Induced endometrial inflammation compromises conceptus development in dairy cattle†.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26 days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Bulls fed a high-gain diet decrease blastocyst formation after in vitro fertilization.

In brief: Paternal high-gain diet reduces blastocyst development following in vitro fertilization and embryo culture but does not affect gene expression or cellular allocation of resultant blastocysts. Abstract: Bulls used in cattle production are often overfed to induce rapid growth, early puberty, and increase sale price. While the negative consequences of undernutrition on bull sperm quality are known, it is unclear how a high-gain diet influences embryo development. We hypothesized that semen collected from bulls fed a high-gain diet would have a reduced capacity to produce blastocysts following in vitro fertilization. Eight mature bulls were stratified by body weight and fed the same diet for 67 days at either a maintenance level (0.5% body weight per day; n = 4) or a high-gain rate (1.25% body weight per day; n = 4). Semen was collected by electroejaculation at the end of the feeding regimen and subjected to sperm analysis, frozen, and used for in vitro fertilization. The high-gain diet increased body weight, average daily gain, and subcutaneous fat thickness compared to the maintenance diet. Sperm of high-gain bulls tended to have increased early necrosis and had increased post-thaw acrosome damage compared with maintenance bulls, but diet did not affect sperm motility or morphology. Semen of high-gain bulls reduced the percentage of cleaved oocytes that developed to blastocyst stage embryos. Paternal diet had no effect on the number of total or CDX2-positive cells of blastocysts, or blastocysts gene expression for markers associated with developmental capacity. Feeding bulls a high-gain diet did not affect sperm morphology or motility, but increased adiposity and reduced the ability of sperm to generate blastocyst-stage embryos.

Prepartum heat stress in dairy cows increases postpartum inflammatory responses in blood of lactating dairy cows.

Uterine diseases and heat stress (HS) are major challenges for the dairy cow. Heat stress alters host immune resilience, making cows more susceptible to the development of uterine disease. Although HS increases the incidence of uterine disease, the mechanisms by which this occurs are unclear. We hypothesize that evaporative cooling (CL) to alleviate HS in prepartum cows has carry-over effects on postpartum innate immunity. Nulliparous pregnant Holstein heifers were assigned to receive either forced CL that resulted in cool conditions (shade with water soakers and fans; n = 14) or to remain under HS conditions (barn shade only; n = 16) for 60 d prepartum. Postpartum, all cows were housed in a freestall barn equipped with shade, water soakers, and fans. Respiratory rate and rectal temperature during the prepartum period were greater in HS heifers compared with CL heifers, indicative of HS. Although milk production was decreased in HS cows compared with CL cows, the incidence of uterine disease and content of total or pathogenic bacteria in vaginal mucus on d 7 or d 21 postpartum was not affected by treatment. Whole blood was collected on d 21 and subjected to in vitro stimulation with lipopolysaccharide. Lipopolysaccharide-induced accumulation of IL-1ß, IL-10, and MIP-1α was greater in blood collected from HS cows compared with CL cows. Our results imply that prepartum HS during late pregnancy has carry-over effects on postpartum innate immunity, which may contribute to the increased incidence of uterine disease observed in cows exposed to prepartum HS.

Inflammatory responses of bovine endometrial epithelial cells are increased under in vitro heat stress conditions.

Cattle exposed to heat stress have reduced fertility, reduced milk production and increased incidence of postpartum uterine infection. Heat stress is suggested to alter immune function of cattle; however, the mechanisms underlying heat stress mediated uterine infection are unknown. We hypothesized that exposure of endometrial cells to heat stress would further increase expression of inflammatory mediators in response to bacterial components due to altered heat-shock protein expression. Bovine endometrial epithelial cells (BEND) were exposed to Escherichia coli lipopolysaccharide (LPS) or a synthetic triacylated lipopeptide (Pam3CSK4) under heat stress (41.0 °C) or thermoneutral (38.5 °C) conditions for 24 h. Exposure of BEND cells to LPS or Pam3CSK4 increased the expression of the proinflammatory mediators IL1B, IL6, and CXCL8 compared to control medium. However, exposure of BEND cells to heat stress increased LPS and Pam3CSK4 induced expression of IL1B compared to cells exposed to thermoneutral conditions, and expression of LPS induced IL6 was also increased when BEND cells were exposed to heat stress. To determine if heat shock proteins increased BEND cell expression of inflammatory mediators, HSP1A1 and HSF1 were targeted by siRNA knock down. Expression of HSP1A1 and HSF1 were reduced following siRNA knockdown; however, knockdown of HSP1A1 or HSF1 further increased heat stress mediated increased expression of inflammatory mediators. These data suggest that heat stress increased BEND cell inflammatory responses to bacterial components, while heat shock proteins HSP1A1 and HSF1 help to restrain inflammatory responses. These mechanisms may contribute to the increased incidence of uterine infection observed in cows under heat stress conditions.

