Uterine Flushings Research Articles

386 Pregnancy supportive metabolites are enhanced in Brahman cows on day 15 of the estrous cycle

Abstract Bos indicus-influenced cattle are more resilient to pregnancy loss while receiving a restricted nutrient diet compared with Bos taurus cattle, but the mechanism behind this resilience is still unknown. To undercover this mechanism, the present study was designed with Angus (Bos taurus), Brangus (Bos indicus-influenced), and Brahman (Bos indicus) cows randomly assigned (d -30) to either a 100% maintenance diet (n = 22), or a 70% maintenance diet (n = 23) for 45 d. All cows were submitted to a 5-d CO-Synch + CIDR synchronization protocol on d -7 with ovulation induction representing d 0. A cervically navigated foley catheter was placed in the uterine horn ipsilateral to the corpus luteum on d 15 of the estrous cycle. Approximately 50 mL of phosphate buffered saline was infused into the uterus then recovered for uterine flush analysis. Recovered uterine fluid was subjected to metabolomic analysis by ultra-high performance liquid chromatography - high resolution mass spectrometry. A significant breed by metabolite interaction occurred (P < 0.001). A treatment by metabolite interaction was also observed (P = 0.015) in which maintenance and restricted cows clustered within their respective treatment when plotted in an orthogonal PCA graph. Of the 101 metabolites analyzed, 7 metabolites (aspartate, glutamate, hydroxyphenylacetate, taurine, uric acid, uridine, and xanthine) differed (P < 0.05) among breeds. Brahman cows had the greatest value of all 7 metabolites (P < 0.05) except hydroxyphenylacetate (P > 0.10). Angus and Brangus metabolomic values were similar for aspartate, taurine, and uridine (P > 0.10) but differed for glutamate, hydroxyphenylacetate, uric acid, and xanthine (P < 0.05). Two metabolites that were greatest in Brahman cows (aspartate and glutamate) contribute significantly to the Kreb Cycle, indicating that Brahman cows have the ability to maintain proper amounts of substrates for energy production compared with Angus cows. Both glutamate and aspartate also aid in the removal of toxic ammonium as they are substrates for a-ketoglutarate and oxaloacetate, respectively, which both consume ammonium in their respective reactions. Furthermore, uric acid (which has a precursor of xanthine) was also greatest in Brahman cows and is a potent antioxidant with strong capabilities of scattering reactive oxygen species. Finally, aspartate also aids in the detoxification of ammonium within the urea cycle. Taken together, these four metabolites (aspartate, glutamate, uric acid, and xanthine) are working together but separately with the overall goal to provide more functional energy, while removing unnecessary toxins which may be two mechanisms in how Bos indicus-influenced cows are better able to sustain optimal reproductive efficiency than Angus cows.

Complement system molecules: Expression and regulation at the maternal-conceptus interface during pregnancy in pigs

The complement system, composed of complement components and complement control proteins, plays an essential role in innate immunity. Complement system molecules are expressed at the maternal-conceptus interface, and inappropriate activation of the complement system is associated with various adverse pregnancy outcomes in humans and rodents. However, the expression, regulation, and function of the complement system at the maternal-conceptus interface in pigs have not been studied. In this study, we investigated the expression, localization, and regulation of complement system molecules at the maternal-conceptus interface in pigs. Complement components and complement control proteins were expressed in the endometrium, early-stage conceptus, and chorioallantoic tissues during pregnancy. The expression of complement components acting on the early stage of complement activation increased in the endometrium on Day 15 of pregnancy, with greater levels on that day compared with the estrous cycle. Localization of several complement components and complement control proteins was cell-type specific in the endometrium. The expression of C1QC, C2, C3, C4A, CFI, ITGB2, MASP1, and SERPING1 was increased by IFNG in endometrial explant tissues. Furthermore, cleaved C3 fragments were detected in endometrial tissues and uterine flushings on Day 15 of the estrous cycle and Day 15 of pregnancy, with greater levels on Day 15 of pregnancy. These results suggest that complement system molecules in pigs expressed at the maternal-conceptus interface play important roles in the establishment and maintenance of pregnancy by regulating innate immunity and modulating the maternal immune environment during pregnancy.

