Abstract Reproductive failure continues to exacerbate economic losses within both the dairy and beef cattle industries. Alterations of the vaginal and uterine environments in response to reproductive hormones and/or conceptus development may coincide with shifts in reproductive microbiota populations to influence fertility. To address this, our objectives aimed to 1) characterize shifts in the vaginal microbiota from time of artificial insemination to maternal recognition of pregnancy and analyze its relationship with interferon-stimulated gene (ISG) 15 in lactating dairy cows; 2) determine the impact of endogenous and exogenous estradiol at the time of ovulation on uterine microbiota during early gestation in beef cattle; and 3) detect and localize ISG15 at the bovine uteroplacental interface during early gestation in beef cattle. For objective 1, lactating Holstein cows (n = 27) were sampled on day (day [d] 0) of artificial insemination (AI) and day 18 post-AI. Sterile swabs were inserted into the vagina and utilized for subsequent bacterial abundance analyses targeting the V4 hypervariable region of the 16S rRNA gene. Blood was collected and resulting buffy coats utilized for real-time quantitative polymerase chain reaction (RT-qPCR) analysis targeting ISG15. Cows underwent transrectal ultrasonography on day 32 post-AI for pregnancy determination. All statistical analysis for relative abundance was conducted using nonparametric ANOVA, diversity using Wilcoxon or Kruskal-Wallis, hormone concentrations using PROC GLM, and correlations with bacterial communities using PROC CORR in SAS. Interestingly, there were no major differences due to the main effect of pregnancy status. However, between days, Bacteroidetes (13.80 ± 1.29 vs. 8.96 ± 1.24; P = 0.009) had a greater relative abundance on d0 compared to d18 in Open cows. Upon evaluation of diversity matrices within days, there was a difference in α-diversity between d0 and d18 for Simpson’s diversity index (P = 0.02) and Shannon’s diversity index (P = 0.009), and a difference between days for the weighted UniFrac β-diversity matrix (P = 0.005). When evaluated with ISG15, there were no significant correlations between the mean relative mRNA expression of ISG15 on d18 with phyla bacterial abundance. This uncertainty of progesterone’s role in shifting the vaginal microbiota during early gestation warranted further consideration as to whether these bacterial shifts are influenced by hormone concentrations. Thus, for objectives 2 and 3, crossbred beef cows (n = 62) were synchronized utilizing the 7-day CO-Synch + CIDR protocol. Two days post-CIDR removal (d0), cows were allocated to one of three treatment groups: 1) Estrus Expression, 2) No Estrus + induced ovulation with gonadotropin releasing hormone, 3) No Estrus + induced ovulation with GnRH and 0.1 mg estradiol 17β. All cows were artificially inseminated on d0 with semen from a single sire, and blood was collected for estradiol, progesterone, and pregnancy associated glycoproteins (PAGs). Cows were harvested and ovariohysterectomized on d20, d26, and d32. An incision was made in each uterine horn (ipsilateral [IPS] and contralateral [CON]), and sterile swabs were rotated along the endometrial mucosa for microbiota analysis. Cross-sections of the ipsilateral uterine horn of suspected pregnant tracts were collected for immunofluorescence analyses to colocalize ISG15 with epithelial cadherin (E-Cad). Interestingly, an increase in PAGs appears to be correlated with a decrease in phylum Firmicutes (r=-0.340; P = 0.03). This coincides with greater relative abundance of genera Blautia (P = 0.03), Caloramator (P = 0.05), Oscillospira (P = 0.03), and Ruminococcus (P = 0.04) on d20 compared to later days of gestation within the IPS horn. Similarly to our first study, there were differences for α- and β-diversity matrices compared between days (P ≤ 0.05), yet there were no differences between treatments, pregnancy status, and uterine horns (P > 0.05). Thus, the uterine microbiota appears to shift during early gestation in a time-dependent manner and may be driven by PAGs production by the developing conceptus. This potential role of the conceptus was further noted on d20, as ISG15 was localized to endothelial cells within the stratum compactum stroma of both caruncular and intercaruncular regions. Similar expression of ISG15 was detected on d26, with distinct localization in stromal areas beneath the uterine luminal epithelium (LE) more proximal to the developing conceptus. With conflicting results across the literature, additional research is needed to further elucidate the complex relationship between vaginal microbiota and interferon-stimulated genes during early pregnancy establishment. Understanding the requirements necessary in the establishment of healthy reproductive tract microbiota that are associated with successful pregnancy establishment is also essential for the future improvement of reproductive efficiency in cattle.

