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 

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