Estrumate

Embryos were loaded into embryo transfer straws in the laboratory and transported in a portable incubator at 38.5 °C. When transferring fresh embryos, adult recipient animals were synchronized to be in heat on the same day as LOPU, for embryo transfer to occur eight days later. Twenty-one days before ET, a bovine progesterone implant was inserted (EAZI-BREED^TM CIDR^® 1380; Zoetis Canada), and it was removed ten days later. Animals received 400 IU eCG (Folligon, Intervet Canada, Kirkland, QC, Canada) and 375 μg cloprostenol (Estrumate^®, Merck Animal Health, Kirkland, QC, Canada) at the time of CIDR removal. Forty-eight hours later, animals received 50 μg gonadorelin GnRH (Cystorelin^®, Merial Canada). The day before ET, recipients were assessed for the presence of corpora lutea. One to two embryos were transferred non-surgically into the uterine horn ipsilateral to the corpus luteum. Pregnancy was assessed 30–40 days following ET using trans-rectal ultrasonography.

Virgin Angus heifers across two different years (year 1, n = 38; year 2, n = 28; total n = 66) during the month of April and located at a University of Tennessee AgResearch and Education Center were grazed on mixed grass pastures and had ad libitum access to hay. Average heifer age was 1.2 ± 0.1 yr. Estrus was synchronized according to Figure 1A and defined as the time when a heifer first stood to be mounted by another. Depending on the year, prostaglandin F_2α (PGF_2α) administered was dinoprost tromethamine (25 mg i.m.; Lutalyse, Zoetis, Parsippany, NJ, USA) or cloprostenol (500 μg i.m., Estrumate, Merck, Rahway, NJ, USA). Eleven days later, gonadotropin-releasing hormone (GnRH) was administered (100 μg i.m., gonadorelin hydrochloride, Factrel, Zoetis or 100 μg i.m., gonadorelin diacetate tetrahydrate, Cystorelin, Boehringer Ingelheim, Duluth, GA, USA). A controlled internal drug release (CIDR) device was placed intravaginally (1.38 g progesterone, Eazi-Breed CIDR, Zoetis). Seven days later, the CIDR was removed, PGF_2α was administered, and an iButton that had been previously affixed to a blank CIDR (containing no progesterone) was placed intravaginally. Beginning ~24 h after PGF_2α, estrus activity was visually assessed hourly until the first heifer displayed signs of estrus at which point monitoring was continuous by a team of individuals on a rotating schedule.

The aim of this experiment was to broadly characterize and compare changes in immune cell populations and expression of immunoregulatory molecules of heifers during different days of the estrous cycle and pregnancy. From 12/2018 to 04/2019, Holstein dairy heifers (N = 7, nulliparous, 13–14 months of age) were synchronized to estrus with an intramuscular injection of prostaglandin F2 alpha (PGF2A) analog (Estrumate, 500 μg cloprostenol sodium, Merck Animal Health) and monitored for estrus (day 0). Blood was collected between 8–10 am (during feeding) from the tail vein into K3 vacuette tubes (Greiner Bio-one) containing ethylenediaminetetraacetic acid (EDTA) on days 14, 17 and 20 of the estrous cycle. Later, the same animals were synchronized to estrus and inseminated, followed by blood collection on days 14, 17, 20 and 23 of pregnancy. Blood samples were used for PBL isolation and hormone assays. Pregnancy was confirmed on day 30 after insemination by transrectal ultrasonography. Two inseminated heifers became pregnant after the second service while the others carried first-service pregnancies. All pregnant animals delivered healthy calves.