Immunoglobulin Glycosylation - An Unexploited Potential for Immunomodulatory Strategies in Farm Animals.

The function of antibodies, namely the identification and neutralization of pathogens, is mediated by their antigen binding site (Fab). In contrast, the subsequent signal transduction for activation of the immune system is mediated by the fragment crystallizable (Fc) region, which interacts with receptors or other components of the immune system, such as the complement system. This aspect of binding and interaction is more precise, readjusted by covalently attached glycan structures close to the hinge region of immunoglobulins (Ig). This fine-tuning of Ig and its actual state of knowledge is the topic of this review. It describes the function of glycosylation at Ig in general and the associated changes due to corresponding glycan structures. We discuss the functionality of IgG glycosylation during different physiological statuses, like aging, lactation and pathophysiological processes. Further, we point out what is known to date about Ig glycosylation in farm animals and how new achievements in vaccination may contribute to improved animal welfare.

Prebiotic Supplementation During Pregnancy Modifies the Gut Microbiota and Increases Metabolites in Amniotic Fluid, Driving a Tolerogenic Environment In Utero.

The gut microbiota is influenced by environmental factors such as food. Maternal diet during pregnancy modifies the gut microbiota composition and function, leading to the production of specific compounds that are transferred to the fetus and enhance the ontogeny and maturation of the immune system. Prebiotics are fermented by gut bacteria, leading to the release of short-chain fatty acids that can specifically interact with the immune system, inducing a switch toward tolerogenic populations and therefore conferring health benefits. In this study, pregnant BALB/cJRj mice were fed either a control diet or a diet enriched in prebiotics (Galacto-oligosaccharides/Inulin). We hypothesized that galacto-oligosaccharides/inulin supplementation during gestation could modify the maternal microbiota, favoring healthy immune imprinting in the fetus. Galacto-oligosaccharides/inulin supplementation during gestation increases the abundance of Bacteroidetes and decreases that of Firmicutes in the gut microbiota, leading to increased production of fecal acetate, which was found for the first time in amniotic fluid. Prebiotic supplementation increased the abundance of regulatory B and T cells in gestational tissues and in the fetus. Interestingly, these regulatory cells remained later in life. In conclusion, prebiotic supplementation during pregnancy leads to the transmission of specific microbial and immune factors from mother to child, allowing the establishment of tolerogenic immune imprinting in the fetus that may be beneficial for infant health outcomes.

Toll-like receptor-4 null mutation causes fetal loss and fetal growth restriction associated with impaired maternal immune tolerance in mice.

Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4-/-) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4-/- mice. CD4+ T cells were expanded after mating in Tlr4+/+ but not Tlr4-/- mice, with failure to expand peripheral CD25+FOXP3+ NRP1- or thymic CD25+FOXP3+ NRP1+ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.

Toll-like receptor signaling is changed in ovine lymph node during early pregnancy.

Toll-like receptors (TLRs) participate in regulation of adaptive immune responses, and lymph nodes play key roles in the initiation of immune responses. There is a tolerance to the allogenic fetus during pregnancy, but it is unclear that expression of TLR signaling is in ovine lymph node during early pregnancy. In this study, lymph nodes were sampled from day 16 of nonpregnant ewes and days 13, 16, and 25 of pregnant ewes, and the expressions of TLR family (TLR2, TLR3, TLR4, TLR5 and TLR9), adaptor proteins, including myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), and interleukin-1-receptor-associated kinase 1 (IRAK1), were analyzed through real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry analysis. The results showed that mRNA and protein levels of TLR2, TLR3, TLR4, TRAF6, and MyD88 were upregulated in the maternal lymph node, but TLR5, TLR9, and IRAK1 were downregulated during early pregnancy. In addition, MyD88 protein was located in the subcapsular sinus and lymph sinuses. Therefore, it is suggested that early pregnancy induces changes in TLR signaling in maternal lymph node, which may be involved in regulation of maternal immune responses in sheep.

Kisspeptin/Kiss1r system and angiogenic and immunological mediators at the maternal-fetal interface of domestic cats.

