Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0031
J. Vallet, J. Miles, B. Freking
Placental insufficiency results in fetal loss, low birth weight, stillbirth, preweaning mortality and poor growth. Placental development begins at conceptus elongation, which is a primary factor controlling the size of the placenta. After elongation, the allantois develops outward from the embryo to establish the allantochorion, which defines the size of the functional placenta. During implantation, chorionic trophoblasts adhere to endometrial epithelial cells. Placental structures known as areolae develop at the openings of the endometrial glands and take up endometrial gland secreted products (histotrophe). Between day 30 and 35 of gestation, the adhered trophoblast-endometrial epithelial bilayer undergoes microscopic folding. Fetal and maternal capillaries develop adjacent to the bilayer and blood flows are arranged in a cross-countercurrent manner. Except for nutrients secreted by the glands, nutrient exchange takes place between these capillaries within these folds. By day 85, the folds deepen and become more complex, increasing surface area. The epithelial bilayer thins and capillaries indent the plane of each layer (but do not penetrate), reducing distance between capillaries. The folded bilayer is surrounded by endometrial stroma on the maternal side and placental stroma on the fetal side. The fetal-placental stroma is partially composed of glycosaminoglycans, the most abundant being hyaluronan and heparan sulfate. Changes in both hyaluronoglucosaminidase and heparanase during placental development suggest that these enzymes play a role in placental development. In addition to structural modifications, various nutrient specific transport mechanisms exist. These mechanisms are likely to be as important to transport of specific nutrients as placental size or structure.
{"title":"Development of the pig placenta.","authors":"J. Vallet, J. Miles, B. Freking","doi":"10.1530/biosciprocs.18.0031","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0031","url":null,"abstract":"Placental insufficiency results in fetal loss, low birth weight, stillbirth, preweaning mortality and poor growth. Placental development begins at conceptus elongation, which is a primary factor controlling the size of the placenta. After elongation, the allantois develops outward from the embryo to establish the allantochorion, which defines the size of the functional placenta. During implantation, chorionic trophoblasts adhere to endometrial epithelial cells. Placental structures known as areolae develop at the openings of the endometrial glands and take up endometrial gland secreted products (histotrophe). Between day 30 and 35 of gestation, the adhered trophoblast-endometrial epithelial bilayer undergoes microscopic folding. Fetal and maternal capillaries develop adjacent to the bilayer and blood flows are arranged in a cross-countercurrent manner. Except for nutrients secreted by the glands, nutrient exchange takes place between these capillaries within these folds. By day 85, the folds deepen and become more complex, increasing surface area. The epithelial bilayer thins and capillaries indent the plane of each layer (but do not penetrate), reducing distance between capillaries. The folded bilayer is surrounded by endometrial stroma on the maternal side and placental stroma on the fetal side. The fetal-placental stroma is partially composed of glycosaminoglycans, the most abundant being hyaluronan and heparan sulfate. Changes in both hyaluronoglucosaminidase and heparanase during placental development suggest that these enzymes play a role in placental development. In addition to structural modifications, various nutrient specific transport mechanisms exist. These mechanisms are likely to be as important to transport of specific nutrients as placental size or structure.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"16 2","pages":"265-79"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41303622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0036
A. Waclawik, A. Blitek, M. Kaczmarek, J. Kiewisz, A. Ziecik
Extended exposure of progesterone and conceptus estrogen influences the vascular compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth factors, extracellular matrix and adhesion molecules, cytokines and transcription factors. One of the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is involved in the process of maternal recognition of pregnancy. However, complex (direct and indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10 and Wnt in the endometrium, to create the appropriate uterine environment for establishment of pregnancy and implantation.
{"title":"Antiluteolytic mechanisms and the establishment of pregnancy in the pig.","authors":"A. Waclawik, A. Blitek, M. Kaczmarek, J. Kiewisz, A. Ziecik","doi":"10.1530/biosciprocs.18.0036","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0036","url":null,"abstract":"Extended exposure of progesterone and conceptus estrogen influences the vascular compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth factors, extracellular matrix and adhesion molecules, cytokines and transcription factors. One of the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is involved in the process of maternal recognition of pregnancy. However, complex (direct and indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10 and Wnt in the endometrium, to create the appropriate uterine environment for establishment of pregnancy and implantation.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"307-20"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42994320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0011
P. Sutovsky
Proteomic analysis occupies an increasingly important place in gamete and embryo biology as an independent tool of discovery and as a means of follow-up to transcriptional profiling. Proteomics have been and will be increasingly helpful in many areas of reproductive biology, including applied science and technology development. Areas likely to be impacted most rapidly by proteomic knowledge include fertility evaluation in male farm animals, male infertility diagnostics in humans, assessment and optimization of oocyte and embryo culture protocols, selection of fittest oocytes for assisted fertilization and selection of most competent embryos for embryo transfer. Oocyte proteomics will help us understand the process of oogenesis and oocyte maturation, and to discover non-invasive markers of oocyte quality. Sperm proteomics correlate with normal sperm structure and function and can be applied to discover novel biomarkers of farm animal fertility and diagnostic markers of human male infertility. Putative receptors participating in fertilization, as well as proteins acquired onto sperm surface from epididymal fluid and seminal plasma, have been discovered by proteomic analysis. An added level of information is provided by advanced proteomic approaches, capable of identifying posttranslational modifications such as phosphorylation, glycosylation and ubiquitination which play important functions in gametogenesis, fertilization and embryo development. By no means exhaustive, the present paper reviews some of the most interesting proteomic studies of mammalian gametes and embryos published in the last decade.