Intrauterine growth restriction (IUGR), caused by maternal undernutrition, impairs fetal growth and increases the risk for postnatal metabolic dysfunction. L-arginine can mitigate these effects; however, its use in sheep is limited by ruminal microbial degradation. Interestingly, L-citrulline, the precursor for arginine synthesis, bypasses ruminal catabolism and may be a practical alternative. This study evaluated if maternal L-citrulline supplementation to nutrient restricted ewes from gestational days (GD) 28 to 140 (term = 147) enhances fetal growth in lambs. Gestating ewes were fed 50% of National Research Council (NRC) nutritional recommendations to induce IUGR and received either L-citrulline (0.40% of diet) or an isonitrogenous alanine control (0.61% of diet). Birth weight and pre-suckling blood samples were collected, and lambs remained with dams until postnatal day 60 (PND60) (citrulline: n = 13; alanine: n = 10). Lambs from L-citrulline treated ewes were heavier at birth (P = 0.05) and PND60 (P < 0.05), with greater (P < 0.05) absolute weights of the pancreas, brain, liver, and small intestine. Pancreatic mass per gram of body weight was greater (P < 0.05) in citrulline lambs. The relative proportion of endocrine and exocrine pancreas were not different between treatments. Circulating insulin concentrations were greater (P = 0.05) at birth and circulating glucose concentrations were increased (P < 0.05) in the citrulline lambs on PND60. These results suggest that maternal L-citrulline supplementation is a viable alternative to arginine for improving fetal growth during maternal malnutrition, with benefits persisting through weaning.

Mucosal vaccines are promising for protecting fish from infectious diseases by stimulating immunity, but their success relies on understanding mucosal immunity's metabolic pathways. This study determined the ability of intestinal leukocytes to metabolize glucose through the pentose cycle (PC) as a potential energy source to support cellular functions required for mucosal immunity. Leukocytes were isolated from hybrid striped bass (HSB) at different development stages with mean body weights (BWs) of 14.2, 41.0, and 215.4 g. In addition, intestines from HSB with a mean BW of 41.0 g were divided into equal segments for proximal, middle, and distal regions. Leukocytes were incubated with oxygenated Krebs-Henseleit bicarbonate buffer containing either 5 mM D-[1-14C]glucose or 5 mM D-[6-14C]glucose at 26 ℃ for 2 h, and the 14CO2 produced from the leukocytes was collected to measure metabolism of glucose through the PC pathway. The glucose flux to the PC (nmol/2 h/106 cells) in leukocytes from HSB with mean BWs of 14.2, 41.0, and 215.4 g ranged from 40.7 to 47.5, 35.3–44.0, and 32.7–46.5, respectively. The glucose flux to the PC across different intestinal segments ranged from 35.2 to 41.1 in the proximal, 45.0–52.8 in the middle, and 39.9–46.4 in the distal. Both analyses reveal the preferential utilization of the PC for glucose oxidation (P < 0.001). Collectively, these findings suggest that intestinal mucosal leukocytes of juvenile HSB support mucosal immunity by metabolizing glucose through the PC pathway as they develop from 40.7 to 215.4 g, and that the entire length of the intestine contributes equally to this metabolism.

Abstract In eutherian mammals, blastocyst implantation is often associated with a quasi-inflammatory reaction in the endometrium, which is resolved with the establishment of the definitive placenta. This is understandable in the case of invasive placentation, since implantation entails a nidatory injury to the maternal tissue due to the invading blastocyst. Quasi-inflammatory processes have also been documented in pregnant pigs, even though the blastocyst only attaches to, rather than invades into, the endometrium of the uterus. In this study, we asked what processes in early porcine pregnancy lead to the resolution of attachment-associated inflammation. In generic wound healing the transition from a pro- to an anti-inflammatory state is caused by a corresponding transition from M1 to M2 polarized macrophages following efferocytosis by macrophages of apoptotic neutrophils. In order to determine whether this scenario applies to the pregnancy-related resolution of inflammation in the porcine uterus, we produced a series of bulk transcriptome samples spanning days (D) 13 to 25 of gestation. This time span corresponds to the transition from pre- to post-attachment stages of pregnancy. We found slower changes in the transcriptome between D20 and D25 than prior to D20, suggesting a turning point in pregnancy-related reprogramming. The turning point at D20 corresponds to the time of firm attachment of trophectoderm to uterine luminal epithelium and the cessation of IFNG signaling from the blastocyst. This transition coincides with increased expression of RNAs of genes implicated in resolution of inflammation and M2 polarization such as ARG1, MRC1/CD206, CD86, TGFb1 and IL10, as well as a significant increase in expression of HGPD, the enzyme that metabolizes prostaglandins. While immunoreactivity for ARG1 was found in putative macrophages in the sub-epithelial stratum compactum, other markers of M2 polarized macrophages were localized to non-immune cells: MRC1 was found on fibroblast-like stromal cells, CD86 on trophoblast cells, and IL10 in luminal and glandular epithelia. These results suggest that intrauterine immune regulation is decoupled from that of the rest of the body by engaging non-immune cell types as anti-inflammatory mediators during the peri-attachment period of pregnancy.

