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1.
Int J Mol Sci ; 22(5)2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33668187

RESUMO

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.


Assuntos
Animais Geneticamente Modificados/genética , Antígenos Heterófilos/biossíntese , Sistemas CRISPR-Cas , Galactosiltransferases/antagonistas & inibidores , Oxigenases de Função Mista/antagonistas & inibidores , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , Zigoto/fisiologia , Animais , Feminino , Edição de Genes , Suínos
2.
Data Brief ; 35: 106939, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33686370

RESUMO

The COVID-19 pandemic has hit humanity, straining health care systems, economies, and governments worldwide. In one of the responses to the pandemic, a big global effort has been mounted to collect, analyze, and make data publicly available. However, many of the existing COVID-19 public datasets are (i) aggregated at country level, and (ii) tend not to bring the COVID-19-specific data coupled with socio-demographic, economic, public policy, health, pollution and environmental factors, all of which may be key elements to study the transmission of the SARS-CoV-2 and its severity. To aid the evaluation of the determinants and impact of the COVID-19 pandemic at a large scale, we present here a new dataset with socio-demographic, economic, public policy, health, pollution and environmental factors for the European Union at the small regions level (NUTS3). The database is freely accessible at http://dx.doi.org/10.17632/2ghxnrkr9p.4. This dataset can help to monitor the COVID-19 mortality and infections at the sub-national level and enable analysis that may inform future policymaking.

3.
Anim Sci J ; 92(1): e13534, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33638256

RESUMO

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.


Assuntos
Sistemas CRISPR-Cas , Eletroporação/métodos , Fertilização In Vitro/veterinária , Edição de Genes/métodos , Edição de Genes/veterinária , Oligodesoxirribonucleotídeos , Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras)/genética , Suínos/embriologia , Suínos/genética , Zigoto , Animais , Blastocisto
4.
Anim Biotechnol ; 32(2): 147-154, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31558095

RESUMO

CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.

5.
Adv Healthc Mater ; 10(6): e2001384, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33274846

RESUMO

The use of conductive materials to promote the activity of electrically responsive cells is an effective means of accelerating wound healing. This article focuses on recent advancements in conductive materials, with emphasis on overviewing their incorporation with non-conducting polymers to fabricate electroactive wound dressings. The characteristics of these electroactive dressings are deliberated, and the mechanisms on how they accelerate the wound healing process are discussed. Potential directions for the future development of electroactive wound dressings and their potential in monitoring the course of wound healing in vivo concomitantly are also proposed.


Assuntos
Bandagens , Cicatrização , Condutividade Elétrica , Polímeros
6.
Acta Vet Hung ; 68(3): 298-304, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33221737

RESUMO

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.


Assuntos
Blastocisto/metabolismo , Curcumina/farmacologia , Técnicas de Cultura Embrionária/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Sus scrofa/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária/métodos , Peróxido de Hidrogênio/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
7.
BMC Biotechnol ; 20(1): 40, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811500

RESUMO

BACKGROUND: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. RESULTS: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. CONCLUSIONS: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.


Assuntos
Sistemas CRISPR-Cas , Eletroporação/métodos , Fertilização In Vitro/métodos , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Edição de Genes/métodos , Zigoto/metabolismo , Animais , Animais Geneticamente Modificados , Blastocisto , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dissacarídeos , Feminino , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , RNA Guia , Suínos , Transplante Heterólogo
8.
Mol Biol Rep ; 47(7): 5073-5079, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32519310

RESUMO

The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.


Assuntos
Sistemas CRISPR-Cas , Eletroporação/métodos , Marcação de Genes/métodos , Suínos/genética , Animais , Blastocisto/metabolismo , Células Cultivadas , Eletroporação/veterinária , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Galactosiltransferases/genética , Marcação de Genes/veterinária , Grelina/genética , Proteínas de Homeodomínio/genética , Oxigenases de Função Mista/genética , RNA Guia/genética , Suínos/fisiologia , Transativadores/genética , Zigoto/metabolismo
9.
Anim Sci J ; 91(1): e13386, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32512638

RESUMO

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.


