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1.
Animal ; 13(S1): s11-s19, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31280748

RESUMO

Milk production by the sow is a major factor limiting the growth and survival of her litter. Understanding the process of morphogenesis of the sow's mammary gland and the factors that regulate mammary development are important for designing successful management tools that may enhance milk production. Primordia of the mammary glands are first observable in the porcine embryo at approximately 23 days of gestation. The glands then progress through a series of morphologically distinct developmental stages such that, at birth, each mammary gland is composed of the teat, an organized fat pad and two separate lactiferous ducts each with a few ducts branching into the fat pad. The glands continue to grow slowly until about 90 days of age when the rate of growth increases significantly. The increased rate of mammary gland growth coincides with the appearance of large ovarian follicles and an increase in circulating estrogen. After puberty, the continued growth of the gland and elongation and branching of the duct system into the fat pad takes place in response to the elevated levels of estrogen occurring as part of the estrous cycles. After conception, parenchymal mass of each gland increases slowly during early pregnancy and then grows increasingly rapidly during the final trimester. This growth is in response to estrogen, progesterone, prolactin and relaxin. Lobuloalveolar development occurs primarily during late pregnancy. By parturition, the fat pad of the mammary gland has been replaced by colostrum-secreting epithelial cells that line the lumen of the alveoli, lobules and small ducts. All mammary glands develop during pregnancy, however, the extent of development is dependent on the location of the mammary gland on the sow's underline. The mammary glands undergo significant functional differentiation immediately before and after farrowing with the formation of colostrum and the transition through the stages of lactogenesis. Further growth of the glands during lactation is stimulated by milk removal. Individual glands may grow or transiently regress in response to the intensity of suckling during the initial days postpartum. Attempts to enhance milk production by manipulation of mammary development at stages before lactation generally have met with limited success. A more in depth understanding of the processes regulating porcine mammary gland morphogenesis at all stages of development is needed to make further progress.


Assuntos
Colostro/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Leite/metabolismo , Suínos/crescimento & desenvolvimento , Animais , Células Epiteliais/metabolismo , Estrogênios/metabolismo , Ciclo Estral , Feminino , Desenvolvimento Fetal , Lactação , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/fisiologia , Parto , Gravidez , Progesterona/metabolismo , Prolactina/metabolismo , Suínos/embriologia , Suínos/fisiologia
2.
J Anim Sci ; 97(5): 1967-1978, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31222274

RESUMO

Pig is one of the major dietary protein sources for human consumption, from which muscle is the largest protein origin. However, molecular mechanisms concerning early porcine embryonic muscle development distinctions between pig breeds are still unclear. In this study, an integrated analysis of transcriptome and miRNAome was conducted using longissimus dorsi muscle of 4 early embryonic stages around the primary myofiber formation time (18-, 21-, 28-, and 35-d post coitus) from 2 pig breeds (Landrace [LR] and Wuzhishan [WZS]) differing in meat mass. The global miRNA/mRNA expression profile showed that WZS prepared for myogenic developmental processes earlier than LR. After identifying and analyzing the interaction network of top 100 up-/down-regulated miRNA and their target genes, we were able to find 3 gene clusters: chromatin modification-related (Chd2, H3f3a, Chd6, and Mll1), myogenesis-related (Pax3, Pbx1, Mef2a, and Znf423), and myosin component-related (Mylk, Myo5a, Mylk4, Myh9, and Mylk2) gene clusters. These genes may involve in miRNA-gene myogenic regulatory network that plays vital role in regulating distinct early porcine embryonic myogenic processes between LR and WZS. In summary, our study reveals an epigenetic-mediated myogenic regulatory axial that will help us to decipher molecular mechanisms concerning early porcine embryonic muscle development distinctions between pig breeds.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , RNA Mensageiro/genética , Suínos/genética , Transcriptoma , Animais , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica/veterinária , Redes Reguladoras de Genes , Masculino , Desenvolvimento Muscular/genética , Especificidade da Espécie , Suínos/embriologia , Suínos/crescimento & desenvolvimento
3.
Anim Reprod Sci ; 205: 150-155, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31076217

RESUMO

As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 µM, 25.0 µM and 50.0 µM). In Experiment 2, inclusion of 50 µM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 µM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P < 0.05). Compared to the control, inclusion of 50 µM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P < 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Embrião de Mamíferos/química , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Suínos/embriologia , Animais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Relação Dose-Resposta a Droga , Ácido Eicosapentaenoico/administração & dosagem , Feminino , Metabolismo dos Lipídeos , Partenogênese , Suínos/fisiologia
4.
Theriogenology ; 132: 95-105, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004879

RESUMO

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.


Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Técnicas de Silenciamento de Genes/veterinária , Partenogênese , Suínos/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Suínos/genética
5.
J Vet Sci ; 20(2): e3, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30944526

RESUMO

The development of long-term surviving fetal cell cultures from primary cell tissue from the developing brain is important for facilitating studies investigating neural development and for modelling neural disorders and brain congenital defects. The field faces current challenges in co-culturing both progenitors and neurons long-term. Here, we culture for the first time, porcine fetal cells from the dorsal telencephalon at embryonic day (E) 50 and E60 in conditions that promoted both the survival of progenitor cells and young neurons. We applied a novel protocol designed to collect, isolate and promote survival of both progenitors and young neurons. Herein, we used a combination of low amount of fetal bovine serum, together with pro-survival factors, including basic fibroblast growth factor and retinoic acid, together with arabinofuranosylcytosine and could maintain progenitors and facilitate in vitro differentiation into calbindin 1+ neurons and reelin+ interneurons for a period of 7 days. Further improvements to the protocol that might extend the survival of the fetal primary neural cells would be beneficial. The development of new porcine fetal culture methods is of value for the field, given the pig's neuroanatomical and developmental similarities to the human brain.


Assuntos
Neurônios/citologia , Células-Tronco/citologia , Telencéfalo/embriologia , Animais , Contagem de Células/veterinária , Células Cultivadas , Feminino , Masculino , Suínos/embriologia , Telencéfalo/citologia
6.
J Anim Physiol Anim Nutr (Berl) ; 103(3): 858-867, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900324

RESUMO

DNA methylation is an important epigenetic strategy for embryo development and survival. The one-carbon metabolism can be disturbed by inadequate provision of dietary methyl donors. Because of the continuous selection for larger litters, it is relevant to explore if highly prolific sows might encounter periods of methyl donor deficiency throughout their reproductive cycles. This study, therefore, assesses the fluctuation(s) in methylation potential (MP) and aims to link possible methyl donor deficiencies to nutrient metabolism. In total, 15 hybrid sows were followed from weaning of the previous reproductive cycle (d-5) to weaning of the present cycle. Blood samples were taken at d-5, 0, 21, 42, 63, 84 and d108 of gestation, the day of parturition (d115), two weeks of lactation (d129) and at weaning (d143). Blood plasma samples were analysed for S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), free methionine, free glycine, acetylcarnitine and 3-hydroxybutyrylcarnitine. Serum samples were analysed for urea and creatinine. Generally, MP (i.e. ratio SAM:SAH) increased throughout gestation (p = 0.009), but strongly fluctuated in the period around parturition and weaning. From d108 to parturition, absolute plasma levels of SAM (p < 0.001), SAH (p = 0.031) and methionine (p = 0.001) increased. The first two weeks of lactation were characterised by an increase in MP (p = 0.039) due to a remaining high value of SAM and a distinct decrease in SAH (p = 0.008). During the last two weeks of lactation, MP decreased (p = 0.038) due to a decrease in SAM (p < 0.001) and a stable value for SAH. The methylation reactions seem to continue after weaning, a period crucial for the follicular and embryonic development of the subsequent litter. This study thus demonstrates that the methylation status fluctuates substantially throughout a sow's reproductive cycle, and further research is needed to identify the factors affecting methylation status.


Assuntos
Ração Animal/análise , Metilação de DNA/fisiologia , Dieta/veterinária , Nutrientes/metabolismo , Suínos/fisiologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Composição Corporal , Peso Corporal , Feminino , Nutrientes/sangue , Gravidez , Suínos/sangue , Suínos/embriologia
7.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
8.
Theriogenology ; 129: 70-76, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30825707

RESUMO

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-ß family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Miostatina/farmacologia , Suínos/embriologia , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/metabolismo
9.
Theriogenology ; 129: 82-89, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30826721

RESUMO

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R®) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis. To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and ß-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 µg/ml). The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 µg/ml, while TRE 20 µg/ml significantly reduced the penetration rate after IVF (p < 0.05) and the blastocyst rate after parthenogenetic activation (p < 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p < 0.001) and increased ROS (p < 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p < 0.001) and the developmental competence after parthenogenetic activation (p < 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 µg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 µg/ml TRE) compared to IVM B (p < 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 µg/ml significantly improved (p < 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels. TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis. In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and ß-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/embriologia , Taninos/farmacologia , Animais , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Quercus/química , Espécies Reativas de Oxigênio/metabolismo
10.
Cell Reprogram ; 21(1): 26-36, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735078

RESUMO

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células , Clonagem de Organismos , Regulação da Expressão Gênica no Desenvolvimento , Suínos/embriologia , Animais , Animais Geneticamente Modificados , Blastocisto/fisiologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Cultivadas , Desenvolvimento Embrionário , Fibroblastos , Técnicas de Transferência Nuclear/veterinária , Células-Tronco Pluripotentes/citologia , Transfecção
11.
Theriogenology ; 123: 185-193, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312936

RESUMO

Maternal undernutrition during the periconceptional period alters the transcriptomic profile of pig endometrium and embryos. Herein, we tested the hypothesis that restricted maternal consumption by females during the periconceptional period impairs the pattern of DNA methylation in both the endometrium and embryos during the peri-implantation period (Day 15-16 of gestation). Affected genes in restricted-diet-fed pig endometrium and embryos were identified using quantitative methylation-specific PCR and comprised those genes which are known to be important in reproductive, metabolic and epigenetic function, thereby exhibiting altered transcriptomic expression in endometrium and embryos of restricted-diet-fed gilts. Specifically, levels of DNA methylation of selected genes with altered expression in the endometrium included acid phosphatase type 2C (PPAP2C), salivary lipocalin (SAL1), endothelin receptor type B (EDNRB), regulator of G-protein signalling 12 (RGS12), type 4 17ß-hydroxysteroid dehydrogenase (HSD17B4), toll-like receptor 3 (TLR3), and adiponectin receptor 1 (ADIPOR1). In embryos, adiponectin receptor 2 (ADIPOR2), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), progestin and adipoQ receptor family member 7 (PAQR7), progesterone receptor membrane component 2 (PGRMC2), steroidogenic acute regulatory protein (STAR), and serpin family A member 1 (SERPINA1) were altered. Finally, 5 acid phosphatase tartrate resistant (ACP5), high mobility group box 2 (HMGB2), and DNA (cytosine-5)-methyltransferase 1 (DNMT1) were altered in both the endometrium and in embryos. In the endometrium, the methylation levels of ACP5 (regulation of endometrial-conceptus iron transport), RGS12 (protein-coupled receptor signalling), and TLR3 (immune response) were increased, while that of EDNRB (corpus luteum maintenance) was decreased. In embryos, the methylation levels of ADIPOR2 (metabolic homeostasis) and DNMT1 (DNA methylation maintenance) were increased. The levels of methylation in other studied endometrial and embryonic genes were unchanged. DNA methylation levels in both the peri-implantation pig endometrium and embryos may be altered in response to female nutritional restriction.


Assuntos
Metilação de DNA/fisiologia , Implantação do Embrião/fisiologia , Endométrio/fisiologia , Privação de Alimentos , Suínos/embriologia , Suínos/fisiologia , Animais , Embrião de Mamíferos/fisiologia , Feminino
12.
Reprod Domest Anim ; 54(3): 520-530, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30578596

RESUMO

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU-23 embryonic culture medium was used for a control group, while NCSU-23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4- and 8-cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2-, 4-, 8-cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p < 0.01). The differentially expressed miRNAs in the 8-cell versus the 4-cell stage in control group as well as in the 8-cell Ber group versus the 8-cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber-regulated miRNAs at the 8-cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8-cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.


Assuntos
Berberina/farmacologia , Desenvolvimento Embrionário/genética , Fertilização In Vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , MicroRNAs/genética , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro/métodos , MicroRNAs/metabolismo , Gravidez , Suínos/embriologia , Zigoto/efeitos dos fármacos , Zigoto/metabolismo
13.
Theriogenology ; 126: 75-80, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30537656

RESUMO

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus , Fertilização In Vitro/veterinária , Oócitos/virologia , Doenças dos Suínos/embriologia , Suínos/embriologia , Animais , Infecções por Circoviridae/embriologia , Meios de Cultura , DNA Viral/isolamento & purificação , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/crescimento & desenvolvimento , Suínos/virologia
14.
Pol J Vet Sci ; 21(3): 609-614, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30468346

RESUMO

Culture gas atmosphere is one of the most important factors affecting embryo development in vitro. The main objective of this study was to compare the effects of CO concentration on the subsequent pre-implantation developmental capacity of pig embryos in vitro, including embryos obtained via parthenogenesis, in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Pig embryos were developed in four different CO2 concentrations in air: 3%, 5%, 10%, or 15%. The cleavage rate of pig parthenogenetic, IVF, or ICSI embryos developed in CO2 concen- trations under 5% was the highest. There were no significant differences in the oocyte cleavage rate in ICSI embryos in CO2 concentrations under 3% and 5% (p>0.05). However, as CO2 levels increased (up to 15%) the blastocyst output on day 7, from parthenogenetic, IVF, and ICSI em- bryos, decreased to 0%. These findings demonstrate that CO2 positively affects the developmen- tal capacity of pig embryos. However, high or low CO2 levels do not significantly improve the developmental capacity of pig embryos. The best results were obtained for all of the pig embryos at a 5% CO2 concentration.