Effect of a novel copper chloride gel on endometrial growth and function in healthy volunteers

Research questionDoes the application of a micro-dose of copper chloride gel increase endometrial production of vascular endothelial growth factor (VEGF) without compromising endometrial function or producing embryo toxicity? DesignAn estimate of optimal dose was made based on cell culture studies. Ten healthy participants received an initial uterine application of placebo gel, followed by copper chloride gel (37.5 μM, 75 μM, or 150 μM dose) in a later hormone replacement cycle. Endometrial biopsies (day 5.5 luteal) and pelvic ultrasound were carried out during each cycle to evaluate endometrial function and growth. Uterine fluid was assessed for residual copper levels on the day of biopsy, and copper chloride gel underwent mouse embryos assay assessment for potential embryo toxicity. ResultsThe copper gel significantly increased endometrial VEGF expression (quantitative polymerase chain reaction), and also increasing endometrial thickness by an average of 2.2 mm compared with matched control cycles. The copper gel did not adversely affect endometrial morphology or maturation (histological dating and molecular receptivity testing), and mouse embryos assay studies showed no evidence of embryo toxicity. Furthermore, uterine cavity flush samples mostly lacked copper, with only negligible amounts present in one sample. ConclusionApplying copper chloride gel to the uterine cavity upregulated endometrial VEGF and significantly increased endometrial thickness and volume. No adverse effects on the endometrium or embryos were observed. Copper chloride gels show promise for treating suboptimal endometrial thickness if the results of this study are confirmed by larger randomized controlled trials.

A TriAdj-Adjuvanted Chlamydia trachomatis CPAF Protein Vaccine Is Highly Immunogenic in Pigs.

Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options-twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 μL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at −20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

Profile of uterine flush lipid mediators in cows with subclinical endometritis: pilot study.

Subclinical endometritis affects reproductive outcomes and causes economic losses in dairy cows, thus, it is important to understand disease progression mechanisms and develop diagnostic procedures for better disease management. We measured the levels of 146 lipid mediators in uterine flush samples using lipid chromatography-mass spectrometry. We detected 25 lipid mediators in the uterine flush of both the control and subclinical endometritis cows; 15 of the 25 lipid mediators were AA-derived metabolites. Among the AA-derived metabolites, cyclooxygenase (COX)-generated mediators were the most abundant. Specifically, levels of 11β-13,14-dihydro-15-keto prostaglandin (PG) F2α, PGE2, PGA2, 13-hydroxyoctadecadienoic acid, and PGD1 were elevated in all the cows with subclinical endometritis. This study may provide new insights for the management of subclinical bovine endometritis.

Regulation of synthesis of polyamines by progesterone, estradiol, and their receptors in uteri of cyclic ewes†.

Progesterone (P4), estradiol (E2), and expression of their receptors (PGR and ESR1, respectively) by cells of the uterus regulate reproductive performance of mammals through effects on secretion and transport of nutrients into the uterine lumen. This study investigated the effect of changes in P4, E2, PGR, and ESR1 on expression of enzymes for the synthesis and secretion of polyamines. Suffolk ewes (n = 13) were synchronized to estrus (Day 0) and then, on either Day 1 (early metestrus), Day 9 (early diestrus), or Day 14 (late diestrus) of the estrous cycle, maternal blood samples were collected, and ewes were euthanized before obtaining uterine samples and uterine flushings. Endometrial expression of MAT2B and SMS mRNAs increased in late diestrus (P < 0.05). Expression of ODC1 and SMOX mRNAs decreased from early metestrus to early diestrus, and expression of ASL mRNA was lower in late diestrus than in early metestrus (P < 0.05). Immunoreactive PAOX, SAT1, and SMS proteins were localized to uterine luminal, superficial glandular, and glandular epithelia, stromal cells, myometrium, and blood vessels. Concentrations of spermidine and spermine in maternal plasma decreased from early metestrus to early diestrus and decreased further in late diestrus (P < 0.05). The abundances of spermidine and spermine in uterine flushings were less in late diestrus than early metestrus (P < 0.05). These results indicate that synthesis and secretion of polyamines are affected by P4 and E2, as well as the expression of PGR and ESR1 in the endometria of cyclic ewes.

