Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Evaluation of runs of homozygosity and genomic inbreeding in Holstein cattle from Colombia / Evaluación de tramos de homocigosidad y consanguinidad genómica en bovinos Holstein de Colombia

Traditional selection programs for dairy cattle, based on quantitative principles, have worked well and allowed strong selection processes in the world over many decades. The objectives of this work were to estimate linkage disequilibrium (LD) levels at varying SNPs densities, to evaluate the effective population size of Holstein cattle, to characterize runs of homozygosity (ROH) distribution through Holstein cattle from Nariño and, to estimate and compare inbreeding coefficient (F) based on genomic markers information, runs of homozygosity (FROH), genomic relationship matrix (FGRM), and excess of homozygous (FSNP). After quality control, the dataset used was composed of 606 Holstein animals and 22200 SNP markers. PLINK program was used to identify LD, Ne, ROH segment and FROH and FSNP, FGRM was calculated with BLUPF90 family of programs. The average of r2 in all chromosomes was 0.011, the highest r2 was found in BTA3 (0.0323), and the lowest in BTA12 (0.0039). 533 ROH segments were identified in 319 animals; findings obtained in this study suggest that on average 0,28% of Holstein genome is autozygous. Total length of ROH was composed mostly of small segments (ROH1-4Mb and ROH4-8Mb). These segments accounted for approximately 96%, while larger ROH (ROH>8Mb) were 3.37% of all ROH detected. Inbreeding averages FROH, FSNP and FGRM methodologies were 0.28%, 3.11% and 3.36% respectively. The Pearson's correlation among these different F values was: 0.49 (FROH-FSNP), 0.25 (FROH-FGRM), 0.22 (FSNP-FGRM). The distribution of ROH shared regions identified on 19 autosome chromosomes, cover a relevant number of genes inside these ROH. Our result evidenced lowest LD extension levels compared with other [...].

Los programas de selección tradicionales para ganado lechero, basados en principios cuantitativos, han funcionado bien y han permitido grandes procesos de selección en el mundo durante muchas décadas. Los objetivos de este trabajo fueron estimar los niveles de desequilibrio de ligamiento (LD) en diferentes densidades de SNPs, evaluar el tamaño efectivo de la población de ganado Holstein, caracterizar tramos de homocigosidad (ROH) distribuidas a través de ganado Holstein de Nariño y, estimar y comparar coeficiente de consanguinidad (F) basado en información de marcadores genómicos, tramos de homocigosidad (FROH), matriz de relación genómica (FGRM) y exceso de SNP homocigotos (FSNP). Después del control de calidad, el conjunto de datos utilizado estaba compuesto por 606 animales Holstein y 22200 marcadores SNP. Se utilizó el programa PLINK para identificar LD, Ne, segmentos de ROH, FROH y FSNP, FGRM se calculó con la familia de programas BLUPF90. El promedio de r2 en todos los cromosomas fue de 0.011, el r2 más alto se encontró en BTA3 (0.0323) y el más bajo en BTA12 (0.0039). Se identificaron 533 segmentos de ROH en 319 animales; los hallazgos obtenidos en este estudio sugieren que, en promedio, el 0,28% del genoma de Holstein es autocigoto. La longitud total de ROH se compuso principalmente de pequeños segmentos (ROH1-4Mb y ROH4-8Mb). Estos segmentos representaron aproximadamente el 96%, mientras que los ROH más grandes (ROH > 8Mb) representaron el 3.37% de todos los ROH detectados. Los promedios de consanguinidad de las metodologías FROH, FSNP y FGRM fueron 0.28%, 3.11% y 3.36% respectivamente. La correlación de Pearson entre estos diferentes valores de F fue: 0.49 (FROH-FSNP), 0.25 (FROH-FGRM), 0.22 (FSNP-FGRM). La distribución de las regiones compartidas de ROH identificadas en 19 cromosomas autosómicos cubre un número importante de genes dentro de estos ROH. Nuestro resultado evidenció niveles más bajos de extensión de LD [...].