{"title":"Proteomic analysis of mammalian gametes and sperm-oocyte interactions.","authors":"P. Sutovsky","doi":"10.1530/biosciprocs.18.0011","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0011","url":null,"abstract":"Proteomic analysis occupies an increasingly important place in gamete and embryo biology as an independent tool of discovery and as a means of follow-up to transcriptional profiling. Proteomics have been and will be increasingly helpful in many areas of reproductive biology, including applied science and technology development. Areas likely to be impacted most rapidly by proteomic knowledge include fertility evaluation in male farm animals, male infertility diagnostics in humans, assessment and optimization of oocyte and embryo culture protocols, selection of fittest oocytes for assisted fertilization and selection of most competent embryos for embryo transfer. Oocyte proteomics will help us understand the process of oogenesis and oocyte maturation, and to discover non-invasive markers of oocyte quality. Sperm proteomics correlate with normal sperm structure and function and can be applied to discover novel biomarkers of farm animal fertility and diagnostic markers of human male infertility. Putative receptors participating in fertilization, as well as proteins acquired onto sperm surface from epididymal fluid and seminal plasma, have been discovered by proteomic analysis. An added level of information is provided by advanced proteomic approaches, capable of identifying posttranslational modifications such as phosphorylation, glycosylation and ubiquitination which play important functions in gametogenesis, fertilization and embryo development. By no means exhaustive, the present paper reviews some of the most interesting proteomic studies of mammalian gametes and embryos published in the last decade.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"103-16"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47327837","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0008
U. Taylor, H. Zerbe, H. Seyfert, D. Rath, H. Schuberth
Inseminations in pigs are characterized by the tremendous amount of spermatozoa needed for successful fertilisation. lf, in contrast, spermatozoa are deposited at the tip of the uterine horn a fraction of the usual porcine insemination dose suffices (Johnson 1991, Vazquez et al. 2005). It thus seems to be the uterine passage where the need for such high sperm numbers arises. In the past the provision of sufficient sperm numbers to reach acceptable fertility rates did not pose a problem due to the abundance of spermatozoa in one single boar ejaculate. However, modern biotechnological procedures, such as sex sorting of spermatozoa, require insemination of sperm portions containing no more then 50 x 106spermatozoa. To facilitate insemination with such small sperm doses also for conventional Al-techniques, more knowledge has to be gathered about fundamental sperm transport and selection mechanisms within the uterus. Previous studies on the pig uterus (Lovell & Getty 1968), the utero-tubal junction (Rodriguez-Martinez et al. 1990) and the oviduct (Wagner et al. 2002) suggested that spermatozoa are indeed subject to close interaction and even binding with the epithelial structures of the female genital tract. The present study aimed to further our understanding of such interactions specifically in the porcine uterus. For this purpose an ex vivo model was developed using uterine segments of 10 cm derived from 50 freshly slaughtered peri-ovulatory German Landrace gilts. In each segment 100 x 106 spermatozoa were incubated for 60 min at 38°C. The sperm cells originated from ejaculates provided by 4 boars of the same breed and of proven fertility. Previous to incubation spermatozoa were either washed and diluted in the semen extender Androhep or diluted with autologous seminal plasma without further washing. The spermatozoa were subsequently flushed out of the segments, counted and their viability parameters were established flow cytometrically using the stains PI and JC1 for membrane integrity and mitochondrial membrane potential respectively. The results indicated a retention of viable spermatozoa within the uterine cavity since only 55 ± 7% of the intact spermatozoa (PI-4C1+) were rediscovered in the flushing, while the damaged sperm population (PI+ /JC1-) was flushed out almost in its entity (93 ± 12%; p < 0.03). The effect was more emphasised in the sperm population, which had been washed and diluted in Androhep' (p < 0.05). The location of the uterine segments in relation to the cervix had no effect on the numbers of recovered sperm cells. Neither were differences observed between segments from the right and left uterine horn. In order to determine the physiological reasons behind the observed uterine sperm retention an in vivo experiment was performed looking for changes in uterine gene expression in response to insemination by measuring endometrial mRNA concentration. Because many processes concerning reproduction such as ovulation and implantation
猪受精的特点是成功受精需要大量精子。相反，如果精子沉积在子宫角的顶端，那么通常猪受精剂量的一小部分就足够了（Johnson 1991，Vazquez等人2005）。因此，似乎是子宫通道需要如此高的精子数量。在过去，提供足够的精子数量以达到可接受的生育率并不是一个问题，因为一次公猪精液中有大量精子。然而，现代生物技术程序，如精子的性别分类，需要对不超过50 x 106精子的精子部分进行受精。为了便于用这种小剂量的精子进行人工授精，也用于传统的人工授精技术，必须收集更多关于子宫内基本精子运输和选择机制的知识。先前对猪子宫（Lovell&Getty 1968）、子宫-输卵管连接处（Rodriguez-Martinez等人，1990）和输卵管（Wagner等人，2002）的研究表明，精子确实与女性生殖道的上皮结构密切相互作用，甚至结合。本研究旨在进一步了解这种相互作用，特别是在猪子宫中。为此，使用50只新鲜屠宰的围排卵期德国长白猪的10cm子宫段建立了离体模型。在每个节段中，将100 x 106个精子在38°C下孵育60分钟。精子细胞来源于同一品种的4头公猪提供的精液，并已证明具有生育能力。孵育前，精子要么在精液扩展器Androhep中洗涤和稀释，要么在不进一步洗涤的情况下用自体精浆稀释。随后将精子从节段中冲洗出来，进行计数，并分别使用PI和JC1染色对其膜完整性和线粒体膜电位进行流式细胞术测定其活力参数。结果表明，活精子保留在子宫腔内，因为只有55±7%的完整精子（PI-4C1+）在冲洗中被重新发现，而受损的精子群体（PI+/JC1-）几乎在其实体中被冲洗掉（93±12%；p＜0.03）。这种影响在精子群体中更为突出，子宫节段相对于宫颈的位置对回收的精子细胞数量没有影响。右侧和左侧子宫角的节段之间也没有观察到差异。为了确定观察到的子宫精子滞留背后的生理原因，进行了一项体内实验，通过测量子宫内膜mRNA浓度来寻找受精后子宫基因表达的变化。因为许多与生殖有关的过程，如排卵和着床，都涉及免疫系统