Highly pathogenic avian influenza virus (HPAIV) of the H5N1 type has recently emerged as a major concern in livestock, with widespread outbreaks now confirmed in U.S. dairy cattle. This raises critical questions about the susceptibility of bovine reproductive tissues to viral entry, replication, and potential transmission. Influenza A viruses (IAV) initiate infection through hemagglutinin (HA) binding to host cell surface sialic acid residues, avian-adapted strains preferentially bind α2,3-linked sialic acids, while human-adapted strains bind α2,6-linked residues. This study aimed to characterize the spatial distribution of α2,6-linked sialic acids (human-like receptors), α2,3-Galβ1-4, and α2,3-Galβ1-3 (avian-like receptors) in male and female bovine reproductive tissues using lectin-based histochemistry. Post-mortem reproductive tissues were collected from bulls (n = 4) and multiparous cows (n = 3) and stained with biotinylated lectins. Human-like receptors were detected in the luminal epithelium of the penile urethra, vas deferens, epididymis, seminiferous tubules, vagina, cervix, uterus, oviduct, and mammary gland. Avian-like receptors were also detected in the penile urethra, epididymis, vagina, cervix, oviduct, and mammary gland, though α2,3-Galβ1-4 and α2,3-Galβ1-3 localization varied by tissue. These findings represent the first comprehensive spatial mapping of IAV receptors in bovine reproductive tissues and highlight potential sites for viral entry or shedding.

Glycine has the greatest rate of deposition in whole-body proteins among all amino acids in neonates, but its provision from sow’s milk meets only 20% of the requirement of suckling piglets. The results of our recent studies indicate that piglets with intrauterine growth restriction (IUGR) have a reduced ability to synthesize glycine. The present study determined the role of glycine in the growth of sow-reared IUGR piglets. In Experiment 1, 56 newborn piglets (postnatal day 0) with a low birth weight (<1.10 kg) were selected from 14 litters, providing 4 IUGR piglets/litter that were allotted randomly into one of four treatment groups (14 piglets/group). Piglets received oral administration of either 0, 0.1, 0.2 or 0.4 g glycine/kg body weight (BW) twice daily (i.e., 0, 0.2, 0.4 or 0.8 g glycine/kg BW/day) between 0 and 14 days of age. L-Alanine was used as the isonitrogenous control. The BWs of all piglets were recorded each week during the experiment. Two weeks after the initiation of glycine supplementation, blood and tissue samples were collected for biochemical analyses. In Experiment 2, rates of muscle protein synthesis in tissues were determined on day 14 using the 3H-phenylalanine flooding dose technique. Compared with piglets in the control group, oral administration of 0.2, 0.4 and 0.8 g glycine/kg BW/day did not affect their milk intake (p > 0.05) but increased (p < 0.05) concentrations of glycine in plasma by 1.52-, 1.94-, and 2.34-fold, respectively, and body weight by 20%, 37%, and 34%, respectively. The dose of 0.4 g glycine/kg BW/day was the most cost-effective. Consistent with its growth-promoting effect, glycine supplementation stimulated (p < 0.05) the phosphorylation of mechanistic target of rapamycin (MTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and ribosomal protein S6 kinase beta-1 (p70S6K) as well as protein synthesis in skeletal muscle, compared with the control group. Collectively, oral administration of glycine activated the MTOR signaling pathway in skeletal muscle and enhanced the growth performance of IUGR piglets. These results indicate that endogenous synthesis of glycine is inadequate to meet the needs of IUGR piglets during the suckling period and that oral supplementation with glycine to these compromized neonates can improve their growth performance.

Background The creatine-creatine kinase-phosphocreatine(Cr-CK-PCr) system maintains intracellular ratios of ATP/ADP for support of cellular functions and has been characterized at the placental-uterine interface of rodents, primates, swine and sheep, and thus may support fetal development. This study determined effects of dietary supplementation of creatine(Cr) to gestating gilts on fetal development, the number and ratio of primary and secondary muscle fibers, and on protein expression in endometrium and fetal biceps-femoris muscle, respectively in fetal pigs on d 60 and d 90 of gestation.Methods Reproductively mature gilts were synchronized to estrus using Matrix, observed for estrus(d 0), and artificially inseminated 12 h and 36 h later. Gilts were individually housed and fed 0.86 kg of 14% crude protein diet twice daily that meets nutritional requirements for pregnant gilts. Gilts were assigned to either basal diet control(CON) group, or Cr supplemented group(provided 30 g Cr monohydrate daily) from d 10 to either d 60 or d 90 of gestation. Gilts were euthanized and hysterectomized on either d 60 or d 90 of gestation. These protocols were completed in two replicates, as gilts were bred in spring and euthanized in summer or bred in fall and euthanized in winter(n = 20 gilts/replicate). Litter size, crown-rump length, sex, and fetal weight was recorded. Three female and male fetuses closest to mean litter weight were selected to assess effects of treatment on weight of fetal brain, kidney, liver, spleen, and biceps-femoris muscle. Data were analyzed to determine effects of treatment, days of gestation, replicate, and sex on litter size, fetal measurements, and incidence of intrauterine growth restriction.Results Dietary Cr supplementation increased fetal brain weight to body weight ratios on d 90 of gestation(P < 0.05) and fetal kidney weight to body weight ratios on d 60 of gestation(P < 0.01), while days of gestation had significant effect on expression of mitochondrial CK isoform in gilt endometria(P < 0.05).Conclusions Results suggest that dietary supplementation of Cr in gestating gilts enhanced development of select fetal organs and contribute to understanding roles of the Cr-CK-PCr system in pregnancy.

