Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

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)

Effect of porcine somatotropin on metabolism, testicular size and sperm characteristics in young boars / Efeito da somatotrofina suína sobre o metabolismo, o tamanho testicular e a qualidade espermática de cachaços jovens

The aim of this study was to evaluate the effect of pST injections on metabolism, testicular size, and sperm characteristics in young boars. Sixty 22-day old piglets were divided into two groups: pST (n=30) and Control (n=30). The pST group was submitted to pST injections (90µg/kg body weight) every three days up to 330 days of age. Blood collections were performed weekly. Testicular weight was measures at 22, 82, 142, 202 and 365 days of age. Libido and fresh semen characteristics were evaluated between 150 and 210 days of age. Semen characteristics were also evaluated during a 72h storage period (15ºC). Testosterone, albumin, and phosphorus blood concentrations were higher in the pST group (P<0.05). The pST group had a higher IGF-I concentration in seminal plasma (P=0.05) and higher testicular weight (P<0.001) compared to the Control group. The pST group had higher ejaculate volume (P<0.001), total sperm count (P=0.047) and number of inseminating doses/ejaculate (P=0.047). During the 72h storage period, the pST group had a lower number of morphological alterations (P<0.001) compared to the Control group. In sum, pST injection in young boars increased testosterone concentration, testicular size, and sperm quality.(AU)

O objetivo deste estudo foi determinar o efeito da administração de pST sobre o metabolismo, o tamanho testicular e a qualidade espermática de cachaços jovens. Foram usados leitões com 22 dias de idade, divididos em dois grupos: pST (n=30) e controle (n=30). O grupo pST foi submetido a injeções de pST (90µg/kg de peso vivo) a cada três dias até 330 dias de idade. Peso testicular foi avaliado aos 22, 82, 142, 202 e 365 dias de idade. Libido e qualidade do sêmen fresco foram avaliados entre 150 e 210 dias de idade. Qualidade espermática foi avaliada durante refrigeração (15ºC) por um período de 72 horas. Concentrações sanguíneas de testosterona, albumina e fósforo foram maiores no grupo pST (P<0,05). O grupo pST apresentou maior concentração de IGF-I no plasma seminal (P=0,05) e maior peso testicular, quando comparado ao grupo controle (P<0,001). O grupo pST apresentou maior volume espermático (P<0,001), concentração espermática (P=0,047) e número de doses espermáticas por ejaculado (P=0,047). Durante o período de 72 horas de refrigeração, o grupo pST teve menor número de patologias espermáticas (P<0,001). Assim, conclui-se que a administração de pST aumenta a concentração sanguínea de testosterona, o tamanho testicular e a qualidade espermática de cachaços jovens.(AU)

Expressão gênica em ovócitos suínos de diferentes classificações morfológicas / Gene expression in swine oocytes in different morphological classifications

A produção in vitro de embriões suínos tem alcançado resultados insatisfatórios: ovócitos maturados in vivo produzem uma porcentagem maior de embriões em relação aos maturados in vitro. O sucesso da maturação in vitro está diretamente relacionado com a competência ovocitária. Somente ovócitos competentes são capazes de serem fecundados e terem desenvolvimento embrionário normal. A competência ovocitária pode ser avaliada por vários parâmetros. Recentemente têm sido utilizados como parâmetro os estudos da expressão de genes associados com a competência. O presente trabalho teve por objetivo avaliar diferenças na expressão dos genes BMP15, RYBP, MATER e ZAR1 em ovócitos imaturos de diferentes classes morfológicas, sendo elas: 1, 2, 3 e 4, com a finalidade de proporcionar importantes marcadores moleculares relacionados com a capacidade ovocitária. O RNA total dos ovócitos foi extraído e utilizado como molde para a síntese da primeira fita de cDNA. Os resultados da expressão gênica foram analisados utilizando-se modelo misto, considerando os dados de expressão gênica variável dependente e as classes ovocitárias variáveis independentes. Os genes BMP15, ZAR1 e RYBP apresentaram expressão semelhante nas classes ovocitárias 1, 2 e 3; somente a categoria 4 diferiu na expressão desses genes (P<0,05). O gene MATER foi expresso de forma semelhante em todas as classes ovocitárias estudadas (P>0,05). A técnica de RT-qPCR foi eficiente para detecção desses transcritos em ovócitos de diferentes classes. No entanto, para melhor entendimento do envolvimento desses transcritos na aquisição da competência ovocitária, são necessários mais estudos avaliando ovócitos de diferentes classes morfológicas, em diferentes fases de desenvolvimento, e implicação de outros genes envolvidos com a competência ovocitária.