{"title":"Binding of porcine spermatozoa to uterine epithelial cells modulates the female immune response and might indicate the formation of a pre-oviductal sperm reservoir.","authors":"U. Taylor, H. Zerbe, H. Seyfert, D. Rath, H. Schuberth","doi":"10.1530/biosciprocs.18.0008","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0008","url":null,"abstract":"Inseminations in pigs are characterized by the tremendous amount of spermatozoa needed for successful fertilisation. lf, in contrast, spermatozoa are deposited at the tip of the uterine horn a fraction of the usual porcine insemination dose suffices (Johnson 1991, Vazquez et al. 2005). It thus seems to be the uterine passage where the need for such high sperm numbers arises. In the past the provision of sufficient sperm numbers to reach acceptable fertility rates did not pose a problem due to the abundance of spermatozoa in one single boar ejaculate. However, modern biotechnological procedures, such as sex sorting of spermatozoa, require insemination of sperm portions containing no more then 50 x 106spermatozoa. To facilitate insemination with such small sperm doses also for conventional Al-techniques, more knowledge has to be gathered about fundamental sperm transport and selection mechanisms within the uterus. Previous studies on the pig uterus (Lovell & Getty 1968), the utero-tubal junction (Rodriguez-Martinez et al. 1990) and the oviduct (Wagner et al. 2002) suggested that spermatozoa are indeed subject to close interaction and even binding with the epithelial structures of the female genital tract. The present study aimed to further our understanding of such interactions specifically in the porcine uterus. For this purpose an ex vivo model was developed using uterine segments of 10 cm derived from 50 freshly slaughtered peri-ovulatory German Landrace gilts. In each segment 100 x 106 spermatozoa were incubated for 60 min at 38°C. The sperm cells originated from ejaculates provided by 4 boars of the same breed and of proven fertility. Previous to incubation spermatozoa were either washed and diluted in the semen extender Androhep or diluted with autologous seminal plasma without further washing. The spermatozoa were subsequently flushed out of the segments, counted and their viability parameters were established flow cytometrically using the stains PI and JC1 for membrane integrity and mitochondrial membrane potential respectively. The results indicated a retention of viable spermatozoa within the uterine cavity since only 55 ± 7% of the intact spermatozoa (PI-4C1+) were rediscovered in the flushing, while the damaged sperm population (PI+ /JC1-) was flushed out almost in its entity (93 ± 12%; p < 0.03). The effect was more emphasised in the sperm population, which had been washed and diluted in Androhep' (p < 0.05). The location of the uterine segments in relation to the cervix had no effect on the numbers of recovered sperm cells. Neither were differences observed between segments from the right and left uterine horn. In order to determine the physiological reasons behind the observed uterine sperm retention an in vivo experiment was performed looking for changes in uterine gene expression in response to insemination by measuring endometrial mRNA concentration. Because many processes concerning reproduction such as ovulation and implantation","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"83-4"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44694320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0005
W. Flowers
Critical needs for the swine industry in terms of boar fertility evaluations are validation of semen quality estimates with in vivo reproductive data; estimation of the relative fertility of boars; and elimination of sub-fertile ejaculates. Single sire matings are the best way to validate semen quality estimates with reproductive performance. Sampling about 20% of the population provides an accurate estimation of the variability among boars and should be sufficient for this purpose. In vitro tests that measure univariate characteristics of ejaculates including motility and morphology appear to be just as accurate as those that measure multivariate traits such as in vitro fertilization in terms of predicting boar fertility. Reasons for this observation may be related to how properties of sperm cells are influenced by the sow reproductive tract. Several seminal plasma proteins show strong correlations with boar fertility and hold potential for being developed into tests that can rank the relative fertility of boars. Almost 90% of the variation in boar fertility was explained when the proportion of motile and acrosome-reacted spermatozoa was combined with relative amounts of 28 kDa, pl 6.0 and 55 kDa, pl 4.5 seminal plasma proteins. Consequently, combining different complementary tests improves estimations of boar fertility. Motility estimates routinely performed in most A.I. centres are a reasonable technique for identification and elimination of sub-fertile ejaculates. However, the accuracy with which they currently are conducted within the swine industry needs improvement.