The Kisspeptin/Kiss1r system is a key regulator of reproduction by stimulating gonadotrophin-releasing hormone and luteinizing hormone release, and in vitro studies have shown that Kisspeptin can modulate angiogenesis and immune function, factors that are also essential for reproduction However, there are no studies on the expression of Kisspeptin/Kiss1r at the maternal-fetal interface in domestic cats and its relationship with angiogenic and immunological mediators. Thus, our objective was to evaluate the spatiotemporal expression profile of Kisspeptin/Kiss1r and angiogenic and immunological mediators in the uterus and placenta of domestic cats during pregnancy. Uterus and placenta samples were collected from cats in mid pregnancy (N = 6) and late pregnancy (N = 6), in addition to uterus from non-pregnant cats in diestrus (N = 7), to evaluate protein and gene expression of kisspeptin (Kiss1), kisspeptin receptor (Kiss1r), vascular endothelial growth factor (VEGF), tyrosine kinase receptor (Flk-1), placental growth factor (PLGF), interferon gamma (INFγ), migration inhibiting factor (MIF), tumor necrosis factor (TNFα), interleukins (IL6 and IL10) by immunohistochemistry and quantitative polymerase chain reaction. Pregnancy increased the uterine expression of Kiss1 and Kiss1r, especially at the late pregnancy, in addition to upregulating INFy, MIF, Vegf, Il10, and Tnf and downregulating Plgf. Higher placental expression of Kiss1r and Plgf mRNA occurred at the late pregnancy, while the expression of Kiss1, VEGF, Flk-1, INFy, TNFα, Il6, and IL10 was higher in the mid of pregnancy. A positive correlation between Kiss1 and Tnf was observed in the placenta, while Kiss1r had a negative correlation with Infγ, Il6, and Il10. The findings reveal that Kisspeptin/Kiss1r and angiogenic and immunological mediators at the maternal-fetal interface of pregnant cat have a gene correlation and are modulated by the gestational age. These data suggest possible functional links of Kisspeptin in placental angiogenesis and immunology.

ß3-Adrenoceptors as Putative Regulator of Immune Tolerance in Cancer and Pregnancy.

Understanding the mechanisms of immune tolerance is currently one of the most important challenges of scientific research. Pregnancy affects the immune system balance, leading the host to tolerate embryo alloantigens. Previous reports demonstrated that ß-adrenergic receptor (ß-AR) signaling promotes immune tolerance by modulation of NK and Treg, mainly through the activation of ß2-ARs, but recently we have demonstrated that also ß3-ARs induce an immune-tolerant phenotype in mice bearing melanoma. In this report, we demonstrate that ß3-ARs support host immune tolerance in the maternal microenvironment by modulating the same immune cells populations as recently demonstrated in cancer. Considering that ß3-ARs are modulated by oxygen levels, we hypothesize that hypoxia, through the upregulation of ß3-AR, promotes the biological shift toward a tolerant immunophenotype and that this is the same trick that embryo and cancer use to create an aura of immune-tolerance in a competent immune environment. This study confirms the analogies between fetal development and tumor progression and suggests that the expression of ß3-ARs represents one of the strategies to induce fetal and tumor immune tolerance.

Maternal Supplementation of Food Ingredient (Prebiotic) or Food Contaminant (Mycotoxin) Influences Mucosal Immune System in Piglets.

The early life period is crucial for the maturation of the intestinal barrier, its immune system, and a life-long beneficial host-microbiota interaction. The study aims to assess the impact of a beneficial dietary (short-chain fructooligosaccharides, scFOS) supplementation vs. a detrimental dietary environment (such as mycotoxin deoxynivalenol, DON) on offspring intestinal immune system developmental profiles. Sows were given scFOS-supplemented or DON-contaminated diets during the last 4 weeks of gestation, whereas force-feeding piglets with DON was performed during the first week of offspring life. Intestinal antigen-presenting cell (APC) subset frequency was analyzed by flow cytometry in the Peyer's patches and in lamina propria and the responsiveness of intestinal explants to toll-like receptor (TLR) ligands was performed using ELISA and qRT-PCR from post-natal day (PND) 10 until PND90. Perinatal exposure with scFOS did not affect the ontogenesis of APC. While it early induced inflammatory responses in piglets, scFOS further promoted the T regulatory response after TLR activation. Sow and piglet DON contamination decreased CD16+ MHCII+ APC at PND10 in lamina propria associated with IFNγ inflammation and impairment of Treg response. Our study demonstrated that maternal prebiotic supplementation and mycotoxin contamination can modulate the mucosal immune system responsiveness of offspring through different pathways.