O-282 Role of extra-cellular vesicles in reproduction

Abstract Extracellular vesicles (EVs) are bilayer-membrane-bound vesicles isolated from biofluids and tissues. Recently, these organelles have been recognized as a novel mode for inter and intra-cellular communication. The three critical subtypes of EVs include exosomes, microvesicles, and apoptotic bodies. They can be discriminated according to their size, cargo, biogenesis, release pathways, and functions. These organelles serve as vehicles for transferring bioactive molecules as lipids, proteins, cytokines, and regulatory molecules as RNAs, locally and remotely. A wide range of cell types releases EVs, and their cargo differs under normal and pathological conditions. These facts make them possible biomarkers for diagnosing and treating different medical conditions. Isolation of EVs from various female and male reproductive tissues such as prostate, epididymis, seminal fluid, fallopian tube, cumulus, and mural granulosa cells, oocytes, embryos at various developmental stages, endometrium, and decidua has been described. Oogenesis, sperm maturation, fertilization, embryo development, and implantation are composite processes highly reliant on the interaction between tissues and cells. Existing data show that miRNAs and proteins in epididymal fluid associated with testicular maturation are transferred to the sperm by EVs Ovarian follicular growth is a synchronized process comprising bidirectional communication between the oocyte, cumulus, and mural granulosa cells. Some studies have identified EVs from several species' follicular fluid (FF), including bovine, equine, and human. To date, data linking EVs with oogenesis are primarily descriptive. EV micro-RNA (EV-miRNA) isolated from FF target pathways as mitogen-activated protein kinase (MAPK), wingless signaling pathway (WNT), epidermal growth factor receptor (ErbB), and transforming growth factor beta (TGFß). These pathways are related to folliculogenesis, meiotic resumption, and ovulation. EV-miRNAs isolated from follicles that contained mature oocytes were associated with fertilization potential and embryo quality. In addition, EVs derived from microvillous-rich oolemma may neutralize acrosome reaction and prevent polyspermy. Animal and human studies have confirmed that embryos at various developmental stages secrete EVs into the extracellular medium, and embryos internalize EVs. The number, miRNA profile, and size of EVs secreted vary across the cycle and according to embryos’ developmental potential. Endometrial EVs release miRNAs essential for the blastocyst's adhesion. In ovine, EVs were identified from the uterine lumen and uterine flushing during implantation. Uterine lumen flush EVs were internalized by the trophectoderm cells in the conceptus, reducing trophectoderm cell proliferation. The study of EVs in reproduction has increased exponentially in the last few years. Evidence support that these organelles contribute to oogenesis, fertilization, embryo development, and implantation. Further research in EVs will help us expand noninvasive methods and identify new biomarkers in the reproductive field.

Induced ovulation and timed recovery of suitably sized embryos for transfer in mares

Studying the size of equine embryos recovered from uterine flushes 8-9d after ovulation has established that size and age of embryos post-ovulation are directly related (Cuervo-Arango et al., Theriogenology. 2018; 115: 90-93). In the field, daily tracking and detection of ovulation are not practical in a commercial embryo transfer program. Considering that induced ovulation with deslorelin acetate (DES) occurs in most mares 38-44h after administration (Spencer et al., Theriogenology. 2022: 140-148), estimated time of ovulation induction may be used to predict the time of embryo recovery. The objective herein is to evaluate embryo size at the time of recovery after DES administration and ovulation and determine its fit within a 0.25mL or 0.5mL embryo transfer straw. Healthy, adult cycling mares (n=15 total) were subjected to ovulation induction with 1mg of DES i.m. when the dominant follicle first reached 35mm. Mares were artificially inseminated 24h later and monitored daily to detect ovulation (D0) with uterine flushes done 8 (D8) or 9 (D9) d later. Recovered embryos were measured by a micrometric ruler and compared: 1) D8 vs D9 after ovulation, 2) <250h vs >250h after DES, and 3) fit into 0.25mL (embryos <1500µm) vs 0.5mL (embryos from 1500 to 2500 µm)straws. Student's t-tests were used to analyze the size of embryos on D8 (n=8) vs D9 (n=5) after ovulation and <250h (n=8) vs >250h (n=7) after DES. Z-score test was used to analyze the probability of fit into a 0.25mL vs 0.50mL straw. All mares ovulated 24-48h after DES. There was no difference (P=0.25) in size of embryos recovered on D8 (mean diameter = 828±193.4µm) vs D9 (mean diameter = 1320±411µm) after ovulation. Relative to induced ovulation, embryos recovered <250h after DES (mean diameter = 828±193.4µm) were smaller (P=0.0474) than those recovered >250h after DES (mean diameter = 1971±514µm). The probability of embryos fitting into a 0.25mL straw was 89% for D8, 57.8% for D9, 89% for <250h, and 36.5% for >250h. Correspondingly, the probability of embryos fitting into a 0.5mL straw was 99.9% for D8, 90% for D9, 99.9% for <250h, and 65.1% for >250h. Thus, these preliminary results indicate that, without daily tracking and detection of ovulation, the predicated time from induced ovulation to recovery of embryos at 236-247h (i.e. 10 days) to 258-264h (i.e. 11 days) after DES yield suitably sized embryos for transfer using a 0.25 or 0.5mL straw.

