Our objectives were to evaluate ovarian dynamics and fertility comparing 2 treatments at the start of a progesterone (P4)-based fixed-time artificial insemination (FTAI) protocol and 2 treatments at the end of the protocol. Thus, 1,035 lactating Holstein cows were assigned in a random phase of the estrous cycle to 1 of 4 treatments using a completely randomized design with a 2×2 factorial arrangement. At the beginning of the protocol (d -10), cows received GnRH or estradiol benzoate (EB) and, at the end, EB (d -1) or estradiol cypionate (ECP; d -2), resulting in 4 treatments: GnRH-EB, GnRH-ECP, EB-EB, and EB-ECP. All cows received an intravaginal P4 device on d -10, which was removed on d -2. Cows also received PGF on d -3 and -2. The FTAI was performed on d 0. Ovaries were evaluated by ultrasound for corpus luteum (CL) presence and regression (d -10 and -3) and follicle measurements (d -10 and 0), as well as the uterus for percentage pregnant per AI (P/AI; d 32 and 60). Blood samples were collected (d -10 and -3) for P4 measurements. Treatment with GnRH rather than EB tended to increase P/AI on d 32 (38.2 vs. 33.7%) and on d 60 (32.9 vs. 28.9%). More cows treated with GnRH had CL on d -3 compared with EB-treated cows (77.3 vs. 58.3%), due to less CL regression between d -10 and -3 (24.7 vs. 43.8%) and more cows with a new CL on d -3 (35.9 vs. 25.0%). Cows treated with GnRH also had greater P4 concentrations on d -3 than EB cows (3.4 vs. 2.0 ng/mL). Increased circulating P4 at the start of the protocol (d -10) decreased the probability of ovulation to EB or GnRH at that time. Cows from GnRH group also ovulated a larger-diameter follicle at the end of the protocol (15.5 vs. 14.7mm). No difference between EB and ECP in P/AI on d 32 (34.8 vs. 37.0) and 60 (30.8 vs. 31.0%) or in pregnancy loss (11.1 vs. 15.4%) was detected and we found no interaction between treatments for P/AI. Independent of treatment, cows with CL on d -10 and -3 had the greatest P/AI on d 60 (36.9%). In conclusion, treatments at the end of the protocol were similar for ECP or EB and we found no additive effect or interactions on P/AI between treatments. However, cows treated with GnRH rather than EB on d -10 had less luteolysis and tended to have greater P/AI, probably because P4 concentrations were greater during the protocol. Finally, regardless of treatments, cows with CL at the beginning of the protocol as well as at the time of PGF had greater fertility.

Interferon-tau (IFNT), serves as a signal to maintain the corpus luteum (CL) during early pregnancy in domestic ruminants. We investigated here whether IFNT directly affects the function of luteinized bovine granulosa cells (LGCs), a model for large-luteal cells. Recombinant ovine IFNT (roIFNT) induced the IFN-stimulated genes (ISGs; MX2, ISG15, and OAS1Y). IFNT induced a rapid and transient (15–45 min) phosphorylation of STAT1, while total STAT1 protein was higher only after 24 h. IFNT treatment elevated viable LGCs numbers and decreased dead/apoptotic cell counts. Consistent with these effects on cell viability, IFNT upregulated cell survival proteins (MCL1, BCL-xL, and XIAP) and reduced the levels of gamma-H2AX, cleaved caspase-3, and thrombospondin-2 (THBS2) implicated in apoptosis. Notably, IFNT reversed the actions of THBS1 on cell viability, XIAP, and cleaved caspase-3. Furthermore, roIFNT stimulated proangiogenic genes, including FGF2, PDGFB, and PDGFAR. Corroborating the in vitro observations, CL collected from day 18 pregnant cows comprised higher ISGs together with elevated FGF2, PDGFB, and XIAP, compared with CL derived from day 18 cyclic cows. This study reveals that IFNT activates diverse pathways in LGCs, promoting survival and blood vessel stabilization while suppressing cell death signals. These mechanisms might contribute to CL maintenance during early pregnancy.