During early gestation, the conceptus is undergoing elongation and begins to signal maternal recognition of pregnancy (day 12) with implantation occurring at approximately day 15. Additionally, luteolysis occurs in cyclic gilts at approximately day 13 of their estrous cycle. Further, there is limited research examining the reproductive microbiomes during these periods. Therefore, this study characterized the bacterial communities associated with the mucosal surfaces of the vagina, cervix, and uterus in both pregnant and cycling gilts. Duroc x Landrace x Yorkshire gilts (n=17) free of physical, health or reproductive-related issues were euthanized and hysterectomized during the preimplantation (D11) and implantation (D15) stages of pregnancy as well as cyclic (Pregnant D11, n=7; Pregnant D15, n=5; Cyclic D14/15, n=5). Sterile swabs were used to collect, in duplicate, samples from the mucosal surface of each tissue for storage in microcentrifuge tubes at -80°C until sequencing. For uterine and cervical tissues, a 1 cm incision was made in the same location, exposing the endometrium and cervical mucosa. The sterile swab was rotated 8 times and immediately placed in a microcentrifuge tube. For vaginal sampling, a sterile swab was inserted 6 inches past the vulva and rotated 8 times. Bacterial DNA extraction and genome sequencing targeting the V4 hypervariable region of the 16S rRNA gene was conducted by FERA Diagnostics and Biologicals Corp. Statistical analyses were conducted using the GLM procedure in SAS. Across all reproductive tissues, the phylum Bacteroidetes differed by pregnancy status with less relative abundance in cyclic gilts compared to D11 and D15 gilts (9.68 ± 1.71% vs 11.51 ± 1.11% and 15.91 ± 1.17%, respectively; P< 0.01). Within this phylum, the relative abundance of genus Prevotella was the greatest at D15 compared to D11 and cyclic (4.91 ± 0.59% vs. 3.27 ± 0.57% and 2.15 ± 0.87%, respectively; P< 0.05). The phylum Actinobacteria differed by pregnancy status with a greater relative abundance in cyclic gilts compared to D11 and D15 gilts (13.1 ± 1.66% vs. 5.53 ± 1.08% and 6.72 ± 1.13%, respectively; P< 0.01). Within this phylum, the relative abundance of genus Corynebacterium was greater in cyclic gilts compared to D11 and D15 gilts (5.22 ± 0.60% vs. 2.52 ± 0.39% and 2.83 ± 0.41%, respectively; P< 0.01). There were numerous bacteria that differed by tissue type with Campylobacter, Bacteroides, Porphyromonas, Actinobacillus, Anaerococcus, Peptoniphilus, and Fusobacterium being greater in relative abundance in the vagina compared to the cervix and uterus (P< 0.05). These results suggest that microbial communities differ among components of the reproductive tract and by pregnancy status. The differences observed between the reproductive microbiome of pregnant versus cyclic gilts could be influenced by signals from the conceptus.