Assuntos
Proteína 9 Associada à CRISPR , Eletroporação/métodos , Eletroporação/veterinária , Desenvolvimento Embrionário/genética , Edição de Genes , Marcação de Genes/veterinária , Miostatina/genética , RNA Guia , Suínos/embriologia , Suínos/genética , Zigoto , Animais , Blastocisto , Proteína 9 Associada à CRISPR/farmacologia , Relação Dose-Resposta a Droga
11.
Mol Reprod Dev ; 87(4): 471-481, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32166879

RESUMO

Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.


Assuntos
Edição de Genes/métodos , Proteínas de Homeodomínio/genética , Mosaicismo , Fenótipo , Suínos/genética , Transativadores/genética , Alelos , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Diabetes Mellitus , Modelos Animais de Doenças , Transferência Embrionária , Feminino , Proteínas de Homeodomínio/metabolismo , Masculino , Taxa de Mutação , Pâncreas/metabolismo , Pâncreas/patologia , Sêmen/metabolismo , Espermatozoides/metabolismo , Transativadores/metabolismo , Zigoto/metabolismo
12.
Nutrients ; 11(9)2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540208

RESUMO

Anorexia nervosa (AN) is a psychiatric disorder affected by psychological, environmental, and biological factors. Individuals with AN avoid high-fat, high-calorie diets and have shown abnormal metabolism of fatty acids (FAs), which are essential for brain and cognitive/neuropsychiatric health. To clarify the relationship between FAs and AN, fasting and postprandial plasma FAs in AN patients and age-matched control women were analyzed via mass-spectrometry. Clinical phenotypes were assessed using Becker Anxiety Inventory and Becker Depression Inventory. AN patients and controls exhibited different FA signatures at both fasting and postprandial timepoints. Lauric acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and alpha-linoleic acid (ALA) were higher in AN than in controls (lauric acid: 15,081.6 ± 14,970.2 vs. 8257.4 ± 4740.2 pmol/mL; ALA at fasting: 2217.7 ± 1587.6 vs. 1087.9 ± 821.2 pmol/mL; ALA at postprandial: 1830.9 ± 1115.6 vs. 1159.4 ± 664.7 pmol/mL. EPA: 33,788.3 ± 17,487.5 vs. 22,860.6 ± 12,642.4 pmol/mL; DPA: 32,664.8 ± 16,215.0 vs. 20,969.0 ± 12,350.0 pmol/mL. FDR-adjusted p-values < 0.05). Food intake and AN status modified the correlations of FAs with body mass index (BMI), depression, and anxiety. Desaturases SCD-18 and D6D showed lower activities in AN compared to controls. Altered FA signature, specifically correlations between elevated n-3 FAs and worsened symptoms, illustrate metabolic underpinnings in AN. Future studies should investigate the mechanisms by which FA dysregulation, specifically elevated n-3 FAs, affects AN risk and outcome.


Assuntos
Anorexia Nervosa/sangue , Ingestão de Alimentos/fisiologia , Ácidos Graxos/sangue , Adulto , Anorexia Nervosa/psicologia , Ansiedade/sangue , Depressão/sangue , Ácido Eicosapentaenoico/sangue , Jejum , Ácidos Graxos Dessaturases , Elongases de Ácidos Graxos , Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-6/sangue , Ácidos Graxos Insaturados/sangue , Feminino , Humanos , Período Pós-Prandial
13.
Animals (Basel) ; 9(9)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31443357

RESUMO

Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3-alone and in combination-were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.