Obesity and age on post-breeding endometritis in the mare

Obesity has been associated with uterine dysfunctions in the pregnant mare (Sessions-B et al, Biology of Reproduction, 2018: 99(4), 761-772). This study hypothesized that obesity itself (body condition score, BCS) affected parameters associated with post-breeding endometritis. Mares (n=17) were classified by their BCS (Henneke et al., 1983) in obese (>7, n=6) and normal-weight (<7, n=11) and age group in mature (5 to 14 years, n=9) and aged mares (15 to 21 years, n=8). Transrectal ultrasonography was performed every day and oedema score and intrauterine fluid diameter were determined at insemination and 8 hours afterwards when a low volume (50 mL) uterine flush with PBS was performed (lavage). Neutrophils were counted using a Neubauer camera and the flush was centrifuged; the supernatant was frozen for metabolite determination by spectrophotometry. Blood samples were taken at this moment also for metabolite determination. Data were analyzed with PROC MIXED (SAS) including BCS and age as fixed effects. Data not normal was log-transformed. Obese mares tended to present greater neutrophil counts than normal-weight mares (43.6±8.3 vs 28.8±5.7 × 106/mL P=0.08); aged mares had greater counts than mature mares(46.7±7.7 vs 21.0±6.3 × 106/mL P=0.02). No effect of age or BCS was found on oedema and IUF at insemination or lavage. In serum, glucose, triglycerides, albumin, and urea concentrations were unaffected. Serum cholesterol concentrations were affected by BCS (P<0.01) and tended to be affected by age (P<0.09). Serum globulin concentrations were not affected by BCS, but aged mares had greater concentrations than mature mares (P=0.05). In the intrauterine fluid, BCS did not affect albumin or glucose concentration nor did age affect them. No effect was observed in cholesterol or triglyceride concentrations in the IUF. Although urea concentrations in the IUF were not affected by BCS, aged mares had greater concentrations than mature mares (1.22±0.23 vs 0.37±0.19 mM, P=0.016). Although the number of animals is limited, data shows that obesity and age are associated with greater neutrophil counts in the uterine lumen 8 hours after insemination, suggesting an altered uterine clearance in these mares. As far as we know this is the first description of metabolites in IUF according to obesity and age, showing significantly greater urea concentrations in the aged group.

The mystery of the capsular vesicles: preliminary studies

An unexpected experimental finding was the presence of capsular vesicles (CVs) in the uterine flushings of mares 48h after transfer of a punctured Day 8 embryo. These CVs were thought to originate from fragments of capsule, in the same way that trophoblast fragments readily form vesicles in vitro. However, recovery of the capsule with the puncture hole alongside numerous CVs showed this was not the case. A preliminary study was undertaken to see if CVs formed in recipient mares following transfer of trophoblast vesicles (TVs) for 24h and if CV formation was uterine stage specific. Day 11 embryos were recovered from donor mares and the capsule removed before fragmenting the trophoblast. Small fragments of trophoblast were incubated in DMEMF12 (+ 5% FBS and pen/strep) for 48h in a Tri-gas incubator at 38°C. After 48h any degenerate TVs were discarded, and the remainder were washed in H199 (+ 5% FBS + pen/strep). Twenty to 50 TVs were transferred to the uteri of 25 recipient mares divided into 4 groups; G1, Days 3–5 post ovulation (n=6); G2, Days 6–8 (n=7); G3, Days 9–11 (n=6); Days 12–14 (n=6). The recipient mares’ uteri were flushed 24h after TV transfer and the recovered fluid searched. In all groups TVs were recovered with varying degrees of degeneration. Data were analyzed using aOne Way ANOVA on ranks with a post-hoc Dunn's test. The median number of CVs varied between G1–4 (0 vs. 68 vs. 17.5 vs. 6.5, respectively), with G1 versus G2 varying significantly (P<0.05). When adjusted for CVs per transferred TV, median values for G1–4 were (0 vs. 1.6 vs. 0.51 vs. 0.225), with G1 versus G2 remaining significantly different (P<0.05). The highest number of CVs recovered was 148 (2.96 per TV) from a Day 8 recipient, with a trend for a reduction in CV production after Day 8. These results suggest that CVs can form in the uterus following TV transfer providing the uterus is at a stage when an embryo would normally be forming a capsule, since transfers before Day 6 failed to produce CVs. Immunohistochemistry needs to be undertaken to confirm the CVs are identical in composition to in vivo produced capsules. Capsule formation in the mare is believed to involve interaction of the embryo with the endometrium, as in vitro produced embryos fail to produce a proper capsule. In vivo, developmentally regulated changes in composition of the capsule occur as its thickness increases up until at least Day 11. The mechanism of the formation of capsular vesicles remains a mystery as they are free in the uterus and do not appear to form around the trophoblastic vesicles.

