Humberto Cisale

... or even a sow in oestrus is not always feasible under field conditions, particularly when the subject is a mature, intractable boar (Fraser 1971). ... to the animals in two phases: first, xylazine (Sedomin 10 per cent; Konig) and diazepam (Diazepan 5 mg/ml; Lamar) were administered ...

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M-0.25 M - 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and...

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M-0.25 M - 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and...

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 ml of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 Me0.25 M e 0.75 M or 1 M su-crose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopres-ervation system-vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently , its use at 0.75 M or 1 M wouldn't be recommended.

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 μm2, perimeter 13.16 μm, length 5.12 μm, width 2.81 μm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.

Sperm &amp;#39;tail stump&amp;#39; defect was found in ejaculates of a wild boar maintained in captivity. It was in good physical condition, the testes and genital tract were found to be of normal size and consistency. There was no evidence of macroscopic abnormalities at the clinical analysis and at necropsy. The volume and concentration of the semen samples obtained by electroejaculation were lower than normal. The slides examined contained a high level of abnormal spermatozoa (52.7%). The most frequent morphological finding was a droplet-like form attached to the base of the head or a very short stump. The non-stumped spermatozoa had no normal tail but a shortened one. Analysing the histological structure with light microscopy, no ring of spermatozoa was observed lining the lumen of the seminiferous tubules and the characteristically cellular structure was not conserved. The ultrastructural examination evidenced a disorganisation of the normal tubular structure of the flagellum, with lost of regular pattern of the axial bundle of fibrils and the mitochondrial helix. The origin of this abnormality is unknown.

The aim of this study was to describe the morphology, morphometry and determine the DNA content of yak sperm nuclei (Bos grunniens). Nuclear morphology observations were performed using light microscopy on slides stained with Feulgen reaction. Abnormalities percentages were determined: pyriform (1.2%), globose (0.2%), small (0.2%), elongated (0.1%). Morphometric measurements were made from digitized images of sperm nuclei stained with the Feulgen reaction. Mean values and standard deviations were obtained: area 21.98 ± 2.60 μm2, length 7.34 ± 0.33 μm, width 3.78 ± 0.27 μm, perimeter 88 ± 19.26 μm, roundness 1.27 ± 0.04, elongation 1.95 ± 0.11 and equivalent diameter 5.28 ± 0.27 μm. According to our knowledge this is the first time that haploid DNA content is determined. In this species 3.42 pg value that was similar to that measured in other species of the family Bovidae and other higher mammals.

RESUMEN El potencial fecundante del semen depende tanto de la movilidad espermática como de la integridad y funcionamiento de sus membranas. El objetivo de este trabajo fue determinar mediante una triple tinción fluorocrómica (Hoechst 33342, Ioduro de Propidio, Pisum Sativum-Isotiocianato de Fluoresceína) la calidad de las membranas espermáticas en semen porcino fresco y refrigerado a 17ºC. Se analizaron 14 muestras de semen fresco y de semen refrigerado provenientes de cuatro verracos terminales híbridos cruza Austral. Se incubó el semen diluido con Pisum Sativum-Isotiocianato de Fluoresceína (0,01%), Ioduro de Propidio (0,05%) y Hoechst (0,1%). Los resultados se analizaron mediante estadística descriptiva. El porcentaje de células con acrosoma intacto fue inferior en el semen refrigerado tanto para espermatozoides con membrana plasmática intacta (Fresco: 59,6%; Refrigerado: 49,92%) como lesionada (Fresco: 16,74%; Refrigerado: 10,17%). Se pudo evidenciar que durante el proceso de refrigeración la membrana más afectada es la acrosomal. ABSTRACT The fertilizing potential of sperm depends on both motility and the integrity and function of their membranes. The aim of this work was the assessment of sperm membranes in fresh and chilled boar semen and chilled to 17°C using a triple fluorochromicstain (Hoechst 33342, Propidium Iodide, Pisum Sativum-Fluorescein Isothiocyanate). We studied 14 samples of fresh and chilled semen and chilled semen. The samples