{"title":"Selection for boar fertility and semen quality--the way ahead.","authors":"W. Flowers","doi":"10.1530/biosciprocs.18.0005","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0005","url":null,"abstract":"Critical needs for the swine industry in terms of boar fertility evaluations are validation of semen quality estimates with in vivo reproductive data; estimation of the relative fertility of boars; and elimination of sub-fertile ejaculates. Single sire matings are the best way to validate semen quality estimates with reproductive performance. Sampling about 20% of the population provides an accurate estimation of the variability among boars and should be sufficient for this purpose. In vitro tests that measure univariate characteristics of ejaculates including motility and morphology appear to be just as accurate as those that measure multivariate traits such as in vitro fertilization in terms of predicting boar fertility. Reasons for this observation may be related to how properties of sperm cells are influenced by the sow reproductive tract. Several seminal plasma proteins show strong correlations with boar fertility and hold potential for being developed into tests that can rank the relative fertility of boars. Almost 90% of the variation in boar fertility was explained when the proportion of motile and acrosome-reacted spermatozoa was combined with relative amounts of 28 kDa, pl 6.0 and 55 kDa, pl 4.5 seminal plasma proteins. Consequently, combining different complementary tests improves estimations of boar fertility. Motility estimates routinely performed in most A.I. centres are a reasonable technique for identification and elimination of sub-fertile ejaculates. However, the accuracy with which they currently are conducted within the swine industry needs improvement.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"67-78"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43249493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0022
S. Novak, H. Moore, F. Paradis, G. Murdoch, M. Dyck, W. Dixon, G. Foxcroft
The trend towards high ovulation rates in mature commercial sows has resulted in intra-uterine crowding in the immediate post-implantation period, with negative impacts on placental development and later fetal development (Town et al. 2004). Factors that improve placental angiogenesis could offset the effects of intra-uterine crowding by supporting placental development at critical times in gestation. Feeding of L-arginine has been shown to have beneficial effects on placental vascularization in gilts (Hazeleger et al. 2007) and on litter size born in gilts (Mateo et al. 2007) and sows (Ramaekers et a/. 2006). In the present study, we investigated the effects of L-arginine supplementation to commercial sows on global placental gene expression, and on temporal changes in the expression of a panel of eight candidate genes known to be involved in angiogenesis, in early pregnancy. Multiparous sows (n —48) were either non-supplemented Controls or were fed an L-arginine supplement (20g.d) from d 15 through 29 of gestation. A representative number of sows were euthanized on days 16 through 49 of gestation and embryonic and placental tissues were collected from two average-sized conceptuses from each uterine horn and placed in RNAlater for later analysis. To obtain temporal expression profiles for specific genes involved in placental angiogenesis, total placental RNA was extracted from all Control samples collected, reverse transcribed and real-time PCR used to determine the transcript abundance of: vascular endothelial growth factor (VEGF) -A; the two VEGF receptors, fms-related tyrosine kinase 1 (FLT1) and fetal liver kinase-1(flk-1/KDR); hypoxia-inducible factor (HIF)1A; the Angiopoietins (ANGPT) -1 and -2 and their receptor, TEK tyrosine kinase; and finally Angiogenin (ANG) -1. The delta ACt values were calculated using 185 as an internal control, and data were analyzed using regression analysis (SAS Institute Inc., Cary, NC). To determine the cumulative effect of L-arginine treatment, real-time PCR for these same candidate genes was also performed on the d 30 placental samples from both Control and L-arginine sows. The relative ACt values for d 30 samples were again calculated using 185 as an internal control and data were analyzed using MIXED models (SAS Institute Inc., Cary, NC). Effects of L-arginine on global placental gene expression (n =4 representative sows per treatment) were also analyzed using PigOligoArray slides and placental tissues collected at d 30 of gestation. Total RNA was extracted, purified using mRNA mini kits (lnvitrogen), amplified with aminoallyl mRNA amplification kit (Ambion), and labeled with Cy3 or Cy5 in a random block dye-swap design. The hybridized slide images were captured with Genepix software and an Axon Scanner set for optimized PMT for each dye. Median spot intensities underwent Loess and quantile normalization and were analyzed using linear models, all in limma (Smyth, 2004).