Evaluation of runs of homozygosity and genomic inbreeding in Holstein cattle from Colombia / Evaluación de tramos de homocigosidad y consanguinidad genómica en bovinos Holstein de Colombia

Traditional selection programs for dairy cattle, based on quantitative principles, have worked well and allowed strong selection processes in the world over many decades. The objectives of this work were to estimate linkage disequilibrium (LD) levels at varying SNPs densities, to evaluate the effective population size of Holstein cattle, to characterize runs of homozygosity (ROH) distribution through Holstein cattle from Nariño and, to estimate and compare inbreeding coefficient (F) based on genomic markers information, runs of homozygosity (FROH), genomic relationship matrix (FGRM), and excess of homozygous (FSNP). After quality control, the dataset used was composed of 606 Holstein animals and 22200 SNP markers. PLINK program was used to identify LD, Ne, ROH segment and FROH and FSNP, FGRM was calculated with BLUPF90 family of programs. The average of r2 in all chromosomes was 0.011, the highest r2 was found in BTA3 (0.0323), and the lowest in BTA12 (0.0039). 533 ROH segments were identified in 319 animals; findings obtained in this study suggest that on average 0,28% of Holstein genome is autozygous. Total length of ROH was composed mostly of small segments (ROH1-4Mb and ROH4-8Mb). These segments accounted for approximately 96%, while larger ROH (ROH>8Mb) were 3.37% of all ROH detected. Inbreeding averages FROH, FSNP and FGRM methodologies were 0.28%, 3.11% and 3.36% respectively. The Pearson's correlation among these different F values was: 0.49 (FROH-FSNP), 0.25 (FROH-FGRM), 0.22 (FSNP-FGRM). The distribution of ROH shared regions identified on 19 autosome chromosomes, cover a relevant number of genes inside these ROH. Our result evidenced lowest LD extension levels compared with other [...].(AU)

Los programas de selección tradicionales para ganado lechero, basados en principios cuantitativos, han funcionado bien y han permitido grandes procesos de selección en el mundo durante muchas décadas. Los objetivos de este trabajo fueron estimar los niveles de desequilibrio de ligamiento (LD) en diferentes densidades de SNPs, evaluar el tamaño efectivo de la población de ganado Holstein, caracterizar tramos de homocigosidad (ROH) distribuidas a través de ganado Holstein de Nariño y, estimar y comparar coeficiente de consanguinidad (F) basado en información de marcadores genómicos, tramos de homocigosidad (FROH), matriz de relación genómica (FGRM) y exceso de SNP homocigotos (FSNP). Después del control de calidad, el conjunto de datos utilizado estaba compuesto por 606 animales Holstein y 22200 marcadores SNP. Se utilizó el programa PLINK para identificar LD, Ne, segmentos de ROH, FROH y FSNP, FGRM se calculó con la familia de programas BLUPF90. El promedio de r2 en todos los cromosomas fue de 0.011, el r2 más alto se encontró en BTA3 (0.0323) y el más bajo en BTA12 (0.0039). Se identificaron 533 segmentos de ROH en 319 animales; los hallazgos obtenidos en este estudio sugieren que, en promedio, el 0,28% del genoma de Holstein es autocigoto. La longitud total de ROH se compuso principalmente de pequeños segmentos (ROH1-4Mb y ROH4-8Mb). Estos segmentos representaron aproximadamente el 96%, mientras que los ROH más grandes (ROH > 8Mb) representaron el 3.37% de todos los ROH detectados. Los promedios de consanguinidad de las metodologías FROH, FSNP y FGRM fueron 0.28%, 3.11% y 3.36% respectivamente. La correlación de Pearson entre estos diferentes valores de F fue: 0.49 (FROH-FSNP), 0.25 (FROH-FGRM), 0.22 (FSNP-FGRM). La distribución de las regiones compartidas de ROH identificadas en 19 cromosomas autosómicos cubre un número importante de genes dentro de estos ROH. Nuestro resultado evidenció niveles más bajos de extensión de LD [...].(AU)

Lethal effect of high concentrations of Parecoxib and Flunixin meglumine on the in vitro culture of bovine embryos

Since cyclooxygenase (COX) inhibitors have been pointed out as potent ial treatments to increase pregnancy rates after embryo transfer, the present experiment aimed to evaluate the effects of flunixin meglumine (FM) and parecoxib (P), a COX-1 and 2 or COX-2 specific inhibitor, respectively, on the development of bovine embryos until the hatched blastocyst stage. In vitro produced bovine embryos were cultured in media with different concentrations of FM (0.14; 1.4; 14; 140 or 1400 μg/ml) or P (0.09; 0.9; 9; 90 or 900 μg/ml) and the production rates were evaluated. Concentrations of FM ≤ 14 μg/ml and P ≤ 90 μg/ml did not impair embryo development, although compiled data from non-lethal FM concentrations ( ≤ 14 μg/ml) indicated a toxic effect enough to decrease the hatching rate of blastocysts. Concentrations of FM at 140 and 1400 μg/ml and P at 900 μg/ml were lethal as no cleavage was detected on presumptive zygotes.

