserelin acetate (SincroForte®, Ourofino, Animal Health, Brazil) was used as an ovulation inductor. On day 0, the animals at each ranch were randomly divided into two experimental groups balanced by body condition score (BCS) and postpartum days. Females in the control group did not receive any additional treatment beyond synchronization, while the cows in the rbST group received a subcutaneous injection of 500 mg of rbST (Elanco MT , Argentina). On day 10, the cows were exposed to bull mating at a bull:cow ratio of 1:20 for a breeding season of 63 days. Ovarian dynamics The ovarian structures were examined by transrectal ultrasonography (Aloka SSD-500, Aloka Hitachi, Tokyo, Japan), using a 7.5-MHz linear transrectal probe. Ultrasounds were performed at the beginning of the hormonal treatment (day 0) and when the intravaginal device was removed together with the injection of PGF2α on day 8 (Fig. 1 ). At both times, the diameter of the largest follicle in the ovaries was measured, and the population of antral follicles (> 2 mm) was recorded. The DF diameter at follicular selection in B. indicus is approximately 6 mm, and since we used B. indicus × B. taurus animals in this experiment, the status of the follicular wave was classified as either in the recruitment phase when both ovaries had follicles ≤ 6 mm in diameter or the selection/dominance phase when there was a follicle > 6 mm in diameter in one of the ovaries. Estrus detection and pregnancy diagnosis At the time of intravaginal device withdrawal, the cow’s tailhead was painted with marker crayons. At the time of AI, the presence or absence of crayon paint was recorded to determine if the females had or had not been mounted by an other cows. To determine the P/AI rate and the pregnancy rate during the breeding season, transrectal ultrasonography, using a 7.5-MHz linear transrectal probe, was performed at 35 days after AI and 45 after the end of the breeding period, respectively. Determination of IGF-1 serum concentrations To determine the concentrations of IGF-1 in serum, five randomly selected animals per group were sampled at the time of the insertion (day 0) and withdrawal (day 8) of the intravaginal progesterone device. From each animal 5–10 mL of blood from the coccygeal vein were drawn in a vacuum tube. The samples were placed on ice immediately after collection and then centrifuged at 1,000×g for 15 min. The serum was stored at − 20ºC until the IGF-1 serum concentration was assayed. IGF-1 serum concentration was determined using a commercial competitive enzyme-linked immunosorbent assay kit (Catalog No. EB0068, Fine Biotech Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Samples from days 0 and 8 were analyzed in duplicate in a dilution 1:2; the intra-assay variation coefficient was 8%. Statistical analysis BCS on day 0 and DF diameter on days 0 and 8 were analyzed by Student’s t-test, whereas cows with CL on days 0 or 8, percentages of animals in estrus and pregnant in the breeding season were analyzed by Chi-square. To determine the effect of the independent variables (treatment, estrus behavior, status of follicular wave, location, CL presence, BCS, and DF diameter on day 8) on P/AI rate, we used the JMP-13 logistic regression procedure. To determine the factors that affect P/AI rate, two models were produced using the stepwise selection method. Model 1 included treatment, estrus behavior, status of follicular wave (days 0 and 8), location, and DF diameter on day 8. Model 2 included animals with CL on days 0 and 8 and BCS on day 8. IGF-1 serum concentrations on day 8 were analyzed by ANOVA using treatment as the fixed effect, IGF-1 serum concentration on day 0 as a covariate, and experimental replicate nested within cow as the random effect. Differences with P values < 0.05 were considered statistically different, and differences with P values from 0.05 to 0.10) in BCS, DF diameter on day 0, percentage of animals with CL or DF on days 0 and 8, estrus presentation and pregnancy in the breeding season between control and rbST-treated cows (Table 1 ). However, the DF diameter and the IGF-1 serum concentrations on day 8 were higher (P < 0.05) in rbST-treated cows than in control cows (Fig. 2 ). Table 1 Ovarian dynamic and breeding season pregnancy rate in beef cows subject to a fixed timed-artificial insemination protocol and treated with recombinant bovine somatotropin (rbST). Control rbST SE P value n 54 53 BCS on day 0 4.4 4.5 0.10 0.7567 DF diameter on day 0 (mm) 9.4 10.2 0.49 0.2402 Cows with CL on day 0 (%) 11 (20.4) 16 (30.2) 0.1720 Cows with DF on day 0 (%) 42 (78.0) 39 (74.0) 0.7733 Cows with CL on day 8 (%) 12 (22.2) 14 (26.4) 0.4103 Cows with DF on day 8 (%) 30 (56.0) 34 (64.0) 0.2696 Cows in estrus (%) 34 (63.0) 35 (66.0) 0.3918 Number pregnant in breeding season (%) 27 (50.0) 29 (54.7) 0.3841 SE = standard error; n = sample size; BCS = body condition score; DF = dominant follicle; CL = corpus luteum. In Model 1 (Table 2 ), cows treated with rbST, cows with a selected or dominant follicle at day 0 and from location 2 tend to have (P < 0.10) higher P/AI rates than cows in the control group, in recruitment phase, and from location 1 respectively. Cows in estrus and with a selected or dominant follicle at day 8 had higher (P 0.10) the P/AI rate. The odd ratios indicated that cows treated with rbST tended to have 2.4 times greater probability of pregnancy per AI than non-treated cows, while females that had a selected or dominant follicle at day 8 have 8.2 times higher probability of pregnancy per AI than females with recruited follicles (P < 0.05). In Model 2 (Table 3 ), only the presence of CL on day 0 significantly affected (P < 0.05) the P/AI rate, whereas there was no effect by CL presence on day 8 or BCS on day 0. Table 2 Influence of recombinant bovine somatotropin (rbST), estrus behavior, status of follicular wave, location and diameter of the dominant follicle (DF) on rate of pregnancy per artificial insemination (P/AI) in beef cows subjected to a fixed timed-artificial insemination protocol. Constant P/AI Odd ratio Estimate SE P value -0.3 1.1 0.8193 Treatment Control (%) 18/54 (33.3) 1 0.4 0.3 0.0823 rbST (%) 24/53 (45.3) 2.4 Estrus behavior No (%) 8/37 (21.6) 1 0.7 0.3 0.0055 Yes (%) 33/70 (47.1) 4.3 Status of follicular wave on day 0 Recruitment (%) 5/24 (20.8) 1 0.6 0.3 0.0568 Selection or Dominance (%) 36/81 (44.4) 3.3 Status of follicular wave on day 8 Recruitment (%) 8/38 (21.1) 1 1.1 0.5 0.0189 Selection or Dominance (%) 31/64 (48.4) 8.2 Location Location 1 (%) 14/42 (33.3) 1 0.5 0.3 0.0738 Location 2 (%) 28/65 (43.1) 2.6 Diameter of DF on day 8 0.2 0.1 0.2652 SE = standard error. Table 3 Influence of the corpus luteum (CL) and body condition score (BCS) on the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol. Variable Estimate SE P value Intercept 1.65 1.50 0.2717 Animal with CL on day 0 (%) 0.59 0.27 0.0325 Animal with CL on day 8 (%) -0.06 0.28 0.8165 BCS on day 0 -0.44 0.34 0.1857 SE = standard error. Discussion Our experiment showed that the injection of rbST on day 0 in cows subjected to an estradiol-based FTAI protocol had enhanced P/AI rates. This was likely due to an increase in the IGF-1serum concentrations and the diameter of the preovulatory follicle. Cows treated with rbST on day 0 of the FTAI protocol had higher IGF-1 serum concentrations than untreated cows eight days later. Similar to our results, injection of rbST into Angus-based, crossbred beef cows at the beginning of an FTAI protocol increased IGF-1 serum concentrations compared to control cows (Oosthuizen et al. 2018). In beef heifers, IGF-1 serum concentrations were higher 22 days after, rbST treatment compared to control animals (Sanford et al. 2021). It is known that rbST, after absorption, binds to the growth hormone (GH) receptor in the liver to promote IGF-1 synthesis and secretion from the hepatocytes via the GH receptor/JAK2/PLC/Ca2 + pathway (Kimura et al. 2023; Our study also showed that rbST administration on day 0 of the FTAI protocol enhances the DF diameter on day 8. Other studies have reported that cows treated with rbST had large DF 8–10 days after treatment compared to