Comparison of Intra-CL Injection and Peripheral Application of Prostaglandin F2α Analog on Luteal Blood Flow and Secretory Function of the Bovine Corpus Luteum

We investigated the effects of different doses of dinoprost injected directly into the bovine corpus luteum (CL) on (i) concentrations of progesterone (P4) and oxytocin (OT) in peripheral blood and (ii) mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 1 (HSD3B), and receptor-interacting protein kinases 1 and 3 (RIPK1, RIPK3) in CL tissue. Moreover, we examined the effects of dinoprost, injected intra-CL or administered intramuscularly (IM), on CL secretory function and on indicators of CL vascular network status: luteal tissue area (LTA), CL blood flow (CLBF), and the CLBF:LTA ratio (Adj. CLBF), in cows at the early and mid-luteal phases. In the Experiment 1, cows (day 10 of the cycle) were allocated to (i) an intra-CL injection of saline (control; n = 3); (ii) an intra-CL injection of dinoprost (1.25 mg; 2.5 mg, or 5 mg; n = 3 for each dose); (iii) an IM administration of saline (control; n = 3); or (iv) an IM administration of dinoprost (25 mg; positive control; n = 3). Concentrations of OT and P4 were measured in plasma samples. The mRNA expression of steroidogenesis- or necroptosis-related factors was determined in CL tissue 4 h after treatments. In Experiment 2, cows on day 4 (n = 12) or day 10 (n = 12) were allocated to (i) an intra-CL injection of dinoprost (2.5 mg/0.5 ml; n = 6), or (ii) IM administration of dinoprost (25 mg/5 ml; n = 6). Concentrations of P4 were measured in plasma samples. Luteal tissue area, CLBF, and Adj. CLBF were assessed based on color Doppler ultrasonography. An intra-CL injection of dinoprost increased OT and decreased P4 levels in the jugular vein (JV) in a dose-dependent manner in cows at the mid-luteal phase. Increased CLBF and Adj. CLBF, accompanied by reduced P4 levels, were observed 2 h after intra-CL dinoprost injection in middle-stage CL. Decreased STAR and increased RIPK1 and RIPK3 mRNA levels confirmed that 2.5 mg dinoprost injected directly into CL is the minimum dose that induces luteolytic cascade. Injection of dinoprost directly into the CL (at a dosage lower than recommended for peripheral application) results in a pattern similar to IM dinoprost administration.

Effects of increased ambient temperature and supplemental altrenogest prior to pregnancy establishment in gilts

Heat stress (HS) mitigation strategies are critically needed to combat the substantial economic effects on animal agriculture. The manifestations of seasonal infertility include delayed puberty onset, reduced conception rates, decreased litter size, and increased wean to estrus interval. To assess the effects of HS during early gestation and evaluate a benefit of supplemental altrenogest (ALT) as a mitigation strategy, thirty crossbred post-pubertal gilts (157 ± 11 kg) were subjected to estrous synchronization via 14 d oral administration of ALT. Artificial insemination during estrus was performed and gilts were then placed into one of four treatment groups; heat stress (HS; 35 ± 1 οC for 12h/31.60 ± 1 οC for 12h) with (HSALT, n = 7) or without (HSCON, n = 7) 15 mg/d ALT supplementation or thermal neutral (TN; 20 ± 1 οC) conditions with (TNALT, n = 8) or without (TNCON, n = 8) 15 mg/d ALT supplementation until 12 d post-estrus (dpe). Administrating ALT occurred at 0600 h from 3-12 dpe and rectal temperatures (TR) and respiration rates (RR) were recorded. Blood was collected via jugular venipuncture on 0, 4, 8 and 12 dpe. Gilts were euthanized humanely at 12 dpe followed by collection of ovarian tissue, and uterine flushing for conceptus collection. In HS compared to TN gilts, RR and TR were increased (P < 0.01) but unaffected by ALT supplementation. Feed intake (FI) was reduced (P < 0.01) by HS but unaltered by ALT treatment. Corpora lutea (CL) weight was reduced (P < 0.01) in HSCON gilts when compared to TNCON and HSALT gilts despite progesterone (P4) concentrations in serum and luteal tissue not being affected by treatment (P ≥ 0.10). CL diameter was reduced (P ≤ 0.05) in HSALT gilts compared to other treatments. Interleukin-1β (IL1B) uterine flush concentration was not affected (P > 0.20) by environment or ALT supplementation, although moderate (P = 0.06) interaction between environment and ALT existed, as IL1B concentration in TNALT was increased (P = 0.03) compared to TNCON gilts. While environment did not affect conceptus development (P = 0.90), ALT supplementation advanced conceptus elongation (P < 0.01). Collectively, these data demonstrate that HS may affect luteal development prior to pregnancy establishment, and ALT increases conceptus elongation by12 dpe.

Protein Kinase A and 5′ AMP-Activated Protein Kinase Signaling Pathways Exert Opposite Effects on Induction of Autophagy in Luteal Cells

In the absence of pregnancy the ovarian corpus luteum undergoes regression, a process characterized by decreased production of progesterone and structural luteolysis involving apoptosis. Autophagy has been observed in the corpus luteum during luteal regression. Autophagy is a self-degradative process important for balancing sources of cellular energy at critical times in development and in response to nutrient stress, but it can also lead to apoptosis. Mechanistic target of rapamycin (MTOR) and 5′ AMP-activated protein kinase (AMPK), key players in autophagy, are known to inhibit or activate autophagy, respectively. Here, we analyzed the signaling pathways regulating the initiation of autophagy in bovine luteal cells. In vivo studies showed increased activating phosphorylation of AMPKα (Thr172) and elevated content of LC3B, a known marker of autophagy, in luteal tissue during PGF2α-induced luteolysis. In vitro, AMPK activators 1) stimulated phosphorylation of regulatory associated protein of MTOR (RPTOR) leading to decreased activity of MTOR, 2) increased phosphorylation of Unc-51-Like Kinase 1 (ULK1) and Beclin 1 (BECN1), at sites specific for AMPK and required for autophagy initiation, 3) increased levels of LC3B, and 4) enhanced colocalization of autophagosomes with lysosomes indicating elevated autophagy. In contrast, LH/PKA signaling in luteal cells 1) reduced activation of AMPKα and phosphorylation of RPTOR, 2) elevated MTOR activity, 3) stimulated phosphorylation of ULK1 at site required for ULK1 inactivation, and 4) inhibited autophagosome formation as reflected by reduced content of LC3B-II. Pretreatment with AICAR, a pharmacological activator of AMPK, inhibited LH-mediated effects on RPTOR, ULK1 and BECN1. Our results indicate that luteotrophic signaling via LH/PKA/MTOR inhibits, while luteolytic signaling via PGF2α/Ca2+/AMPK activates key signaling pathways involved in luteal cell autophagy.

Accessory CL regression during pregnancy I: Timing, physiology, and P4 profiles

Inappropriate corpus luteum (CL) regression can produce pregnancy loss. An experimental model was utilized to investigate regression of accessory CL during pregnancy in dairy cows. Cows were bred (Day 0) and treated with GnRH 6 d later to form accessory CL. Transrectal ultrasound (every other d) and blood samples for progesterone (P4; daily) were performed until Day 56 of pregnancy. On Day 28, 13 cows were confirmed pregnant, and accessory CL were found contralateral (n=9) or ipsilateral (n=4) to previous ovulation. On Day 18, CL biopsy was performed to analyze mRNA expression for interferon-stimulated genes (ISGs). Luteolysis occurred more frequently in cows that had contralateral accessory CL [88.9% (8/9)] than cows with ipsilateral accessory CL [0% (0/4)]. Luteolysis of contralateral accessory CL occurred either earlier (Day 19-23; 2/8) or later (Day 48-53; 6/8) in pregnancy and occurred rapidly (24 h), based on daily P4. After onset of earlier or later accessory CL regression, circulating P4 decreased 41.2%. There was no difference in luteal tissue mRNA expression for ISGs on Day 19 between accessory and original CL and between CL that subsequently regressed or did not regress. On Day 56, an oxytocin challenge dramatically increased PGFM in all cows but produced no pregnancy losses, although cows with previous accessory CL regression had greater PGFM. In summary, ipsilateral accessory CL did not regress during pregnancy, whereas most contralateral CL regressed by 63 d of pregnancy, providing evidence for local mechanisms in regression of accessory CL and protection of CL of pregnancy.

The mutual influence of the uterus and ovaries

Takakusu (Arch, fm A. and. Entwicklmech., Bd. 102, H), putting on mice a series of experiments with the extirpation of the uterus, injections of extracts from this organ, with parabiosis and reinforcement of the uterus, came to the conclusion that both the uterus and and the ovary have a hormonal effect on each other 'The influence of the uterus is expressed in the fact that it delays the development of luteal tissue and leads to the reverse development of already formed yellow bodies

​Color Doppler Ultrasonographic Characteristics of Embryonic Development and Mortality in Dairy Cows

