Néstor Sepúlveda

Migration/sedimentation sperm selection method used in bovine in vitro fertilization: Comparison with washing/centrifugation

Sperm selection methods are usually considered for in vitro fertilization (IVF) programs. To achieve a population of viable, motile and morphologically normal spermatozoa, seminal plasma and contaminants must be removed. In this study 2 sperm selection methods were compared with regard to conventional parameters: 1) repeated washing/centrifugation, and 2) migration/sedimentation. Their effect on the fertilization of oocytes aspired from ovaries of slaughtered cows was determined. Frozen-thawed semen was used for all assays (n = 8). The sperm concentration was adjusted to 1.0 × 106 cells/ml for in vitro insemination. Selected oocytecumulus complexes were matured in vitro for 24 h and were co-incubated with spermatozoa prepared by the 2 different methods. Presumptive zygotes were co-cultered in TCM-199. The percentage of motile, viable and morphologically normal spermatozoa was markedly higher (p < 0.05) with the migration-sedimentation method. Triple stain showed that the integrity of the acrosome was significantly better maintained after migration/sedimentation (68.3%) than after washing/centrifugation (53.2%; p < 0.05). In accordance with these results, a high fertilisation rate was achieved after application of the migration/sedimentation method (92.5 vs 69.1%;p < 0.05). It is concluded, that this method is very promising for obtaining spermatozoa with optimal fertilization capacity.

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa.

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.

Índices zoométricos en ovejas criollas araucanas

El ovino criollo Araucano presente en la region de La Araucania, Chile, actualmente se encuentra amenazado por el mestizaje de su material genetico con razas productoras de carne como la Suffolk. Con el objetivo de realizar su caracterizacion racial, se evaluaron 94 hembras provenientes de diferentes rebanos de la region de la Araucania, Chile. Se determino el peso vivo y 14 medidas morfologicas a partir de las cuales se calcularon 9 indices zoometricos: cefalico (ICE), pelviano (IPE), corporal (ICO), proporcionalidad (IPRO), profundidad relativa del torax (IPRP), dactilo toracico (IDT), pelviano transversal (IPET), pelviano longitudinal (IPEL) y espesor relativo de la cana (IERC). Ademas de cada animal se obtuvieron caracteristicas morfologicas y faneropticas. Del analisis estadistico descriptivo se observo que de las medidas zoometricas evaluadas presentaron un rango de variacion entre 4,97% (para la ALC) y 14,9% (para el AGP), reflejando que la muestra evaluada presento un grado medio a alto de homogeneidad. Respecto a los indices zoometricos las ovejas Araucanas se caracterizaron por ser brevilineas (ICO=75,40±6,27), de formatos grandes de acuerdo al indice dactilo-toracico (10,63±0,91) y presentan una grupa convexilinea (IPE<100). De acuerdo a los indices IPET e IPEL existe una tendencia al desarrollo del tejido muscular en la zona de cortes mas valiosos. Respecto las caracteristicas morfologicas y faneropticas se destaca que los ovinos Araucanos son animales que presenta un perfil cefalico recto, mucosas generalmente negras, orejas medianas y horizontales, son animales ventrudos, de ubre pequena sin pigmentacion, y de extremidades con pezunas oscuras. Los resultados obtenidos desprenden indices zoometricos elevados respecto a otros genotipos ovinos, presentando la oveja Araucana tendencia a la aptitud carnica, capacidad que podria asegurar su conservacion al permitir aplicar criterios selectivos que conduzcan a una menor variabilidad en los caracteres etnologicos y funcionales.

Polyunsaturated fatty acid induces cardioprotection against ischemia-reperfusion through the inhibition of NF-kappaB and induction of Nrf2

The mechanistic evidence to support the cardioprotective effects of polyunsaturated fatty acids (PUFA) are controversial. The aim was to test cardioprotective mechanisms induced by PUFA supplementation against cardiac ischemia-reperfusion (IR) injury. Ten-week-old male Wistar rats (225 ± 14 g, n = 14) were divided in two groups: rats without supplementation (n = 7) and a PUFA group, supplemented by PUFA (0.6 g/kg/day; DHA:EPA = 3:1) for eight weeks (n = 7). Hearts were perfused with Krebs–Henseleit buffer for 20 min (control conditions); others were subjected to control conditions, 30 min of global ischemia and 120 min of reperfusion (IR group). Infarct size (IS) and left ventricular developed pressure (LVDP) were measured at 120 min of reperfusion. Oxidative stress biomarkers (TBARS, total carbonyls), antioxidant status (CAT, catalase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase activity and GSH/GSSG ratio), myeloperoxidase activity, ATP levels and nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappaB (NF-κB) were determined in both experimental conditions. At the end of reperfusion, hearts supplemented with PUFA showed lower IS and a higher LVDP compared with the nonsupplemented rats. Hearts in the group supplemented with PUFA showed lower levels of oxidative stress markers and higher antioxidant activity, decreased MPO activity and NF-κB and Nrf2 activation compared with the nonsupplemented group. Cardioprotective effects of PUFA are exerted through induction of anti-inflammatory and antioxidant mechanism at tissue level.

