Abstract Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), disease that causes a syndrome of bad nutrient absorption, weight loss and eventually death. The intestine is the main target organ where the infection develops; however, there is evidence of infection by MAP in extra-intestine sites of sheep, including mesenteric nodes and semen. The aim of the study was to identify the presence of MAP in reproductive tissue and semen of infected Pelibuey rams in clinical state of PTB. Seven rams were used in clinical PTB state and a non-infected ram by MAP of the Pelibuey breed, confirmed by serology, nPCR and bacteriological culture, with average weight and age of 57.23 ± 1.73 kg and 2.91 ± 0.17 years, respectively. The presence of MAP was identified in different tissue samples: spleen (1/7, 14.3% and 2/7, 28.6%), small intestine (3/7, 42.9% and 4/7, 57.1%) and mesenteric lymph nodes (3/7, 42.9% and 3/7, 42.9%), with nPCR and culture, respectively. It was also identified in epididymis tissue (1/7, 14.3%), Cowper gland (2/7, 28.6%) and prostate (1/7, 14.3%), using nPCR, although without detection in culture. It was identified in testicular tissue in 42.8% (3/7; culture or nPCR technique), but in 28.6% (2/7) with both techniques. Finally, the presence of MAP was identified in 42.9% (3/7) of semen samples with nPCR; however, it was not detected through culture. In conclusion, the presence of MAP was identified in lymphatic, digestive tissue, and semen; the presence of MAP was reported for the first time in epididymis, Cowper gland, prostate and testicles of infected Pelibuey rams.

Abstract: The effect of resveratrol on the in vitro maturation (IVM) of ovine (Ovis aries) oocytes and the development of handmade cloned embryos was evaluated. The nuclear maturation and reactive oxygen species (ROS) levels in the oocytes, as well as the early development and morphological cloned embryo quality, were evaluated under different resveratrol concentrations (0, 0.5, 2 and 5 µM). After IVM, no significant difference was observed in the maturation rate of oocytes treated with 0.5 µM (81.3 %) and 2 µM (72 %) resveratrol compared to that of the control group (0 µM) (74.2 %), but the rate significantly decreased at 5 µM (56 %) (p < 0.05). When the oocyte ROS levels were determined, no significant differences among the groups were observed (p > 0.05). For cloned embryo development, the embryos obtained from the oocytes treated with 0.5 µM resveratrol showed higher (p < 0.05) compacted morula rates (10.7 %) compared to the embryos obtained from the oocytes treated with 0, 2 and 5 µM (6.2, 0 and 0 %, respectively). Regarding embryo morphological quality, the embryos from the oocytes treated with 0.5 µM resveratrol showed a lower rate of poor quality morulae (4.7 %) in comparison to those treated with 0, 2 and 5 µM (23.8, 23.3 and 33.3 %, respectively) (p < 0.05). In conclusion, resveratrol showed no significant improvement on the IVM or ROS levels in domestic ovine oocytes. However, treatment with 0.5 µM resveratrol during IVM improved embryo quality and promoted morulae compaction of Ovis aries handmade cloned embryos.

ABSTRACT The oocyte is indispensable for the embryonic development of an organism. Its quality is currently evaluated considering the morphological parameters that allow identifying the alterations that compromise the development of the embryo. So far, no morphological abnormalities have been reported in sheep oocytes. The objective of this study was to determine the frequency and type of morphological alterations in them. For this, 10 collections were carried out, of which 567 ovaries were obtained from slaughtered females and 2 374 oocytes were obtained by follicular aspiration; all of them matured in vitro. According to the criteria of the Association for the Study of Reproductive Biology (ASEBIR), 798 oocytes (33.6 %) were classified as normal morphology and 1 576 (66.4 %) presented different morphological alterations, as granular cytoplasm (32.6 %), incomplete cytoplasm (21.1 %), increased perivitelline space (11.7 %), presence of vacuoles (11.6 %), degenerated oocytes (8.9 %), abnormalities of the pellucid zone ( 7 %), cytoplasm exudates (4.7 %), polar body dimorphism (0.3 %), and alterations produced by the management technique, like the absence of pellucid zone (2.1 %). It is concluded that, given the high rate of morphological abnormalities in the in vitro maturation of oocytes, their detailed selection is important to obtain better results in the in vitro embryonic development.

