Performing Department
Dairy Science
Non Technical Summary
Pregnancy loss represents a substantial economic cost for dairy farms due to critical delays in time to pregnancy and decreased offspring (Cabrera, 2012; Giordano et al., 2012; Ribero et al., 2012) and, among domestic livestock species, lactating dairy cows experience relatively high rates of pregnancy loss after establishing a viable pregnancy. In a summary of 14 studies (Santos et al., 2004), pregnancy loss from an initial pregnancy diagnosis based on transrectal ultrasonography at 27 to 31 d after artificial insemination (AI) to a pregnancy reconfirmation at 38 to 50 d of gestation averaged 13%. Vasconcelos et al. (1997) characterized pregnancy loss throughout gestation and reported pregnancy losses of 11% from 28 to 42 d, 6% from 42 to 56 d, and 2% from 56 to 98 d after AI, supporting that the rate of pregnancy loss is greater early during gestation, and then decreases as gestation proceeds. Progesterone (P4) produced by the corpus luteum (CL) in the ovary is the most biologically active progestogen in cattle, and there is an unequivocal requirement for P4 to support embryo development as well as for establishment and maintenance of pregnancy (reviewed by Spencer et al., 2016).The overall objective of this proposal is to understand the underlying mechanisms causing pregnancy loss and to develop strategies to decrease pregnancy loss in lactating dairy cows.Maintenance of the CL to provide continued P4 production during pregnancy occurs during two critical periods after artificial insemination and fertilization. The initial and most studied signal for maternal recognition of pregnancy in cattle occurs around Day 16 of the estrous cycle. In nonpregnant cows, prostaglandin F2α (PGF2α) pulses produced by the uterine epithelium act on the CL in the ovary to induce luteolysis thereby initiating a new estrous cycle (Spencer and Hansen, 2015; Arosh et al., 2016). By contrast, in pregnant cows, secretion of interferon-tau (IFNτ) from the trophectoderm of the developing embryo is the initial signal that the embryo has undergone elongation (Brooks et al., 2014; Bazer et al., 2015). Production of IFNτ from the embryo blocks PGF2α pulses from the uterine endometrium, the CL is maintained, and the pregnancy advances to the second month of gestation. Further, the mechanism for luteal regression and maintenance in ruminants occurs through a local, rather than a systemic pathway. Based on early experiments in ewes, ipsilateral hysterectomy prolonged CL lifespan, whereas contralateral hysterectomy failed to affect CL lifespan (Inskeep and Butcher, 1966). The ovarian artery in ruminants is convoluted and lies in close apposition to the uterine vein (Ginther and Delcampo, 1974). Studies of vascular anastomoses demonstrated that the uterine horn secreted PGF2α into the uterine vein that diffused to the ovarian artery thereby delivering PGF2α to the ovary bearing the CL and causing luteolysis (Mapletoft et al., 1976).It has recently been proposed that a second critical period of CL maintenance occurs during the second month of pregnancy in cattle after the initial signal for maternal recognition of pregnancy (Wiltbank et al., 2016). A recent study (Baez et al., 2017) reported that during the second month of pregnancy, cows with induced accessory CL located on the contralateral ovary (opposite side to pregnancy) regress, whereas accessory CL located ipsilateral to the pregnancy are maintained. Surprisingly, only 25% of accessory CL regressed during the normal "maternal recognition" period during the first month of pregnancy, whereas 75% of accessory CL regressed between day 30 and 60 of pregnancy (Baez et al., 2017), a period of gestation when substantial pregnancy loss occurs in dairy cows (Vasconcelos et al., 1997; Santos et al., 2004). The authors concluded that there must be a second pivotal period for CL maintenance that occurs during the second month of pregnancy (Wiltbank et al., 2016). That there is a second period of maternal recognition of pregnancy during the second month of pregnancy is completely novel idea, and this phenomenon as well as potential mechanisms underlying it has not been studied.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Goals / Objectives
Increase the efficiency and predictability of sustainable reproductive technologies and management programs for cattle.
Evaluate mechanisms that regulate reproductive processes impacting production efficiency in cattle.
Disseminate reproductive management information to stakeholders to improve sustainability of cattle enterprises.