Effects of early pregnancy on expression of interferon-stimulated gene 15, STAT1, OAS1, MX1, and IP-10 in ovine liver.

Interferon-tau (IFNT) regulates maternal recognition during early pregnancy in ruminants. The liver can serve as a hematopoietic organ, and it has immune functions. This study hypothesized whether mRNA and proteins of interferon-stimulated genes (ISGs) induced by early pregnancy are upregulated in maternal liver. Therefore, we determined the expression of interferon-stimulated gene 15-kDa protein (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance protein 1 (MX1), interferon-gamma-inducible protein 10 (IP-10), and signal transducer and activator of transcription 1 (STAT1) in maternal livers during early pregnancy in sheep. Ovine livers were sampled on day 16 of the estrous cycle, and days 13, 16, and 25 of pregnancy, and expression of ISGs was detected by quantitative real-time PCR, Western blot, and immunohistochemistry analysis. Our results showed that there were increases in expression of the mRNA and proteins of ISG15, OAS1, IP-10, STAT1, and MX1 during early pregnancy. STAT1 protein was limited to the hepatocytes, and endothelial cells of proper hepatic arteries and hepatic portal veins. In conclusion, the upregulation of ISG15, OAS1, IP-10, STAT1, and MX1 proteins may be implicated in maternal hepatic immune adjustment and other functions during early pregnancy in sheep.

Immunological memory and tolerance at the maternal-fetal interface: Implications for reproductive management of mares.

The development of placentation that coincided with the evolution of mammals presented new challenges to the transmission of life from one generation to the next, particularly with regard to the possibility of maternal immunological recognition and destruction of the developing conceptus. The balance between immunity and tolerance dominates the immunological relationship between mother and fetus during mammalian pregnancy, and the focal point of this relationship lies at the interface between the trophoblast cells that comprise the outermost layer of the placenta and the maternal endometrial tissues. Immune memory and tolerance are two of the cardinal characteristics of the immune system. Immune memory is essential in preventing or lessening the effect of infections to the mother or conceptus, but may also be a threat to the semi-allogeneic tissues of the fetus and placenta. The mother must develop functional immune tolerance to her fetus, but at the same time retain her ability to combat infections while pregnant. To address this imperative, mammals have developed overlapping and independent mechanisms for evading maternal anti-fetal immune responses that could result in pregnancy loss. Studies of the unusual component of equine invasive trophoblast in the epitheliochorial placenta have illuminated aspects of immune memory and tolerance that have relevance to fertility in the horse and other mammalian species.

Embryo-maternal communication in dogs: Immune system related factors.

In the bitch, establishment of pregnancy is believed to be mainly initiated by the free-floating embryo in the uterus that is under progesterone influence. As in other species, the active participation of the embryo is no longer questioned. Secretory products are transported to the embryo-maternal interface and contribute to extra-cellular matrix (ECM) degradation, a change in the intrauterine immune milieu towards a reduction of immune cells and a change in lymphocyte subsets, cell differentiation, angiogenesis, and the balance between proliferation and apoptosis. For cell-to-cell communication between embryo and maternal tissue, biomolecules inclusive microRNAs might be transported and exchanged via extracellular vesicles (EVs) as in other species. Maternal acceptance of the fetal allograft is vital for the establishment of pregnancy. Findings so far indicate that the embryo avoids attacks from the maternal system via passive and active mechanisms. One hypothesis is that expression or suppression of surface molecules help the canine embryo to hide from the maternal immune system on one side and to actively destroy cytotoxic immune cells on the other side; there are further clues that the canine embryo blocks activation of intrauterine leukocytes. Intracellular repair mechanisms via heat shock proteins (HSP) are candidates under investigation. The presence and function of immunomodulatory intrauterine cells like Treg cells and their interaction with the embryo have been intensely studied in other species but remains to be investigated in the canine preimplantation uterus.

Expression of melatonin receptors and CD4 in the ovine thymus, lymph node, spleen and liver during early pregnancy.

