Congelación y conservación del semen en la especie caprina mediante la utilización de ultracongeladores de -152ºC: Tasa de fertilidad tras inseminación con semen congelado por diferentes protocolos de crioconservación

[ES] El análisis de los distintos parámetros evaluados para determinar la calidad seminal de siete machos cabríos de raza Majorera, demuestra que la congelación del semen mediante ultracongeladores de -152 ºc es igual de efectiva que la congelación mediante nitrógeno líquido, técnica que se ha venido usando desde hace ya muchos años. se observa cómo la calidad seminal in vitro tras la descongelación no varía en los diferentes períodos de congelación testados (1, 30 y 180 días), así mismo, el porcentaje de gestaciones obtenido a partir de la inseminación artificial en 40 hembras, es similar en ambos protocolos de congelación para muestras congeladas durante un período de un mes

Aportaciones tecnológicas en la preservación del semen en la raza caprina

Programa de doctorado: Clínica e investigación terapeútica.

Effect of antioxidant supplementation on pig and horse gamete storage

According to recent studies, antioxidant supplementation on gamete processing and/or storage solutions improvesgamete quality parameters, after cooling or storage at sub zero temperature. The aim of the present study was to investigate the effects of antioxidant supplementation on pig and horse gamete storage. The first study aimed to determine the effects of resveratrol (RESV) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. RESV(2µM) was added during: IVM (A); 2 h post-warming incubation (B); vitrification/warming and 2 h post-warming incubation (C); all previous phases (D). The obtained data demonstrate that RESV supplementation in the various steps of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage. In the second work different concentrations of RESV (10, 20, 40, and 80µM) were added during liquid storage of stallion sperm for 24 hours at either 10°C or 4°C, under anaerobic conditions. Our findings demonstrate that RESV supplementation does not enhance sperm quality of stallion semen after 24 hours of storage. Moreover, the highest RESV concentrations tested (40 and 80µM) could damage sperm functional status, probably acting as pro-oxidant. Finally, in the third work other two antioxidants, ascorbic acid (AA) (100 µM) and glutathione (GSH) (5mM) were added on boar freezing and/or thawing solutions. In our study different sperm parameters were evaluated before freezing and at 30 and 240 minutes after thawing. Our results showed that GSH and AA significantly improved boar sperm cryotolerance, especially when supplemented together to both freezing and thawing media. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels.

Entwicklung einer Kryokonservierungsmethode für Spermien der Honigbiene, Apis mellifera

Die westliche Honigbiene (Apis mellifera) ist von großer ökologischer und ökonomischer Bedeutung. Seit Jahren zeichnen sich in Nordamerika sowie in manchen Teilen Europas rückläufige Bienenvölkerzahlen und eine abnehmende Artenvielfalt innerhalb der Bienenfamilie ab. Mittlerweile ist von einer globalen Bestäuberkrise die Rede und es wird verstärkt nach Möglichkeiten gesucht, um dieser Krise entgegenzuwirken. Eine Konservierung von Bienenspermien in flüssigem Stickstoff ohne Fruchtbarkeitsreduzierung würde die Bienenzucht revolutionieren und stark beschleunigen, da räumliche und zeitliche Restriktionen bei der Wahl des Bienenspermas wegfielen. