A single-cell gene expression atlas of human follicular aspirates: Identification of leukocyte subpopulations and their paracrine factors.

Leukocytes are in situ regulators critical for ovarian function. However, little is known about leukocyte subpopulations and their interaction with follicular cells in ovulatory follicles, especially in humans. Single-cell RNA sequencing (scRNA-seq) was performed using follicular aspirates obtained from four IVF patients and identified 13 cell groups: one granulosa cell group, one thecal cell group, 10 subsets of leukocytes, and one group of RBC/platelet. RNA velocity analyses on five granulosa cell populations predicted developmental dynamics denoting two projections of differentiation states. The cell type-specific transcriptomic profiling analyses revealed the presence of a diverse array of leukocyte-derived factors that can directly impact granulosa cell function by activating their receptors (e.g., cytokines and secretory ligands) and are involved in tissue remodeling (e.g., MMPs, ADAMs, ADAMTSs, and TIMPs) and angiogenesis (e.g., VEGFs, PGF, FGF, IGF, and THBS1) in ovulatory follicles. Consistent with the findings from the scRNA-seq data, the leukocyte-specific expression of CD68, IL1B, and MMP9 was verified in follicle tissues collected before and at defined hours after hCG administration from regularly cycling women. Collectively, this study demonstrates that this data can be used as an invaluable resource for identifying important leukocyte-derived factors that promote follicular cell function, thereby facilitating ovulation and luteinization in women.

Calcium-binding proteins S100A8, S100A9, and S100A12: expression and regulation at the maternal-conceptus interface in pigs†.

Among the many calcium-binding proteins, S100A8, S100A9, and S100A12 play important roles in inflammation, innate immunity, and antimicrobial function, but their expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression and regulation of S100A8, S100A9, S100A12, and their receptor AGER at the maternal-conceptus interface in pigs. We found that S100A8, S100A9, and S100A12 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy, with the greatest levels on Day (D) 12 of pregnancy, and AGER appeared at greater levels on D15 and D30 of pregnancy than on other days. The expression of S100A8, S100A9, and S100A12 was predominantly localized to epithelial cells in the endometrium, and they were detected in early-stage conceptus and later chorioallantoic tissues during pregnancy. AGER expression was localized to endometrial epithelial and stromal cells and chorionic epithelial cells. In endometrial explant tissues, the expression of S100A8, S100A9, and S100A12 was induced by estrogen, S100A8 by interleukin-1ß, and AGER by interferon-γ. We further found that on D12 of pregnancy, the expression of S100A8, S100A9, and S100A12 decreased significantly in the endometria of gilts carrying conceptuses derived from somatic cell nuclear transfer. These results indicate that the expression of S100A8, S100A9, and S100A12 is dynamically regulated in response to conceptus-derived signals at the maternal-conceptus interface, suggesting that S100A8, S100A9, and S100A12 could play a critical role in regulating endometrial epithelial cell function and conceptus implantation to support the establishment and maintenance of pregnancy in pigs.

Neurotensin: a neuropeptide induced by hCG in the human and rat ovary during the periovulatory period†.

Neurotensin (NTS) is a tridecapeptide that was first characterized as a neurotransmitter in neuronal cells. The present study examined ovarian NTS expression across the periovulatory period in the human and the rat. Women were recruited into this study and monitored by transvaginal ultrasound. The dominant follicle was surgically excised prior to the luteinizing hormone (LH) surge (preovulatory phase) or women were given 250 µg human chorionic gonadotropin (hCG) and dominant follicles collected 12-18 h after hCG (early ovulatory), 18-34 h (late ovulatory), and 44-70 h (postovulatory). NTS mRNA was massively induced during the early and late ovulatory stage in granulosa cells (GCs) (15 000 fold) and theca cells (700 fold). In the rat, hCG also induced Nts mRNA expression in intact ovaries and isolated GCs. In cultured granulosa-luteal cells (GLCs) from IVF patients, NTS expression was induced 6 h after hCG treatment, whereas in cultured rat GCs, NTS increased 4 h after hCG treatment. Cells treated with hCG signaling pathway inhibitors revealed that NTS expression is partially regulated in the human and rat GC by the epidermal-like growth factor pathway. Human GLC, and rat GCs also showed that Nts was regulated by the protein kinase A (PKA) pathway along with input from the phosphotidylinositol 3- kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The predominat NTS receptor present in human and rat GCs was SORT1, whereas NTSR1 and NTSR2 expression was very low. Based on NTS actions in other systems, we speculate that NTS may regulate crucial aspects of ovulation such as vascular permeability, inflammation, and cell migration.