This experiment aimed to compare circulating progesterone (P4), follicular dynamics, and fertility during reuse of intravaginal P4 implants that were sanitized by autoclave or chemical disinfection in lactating Holstein cows submitted to fixed-time artificial insemination (FTAI). For this, 123 primiparous and 226 multiparous cows from 2 farms, averaging (mean ± standard deviation) 163.9 ± 141.9 d in milk, 35.7 ± 11.3 kg of milk/d, and a body condition score of 2.9 ± 0.5, were enrolled in the study. Cows were randomly assigned to 1 of 2 treatments using a completely randomized design and each cow received a reused implant (1.9 g of P4; previously used for 8 d) that was either autoclaved (AUT; n = 177) or chemically disinfected (CHEM; n = 172) on d -10. Also on d -10, cows received 2 mg of estradiol benzoate and 100 μg of GnRH. On d -3, cows received 25 mg of dinoprost (PGF). A second PGF was given on d -2, along with 1 mg of estradiol cypionate and P4 implant removal. Cows received FTAI on d 0. A subset of cows (n = 143) was evaluated by ultrasound on d -10, -8, -6, -3, -2, 0, and 5 to identify ovarian structures, and blood was sampled on d -10, -3, and -2 for P4 concentrations by RIA. Pregnancy diagnoses were performed at d 32 and 60. Statistical analyses was performed using PROC-MIXED for continuous variables and PROC-GLIMMIX of SAS 9.4 (SAS Institute Inc., Cary, NC) for binomial variables. The treatments did not differ in circulating P4 on d -10 or -3, but P4 was greater on d -2 in CHEM cows. Ovulation to the treatments on d -10 was associated with lower circulating P4 on d -10 (2.0 vs. 3.1 ng/mL) and resulted in greater P4 on d -3 (4.0 vs. 2.4 ng/mL) and more cows with a corpus luteum on d -3 (100 vs. 40%) than nonovulating cows. Cows that ovulated to d -10 treatments were more likely to have a synchronized new follicular wave (97.9 vs. 63.2%) and had an earlier wave emergence (1.9 vs. 2.6 d), resulting in less cows ovulating a persistent follicle (0.0 vs. 35.7%). Type of P4 implant, corpus luteum presence on d -10, and ovulation to d -10 treatments did not affect fertility (pregnancy per AI; P/AI). However, P/AI on farm A was greater than on farm B at 32 (40.8 vs. 27.8%) and 60 d (35.8 vs. 24.3%), independent of treatment. In conclusion, P4 implants with different P4 release patterns did not produce detectable differences in follicular dynamics, synchronization rate, or P/AI. Nevertheless, presence of corpus luteum or ovulation at the beginning of the FTAI protocol affected reproductive variables, such as timing and synchronization of follicular wave emergence, and size of the ovulatory follicle. Beyond that, more overall synchronized cows became pregnant to the FTAI protocol.

Maintenance of the corpus luteum (CL) during pregnancy is essential for continuing the elevated circulating progesterone (P4) that is required to maintain pregnancy. The mechanisms that protect the CL during early pregnancy when the non-pregnant animal would typically undergo CL regression have been extensively investigated. It is clear uterine prostaglandin F2α (PGF) causes regression of the CL in non-pregnant ruminants and that maintenance of the CL during early pregnancy is dependent upon secretion of interferon-tau (IFNT) from the elongating embryo. A number of specific mechanisms appear to be activated by IFNT. Most studies indicate that there is an inhibition of oxytocin-induced secretion of uterine PGF. There is also evidence for increased resistance to PGF action, perhaps due to secretion of PGE2 and PGE1 or direct endocrine actions of circulating IFNT. These mechanisms occur concurrently and each may help to maintain the CL during the first month of pregnancy. However, during the second month of pregnancy, IFNT is no longer secreted by the embryo. Attachment of the embryo to the uterus and subsequent placentome development have been linked to silencing of expression from the IFNT gene. In addition, there is some evidence that oxytocin responsiveness of the uterus returns during the second month of pregnancy leading to substantial basal secretion of PGF and perhaps PGF pulses. There is also no evidence that the CL during the second month of pregnancy is resistant to the actions of PGF as observed during the first month. Thus, this manuscript attempts to compare the mechanisms that maintain the CL during the first and second months of pregnancy in ruminants and provides a new, speculative, physiological model for maintenance of the CL during month two of pregnancy that is distinct from the previously-described mechanisms that maintain the CL during the first month of pregnancy.

The aim of this study was to compare plasma progesterone (P4) concentrations in nonlactating, multiparous Holstein cows (n = 24) treated with 2 types of intravaginal implants containing either 1.0 or 1.9 g of P4 either at the first use or during reuse of the implants after sanitizing the implant by autoclave or chemical disinfection. In a completely randomized design with a 2 × 3 factorial arrangement and 2 replicates, every cow underwent 2 of 6 treatments. Two sources of P4 [controlled internal drug release (1.9 g of P4) from Zoetis (São Paulo, Brazil), and Sincrogest (1.0 g of P4) from Ourofino (Cravinhos, Brazil)] and 3 types of processing, new (N), reused after autoclave (RA), and reused after chemical disinfection (RC), were used. After inducing luteolysis to avoid endogenous circulating P4, the cows were randomized in 1 of 6 treatments (1.9 g of N, 1.9 g of RA, 1.9 g of RC, 1.0 g of N, 1.0 g of RA, and 1.0 g RC). Cows were treated with the implants for 8 d and during this period blood samples were collected at 0, 2, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h. Statistical analyses were performed using Proc-Mixed and the mean ± standard error of the mean P4 concentrations were calculated using the Proc-Means procedures of SAS 9.4 (SAS Institute Inc., Cary, NC). No interaction between treatments was observed. Comparing types of implant, average P4 concentrations during treatments were greater for 1.9 g than 1.0 g (1.46 vs. 1.14 ± 0.04 ng/mL). When types of processing were compared, average P4 concentrations did not differ between autoclaved and new inserts (1.46 vs. 1.37 ± 0.05 ng/mL; respectively), but both were greater than chemically disinfected implants (1.09 ± 0.04 ng/mL). Within 1.9-g P4 inserts, P4 concentrations from autoclaved implants were greater than new, which were greater than chemically disinfected (1.67 ± 0.06 vs. 1.49 ± 0.07 vs. 1.21 ± 0.05 ng/mL; respectively). For 1.0-g P4 implants, P4 concentrations from autoclaved did not differ from new, but both were greater than chemically disinfected (1.20 ± 0.08 vs. 1.24 ± 0.06 vs. 0.97 ± 0.05 ng/mL; respectively). In conclusion, the mean plasma P4 concentration in nonlactating Holstein cows was greater for 1.9 than 1.0 g of P4 and regardless of the type of implant, the autoclaving process provided greater circulating P4 in relation to chemical disinfection, and similar or greater P4 concentrations compared with a new implant.