Cholesterol supports bovine granulosa cell inflammatory responses to lipopolysaccharide.

In brief: Bovine granulosa cells need to be cultured with serum to generate inflammation in response to bacterial lipopolysaccharide. This study shows that it is cholesterol that facilitates this lipopolysaccharide-stimulated cytokine secretion. Abstract: During bacterial infections of the bovine uterus or mammary gland, ovarian granulosa cells mount inflammatory responses to lipopolysaccharide (LPS). In vitro, LPS stimulates granulosa cell secretion of the cytokines IL-1α and IL-1ß and the chemokine IL-8. These LPS-stimulated inflammatory responses depend on culturing granulosa cells with serum, but the mechanism is unclear. Here, we tested the hypothesis that cholesterol supports inflammatory responses to LPS in bovine granulosa cells. We used granulosa cells isolated from 4 to 8 mm and >8.5 mm diameter ovarian follicles and manipulated the availability of cholesterol. We found that serum or follicular fluid containing cholesterol increased LPS-stimulated secretion of IL-1α and IL-1ß from granulosa cells. Conversely, depleting cholesterol using methyl-ß-cyclodextrin diminished LPS-stimulated secretion of IL-1α, IL-1ß and IL-8 from granulosa cells cultured in serum. Follicular fluid contained more high-density lipoprotein cholesterol than low-density lipoprotein cholesterol, and granulosa cells expressed the receptor for high-density lipoprotein, scavenger receptor class B member 1 (SCARB1). Furthermore, culturing granulosa cells with high-density lipoprotein cholesterol, but not low-density lipoprotein or very low-density lipoprotein cholesterol, increased LPS-stimulated inflammation in granulosa cells. Cholesterol biosynthesis also played a role in granulosa cell inflammation because RNAi of mevalonate pathway enzymes inhibited LPS-stimulated inflammation. Finally, treatment with follicle-stimulating hormone, but not luteinising hormone, increased LPS-stimulated granulosa cell inflammation, and follicle-stimulating hormone increased SCARB1 protein. However, changes in inflammation were not associated with changes in oestradiol or progesterone secretion. Taken together, these findings imply that cholesterol supports inflammatory responses to LPS in granulosa cells.

Oxysterols protect bovine endometrial cells against pore-forming toxins from pathogenic bacteria.

Many species of pathogenic bacteria secrete toxins that form pores in mammalian cell membranes. These membrane pores enable the delivery of virulence factors into cells, result in the leakage of molecules that bacteria can use as nutrients, and facilitate pathogen invasion. Inflammatory responses to bacteria are regulated by the side-chain-hydroxycholesterols 27-hydroxycholesterol and 25-hydroxycholesterol, but their effect on the intrinsic protection of cells against pore-forming toxins is unclear. Here, we tested the hypothesis that 27-hydroxycholesterol and 25-hydroxycholesterol help protect cells against pore-forming toxins. We treated bovine endometrial epithelial and stromal cells with 27-hydroxycholesterol or 25-hydroxycholesterol, and then challenged the cells with pyolysin, which is a cholesterol-dependent cytolysin from Trueperella pyogenes that targets these endometrial cells. We found that treatment with 27-hydroxycholesterol or 25-hydroxycholesterol protected both epithelial and stomal cells against pore formation and the damage caused by pyolysin. The oxysterols limited pyolysin-induced leakage of potassium and lactate dehydrogenase from cells, and reduced cytoskeletal changes and cytolysis. This oxysterol cytoprotection against pyolysin was partially dependent on reducing cytolysin-accessible cholesterol in the cell membrane and on activating liver X receptors. Treatment with 27-hydroxycholesterol also protected the endometrial cells against Staphylococcus aureus α-hemolysin. Using mass spectrometry, we found 27-hydroxycholesterol and 25-hydroxycholesterol in uterine and follicular fluid. Furthermore, epithelial cells released additional 25-hydroxycholesterol in response to pyolysin. In conclusion, both 27-hydroxycholesterol and 25-hydroxycholesterol increased the intrinsic protection of bovine endometrial cells against pore-forming toxins. Our findings imply that side-chain-hydroxycholesterols may help defend the endometrium against pathogenic bacteria.