RESUMEN El ovocito es indispensable para el desarrollo embrionario de un organismo. Actualmente, su calidad se evalúa considerando parámetros morfológicos que permiten identificar alteraciones que comprometen el desarrollo del embrión. Sin embargo, en ovocitos de ovino, hasta el momento, no se han reportado anormalidades morfológicas. El objetivo del presente trabajo fue determinar la frecuencia y el tipo de alteraciones morfológicas en ovocitos de borrega. Para ello se realizaron 10 colectas, de las que se obtuvieron 567 ovarios procedentes de hembras sacrificadas en rastro y, mediante aspiración folicular, se lograron 2 374 ovocitos; todos ellos fueron madurados in vitro. De acuerdo a los criterios de la Asociación para el Estudio de la Biología de la Reproducción (ASEBIR), 798 ovocitos (33,6 %) se clasificaron de morfología normal y 1 576 (66,4 %) presentaron diferentes alteraciones morfológicas, como citoplasma granuloso (32,6 %), citoplasma incompleto (21,1 %), espacio perivitelino aumentado (11,7 %), presencia de vacuolas (11,6 %), ovocitos degenerados (8,9 %), alteraciones de la zona pelúcida (7 %), exudados citoplasmáticos (4,7 %), dimorfismo del cuerpo polar (0,3 %) y alteraciones producidas por la técnica de manejo, como la ausencia de zona pelúcida (2,1 %). Se concluye que, dada la alta tasa de anormalidades morfológicas en los ovocitos de ovinos madurados in vitro, es importante su minuciosa selección para obtener mejores resultados en el desarrollo embrionario in vitro.

Forty sheep ejaculates were used to evaluate the vitrification advantages in relation to the conventional freezing. From each ejaculate, the volume, concentration and normal sperm were determined, with values of 0.8 mL, 1519X10(6) and 95 %, respectively. Subsequently, each ejaculate was divided into two fractions of equal concentration, one to freeze and another to vitrify. Zero point five mL straws were used for freezing and 60 μL drops for vetrification. Sperm concentration in both methods was 50x10(6) sperm/mL. Before and after cryopreservation, the progressive mobility (PM) was evaluated in fresh semen and it was 81 %. There was a difference (p<0.05) in relation to the cryopreserved semen, since mobility was greater (p<0.05) in thawed sperm (50 %) than in heated sperm (36 %). When analyzing viability results, a significant decrease (p<0.05) was observed in fresh semen (70 %), compared to the thawed (51 %) and heated sperm (54 %). When evaluating the acrosomal status and sperm viability, there were differences (p<0.05) between the fresh sperm (64 %) with respect to the thawed (45 %) and heated (47 %) in the live sperm with acrosomal reaction (VCA). This did not happened in the live sperm without acrosomal reaction (VSA) (6 %, 6 % and 7 %, respectively), in the dead acrosome-reacted sperm (MCA) (19 %, 29 % and 27 %, respectively), and in the dead sperm without acrosomal reaction (MSA) (11 %, 19 % and 18 %, respectively). It is concluded that, although both cryopreservation techniques showed similar results, vitrification has certain advantages such as that it requires less work time, material and equipment, decreasing the cost of the process and it can be applied in any laboratory

Para evaluar las ventajas de la vitrificación, con relación a la congelación convencional, se utilizaron 40 eyaculados de ovino. De cada eyaculado se determinó el volumen, la concentración y los espermatozoides normales, con valores de 0,8 mL, 1519X10(6) y 95 %, respectivamente. Cada eyaculado se dividió en una fracción para congelar y otra para vitrificar. Para la congelación se utilizaron pajillas de 0,5 mL y para la vitrificación gotas de 60 µL. La concentración en ambos métodos fue de 50x10(6) espermatozoides/mL. Antes y después de la criopreservación se evaluó la movilidad progresiva (MP); en semen fresco fue de 81 % y se observó una diferencia (p<0,05) con relación al semen criopreservado, pues fue mayor la movilidad (p<0,05) en semen descongelado (50 %) que en el calentado (36 %). Al analizar los resultados de la viabilidad se observó una disminución importante (p<0,05) del semen fresco (70 %) en comparación con el semen descongelado (51 %) y calentado (54 %). Cuando se evaluó la viabilidad y el estado acrosomal de los espermatozoides, solo en los vivos con acrosoma intacto (VCA) se observaron diferencias (p<0,05) entre el semen fresco (64 %) con respecto al semen descongelado (45 %) y calentado (47 %), pero no así en los espermatozoides vivos sin acrosoma (VSA) (6 %, 6 % y 7 %, respectivamente), en los espermatozoides muertos con acrosoma (MCA) (19 %, 29 % y 27 %, respectivamente) y en los espermatozoides muertos sin acrosoma (MSA) (11 %, 19 % y 18 %, respectivamente). Las dos técnicas de criopreservación mostraron resultados similares; sin embargo, la vitrificación requiere menor tiempo de trabajo, de material y de equipo, lo cual disminuye el costo del proceso y puede realizarse en cualquier laboratorio