As a pineal gland hormone, melatonin acts through its receptors to modulate the immune system. The immune system is composed of primary and secondary organs, and immune organs are adapted to the presence of the fetal alloantigen during pregnancy. However, it is unclear whether melatonin affects maternal immune organs during early pregnancy in sheep. In this study, the ovine thymus, lymph node, spleen and liver were sampled at day 16 of the oestrous cycle, and at days 13, 16 and 25 of pregnancy. The expression of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2) and cluster of differentiation 4 (CD4) was detected by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry experiments. Our results showed that during early pregnancy there was an upregulation of MT1 mRNA and protein in the thymus, lymph node and liver, and there was a downregulation in the spleen. The expression of MT2 mRNA and protein was increased in the thymus but decreased in the spleen and liver, and there was no significant change in the lymph node during early pregnancy. CD4 protein was upregulated in the thymus, lymph node and liver, but there were no significant changes in the spleen during early pregnancy. In conclusion, early pregnancy induces tissue-specific expression of MT1, MT2 and CD4, which may be due to the different functions of the thymus, lymph node, spleen and liver. Further, melatonin is involved in immune regulation of the maternal thymus, lymph node, spleen and liver during early pregnancy in sheep.

Pregnancy status alters IL-21-mediated effects on murine B lymphocytes.

A favorable outcome of pregnancy depends greatly on an adequate balance of immune protection and fetal tolerance at the fetomaternal interface. IL-21 is a pro-inflammatory cytokine associated with altering immune responses in autoimmune diseases. IL-21 has pleiotropic functions, including induction of Th17 T cells, inhibition of Treg development, and modulation of antibody responses of B lymphocytes. Genetic polymorphisms of IL21 have been associated to poor pregnancy outcomes. However, the mechanism of IL-21 actions needs further evaluation. Here, we postulate that IL-21 affects splenic B cell function during pregnancy and shapes immune responses. We show that splenic B cells from CBA/J × BALB/c mice with favorable pregnancy outcome expressed lower IL21R levels than in CBA/J × DBA/2J mice, a mouse model for immune-induced bad pregnancy outcome. As a consequence, B cells from CBA/J × BALB/c mice reacted less sensitively to IL-21 than B cells from non-pregnant mice (NPM) or from CBA/J × DBA/2J mice. Also, LPS-induced apoptotic rates were altered in NPM and CBA/J × DBA/2J but not in CBA/J × BALB/c mice. This is accompanied by improved survival of B cells that produce the anti-inflammatory cytokine IL-10 upon stimulation with LPS. We also observed lower numbers of CD4+CXCR5+Bcl-6+ follicular T-helper cells (Tfh) in normal pregnant mice, compared to non-pregnant and mice with disturbed pregnancies. Our data indicate that alterations of the Tfh/IL-21/IL-10 axis may have important influence on pregnancy outcome.

Maternal plasma proteomics in a rat model of pregnancy complications reveals immune and pro-coagulant gene pathway activation.

PROBLEM: The Brown Norway (BN) rat is a model of T-helper 2 immune diseases, and also a model of pregnancy disorders that include placental insufficiency, fetal loss, and pre-eclampsia-like symptoms. The aim of this study was to investigate the plasma proteomic/cytokine profile of pregnant BN rats in comparison to that of the Lewis (LEW) rat strain. METHOD OF STUDY: Plasma proteomics differences were studied at day 13 of pregnancy in pooled plasma samples by differential in-gel electrophoresis, and protein identification was performed by mass spectrometry. Key protein findings and predicted cytokine differences were validated by ELISA using plasma from rats at various pregnancy stages. Proteomics data were used for ingenuity pathway analysis (IPA). RESULTS: In-gel analysis revealed 74 proteins with differential expression between BN and LEW pregnant dams. ELISA studies confirmed increased maternal plasma levels of complement 4, prothrombin, and C-reactive protein in BN compared to LEW pregnancies. LEW pregnancies showed higher maternal plasma levels of transthyretin and haptoglobin than BN pregnancies. Ingenuity pathway analysis revealed that BN pregnancies are characterized by activation of pro-coagulant, reactive oxygen species, and immune-mediated chronic inflammation pathways, and suggested increased interleukin 6 and decreased transforming growth factor-ß1 as potential upstream events. Plasma cytokine analysis revealed that pregnant BN dams have a switch from anti- to pro-inflammatory cytokines with the opposite switch observed in pregnant LEW dams. CONCLUSION: Brown Norway rats show a maternal pro-inflammatory response to pregnancy that likely contributes to the reproductive outcomes observed in this rat strain.

Immune Regulation in Eutherian Pregnancy: Live Birth Coevolved with Novel Immune Genes and Gene Regulation.