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The objective of the current study was to evaluate the relationship of body condition score (BCS) at 35 d in milk (DIM), milk production, diseases, and duration of the dry period with prevalence of anovulation at 49 DIM and then, specifically, with the prevalence of each anovular phenotype. We hypothesized that anovular follicular phenotypes, classified based on maximal size of the anovular follicle, have different etiologies. A total of 942 lactating Holstein cows (357 primiparous and 585 multiparous) from 1 herd had ovaries evaluated by ultrasonography at 35 ± 3 and 49 ± 3 DIM to detect the absence of a corpus luteum (CL), and to measure the diameter of the largest follicle. Cows were classified as cyclic at 49 DIM if a CL was observed in at least 1 of the 2 examinations, or anovular if no CL was observed at either examination. Cows considered anovular were divided into 3 groups based on the largest diameter of the largest follicle as follows: ranging from 8 to 13 mm, 14 to 17 mm, or ≥18 mm. Cows were evaluated for the following diseases: retained placenta, metritis, hyperketonemia, mastitis, lameness, respiratory problem, and digestive problem. At 35 DIM, BCS was determined, and milk yield for individual cows was recorded. A total of 28.5% (268/942) of cows were classified as anovular. Anovular cows had longer dry periods (90 vs. 71 d) and smaller BCS than cyclic cows (2.83 vs. 2.99). Cows with a single disease or multiple diseases had 2 and 3-fold increase in odds of being anovular, respectively. Anovular cows had follicles that ranged from 4 to 50 mm. The prevalence of anovular phenotype, among anovular cows, that had the diameter of the largest follicle ranging from 8 to 13 mm, 14 to 17 mm, and ≥18 mm was 29.9 (79/264), 37.5 (99/264), and 32.6% (86/264), respectively. Anovular cows with follicles of 8 to 13 mm had longer dry periods than those with follicles ≥18 mm (104 vs. 74 d), whereas anovular cows with medium size follicles had intermediate days dry (99 d). Cows with small and medium anovular follicles had smaller BCS and greater prevalence of multiple diseases than cyclic cows. For almost all risk factors, the cows with large anovular follicles (≥18 mm) were similar to cyclic cows and different from cows with smaller anovular follicles (8-13 mm). Thus, longer dry periods, less BCS at 35 DIM, and diseases were risk factors for anovulation. Moreover, the risk factors for the 3 distinct anovular follicle phenotypes differed.

Changes in circulating progesterone (P4) and estradiol (E2) during proestrus produce dynamic changes in endometrial function and pituitary release of gonadotropins. Independent and combined effects of P4 and E2 on endometrium and pituitary were evaluated. In a preliminary study, an exogenous hormone model of proestrus was created by removal of corpus luteum and follicles ≥5 mm followed by gradual removal of intravaginal P4 implants during 18 h and treatment with increasing doses of estradiol benzoate during 48 h to mimic proestrus using high E2 (n = 9) or low E2 (n = 9). Decreased P4, increased E2, and increased endometrial area (EA) simulated proestrus in high-E2 cows and this was used subsequently. The main experiment used a 2 × 2 factorial design with: high E2 and low P4 (n = 11); high E2 and high P4 (n = 11); low E2 and high P4 (n = 11); low E2 and low P4 (n = 10). At 48 h, gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle stimulating hormone (FSH) release was determined. Variables were analyzed using PROCMIXED of Statistical Analysis System. The EA increased dramatically during 48 h only in high-E2 and low-P4 cows. For FSH, high-E2 cows had greater area under the curve (AUC) and FSH peak after GnRH than low E2, with mild negative effects of high P4. For LH, concentration at peak and AUC were 2-fold greater in high E2 compared to low-E2 groups, with low P4 also 2-fold greater than high-P4 groups. Thus, maximal changes in uterus and pituitary during proestrus depend on both low P4 and high E2, but different physiologic responses are regulated differently by E2 and P4. Changes in endometrium depend on low P4 and high E2, whereas GnRH-induced FSH secretion primarily depends on high E2, and GnRH-induced LH secretion is independently increased by high E2 or reduced by high P4.