Analysis of interferon-γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal-conceptus interface and the role of interferon-γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs.

It has long been known that pig conceptuses produce interferon-γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal-conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin-1ß did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy- and cell-type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs.

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal-conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal-conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid- to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal-conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen-responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal-conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.

Analysis of cysteine-X-cysteine motif chemokine ligands 9, 10, and 11, their receptor CXCR3, and their possible role on the recruitment of immune cells at the maternal-conceptus interface in pigs.

Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period in pigs.

The expression of CXCR4 is induced by the luteinizing hormone surge and mediated by progesterone receptors in human preovulatory granulosa cells.

The chemokine CXC motif ligand 12 (CXCL12) and its cognate receptor, CXCR4, have been implicated in the ovulatory process in various animal models. However, little is known about the expression and regulation of CXCL12 and CXCR4 and their functions during the ovulatory period in the human ovary. In this study, we characterized the expression patterns of CXCL12 and CXCR4 in preovulatory follicles collected before the luteinizing hormone (LH) surge and at defined hours after hCG administration in women with the regular menstrual cycle. The levels of mRNA and protein for CXCR4 were increased in granulosa cells of late ovulatory follicles, whereas CXCL12 expression was constant in follicles throughout the ovulatory period. Both CXCR4 and CXCL12 were localized to a subset of leukocytes around and inside the vasculature of human preovulatory follicles. Using a human granulosa cell culture model, the regulatory mechanisms and functions of CXCL12 and CXCR4 expression were investigated. Human chorionic gonadotropin (hCG) stimulated CXCR4 expression, whereas CXCL12 expression was not affected, mimicking in vivo expression patterns. Both RU486 (progesterone receptor antagonist) and CoCl2 (HIFs activator) blocked the hCG-induced increase in CXCR4 expression, whereas AG1478 (EGFR inhibitor) had no effect. The treatment with CXCL12 had no effect on granulosa cell viability but decreased hCG-stimulated CXCR4 expression.

Expression and regulation of prostaglandin transporters, ATP-binding cassette, subfamily C, member 1 and 9, and solute carrier organic anion transporter family, member 2A1 and 5A1 in the uterine endometrium during the estrous cycle and pregnancy in pigs.

OBJECTIVE: Prostaglandins (PGs) function in various reproductive processes, including luteolysis, maternal pregnancy recognition, conceptus development, and parturition. Our earlier study has shown that PG transporters ATP-binding cassette, subfamily C, member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) are expressed in the uterine endometrium in pigs. Since several other PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 are known to be present in the uterine endometrium, this study investigated the expression of these PG transporters in the porcine uterine endometrium and placenta. METHODS: Uterine endometrial tissues were obtained from gilts on day (D) 12 and D15 of the estrous cycle and days 12, 15, 30, 60, 90, and 114 of pregnancy. RESULTS: ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs were expressed in the uterine endometrium, and levels of expression changed during the estrous cycle and pregnancy. Expression of ABCC1 and ABCC9 mRNAs was localized mainly to luminal and glandular epithelial cells in the uterine endometrium, and chorionic epithelial cells during pregnancy. Conceptuses during early pregnancy and chorioallantoic tissues from mid to late pregnancy also expressed these PG transporters. Estradiol-17ß increased the expression of ABCC1 and SLCO5A1, but not ABCC9 and SLCO4C1 mRNAs and increasing doses of interleukin-1ß induced the expression of ABCC9, SLCO4C1, and SLCO5A1 mRNAs in endometrial explant tissues. CONCLUSION: These data showed that several PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 were expressed at the maternal-conceptus interface, suggesting that these PG transporters may play an important role in the establishment and maintenance of pregnancy by regulating PG transport in the uterine endometrium and placenta in pigs.

Evaluation of high nutrient diets on litter performance of heat-stressed lactating sows.