Novel regulatory elements that enabled expression of pre-existing immune genes in reproductive tissues and novel immune genes with pregnancy-specific roles in eutherians have shaped the evolution of mammalian pregnancy by facilitating the emergence of novel mechanisms for immune regulation over its course. Trade-offs arising from conflicting fitness effects on reproduction and host defenses have further influenced the patterns of genetic variation of these genes. These three mechanisms (novel regulatory elements, novel immune genes, and trade-offs) played a pivotal role in refining the regulation of maternal immune systems during pregnancy in eutherians, likely facilitating the establishment of prolonged direct maternal-fetal contact in eutherians without causing immunological rejection of the genetically distinct fetus.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

PROBLEM: Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. METHODS: Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. RESULTS: In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1ß in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1ß expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. CONCLUSION: P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

IL-1ß, IL-2 and IL-4 concentration during porcine gestation.

In pigs, given the type of epitheliochorial and non-invasive placenta, the trophoblast is in intimate contact with maternal tissues. The dialogue established between the conceptus and the endometrium involves, among others, the immune system, which minimizes the chances of rejection of the embryo and promotes the establishment of pregnancy. The aim of this work was to determine the concentration of IL-1ß, IL-2 and IL-4 in sera and in extracts of maternal and fetal placenta from sows of different gestational periods. Reproductive tracts from 23 crossbreed sows, between 30 and 114 days of gestation (dg), and from 8 non-pregnant sows were used. The concentration of the cytokines was determined by ELISA. IL-1ß, IL-2 and IL-4 demonstrated a similar pattern of concentration at the placental interface and serum; they were found elevated in tissues at 30 and 60-70 dg, and significantly decreased at term, period in which the cytokines were significantly increased in serum. These results show that IL-1ß, IL-2, and IL-4 are differentially modulated during pregnancy and at term, and suggest an important role of these cytokines in defining the proinflammatory stage of these periods.

Spatio-temporal distribution of eosinophils in the mouse uterus during peri-implantation period.

Embryo implantation is an immunologically paradoxical event. In humans and rodents, blastocysts adhere to uterine epithelium and then invade into endometrial stroma, while maternal body is protected from extraneous materials by its immune system. Eosinophils, a kind of leucocytes involving parasitic infections and allergic response, increase in number in uterus when serum estrogen level is elevated during estrus cycles. However, response of uterine eosinophils to ovarian estrogen during peri-implantation period is not clear. Therefore, we investigated the distribution of eosinophils in murine peri-implantation uterus. On day 0.5 of pregnancy, eosinophils were found primarily in endometrial stroma near the luminal epithelium, whereas they were primarily distributed in basal endometrium and myometrium on day 3.5 of pregnancy. The number of uterine eosinophils on day 4.5 of pregnancy was significantly increased by inhibition of maternal estrogen action. Collectively, our results indicate that the ovarian estrogen negatively regulates uterine eosinophil distribution during peri-implantation period and provide insight into a role of maternal immune system in embryo implantation.

Effect of single dose follicle stimulating hormone on follicular aspiration, in vitro fertilization and pregnancy rate / Efeito do hormônio folículo estimulante em dose única na aspiração folicular, fertilização in vitro e taxa de prenhez

The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in v itrofertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p<0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p>0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p>0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage.

O presente estudo avaliou vacas Brangus tratadas com doses únicas de hormônio folículo estimulante (FSH) submetidas a aspiração folicular após vinte e quatro horas, para avaliação da recuperação oocitária, fertilização in vitro e taxa de prenhez. Folículos superiores a três milímetros de diâmetro foram aspirados, 200 mg de FSH foram administrados dois dias depois e uma nova aspiração folicular foi realizada 24 horas após. Esses métodos foram efetivados três vezes a cada três dias. No controle, as aspirações foliculares ocorreram em intervalos de uma semana sem administração de FSH. Os oócitos aspirados foram avaliados, submetidos à fertilização in vitro e os embriões foram transferidos em receptoras. A recuperação média dos oócitos foi superior (p<0,05) nas vacas controle (12,4±1,8) do que nas vacas tratadas (9,4±1,3). Não houve diferença (p>0,05) na porcentagem média de oócitos viáveis (52,0±3,9 e 62,7±4,7%) ou na porcentagem média de embriões (41,4±4,8 e 41,5±4,2%) entre vacas controle e vacas tratadas, respectivamente. A porcentagem média de prenhez não diferiu (p>0,05) para as vacas controle (43,8±2,7%) e as tratadas (40,9±6,8%). Em conclusão, o tratamento com FSH não melhorou a recuperação de oócitos, a fertilização in vitro e o percentual de prenhez. No entanto, existe a possibilidade de várias aspirações foliculares consecutivas a cada três dias, concentrando a fertilização in vitro e o percentual de prenhez.