Abstract: Effect of sequential human medium use in the production of blastocysts of female Ovis canadensis mexicana by interspecies handmade cloning. Faced to the notable problem that a significant number of world wild species are endangered, some biotechnologies such as the interspecific somatic cell nuclear transfer (SCNT), have been applied to raise their reproduction and conservation. This technology has been successfully applied by producing embryos and live offspring in some of these species. However, the interspecific SCNT still has to solve problems such as the low embryo production and offspring survival rates (3-6%). These problems, among others, involve a high incidence of embryo fragmentation and in order to solve them, most studies are now aimed to determine the effect of different culture media or supplements used for in vitro embryo production. Therefore, the aim of the present study was to use a variant of the interspecific SCNT technique (Handmade cloning-HMC) for embryo production, determining the effect of human sequential media in the endangered Mexican wild sheep O. c. mexicana. Results indicate that the embryo production increased when using human sequential media, allowing the embryos to reach the blastocyst stage (16% vs. 0%, P < 0.05). This is the first study that reports the successful cloning of female O. c. Mexicana embryos in Mexico.

Resumen: Efecto del uso de medio secuencial humano en la producción de blastocistos de hembra Ovis canadensis mexicana por clonación manual interespecies. Desde el evidente problema del peligro de extinción que enfrenta un número significativo de especies silvestres a nivel mundial, se han aplicado biotecnologías encaminadas a promover su conservación y reproducción. Tal es el caso de la transferencia nuclear de células somáticas (TNCS) interespecies que ha sido aplicada y ha producido exitosamente embriones y crías nacidas vivas en algunas de estas especies. Sin embargo, la TNCS interespecies aún debe lidiar con otros problemas como la baja tasa de producción de embriones y de supervivencia de las crías nacidas (3-6%). Problemas que, entre otros, involucran la alta incidencia de fragmentación embrionaria. Los estudios son ahora dirigidos a resolver estos obstáculos, determinando el efecto de diferentes medios o suplementos utilizados en la producción in vitro de embriones. El objetivo de este estudio fue aplicar una variante de la TNCS interespecies (Clonación Manual-HMC) para la producción de embriones, determinando el efecto del uso de medios secuenciales humanos, en el borrego salvaje mexicano amenazado de extinción O. c. mexicana. Los resultados muestran que la producción de embriones incrementa con el uso de medios secuenciales humanos, permitiendo al embrión desarrollarse hasta la etapa de blastocisto (16% vs. 0%, P < 0.05). Este es el primer reporte de clonación exitosa de embriones de hembra O. c. mexicana en México.

Several mammal species of ecological interest in our country boast some category of risk or danger of extinction. With the purpose of guarding the genetic information of these species, researchers have developed techniques of ex situ conservation, which are key components of global conservation programs that contribute for the storage of the genome. This storage is carried out through the collection of cells and tissues embedded in germplasm banks, which are the most efficient solution to the critical panorama of the threatened wild fauna. In the present work, for the very first time in Mexico, epithelial (keratinocytes) and connective tissue (fibroblasts) cells were derived in culture on day 28, from Ovis canadensis mexicana after 54 days thawed skin tissue, to create a germplasm bank.

En nuestro país existen diversas especies de mamíferos de interés ecológico que ostentan alguna categoría de riesgo o peligro de extinción. Con la finalidad de resguardar la información genética de estas especies se han desarrollado técnicas de conservación ex situ, las cuales son componentes fundamentales de programas de conservación global que contemplan operaciones de almacenamiento del genoma. El almacenamiento se lleva a cabo mediante las colecciones de células y tejidos incorporados a bancos de germoplasma, que son una alternativa eficaz y próxima para hacerle frente al panorama crítico que sufre la fauna silvestre amenazada. En el presente trabajo se derivaron por vez primera en México, células epiteliales (queratinocitos) y de tejido conectivo (fibroblastos) a los 28 días de cultivo, a partir de piel de Ovis canadensis mexicana post-congelación de 54 días, para la formación de bancos de germoplasma.