{"title":"Temporal candidate gene expression patterns in the sow placenta during early gestation and the effect of maternal L-arginine supplementation.","authors":"S. Novak, H. Moore, F. Paradis, G. Murdoch, M. Dyck, W. Dixon, G. Foxcroft","doi":"10.1530/biosciprocs.18.0022","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0022","url":null,"abstract":"The trend towards high ovulation rates in mature commercial sows has resulted in intra-uterine crowding in the immediate post-implantation period, with negative impacts on placental development and later fetal development (Town et al. 2004). Factors that improve placental angiogenesis could offset the effects of intra-uterine crowding by supporting placental development at critical times in gestation. Feeding of L-arginine has been shown to have beneficial effects on placental vascularization in gilts (Hazeleger et al. 2007) and on litter size born in gilts (Mateo et al. 2007) and sows (Ramaekers et a/. 2006). In the present study, we investigated the effects of L-arginine supplementation to commercial sows on global placental gene expression, and on temporal changes in the expression of a panel of eight candidate genes known to be involved in angiogenesis, in early pregnancy. Multiparous sows (n —48) were either non-supplemented Controls or were fed an L-arginine supplement (20g.d) from d 15 through 29 of gestation. A representative number of sows were euthanized on days 16 through 49 of gestation and embryonic and placental tissues were collected from two average-sized conceptuses from each uterine horn and placed in RNAlater for later analysis. To obtain temporal expression profiles for specific genes involved in placental angiogenesis, total placental RNA was extracted from all Control samples collected, reverse transcribed and real-time PCR used to determine the transcript abundance of: vascular endothelial growth factor (VEGF) -A; the two VEGF receptors, fms-related tyrosine kinase 1 (FLT1) and fetal liver kinase-1(flk-1/KDR); hypoxia-inducible factor (HIF)1A; the Angiopoietins (ANGPT) -1 and -2 and their receptor, TEK tyrosine kinase; and finally Angiogenin (ANG) -1. The delta ACt values were calculated using 185 as an internal control, and data were analyzed using regression analysis (SAS Institute Inc., Cary, NC). To determine the cumulative effect of L-arginine treatment, real-time PCR for these same candidate genes was also performed on the d 30 placental samples from both Control and L-arginine sows. The relative ACt values for d 30 samples were again calculated using 185 as an internal control and data were analyzed using MIXED models (SAS Institute Inc., Cary, NC). Effects of L-arginine on global placental gene expression (n =4 representative sows per treatment) were also analyzed using PigOligoArray slides and placental tissues collected at d 30 of gestation. Total RNA was extracted, purified using mRNA mini kits (lnvitrogen), amplified with aminoallyl mRNA amplification kit (Ambion), and labeled with Cy3 or Cy5 in a random block dye-swap design. The hybridized slide images were captured with Genepix software and an Axon Scanner set for optimized PMT for each dye. Median spot intensities underwent Loess and quantile normalization and were analyzed using linear models, all in limma (Smyth, 2004).","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"201-2"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41920355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0033
A. Clutter
Genetic improvement of sow lifetime reproductive performance has value from both the economic perspectives of pork producers and the pork industry, but also from the perspective of ethical and animal welfare concerns by the general public. Genetic potential for piglets produced from individual litters is a primary determinant of lifetime prolificacy, but females must be able to sustain productivity without injury or death beyond the achievement of positive net present value. Evidence exists for between- and within-line genetic variation in sow lifetime performance, suggesting that improvements may be made by both line choices and genetic selection within lines. However, some of the same barriers to accurate within-line selection that apply to individual litter traits also present challenges to genetic selection for sow lifetime prolificacy: generally low heritabilites, sex-limited expression, expression after the age that animals are typically selected, and unfavorable genetic correlations with other traits in the profit function. In addition, there is an inherent conflict within the genetic nucleus herds where selections take place between the goal of shortened generation interval to accelerate genetic progress and the expression of sow lifetime traits. A proliferation in the industry of commercial multipliers with direct genetic ties and routine record flows to genetic nucleus herds provides a framework for accurate estimates of relevant genetic variances and covariances, and estimation of breeding values for sow lifetime traits that can be used in genetic selection.