untreated animals (Kaminski et al. 2019; Valle et al. 2024) indicating that rbST promote follicular growth. The somatotropin-IGF-1 axis is an important regulator of follicular development in several species, including bovines (Benetis-Piauet et al. 2023). Ovarian follicular cells have receptors for both somatotropin and IGF-1, and GH directly or via IGF-1 stimulates granulosa cell proliferation, synthesis of steroidogenic hormones, and synthesis of gonadotropin receptors (Chang et al. 2022). Therefore, in our experiment it is likely that rbST by itself or the increase in IGF-1 serum concentration stimulated proliferation of granulosa cells promote estradiol secretion and synthesis of gonadotropin receptors to increase the diameter of the preovulatory follicle. Overall, the beef cows treated with rbST in our study had 36% higher P/AI rate than untreated cows. When females were treated with rbST on the first day of a FTAI protocol, there was a 58% increase in the P/AI rate in B. taurus and crossbred cows (Gasseferth et al. 2023) and a 37% increase in B. indicus cows (Vale et al. 2024). One of the most important factors that affects the P/AI rate in beef cows subjected to a FTAI protocols is the diameter of the preovulatory follicle because this parameter is highly correlated with oocyte quality and the formation of a large CL that produces enough progesterone to maintain pregnancy (Baruselli et al. 2023 ). Therefore, an increase in the diameter of the preovulatory follicle induced by rbST likely explains the higher P/AI rate in the treated cows in our study. The results of our experiment also showed that ovarian dynamics and estrus behavior affect the P/AI rate. Cows with a follicle in the selection/dominance phase on day 0 of the FTAI protocol had at least three times higher probability of becoming pregnant after AI than animals with recruited follicles. Treatment with estradiol benzoate plus the insertion of a progesterone implant on day 0 has the objective to reduce peripheric concentration of gonadotropins and induce follicular atresia to synchronize the onset of a new follicular wave (Bó et al. 2023; Hernández-Coronado et al. 2023). Large antral follicles could be more susceptible to becoming atretic due to a reduction in trophic factors. Therefore, in animals with follicles in the selection/dominance phase on day 0 of the FTAI, it is more likely to initiate and synchronize a new follicular wave to have a preovulatory follicle days later when the IA is performed. Similar to our results, Oosthuizen et al. (2020), Rodrigues et al. (2018) and Silva et al. (2024) observed that cows with follicles in the selection/dominance phase on day 8 of the FTAI protocol had higher probability of pregnancy than cows with recruited follicles. Thus, it is likely that the selection/dominance phase at the beginning of proestrus assures that a preovulatory follicle will ovulate with the injection of gonadotropin-releasing hormone to improve the P/AI rate. As in previous reports (Silva et al. 2018; Vázquez-López et al. 2024), the cows with CL at the beginning of the FTAI protocol in our study had higher probability of P/AI than animals without CL. The presence of a CL is associated with cow cyclicity, which indicates that postpartum cows are producing viable oocytes (Crowe 2008). This association explains the positive relationship between the presence of CL and P/AI rate that we observed. The cows in estrus at the time of insemination in our study had higher P/AI rates than the cows not displaying estrus behavior, which corroborates the findings in studies by Nogueira et al. (2019). It has been suggested that cows in estrus produce a better-quality oocyte (Vázquez-López et al. 2024), thus explaining the positive association between estrus behavior and fertility. In conclusion, our research indicates that beef cows treated with rbST at the beginning of an estradiol-based FTAI protocol have improved P/AI rates because rbST increases the IGF-1 serum concentration that enhances the diameter of the preovulatory follicle and likely the quality of the oocyte, and the CL derived from it. In addition, cows with follicles in the selection/dominance phase on day 8 of the protocol have 8.2 times higher probability of pregnancy per IA than cows with recruited follicles. Our research confirms that cows with a CL at the beginning of the FTAI protocol have a higher probability of P/AI than cows without a CL because the presence of a CL is associated with cow cyclicity. Declarations Ethical approval All animal procedures were approved by the Research Ethics Committee of the División de Ciencias Biológicas y de la Salud from the Universidad Autónoma Metropolitana-Xochimilco under protocol number CEI. 2022.014 Consent to participate The consent was obtained from all authors. Informed consent Not applicable. Conflict of interest The authors declare no conflicts of interest Author contributions Cyndi G. Hernández-Coronado, Ana Ma. Rosales-Torres: Writing-review and editing. Rene C. Calderón-Robles, Ricardo Faustino-Carmona, Mariel Alejandra Ramos-Pérez: Investigation, Data Collection, and Methodology. Zaire B. Medina-Moctezuma, David. González-Aretia: Hormanal determination and data collection. Adrián Guzmán-Sánchez: Writing Original Draft, Methodology, Investigation, Supervision, Funding acquisition, and Project administration. Acknowledgements The authors are grateful for the financial support provided by the Universidad Autónoma Metropolitana-Xochimilco via the divisional project CBS-34402080 and the program Fortalecimiento de la Investigación 2022. Grant number R.X.155.22. Data Availability Statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Baruselli PS, Abreu LA, Catussi BLC et al (2023) Use of new recombinant proteins for ovarian stimulation in ruminants. Anim Reprod 20(2):e20230092. https://doi.org/10.1590/1984-3143-ar2023-0092 Baruselli PS, Ferreira RM, Filho MFS, Bó GA (2018) Review: Using artificial insemination v. natural service in beef herds. Animal 12(s1):s45-s52. https://doi.org/10.1017/S175173111800054X Benetis-Piau TB, de Queiroz-Rodrigues A, Paulini F (2023) Insulin-like growth factor (IGF) performance in ovarian function and applications in reproductive biotechnologies. Growth Horm IFG Res. 72–73:101561. https://doi.org/10.1016/j.ghir.2023.101561 Bishop BE, Thomas JM, Abel JM. et al (2017) Split-time artificial insemination in beef cattle: III. Comparing fixed-time artificial insemination to split-time artificial insemination with delayed administration of GnRH in postpartum cows. Theriogenology, 99:48–52. https://doi.org/10.1016/j.theriogenology.2017.04.046 Bó GA, Paucar F, Mendoza BL, Pinagorte L, Ocampo V, Cedeño V (2023) Pregnancy rates in Bos indicus × Bos taurus recipients synchronized with a GnRH/progesterone-based or an estradiol/progesterone-based protocol with prolonged proestrus. Reproduction Fertility and Development 36(2):207–208. https://doi.org/10.1071/RDv36n2Ab110 Bó GA, de la Mata JJ, Baruselli PS, Menchaca A (2016) Alternative programs for synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 86(1):388–396. https://doi.org/10.1016/j.theriogenology.2016.04.053 Bó GA, Menchaca A (2023) Prohibition of hormones in animal reproduction: what to expect and what to do?. Anim Reprod 20(2):20230067. https://doi.org/10.1590/1984-3143-AR2023-0067. Chang CW, Sung YW, Hsueh YW et al (2022) Growth hormone in fertility and infertility: Mechanisms of action and clinical applications. Front in Endocrinol 13:1040503. https://doi.org/10.3389/fendo.2022.1040503 Crowe, M (2008) Resumption of Ovarian Cyclicity in Post-partum Beef and Dairy Cows. Reprod Domest Anim 43:20–28. https://doi.org/10.1111/j.1439-0531.2008.01210.x Gasseferth G, Gaievski FR, Bergstein-Galan TG et al (2023) Effect of recombinant bovine somatotropin on the reproductive efficiency of beef cows subjected to differently timed-artificial insemination protocols. Reprod Domest Anim. 58(12):1654–1661. https://doi.org/10.1111/rda.14479 Guzmán A, Hernández-Coronado CG, Gutiérrez CG, Rosales-Torres AM (2023) The vascular endothelial growth factor (VEGF) system as a key regulator of ovarian follicle angiogenesis and growth. Mol Reprod Dev 90(4):201–217. https://doi.org/10.1002/mrd.23683 Hernández-Cerón J Gutierrez-Aguilar CG (2013) Recombinant bovine somatotropin and reproduction in cattle, sheep and goat. Agrociencia 47(1):35–45. Hernández-Coronado C, Rosales-Torres A, Vázquez-López S, Guzmán-Sánchez A (2023) Synchronization of estrus and ovulation in bovine females. Endocrine bases and treatments used. Abanico Veterinario 13:1–37 https://doi.org/10.21929/abavet2023.16 Kaminski AP, Andrade-Carvalho ML, Segui MS et al (2019) Impact of recombinant bovine somatotropin, progesterone, and estradiol benzoate on ovarian follicular dynamics in Bos taurus taurus cows using a protocol for estrus and ovulation synchronization. Theriogenology 140:58–61. https://doi.org/10.1016/j.theriogenology.2018.11.009 Kimura M, Moteki H, Ogihara M (2023) Role of Hepatocyte Growth Regulators in Liver Regeneration. Cells 12(2):208. https://doi.org/10.3390/cells12020208 Liu T, Huang Y, Lin H (2021) Estrogen disorders: Interpreting the abnormal regulation of aromatase in granulosa cells (Review). Int J Mol Med 47(5):73. https://doi.org/10.3892/ijmm.2021.4906 Nogueira E, Silva MR, Silva JCB (2019) Timed artificial insemination plus heat I: effect of estrus expression scores on pregnancy of cows subjected to progesterone–estradiol-based protocols. Animal 13(10):2305–2312. https://doi.org/10.1017/s1751731119000442 Oosthuizen N, Fontes PLP, Henry DD et al (2018) Administration of recombinant bovine somatotropin prior to fixed-time artificial insemination and the effects on fertility, embryo, and fetal size in beef heifers. J Anim Sci 96(5):1894–1902. https://doi.org/10.1093/jas/sky077 Oosthuizen N, Cooke RF, Schubach KM et al (2020) Effects of estrous expression and intensity of behavioral estrous symptoms on variables associated with fertility in beef cows treated for fixed-time artificial insemination. Anim Reprod Sci 214:106308–106308. https://doi.org/10.1016/j.anireprosci.2020.106308 Pessoa GA, Martini AP, Carloto GW et al (2016) Different doses of equine chorionic gonadotropin on ovarian follicular growth and pregnancy rate of suckled Bos taurus beef cows subjected to timed artificial insemination protocol.” Theriogenology 85(5):792–799. https://doi.org/10.1016/j.theriogenology.2015.09.05 Raux A, Bichon E, Benedetto A et al (2022) The Promise and Challenges of Determining Recombinant Bovine Growth Hormone in Milk. Foods 11(3):274. https://doi.org/10.3390/foods11030274 Rodrigues AD, Cooke RF, Cipriano RS et al (2018) Impacts of estrus expression and intensity during a timed-AI protocol on variables associated with fertility and pregnancy success in Bos indicus-influenced beef cows. J Anim Sci96(1):236–249. https://doi.org/10.1093/jas/skx043 Rosales-Torres AM, Guzmán-Sánchez A, Aguilar CG (2012) Follicular development in dosmestic ruminants. Tropic SubtropAgroeco15(1):s147–s160. Sanford CD, Owen MPT, McCarty K et al (2021) Effects of Administering Exogenous Bovine Somatotropin During the First Trimester of Pregnancy Altered Uterine Hemodynamics in Suckled Beef Cows. Front AnimSci 2:744790. https://doi.org/10.3389/fanim.2021.744790 Silva EP, Wiltbank MC, Machado AB et al (2018) Optimizing timed AI protocols for Angus beef heifers: Comparison of induction of synchronized ovulation with estradiol cypionate or GnRH. Theriogenology 121:7–12. https://doi.org/10.1016/j.theriogenology.2018.07.019 Silva GC, Silveira MV, Silva A et al (2024) Identification of parameters that affect the conception rate of precocious Nelore heifers before the start of the breeding season. Revista Brasileira de Zootecnia 53:e20230012. https://doi.org/10.37496/rbz5320230012 Valle VM, Fazzano CJ, Segui MS et al (2024) Bovine somatotropin increases the pregnancy rate in fixed-time artificial insemination in beef cattle. Reprod Domest Anim 59(6):e14642. https://doi.org/10.1111/rda.14642 Vázquez-López S, Hernández-Coronado CG, Calderón-Robles RC et al (2024) CO-Synch as a cheap fixed-time artificial insemination protocol to improve pregnancy rate in cow-calf production systems. Larg Anim Rev 30(2):85–92. https://www.largeanimalreview.com/index.php/lar/article/view/751 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-7745904","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":532917192,"identity":"7f3bac02-889f-4a1d-bce0-f2836914f89f","order_by":0,"name":"Cyndi G. Hernández-Coronado","email":"","orcid":"","institution":"Universidad Autónoma Metropolitana: Universidad Autonoma Metropolitana","correspondingAuthor":false,"prefix":"","firstName":"Cyndi","middleName":"G.","lastName":"Hernández-Coronado","suffix":""},{"id":532917193,"identity":"f16362a0-45f0-4a5b-9115-46262e600514","order_by":1,"name":"Rene C. Calderón-Robles","email":"","orcid":"","institution":"Instituto Nacional de Investigaciones Forestales Agricolas y Pecuarias","correspondingAuthor":false,"prefix":"","firstName":"Rene","middleName":"C.","lastName":"Calderón-Robles","suffix":""},{"id":532917194,"identity":"82c49cb6-aa03-446b-9d39-92c32e50166e","order_by":2,"name":"Ana María Rosales-Torres","email":"","orcid":"","institution":"Universidad Autonoma Metropolitana","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"María","lastName":"Rosales-Torres","suffix":""},{"id":532917195,"identity":"246b4104-749c-4dd1-a93a-e75ed8c7decf","order_by":3,"name":"Mariel Alejandra Ramos-Pérez","email":"","orcid":"","institution":"Universidad Autónoma Metropolitana: Universidad Autonoma Metropolitana","correspondingAuthor":false,"prefix":"","firstName":"Mariel","middleName":"Alejandra","lastName":"Ramos-Pérez","suffix":""},{"id":532917196,"identity":"e1f02320-c18b-45b1-83c8-433709d28c89","order_by":4,"name":"Ricardo Faustino-Carmona","email":"","orcid":"","institution":"Private Practice","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Faustino-Carmona","suffix":""},{"id":532917197,"identity":"26d6436a-46cd-4d29-b7bb-eb9ffe7d474f","order_by":5,"name":"Zaire B. Medina-Moctezuma","email":"","orcid":"","institution":"Universidad Autonoma Metropolitana","correspondingAuthor":false,"prefix":"","firstName":"Zaire","middleName":"B.","lastName":"Medina-Moctezuma","suffix":""},{"id":532917198,"identity":"bb9f7463-217d-49de-b85a-99085d31a0b5","order_by":6,"name":"David González-Aretia","email":"","orcid":"","institution":"Universidad Autonoma Metropolitana","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"González-Aretia","suffix":""},{"id":532917199,"identity":"e29c11a2-cec1-4a87-bca1-5ce0515d5baf","order_by":7,"name":"Adrian Guzmán-Sánchez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBADOdK1GJOuJbGBaKX8/WuMP92ouZPe39777MOHCobEfvYDbBI/cxiiDQ5g1yJx442Bcc6xZ7kzzhw3njnjDIOxZE8Cm2TvNobcmbgsvnHGIDmH7XDuBok0ZmbeNgY5gxsMbBK8QC39OHTIA7Uczvl3ON0ApOVvGwMPSIvkX6CWNhxaDM73GDbnth1OAGthhNoijc8Wwxtsxcy5fYcNZ5w5xszYc0YC6JfEZmvZbRI4/SJ3/vDmzznfDsvzt7cxM/yosAGG2OGDN99us8ndgCPEGCQSULlAzNgAZeAA/LjMGgWjYBSMglEAAwDI7VZYasYX0gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1104-882X","institution":"Universidad Autónoma Metropolitana: Universidad Autonoma Metropolitana","correspondingAuthor":true,"prefix":"","firstName":"Adrian","middleName":"","lastName":"Guzmán-Sánchez","suffix":""}],"badges":[],"createdAt":"2025-09-30 01:13:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7745904/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7745904/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94985833,"identity":"2cad3ff9-9f7f-4447-a455-ce98e891fa27","added_by":"auto","created_at":"2025-11-03 06:59:04","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139862,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/92dfc14277215208d286effc.tif"},{"id":94872959,"identity":"bbf1ba44-a62a-4a02-b9af-88d5e26c3bf7","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":237168,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/be157b2ac2e243a6b63cf11d.tif"},{"id":94872962,"identity":"68774d56-3ade-47ad-8695-aa1a648c64c5","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20465,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/9ac2ec25d135a3358310e1f9.docx"},{"id":94872952,"identity":"d779bbe5-57be-4d8d-b87e-1c2aeb005cb8","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12585,"visible":true,"origin":"","legend":"","description":"","filename":"tropTROPD2501886.xml","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/fd96f89bea3d88ada9be357f.xml"},{"id":94872953,"identity":"98db92a0-08ce-445a-8612-2b6ec8ab81ce","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1030,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD250188651247.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/9a89cea113cd33f45f99e9a9.xml"},{"id":94986800,"identity":"ae03ca6b-4c08-4e1d-9971-6e89832eb6f2","added_by":"auto","created_at":"2025-11-03 07:00:48","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1015,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD2501886Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/ea04da135df0d8a64c48fe8e.xml"},{"id":94872958,"identity":"8e8c71f1-16a6-4351-a30c-768e98ebcf32","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66190,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD25018860enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/cb38dcd32db92a39992930e6.xml"},{"id":94987019,"identity":"c81a2703-5dda-44c4-a2bc-032d24fd2f09","added_by":"auto","created_at":"2025-11-03 07:01:06","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139862,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/8089f0b575c475cae62dbe88.tif"},{"id":94872961,"identity":"fccb452a-4d87-4956-abb1-f99ed7207772","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":237168,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/39bafac6b60321b253d4bfe7.tif"},{"id":94872954,"identity":"f5baf787-584b-48c3-8583-bd916c42205c","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65147,"visible":true,"origin":"","legend":"","description":"","filename":"TROPD25018860structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/6b9d0f2f5c157f2091056f8a.xml"},{"id":94986198,"identity":"e7a68113-28c3-4911-88ca-860eb795b7a8","added_by":"auto","created_at":"2025-11-03 07:00:03","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":71700,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/d7db7c224f324ee19a03cb24.html"},{"id":94872950,"identity":"32ebbfb6-7186-40d0-8106-5bf95ef0d70c","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46985,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of an estradiol-based fixed-time artificial insemination protocol with the use of recombinant bovine somatotropin (rbST) on day 0 in beef cows\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/0e7a9d029e6495589095c32a.png"},{"id":94872951,"identity":"3c9a1b95-4efa-4530-a0ec-79c752a17ebe","added_by":"auto","created_at":"2025-10-31 15:21:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34855,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of recombinant bovine somatotropin (rbST) on dominant follicle diameter and IGF-1 serum concentrations at day 8 of a fixed-time artificial insemination protocol in beef cows\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/982d1c528758035bf6aee8ca.png"},{"id":96913113,"identity":"6a5a47a2-bd57-4eae-8267-baf4d935f3ed","added_by":"auto","created_at":"2025-11-27 13:52:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":880831,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7745904/v1/2d36d7da-1182-44d9-bcee-70ee1a4c4ced.pdf"}],"financialInterests":"","formattedTitle":"Follicular status, estrus behavior, and recombinant bovine somatotropin improve the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe use of fixed-time artificial insemination (FTAI) protocols in beef cows worldwide has increased recently because they facilitate the use of artificial insemination and improve reproductive performance in cow-calf production systems (B\u0026oacute; and Menchaca 2023). Even when use of these protocols have acceptable rates of pregnancy per artificial insemination (P/AI), the results are highly variable (Baruselli et al. 2018; Hern\u0026aacute;ndez-Coronado et al. 2023). For example, B\u0026oacute; et al. (2016) mentioned that P/AI ranged 6\u0026ndash;100%, whereas females treated with a CO-Synch protocol had very low P/AI (17.5%), which was improved to 35.3% by an estradiol-progesterone-based protocol (V\u0026aacute;zquez-L\u0026oacute;pez et al. 2024). However, these P/AI rates are lower than the expected 50\u0026ndash;60% P/AI reported by Hern\u0026aacute;ndez-Coronado et al. (2023). For this reason, it is important to develop strategies that may improve the P/AI rate in animals subjected to FTAI protocols.\u003c/p\u003e\u003cp\u003eSeveral reports on beef cows subjected to different protocols of FTAI have shown that the increased diameter of the dominant follicle (DF) after administration of prostaglandin F2α (PGF2α) or the diameter of the preovulatory follicle increased the rate of P/AI (Bishop et al. 2017; Pessoa et al. 2016). These results support the idea that the diameter of the preovulatory follicle is one of the most important factors that determines P/AI, which could be associated with a better oocyte quality and with the formation of a large corpus luteum (CL) capable of producing a large amount of progesterone (V\u0026aacute;zquez-L\u0026oacute;pez et al. 2024). Therefore, treatments that promote preovulatory follicle growth may favor success of FTAI protocols.\u003c/p\u003e\u003cp\u003eFollicular growth is regulated by several signals, including hormones and growth factors (Guzman et al. 2023). Among these molecules, insulin-like growth factor-I (IGF-1) and gonadotropins are the most important promoters of antral follicular growth (Rosales-Torres et al. 2012). IGF-1 is secreted by almost all body cells, but the main source of IGF-1 is the liver, which secretes large amounts of the growth factor in response to growth hormone (Hern\u0026aacute;ndez-Cer\u0026oacute;n and Gutierrez-Aguilar 2013). IGF-1 coming from the liver or synthesized locally stimulates proliferation of granulosa and theca cells and increases aromatase expression and estradiol secretion (Liu et al. 2021). Following its development in the 1980s, recombinant bovine somatotropin (rbST) has been widely used in several countries to stimulate milk production in dairy animals (Raux et al. 2022). However, because of the effect somatotropin has on IGF-1 secretion by the liver, it is likely that the use of rbST during the development of the preovulatory follicle may promote its growth and increase the P/AI rate in cows in a FTAI protocol. In non-lactating, multiparous cows in a FTAI protocol, the use of rbST increased the diameter of the preovulatory follicle and the CL diameter (Kaminski et al. 2019). Whereas an increased P/AI rate was seen in \u003cem\u003eBos taurus\u003c/em\u003e, but not \u003cem\u003eBos indicus\u003c/em\u003e, treated with rbST on the first day of a FTAI protocol (Gasseferth et al. 2023).\u003c/p\u003e\u003cp\u003eTherefore, the objective of the present study was to evaluate the effect of rbST on IGF-1 serum concentration, diameter of preovulatory follicle, and the reproductive response of beef cows subject to an estradiol/progesterone FTAI protocol.