Introduction

In order to achieve maximum success at artificial insemination (AI), capacitated sperm should be present in the female reproductive tract at ovulation time. Then, AI should be carried out close to the ovulation time.

The hormonal fluctuations during the oestrous cycle induce changes on the female reproductive tract at both uterine and ovary level (Carrière et al., 2010Carrière PD, Gnemmi G, DesCôteaux L, Matsui M, Miyamoto A, Colloton J, 2010. Bovine ovary. In: Practical atlas of ruminant and camelid reproductive ultrasonography; DesCôteaux L, Gnemmi G, Colloton J (eds). pp: 35-59. Wiley Blackwell Publ, Ames. https://doi.org/10.1002/9781119265818.ch4; DesCôteaux et al., 2010DesCôteaux L, Chastant-Maillard S, Gnemmi G, Colloton J, Bollwein H, 2010. Bovine uterus. In: Practical atlas of ruminant and camelid reproductive ultrasonography; DesCôteaux L, Gnemmi G, Colloton J (eds). pp: 61-80. Wiley Blackwell Publishing, Ames. https://doi.org/10.1002/9781119265818.ch5). The uterine wall presents oedema, and a variable quantity of mucus in the uterine lumen can be observed, which may be greater during oestrus. Besides, uterine blood flow velocity increases during proestrus and oestrus and returns to its lowest values after ovulation (Bollwein et al., 2000Bollwein H, Meyer HHD, Maierl J, Weber F, Baumgartner U, Stolla R, 2000. Transrectal doppler sonography of uterine blood flow in cows during the estrous cycle. Theriogenology 53: 1541-1552. https://doi.org/10.1016/S0093-691X(00)00296-X). Regarding the ovary, not only an increase in size of the preovulatory follicle can be observed, but also an increase in blood flow around the initiation of the luteinizing hormone (LH) surge that reaches its maximum just before ovulation (Carrière et al., 2010Carrière PD, Gnemmi G, DesCôteaux L, Matsui M, Miyamoto A, Colloton J, 2010. Bovine ovary. In: Practical atlas of ruminant and camelid reproductive ultrasonography; DesCôteaux L, Gnemmi G, Colloton J (eds). pp: 35-59. Wiley Blackwell Publ, Ames. https://doi.org/10.1002/9781119265818.ch4).

In addition, hormonal environment is responsible for the oestrus behaviour, which is characterized by standing immobile while being mounted, restlessness, sniffing the vulva of another cow, resting chin, and mounting (Roelofs et al., 2010Roelofs J, López-Gatius F, Hunter RHF, van Eerdenburg FJCM, Hanzen C, 2010. When is a cow in estrus? Clinical and practical aspects. Theriogenology 74: 327-344. https://doi.org/10.1016/j.theriogenology.2010.02.016). Following these signs, the well-known rule “AM-PM” has been established as a recommendation for AI after oestrus detection (Trimberger, 1948Trimberger GW, 1948. Breeding efficiency in dairy cattle from artificial insemination at various intervals before and after ovulation. Bull Agr Exp Stat Nebraska 153: 117.). Overall, it has been stated that the optimal time of insemination is 24 to 12 h before ovulation (Pursley et al., 1998Pursley RJ, Silcox RW, Wiltbank MC, 1998. Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. J Dairy Sci 81: 2139-2144. https://doi.org/10.3168/jds.S0022-0302(98)75790-X; Roelofs et al., 2006aRoelofs JB, Graat EAM, Mullaart E, Soede NM, Voskamp-Harkema W, Kemp B, 2006a. Effects of insemination-ovulation interval on fertilization rates and embryo characteristics in dairy cattle. Theriogenology 66: 2173-2181. https://doi.org/10.1016/j.theriogenology.2006.07.005).

Nowadays, automatic milking machines and monitoring devices are the new tools to perform oestrus detection, and each day more farms are switching to this approach (Roelofs & Van Erp-Van Der Kooij, 2015Roelofs JB, Van Erp-Van Der Kooij E, 2015. Estrus detection tools and their applicability in cattle: recent and perspectival situation. Anim Reprod 12(3): 498-504.; Saint-Dizier & Chastant-Maillard, 2018Saint-Dizier M, Chastant-Maillard S, 2018. Potential of connected devices to optimize cattle reproduction. Theriogenology 112: 53-62. https://doi.org/10.1016/j.theriogenology.2017.09.033). These technologies can discern between the onset, middle and end of oestrus, and thus determine its duration and even the maximum heat activity.