{"title":"Genetic selection for lifetime reproductive performance.","authors":"A. Clutter","doi":"10.1530/biosciprocs.18.0033","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0033","url":null,"abstract":"Genetic improvement of sow lifetime reproductive performance has value from both the economic perspectives of pork producers and the pork industry, but also from the perspective of ethical and animal welfare concerns by the general public. Genetic potential for piglets produced from individual litters is a primary determinant of lifetime prolificacy, but females must be able to sustain productivity without injury or death beyond the achievement of positive net present value. Evidence exists for between- and within-line genetic variation in sow lifetime performance, suggesting that improvements may be made by both line choices and genetic selection within lines. However, some of the same barriers to accurate within-line selection that apply to individual litter traits also present challenges to genetic selection for sow lifetime prolificacy: generally low heritabilites, sex-limited expression, expression after the age that animals are typically selected, and unfavorable genetic correlations with other traits in the profit function. In addition, there is an inherent conflict within the genetic nucleus herds where selections take place between the goal of shortened generation interval to accelerate genetic progress and the expression of sow lifetime traits. A proliferation in the industry of commercial multipliers with direct genetic ties and routine record flows to genetic nucleus herds provides a framework for accurate estimates of relevant genetic variances and covariances, and estimation of breeding values for sow lifetime traits that can be used in genetic selection.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"293-302"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42435291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0039
F. Bazer, H. Gao, G. Johnson, G. Wu, D. W. Bailey, R. Burghardt
Glucose present in the intrauterine environment can be metabolized, activate cell signaling pathways or be converted to a "storage" form. Total recoverable glucose in uterine fluid of pregnant, but not cyclic pigs increases from Day 12 after onset of estrus in concert with conceptus elongation (Bazer et al. 1991). Transport of glucose into the ovine uterus and its uptake by conceptuses involves sodium-dependent and facilitative glucose transporters (Gao et al. 2009). Glucose can activate FRAP1/mTOR "nutrient sensing" pathway in which protein kinases activate p70S6 through phosphorylation to increase translation of 5'TOP mRNAs (terminal oligopyrimidine tract) (Wen et a/. 2005). Activated FRAP1 also regulates differentiation of trophectoderm (Tr) via Ras transformation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (eIF4EBP1), a translational repressor of CAP-dependent translation (De Benedetti & Rhoads 1990). Select nutrients that stimulate FRAP1 activity in Tr include glucose, arginine (Arg), leucine (Leu) and glutamine (Gln) which may increase expression of IGF2, ODC and NOS mRNAs (Nielsen et al. 1995; Kimball et al. 1999; Martin & Sutherland 2001) which are required for conceptus development, differentiation and implantation through effects on production of NO (Kaliman et al. 1999) and polyamines (Van Winkle & Campione 1983). FRAP1 null mice die shortly after implantation due to impaired cell proliferation and hypertrophy in both the embryonic disc and Tr (Murakami et al. 2004). There are 14 isoforms of facilitative glucose transporters and 6 sodium-dependent glucose transporters. Of these, SLC2A I, SLCSA1 and SLCSA1 I mRNAs are most abundant in endometria and SLC2A3 is uniquely expressed by ovine conceptus Tr and endoderm (Gao et al. 2009). The objective of this study with sexually mature giks was to identify effects of pregnancy, long-term treatment of ovariectomized gilts with progesterone (P4) and estradiol-induced pseudopregnancy (PP) on changes in amounts of select nutrients (glucose, Arg, Leu and Gln) in uterine fluid and expression of glucose transporters in endometria and conceptuses. Experiment 1 determined effects of day of the estrous cycle and pregnancy on total recoverable glucose, Arg, Leu and Gln in uterine flushings from gilts on Days 5, 9, 12 and 15 of the estrous cycle (Cy) and Days 9, 10, 12, 13, 14 and 15 of pregnancy (Px). Total recoverable glucose, Arg, Leu and Gln increased (P< 0.05) with day in Cy and Px gilts, but only Arg increased more in Px than Cy ewes (day x pregnancy status; P<0.05) between Days 12 and 15. Experiment 2 determined recoverable amounts of selected nutrients in uterine flushings of gilts ovariectomized on Day 12 and treated daily with either corn oil (OVX-CO; n —4) or 200 mg progesterone (OVX-P4;n —5) to Day 39 and hysterectomized on Day 40. Values (mean +SEM; nmol) were greater for OVX-P4 than OVX-CO gilts for glucose (4,955+2,534 vs 726+ 133), Arg (207,112 + 160,979 vs 7,409+
宫内环境中存在的葡萄糖可以被代谢、激活细胞信号通路或转化为“储存”形式。妊娠期（但不是周期性）猪子宫液中的总可回收葡萄糖从发情期开始后第12天起随着妊娠期延长而增加（Bazer等人，1991）。葡萄糖进入绵羊子宫的运输及其通过受孕物的摄取涉及钠依赖性和促进性葡萄糖转运蛋白（Gao等人，2009）。葡萄糖可以激活FRAP1/mTOR“营养感应”途径，其中蛋白激酶通过磷酸化激活p70S6，以增加5’TOP-mRNA（末端寡嘧啶通道）的翻译（Wen等人，2005）。活化的FRAP1还通过磷酸化真核起始因子4E结合蛋白1（eIF4EBP1）（CAP依赖性翻译的翻译阻遏物），通过Ras转化调节滋养外胚层（Tr）的分化（De Benedetti&Rhoads 1990）。刺激Tr中FRAP1活性的选择营养素包括葡萄糖、精氨酸（Arg）、亮氨酸（Leu）和谷氨酰胺（Gln），它们可能增加IGF2、ODC和NOS mRNA的表达（Nielsen等人1995；Kimball等人1999；Martin和Sutherland 2001），这是受孕发育所需的，通过对NO（Kaliman等人，1999）和多胺（Van Winkle和Campione 1983）的产生的影响进行分化和植入。FRAP1缺失小鼠在植入后不久由于胚胎椎间盘和Tr中的细胞增殖受损和肥大而死亡（Murakami等人，2004）。促进性葡萄糖转运蛋白有14种异构体，钠依赖性葡萄糖转运因子有6种。其中，SLC2A I、SLCSA1和SLCSA1 I mRNA在子宫内膜中最为丰富，并且SLC2A3在绵羊孕体Tr和内胚层中唯一表达（Gao等人，2009）。本研究的目的是对性成熟giks进行研究，以确定妊娠、用孕酮（P4）和雌二醇诱导的假妊娠（PP）长期治疗去卵巢母猪对子宫液中选择营养素（葡萄糖、精氨酸、亮氨酸和谷氨酰胺）含量变化以及子宫内膜和孕体中葡萄糖转运蛋白表达的影响。实验1测定了发情周期的第5、9、12和15天以及妊娠的第9、10、12、13、14和15天（Px），发情周期的天数和妊娠对来自母猪的子宫冲洗中的总可回收葡萄糖、Arg、Leu和Gln的影响。Cy和Px母猪的总可恢复葡萄糖、Arg、Leu和Gln随着天数的增加而增加（P<0.05），但在第12天和第15天之间，只有Px的Arg比Cy母羊增加更多（第x天妊娠状态；P<0.05）。实验2测定了在第12天切除卵巢并每天用玉米油（OVX-CO；n-4）或200mg孕酮（OVX-P4；n-5）处理至第39天并在第40天切除子宫的母猪的子宫冲洗中所选营养素的可回收量。OVX-P4的葡萄糖（4955+2534 vs 726+133）、精氨酸（207112+160979 vs 7409+2877）和亮氨酸（248255+178599 vs 13983+5225）的值（平均值+SEM；nmol）高于OVX-CO镀金，但由于高变异性和小样本量，差异不显著。实验3测定了由5 mg/天苯甲酸雌二醇治疗诱发的假妊娠（PP）第90天的母猪子宫冲洗中选定营养素的量