Manipulating bovine granulosa cell energy metabolism limits inflammation.

Bovine granulosa cells are often exposed to energy stress, due to the energy demands of lactation, and exposed to lipopolysaccharide from postpartum bacterial infections. Granulosa cells mount innate immune responses to lipopolysaccharide, including the phosphorylation of mitogen-activated protein kinases and production of pro-inflammatory interleukins. Cellular energy depends on glycolysis, and energy stress activates intracellular AMPK (AMP-activated protein kinase), which in turn inhibits mTOR (mechanistic target of rapamycin). Here, we tested the hypothesis that manipulating glycolysis, AMPK or mTOR to mimic energy stress in bovine granulosa cells limits the inflammatory responses to lipopolysaccharide. We inhibited glycolysis, activated AMPK or inhibited mTOR in granulosa cells isolated from 4-8mm and from > 8.5 mm diameter ovarian follicles, and then challenged the cells with lipopolysaccharide and measured the production of interleukins IL-1α, IL-1ß, and IL-8. We found that inhibiting glycolysis with 2-deoxy-d-glucose reduced lipopolysaccharide-stimulated IL-1α > 80%, IL-1ß > 90%, and IL-8 > 65% in granulosa cells from 4-8 mm and from > 8.5 mm diameter ovarian follicles. Activating AMPK with AICAR also reduced lipopolysaccharide-stimulated IL-1α > 60%, IL-1ß > 75%, and IL-8 > 20%, and shortened the duration of lipopolysaccharide-stimulated phosphorylation of the mitogen-activated protein kinase ERK1/2 and JNK. However, only the mTOR inhibitor Torin 1, and not rapamycin, reduced lipopolysaccharide-stimulated IL-1α and IL-1ß. In conclusion, manipulating granulosa cell energy metabolism with a glycolysis inhibitor, an AMPK activator, or an mTOR inhibitor, limited inflammatory responses to lipopolysaccharide. Our findings imply that energy stress compromises ovarian follicle immune defences.

Experimentally Induced Endometritis Impairs the Developmental Capacity of Bovine Oocytes†.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Uterine infection alters the transcriptome of the bovine reproductive tract three months later.

Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract 3 months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles >8 mm diameter were profiled by RNA sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, 3 months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.

MILK Symposium review: Identifying constraints, opportunities, and best practices for improving milk production in market-oriented dairy farms in Sri Lanka.

Dairy is the most important subsector in the Sri Lankan livestock industry, due to the need to address the growing demand for fresh milk and milk products, and because of its potential influence on the rural economy. The USDA Food for Progress program awarded a 4.5-year Market-Oriented Dairy project to International Executive Service Corps, a not-for-profit organization based in Washington, DC. The objective of the Market-Oriented Dairy project is to support Sri Lanka's dairy sector and catalyze sustainable growth by strengthening the dairy sector through better technological, financial, and management practices benefiting all stakeholders and consumers along the dairy value chain. The University of Florida is working with International Executive Service Corps as technical experts in conducting dairy value chain assessments, identifying gaps and challenges in dairy management practices, extension services, milk quality management standards, and artificial insemination services. Assessment of the dairy value chain in 2018 identified a lack of good quality and quantity of feed, along with poor dairy management practices and ineffective extension services as major constraints to improving dairy productivity in Sri Lanka. In addition, lack of national milk quality standards that are consistent with international benchmarks and inadequate cooling facilities are significant challenges to improving milk quality. The nutritional status of cows is not suitable for optimal reproductive performance, compromising the success of artificial insemination in Sri Lanka. Based on these findings, we developed a dairy assessment tool and provided comprehensive training sessions targeting extension agents, veterinarians, and farmers to promote best practices in dairy management. Beyond training, however, industry support for standardization and monitoring of milk and feed quality are needed, providing opportunities for private investment to support the dairy industry. Similar opportunities are available for forage production and delivery to producers. The broader aim of the Market-Oriented Dairy project intervention is to reduce Sri Lanka's dependency on imported milk and contribute toward the goal of a safe, self-sufficient fresh milk supply.