Endocrinology and metabolomics of uterine fluid after breeding in the mare

Understanding the endocrinology and metabolome variations in the uterine milieu is essential for pregnancy success (Assman and Finlay; Journal of Clinical Investigation. 2016; 126(6):2031-2039). The aim of the study was to determine adipokines and metabolites in uterine fluid after breeding. Healthy oestrous mares (n=6; age 5 to 12 years), monitored by ultrasound for follicle development and screened for uterine inflammation were inseminated with fresh semen. Eight hours after insemination, a low volume uterine flush (50 ml of PBS) was performed and the supernatant was obtained for analysis. Adiponectin and leptin were assessed by radioimmunoassays, while metabolites were determined by spectrophotometry and 1H nuclear magnetic resonance (NMR) spectroscopy. The concentration of adiponectin ranged from 5.6 to 17.6 ng/mL and leptin from 0.11 to 16.1 ng/mL. Albumin concentration ranged from 0.4 to 0.9 g/L, glucose 40 to 70 umol/L, globulins 0.8 to 1.5 g/L, and urea 0.4 to 1.8 mmol/L. Triglycerides and cholesterol were not detected in some samples or maximum concentrations were up to 12.4 mg/dL and 0.08 mmol/L respectively. NMR spectroscopy detected amino acids such as creatine, valine, isoleucine and taurine in the uterine fluid. High concentrations of lactate were observed, while lower contents of pyruvate, citrate, beta-hydroxybutirate and acetate were found (Figure 1). While albumin and globulins are present in equine uterine fluid (Tunón. et.al. Theriogenology.1998; 50(6): 821-831), this is the first report on adipokines and metabolites in uterine fluidafter breeding. Many of the metabolites are considered biomarkers of cell signaling regulation (Tríbulo.et.al. Molecular Reproduction and Development. 2019; 86(1):75-87) and metabolic stress in cattle (López Radcenco.et.al. Dairy 2021; 2:356-366) and may affect sperm transport and bacterial removal from the uterus. Adipokines and metabolome variations in uterine fluid as diagnostic markers may allow to investigate therapeutic strategies and thus improve reproductive performance in the mare.

31 Correlation of the Reproductive Microbiome and Cytokine Profiles with Plasma Progesterone Concentrations in Postpartum Beef Cows Prior to Timed Artificial Insemination

Abstract Female sex steroid hormones have been shown to interact with cytokines within the immune system to help influence fertility. Previous research has indicated that shifts in commensal bacteria are associated with compromised reproductive performance during the postpartum period, yet evaluation of how the female sex hormone progesterone (P4) influences these microbial shifts is not fully understood. The objectives of this study were to determine 1) the relationship between P4 concentrations and cytokine concentrations, and 2) the relationship between P4 concentrations and the reproductive microbiome. Twenty Bos indicus-influenced beef cows (~60 days postpartum and free of health issues) were weighed, body condition scored and then subjected to the Bos indicus prostaglandin 5 day (d) + CIDR protocol beginning on d -8 and timed artificially inseminated on d 0. Blood samples were collected on d-3, -1, 0, and 28. Cytokines and reproductive microbiome were evaluated using vaginal and uterine flushes collected on d -3 and d-1. Experimental groups were assigned by d 28 pregnancy status, determined by transrectal ultrasonography, resulting in Open (n = 13) and Pregnant (n = 7) females. Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Plasma P4 concentrations on d -3 and d -1 were quantified per manufacturers’ instructions using a double-antibody RIA kit. Cytokine analyses of plasma and flushes were conducted using the RayBiotech Quantibody Bovine Cytokine Array Q1 kit per the manufacturer’s instructions. Statistical analyses were conducted using the GLM procedure and CORR procedure in SAS. Plasma P4 concentrations did not differ by pregnancy status (P &amp;gt; 0.05) but did decrease from d -3 to d -1 (0.89 vs. 0.08 ± 0.23 ng/mL, respectively; P = 0.02). There were no significant correlations between plasma P4 and plasma cytokines (P &amp;gt; 0.05). Interferon-alpha (IFNα) and P4 concentrations were positively correlated in Open and Pregnant uterine flushes (r = 0.41; P = 0.01). Concentrations of P4 interleukin (IL)-1F5 were negatively correlated in Open and Pregnant vaginal flushes (r = -0.32; P = 0.04). The relative abundance of genus Methanobrevibacter in uterine flushes was positively correlated with P4 concentrations in both experimental groups (r = 0.35; P = 0.02). Within vaginal flushes, there was a tendency for a negative correlation between the relative abundance of genus Bacteroides and P4 concentrations (r = -0.31; P = 0.05). Alternatively, there is a positive correlation between P4 concentration and relative abundance of genus Streptococcus (r = 0.47; P = 0.01) and genus Methanobrevibacter (r = 0.37; P = 0.02) in vaginal flushes. These results suggest there may be relationships between several cytokines and circulating P4 concentrations; therefore, potentially suggesting immunomodulatory activities of P4. Overall, this research alludes to P4 having a vital role in reproductive microbial shifts and associated immune responses. Further investigation on the local effects of P4 within the reproductive tract on immune responses and bacterial shifts could provide greater insight into mechanisms to improve fertility in beef cattle.