Therefore, whether using observational oestrus detection or electronic devices, it is necessary to determine which oestrus characteristic features are better indicators of ovulation, aiming to perform AI at the appropriate time. Some studies have been conducted using behavioural signs (Layek et al., 2011Layek SS, Mohanty TK, Kumaresan A, Behera K, Chand S, 2011. Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle. Anim Reprod Sci 129: 140-145. https://doi.org/10.1016/j.anireprosci.2011.11.006), ultrasonographic features of the preovulatory follicle (Siddiqui et al., 2010Siddiqui MAR, Ferreira JC, Gastal EL, Beg MA, Cooper DA, Ginther OJ, 2010. Temporal relationships of the LH surge and ovulation to echotexture and power Doppler signals of blood flow in the wall of the preovulatory follicle in heifers. Reprod Fertil Dev 22: 1110. https://doi.org/10.1071/RD09251), and progesterone (P4) concentrations (Roelofs et al., 2006bRoelofs JB, Van Eerdenburg FJCM, Hazeleger W, Soede NM, Kemp B, 2006b. Relationship between progesterone concentrations in milk and blood and time of ovulation in dairy cattle. Anim Reprod Sci 91: 337-343. https://doi.org/10.1016/j.anireprosci.2005.04.015). Additionally, investigations on the relation between uterine wall changes and hormone concentration and fertility have been reported (Souza et al., 2011Souza AH, Silva EPB, Cunha AP, Gümen A, Ayres H, Brusveen DJ, et al., 2011. Ultrasonographic evaluation of endometrial thickness near timed AI as a predictor of fertility in high-producing dairy cows. Theriogenology 75: 722-733. https://doi.org/10.1016/j.theriogenology.2010.10.013; Sugiura et al., 2018Sugiura T, Akiyoshi S, Inoue F, Yanagawa Y, Moriyoshi M, Tajima M, et al., 2018. Relationship between bovine endometrial thickness and plasma progesterone and estradiol concentrations in natural and induced estrus. J Reprod Dev 64: 135-143. https://doi.org/10.1262/jrd.2017-139). To our knowledge, research on the combined evaluation of uterine features and size and vascularization of the preovulatory follicle to predict ovulation time, with the aid of monitoring devices to perform oestrus detection, has not been published. In addition, another value of this study is that it aims to simulate as closely as possible the conditions veterinarians have to face on farm work in a daily basis, so the results can have a direct application.

Consequently, the objective of this study was to assess the utility of B-mode ultrasonography to predict ovulation time by assessment of uterine and follicular measurements; additionally, we aimed to determine the usefulness of Power Doppler ultrasonography compared to B-mode to predict ovulation, by the assessment of blood flow to the preovulatory follicle.

Material and methods

Results and discussion

Of the 33 cows, 8 (24%) did not respond to the treatment. In addition, 2 cows (6%) ovulated before 52 h and were excluded from de analysis. The distribution of ovulation times of the remaining cows (n = 23) is displayed in Fig. 1. Additionally, descriptive statistics are shown in Table 1. No statistically significant differences were observed in this experiment regarding 2 or 3 PGF2α administrations for the variables of interest.

Results for the one-way ANOVA test showed that, for the variable UL, time -6 (1.53 mm) significantly differed from time -42 (5.69 mm, p = 0.016). Concerning DF, significant differences were observed between time -6 (20.48 mm) and time -54 (16.60 mm, p = 0.038). As for P4, significant differences were found between time -6 (0.34 ng/mL) and time -54 (1.03 ng/mL, p = 0.013). No significant differences were found between examination times for END/MYO (p = 0.707). Considering MHA, statistically significant differences were observed for the UL between group AFT (1.60 mm) and groups BEF and DUR (4.90 mm (p = 0.002) and 4.84 mm (p = 0.009), respectively); similar results were obtained for DF, with significant differences between group BEF (18.63 mm) and group AFT (20.90 mm, p = 0.043). As for heat, the UL significantly differed between group AFT (1.57 mm) and groups BEF and DUR (5.09 and 4.25 mm, respectively, p = 0.002).

Results for the Pearson’s χ² test showed that the percentage of cows with Doppler signal in the preovulatory follicle at examination times -54, -42, -30, -18, and -6 h was 0%, 23.1%, 14.3%, 36.4%, and 54.2%, respectively (p = 0.011). For the variable PDCL, the percentage of cows with Doppler signal was 60.0%, 23.1%, 19.0%, 8.7%, and 0% (p < 0.001). Concerning MHA and heat, statistically significant differences were found for both variables (Table 2).

One important finding was the presence of Doppler signal in most preovulatory follicles few hours before ovulation, and the increase in the percentage of follicles with blood flow during and after MHA and heat. Blood flow to the preovulatory follicle is correlated to the increase concentration of oestradiol and the LH surge (Acosta et al., 2003Acosta TJ, Hayashi KG, Ohtani M, Miyamoto A, 2003. Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows. Reproduction 125: 759-767. https://doi.org/10.1530/rep.0.1250759; Pancarci et al., 2012Pancarcı ŞM, Ari UÇ, Atakisi O, Güngör Ö, Çiğremiş Y, Bollwein H, 2012. Nitric oxide concentrations, estradiol-17β progesterone ratio in follicular fluid, and COC quality with respect to perifollicular blood flow in cows. Anim Reprod Sci 130: 9-15. https://doi.org/10.1016/j.anireprosci.2011.12.013). Moreover, Siddiqui et al. (2010Siddiqui MAR, Ferreira JC, Gastal EL, Beg MA, Cooper DA, Ginther OJ, 2010. Temporal relationships of the LH surge and ovulation to echotexture and power Doppler signals of blood flow in the wall of the preovulatory follicle in heifers. Reprod Fertil Dev 22: 1110. https://doi.org/10.1071/RD09251) reported a biphasic increase and decrease in blood flow to the preovulatory follicle, with the first peak occurring 3 h after GnRH treatment and the second peak occurring 8-6 h before ovulation. Probably due to our study design (examinations carried out every 12 h), we were not able to identify the two peaks, but a progressive increase in follicles with detectable peripheral vascularization instead. Our results suggest that the identification of Doppler signal on the follicular wall might be of use as an indicator of ovulation within the following 6 to 18 h. Additionally, this identification took place mostly after MHA and heat, determined by electronic monitoring devices, which offers the possibility of a complementary use of these two approaches.