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eLocation and animals\u003c/h2\u003e\u003cp\u003eAll animal handling procedures were approved by the Research Ethics Committee of the Divisi\u0026oacute;n de Ciencias Biol\u0026oacute;gicas y de la Salud at the Universidad Aut\u0026oacute;noma Metropolitana-Xochimilco under protocol number CEI. 2022.014 in accordance with Section 2, Article 20, 3rd Title of the Federal Law on Animal Health (2018), Mexico. The study was performed on two commercial ranches. One ranch was located between 19\u0026deg;59\u0026acute;39\u0026rdquo; and 20\u0026deg;08\u0026acute;39\u0026rdquo; north latitude and 97\u0026deg;21\u0026acute;39\u0026rdquo; and 97\u0026deg;27\u0026acute;39\u0026rdquo; west longitude in Ayotoxco de Guerrero, Puebla, Mexico, and the other was located between 20\u0026deg;08\u0026rsquo; and 20\u0026deg;18\u0026rsquo; north latitude and 96\u0026deg;46\u0026rsquo; and 97\u0026deg; 02\u0026rsquo; west longitude in San Rafael, Veracruz, Mexico. A total of 107 \u003cem\u003eB. indicus \u0026times; B. taurus\u003c/em\u003e multiparous, postpartum beef cows were allocated in pastureland and fed common Star of Africa grass (\u003cem\u003eCynodon plectostachyus\u003c/em\u003e (K. Schum.) Pilg.) mixed with native grasses (\u003cem\u003eAxonopus\u003c/em\u003e spp. and \u003cem\u003ePaspalum\u003c/em\u003e spp.) as a basic diet supplemented with mineral salts (Ganafos 12%; Mexico) containing calcium (12%), phophorus (12%), and manganese (12,000 ppm) offered \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eAll the cows were subjected to an estradiol-based FTAI protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the first day of the protocol (day 0), the animals received an injection of 2 mg of estradiol benzoate (EB; Sincrodiol\u0026reg;, Ourofino Animal Health, Brazil), and an intravaginal progesterone device (Sincrogest\u0026reg;, Ourofino Animal Health, Brazil). The intravaginal device was removed eight days later, and 0.5 mg of cloprostenol (Sincrocio\u0026reg;, Ourofino Animal Health, Brazil) was injected. Between 48 and 56 hours after intravaginal device removal, all the cows were inseminated, and an injection of 150 mg of buserelin acetate (SincroForte\u0026reg;, Ourofino, Animal Health, Brazil) was used as an ovulation inductor. On day 0, the animals at each ranch were randomly divided into two experimental groups balanced by body condition score (BCS) and postpartum days. Females in the control group did not receive any additional treatment beyond synchronization, while the cows in the rbST group received a subcutaneous injection of 500 mg of rbST (Elanco\u003csup\u003eMT\u003c/sup\u003e, Argentina). On day 10, the cows were exposed to bull mating at a bull:cow ratio of 1:20 for a breeding season of 63 days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eOvarian dynamics\u003c/h3\u003e\n\u003cp\u003eThe ovarian structures were examined by transrectal ultrasonography (Aloka SSD-500, Aloka Hitachi, Tokyo, Japan), using a 7.5-MHz linear transrectal probe. Ultrasounds were performed at the beginning of the hormonal treatment (day 0) and when the intravaginal device was removed together with the injection of PGF2α on day 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At both times, the diameter of the largest follicle in the ovaries was measured, and the population of antral follicles (\u0026gt;\u0026thinsp;2 mm) was recorded. The DF diameter at follicular selection in \u003cem\u003eB. indicus\u003c/em\u003e is approximately 6 mm, and since we used \u003cem\u003eB. indicus\u003c/em\u003e \u0026times; \u003cem\u003eB. taurus\u003c/em\u003e animals in this experiment, the status of the follicular wave was classified as either in the recruitment phase when both ovaries had follicles\u0026thinsp;\u0026le;\u0026thinsp;6 mm in diameter or the selection/dominance phase when there was a follicle\u0026thinsp;\u0026gt;\u0026thinsp;6 mm in diameter in one of the ovaries.\u003c/p\u003e\n\u003ch3\u003eEstrus detection and pregnancy diagnosis\u003c/h3\u003e\n\u003cp\u003eAt the time of intravaginal device withdrawal, the cow\u0026rsquo;s tailhead was painted with marker crayons. At the time of AI, the presence or absence of crayon paint was recorded to determine if the females had or had not been mounted by an other cows. To determine the P/AI rate and the pregnancy rate during the breeding season, transrectal ultrasonography, using a 7.5-MHz linear transrectal probe, was performed at 35 days after AI and 45 after the end of the breeding period, respectively.\u003c/p\u003e\n\u003ch3\u003eDetermination of IGF-1 serum concentrations\u003c/h3\u003e\n\u003cp\u003eTo determine the concentrations of IGF-1 in serum, five randomly selected animals per group were sampled at the time of the insertion (day 0) and withdrawal (day 8) of the intravaginal progesterone device. From each animal 5\u0026ndash;10 mL of blood from the coccygeal vein were drawn in a vacuum tube. The samples were placed on ice immediately after collection and then centrifuged at 1,000\u0026times;g for 15 min. The serum was stored at \u0026minus;\u0026thinsp;20\u0026ordm;C until the IGF-1 serum concentration was assayed. IGF-1 serum concentration was determined using a commercial competitive enzyme-linked immunosorbent assay kit (Catalog No. EB0068, Fine Biotech Co., Ltd., Wuhan, China) according to the manufacturer\u0026rsquo;s instructions. Samples from days 0 and 8 were analyzed in duplicate in a dilution 1:2; the intra-assay variation coefficient was 8%.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eBCS on day 0 and DF diameter on days 0 and 8 were analyzed by Student\u0026rsquo;s t-test, whereas cows with CL on days 0 or 8, percentages of animals in estrus and pregnant in the breeding season were analyzed by Chi-square. To determine the effect of the independent variables (treatment, estrus behavior, status of follicular wave, location, CL presence, BCS, and DF diameter on day 8) on P/AI rate, we used the JMP-13 logistic regression procedure. To determine the factors that affect P/AI rate, two models were produced using the stepwise selection method. Model 1 included treatment, estrus behavior, status of follicular wave (days 0 and 8), location, and DF diameter on day 8. Model 2 included animals with CL on days 0 and 8 and BCS on day 8. IGF-1 serum concentrations on day 8 were analyzed by ANOVA using treatment as the fixed effect, IGF-1 serum concentration on day 0 as a covariate, and experimental replicate nested within cow as the random effect. Differences with P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically different, and differences with P values from 0.05 to \u0026lt;\u0026thinsp;0.10 were considered as tendencies.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThere were no significant differences (P\u0026thinsp;\u0026gt;\u0026thinsp;0.10) in BCS, DF diameter on day 0, percentage of animals with CL or DF on days 0 and 8, estrus presentation and pregnancy in the breeding season between control and rbST-treated cows (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the DF diameter and the IGF-1 serum concentrations on day 8 were higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in rbST-treated cows than in control cows (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\u003eOvarian dynamic and breeding season pregnancy rate in beef cows subject to a fixed timed-artificial insemination protocol and treated with recombinant bovine somatotropin (rbST).\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=\"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=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\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\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003erbST\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBCS on day 0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.7567\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDF diameter on day 0 (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.2402\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCows with CL on day 0 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (20.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16 (30.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.1720\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCows with DF on day 0 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42 (78.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39 (74.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.7733\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCows with CL on day 8 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (22.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14 (26.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.4103\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCows with DF on day 8 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 (56.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34 (64.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.2696\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCows in estrus (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34 (63.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e35 (66.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.3918\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber pregnant in breeding season (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27 (50.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29 (54.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.3841\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eSE\u0026thinsp;=\u0026thinsp;standard error; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;sample size; BCS\u0026thinsp;=\u0026thinsp;body condition score; DF\u0026thinsp;=\u0026thinsp;dominant follicle; CL\u0026thinsp;=\u0026thinsp;corpus luteum.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Model 1 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), cows treated with rbST, cows with a selected or dominant follicle at day 0 and from location 2 tend to have (P\u0026thinsp;\u0026lt;\u0026thinsp;0.10) higher P/AI rates than cows in the control group, in recruitment phase, and from location 1 respectively. Cows in estrus and with a selected or dominant follicle at day 8 had higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) P/AI rates than cows without estrus behavior and with recruited follicles. The DF diameter on day 8 did not influence (P\u0026thinsp;\u0026gt;\u0026thinsp;0.10) the P/AI rate. The odd ratios indicated that cows treated with rbST tended to have 2.4 times greater probability of pregnancy per AI than non-treated cows, while females that had a selected or dominant follicle at day 8 have 8.2 times higher probability of pregnancy per AI than females with recruited follicles (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In Model 2 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), only the presence of CL on day 0 significantly affected (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the P/AI rate, whereas there was no effect by CL presence on day 8 or BCS on day 0.\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\u003eInfluence of recombinant bovine somatotropin (rbST), estrus behavior, status of follicular wave, location and diameter of the dominant follicle (DF) on rate of pregnancy per artificial insemination (P/AI) in beef cows subjected to a fixed timed-artificial insemination protocol.\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=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eConstant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eP/AI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eOdd ratio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eEstimate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-0.3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.8193\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eControl (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e18/54 (33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0823\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003erbST (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e24/53 (45.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEstrus behavior\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eNo (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e8/37 (21.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0055\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eYes (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e33/70 (47.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e4.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStatus of follicular wave on day 0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eRecruitment (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e5/24 (20.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0568\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSelection or Dominance (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e36/81 (44.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStatus of follicular wave on day 8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eRecruitment (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e8/38 (21.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0189\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSelection or Dominance (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e31/64 (48.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e8.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLocation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLocation 1 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e14/42 (33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.0738\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLocation 2 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e28/65 (43.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDiameter of DF on day 8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.2652\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eSE\u0026thinsp;=\u0026thinsp;standard error.\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\u003eInfluence of the corpus luteum (CL) and body condition score (BCS) on the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol.\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\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEstimate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntercept\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.2717\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnimal with CL on day 0 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0325\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnimal with CL on day 8 (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.8165\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBCS on day 0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.1857\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSE\u0026thinsp;=\u0026thinsp;standard error.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur experiment showed that the injection of rbST on day 0 in cows subjected to an estradiol-based FTAI protocol had enhanced P/AI rates. This was likely due to an increase in the IGF-1serum concentrations and the diameter of the preovulatory follicle.\u003c/p\u003e\u003cp\u003eCows treated with rbST on day 0 of the FTAI protocol had higher IGF-1 serum concentrations than untreated cows eight days later. Similar to our results, injection of rbST into Angus-based, crossbred beef cows at the beginning of an FTAI protocol increased IGF-1 serum concentrations compared to control cows (Oosthuizen et al. 2018). In beef heifers, IGF-1 serum concentrations were higher 22 days after, rbST treatment compared to control animals (Sanford et al. 2021). It is known that rbST, after absorption, binds to the growth hormone (GH) receptor in the liver to promote IGF-1 synthesis and secretion from the hepatocytes via the GH receptor/JAK2/PLC/Ca2\u0026thinsp;+\u0026thinsp;pathway (Kimura et al. 2023;\u003c/p\u003e\u003cp\u003eOur study also showed that rbST administration on day 0 of the FTAI protocol enhances the DF diameter on day 8. Other studies have reported that cows treated with rbST had large DF 8\u0026ndash;10 days after treatment compared to untreated animals (Kaminski et al. 2019; Valle et al. 2024) indicating that rbST promote follicular growth. The somatotropin-IGF-1 axis is an important regulator of follicular development in several species, including bovines (Benetis-Piauet et al. 2023). Ovarian follicular cells have receptors for both somatotropin and IGF-1, and GH directly or via IGF-1 stimulates granulosa cell proliferation, synthesis of steroidogenic hormones, and synthesis of gonadotropin receptors (Chang et al. 2022). Therefore, in our experiment it is likely that rbST by itself or the increase in IGF-1 serum concentration stimulated proliferation of granulosa cells promote estradiol secretion and synthesis of gonadotropin receptors to increase the diameter of the preovulatory follicle.\u003c/p\u003e\u003cp\u003eOverall, the beef cows treated with rbST in our study had 36% higher P/AI rate than untreated cows. When females were treated with rbST on the first day of a FTAI protocol, there was a 58% increase in the P/AI rate in \u003cem\u003eB. taurus\u003c/em\u003e and crossbred cows (Gasseferth et al. 2023) and a 37% increase in \u003cem\u003eB. indicus\u003c/em\u003e cows (Vale et al. 2024). One of the most important factors that affects the P/AI rate in beef cows subjected to a FTAI protocols is the diameter of the preovulatory follicle because this parameter is highly correlated with oocyte quality and the formation of a large CL that produces enough progesterone to maintain pregnancy (Baruselli et al. 2023 ). Therefore, an increase in the diameter of the preovulatory follicle induced by rbST likely explains the higher P/AI rate in the treated cows in our study.\u003c/p\u003e\u003cp\u003eThe results of our experiment also showed that ovarian dynamics and estrus behavior affect the P/AI rate. Cows with a follicle in the selection/dominance phase on day 0 of the FTAI protocol had at least three times higher probability of becoming pregnant after AI than animals with recruited follicles. Treatment with estradiol benzoate plus the insertion of a progesterone implant on day 0 has the objective to reduce peripheric concentration of gonadotropins and induce follicular atresia to synchronize the onset of a new follicular wave (B\u0026oacute; et al. 2023; Hern\u0026aacute;ndez-Coronado et al. 2023). Large antral follicles could be more susceptible to becoming atretic due to a reduction in trophic factors. Therefore, in animals with follicles in the selection/dominance phase on day 0 of the FTAI, it is more likely to initiate and synchronize a new follicular wave to have a preovulatory follicle days later when the IA is performed. Similar to our results, Oosthuizen et al. (2020), Rodrigues et al. (2018) and Silva et al. (2024) observed that cows with follicles in the selection/dominance phase on day 8 of the FTAI protocol had higher probability of pregnancy than cows with recruited follicles. Thus, it is likely that the selection/dominance phase at the beginning of proestrus assures that a preovulatory follicle will ovulate with the injection of gonadotropin-releasing hormone to improve the P/AI rate. As in previous reports (Silva et al. 2018; V\u0026aacute;zquez-L\u0026oacute;pez et al. 2024), the cows with CL at the beginning of the FTAI protocol in our study had higher probability of P/AI than animals without CL. The presence of a CL is associated with cow cyclicity, which indicates that postpartum cows are producing viable oocytes (Crowe 2008). This association explains the positive relationship between the presence of CL and P/AI rate that we observed.\u003c/p\u003e\u003cp\u003eThe cows in estrus at the time of insemination in our study had higher P/AI rates than the cows not displaying estrus behavior, which corroborates the findings in studies by Nogueira et al. (2019). It has been suggested that cows in estrus produce a better-quality oocyte (V\u0026aacute;zquez-L\u0026oacute;pez et al. 2024), thus explaining the positive association between estrus behavior and fertility.\u003c/p\u003e\u003cp\u003eIn conclusion, our research indicates that beef cows treated with rbST at the beginning of an estradiol-based FTAI protocol have improved P/AI rates because rbST increases the IGF-1 serum concentration that enhances the diameter of the preovulatory follicle and likely the quality of the oocyte, and the CL derived from it. In addition, cows with follicles in the selection/dominance phase on day 8 of the protocol have 8.2 times higher probability of pregnancy per IA than cows with recruited follicles. Our research confirms that cows with a CL at the beginning of the FTAI protocol have a higher probability of P/AI than cows without a CL because the presence of a CL is associated with cow cyclicity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cp\u003eAll animal procedures were approved by the Research Ethics Committee of the Divisi\u0026oacute;n de Ciencias Biol\u0026oacute;gicas y de la Salud from the Universidad Aut\u0026oacute;noma Metropolitana-Xochimilco under protocol number CEI. 2022.014\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cp\u003eThe consent was obtained from all authors.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eCyndi G. Hern\u0026aacute;ndez-Coronado, Ana Ma. Rosales-Torres: Writing-review and editing. Rene C. Calder\u0026oacute;n-Robles, Ricardo Faustino-Carmona, Mariel Alejandra Ramos-P\u0026eacute;rez: Investigation, Data Collection, and Methodology. Zaire B. Medina-Moctezuma, David. Gonz\u0026aacute;lez-Aretia: Hormanal determination and data collection. Adri\u0026aacute;n Guzm\u0026aacute;n-S\u0026aacute;nchez: Writing Original Draft, Methodology, Investigation, Supervision, Funding acquisition, and Project administration.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors are grateful for the financial support provided by the Universidad Aut\u0026oacute;noma Metropolitana-Xochimilco via the divisional project CBS-34402080 and the program Fortalecimiento de la Investigaci\u0026oacute;n 2022. Grant number R.X.155.22.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaruselli PS, Abreu LA, Catussi BLC et al (2023) Use of new recombinant proteins for ovarian stimulation in ruminants. Anim Reprod 20(2):e20230092. https://doi.org/10.1590/1984-3143-ar2023-0092\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaruselli PS, Ferreira RM, Filho MFS, B\u0026oacute; GA (2018) Review: Using artificial insemination v. natural service in beef herds. Animal 12(s1):s45-s52. https://doi.org/10.1017/S175173111800054X\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBenetis-Piau TB, de Queiroz-Rodrigues A, Paulini F (2023) Insulin-like growth factor (IGF) performance in ovarian function and applications in reproductive biotechnologies. Growth Horm IFG Res. 72\u0026ndash;73:101561. https://doi.org/10.1016/j.ghir.2023.101561\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBishop BE, Thomas JM, Abel JM. et al (2017) Split-time artificial insemination in beef cattle: III. Comparing fixed-time artificial insemination to split-time artificial insemination with delayed administration of GnRH in postpartum cows. Theriogenology, 99:48\u0026ndash;52. https://doi.org/10.1016/j.theriogenology.2017.04.046\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB\u0026oacute; GA, Paucar F, Mendoza BL, Pinagorte L, Ocampo V, Cede\u0026ntilde;o V (2023) Pregnancy rates in Bos indicus \u0026times; Bos taurus recipients synchronized with a GnRH/progesterone-based or an estradiol/progesterone-based protocol with prolonged proestrus. Reproduction Fertility and Development 36(2):207\u0026ndash;208. https://doi.org/10.1071/RDv36n2Ab110\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB\u0026oacute; GA, de la Mata JJ, Baruselli PS, Menchaca A (2016) Alternative programs for synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 86(1):388\u0026ndash;396. https://doi.org/10.1016/j.theriogenology.2016.04.053\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB\u0026oacute; GA, Menchaca A (2023) Prohibition of hormones in animal reproduction: what to expect and what to do?. Anim Reprod 20(2):20230067. https://doi.org/10.1590/1984-3143-AR2023-0067.