Expression of interferon-stimulated gene 15-kDa protein, cyclooxygenase (COX) 1, COX-2, aldo-keto reductase family 1, member B1, and prostaglandin E synthase in the spleen during early pregnancy in sheep.

The spleen is a unique lymphoid organ that plays a key role in immune regulation. Interferon-tau induces upregulation of interferon-stimulated gene 15-kDa protein (ISG15) in the uterus during early pregnancy in sheep. Prostaglandins (PGs) have important effects in both the activation and the inhibition of immune response through an autocrine and paracrine manner. In this study, splenic samples were obtained at Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy from ewes, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, PGE synthase (PTGES), and PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1), was detected through quantitative real-time PCR, western blot, and immunohistochemistry analysis. Our results showed that there was upregulation of COX-2 mRNA and protein at Day 25 of pregnancy, and ISG15 mRNA and conjugated proteins, and AKR1B1 mRNA and dimer at Days 16 and 25 of pregnancy. COX-2 and AKR1B1 proteins were limited to the capsule, trabeculae, and splenic cords. However, the expression of COX-1 and PTGES was not affected by early pregnancy. In conclusion, ISG15-conjugated proteins, COX-2 and AKR1B1 upregulated in maternal spleen during early pregnancy, which may be beneficial for the placentation in sheep.

Maternal supply of methionine during late pregnancy is associated with changes in immune function and abundance of microRNA and mRNA in Holstein calf polymorphonuclear leukocytes.

Pregnancy and early life are critical periods during which environmental factors such as nutrition can affect development. Rumen-protected methionine (Met; RPM) supplementation during the prepartum period improves not only performance but immune responses in dairy cows. We investigated the effects of enhanced maternal supply of Met via feeding RPM on whole-blood in vitro lipopolysaccharide (LPS; 0, 0.01, or 5 µg/mL of blood) challenge and targeted microRNA and mRNA abundance in calf blood polymorphonuclear leukocytes (PMNL). Calves (n = 12/maternal diet) born to cows fed RPM at 0.08% of diet dry matter (DM)/d (MET) for the last 21 ± 2 d before calving or fed a control diet with no added Met (CON) were used. The PMNL were isolated at birth (before colostrum feeding) and d 1 (24 h after colostrum intake), 14, 28, and 50 of age. Maternal blood was collected at -10 ± 1.3 d relative to calving. Cows in the MET group had greater DM intake and lower prepartal haptoglobin concentration. In CON cows, haptoglobin was positively correlated with proinflammatory and host-defense mRNA abundance in CON calves. Except for NOS2 and NFE2L2, abundance of CASP8, MPO, ZBP1, and TNF was lower at birth in MET calves. Interleukin 1ß concentration in response to LPS challenge in CON and MET calves was greatest at birth, underscoring the role of this cytokine for lymphocyte activation. Compared with 1 d of age, the interleukin-1ß response to incremental doses of LPS was greater at 14 through 28 d, suggesting that the neonatal calf can mount a robust response to inflammatory stimuli. Greater abundance in CON calves of NOS2, CADM1, and TLR2 coupled with lower SELL from 1 through 50 d of age suggested a chronic activation of the PMNL. There was a marked upregulation over time of MIR125b, MIR146a, MIR155, and MIR9 in both CON and MET calves, suggesting that these microRNA could affect gene transcription associated with differentiation and inflammatory function in PMNL. Regardless of maternal diet, the gradual downregulation of MIR223 (the most abundant microRNA in PMNL) is in line with the progressive increase over time in the proinflammatory signature of the PMNL. Data revealed the potential for maternal supply of Met during late pregnancy through either greater DM intake or Met to elicit some changes in PMNL function during early postnatal life, partly through changes in mRNA expression encompassing cell adhesion and chemotaxis, oxidative stress, Toll-like receptor signaling, and Met metabolism.