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Doppler sonography of the corpus luteum during the oestrus cycle in dairy cows

A reliable assessment of the age and functional status of the bovine corpus luteum (CL) is critical for different field applications such as early pregnancy diagnosis, use of prostaglandins in oestrus synchronizing programs, or selection of donors and recipients in embryo transfer programs. Under field conditions, the functional status of a CL is determined by transrectal palpation, which is based on the positive correlation between luteal size (LS) and progesterone (P4) levels (12, 15), although even experienced practitioners can make mistakes in CL manual assessment. Nowadays, ultrasound is the gold standard for a more reliable CL diagnosis,permitting evaluation of LS throughout the measure of the transverse diameter of the CL (9, 11, 15). It has been debated whether LS is the most appropriate parameter for assessing the functional status of a CL (3, 9, 12, 15, 19). During the regression phase, large CLs may be already inactive (9, 15, 18). P4 levels are strictly correlated with luteal blood flow (LBF) that can be assessed by transrectal colour Doppler sonography (1). In cows, LBF has been examined during short phases of the oestrous cycle (1, 10, 14) and during an entire oestrus cycle (6). There are many differences among these studies regarding the enrolled animals, the ultrasound scanner and its setting, the kind of Doppler, the technique to capture representative scans of the CL and the system of evaluation of the blood flow. For this reason, data are not easily repeatable. Furthermore, the number of follicular waves affects the duration of the oestrus cycle in bovines. About 80% of dairy cows has two follicular waves and an interoestrus of about 20 days, while the remaining 20% has 3 follicular waves and an interoestrus of about 23 days; the timing of luteolysis is different and the luteal phase is 2-4 days longer in 3 follicular wave cows (5). The aim of the study was to evaluate by two alternative systems (image analysis and visual score) changes of LBF in the different phases of the oestrous cycle in cows with two waves of follicular growth dairy cattle by applying Colour and Power Doppler ultrasound.

Material and methods

The study was approved by the Ethics Committee of the Department of Veterinary Sciences of the University of Messina (reference number 010/2016). Friesian cows from a high standard farm in Ragusa, Italy, were selected for the following criteria: age (3-5 years), over 70 days post-partum, good reproductive anamnesis, body condition score greater than or equal to 3, healthy, lack of abnormality and disease of the genital tract, anatomical conformation of uterus and ovaries (pelvic displacement), and docile temperament. Ultrasound and Doppler examination were performed using an Esaote My Lab Vet 30 Gold (Genoa, Italy) equipped with a 5-7.5 MHz endocavitary linear probe. Only Cows with a CL larger than 2 cm in diameter and a dominant follicle over 8 mm were synchronized with a single administration of the synthetic prostaglandin F2alpha (cloprostenol 500 mcg im, Estrotek, Fatro). The visual monitoring of the oestrous manifestations was performed twice a day until the onset of the standing oestrus. Only cows showing standing oestrus from 48 (day 0) to 72 h (day 1) after prostaglandin injection were enrolled. The ultrasound sessions started from day 4, when the newly formed CLs could be evaluated. The procedure was repeated at day 8, 12, 16 and 20.Furthermore, the follicular wave emergence at the 4th day (1st follicular wave) and at 12th day (2nd follicular wave) was recorded and the dominant follicle of the 2nd follicular wavewas followed until ovulation. The cows were restrained by means of self-tapping racks and at the same time palatable food was supplied. The Doppler scans were performed by slowly moving the transducer on the ovary surface, from the cranial to the caudal pole until identifying the luteal spiral artery and visualize the maximum transverse diameter of the CL, in order to scan a vertical plane including the apex and the basis of the CL (1). The transducer was kept at a distance of about 1-2 cm from the CL. The ultrasound setting for each session in colour flow mode (CFM) was: frequency 5.0 MHz, gain 70%, pulse repetition frequency (PRF) 2.1 KHz, dynamic range, enhancement and density (PRC) 3-BA, persistence (PRS) 4, velocity ± 16 m/s; and in power flow mode (PFM): frequency 5.0 MHz, gain 70%, PRF 2.8 KHz, PRC 3-BA, PRS 4. In absence of flash and blooming artefacts, at least 3 images were recorded for each session and for all the functions: B-mode, CFM and PFM. The real area of the CL (RACL) was calculated with the formula RACL = Π(D/2)2 –Π(d/2)2 , where D was the diameter of the CL and d the diameter of the eventual inner cavity. The LBF was quantified off-line by means of a visual score and of an image analysis system (Digimizer 4.1) (Fig. 1). The LBF was expressed in cm 2 . A visual score developed for follicles (4) was also used, estimating the percentage of coloured luteal tissue respect to the complete circumference delimiting the CL (Fig. 2). The following grading was employed: 1 if less of 25%; 2 if between 25 and 50%; 3 if between 50 and 75%; 4 if over 75%. Each ultrasound session had an average duration of about 7 minutes and was performed from 9:30 to 11:30 am by the same operator. After verifying the normality of the data, the repeatability

Material and methods

Fig. 1. Image analysis (Digimizer 4.0) for the calculation of the area of corpus luteum and inner cavity. Luteal blood flow area was obtained by the addiction of the coloured pixels inside the luteal parenchyma

coloured peripherical parenchym

Fig. 2. Visual score was based on the percentage of coloured peripherical parenchyma of the corpus luteum. Score 1: less than 25%. Score 2: from 25 to 50%. Score 3 from 50 to 75%. Score 4 more than 75%

A total of 548 scans were performed, 160 in B-mode, 209 in CFM and 179 in PFM with a mean of 3.2, 4.2 and 3.6 scans for session and cow. RACL and LBF were calculated for each scan. The probability of an erroneous capture of the CL and, consequently, measurement of RACL and LBF was 0.00 for B-mode, 0.01 for CFM and 0.01 for PFM. An Anova one-way was employed to exclude individual variability. The Student unpaired test was finally employed to compare the means for each detected parameter (RACL, LBF in CFM, PFM, visual score CFM, visual score PFM) in the days of the oestrus cycle. The significance was set at 0.05. The employed software was “goodcalculators”.


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