43 Metabolomic Analysis of Uterine Luminal Fluid During the Peri-Implantation Period of Pregnancy in Pigs

Abstract During the peri-implantation period of pregnancy in pigs, the rapid elongation of conceptuses (embryonic/fetus and its extraembryonic membranes) is highly dependent on the composition of histotroph secreted from uterine luminal (LE) and glandular (GE) epithelial and stroma cells, as well as selective transport of nutrients. However, little is known about the metabolites in the uterine luminal fluid (ULF) associated with the uterine-conceptus communication during early pregnancy in pigs. Thus, this study was conducted to profile the metabolome in porcine ULF between days 10 and 16 of estrus cycle (C) and pregnancy (P). Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (day 0). On days 10, 12, 14, or 16 of the estrous cycle and pregnancy (n = 6 gilts per day and status), uteri were flushed with 20 mL sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Recoverable uterine flushings (i.e., ULF) were then subjected to metabolomic analysis by LC-MS. Overall, 222 metabolites were detected in the ULF of which 102 were altered by status and 63 were altered by days (P &amp;lt; 0.05; fold change&amp;gt;1.5). Comparing to cyclic day 10 (10C), pregnancy stimulated increases in citric acid, lysine, arginine and other 19 metabolites in the ULF at pregnant day 10 (10P). At 12P when porcine conceptus initiates its morphological changes, ornithine, α-D-glucose, phenylalanine and other 13 metabolites were up-regulated as compared with 12C. At 14P when conceptus initiates implantation, 15 metabolites changed in ULF. At 16P, 123 metabolites were altered in ULF as compared with 16C. Interestingly, of 15 altered metabolites in ULF, 12 were downregulated including asparagine, nicotinamide riboside, and citrulline at 14P. Whereas 102 of 123 altered metabolites were upregulated including phosphorylcholine, 6-phosphate fructose and 6-phosphate glucose in ULF at 16P. To determine the absolute amount of amino acids in the ULF, we performed the targeted metabolomic analyses for 19 amino acids. As a result, arginine, asparagine, glutamic acid, glutamine, histidine, leucine/isoleucine, lysine, phenylalanine, proline, and valine were increased between 10P and 16P. They were less expressed in the ULF of estrus cycle as compared with pregnancy, and remained unchanged between 10C and 16C. Pathway analysis based on KEGG database indicating that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis have the highest degree of enrichment. These novel findings provide the foundation for future investigation of metabolomic and functional studies on both endometrium and conceptus required for elongation of porcine conceptus during peri-implantation period of pregnancy. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture

Impact of intrauterine infusion of Platelets-Rich plasma on endometritis and reproductive performance of Arabian mare.

Equine endometritis is one of the most common causes of reproduction failure. To achieve better treatment outcomes, different diagnostic methods should be combined. In the current study, 39 repeat breeder mares were subjected to ultrasonography examination to detect excessive accumulation of intrauterine fluids and an abnormal oedema pattern, which revealed that 61.5% of mares were positive. Combined with endometrial cytology by low-volume uterine flush, 47.7% of smears contain neutrophils (more than 2-3 per HPF X100), and microbial culture. 92.3% of mares were infected with different bacterial isolates, such as Escherichia coli, Streptococci, Staphylococcus, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Providencia alcalifaciens, and Proteus mirabilis. All mares were given saline solution and gravity withdrawal before being given 20 IU of oxytocin (as ecbolic agents). Mares were divided into three groups; Group one (n=15) received an intrauterine infusion of 20 mL of freshly prepared autologous platelet-rich plasma (PRP) 6 h after breeding, Group two (n=15) was treated with three doses of systemic Enrofloxacin 5% during the estrus period, and Group three (n=9) received only uterine lavage and 20 IU of oxytocin. PRP and Enrofloxacin resulted in a significant (p < .05) reduction in endometrial thickness (oedema; 5.05 and 6.74 mm, respectively) and disappearance of intrauterine fluids compared to the control (10.98 mm). Furthermore, PRP (days) and Enrofloxacin (17.89 days) reduced the days to the next oestrus compared to the control (18.58 and 17.89 vs. 21.19 days, respectively). Furthermore, the pregnancy rate improved to reach 70% in the PRP group and 60% in the Enrofloxacin group, while the control remained low at 22%. In conclusion, autologous PRP can be used as a low-cost alternative therapy for modulating the inflammatory process and effectively treating mares' endometritis.