Genotyping of BMPR1B, BMP15 and GDF9 genes in Chilean sheep breeds and association with prolificacy

References1 Barzegari A. et al. (2010) Reprod Domest Anim 45, 666–9.2 Galloway S.M. et al. (2000) Nat Genet 25, 279–83.3 Hanrahan J.P. et al. (2004) Biol Reprod 70, 900–9.4 Wilson T. et al. (2001) Biol Reprod 64, 1225–35.5 Paz E. et al. (2014) Arch Med Vet 46, 327–31.6 Silva B.D. et al. (2010) Anim Genet 42,89–92.7 Drummond A. et al. (2012) Geneious v5.6.Correspondence: E. Paz (e.paz01@ufromail.cl)

Use of Morphometric Variables for Differentiating Spanish Hound Breeds

El termino Podencos se refiere a un grupo de razas de perros originarios de Espana y clasificados en el grupo 5 de razas caninas por la Federacion Cinologica Internacional (FCI). El objetivo del presente trabajo fue comparar caracteristicas morfometricas de las razas caninas de Podencos; Canario, Ibecenco, Andaluz (pequeno, medio y grande) y Orito. Se midieron diez variables morfometricas en 385 animales (245 hembras y 140 machos); longitud de la cabeza, la longitud hocico, altura a la cruz, alzada a la grupa, la longitud del cuerpo, ancho de la cabeza, ancho de la grupa, la profundidad del pecho, la circunferencia del pecho y la circunferencia de la cana. Las variables morfoestructurales fueron mas homogeneas en el Podenco Ibicenco y el Podenco Canario, porque por muchos anos fueron fijados gradualmente los rasgos distintivos para los estandares de cada raza. El analisis discriminante manifesto diferencias morfoestructurales entre todas las razas, lo que se refleja en la significativa distancia de Mahalanobis, y confirmo que el podenco Orito puede ser aceptado como una poblacion de perros diferentes.

Attitude and Willingness to Pay for National and Store Brands of Milk in the South of Chile

ABSTRACT In Chile the milk consumption has maintained between 124 and 134 Ls per capita since 1996. To determine the attitude and willingness to pay for national and store brands of fluid milk in the south of Chile, a survey of 400 people was conducted. A conjoint analysis determined that the consumers experienced a positive reaction toward two national brands (traditional and those emergent in the market) and expressed negative reaction to a store brand. The consumers were determined to pay lower prices in relation to the market value for the store brand and higher prices for national brands.

Determination of fatty acid profile in ram spermatozoa and seminal plasma

Fatty acids are important in male reproductive function because they are associated with membrane fluidity, acrosome reaction, sperm motility and viability, but limited information exists about the fatty acid profile of ram semen. Our aim was to determine the fatty acid composition in ram spermatozoa and seminal plasma. Sixty ejaculates were obtained from three ram (20 ejaculates/ram) using artificial vagina. Ram spermatozoa (RS) and seminal plasma (SP) were separated using centrifugation, and the fatty acids were analysed by gas chromatography. Total lipids obtained in ram spermatozoa were 1.8% and 1.6% in seminal plasma. Saturated fatty acid (SFA) was proportionally major in SP (66.6%) that RS (49.9%). The highest proportions of SFA corresponded to C4:0 (RS = 16.3% and SP = 28.8%) and C16:0 (RS = 16.3% and PS = 20%). The most important unsaturated fatty acid (UFA) was docosahexaenoic acid (DHA), 44.9% in RS and 31.5% in SP. The profile of fatty acid and their proportions showed differences between spermatozoa and seminal plasma.

Detección del Polimorfismo en el Gen del Receptor de Melatonina (MT1) en la Oveja Criolla Araucana

La melatonina es una hormona que regula los ciclos circadianos y muchos de los aspectos reproductivos de los mamiferos y es secretada por la glandula pineal en las horas de ausencia de luz. Esta hormona posee receptores de alta afinidad acoplados a proteinas de tipo G, denominados MT1. Un polimorfismo de la secuencia que codifica para estos receptores estaria involucrado en el control de la reproduccion estacional de los ovinos. El proposito de este estudio busca determinar la presencia del polimorfismo del receptor MT1 en la oveja criolla Araucana, un ovino local en el que se ha registrado un corto anestro reproductivo. Para poder realizar este trabajo se utilizo la tecnica denominada reaccion en cadena de la polimerasa para polimorfismo en el tamano de los fragmentos de restriccion PCR-RFLP, para lo cual, se obtuvieron muestras de ADN genomico de 50 ovejas Araucanas, las cuales fueron digeridas con la endonucleasa de restriccion Mnl1. Se logro identificar la presencia del polimorfismo del receptor MT1 en la oveja Araucana. Los genotipos se hallaron en una frecuencia de 68% para el genotipo +/+, 28% para el genotipo +/- y4% para el genotipo -/-. Este alto porcentaje de animales con genotipo +/+ podria explicar el corto anestro reproductivo que presenta esta raza.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.