RESUMEN La utilización de la inseminación artificial en la especie porcina se ha incrementado enormemente en los últimos años. Cuando se utiliza semen congelado se obtienen bajos porcentajes de concepción en comparación con los obtenidos con semen fresco o refrigerado. El objetivo de este trabajo fue comparar el efecto de diferentes diluyentes comerciales para semen refrigerado en el proceso de congelado y descongelado. Se utilizaron tres diluyentes de larga duración (Mr-A ® , Androstar ® y Porci-Star ®) y uno de mediana duración (M III ®). Se evaluaron: movilidad, integridad y funcionalidad de membrana plasmática e integridad acrosómica, en cuatro etapas diferentes del proceso. No se encontraron diferencias significativas en los parámetros de calidad evaluados por lo que los diferentes prediluyentes no afectarían el proceso de congelado ABSTRACT Artificial insemination in pigs has increased enormously in the last years. When performed using frozen/thawed semen low conception rates are obtained, compared with those of fresh or cooled semen. The aim of this study was to compare the effect of different commercial extenders for cooled semen in the freezing/thawing process. Three long term extenders (MR-A ® , Androstar ® and Porci-Star ®) and one medium term (M III ®) were used. We evaluated: motility, integrity and functionality of the plasmatic membrane and acrosomal integrity in four different stages of the process. No significant differences in the quality parameters were found, so the different extenders do not affect the freezing process.

El potencial fecundante del semen depende tanto de la movilidad espermática como de la integridad y funcionamiento de sus membranas. El objetivo de este trabajo fue determinar mediante una triple tinción fluorocrómica (Hoechst 33342, Ioduro de Propidio, Pisum Sativum-Isotiocianato de Fluoresceína) la calidad de las membranas espermáticas en semen porcino fresco y refrigerado a 17ºC. Se analizaron 14 muestras de semen fresco y de semen refrigerado provenientes de cuatro verracos terminales híbridos cruza
Austral. Se incubó el semen diluido con Pisum Sativum-Isotiocianato de Fluoresceína (0,01%), Ioduro de Propidio (0,05%) y Hoechst (0,1%). Los resultados se analizaron mediante estadística descriptiva. El porcentaje de células con acrosoma intacto fue inferior en el semen
refrigerado tanto para espermatozoides con membrana plasmática
intacta (Fresco: 59,6%; Refrigerado: 49,92%) como lesionada
(Fresco: 16,74%; Refrigerado: 10,17%). Se pudo evidenciar que
durante el proceso de refrigeración la membrana más afectada es la acrosomal.

La utilización de la inseminación artificial en la especie porcina se ha incrementado enormemente en los últimos años. Cuando se utiliza semen congelado se obtienen bajos porcentajes de concepción en comparación con los obtenidos con semen fresco o refrigerado. El objetivo de este trabajo fue comparar el efecto de diferentes diluyentes comerciales para semen refrigerado en el proceso de congelado y descongelado. Se utilizaron tres diluyentes de larga duración (Mr-A®, Androstar® y Porci-Star®) y uno de mediana duración (M III®). Se evaluaron: movilidad, integridad y funcionalidad de membrana
plasmática e integridad acrosómica, en cuatro etapas diferentes del proceso. No se encontraron diferencias significativas en los parámetros de calidad evaluados por lo que los diferentes prediluyentes no afectarían el proceso de congelado.

La vitrificación de tejido ovárico permite conservar gran cantidad de ovocitos contenidos en folículos preantrales (FPA). El objetivo del presente trabajo fue analizar el efecto de distintas combinaciones de crioprotectores en la preservación de la estructura histológica de FPA porcinos durante la exposición (toxicidad) y en el proceso de vitrificación. Se analizó la respuesta frente a etilenglicol, dimetilsulfóxido (DMSO), y etilenglicol + DMSO, en presencia de sacarosa (0,25M); y la respuesta a etilenglicol asociado a concentraciones crecientes de sacarosa (0M; 0,25M; 0,75M y 1,0M). Los FPA primordiales
tratados con etilenglicol presentaron menor cantidad de alteraciones morfológicas que los expuestos a DMSO y a la combinación de etilenglicol + DMSO (Control: 87%; etilenglicol: 52%; DMSO: 17% y etilenglicol + DMSO: 26%; Friedman, p<0,05). En los FPA primarios el etilenglicol resultó menos tóxico (Control: 70%; etilenglicol: 34%), mientras que la respuesta a la vitrificación del grupo etilenglicol + DMSO fue superior (20%). La vitrificación en presencia de concentraciones crecientes de sacarosa disminuyó significativamente el porcentaje de folículos primordiales normales (Control:64%; 0M: 48%; 0,25M: 42%; 0,75M: 12%; 1M: 9%). Para todos los tratamientos, los folículos primarios presentaron más daños. En conclusión, el medio de elección para la vitrificación resultó ser TCM 199-Hepes con 30% etilenglicol y 0 o 0,25M de sacarosa.