Uterine Flushing Fluid-Derived Let-7b Targets CXCL10 to Regulate Uterine Receptivity in Goats during Embryo Implantation.

Exosomes have the ability to carry a wide range of chemicals, convey them to target cells or target regions, and act as "messengers." For the purpose of investigating embryo attachment, it is helpful to comprehend the range of exosomal mRNAs and miRNAs derived from the uterine flushing fluid before and after embryo attachment. In this study, we recovered exosomes from goat uterine rinsing fluid at 5, 15, and 18 days of gestation and used RNA-Seq to identify the mRNA and miRNA profiles of exosomes obtained from uterine rinsing fluid before and after embryo implantation. In total, 91 differently expressed miRNAs and 27,487 differentially expressed mRNAs were found. The target genes predicted by the differentially expressed miRNAs and the differentially expressed mRNAs were mainly membrane-related organelles with catalytic activity, binding activity, transcriptional regulation activity, and involved in metabolism, biological regulation, development, and other processes. This was revealed by GO analysis. Furthermore, KEGG analysis revealed that they were abundant in signaling pathways associated with embryo implantation, including the "PI3K-Akt signaling pathway," "Toll-like receptor signaling pathway," "TGF-beta signaling route," "Notch signaling pathway," and others. Moreover, our research has demonstrated, for the first time, that chi-let-7b-5p specifically targets the 3'UTR of CXCL10. Our research offers a fresh viewpoint on the mechanics of embryo attachment.

Reproductive microbiome and cytokine profiles associated with fertility outcomes of postpartum beef cows.

Shifts from commensal bacteria (for example, Lactobacillus in the phylum Firmicutes) within the reproductive tract have been associated with changes in local reproductive immune responses and decreased fertility in humans. The objective of this study was to characterize the microbiome and cytokine concentrations before artificial insemination (AI) in vaginal and uterine flushes from postpartum beef cows. Twenty Bos indicus-influenced beef cows (approximately 60 d postpartum and free of reproductive, health, or physical issues) were enrolled. The B. indicus prostaglandin (PG) 5-d + controlled intervaginal drug-releasing estrus synchronization protocol was initiated on day -8 of the study with timed AI on d0. Blood samples were collected on days -3, -1, and 28 via coccygeal venipuncture. Vaginal and uterine flushes were collected on days -3 and -1. Based on days 28 pregnancy status determined by transrectal ultrasonography, cows were identified as either Open (n = 13) or Pregnant (n = 7). Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Cytokine analyses were performed using the RayBiotech Quantibody Bovine Cytokine Array Q1 and MyBioSource ELISA kits per the manufacturer's instructions. Statistical analyses for bacteria relative abundance were conducted using PROC NPAR1WAY and for cytokine concentrations using PROC GLM in SAS 9.4. Uterine concentrations of interferon γ, interleukin (IL)1α, and IL21 were greater in Open than in Pregnant cows (P < 0.05). Regardless of pregnancy status, uterine IL13 increased from days -3 to -1 (9.76 vs. 39.48 ± 9.28 pg/mL, respectively; P < 0.05). Uterine relative abundance of the phylum Firmicutes decreased from days -3 to -1 in Open cows (60.4% ± 0.9% vs. 48.5% ± 3.2%; P = 0.004). In Open cows, the genus Blautia decreased in relative abundance within the uterus from days -3 to -1 (2.1% ± 0.2% vs. 0.9% ± 0.1%; P = 0.002). Uterine relative abundance of the phylum Tenericutes increased from days -3 to -1 in Pregnant cows (1.0% ± 0.1% vs. 7.6% ± 4.1%; P = 0.002). In Pregnant cows, the genus Ureaplasma tended to increase within the uterus from days -3 to -1 (0.08% ± 0.06% vs. 7.3% ± 4.1%; P = 0.054). These findings suggest a distinct difference in the reproductive microbiome and cytokine profiles before AI for resulting Open vs. Pregnant cows.

