Uterine biology in pigs and sheep

There is a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus which must be established during the peri-implantation period for pregnancy recognition signaling, implantation, regulation of gene expression by uterine epithelial and stromal cells, placentation and exchange of nutrients and gases. The uterus provide a microenvironment in which molecules secreted by uterine epithelia or transported into the uterine lumen represent histotroph required for growth and development of the conceptus and receptivity of the uterus to implantation. Pregnancy recognition signaling mechanisms sustain the functional lifespan of the corpora lutea (CL) which produce progesterone, the hormone of pregnancy essential for uterine functions that support implantation and placentation required for a successful outcome of pregnancy. It is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. With proper placentation, the fetal fluids and fetal membranes each have unique functions to ensure hematotrophic and histotrophic nutrition in support of growth and development of the fetus. The endocrine status of the pregnant female and her nutritional status are critical for successful establishment and maintenance of pregnancy. This review addresses the complexity of key mechanisms that are characteristic of successful reproduction in sheep and pigs and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of livestock species.     

Uterine biology in pigs and sheep

ABSTRACT: There is a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus which must be established during the peri-implantation period for pregnancy recognition signaling, implantation, regulation of gene expression by uterine epithelial and stromal cells, placentation and exchange of nutrients and gases. The uterus provide a microenvironment in which molecules secreted by uterine epithelia or transported into the uterine lumen represent histotroph required for growth and development of the conceptus and receptivity of the uterus to implantation. Pregnancy recognition signaling mechanisms sustain the functional lifespan of the corpora lutea (CL) which produce progesterone, the hormone of pregnancy essential for uterine functions that support implantation and placentation required for a successful outcome of pregnancy. It is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. With proper placentation, the fetal fluids and fetal membranes each have unique functions to ensure hematotrophic and histotrophic nutrition in support of growth and development of the fetus. The endocrine status of the pregnant female and her nutritional status are critical for successful establishment and maintenance of pregnancy. This review addresses the complexity of key mechanisms that are characteristic of successful reproduction in sheep and pigs and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of livestock species.     

精液对母兔子宫免疫及孕向发育的影响

在结扎子宫颈的情况下,在同一子宫的两侧子宫角分别注入生理盐水（对照组）和精液（试验组）以研究精液对子宫免疫和孕向发育的影响。试验共用母兔8只。子宫注入后48 h处死试验用兔,取子宫进行测量,并制片染色,用图像采集分析系统进行研究。结果显示：1）与对照组相比,兔子宫注入精液后可以显著（P〈0.05）增加子宫的长度、直径和子宫角指数;可明显增加子宫壁（P〈0.05）、黏膜上皮层（P〈0.01）和黏膜层（P〈0.05）的厚度;可显著增加黏膜中腺管（P〈0.05）和血管（P〈0.05）数量,可明显增加腺上皮的厚度（P〈0.05）。2）子宫注入精液后,子宫冲洗液中的细胞数（53.3±4.03×109/L）较对照组极显著增加（P〈0.01）;单位面积子宫黏膜中肥大细胞的数量（2.85±0.05个/mm2）较对照组（3.72±0.04个/mm2）显著减少（P〈0.05）。子宫冲洗液涂片染色检查发现,子宫冲洗液中的细胞主要为嗜中性粒细胞。本研究表明,兔精液确实能够促进母兔子宫的孕向发育,能够改变子宫的免疫状态。     

Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus (embryo and its associated extra-embryonic membranes) to produce a hormone that acts on the uterus and/or corpus luteum (CL) to ensure maintenance of a functional CL for production of progesterone; the hormone required for pregnancy in most mammals. The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy. In ruminants, interferon tau (IFNT) is the pregnancy recognition signal. IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha (PGF) by uterine epithelia; therefore, the CL continues to produce progesterone required for pregnancy. Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy, but there is no evidence that they are involved in pregnancy recognition signaling. Rather, pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy. Estrogen, likely in concert with prolactin, prevents secretion of PGF into the uterine venous drainage (endocrine secretion), but maintains secretion of PGF into the uterine lumen (exocrine secretion) where it is metabolized to a form that is not luteolytic. Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis, functional CL are maintained for production of progesterone. In addition to effects of chorionic gonadotrophin, IFNT and estrogens to signal pregnancy recognition, these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus（embryo and its associated extraembryonic membranes） to produce a hormone that acts on the uterus and/or corpus luteum（CL） to ensure maintenance of a functional CL for production of progesterone;the hormone required for pregnancy in most mammals.The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy.In ruminants,interferon tau（IFNT） is the pregnancy recognition signal.IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha（PGF） by uterine epithelia;therefore,the CL continues to produce progesterone required for pregnancy.Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy,but there is no evidence that they are involved in pregnancy recognition signaling.Rather,pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy.Estrogen,likely in concert with prolactin,prevents secretion of PGF into the uterine venous drainage（endocrine secretion）,but maintains secretion of PGF into the uterine lumen（exocrine secretion） where it is metabolized to a form that is not luteolytic.Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis,functional CL are maintained for production of progesterone.In addition to effects of chorionic gonadotrophin,IFNT and estrogens to signal pregnancy recognition,these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.     

Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Detection of Coxiella burnetii, the agent of Q fever, in oviducts and uterine flushing media and in genital tract tissues of the non pregnant goat

The aim of the present study was the detection and quantification of Coxiella burnetii DNA in the flushing media (oviducts and uterine horns) and genital tract tissues of non pregnant goats from 20 goats chosen at random from 86 goats originating from 56 different breeding herds in south-west France. The serological prevalence rate of C. burnetii in the study population was 70.3%.The DNA of C. burnetii was identified using conventional PCR in the flushing media from the oviducts and uterus in 8/20 goats (40%) and in genital tract tissues (oviduct, uterus and ovary) in 5/20 goats (25%). This study clearly shows for the first time that the media used to flush the oviducts or uterine horns, collected using the standard embryo harvesting technique in goats, are susceptible to infection with C. burnetii. The 16 conventional PCR-positive samples were also analyzed using real-time PCR. The bacterial load of the oviduct and uterine flushing media varied from 2.9 × 10⁴ to 7.5 × 10⁶ bacteria per flushing medium, while the bacterial load of the tissue samples varied from 1.0 × 10² to 1.5 × 10⁵ bacteria per mg of tissue. The infection of genital tract flushing media and tissues is a risk factor for the transmission of C. burnetii from donor to recipient during embryo transfer or to the embryo and fetus when gestation is pursued to term.     

Antimicrobial susceptibilities and population structure of Staphylococcus epidermidis associated with ovine mastitis

Intramammary infections are a serious problem for dairy sheep farms, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 131 S. epidermidis isolates, collected from 2201 dairy Sarda sheep belonging to 14 flocks with high somatic cell count scores, were studied. The flocks were located in diverse geographical areas of Sardinia, Italy. The aim of study was to assess the susceptibility of isolates to 13 antimicrobial agents, many of which are frequently used in mastitis therapy. Oxacillin was used for detecting methicillin-resistant S. epidermidis (MRSE) by disk diffusion test. Thirty-eight percent of the isolates (n = 50) were resistant to penicillin, 7.6% (n = 10) were resistant to tetracycline, and 2.3% (n = 3) were resistant to both penicillin and tetracycline (PTRSE). Two isolates were resistant to five antimicrobials including methicillin. Analysis of staphylococcal cassette chromosome mec (SCCmec) elements showed that both MRSE isolates harbored SCCmec type IVa. Based on pulsed-field gel electrophoresis (PFGE) typing by SmaI macrorestriction, S. epidermidis isolates were grouped into four clusters at 75% similarity level. The two multi-drug resistant MRSE isolates displayed distinct PFGE patters. This study indicates that S. epidermidis isolates from sheep milk samples may accumulate resistance markers for different antimicrobial agents. Furthermore, the occurrence of PTRSE and MRSE suggests to adopt adequate hygienic measures when handling animals with intramammary infections, in order to prevent spreading PTRSE and MRSE strains to humans through direct contact and/or consumption of contaminated food.     

Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.     

Induction of interferon production in mice by delipidated whole cells and fractions of the aerobic corynebacterium, Corynebacterium catarrhalis

The i.v. or i.p. injection of delipidated whole cells of an aerobic Corynebacterium, designated as C. catarrahalis, resulted in the production of a low titre of interferon in mice. Two fractions, a particulate fraction, designated as CBA p40, and a soluble fraction, designated as CBA LS, could be isolated from the delipidated whole cells and were found to lead to the formation of more or less high interferon levels according to the route of administration. Furthermore, the CBA p40 fraction, when injected i.p., elicited the production of a maximum titre of interferon at the 24th hour (viral-like interferon).     

Climate and the epidemiology of gastrointestinal nematode infections of sheep in Europe

The free-living stages of gastrointestinal nematode parasites of sheep are strongly affected by climate. Thus, extreme heat and cold are detrimental to development and survival, while, within tolerable limits, increasing temperatures generally accelerate development but increase mortality. Moisture is needed for development and translation of larvae from faeces to pasture, and so rainfall is a limiting factor for transmission. Together, these factors underpin seasonal patterns of infection in sheep, as well as geographic variation in the epidemiology and relative importance of different species within Europe. Local knowledge and experience enable treatment to be targeted appropriately to prevent dangerous levels of infection. This traditional know-how can be supplemented by predictive epidemiological models, built on thorough understanding of the influence of climate on larval availability. However, management also has a dominant role in determining patterns of infection, and is itself influenced by climate. Current geographic variation in nematode epidemiology across Europe, and knowledge of systems from outside Europe, can provide only limited perspectives on the likely effects of climate change on disease in future. This is because disease arises from complex interaction between host and parasite factors, and the implementation of optimal control strategies to meet new challenges will be slowed by the inertia of current systems. Approaches to nematode control must therefore take account not only of parasite biology, but also the forces that shape sheep farming systems and management decisions.     

Effect of zinc supplementation in pregnant mice during experimental Trypanosoma cruzi infection

Zinc is an essential micronutrient and has significant effects on human growth, development, and immune function. Zinc supplementation or deficiency may affect the course of infection. Zinc enhances immune response against a wide range of viral, bacterial, and parasitic pathogens. In the present study, we investigated the effects of zinc sulphate (ZnSO₄) supplementation (20 mg/kg/day) during pregnancy in mice, Swiss Webster strain infected by the Y strain of Trypanosoma cruzi. Oral supplementation of zinc sulphate in pregnant and non-pregnant infected animals did not affect the count of blood parasites as well as tissue parasitism in the heart, liver, and spleen. Zinc supplementation did not alter female body weight, the length of fetuses and neonates, placental size/weight and mortality rate. Among zinc supplied animals, no significant plasmatic zinc concentrations were observed. Concerning to tissue zinc concentrations, only the liver displayed enhanced values as compared to other organs. For placental parasitism, zinc supplied group displayed a significant decrease in amastigote burdens (P < 0.05). However due to the reduced number of parasite burdens in placenta of animals supplied with zinc, these data suggest that zinc was partially effective in up-regulating the host’s immune response against parasite, probably attenuating the infection in fetuses.     

Expression and regulation of Foxa2 in the rat uterus during early pregnancy

The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.     

Molecular survey of Dirofilaria immitis and Dirofilaria repens by direct PCR for wild caught mosquitoes in the Republic of Korea

Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) x 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.     