La inseminación artificial bovina ha sido la técnica de reproducción asistida de mayor difusión por las ventajas que presenta en el mejoramiento genético, la prevención y
control de enfermedades de transmisión sexual, la posibilidad de emplear toros con facilidad de parto y la optimización del manejo reproductivo del rodeo. La Inseminación Artificial a Tiempo Fijo (IATF) suma beneficios tales como evitar la detección de celo, acortar el período de anestro post-parto y concentrar los partos en un período breve. El éxito de la IATF depende de múltiples factores entre los que se encuentra la calidad seminal. Ningún examen de semen in vitro presenta una correlación alta con la fertilidad, por lo cual debe desarrollarse un protocolo de control de calidad que estudie el mayor número posible de características de los espermatozoides. La selección de mejores muestras para IATF permitiría evitar la repetición en las inseminaciones o el repaso con monta natural. El objetivo del presente trabajo es encontrar indicadores seminales, a través de los análisis de rutina y del núcleo espermático, que estén asociados con tasas de preñez elevadas para IATF y permitan predecir el comportamiento de las dosis a campo. Se tomarán muestras aleatorias de cada partida de las dosis seminales congeladas ad hoc y se realizará el control de calidad espermático de rutina y el control nuclear. Se evaluarán: parámetros de motilidad espermática obtenidos por análisis computarizado (CASA),
morfología, viabilidad, integridad acrosómica y funcionalidad de la membrana espermática. La evaluación del núcleo espermático incluirá: morfología nuclear, maduración de la cromatina, decondensación y fragmentación nuclear. Esta última, se realizará a través de una prueba desarrollada en nuestro laboratorio (FCV-UBA). Los responsables del CIAVT seleccionarán los establecimientos donde se realizarán los
protocolos de IATF, así como los profesionales responsables de implementarlos, quienes recolectarán los resultados de preñez a campo. Sobre la base de estos resultados preliminares de nuestro grupo de trabajo y la experiencia de campo de los responsables del CIAVT, se espera encontrar asociación entre algunos de los parámetros seminales analizados y los porcentajes de preñez obtenidos por la técnica de IATF. Este proyecto intenta encontrar la existencia deindicadores de clasificación de fertilidad y de pérdidas embrionarias tempranas, lo que permitiría realizar una selección de los individuos más aptos para ser empleados como donantes para IATF. De esta forma, se podrían comercializar muestras de semen especialmente seleccionadas, con el valor agregado que
esto conlleva.

To our knowledge, the value of the haploid DNA content (C-value) of Ovis musimon (mouflon) has not been previously published. Therefore, the aim of the present work was to determine the C-value and the nuclear area of O. musimon sperm cells and compare both parameters with those of Ovis aries. Feulgen reaction, which is specific and stoichiometric for DNA, was carried out on semen smears. The C-value and sperm nuclear area were determined using microspectrophotometry and Gallus domesticus erythrocytes as standard species. The C-value of O. musimon was 3.02 ± 0.04 pg, and the sperm nuclear area was 23.92 ± 0.89 μm(2). The C-value and the sperm nuclear area of O. aries were 3.07 ± 0.03 pg and 22.98 ± 0.86 μm(2) respectively. The O. musimon C-value was not significantly different (P &amp;gt; 0.05) from that of O. aries, indicating that both species may have a very close phylogenetic relation.

Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a direct interaction between these vesicles and sperm, whereas inhibition of some capacitation-dependent features suggests a stabilizing function for exosomes in boar semen.

Sperm ‘tail stump’ defect was found in ejaculates of a wild boar maintained in captivity. It was in good physical condition, the testes and genital tract were found to be of normal size and consistency. There was no evidence of macroscopic abnormalities at the clinical analysis and at necropsy. The volume and concentration of the semen samples obtained by electroejaculation were lower than normal. The slides examined contained a high level of abnormal spermatozoa (52.7%). The most frequent morphological finding was a droplet-like form attached to the base of the head or a very short stump. The non-stumped
spermatozoa had no normal tail but a shortened one. Analysing the histological structure with light microscopy, no ring of spermatozoa was observed lining the lumen of the seminiferous tubules and the characteristically cellular structure was not conserved. The ultrastructural examination evidenced a disorganisation of the normal tubular structure of the flagellum, with lost of regular pattern of the axial bundle of fibrils and the mitochondrial helix. The origin of
this abnormality is unknown.