Assuntos
Dióxido de Carbono/farmacologia , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Suínos/embriologia , Animais , Blastocisto/efeitos dos fármacos , Dióxido de Carbono/administração & dosagem , Relação Dose-Resposta a Droga
15.
Yi Chuan ; 40(9): 749-757, 2018 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-30369478

RESUMO

Non-homologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells. It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR in porcine fetal fibroblasts (PFFs), several RNA interference (RNAi) systems were designed to knockdown NHEJ key molecules, such as polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4) and NHEJ1. The results show that siRNA significantly knocked down LIG4, PNKP and NHEJ1 expression. Suppression of PNKP dramatically increased the efficiency of single-strand annealing (SSA), double-strand DNA (dsDNA) and single-strand DNA (ssODN) mediated homology-directed repair (HDR) by 55.7%, 37.4% and 73.1% after transfected with the SSA-GFP reporter, HDR-GFP system or ssODN-GFP system, respectively; whereas knockdown of LIG4 and NHEJ1 repair factors significantly increased dsDNA or ssODN-mediated HDR efficiency by 37.5% and 76.9%, respectively.


Assuntos
Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Interferência de RNA , Suínos/genética , Animais , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Feminino , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Masculino , Reparo de DNA por Recombinação , Suínos/embriologia , Suínos/metabolismo
16.
Anim Reprod Sci ; 197: 305-316, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30197056

RESUMO

Restricted nutritional consumption during the peri-conceptional period affects the potential for DNA methylation and alters endometrial transcriptomic profile during the peri-implantation period. The restricted diet fed to females during the peri-conceptional period may affect the transcriptomic profile in peri-implantation embryos. In the present study, the transcriptome of embryos of normal-diet-fed gilts was determined and compared with that in embryos of restricted-diet-fed gilts during the peri-implantation period. The restricted-diet-fed gilts were fed forage, in which the dose of proteins and energy had been reduced by 30% compared to the normal diet (Polish Norms of Nutrition). To clarify the issue Agilent's Porcine (V2) Two-Color Gene Expression Microarray 4 × 44 was used. Analysis of the microarray data revealed that the expression of 787 genes with known biological function were consistently altered (496 up- and 291 down-regulated) in embryos. The accurately annotated genes were organized into five categories and 18 subcategories containing 62 biological pathways. The qPCR analysis of ten selected genes [i.e., 5 acid phosphatase, tartrate resistant (ACP5), high mobility group box 2 (HMGB2), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), adiponectin receptor 2 (ADIPOR2), DNA (cytosine-5)-methyltransferase 1 (DNMT1), steroidogenic acute regulatory protein (STAR), progesterone receptor membrane component 2 (PGRMC2), progestin and adipoQ receptor family member 7 (PAQR7) and serpin family A member 1 (SERPINA1)] confirmed altered gene expression in embryos of restricted-diet-fed gilts. The insight into embryonic transcriptome indicates that female under-nutrition during the peri-conceptional period may create alterations in the pattern of genes expressed in the peri-implantation embryos.


Assuntos
Implantação do Embrião , Endométrio/fisiologia , Suínos , Transcriptoma , Animais , Metilação de DNA/fisiologia , Implantação do Embrião/fisiologia , Endométrio/metabolismo , Feminino , Fertilização , Suínos/embriologia , Suínos/fisiologia
17.
Genet Sel Evol ; 50(1): 46, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30227828