Lethal effect of high concentrations of Parecoxib and Flunixin meglumine on the in vitro culture of bovine embryos

Since cyclooxygenase (COX) inhibitors have been pointed out as potent ial treatments to increase pregnancy rates after embryo transfer, the present experiment aimed to evaluate the effects of flunixin meglumine (FM) and parecoxib (P), a COX-1 and 2 or COX-2 specific inhibitor, respectively, on the development of bovine embryos until the hatched blastocyst stage. In vitro produced bovine embryos were cultured in media with different concentrations of FM (0.14; 1.4; 14; 140 or 1400 μg/ml) or P (0.09; 0.9; 9; 90 or 900 μg/ml) and the production rates were evaluated. Concentrations of FM ≤ 14 μg/ml and P ≤ 90 μg/ml did not impair embryo development, although compiled data from non-lethal FM concentrations ( ≤ 14 μg/ml) indicated a toxic effect enough to decrease the hatching rate of blastocysts. Concentrations of FM at 140 and 1400 μg/ml and P at 900 μg/ml were lethal as no cleavage was detected on presumptive zygotes.(AU)

Temperature gradients in female reproductive tissues and their potential significance

This review challenges the long-standing dogma that deep-body temperature should be regarded as uniform. Not only may this not be so, but small gradients in temperature in and across reproductive tissues might have assumed functional importance during the evolution of eutherian mammals. Temperature gradients within the Fallopian tubes of estrous animals are interpreted in a context of preovulatory storage of viable spermatozoa and their periovulatory activation and release from the functional reservoir in the caudal isthmus. Proposals concerning the response of potentially-fertilizing spermatozoa to increasing temperature along the isthmus of the Fallopian tube are recalled, and application of the term thermotaxis to this phase of sperm migration is assessed critically. Classical findings on the temperature of Graafian follicles in rabbits and women are highlighted, and more recent work on temperatures in pig preovulatory follicles is considered in detail. Although an experimental approach involving anaesthesia and infrared sensing is open to criticism, the finding that preovulatory follicles are cooler than ovarian stroma cannot be discounted as artifact. Instead, evidence for endothermic reactions that act to lower temperature within pre-ovulatory follicles is presented together with a description of relevant counter-current vascular physiology that enables maintenance of a cooler follicular temperature. As to future experimental work, the possibility is raised that temperatures may not be uniform across the cytoplasm of maturing oocytes nor at different stages of the cell cycle in very young embryos. These proposals lead to speculation that temperature may be exploited at a molecular level to modulate unfolding gene expression in zygotes and early cleavage stage embryos. Modern micro-imaging technology needs to be applied to such concepts.

Temperature gradients in female reproductive tissues and their potential significance

This review challenges the long-standing dogma that deep-body temperature should be regarded as uniform. Not only may this not be so, but small gradients in temperature in and across reproductive tissues might have assumed functional importance during the evolution of eutherian mammals. Temperature gradients within the Fallopian tubes of estrous animals are interpreted in a context of preovulatory storage of viable spermatozoa and their periovulatory activation and release from the functional reservoir in the caudal isthmus. Proposals concerning the response of potentially-fertilizing spermatozoa to increasing temperature along the isthmus of the Fallopian tube are recalled, and application of the term thermotaxis to this phase of sperm migration is assessed critically. Classical findings on the temperature of Graafian follicles in rabbits and women are highlighted, and more recent work on temperatures in pig preovulatory follicles is considered in detail. Although an experimental approach involving anaesthesia and infrared sensing is open to criticism, the finding that preovulatory follicles are cooler than ovarian stroma cannot be discounted as artifact. Instead, evidence for endothermic reactions that act to lower temperature within pre-ovulatory follicles is presented together with a description of relevant counter-current vascular physiology that enables maintenance of a cooler follicular temperature. As to future experimental work, the possibility is raised that temperatures may not be uniform across the cytoplasm of maturing oocytes nor at different stages of the cell cycle in very young embryos. These proposals lead to speculation that temperature may be exploited at a molecular level to modulate unfolding gene expression in zygotes and early cleavage stage embryos. Modern micro-imaging technology needs to be applied to such concepts.(AU)