OBJECTIVE: The present study investigated the litter performance of multiparous sows fed 3% and 6% densified diets at farrowing to weaning during summer with mean maximum room temperature of 30.5°C. METHODS: A total of 60 crossbred multiparous sows were allotted to one of three treatments based on body weight according to a completely randomized design. Three different nutrient levels based on NRC were applied as standard diet (ST; metabolizable energy, 3,300 kcal/kg), high nutrient level 1 (HE1; ST+3% higher energy and 16.59% protein) and high nutrient level 2 (HE2; ST+6% higher energy and 17.04% protein). RESULTS: There was no variation in the body weight change. However, backfat thickness change tended to reduce in HE1 in comparison to ST treatment. Dietary treatments had no effects on feed intake, daily energy intake and weaning-to-estrus interval in lactating sows. Litter size, litter weight at weaning and average daily gain of piglets were significantly greater in sows in HE1 compared with ST, however, no difference was observed between HE2 and ST. Increasing the nutrient levels had no effects on the blood urea nitrogen, glucose, triglyceride, and creatinine at post-farrowing and weaning time. The concentration of follicle stimulating hormone, cortisol and insulin were not affected by dietary treatments either in post-farrowing or weaning time. The concentration of blood luteinizing hormone of sows in ST treatment was numerically less than sows in HE2 treatment at weaning. Milk and colostrum compositions such as protein, fat and lactose were not affected by the treatments. CONCLUSION: An energy level of 3,400 kcal/kg (14.23 MJ/kg) with 166 g/kg crude protein is suggested as the optimal level of dietary nutrients for heat stressed lactating sows with significant beneficial effects on litter size.

Effects of Ecklonia cava as fucoidan-rich algae on growth performance, nutrient digestibility, intestinal morphology and caecal microflora in weanling pigs.

OBJECTIVE: In the present study, role of increasing levels of Ecklonia cava (seaweed) supplementation in diets was investigated on growth performance, coefficient of total tract apparent digestibility (CTTAD) of nutrients, serum immunoglobulins, cecal microflora and intestinal morphology of weanling pigs. METHODS: A total of 200 weaned pigs (Landrace×Yorkshire×Duroc; initial body weight 7.08±0.15 kg) were randomly allotted to 4 treatments on the basis of body weight. There were 5 replicate pens in each treatment including 10 pigs of each. Treatments were divided by dietary Ecklonia cava supplementation levels (0%, 0.05%, 0.1%, or 0.15%) in growing-finishing diets. There were 2 diet formulation phases throughout the experiment. The pigs were offered the diets ad libitum for the entire period of experiment in meal form. RESULTS: The pigs fed with increasing dietary concentrations of Ecklonia cava had linear increase (p<0.05) in the overall average daily gain, however, there were no significant differences in gain to feed ratio, CTTAD of dry matter and crude protein at both phase I and phase II. Digestibility of gross energy was linearly improved (p<0.05) in phase II. At day 28, pigs fed Ecklonia cava had greater (linear, p<0.05) Lactobacillus spp., fewer Escherichia coli (E. coli) spp. (linear, p<0.05) and a tendency to have fewer cecal Clostridium spp. (p = 0.077). The total anaerobic bacteria were not affected with supplementation of Ecklonia cava in diets. Polynomial contrasts analysis revealed that villus height of the ileum exhibited a linear increase (p<0.05) in response with the increase in the level of dietary Ecklonia cava. However, villus height of duodenum and jejunum, crypt depth, villus height to crypt depth ratio of different segments of the intestine were not affected. CONCLUSION: The results suggest that Ecklonia cava had beneficial effects on the growth performance, cecal microflora, and intestinal morphology of weanling pigs.

Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

Many chemokines are present at the maternal-fetal interface and play important roles in the establishment and maintenance of pregnancy. Our study demonstrates that a chemokine, chemokine (C-C motif) ligand 28 (CCL28), is expressed in the uterine endometrium during early pregnancy in pigs. Thus, we investigated expression of CCL28 and its receptors, chemokine (C-C motif) receptor type 3 (CCR3) and 10 (CCR10), in the uterine endometrium during the estrous cycle and pregnancy and the function of CCL28 at the maternal-fetal interface during early pregnancy. Levels of CCL28 mRNAs were highest on Day 10 of pregnancy and decreased thereafter during pregnancy and CCL28 was localized mainly to endometrial glandular epithelial cells. The presence of the CCL28 protein in uterine flushings was confirmed on Day 12 of the estrous cycle and pregnancy. Endometrial tissues expressed CCR3 and CCR10 during pregnancy. The CCR10 protein was localized to endometrial luminal and glandular epithelial cells, chorionic epithelial cells, and the allantoic membrane during pregnancy. Conceptuses during early pregnancy expressed CCL28 and CCR10, but not CCR3, and chorioallantoic tissues expressed CCR10 at increasing levels towards term. Treatment with recombinant CCL28 increased the proliferation and migration of a porcine trophectoderm cell line. These results indicated that the CCL28 chemokine and its receptors, CCR3 and CCR10, are expressed at the maternal-conceptus interface, and CCL28 induces the proliferation and migration of trophectoderm cells through CCR10, suggesting that CCL28 may play a critical role in the establishment and maintenance of pregnancy in pigs.

Prostaglandin transporters ABCC4 and SLCO2A1 in the uterine endometrium and conceptus during pregnancy in pigs.

Prostaglandins (PGs) are involved in many reproductive activities including luteolysis, maternal recognition of pregnancy, endometrial gene expression, conceptus development, and parturition in domestic animals. However, mechanisms by which PGE2 and PGF2alpha are modulated in the uterine endometrium and expression of ABCC4 and SLCO2A1, responsible for efficient transport of PGs across the cell membrane, in the endometrium during the estrous cycle and pregnancy are not fully understood in pigs. Therefore, we determined expression of ABCC4 and SLCO2A1, genes involved in transport of PGE2 and PGF2alpha in the uterine endometrium during the estrous cycle and pregnancy in pigs. ABCC4 and SLCO2A1 mRNAs were expressed in the uterine endometrium, most abundantly on Day 12 of pregnancy and during late pregnancy. Expression of ABCC4 mRNA and protein was localized mainly to uterine luminal epithelial (LE) and glandular epithelial (GE) cells, and expression of SLCO2A1 mRNA and protein was expressed primarily in uterine LE and blood vessels. Expression of ABCC4 and SLCO2A1 mRNAs was also detected in conceptuses during early pregnancy. In addition, explant culture experiments showed that increasing doses of interleukin 1B (IL1B) with estrogen and progesterone increased levels of ABCC4 and SLCO2A1 mRNAs in the uterine endometrium. These results indicate that expression of genes responsible for transport of PGE2 and PGF2alpha are dynamically regulated in the uterine endometrium during pregnancy and that ABCC4 and SLCO2A1 play critical roles in supporting the establishment and maintenance of pregnancy by regulating PG transport at the maternal-fetal interface in pigs.

Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy.

Analysis of legumain and cystatin 6 expression at the maternal-fetal interface in pigs.

Cathepsins (CTSs), a family of lysosomal cysteine proteases, and their inhibitors, cystatins (CSTs), play a critical role in endometrial and placental tissue remodeling during the establishment and maintenance of pregnancy in many species including rodents, sheep, cow, and pigs. In this study, we determined expression of legumain (LGMN), a cathepsinmember, and its inhibitor, CST6, at the maternal-fetal interface in pigs. Expression of both LGMN and CST6 mRNAs increased during mid- to late pregnancy in the uterine endometrium. LGMN and CST6 mRNAs localized to luminal epithelial cells (LE) and glandular epithelial cells (GE) and to the chorionic membrane (CM), with a strong intensity in GE and the CM for LGMN and in the CM for CST6 during pregnancy. LGMN protein was detected at molecular weights (MW) of approximately 50,000 and 37,000, and the abundance of the37,000-MW LGMN protein increased during mid- to latepregnancy. CST6 protein was also highly expressed in the uterine endometrium in mid- to latepregnancy. LGMN protein localized to LE, GE, and the CM during pregnancy. LGMN and CST6 were aberrantly expressed in the uterine endometrium from gilts with somatic cell nuclear transfer-derived conceptuses at term compared to those of gilts carrying conceptuses derived from natural mating. These results demonstrated that LGMN and CST6 were expressed in the uterine endometrium in a cell-type and stage-specific manner, suggesting that the LGMN and CST6 system at the maternal-fetal interface may play an important role in the establishment and maintenance of pregnancy in pigs.