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang CW, Sung YW, Hsueh YW et al (2022) Growth hormone in fertility and infertility: Mechanisms of action and clinical applications. Front in Endocrinol 13:1040503. https://doi.org/10.3389/fendo.2022.1040503\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrowe, M (2008) Resumption of Ovarian Cyclicity in Post-partum Beef and Dairy Cows. Reprod Domest Anim 43:20\u0026ndash;28. https://doi.org/10.1111/j.1439-0531.2008.01210.x\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGasseferth G, Gaievski FR, Bergstein-Galan TG et al (2023) Effect of recombinant bovine somatotropin on the reproductive efficiency of beef cows subjected to differently timed-artificial insemination protocols. Reprod Domest Anim. 58(12):1654\u0026ndash;1661. https://doi.org/10.1111/rda.14479\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuzm\u0026aacute;n A, Hern\u0026aacute;ndez-Coronado CG, Guti\u0026eacute;rrez CG, Rosales-Torres AM (2023) The vascular endothelial growth factor (VEGF) system as a key regulator of ovarian follicle angiogenesis and growth. Mol Reprod Dev 90(4):201\u0026ndash;217. https://doi.org/10.1002/mrd.23683\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-Cer\u0026oacute;n J Gutierrez-Aguilar CG (2013) Recombinant bovine somatotropin and reproduction in cattle, sheep and goat. Agrociencia 47(1):35\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez-Coronado C, Rosales-Torres A, V\u0026aacute;zquez-L\u0026oacute;pez S, Guzm\u0026aacute;n-S\u0026aacute;nchez A (2023) Synchronization of estrus and ovulation in bovine females. Endocrine bases and treatments used. Abanico Veterinario 13:1\u0026ndash;37 https://doi.org/10.21929/abavet2023.16\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaminski AP, Andrade-Carvalho ML, Segui MS et al (2019) Impact of recombinant bovine somatotropin, progesterone, and estradiol benzoate on ovarian follicular dynamics in Bos taurus taurus cows using a protocol for estrus and ovulation synchronization. Theriogenology 140:58\u0026ndash;61. https://doi.org/10.1016/j.theriogenology.2018.11.009\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKimura M, Moteki H, Ogihara M (2023) Role of Hepatocyte Growth Regulators in Liver Regeneration. Cells 12(2):208. https://doi.org/10.3390/cells12020208\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu T, Huang Y, Lin H (2021) Estrogen disorders: Interpreting the abnormal regulation of aromatase in granulosa cells (Review). Int J Mol Med 47(5):73. https://doi.org/10.3892/ijmm.2021.4906\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNogueira E, Silva MR, Silva JCB (2019) Timed artificial insemination plus heat I: effect of estrus expression scores on pregnancy of cows subjected to progesterone\u0026ndash;estradiol-based protocols. Animal 13(10):2305\u0026ndash;2312. https://doi.org/10.1017/s1751731119000442\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOosthuizen N, Fontes PLP, Henry DD et al (2018) Administration of recombinant bovine somatotropin prior to fixed-time artificial insemination and the effects on fertility, embryo, and fetal size in beef heifers. J Anim Sci 96(5):1894\u0026ndash;1902. https://doi.org/10.1093/jas/sky077\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOosthuizen N, Cooke RF, Schubach KM et al (2020) Effects of estrous expression and intensity of behavioral estrous symptoms on variables associated with fertility in beef cows treated for fixed-time artificial insemination. Anim Reprod Sci 214:106308\u0026ndash;106308. https://doi.org/10.1016/j.anireprosci.2020.106308\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePessoa GA, Martini AP, Carloto GW et al (2016) Different doses of equine chorionic gonadotropin on ovarian follicular growth and pregnancy rate of suckled Bos taurus beef cows subjected to timed artificial insemination protocol.\u0026rdquo; Theriogenology 85(5):792\u0026ndash;799. https://doi.org/10.1016/j.theriogenology.2015.09.05\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaux A, Bichon E, Benedetto A et al (2022) The Promise and Challenges of Determining Recombinant Bovine Growth Hormone in Milk. Foods 11(3):274. https://doi.org/10.3390/foods11030274\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRodrigues AD, Cooke RF, Cipriano RS et al (2018) Impacts of estrus expression and intensity during a timed-AI protocol on variables associated with fertility and pregnancy success in Bos indicus-influenced beef cows. J Anim Sci96(1):236\u0026ndash;249. https://doi.org/10.1093/jas/skx043\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRosales-Torres AM, Guzm\u0026aacute;n-S\u0026aacute;nchez A, Aguilar CG (2012) Follicular development in dosmestic ruminants. Tropic SubtropAgroeco15(1):s147\u0026ndash;s160.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanford CD, Owen MPT, McCarty K et al (2021) Effects of Administering Exogenous Bovine Somatotropin During the First Trimester of Pregnancy Altered Uterine Hemodynamics in Suckled Beef Cows. Front AnimSci 2:744790. https://doi.org/10.3389/fanim.2021.744790\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva EP, Wiltbank MC, Machado AB et al (2018) Optimizing timed AI protocols for Angus beef heifers: Comparison of induction of synchronized ovulation with estradiol cypionate or GnRH. Theriogenology 121:7\u0026ndash;12. https://doi.org/10.1016/j.theriogenology.2018.07.019\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva GC, Silveira MV, Silva A et al (2024) Identification of parameters that affect the conception rate of precocious Nelore heifers before the start of the breeding season. Revista Brasileira de Zootecnia 53:e20230012. https://doi.org/10.37496/rbz5320230012\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValle VM, Fazzano CJ, Segui MS et al (2024) Bovine somatotropin increases the pregnancy rate in fixed-time artificial insemination in beef cattle. Reprod Domest Anim 59(6):e14642. https://doi.org/10.1111/rda.14642\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eV\u0026aacute;zquez-L\u0026oacute;pez S, Hern\u0026aacute;ndez-Coronado CG, Calder\u0026oacute;n-Robles RC et al (2024) CO-Synch as a cheap fixed-time artificial insemination protocol to improve pregnancy rate in cow-calf production systems. Larg Anim Rev 30(2):85\u0026ndash;92. https://www.largeanimalreview.com/index.php/lar/article/view/751\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":"beef cows, recombinant bovine somatotropin, IGF-1, ovarian dynamics, estrus synchronization, pregnancy","lastPublishedDoi":"10.21203/rs.3.rs-7745904/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7745904/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo evaluate the effect of rbST on insulin-like growth factor 1 (IGF-1) serum concentration, ovarian dynamics, and the reproductive response of beef cows subjected to an estradiol-based fixed-time artificial insemination (FTAI) protocol, 107 \u003cem\u003eBos indicus\u003c/em\u003e \u0026times; \u003cem\u003eBos taurus\u003c/em\u003e multiparous, postpartum beef cows on two commercial herds were used. The animals were randomly divided into two experimental groups balanced by body condition score. Females in the control group did not receive any additional treatment, whereas cows in the treatment group received a subcutaneous injection of 500 mg of rbST. The ovarian dynamics and pregnancy were determined by transrectal ultrasonography. Ovarian dynamics were recorded at the beginning of the hormonal treatment (day 0) and when the intravaginal device was removed (day 8). IGF-1 serum concentrations were measured by enzyme-linked immunosorbent assay on days 0 and 8. The diameter of the dominant follicle and the IGF-1 serum concentrations on day 8 were higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in rbST-treated cows than in control cows. Logistic regression model indicated that cows with a dominant follicle on days 0 and 8 and in estrus had greater probability of pregnancy (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas use of rbST tended (P\u0026thinsp;\u0026lt;\u0026thinsp;0.10) to increase the rate of pregnancy per artificial insemination (P/AI). The presence of corpus luteum on day 0 positively affected (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the P/AI rate. Our research indicated that beef cows subjected to the estradiol-based FTAI protocol with rbST injected on the first day had improved P/AI rate likely because the increased IGF-1 serum concentrations induced by rbST enlarge the diameter of the preovulatory follicle.\u003c/p\u003e","manuscriptTitle":"Follicular status, estrus behavior, and recombinant bovine somatotropin improve the rate of pregnancy per artificial insemination in beef cows subjected to a fixed timed-artificial insemination protocol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 15:21:00","doi":"10.21203/rs.3.rs-7745904/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":"333de9b3-f55f-4e91-9981-42cea651e128","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-20T10:48:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 15:21:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7745904","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7745904","identity":"rs-7745904","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}