Background: Embryonic mortality is one of the major factors leading to reproductive failure, decreased fertility and delayed pregnancy in cattle. Method: The study was aimed for investigating the incidence of embryonic mortality(EM), differentiate embryonic development and mortality based on color Doppler ultrasonographic characteristics. Different characteristics correlated were corpus luteum(CL) vascularity, embryonic development and EM features, monitored on day 21 post-insemination. Result: The overall incidence of EM was recorded to be 25%, with 15% and 10% mortality occurred between days 21-28 and 28-35 of gestation. Color Doppler analysis stated that, the CL in pregnant cows had a higher percentage of luteal tissue with positive Doppler signals on initial examination (day 21) differing significantly (P less than 0.01) from non-pregnant and consistently increased till the end of the examination. However, cows with EM had a significantly lower CL vascularity than pregnant (P less than 0.05) but significantly higher (P less than 0.05) than non-pregnant cows on day 21. Embryos from pregnant cows (detected by 28 days) increased in size along the days of examination. However, cows diagnosed with EM showed signs (day 28), which included reduced intra-uterine allantoic fluid, absence of embryo proper with an embryonic heartbeat, hyperechoic free-floating embryonic debris in the uterus and distorted allanto-chorionic membrane. In conclusion, the present study firmly supports color Doppler ultrasonography in diagnosis of pregnant cows based on CL vascularity, visualizing the different characteristics signs of EM, and detecting non-pregnant animals undergoing EM as early as day 21.

Immunological aspects of follicle ovulation and corpus luteum formation in cattle

Ovulation has been described as an inflammatory event, characterized by an influx of leukocytes into the ovulatory follicle and changes in the expression of immune factors in both the theca and granulosa tissue layers. Since information on this process is limited in cattle, our objective was to elucidate the contribution of the immune system to dominant follicle luteinization, ovulation and corpus luteum formation in cattle. Beef heifers (n=50) were oestrous synchronized, slaughtered and ovarian follicular or luteal tissue collected during a 96h window around ovulation. Follicular fluid cytokine concentration, temporal immune cell infiltration and inflammatory status were determined by Luminex multiplex analysis, immunohistochemistry and quantitative real time PCR-analysis, respectively, in pre- and peri-ovulatory follicular tissues. The concentrations of CXCL10 and VEGF-A were highest in pre-ovulatory follicular fluid samples. The pre and peri -ovulatory follicles play host to a broad repertoire of immune cells, including T-cells, granulocytes and monocytes. Dendritic cells were the most abundant cells in ovulatory follicular and luteal -tissue at all times. The mRNA expression of candidate genes associated with inflammation was highest in pre- and peri-ovulatory tissue, whereas tissue growth and modelling factors were highest in the post-ovulatory follicular and early luteal tissue. In conclusion, ovulation in cattle is characterized by the presence of neutrophils, macrophages and dendritic cells in the ovulatory follicle, reflected in compartmentalized cytokine and growth factor expression. These findings indicate a tightly regulated sterile inflammatory response to the LH surge in the ovulatory follicle which is rapidly resolved during early corpus luteum formation.

Latest Advances in Sow Nutrition during Early Gestation

In the pig, the establishment and maintenance of luteal function in early gestation is crucial to endometrial function, embryo development, and survival. The level of feed intake has a positive effect on formation of luteal tissue and progesterone secretion by the ovaries in the pre-implantation period, which is important for endometrial remodeling and secretion. These effects are independent of luteinising hormone (LH) and probably driven by metabolic cues, such as insulin and insulin-like growth factor (IGF-1), and seem to support progesterone secretion and delivery to the endometrium, the latter which occurs directly, bypassing the systemic circulation. Even after implantation, a high feed intake seems to improve embryo survival and the maintenance of pregnancy. In this stage, luteal function is LH-dependent, although normal variations in energy intake may not result in pregnancy failure, but may contribute to nutrient supply to the embryos, since in this phase uterine capacity becomes limiting. Feed incidents, however, such as unintended fasting of animals or severe competition for feed, may result in embryo or even pregnancy loss, especially in periods of seasonal infertility. Specific nutrients such as arginine have a role in the vascularisation of the placenta and can improve the uterine capacity in the period after implantation.

Ultrasound Characteristics of the Cavitary Corpus Luteum after Oestrus Synchronization in Heifers in Relation to the Results of Embryo Transfer

The aim of the study was to conduct an ultrasound analysis of quantitative parameters of the corpus luteum (CL) in recipient heifers on days 6–8 after oestrus, and to compare reproduction potential of both types of CL in those females. Analyses were performed on 300 heifers, synchronized with two injections of cloprostenol. Clinical and ultrasound examinations of ovaries were performed and measurements of the CL were recorded. The blood samples were taken to determine progesterone level. Pregnancy examination was conducted after 6–8 weeks from the ET. Cavitary CL was found in 32.7% heifers In 48.0% of the cavitary CL, its luteal tissue area was reduced by 14.3% compared to the compact CL, while 16.3% of the CL had luteal tissue reduced by more than 33.8%. Progesterone level in blood serum was higher in heifers with the cavitary CL (p < 0.001). Pregnancy rate was higher for females with a cavitary CL (52%) than those with compact ones (33%, p < 0.05). The ultrasound assessment of luteal tissue should be included in the evaluation of the functional status of the CL in ET-recipient heifers. The cavitary CL presence may indicate a higher potential of the recipient in maintaining the pregnancy.