Analysis of ENPP2 in the Uterine Endometrium of Pigs Carrying Somatic Cell Nuclear Transfer Cloned Embryos.

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.

Anti-Bordetella bronchiseptica effects of targeted bacteriophages via microbiome and metabolic mediated mechanisms.

Bordetella bronchiseptica poses a significant challenge in the context of respiratory infections, particularly in weanling pigs. In this study, we investigated the impact of a novel targeted bacteriophage in controlling B. bronchiseptica challenge (BBC) in an experimental design involving five distinct treatment groups: NC (no challenge), PC (BBC challenge), BF (108 pfu bacteriophage/kg diet + BBC), BN (2 × 107 pfu/day bacteriophage by nasal spray + BBC), and AT (antibiotic + BBC). The experiment was conducted for 2 weeks. The highest turbinate score was observed in the PC. The BF treatment showed higher plasma IL (interleukine)-1ß and IL-6 compared with the BN and AT treatments. Plasma concentrations of IL-1ß were increased in the BF pigs compared with the BN, AT, and NC. Among the BBC groups, the PC treatment exhibited a higher abundance of Staphylococcus. aureus and B. bronchiseptica in the lung. A lower S. aureus, Streptococcus. suis, and B. bronchiseptica colonization was detected in the AT compared with the BF and BN treatments. The BF showed lower plasma zonulin compared with the BN and AT. A higher plasma concentration of superoxide dismutase was observed in the BF and AT compared with PC and BN. The BN influenced the glycine, serine-threonine metabolism; glycerolipid metabolism; glyoxylate-dicarboxylate metabolism; and arachidonic acid metabolism compared with the NC. In conclusion, nasal-sprayed bacteriophage effectively controlled B. bronchiseptica infection, however, their efficiency was lower than the antibiotic.

A quantum-inspired probabilistic prime factorization based on virtually connected Boltzmann machine and probabilistic annealing.

Probabilistic computing has been introduced to operate functional networks using a probabilistic bit (p-bit), broadening the computational abilities in non-deterministic polynomial searching operations. However, previous developments have focused on emulating the operation of quantum computers similarly, implementing every p-bit with large weight-sum matrix multiplication blocks and requiring tens of times more p-bits than semiprime bits. In addition, operations based on a conventional simulated annealing scheme required a large number of sampling operations, which deteriorated the performance of the Ising machines. Here we introduce a prime factorization machine with a virtually connected Boltzmann machine and probabilistic annealing method, which are designed to reduce the hardware complexity and number of sampling operations. From 10-bit to 64-bit prime factorizations were performed, and the machine offers up to 1.2 × 108 times improvement in the number of sampling operations compared with previous factorization machines, with a 22-fold smaller hardware resource.

Comparison of growth performance and related gene expression of muscle and fat from Landrace, Yorkshire, and Duroc and Woori black pigs.

The purpose of this study was to compare marbling score, meat quality, juiciness, sarcomere length, and skeletal muscle satellite cell (SMSC) growth and related gene expression between Woori black pig (WB) and the Landrace, Yorkshire, and Duroc (LYD) crossbreed at different body weights (b.w.). WB was developed to improve meat quality and growth efficiency by crossbreeding Duroc with Korean native black pig. A total of 24 pigs were sacrificed when their b.w. reached about 50, 75, 100, and 120 kg. SMSC were isolated from the femoris muscles, and muscle and adipose tissues were sampled from the middle and the subcutaneous part of the femoris of hind legs, respectively. Expression levels of genes including Myoblast determination protein 1 (MyoD), Paired box gene 3 (Pax3), Myosin heavy chain (MyHC), and Myogenin, which are responsible for the growth and development of SMSC, were higher in LYD than the WB. Muscle growth inhibitor myostatin (MSTN), however, was expressed more in WB compared to LYD (p < 0.01). Numbers of SMSC extracted from femoris muscle of LYD at 50, 75, 100, and 120 kg b.w. were 8.5 ± 0.223, 8.6 ± 0.245, 7.2 ± 0.249, and 10.9 ± 0.795, and those from WB were 6.2 ± 0.32, 6.2 ± 0.374, 5.3 ± 0.423, and 17.1 ± 0.315, respectively. Expression of adipogenic genes in adipose tissue including CCAAT/enhancer-binding protein (CEBP)-ß, peroxisome proliferator activated receptor (PPAR)-γ, and fatty acid synthase (FASN), were greater in WB when compared with LYD (p < 0.01). Results from the current study suggest that different muscle cell numbers between 2 different breeds might be affected by related gene expression and this warrants further investigation on other growth factors regulating animal growth and development.