Expression study of pro-inflammatory cytokines and acute phase proteins in post-partum crossbred dairy cows with subclinical endometritis

Subclinical endometritis (SCE) is one of the causes of poor reproductive performance in dairy cows. The objective of the study was to evaluate the expression of pro-inflammatory cytokines [PICs; Interleukin -1 beta (IL-1β), Interleukin -6 (IL-6), and Tumor necrosis factor- alpha (TNF-α)] and Acute phase proteins [APPs; Serum Amyloid- A (SAA) and Haptoglobin (HP)] in blood and uterine flushings of postpartum dairy cows with subclinical endometritis (SCE). Animals (n=49) were screened for SCE at 60 days postpartum (dpp) using the endometrial cytobrush cytology, and those with PMN cell percentage ≥ 18% were identified as SCE cows. The blood samples and uterine flushings from SCE (n=15) and the control group (n=15) were assessed for mRNA expressions of PICs and APPs using the Real-Time PCR method (RT-PCR). The PICs and APPs except TNF-α were non-significant (p&gt;0.05) in blood samples whereas a significant difference (p&lt;0.05) was found in uterine flushings. The PICs and APPs were significantly up-regulated in the uterine endometrium indicating that collection of uterine flushings at 60 dpp could be used as a diagnostic method for evaluating gene expressions of inflammatory mediators in dairy cows with SCE.

Global analyses and potential effects of extracellular vesicles on the establishment of conceptus implantation during the peri-implantation period.

Intrauterine extracellular vesicles (EVs) are involved in establishing proper conceptus-endometrial communication, which is essential for conceptus implantation and subsequent successful placentation. Despite several studies on intrauterine EVs, the composition and quantitative changes in conceptus and endometrial EVs, as well as the effects of intrauterine EVs on endometrial epithelial cells (EECs) during the peri-implantation period, have not been well characterized. To elucidate global changes in proteins in EVs extracted from uterine flushings (UFs) during the pre-implantation (P17), just-implantation (P20), and post-implantation (P22) periods, the datasets of the proteome iTRAQ analysis were compared among P17, P20, and P22 EVs. These analyses revealed that the composition and function of proteins in the EVs changed dramatically during peri-implantation in cattle. Notably, intrauterine P17 EVs affected the high expression of "Developmental Biology" and "morphogenesis of an endothelium" compared with those in P20 and P22 EVs. Furthermore, P20 EVs had the functions of the high expression of "mitochondrial calcium ion homeostasis" and "Viral mRNA Translation" compared with those in P17 EVs. Transcripts extracted from EECs treated with P17, P20, or P22 EVs were subjected to RNA-seq analysis. These analyses identified 60 transcripts in EECs commonly induced by intrauterine EVs recovered from P17, P20, and P22, a large number of which were associated with "type I interferon signaling pathway". Collectively, these findings reveal the presence and multiple functions of EVs that are potentially implicated in facilitating conceptus implantation into the uterine epithelium during the peri-implantation period.

A feasible method for llama embryo paraffin embedding

Maternal recognition of pregnancy in llamas would be related to a still-unknown signal secreted by the embryo to extend the corpus luteum lifespan. In order to study llamas reproductive physiology and early pregnancy aspects, research on embryos between 8 and 12 days post-mating is essential. However, there is a lack of information on llama embryo paraffin embedding, a procedure that exhibits difficulties, mostly for embryo manipulation. This work presents an accessible, easy and rapid protocol for embedding llama blastocysts. Uterine flushings from four superovulated llamas were performed to recover embryos 8 days post-mating. Each embryo was fixed in 1 ml of 10 % neutral buffered formalin for 24 h, at 4 °C, and transferred to 70 % ethanol. Later, they were submitted to a series of baths: 500 µl PBS with 5 % bovine foetal serum solution (PBS+BFS) for 1 min; zona pellucida permeabilization with 500 µl PBS with 0.2 % Triton X-100 for 20 min; 500 µl PBS+BFS for 1 min; dehydration on baths with ascendent grades of ethanol (70 %, 96 %, and 100 % twice) for 10 min each; and bath of butanol for 5 min. Finally, embryos were embedded in a pellet of paraffin and afterwards in a paraffin block. Complete sections from 8 of 13 grade I and II embryos were obtained. In conclusion, by applying this method, good quality sections with preserved embryo morphology suitable for histological, histochemical or immunohistochemical approaches would be possible to acquire.