Project Methods
The overall objective of this proposal is to understand the underlying mechanisms causing pregnancy loss and to develop strategies to decrease pregnancy loss in lactating dairy cows. The three experiments in this proposal address questions related to the role of progesterone during the two critical periods of CL maintenance that occur during the first and second month of pregnancy in dairy cows.Specific Objective 1 is to manipulate progesterone early after ovulation to affect luteal function and embryo growth and development.Experiment 1 will evaluate the effect of insemination and treatment with human chorionic gonadotropin (hCG) on the timing of luteal regression. Lactating dairy cows (n=120) will be synchronized for first timed artificial insemination (TAI) using a Double-Ovsynch protocol as per the standard reproductive management protocol of the herd. Cows will be unequally randomized to two treatments, and data collection and laboratory procedures will be conducted as we have described elsewhere. Cows (n=40) in the first treatment will be synchronized but will not be inseminated (negative control), whereas cows (n=80) in the second treatment will receive TAI 16 h after the end of the synchronization protocol. Within each treatment, cows will be further randomized to receive 3,000 IU hCG 7 d after the end of the synchronization protocol to induce ovulation of a first-wave dominant follicle. Blood samples will be collected thrice weekly (MWF) from 4 to 32 d after TAI for analysis of plasma P4 concentrations and at 25 and 32 d after TAI for evaluation of plasma PAG levels, by venipuncture of the median coccygeal artery or vein. Ovarian structures will be evaluated using a portable scanner equipped with a 7.5 MHz linear-array transducer thrice weekly (MWF) from 4 to 32 d after TAI to measure CL and follicle diameters. For cows in the first treatment, and for cows diagnosed not pregnant 32 d after TAI in the second treatment, the day of CL regression will be defined as the day P4 first decreased to < 1.0 ng/mL.Experiment 2 will evaluate the effects of increasing or decreasing progesterone early after insemination on embryo growth and development. Specific Objective 2 is to develop a practical management strategy to increase fertility and/or decrease pregnancy loss in lactating dairy cows after insemination. Lactating Holstein cows (n = 120) will be submitted to a Double-Ovsynch protocol for first TAI (Herlihy et al., 2012) and will be assigned randomly to the following three treatments (n=40 cows/trt): 1) Control: no treatment; 2) High P4: 3,000 IU hCG 7 d after TAI and two intravaginal P4 inserts (CIDR; Zoetis Animal Health) from 7 to 14 d after TAI, and 3) Low P4: 25 mg PGF2α (Dinoprost) 5 d after TAI. This experiment will be conducted using laboratory methods we have described elsewhere (Carvalho et al., 2017). Blood samples will be collected thrice weekly from 5 to 29 d after TAI for all cows and from 32 to 67 d for pregnant cows, and will be analyzed for P4 and pregnancy-specific protein B (PSPB) concentrations. Expression of interferon-tau stimulated gene 15 (ISG15) will be assessed in blood leukocyte mRNA 18 and 20 d after TAI (see letter of collaboration from Dr. Laura Hernandez). Pregnancy diagnosis will be performed weekly using transrectal ultrasonography from 32 to 67 d after TAI, and embryonic crown-rump length will be measured using transrectal ultrasonography 32, 39, and 46 d after TAI.Experiment 3 is designed as a large field trial to evaluate treatment with hCG 7 d after insemination and/or at Day 32 of pregnancy on pregnancy outcomes and/or pregnancy loss in lactating dairy cows. This study will be conducted as a field trial in collaboration with several large commercial dairy farms in Wisconsin. Lactating dairy cows (n = 4,000) will be submitted to a Double-Ovsynch protocol (Herlihy et al., 2012) for submission for first TAI. Cows will be randomized to one of two treatments on 7 d after TAI: 1) Saline (control, C, n = 2,000) or treatment with 3,000 IU hCG (hCG7; n = 2,000). On Day 32, cows diagnosed pregnant will be randomly assigned to one of two treatments: 1) saline (Sal33) or treatment with 3,000 IU hCG (hCG32). This experiment is a 2x2 factorial design and will result in the following 4 treatments: 1) Saline on Day 7 and Day 32 (Control); 2) hCG on Day 7, saline on Day 32 (hCG7-Sal32); 3) Saline on Day 7 and hCG on Day 32 (Sal7-hCG32); 4) hCG on both Day 7 and Day 32 (hCG7-hCG32). All cows will be evaluated using transrectal ultrasonography on Days 0, 7, 14, 32, and 39 to test Hypotheses 1 and 2, whereas a subset of cows (n = 2,000) will be evaluated twice weekly from Days 32 to 60 of pregnancy to evaluate Hypothesis 3 to determine the underlying cause of pregnancy loss. To determine whether CL regression precedes embryo loss (basal P4 [< 1 ng/mL] before basal PAG [< 0.5 ng/mL for PSPB and < 1 ng/mL for PAG ELISA) or, alternatively, whether embryo death precedes CL regression (basal PAG before basal P4) twice weekly blood sampling and ultrasound will be conducted (evaluation of heart-beat) in half of the cows from day 32 to 60 of pregnancy (n = 2,000 cows). Based on 50% P/AI, I anticipate ~1,000 pregnancies will be available for evaluation and from 60 (6% loss) to 120 (12% loss) pregnancy losses will be available for evaluation. In cows that undergo pregnancy loss, we will evaluate all blood samples before loss and for 2 weeks after pregnancy loss. We will use a case-control designby designating two matching cows (matched for week of TAI) that did not experience pregnancy loss for each cows undergoing pregnancy loss. Data on these matching cows will be used as the baseline values to compare cows with pregnancy loss.