{"title":"Select nutrients and glucose transporters in pig uteri and conceptuses.","authors":"F. Bazer, H. Gao, G. Johnson, G. Wu, D. W. Bailey, R. Burghardt","doi":"10.1530/biosciprocs.18.0039","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0039","url":null,"abstract":"Glucose present in the intrauterine environment can be metabolized, activate cell signaling pathways or be converted to a \"storage\" form. Total recoverable glucose in uterine fluid of pregnant, but not cyclic pigs increases from Day 12 after onset of estrus in concert with conceptus elongation (Bazer et al. 1991). Transport of glucose into the ovine uterus and its uptake by conceptuses involves sodium-dependent and facilitative glucose transporters (Gao et al. 2009). Glucose can activate FRAP1/mTOR \"nutrient sensing\" pathway in which protein kinases activate p70S6 through phosphorylation to increase translation of 5'TOP mRNAs (terminal oligopyrimidine tract) (Wen et a/. 2005). Activated FRAP1 also regulates differentiation of trophectoderm (Tr) via Ras transformation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (eIF4EBP1), a translational repressor of CAP-dependent translation (De Benedetti & Rhoads 1990). Select nutrients that stimulate FRAP1 activity in Tr include glucose, arginine (Arg), leucine (Leu) and glutamine (Gln) which may increase expression of IGF2, ODC and NOS mRNAs (Nielsen et al. 1995; Kimball et al. 1999; Martin & Sutherland 2001) which are required for conceptus development, differentiation and implantation through effects on production of NO (Kaliman et al. 1999) and polyamines (Van Winkle & Campione 1983). FRAP1 null mice die shortly after implantation due to impaired cell proliferation and hypertrophy in both the embryonic disc and Tr (Murakami et al. 2004). There are 14 isoforms of facilitative glucose transporters and 6 sodium-dependent glucose transporters. Of these, SLC2A I, SLCSA1 and SLCSA1 I mRNAs are most abundant in endometria and SLC2A3 is uniquely expressed by ovine conceptus Tr and endoderm (Gao et al. 2009). The objective of this study with sexually mature giks was to identify effects of pregnancy, long-term treatment of ovariectomized gilts with progesterone (P4) and estradiol-induced pseudopregnancy (PP) on changes in amounts of select nutrients (glucose, Arg, Leu and Gln) in uterine fluid and expression of glucose transporters in endometria and conceptuses. Experiment 1 determined effects of day of the estrous cycle and pregnancy on total recoverable glucose, Arg, Leu and Gln in uterine flushings from gilts on Days 5, 9, 12 and 15 of the estrous cycle (Cy) and Days 9, 10, 12, 13, 14 and 15 of pregnancy (Px). Total recoverable glucose, Arg, Leu and Gln increased (P< 0.05) with day in Cy and Px gilts, but only Arg increased more in Px than Cy ewes (day x pregnancy status; P<0.05) between Days 12 and 15. Experiment 2 determined recoverable amounts of selected nutrients in uterine flushings of gilts ovariectomized on Day 12 and treated daily with either corn oil (OVX-CO; n —4) or 200 mg progesterone (OVX-P4;n —5) to Day 39 and hysterectomized on Day 40. Values (mean +SEM; nmol) were greater for OVX-P4 than OVX-CO gilts for glucose (4,955+2,534 vs 726+ 133), Arg (207,112 + 160,979 vs 7,409+ ","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"335-6"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47470975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0032
F. P. Bortolozzo, M. Bernardi, R. Kummer, I. Wentz
Optimizing gilt management is a critical point to improve breeding herd efficiency. This review describes the effects of growth rate (GR) and body state at onset of puberty stimulation or at first mating on gilt puberty attainment, productivity and sow longevity. Traditional management practices should be re-evaluated with attention to different modern genotypes. It is difficult to discern the real effects of age, weight, backfat depth and estrus number at first insemination on longevity and reproductive performance, because these variables affect one another. GR interacts with age at boar exposure to influence age at puberty. Higher lifetime GR gilts (>700 g/d) attain puberty earlier and have a lower anoestrus rate. If gilts attain a target weight (135-150 kg), are adapted to herd health status and have at least one previously recorded estrus, they can be inseminated. Overweight at first breeding and throughout gestation should be avoided. There is no advantage in breeding gilts heavier than 150 kg; at first farrowing the target weight is 180-185 kg. Piglet production at first parity may be increased in gilts with a high GR but the number of stillborn piglets can also be increased. The culling rate over 3 parities for locomotion problems, which is one of the major risk factors for reduced herd retention rate, can be increased in overweight gilts at first breeding (>150-170 kg).