Regarding the UL, differences were observed among times of examination. During oestrus, the UL may show an accumulation of anechogenic content inside the uterine horns, that will normally disappear during the dioestrus phase (DesCôteaux et al., 2010DesCôteaux L, Chastant-Maillard S, Gnemmi G, Colloton J, Bollwein H, 2010. Bovine uterus. In: Practical atlas of ruminant and camelid reproductive ultrasonography; DesCôteaux L, Gnemmi G, Colloton J (eds). pp: 61-80. Wiley Blackwell Publishing, Ames. https://doi.org/10.1002/9781119265818.ch5). However, no significant differences were observed for UL at the examination times closer to ovulation (-18 and -6), in contrast with the PDF. Consequently, UL may be useful as a positive sign of heat, but it may not be an adequate parameter to estimate ovulation time. Special attention should be paid to the characteristics of the uterine content to discern between heat and pathological situations like endometritis (Sheldon et al., 2006Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO, 2006. Defining postpartum uterine disease in cattle. Theriogenology 65: 1516-1530. https://doi.org/10.1016/j.theriogenology.2005.08.021).

As for END/MYO, no significant differences were found among examination times. The high level of circulating oestrogens during the perioestrus period is responsible for the greater thickness and oedema of the uterine wall, that will normally decrease between 4 and 5 days after oestrus (DesCôteaux et al., 2010DesCôteaux L, Chastant-Maillard S, Gnemmi G, Colloton J, Bollwein H, 2010. Bovine uterus. In: Practical atlas of ruminant and camelid reproductive ultrasonography; DesCôteaux L, Gnemmi G, Colloton J (eds). pp: 61-80. Wiley Blackwell Publishing, Ames. https://doi.org/10.1002/9781119265818.ch5). A possible explanation for the lack of significance could be the fact that the first examination was delayed 52 h after PGF2α administration. Therefore, cows were already at the onset of heat. Nevertheless, no differences were found at the times closer to ovulation, which suggest that END/MYO could be a good parameter to identify cows in heat, but not to predict the time of ovulation. Additionally, it should be noted that endometrial thickness also increases during endometritis due to inflammation. Consequently, a differential diagnosis should be made taking into account other aspects such as the uterine fluid, time of the cycle and the diagnostic tools available like endometrial cytology (Dubuc et al., 2010Dubuc J, Duffield TF, Leslie KE, Walton JS, LeBlanc SJ, 2010. Definitions and diagnosis of postpartum endometritis in dairy cows. J Dairy Sci 93: 5225-5233. https://doi.org/10.3168/jds.2010-3428).

Finally, we used Doppler ultrasonography and P4 serum levels to assess luteal function, since its accuracy has been previously described by numerous researchers (Siqueira et al., 2013Siqueira LGB, Areas VS, Ghetti AM, Fonseca JF, Palhao MP, Fernandes CAC, et al., 2013. Color Doppler flow imaging for the early detection of nonpregnant cattle at 20 days after timed artificial insemination. J Dairy Sci 96: 6461-6472. https://doi.org/10.3168/jds.2013-6814; Rocha et al., 2019Rocha CC, Martins T, Cardoso BO, Silva LA, Binelli M, Pugliesi G, 2019. Ultrasonography-accessed luteal size endpoint that most closely associates with circulating progesterone during the estrous cycle and early pregnancy in beef cows. Anim Reprod Sci 201: 12-21. https://doi.org/10.1016/j.anireprosci.2018.12.003; Dubuc et al., 2020Dubuc J, Houle J, Rousseau M, Roy JP, Buczinski S, 2020. Short communication: Accuracy of corpus luteum color flow Doppler ultrasonography to diagnose nonpregnancy in dairy cows on day 21 after insemination. J Dairy Sci 103: 2019-2023. https://doi.org/10.3168/jds.2019-17234). In this case, similar results were obtained, as PDCL progressively decreased until heat, and was absent near ovulation.

In conclusion, the use of Power Doppler to predict the time of ovulation needs further research. Additionally, END/MYO and UL could be of use to identify cows in heat; however, their application to determine ovulation time seems to be inaccurate. Therefore, the difficulties to predict the exact moment of ovulation in field conditions have to be considered, and additional studies, including a bigger sample size, are needed.