Abstract:Tissue non-specific alkaline phosphatase (TNSALP) regulates postnatal phosphate homeostasis, but its role in utero-placental phosphate availability remains poorly understood. Gilts were bred and hysterectomized on Day 60 or Day 90 of gestation (n = 6/day). Phosphate was less abundant in allantoic and amniotic fluids on Day 90 compared to Day 60. TNSALP protein was immunolocalized, and enzymatic activity was quantified and localized in endometrial and chorioallantois tissues. Day had no effect on TNSALP activity in the chorioallantois. In contrast, endometrial TNSALP activity was lower on Day 90 compared to Day 60. Phosphate abundance in allantoic fluid correlated positively with endometrial TNSALP activity on Day 60 but not Day 90. TNSALP protein was abundantly expressed in the endometrium and chorioallantois on both days investigated, with localization to the endometrial, chorionic, and areolar epithelia, as well as stromal cells and endothelium. TNSALP activity was detected in the endothelium of the blood vessels in both the endometrium and chorioallantois, and on the basal surface of the endometrial glands on Day 60 but not Day 90. The endometrial stratum compactum stroma had strong TNSALP activity on Day 60. Weak TNSALP activity was present in the areolar epithelium, with a modest increase in activity on Day 90 compared to Day 60. TNSALP activity was present in the columnar chorionic epithelial cells, with an apparent decrease in activity in the chorioallantois on Day 90 compared to Day 60. These data reveal spatiotemporal changes in TNSALP localization and activity, suggesting its involvement in regulating phosphate availability at the utero-placental interface in swine. Lay Summary:Phosphate is an essential nutrient for fetal growth, but how it is managed during pregnancy is not fully understood. This study explored the role of an enzyme called tissue non-specific alkaline phosphatase (TNSALP) in regulating phosphate availability in the uterus and placenta in pigs in mid- and late pregnancy. Phosphate levels decreased in the fluids surrounding the fetus in late pregnancy. TNSALP was present in the uterus and placenta, and the amount of the enzyme varied depending on the tissue and stage of pregnancy and correlated with changes in phosphate levels. These findings suggest that TNSALP plays a key role in managing phosphate transport from the mother to the fetus in pregnancy to support fetal development.

The creatine (Cr) biosynthesis pathway buffers adenosine triphosphate in metabolically active tissues. We investigated whether sex of fetus and day of gestation influence Cr in endometrial and conceptus tissues from gilts on days 60 and 90 (n = 6 gilts/day) of gestation. Uterine and conceptus tissues associated with one male and one female fetus from each gilt were analyzed for creatine, messenger RNAs (mRNAs), and proteins for Cr biosynthesis. Total Cr decreased in amniotic fluid but increased in allantoic fluid between days 60 and 90 of gestation for male (P < 0.05) but not for female fetuses (P > 0.05). Endometrial expression of creatine kinase, muscle (CKM), creatine kinase mitochondrial type 1 (CKMT1), and solute carrier family 6, member 8 (SLC6A8) mRNAs increased (P < 0.05) between days 60 and 90 only for female fetuses. On day 60, expression of creatine kinase, brain (CKB) and CKMT1 mRNAs was greater (P < 0.05) for placentae of female than male fetuses. Livers of male fetuses had greater expression of arginine:glycine amidinotransferase (AGAT) and CKB than for females on day 60, while kidneys of female fetuses had greater expression of guanidinoacetate-N-methyltransferase (GAMT) than male fetuses on day 90 (P < 0.05). Localization of GAMT, CKB, CKMT1, and SLC6A8 proteins to uterine and chorionic epithelium was not influenced by gestational age or fetal sex. Arginine-glycine amidinotransferase localized to fetal kidneys and appeared greater on day 90 than on day 60 in both sexes. Thus, expression of the creatine-creatine kinase-phosphocreatine system at the uterine-conceptus interface is affected by gestational age and fetal sex to influence energy homeostasis in pigs.

Introduction: Secreted protein acidic and rich in cysteine (SPARC) and secreted phosphoprotein 1 (SPP1) are matricellular proteins implicated in many physiological processes including cell migration, tissue remodeling, and angiogenesis. The role of SPP1 in the pregnancies of many species has been studied extensively, however the contributions of SPARC to pregnancy are not well understood. Methods: Here, we examine the localization of Sparc mRNA and protein in mouse fetuses, endometrial decidua, and placentae during various days of pregnancy in the delayed implantation model to identify potential interactions between SPARC and SPP1 in mouse pregnancy. Results: In situ hybridization and immunofluorescence microscopy localized Sparc mRNA and protein to the endometrial stroma at implantation sites on gestational day (GD)5-6, and to the parietal yolk sac, Reichert's membrane, and select trophoblast lineages on GD9-16. Spp1 mRNA is strongly expressed in the endometrial luminal epithelium at inter-implantation sites and SPP1 is detected in uterine natural killer cells (uNKs) and glycogen trophoblast cells of the placenta. SPARC and SPP1 both localized to many regions of the GD16 fetus, including separate chondroblastic lineages within the developing cartilage. Discussion: The placental expression pattern of these two matricellular proteins suggests possible spatio-temporal and mechanistic relationships between SPARC and SPP1 during pregnancy. While future studies are needed to elucidate the functional roles of SPARC, results from this study demonstrate that SPARC is a prominent component of the matricellular milieu throughout gestation.