The in vitro production of pig embryos has achieved unsatisfactory results; in vivo matured oocytes produce a higher percentage of embryos compared to in vitro maturation. The success of in vitro maturation is directly related to oocyte competence. Only competent oocytes are capable of being fertilized and have normal embryonic development. The oocyte competence can be assessed using several parameters. Recently these parameters have been used for gene expression studies associated with competence. This work aimed to evaluate differences in gene expression BMP15, RYBP, MATER, ZAR1 as endogenous control and the constitutive gene GAPDH in immature oocytes of different morphological classes which are: 1, 2, 3 and 4, in order to provide significant molecular markers linked to the ability of development. Oocytes Total RNA was extracted and used as a template for synthesis of the first cDNA strand. The results of gene expression were analyzed using a mixed model, considering the dependent gene expression data and independent ovocitary variable classes. The genes BMP15, RYBP ZAR1 and showed similar ovocitary expression in classes 1, 2 and 3 differ only in category 4 in their expression (P<0.05). The MATER gene was similarly expressed in all ovocitary classes studied (P>0.05). The RTQ-PCR technique was effective for detection of these transcripts in oocytes from different classes. However, for better understanding of the involvement of these transcripts in the acquisition of oocyte competence more studies are needed to evaluate different morphological classes of oocytes at different stages of development and the implication of other genes involved in oocyte competence.

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Lymphangiogenesis: from the pig embryos to cancer: [review] / Linfangiogênese: de embriões de suínos ao câncer: [revisão]

The discovery and the comprehension of lymphatic vessels suffered several historical delays and setbacks. The inherent anatomical problems slowed down the precise identification of the lymphatic system during the development of medical science. Gasparo Aselli, an Italian surgeon and anatomist, was the first to describe the lymphatic vessels in 1627 (De Lacteibus sive Lacteis Venis). However, most original descriptions that report the morphology of the lymphatic system in different organisms were done during the 19th and the 20th centuries. The recent identification of specific lymphatic vasculature molecular markers allows a more accurate identification and characterization of the lymphatic system evolution in different organs, as well as its role in different pathological conditions, including cancer. This study summarizes the current understanding of lymphangiogenesis in tumour progression, as well as it presents a review of the promising data regarding the prognostic value of lymphatic density and the use of therapeutic lymphangiogenic molecules.

A descoberta dos vasos linfáticos e sua compreensão enfrentaram uma série de atrasos e dificuldades históricos. As inerentes dificuldades anatômicas retardaram a identificação precisa da rede vascular linfática durante o desenvolvimento da ciência médica. Gasparo Aselli, um anatomista e cirurgião italiano, foi o primeiro a descrever os vasos linfáticos, em 1627 (De Lacteibus sive Lacteis Venis). Entretanto, a maioria das descrições originais que relatam a morfologia do sistema linfático nos diferentes organismos foi realizada depois, entre os séculos XIX e XX. A recente identificação de marcadores moleculares específicos à vasculatura linfática permite agora identificação e caracterização mais acuradas da evolução da rede linfática nos vários órgãos e em diferentes situações, inclusive no câncer. Esta revisão resume o conhecimento sobre a linfangiogênese na progressão tumoral, bem como apresenta uma síntese dos dados mais promissores em relação ao valor prognóstico da densidade linfática e da utilização das moléculas linfangiogênicas como alvo terapêutico.

Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p > 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 microsec (19 +/- 2% vs. 77 +/- 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 microsec, although this was still lower than the rate of fusion in the CCCs (33 +/- 1% vs. 80 +/- 2%). The rates of cleavage (57 +/- 5%) and blastocyst formation (1 +/- 1%) in the DCC-derived embryos did not differ from those (55 +/- 6% and 3 +/- 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 +/- 2%) showed higher levels of blastocyst formation (p > 0.01) than CCC-derived autologous SCNT embryos (1 +/- 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.

Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

Animal cloning technology with somatic cells provides an alternative tool to conventional methods for producing transgenic animals. Gene targeting in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cells for knocking-out genes. Homologous recombination events in somatic cells are relatively inefficient as compared to those in ES cells, suggesting the need for establishment of efficient gene targeting system in somatic cells. To investigate the efficiency of positive and negative selection for gene targeting in pig fetal fibroblast cells, pig alpha-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neo(r)) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers in this experiment. Following transfection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium containing 5 to 10% serum, Pig fetal fibroblast cells failed to proliferate during drug selection. Increasing serum concentration to 15% of medium yielded less senescent colonies of pig fetal fibroblast cells following drug selection that allowed enough cell colonies to screen genomic DNA. The frequency of gene targeting in pig fetal fibroblast cells with double drug selection was more than 10-fold efficient compared to that with G418 single selection. Double selection method with Neo' and HSV-tk genes could be useful for gene targeting in somatic cells for production of cloned animals carrying targeted endogenous genes.

Expresión de filamentos intermedios durante el desarrollo embrionario de cerdo sus scrofa y bovino bos taurus / Intermediate filaments expression during embryonic development of sus scrofa pig and bos taurus bovine

Utilizando la técnica de inmunoperoxidasa y un panel de anticuerpos monoclonales, se realizó un estudio para caracterizar la expresión de citoqueratinas y vimentina, proteínas constituyentes de los filamentos intermedios, en los tejidos en diferenciación de embriones de cerdo y bovino, en diferentes etapas de la embriogénesis. Ambas especies presentaron características similares en la expresión de citoqueratinas y vimentina. En los embriones prefetales, mayores de 18 mm de longitud cráneo-caudal, ya existe un patrón de citoqueratinas en la mayoría de las células epiteliales, ya que éstas se tiñeron intensamente con los anticuerpos antiqueratinas AE1/AE3 y AE1. En embriones menores, sólo reaccionaron moderadamente con estos anticuerpos, el mesonefros, tubo digestivo y epitelio celómico. La vimentina aparece en embriones más pequeños en el endotelio vascular, en el mesonefros y en algunas células conectivas y del tubo natural. En embriones mayores, además, aparece inmunotinción con este anticuerpo, en el endocardio, dermis, plexos coroídeos, osteoblastos y odontoblasto. La citoqueratina 18 y los controles negativos de las inmunotinciones empleadas, no mostraron reacción positiva en ninguna de las estructuras embrionarias de ambas especies. Nuestros resultados enfatizan la importancia de los filamentos intermedios, especialmente de las citoqueratinas, como marcadores de diferenciación de los tejidos embrionarios y sugieren que en aquellos órganos que comienzan a funcionar tempranamente en el embrión, como el mesonefros y la red vascular, el citoesqueleto se organiza más tempranamente

Morfogénesis externa en el desarrollo embrionario del cerdo / External morphogenesis of the embrionary development of the pig

La embriogénesis en cerdo ha sido estudiada principalmente con énfasis en la anatomía de embriones de longitudes determinadas, o en la evolución de diversos órganos o aparatos. La caracterización del embrión de longitud dada no es útil pues embriones del mismo tamaño suelen presentar diferente grado de desarrollo, lo cual puede inducir a error. Por ello, se estima de utilidad establecer una seriación de estados de desarrollo basados en un conjunto de características. En consecuencia, se emprende el presente trabajo con la finalidad de establecer estados del desarrollo embrionario del cerdo tomando como base los estados del desarrollo embrionario humano definidos por O`Rahilly. Se recolectaron embriones en el matadero SOPROCAR, Temuco, se fijaron en formaldehido 10% y se realizó detallado examen morfológico externo de ellos al microscopio estereoscópico. Cada embrión fue considerado dentro del estado del cual presentaba mayor número de características. Como resultado se definen los estados 14 al 23 del desarrollo embrionario del cerdo