Non Technical Summary
Early pregnancy failures continue to be a major problem in the beef and dairy industries resulting in millions of dollars lost in meat and milk production. The most significant window of pregnancy loss occurs around day 14 - 18 after mating when slow developing embryos fail to prevent onset of the next estrous cycle. Immature embryos are unable to robustly communicate their presence, and thus prostaglandin F2a escapes the pregnant uterus and destroys the corpus luteum (luteolysis) and its essential progesterone support to the nascent pregnancy. Data show that supplementing dietary omega-3 fatty acids from fish oil extends the pregnancy recognition window by de-sensitizing the corpus luteum to uterine prostaglandin F2a, but the underlying mechanisms are still largely unknown. A mature corpus luteum exposed to prostaglandin F2a experiences a rapid (within 6 hours) reduction in blood flow and diminished progesterone production due to downregulation of critical progesterone synthesis genes. It is hypothesized that dietary omega-3 fatty acids from fish oil will prevent prostaglandin F2a-induced reductions in luteal blood flow and progesterone production. The objectives of the current study will be to deliver a natural dose of prostaglandin F2a to cattle synchronized to day 14-18 of the estrous cycle and determine whether dietary omega-3 fatty acids preserve luteal: 1) blood flow, 2) progesterone production, and 3) abundance of key progesterone synthesis proteins. It is anticipated that dietary omega-3 fatty acids from fish oil will reduce sensitivity of the corpus luteum to a natural prostaglandin F2a challenge, preserving luteal blood flow and sustaining progesterone secretion. Outcomes from these studies would suggest that the reproductive efficiency of cattle operations could be improved through development of novel, cost-effective omega-3 fatty acid supplementation strategies that reduce luteal prostaglandin F2a sensitivity and maintain progesterone support for the embryo during the critical window that a pregnancy is trying to establish.
Animal Health Component
Research Effort Categories
Goals / Objectives
Our overarching goal is to reduce early embryonic mortality and improve profitability in farm animals through supplemental feeding strategies. Dietary supplementation of fish oil is luteal protective to intrauterine infusions of prostaglandin F2a. However, there is currently a knowledge gap in our understanding of how fish products affect corpus luteum function in ruminants, specifically luteal sensitivity to prostaglandin F2a. The goal of this project is to bridge this gap of knowledge. The specific objectives of this project will be to 1) determine luteal blood flow following intrauterine infusion of prostaglandin F2a and 2) determine the expression of key genes and abundance of critical proteins that regulate progesterone biosynthesis following prostaglandin F2a treatment.
Beef cows will be individually fed diets that consist of 95% mixed hay and will be randomly allotted to a 5% vegetable or fish oil supplement group. Rations will be delivered daily at a dry matter intake equivalent to 2.0% body weight for 60 days. Supplements will be formulated to be isonitrogenous and isocaloric. Body weights will be determined weekly and diets adjusted as needed to maintain desired supplementation at 5% dry matter intake. Weekly blood samples will be collected to monitor plasma omega-3 fatty acid composition. Estrous cycles will be synchronized using two injections of prostaglandin F2a at 14-day intervals. Data collection will commence between day 10 to 12 following synchronized estrus, which will be approximately day 60 of the supplementation period. Cows will be administered two intrauterine infusions of 0.25 mL saline or 0.5 mg prostaglandin F2a diluted in 0.25 mL saline at 12-h intervals. Jugular blood samples will be collected immediately before infusion and at 3-h intervals for the first 24 h. After which, samples will be collected at 6-h intervals until ovariectomy to determine serum concentrations of progesterone. Ovariectomies will be performed at 30 or 48 h of the experimental period. In experiment 1, color-Doppler ultrasonography will be used to estimate luteal blood flow following prostaglandin F2a treatment. The percentage of luteal area with color-Doppler signals of blood flow will be estimated at the first intrauterine infusion (0 h) and at second intrauterine infusion (12 h). Relative peak systolic velocity and time-averaged maximum velocity at each time point will be estimated. Cows infused with two doses of prostaglandin F2a will be grouped as having a regressed or functional corpus luteum. Cows with regressed corpus luteum will have serum progesterone below 1 ng/mL at the time of ovariectomy. The effects of dietary supplementation (vegetable oil or fish oil), treatment (saline, prostaglandin F2a regressed corpus luteum, or prostaglandin F2a functional corpus luteum), and time on changes in luteal blood flow will be analyzed using the mixed models procedure of SAS with repeated measures. The statistical model will include dietary supplementation, treatment, time and all possible interactions as sources of variation. Animal within dietary supplementation will be considered as a random variable in the statistical model. If main effects or interactions are significant (P < 0.05), means will be separated using Tukey's. The corpus luteum will be cut into four equal sections at the time of ovariectomy and snap frozen in liquid nitrogen. In experiment 2a, one section of tissue will be used to determine fold change in expression of gene that regulate progesterone biosynthesis (steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, 3-beta hydroxysteroid dehydrogenase, and low-density lipoprotein receptor) following prostaglandin treatment using qPCR. In experiment 2b, another section of tissue will be used to determine changes in abundance of protein for these genes using western blotting. Prostaglandin F2a treated animals will be sorted into regressed or functional CL as described above. The effects of dietary supplementation (vegetable oil or fish oil), treatment (saline, prostaglandin F2a regressed CL and prostaglandin F2a functional CL), and time on fold-change in mRNA or protein abundance (increase or decrease) of steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, 3-beta hydroxysteroid dehydrogenase and low-density lipoprotein receptor will be analyzed using the mixed models procedure of SAS. The statistical model will include dietary supplementation, treatment, time, and all possible interactions as sources of variation. Animal within dietary supplementation will be considered as a random variable in the statistical model. If main effects or interactions are significant (P < 0.05), means will be separated using Tukey's.