Effects of time of progesterone supplementation on embryo development and interferon-tau production in the cow

We have investigated the effects of the timing of progesterone supplementation on early embryo development in mature, non-lactating Holstein-Friesian cows. Animals were inseminated 72 h (day 1) and 96 h following prostaglandin injection and were either left as untreated controls (n=6) or received progesterone supplementation from either days 5 to 9 (early; n=6) or from days 12 to 16 (late; n=6). Daily plasma samples were collected until day 16, when cows were slaughtered and reproductive tracts recovered and flushed to collect embryos and to measure interferon-tau activity. Both early and later progesterone supplementation resulted in marked increases in plasma progesterone (P<0.01). Early, but not late, progesterone supplementation resulted in a fourfold increase in trophoblast length (P<0.01) and a sixfold increase in uterine concentration of interferon-tau (P<0.05). The results demonstrate that progesterone supplementation during the postovulatory rise, but not later in the luteal phase, increases embryo development and interferon-tau production.     

Effects of a standardized purified dry extract from Echinacea angustifolia on proliferation and interferon gamma secretion of peripheral blood mononuclear cells in dairy heifers

This study was performed to ascertain whether a standardized extract from Echinacea angustifolia (Polinacea™) affects proliferation and interferon gamma (IFN-γ) secretion in bovine peripheral blood mononuclear cells (PBMC).PBMC from six Holstein heifers were incubated with 0, 6.3, 20, 60, or 180 μg/ml of the tested compound. Proliferation was stimulated by concanavalin A (ConA) or pokeweed-mitogen (PWM). Secretion of IFN-γ was stimulated by ConA.All concentrations of Polinacea™ exerted a mitogenic effect. With respect to control PBMC (0 μg/ml), the lowest and highest increase of proliferation were observed with Polinacea™ at 6.3 (2-fold increase) or 180 (10-fold increase) μg/ml, respectively. Polinacea™ at 180 μg/ml reduced ConA-driven proliferation, whereas at 20 and 60 μg/ml improved proliferation of PWM-stimulated PBMC. IFN-γ secretion was not affected. In conclusion, Polinacea™ modulates bovine PBMC proliferation, and deserves to be tested in vivo to define conditions that may benefit from its utilization.     

Trematode infections in pregnant ewes can predispose to mastitis during the subsequent lactation period

Objective was to investigate if trematode infections predispose ewes to mastitis and/or metritis. We used 80 trematode-infected ewes: primigravidae in group P-A and multigravidae in M-A remained untreated, primigravidae in P-B and multigravidae in M-B were drenched with netobimin and multigravidae in M-C were given rafoxanide. We collected faecal samples for parasitological examination, blood samples for β-hydroxybutyrate concentration measurement and uterine content, teat duct material and milk samples for bacteriological examination. We found significant differences in blood β-hydroxybutyrate concentrations between M-A, M-B and M-C during pregnancy (P ? 0.002). We did not observe significant differences between groups regarding development of metritis (P > 0.83). We found that for M-A, M-B and M-C ewes, respectively, median time to first case of mastitis was 5.75, 21 and 6.75 days after lambing (P = 0.003) and incidence risk of mastitis was 0.308, 0.069 and 0.222 (P = 0.047). We postulate that trematode infections predispose ewes to mastitis; perhaps, increased β-hydroxybutyrate blood concentrations adversely affect mammary cellular defences. This is the first report associating parasitic infections with mastitis in sheep.     

2株枯草芽孢杆菌对大肠杆菌和沙门氏菌的体外抑菌试验研究简

本试验旨在检测2株枯草芽孢杆菌对致病性大肠杆菌和沙门氏菌抑菌作用。以致病性大肠杆菌和沙门氏菌作为指示菌,采用2株枯草芽孢杆菌与大肠杆菌和沙门氏菌同时接种及先后接种的方法,对其与大肠杆菌和沙门氏菌的生物颉颃作用进行了研究。结果表明,先接种枯草芽孢杆菌再接种致病菌抑制作用最明显且在24 h时抑菌作用最强,同时接种枯草芽孢杆菌和致病菌抑制作用次之,先接种致病菌再接种枯草芽孢杆菌的抑制作用最弱。提示,2株枯草芽孢杆菌对致病性大肠杆菌和沙门氏菌都有一定的抑制作用,但抑制作用的强弱有差异,且与枯草芽孢杆菌和致病菌接种的时间长短有关。