Seminal plasma modulates expression of endometrial inflammatory meditators in the bovine†.

Seminal plasma has conventionally been viewed as a transport and survival medium for mammalian sperm; however, its role now extends beyond this process to actively targeting female tissues. Studies in rodents, swine, and humans demonstrate that seminal plasma induces molecular and cellular changes within the endometrium or cervix following insemination. Seminal-plasma-induced alterations to the maternal environment have been theorized to facilitate embryo development, modulate maternal immunity toward the conceptus, and potentially improve pregnancy success. It is unknown if bovine seminal plasma modulates the uterine environment following insemination in the cow, where routine use of artificial insemination reduces maternal exposure to seminal plasma. We hypothesize that seminal plasma modulates the expression of inflammatory mediators in the endometrium, altering the maternal environment of early pregnancy. In vitro, seminal plasma altered intact endometrial explant expression of CSF2, IL1B, IL6, IL17A, TGFB1, IFNE, PTGS2, and AKR1C4. Furthermore, endometrial epithelial cell CSF2, CXCL8, TGFB1, PTGS2, and AKR1C4 expression were increased after seminal plasma exposure, while endometrial stromal cell CSF2, IL1B, IL6, CXCL8, IL17A, TGFB1, PTGS2, and AKR1C4 expression were increased following seminal plasma exposure. Endometrial expression of IL1B was increased in the cow 24 h after uterine infusion of seminal plasma, while other evaluated inflammatory mediators remained unchanged. These data indicate that seminal plasma may induce changes in the bovine endometrium in a temporal manner. Understanding the role of seminal plasma in modulating the maternal environment may aid in improving pregnancy success in cattle.

Effect of seminal plasma or transforming growth factor on bovine endometrial cells.

Semen induces post-coital inflammation of the endometrium in several species. Post-coital inflammation is proposed to alter the endometrial environment of early pregnancy, mediate embryonic development and modulate the maternal immune response to pregnancy. In cattle, it is common for pregnancies to occur in the absence of whole semen due to the high utilization of artificial insemination. Here, we have utilized a cell culture system to characterize semen-induced expression of inflammatory mediators in bovine endometrial cells and test the efficacy of transforming growth factor beta as the active agent in mediating any such change. We hypothesize that seminal plasma-derived transforming growth factor beta increases the expression of inflammatory mediators in bovine endometrial cells. Initially, we describe a heat-labile cytotoxic effect of seminal plasma on BEND cells, and a moderate increase in IL1B and IL6 expression. In addition, we show that transforming growth factor beta is present in bovine semen and can increase the expression of endometrial IL6, whereas blocking transforming growth factor beta in semen ameliorates this effect. However, intra-uterine infusion of seminal plasma, sperm or transforming growth factor beta did not alter the endometrial expression of inflammatory mediators. We conclude that bovine semen can modulate endometrial gene expression in vitro, which is partially due to the presence of transforming growth factor beta. It is likely that additional, unidentified, bioactive molecules in semen can alter the endometrial environment. Characterizing bioactive molecules in bovine semen may lead to the development of additives to improve artificial insemination in domestic species.

Persistent effects on bovine granulosa cell transcriptome after resolution of uterine disease.

Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis, we collected the follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes were ITLN1, NCF2, CLRN3, FSIP2 and ANKRD17, and the most downregulated genes were ACSM1, NR4A2, GHITM, CBARP and NR1I3. Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell-cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NFκB, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease.