RESUMO

BACKGROUND: In polytocous livestock species, litter size and offspring weight act antagonistically; in modern pig breeds, selection for increased litter size has resulted in lower mean birth weights, an increased number of small piglets and an increased number of those affected by varying degrees of intrauterine growth retardation (IUGR). IUGR poses life-long challenges, both mental, with morphological brain changes and altered cognition, and physical, such as immaturity of organs, reduced colostrum intake and weight gain. In pigs, head morphology of newborn piglets is a good phenotypic marker for identifying such compromised piglets. Growth retardation could be considered as a property of the dam, in part due to either uterine capacity or insufficiency. A novel approach to this issue is to consider the proportion of IUGR-affected piglets in a litter as an indirect measure of uterine capacity. However, uterine capacity or sufficiency cannot be equated solely to litter size and thus is a trait difficult to measure on farm. RESULTS: A total of 21,159 Landrace × Large White or Landrace × White Duroc piglets (born over 52 weeks) with recorded head morphology and birth weights were followed from birth until death or weaning. At the piglet level, the estimated heritability for IUGR (as defined by head morphology) was low at 0.01 ± 0.01. Piglet direct genetic effects of birth weight (h2 = 0.07 ± 0.02) were strongly negatively correlated with head morphology (- 0.93), in that IUGR-affected piglets tended to have lower birth weights. At the sow level, analysis of the proportion of IUGR-affected piglets in a litter gave a heritability of 0.20 ± 0.06, with high and negative genetic correlations of the proportion of IUGR-affected piglets with average offspring birth weight (- 0.90) and with the proportion of piglets surviving until 24 h (- 0.80). CONCLUSIONS: This suggests that the proportion of IUGR-affected piglets in a litter is a suitable indirect measure of uterine capacity for inclusion in breeding programmes that aim at reducing IUGR in piglets and improving piglet survival.


Assuntos
Retardo do Crescimento Fetal/genética , Seleção Genética , Seleção Artificial , Doenças dos Suínos/genética , Suínos/genética , Animais , Peso ao Nascer , Feminino , Retardo do Crescimento Fetal/veterinária , Masculino , Característica Quantitativa Herdável , Suínos/embriologia , Útero/fisiologia
18.
Pesqui. vet. bras ; 38(9): 1726-1730, set. 2018. tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-976505

RESUMO

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)


Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)


Assuntos
Animais , Suínos/embriologia , Criopreservação/veterinária , Análise do Sêmen/estatística & dados numéricos
19.
Theriogenology ; 120: 147-156, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30121547

RESUMO

Current research suggests that supplementing in vitro culture (IVC) media with vascular endothelial growth factor (VEGF) may have beneficial effects on the development of porcine embryos in vitro. However, the molecular signaling mechanisms underlying this effect are unclear. Therefore, we aimed to investigate the effects of VEGF on molecular signaling events during in vitro embryonic development of porcine embryos. Porcine oocytes matured in vitro were fertilized, and the resultant zygotes were cultured with 5 ng/mL of VEGF supplemented with or without fetal bovine serum from day 4 till day 7. Without VEGF and/or FBS served as the control group. Real-time quantitative PCR was used to detect expression patterns of apoptosis- and oxidative stress-related genes in day 7 blastocysts (BLs). Early-stage apoptosis was detected by annexin-V assays in day 2 and day 7 embryos. We found that the addition of VEGF throughout the culture period with or without FBS supplementation significantly improved embryo survival and development. Supplementation with VEGF in the IVC medium significantly increased early BL formation (p < 0.05), although addition of FBS on day 4 significantly increased hatched BL formation (p < 0.05) regardless of VEGF supplementation. However, supplementation of media with both VEGF and FBS increased the formation of expanded BLs synergistically. The average total cell numbers per BL were significantly (p < 0.05) higher in embryos supplemented with VEGF and FBS than in those supplemented with either VEGF or FBS alone. We also found that accumulation of reactive oxygen species in VEGF-treated embryos was significantly lower (p < 0.05) than that in untreated embryos. The mRNA levels of caspase-3 were significantly lower (p < 0.05), and those of Bcl-2 and Nrf-2 were significantly higher (p < 0.05) in embryos grown in VEGF-supplemented media than in embryos grown in non-supplemented media. Furthermore, on day 2, the numbers of viable embryos (44.06 ±â€¯3.94%) and blastomeres (67.18 ±â€¯3.60%) were significantly higher (p < 0.05), and the numbers of early apoptotic embryos (55.94 ±â€¯3.94) and blastomeres (23.23 ±â€¯4.22) were significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Furthermore, the numbers of early apoptotic cells in BLs on day 7 were also significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Overall, our results indicate that supplementing IVC media with VEGF during in vitro culture of porcine embryos increases their developmental potential.


Assuntos
Blastocisto/efeitos dos fármacos , Fertilização In Vitro/veterinária , Suínos/embriologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Apoptose , Blastocisto/fisiologia , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Estresse Oxidativo , Transdução de Sinais
20.
Theriogenology ; 121: 160-167, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30165304

RESUMO

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.


Assuntos
Metilação de DNA , Oócitos/citologia , Animais , Betaína/farmacologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Cicloleucina/farmacologia , Meiose/genética , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Suínos/embriologia
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