{"title":"Growth, body state and breeding performance in gilts and primiparous sows.","authors":"F. P. Bortolozzo, M. Bernardi, R. Kummer, I. Wentz","doi":"10.1530/biosciprocs.18.0032","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0032","url":null,"abstract":"Optimizing gilt management is a critical point to improve breeding herd efficiency. This review describes the effects of growth rate (GR) and body state at onset of puberty stimulation or at first mating on gilt puberty attainment, productivity and sow longevity. Traditional management practices should be re-evaluated with attention to different modern genotypes. It is difficult to discern the real effects of age, weight, backfat depth and estrus number at first insemination on longevity and reproductive performance, because these variables affect one another. GR interacts with age at boar exposure to influence age at puberty. Higher lifetime GR gilts (>700 g/d) attain puberty earlier and have a lower anoestrus rate. If gilts attain a target weight (135-150 kg), are adapted to herd health status and have at least one previously recorded estrus, they can be inseminated. Overweight at first breeding and throughout gestation should be avoided. There is no advantage in breeding gilts heavier than 150 kg; at first farrowing the target weight is 180-185 kg. Piglet production at first parity may be increased in gilts with a high GR but the number of stillborn piglets can also be increased. The culling rate over 3 parities for locomotion problems, which is one of the major risk factors for reduced herd retention rate, can be increased in overweight gilts at first breeding (>150-170 kg).","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"281-91"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44636099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-10-12DOI: 10.1530/biosciprocs.18.0014
H. Quesnel
In sows, follicular development is inhibited during lactation, and weaning the piglets allows recruitment and selection of follicles that will undergo preovulatory maturation and ovulate. Lactation inhibits GnRH secretion, and in turn LH secretion, through neuroendocrine stimuli induced by suckling. Pituitary response to GnRH and the sensitivity of the hypothalamo-pituitary unit to oestradiol positive feedback are also reduced. The impact of lactation on the reproductive axis is further complicated by the physiological and metabolic adaptations that are developed for milk production and that depend on nutrient intake, nutrient needs and body reserves. A strongly catabolic state during lactation amplifies the inhibition of LH secretion, thereby inducing a delay of oestrus and ovulation after weaning. Nevertheless, post-weaning ovulation is less delayed nowadays than in the 1970's or 80's. Nutritional deficiency has also deleterious effects on embryo survival, which are likely related to alterations in follicular growth and maturation. The physiological mechanisms by which information on the metabolic changes is transmitted to the hypothalamus-pituitary-ovary axis are not fully understood in the sow. Glucose, insulin and leptin are the most likely signals informing the hypothalamus of the metabolic state, yet their roles have not been definitely established. At the ovarian level, folliculogenesis is likely to be altered by the reduction in insulin and IGF-I concentrations induced by nutritional deficiency. More knowledge is needed at the intrafollicular level to better understand nutritional effects on follicular development, and also on occyte quality and embryo development.
{"title":"Nutritional and lactational effects on follicular development in the pig.","authors":"H. Quesnel","doi":"10.1530/biosciprocs.18.0014","DOIUrl":"https://doi.org/10.1530/biosciprocs.18.0014","url":null,"abstract":"In sows, follicular development is inhibited during lactation, and weaning the piglets allows recruitment and selection of follicles that will undergo preovulatory maturation and ovulate. Lactation inhibits GnRH secretion, and in turn LH secretion, through neuroendocrine stimuli induced by suckling. Pituitary response to GnRH and the sensitivity of the hypothalamo-pituitary unit to oestradiol positive feedback are also reduced. The impact of lactation on the reproductive axis is further complicated by the physiological and metabolic adaptations that are developed for milk production and that depend on nutrient intake, nutrient needs and body reserves. A strongly catabolic state during lactation amplifies the inhibition of LH secretion, thereby inducing a delay of oestrus and ovulation after weaning. Nevertheless, post-weaning ovulation is less delayed nowadays than in the 1970's or 80's. Nutritional deficiency has also deleterious effects on embryo survival, which are likely related to alterations in follicular growth and maturation. The physiological mechanisms by which information on the metabolic changes is transmitted to the hypothalamus-pituitary-ovary axis are not fully understood in the sow. Glucose, insulin and leptin are the most likely signals informing the hypothalamus of the metabolic state, yet their roles have not been definitely established. At the ovarian level, folliculogenesis is likely to be altered by the reduction in insulin and IGF-I concentrations induced by nutritional deficiency. More knowledge is needed at the intrafollicular level to better understand nutritional effects on follicular development, and also on occyte quality and embryo development.","PeriodicalId":87420,"journal":{"name":"Society of Reproduction and Fertility supplement","volume":"66 1","pages":"121-34"},"PeriodicalIF":0.0,"publicationDate":"2020-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47814046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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