Lipopolysaccharide and tumor necrosis factor-alpha alter gene expression of oocytes and cumulus cells during bovine in vitro maturation.

Communication between the oocyte and cumulus facilitates oocyte growth, cell cycle regulation, and metabolism. This communication is mediated by direct contact between oocytes and cumulus cells, and soluble secreted molecules. Secreted molecules involved in this process are known inflammatory mediators. Lipopolysaccharide (LPS) is detected in follicular fluid and is associated with reduced fertility, whereas accumulation of inflammatory mediators in follicular fluid, including tumor necrosis factor-α (TNF-α), is associated with female infertility. Maturation of oocytes in the presence of LPS or TNF-α reduces meiotic maturation and the capacity to develop to the blastocyst. Here we evaluated the abundance of 92 candidate genes involved immune function, epigenetic modifications, embryo development, oocyte secreted factors, apoptosis, cell cycle, and cell signaling in bovine cumulus cells or zona-free oocytes after exposure to LPS or TNF-α during in vitro maturation. We hypothesize that LPS or TNF-α will alter the abundance of transcripts in oocytes and cumulus cell in a cell type dependent manner. Exposure to LPS altered abundance of 31 transcripts in oocytes (including ACVR1V, BMP15, DNMT3A) and 12 transcripts in cumulus cells (including AREG, FGF4, PIK3IP1). Exposure to TNF-α altered 1 transcript in oocytes (IGF2) and 4 transcripts in cumulus cells (GJA1, PLD2, PTGER4, STAT1). Cumulus expansion was reduced after exposure to LPS or TNF-α. Exposing COCs to LPS had a marked effect on expression of targeted transcripts in oocytes. We propose that altered oocyte transcript abundance is associated with reduced meiotic maturation and embryo development observed in oocytes cultured in LPS or TNF-α.

A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes.

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.

Corrigendum to: Paternal priming of maternal tissues to optimise pregnancy success.

The question of 'how does the allogeneic fetus survive gestation in the face of the maternal immune system?' has yet to be definitively answered. Several acceptable mechanisms exist to facilitate survival of the semi-allogeneic fetus in various species; paramount is the immunological separation of maternal and fetal tissues during gestation. However, keen observation of the maternal immune system during pregnancy has noted maternal immune tolerance to paternal-specific antigens. A mechanism by which the maternal immune system tolerates specific paternal antigens expressed on the fetus would be far more beneficial than the previously proposed immune indolence that would leave the mother susceptible to infection. In species like human or rodent, implantation occurs days after fertilisation and, as such, the mechanisms to establish antigen-specific tolerance must be initiated very early during pregnancy. We and others propose that these mechanisms are initiated at the time of insemination when paternal antigens are first introduced to the maternal immune system. Indeed, a new paradigm demonstrating the importance of paternal-maternal communication at the time of insemination is becoming evident as it relates to maternal tolerance to fetal antigen and ultimately pregnancy success.

Paternal priming of maternal tissues to optimise pregnancy success.

The question of 'how does the allogeneic fetus survive gestation in the face of the maternal immune system?' has yet to be definitively answered. Several acceptable mechanisms exist to facilitate survival of the semi-allogeneic fetus in various species; paramount is the immunological separation of maternal and fetal tissues during gestation. However, keen observation of the maternal immune system during pregnancy has noted maternal immune tolerance to paternal-specific antigens. A mechanism by which the maternal immune system tolerates specific paternal antigens expressed on the fetus would be far more beneficial than the previously proposed immune indolence that would leave the mother susceptible to infection. In species like human or rodent, implantation occurs days after fertilisation and, as such, the mechanisms to establish antigen-specific tolerance must be initiated very early during pregnancy. We and others propose that these mechanisms are initiated at the time of insemination when paternal antigens are first introduced to the maternal immune system. Indeed, a new paradigm demonstrating the importance of paternal-maternal communication at the time of insemination is becoming evident as it relates to maternal tolerance to fetal antigen and ultimately pregnancy success.

Maternal tract factors contribute to paternal seminal fluid impact on metabolic phenotype in offspring.