Species have different strategies for implantation and placentation. Much can be learned about general molecular and cellular biology through the examination and comparison of these differences. To varying degrees, implantation in all species includes alterations in epithelial polarity, the transformation of the endometrial stroma, the differentiation of the trophoblast, cell-to-cell and tissue-to-tissue signaling through hormones, cytokines, and extracellular vesicles, and the alteration of the maternal immune system. This review focuses on implantation in pigs, sheep, and cows. These species share with mice/rats and humans/primates the key events of early embryonic development, pregnancy recognition, and the establishment of functional placentation. However, there are differences between the pregnancies of livestock and other species that make livestock unique biomedical models for the study of pregnancy and cell biology in general. Pig, sheep, and cow conceptuses (embryo/fetus and associated placental membranes) elongate prior to implantation, displaying central implantation, extended periods of conceptus attachment to the uterus, and epitheliochorial (pigs) and synepitheliochorial (sheep and cows) placentation. This review will discuss what is understood about how the trophoblast and extraembryonic endoderm of pig, sheep, and cow conceptuses elongate, and how a major goal of current in vitro models is to achieve conceptus elongation. It will then examine the adhesion cascade for conceptus implantation that initiates early placental development in pigs, sheep, and cows. Finally, it will conclude with a brief overview of early placental development in pigs, sheep, and cows, with a listing of some important “omics” studies that have been published.

In brief:Phosphate plays a critical role in conceptus development, yet the mechanisms regulating utero-placental availability remain underinvestigated. This research characterized the spatiotemporal expression and endocrine regulation of XPR1, a phosphate exporter, in ovine utero-placental tissues, suggesting a potential role of XPR1 in the regulation of utero-placental phosphate availability. Abstract:Phosphate is an essential regulator of conceptus development, but there is limited understanding of mechanisms regulating phosphate availability in utero-placental tissues. These experiments characterized the expression of xenotropic and polytropic retrovirus receptor 1 (XPR1), a phosphate exporter, in ovine utero-placental tissues. In Experiment 1, ewes were hysterectomized on day 1, 9, or 14 of the estrous cycle or day 30, 50, 70, 110, or 125 of pregnancy. Day of the estrous cycle did not affect XPR1 mRNA expression or protein localization. Expression of XPR1 mRNA decreased with day of gestation in placentomes, while XPR1 protein was detectable in uterine epithelia, blood vessels, endometrial stromal cells, myometrium, caruncular stroma, and syncytium of the placentome. In Experiment 2, ewes received daily injections of either corn oil vehicle (CO) or 25 mg progesterone (P4) in vehicle for the first 8 days of pregnancy and were hysterectomized on either day 9, 12, or 125. Endometrial stroma from P4-treated ewes had greater XPR1 immunoreactivity than CO-treated ewes on day 9. On day 125, endometria from P4-treated ewes had decreased expression of XPR1 mRNA compared to CO-treated ewes. Greater XPR1 protein immunoreactivity was present in uterine epithelia and stratum compactum stroma of P4-treated than CO-treated ewes. P4-treated ewes with a singleton fetus tended to have greater expression of XPR1 mRNA in placentomes than CO-treated ewes with a singleton fetus. Collectively, these results suggest a potential role of XPR1 in the regulation of phosphate availability in utero-placental tissues in ruminants.

The composition of bacterial communities within the female reproductive tract has been associated with fertility status in mammals; however, limited research has explored reproductive tract microbiota in swine. The objective of this study was to analyze the abundance and diversity of bacterial communities from the mucosal surface of the vagina, cervix, uterus, and chorioallantois, and within the allantoic and amniotic fluids throughout gestation in gilts. Duroc x Landrace x Yorkshire gilts (n=38) free of physical, health or reproductive-related issues were euthanized and hysterectomized on either day 11 (n = 11; peri-implantation), 15 (n = 10; implantation), 60 (n =6; mid-gestation) or 90 (n = 6; late-gestation) of pregnancy, as well as day 15 (n = 5; cyclic) of the estrous cycle. Bacterial analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. In day 15 cyclic gilts, genera Campylobacter, Actinobacillus, Anaerococcus, and Fusobacterium were more abundant in the vagina than in the cervix (P < 0.05). Additionally, Campylobacter, Porphyromonas, and Anaerococcus were more abundant in the vagina than in the endometrium (P < 0.05). In pregnant gilts, the genus Fusobacteria abundance was greater in the vagina than in the cervix and endometrium on days 11 (P < 0.01) and 15 (P < 0.01) of gestation. Additionally, Lactobacillus was more abundant in allantoic fluid than the endometrium on day 60 (P < 0.05). Further, Streptococcus, Campylobacter, and Actinobacillus were more abundant in the vagina than in the amniotic fluid on day 90 (P < 0.05). Alpha diversity metrics indicated lower microbial diversity in amniotic fluid relative to other tissues (P < 0.05). However, alpha diversity did not differ between cyclic and pregnant gilts (P > 0.05). Beta diversity analysis revealed distinct clustering patterns based on tissue type and stage of pregnancy. Specifically, the vagina, cervix, and endometrium clustered separately from placental fluids. Additionally, microbial communities in samples from day 15 cyclic, day 11 pregnant, and day 15 pregnant gilts clustered distinctly from days 60 and 90 of gestation (P < 0.01). These findings suggest that microbial communities within the reproductive tract are dynamic and vary by both anatomical location and stage of pregnancy or estrous cycle. The distinct clustering of microbial populations between early and late stages of gestation indicates shifts in bacterial composition potentially associated with the presence of a conceptus. Additionally, the observed differences in microbial diversity across reproductive tissues and fetal fluids highlight potential functional roles of microbiota in reproductive success.