Effects of dietary supplementation of bacteriophage cocktail on health status of weanling pigs in a non-sanitary environment.

BACKGROUND: The study evaluated the effects of bacteriophage cocktail (BP) and ZnO administered during weaning time for piglets exposed to a non-sanitary environment. The bacteriophages were designed to eliminate Escherichia coli (K88, K99 and F41), Salmonella (typhimurium and enteritidis), and Clostridium perfreingens (types A and C). Forty 21-day-old crossbreed piglets were assigned to four treatments, including the PC (sanitary environment), NC (non-sanitary environment), BP (NC plus 108 pfu/kg BP), and ZO (NC plus 2,500 mg/kg ZnO). Piglets in the NC, BP and ZO were kept in a non-sanitary environment for 14 d, which was contaminated with the feces of infected pigs. RESULTS: Pigs in the BP and ZO treatments had a higher final body weight compared with the NC. The NC treatment showed the highest concentration of inflammatory cytokines including interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α in the plasma. The administration of BP and ZO showed lower myeloperoxidase concentrations compared with the NC. The NC treatment showed a lower concentration of superoxide dismutase in serum compared with the PC. Among the treatments in non-sanitary environment, the NC treatment showed a higher concentration of malondialdehyde compared with the ZO. The PC treatment showed a lower concentration of butyric acid in the feces compared with the BP treatment. Among non-sanitary treatments, the villus height in the duodenum was greater in the BP and ZO compared with the NC. The lower abundance of Proteobacteria phylum was observed in the BP and PC treatments compared with the NC. The highest relative abundance of Eubacterium was recorded in the BP treatment. The abundance of Megasphaera and Schwartzia was higher in the NC pigs compared with the BP piglets. The abundance of Desulfovibrio was lower in the supplemented treatments (BP and ZO) compared with non-supplemented (NC and PC). The abundance of Cellulosilyticum genera was higher in the BP and ZO treatments rather than in the NC. The piglets in the NC treatment had the highest abundance of Escherichia-Shigella, followed by the PC and ZO treatments. CONCLUSION: In conclusion, these results suggest that the supplementation of bacteriophage cocktail could effectively control Proteobacteria phylum, Clostridium spp. and coliforms population and mitigated the adverse influences of weaning stress in piglets.

An intelligent method for pregnancy diagnosis in breeding sows according to ultrasonography algorithms.

Pig breeding management directly contributes to the profitability of pig farms, and pregnancy diagnosis is an important factor in breeding management. Therefore, the need to diagnose pregnancy in sows is emphasized, and various studies have been conducted in this area. We propose a computer-aided diagnosis system to assist livestock farmers to diagnose sow pregnancy through ultrasound. Methods for diagnosing pregnancy in sows through ultrasound include the Doppler method, which measures the heart rate and pulse status, and the echo method, which diagnoses by amplitude depth technique. We propose a method that uses deep learning algorithms on ultrasonography, which is part of the echo method. As deep learning-based classification algorithms, Inception-v4, Xception, and EfficientNetV2 were used and compared to find the optimal algorithm for pregnancy diagnosis in sows. Gaussian and speckle noises were added to the ultrasound images according to the characteristics of the ultrasonography, which is easily affected by noise from the surrounding environments. Both the original and noise added ultrasound images of sows were tested together to determine the suitability of the proposed method on farms. The pregnancy diagnosis performance on the original ultrasound images achieved 0.99 in accuracy in the highest case and on the ultrasound images with noises, the performance achieved 0.98 in accuracy. The diagnosis performance achieved 0.96 in accuracy even when the intensity of noise was strong, proving its robustness against noise.