In vivo Study

The study was conducted on Holstein dairy cows at the dairy farm of the Agricultural Research Organization's Volcani Center according to the Ethics Committee of the Hebrew University. The selected animals (n = 12) were non-pregnant lactating cows, cyclic and healthy, over 100 days in milk. Cows were synchronized according to the “OvSynch” protocol which includes 2 ml intramuscular injection of GnRH analog (day 0), followed by injection of 2.5 ml PGF2α on day 7 and a second injection of 2 ml GnRH 48 h later. The second GnRH injection was defined as day −1 of the synchronized cycle. An additional PGF2α injection was carried out at the end synchronization to confirm ovulation. Follicle and corpus luteum development through the synchronized cycle were recorded by ultrasound monitoring (Aloka, SSD-900, Tokyo, Japan).

Blood and milk samples were collected daily from day 3 pre-estrus to day 2 post-estrus, and from day 17 to day 21 post-estrus. Blood samples were collected from the coccygeal blood vessel into vacuum tubes (Becton Dickinson Systems, Crowley, UK). The plasma was immediately separated by centrifugation for 10 min at 800× g and stored until analysis at −20°C. Plasma was taken to determine progesterone concentration using the Progesterone RIA kit according the manufacturer's protocol. Cows were milked three times a day and the lactation data, including milk yield, fat, protein, and lactose concentrations, were recorded automatically by the Afilab system (Afikim, Israel). Milk samples from the morning milking were stained with Nile red and MFG size was determined (detailed below).