Paternal characteristics and exposures influence physiology and disease risks in progeny, but the mechanisms are mostly unknown. Seminal fluid, which affects female reproductive tract gene expression as well as sperm survival and integrity, provides one potential pathway. We evaluated in mice the consequences for offspring of ablating the plasma fraction of seminal fluid by surgical excision of the seminal vesicle gland. Conception was substantially impaired and, when pregnancy did occur, placental hypertrophy was evident in late gestation. After birth, the growth trajectory and metabolic parameters of progeny were altered, most profoundly in males, which exhibited obesity, distorted metabolic hormones, reduced glucose tolerance, and hypertension. Altered offspring phenotype was partly attributable to sperm damage and partly to an effect of seminal fluid deficiency on the female tract, because increased adiposity was also evident in adult male progeny when normal two-cell embryos were transferred to females mated with seminal vesicle-excised males. Moreover, embryos developed in female tracts not exposed to seminal plasma were abnormal from the early cleavage stages, but culture in vitro partly alleviated this. Absence of seminal plasma was accompanied by down-regulation of the embryotrophic factors Lif, Csf2, Il6, and Egf and up-regulation of the apoptosis-inducing factor Trail in the oviduct. These findings show that paternal seminal fluid composition affects the growth and health of male offspring, and reveal that its impact on the periconception environment involves not only sperm protection but also indirect effects on preimplantation embryos via oviduct expression of embryotrophic cytokines.

Evaluating lipopolysaccharide-induced oxidative stress in bovine granulosa cells.

PURPOSE: The purpose of this study was to evaluate the capacity of bovine granulosa cells to generate reactive oxygen intermediates in response to lipopolysaccharide. We hypothesized that granulosa cells increase reactive oxygen intermediates in response to Gram-negative lipopolysaccharide in a similar manner to immune cells. METHODS: Bovine peripheral blood mononuclear cells and granulosa cells were cultured in the presence of lipopolysaccharide. Oxidative stress was evaluated using the fluorescent marker dye CellROX, and oxidative stress-related genes were measured using real-time RT-PCR. RESULTS: As expected, peripheral blood mononuclear cells increased oxidative stress in response to lipopolysaccharide as measured by accumulation of the fluorescent marker dye CellROX. While granulosa cells demonstrate the capacity to increase accumulation of CellROX dye in response to a positive control menadione, lipopolysaccharide had no effect on accumulation of CellROX dye. The expression of GSR, SOD1, and SOD2 were variable in peripheral blood mononuclear cells treated with lipopolysaccharide but were consistently upregulated when co-incubated with the antioxidant, N-acetyl cysteine. The expression of oxidative stress-related genes was not altered in granulosa cells, with the exception of elevated SOD1 following lipopolysaccharide exposure in the absence of antioxidant. CONCLUSIONS: Combined, these data suggest that while reactive stress is important in pathogen killing and inflammation in immune cells, granulosa cells do not increase oxidative stress in response to lipopolysaccharide.

A role for seminal plasma in modulating pregnancy outcomes in domestic species.

Seminal plasma is a complex fluid produced by the accessory glands of the male reproductive tract. Seminal plasma acts primarily as a transport medium for sperm on its arduous journey through the male and then female reproductive tract following ejaculation. This spermatozoan expedition will hopefully result in the meeting of and resultant fertilization of an oocyte, perpetuating the genetic lineage of both sexes. Whereas seminal plasma has historically been perceived as only a transport medium providing a nutrient-rich fluid environment for sperm during this exchange of genetic material, new insights into a complex communication pathway between males and females has been unraveled in the past 30 years. This new research suggests seminal plasma as a method to promote early pregnancy success by modulating cellular and molecular adaptions of the maternal environment required to facilitate healthy, successful pregnancy outcomes. Whereas much work on this exciting new communication process has focused on mice and translation to human reproduction, here we review the current evidence in domestic species where artificial insemination in the absence of seminal plasma is routine. Improving artificial insemination in domestic species to optimize offspring health and productivity could have far-reaching impacts on agriculturally relevant species such as cattle, sheep, pigs and horses.