In the beef cattle industry, over $1 billion is lost annually due to reproductive failure during early gestation. Shifts in bacterial communities have been associated with endogenous estradiol concentrations and pregnancy status. However, quantification of factors that influence bacterial diversity of the beef cow uterine microbiome has yet to be evaluated. Our objective was to determine the influence of hormone treatment, day, pregnancy status, and sample location on bacterial diversity of the beef cow uterine microbiome. Crossbred beef cows (n=62) were synchronized utilizing the 7-day CO-Synch + CIDR protocol. Two days post-CIDR removal (day [d] 0), cows were allocated to one of three treatment groups: 1) Estrus Expression (ESTRUS), 2) No Estrus + induced ovulation with gonadotropin releasing hormone (GnRH; GNRH), 3) No Estrus + induced ovulation with GnRH and 0.1 mg estradiol 17β (GNRH+E2). All cows were artificially inseminated on d0 with semen from a single sire. A modified version of the IDEXX Bovine Pregnancy ELISA detected pregnancy associated glycoproteins for pregnancy diagnosis (NONPREG=nonpregnant; PREG=pregnant). Cows were ovariohysterectomized at harvest on d20, d26, and d32. An anti-mesometrial incision was made in each uterine horn (ipsilateral [IPS] and contralateral [CON]) and sterile swabs were rotated along the endometrial mucosa, placed in sterile microcentrifuge tubes, and stored at -80°C. Microbiome analysis targeted the V4 hypervariable region of the 16S rRNA bacterial gene. Sequence quality was assessed utilizing DADA2, and taxon analysis was processed in the Qiime2 pipeline with resulting files exported for diversity analysis using the R package ‘phyloseq’. Alpha diversity was determined with Wilcoxon or Kruskal-Wallis tests, and beta diversity was determined using PERMANOVA. Alpha diversity analysis included observed OTUs and Chao1 index (within sample richness), and Shannon and Simpson indices (within sample richness and evenness). Beta diversity matrices included unweighted UniFrac (between sample bacterial presence or absence) and weighted UniFrac (between sample bacterial presence or absence and abundance). Diversity within samples was different for all alpha diversity matrices compared between days (P≤0.05). Specifically, observed OTUs and Chao1 index indicated greater diversity within samples on d32 compared with d26 (P=0.01). Shannon and Simpson indices indicated greater diversity within samples for both d32 and d20 compared to d26 (P≤0.001). There were differences in diversity between samples for both unweighted and weighted Unifrac matrices compared between days (P < 0.01). Specifically, there was a difference in unweighted Unifrac between d26 and d32 (P < 0.01). However, there were no differences for all alpha and beta diversity matrices between treatments, pregnancy status, and uterine horns (P>0.05). Thus, bacterial diversity of the beef cow uterine microbiome appears to shift by day. Additional studies are needed to elucidate the, as yet, undetermined mechanisms that cause time-dependent alterations in bacterial diversity within the uterine microbiome.

Mammals exhibit unique and highly variable mechanisms for the establishment and maintenance of pregnancy. Ruminants (e.g., sheep, cows, and goats) have novel mechanisms whereby the conceptus (embryo and its extra-embryonic membranes) signals for the establishment of pregnancy and exhibits unique metabolic pathways favoring conceptus development. Embryos of ruminants reach the spherical blastocyst stage at 5 to 10 mm in diameter and then elongate rapidly to elongated filamentous conceptuses of greater than 250 mm as they make contact with the uterine luminal epithelium (LE) for implantation. During conceptus elongation the trophectoderm cells secrete interferon tau (IFNT), a novel pregnancy recognition signal for ruminants to ensure maintenance of a functional corpus luteum (CL) to secrete progesterone (P4) required for pregnancy. P4 induces uterine epithelia cells to express the endogenous Jaagsiekte Retrovirus (enJSRV) that may transactivate toll-like receptors 7 and 8 in the conceptus trophectoderm to induce secretion of IFNT, a classical viral–antiviral mechanism. IFNT silences expression of receptors for estradiol (E2) and oxytocin (OXTR), which abrogates the mechanism whereby oxytocin from CL and posterior pituitary would otherwise induce large pulses of prostaglandin F2α (PGF) by uterine epithelia to cause regression of the CL and its secretion of P4. IFNT has another novel role in silencing expression of not only ESR1 and OXTR, but all classical interferon-stimulated genes in the uterine LE and superficial glandular epithelium (sGE), but with P4 increasing expression of genes for transport of nutrients such as glucose and arginine into the uterine lumen to support conceptus development. Ruminant conceptuses convert glucose to fructose, a novel hexose sugar that cannot be transported back to the maternal circulation. Fructose is converted to fructose-1-PO4 for metabolism, not via the pathway for glycolysis but via the novel fructolysis pathway uninhibited by low pH, citrate, or ATP as is the case for glycolysis. Thus, fructose and its metabolites support the pentose cycle, hexosamine biosynthesis pathway, one-carbon metabolism, and the citric acid cycle for all cells of the conceptus. Arginine is another key nutrient transported into the uterine lumen by the uterine LE/sGE in response to P4 and IFNT. Arginine is metabolized to generate nitric oxide, polyamines, and creatine, essential for conceptus growth and development, while enhancing production of IFNT as a novel pregnancy recognition signal, and upregulating expression of genes in the uterine LE/sGE for transport of nutrients. Fructose is the major hexose sugar supporting major metabolic pathways required for conceptus growth and development in ruminants.