14.
In Vitro Cell Dev Biol Anim ; 55(8): 598-603, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297696

RESUMO

The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Eletroporação/métodos , Edição de Genes , Genoma , Mutação/genética , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Bovinos , Embrião de Mamíferos/metabolismo , Taxa de Mutação
15.
J Reprod Dev ; 65(5): 475-479, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31178553

RESUMO

The introduction of exogenous molecules into embryos is required for analyses of molecular dynamics and specific gene functions during early embryonic development. Electroporation is an effective method to transport exogenous molecules into cells, but is rarely used in bovine embryos. First, we evaluated the viability of in vivo-derived bovine blastocysts after electroporation with fluorescein (FAM) labeled-oligonucleotides with varying pulse numbers (3, 5, 7, and 10), while keeping the pulse duration at 1 msec and the electric field of 20 V/mm. Next, we examined the effects of zona pellucida status on blastocyst quality after electroporation, by comparing the average diameter of blastocysts before and after electroporation using blastocysts with intact zona pellucida and hatching/hatched blastocysts. Electroporation successfully introduced exogenous molecules into in vivo-derived bovine blastocysts without loss of viability. Moreover, the status of the zona pellucida may be associated with the quality of blastocysts after electroporation.


Assuntos
Blastocisto/citologia , Eletroporação , Fertilização In Vitro/veterinária , Zona Pelúcida/fisiologia , Animais , Bovinos , Contagem de Células , Sobrevivência Celular , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Fluoresceína/química , Oligonucleotídeos/química
16.
Anim Sci J ; 90(6): 712-718, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30977253

RESUMO

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti-Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross-sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.


Assuntos
Hormônio Antimülleriano/sangue , Cruzamento , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Suínos/sangue , Suínos/fisiologia , Animais , Blastocisto , Feminino , Fertilização , Técnicas de Maturação in Vitro de Oócitos , Tamanho da Ninhada de Vivíparos , Masculino , Reprodução
17.
Acta Vet Hung ; 67(1): 106-114, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30922097

RESUMO

Tris (hydroxymethyl) aminomethane (Tris) has been used as a pH regulator for buffering the pH of dilution extenders for boar semen, such as the Modena extender. The purpose of the present study was to assess the effects of Tris supplementation at different concentrations (0, 8, 24 and 72 µM) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 µM of Tris gave significantly higher percentages of sperm viability and plasma membrane integrity than those of the control group at any time point of assessment (0 h and 3 h post-thawing) (P < 0.05). However, there were no significant differences in the acrosome integrity parameter among the groups. Higher percentages of sperm motility were observed in the spermatozoa cryopreserved with 24 µM of Tris compared to the control groups when the samples were analysed 0 h after thawing (P < 0.05). However, an increase of the Tris concentration to 72 µM did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 µM Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 µM Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Suínos , Trometamina/farmacologia , Animais , Sobrevivência Celular , Masculino
18.
In Vitro Cell Dev Biol Anim ; 55(4): 237-242, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30820813

RESUMO

The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Diferenciação Celular , Eletroporação/métodos , Edição de Genes , Genoma , Mutação/genética , Oócitos/metabolismo , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Desenvolvimento Embrionário , Taxa de Mutação , RNA Guia/metabolismo , Suínos
19.
Reprod Domest Anim ; 54(5): 750-755, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30788874

RESUMO

The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25°C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 hr was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.


Assuntos
Blastocisto/efeitos dos fármacos , Ácido Clorogênico/farmacologia , Técnicas de Cultura Embrionária/veterinária , Zigoto/crescimento & desenvolvimento , Animais , Temperatura Baixa , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro/veterinária , Líquido Folicular/fisiologia , Soroalbumina Bovina/farmacologia , Suínos
20.
J Reprod Dev ; 65(3): 209-214, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-30726783

RESUMO

Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/µl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/µl each (20 ng/µl group) or 100 ng/µl each (100 ng/µl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/µl group was significantly higher (P < 0.05) than that in the 20 ng/µl group. Although no blastocysts from the 20 ng/µl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/µl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.


Assuntos
Sistemas CRISPR-Cas , Citoplasma/genética , Mosaicismo , Alelos , Animais , Animais Geneticamente Modificados , Blastocisto/citologia , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro , Edição de Genes , Masculino , Microinjeções , Mutação , Nucleotídeos/genética , Oócitos/citologia , Suínos , Zigoto
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