Histotroph is regulated by P4 and paracrine factors and contains amino acids essential for porcine conceptus development. We hypothesized P4 contributes to amino acid transport during pregnancy in pigs. Ovariectomized gilts received daily P4 or corn oil (CO) injections from Days 12 through 39 post-estrus. High-performance liquid chromatography (HPLC) determined increased abundances of amino acids including glycine, in uterine flushings from P4-treated gilts. SLCs transport amino acids across membranes. rtPCR analyses revealed endometrial expression of SLC6A9 mRNA increased during the peri-implantation period of pregnancy. SLC7A8 and SLC38A1 mRNA expression also increased during that period but decreased later in pregnancy. SLC1A4, SLC1A5, and SLC7A10 mRNA expression was greatest later in pregnancy. Immunofluorescence analyses localized SLC6A9, which transports glycine exclusively, to the endometrial luminal epithelium from Day 12 through 25, uterine glandular epithelium throughout gestation, conceptus trophectoderm and endoderm between Days 15 and 20, and chorionic epithelia (CE) of interareolar and areolar CE between Days 40 and 60. Expression in interareolar regions decreased by Day 60 but remained high in the areolae. Expression of SLC6A9 was greater for endometrium from P4-treated than CO-treated pigs. These results illustrate the complexity of, and stage-dependent changes in, expression of amino acid transporters in endometria from pregnant pigs. The results suggest that transport of glycine into the uterine lumen and across the chorioallantois by SLC6A9 may be critical for delivery of this amino acid that is essential for conceptus development.

Fluorometers and backscatter sensors are essential tools in oceanography, used to measure various biogeochemical parameters, and are key components of the BGC-Argo program. The RBRtridente integrates these two sensors into a single device. In this study, we detail the calibration procedure for the RBRtridente, a combined fluorescence-backscattering sensor developed by RBR. The calibration process involves three key steps: gain calibration, temperature compensation, and optical calibration. We describe each step in detail including the methodology for generating primary standards for calibration, as well as the primary and secondary reference instruments used for calibration. The calibration procedure is validated in the field by comparing the measurements of the RBRtridente to other fluorometers and backscatter sensors commonly used as part of the BGC-Argo program.

Mammals differ regarding their placentae, but in all species placental trophoblasts interact intimately with the uterine endometrium to mediate the transfer of nutrients from the mother to the embryo/fetus through the closely juxtaposed microcirculatory systems of the uterus and placenta. Placentation in ruminants is intermediate between the non-invasive type, as observed in the epitheliochorial placenta of pigs, and the invasive type, as observed in the haemochorial placentae of mice and humans. In ruminants, placental trophoblast cells invade uterine endometrial tissue, but invasion is believed to be limited to the endometrial luminal epithelium (LE). In the LE there are varying degrees of syncytialisation among species, with syncytialisation being more extensive in sheep than cows. The hallmarks of placentation in ruminants include: (1) an extended period in which conceptuses (embryos and associated placental membranes) elongate and must be supported by secretions (histotroph) from the uterus; (2) a cascade involving an array of adhesion molecules that includes integrin-mediated attachment of the conceptus trophoblast to the endometrial LE for implantation; (3) syncytialisation of the developing early placenta, a process for which there is currently limited understanding; and (4) development of placentomes that define the cotyledonary placentae of cows and sheep, and provide haemotrophic support of fetal development.

Fructose, the most abundant hexose sugar in fetal fluids and the blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway, and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell-specific manner. The KHK-A protein was more abundant in the trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in the endoderm of Day 16 conceptuses and the chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the tricarboxylic cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway, and one-carbon metabolism required for conceptus development throughout pregnancy.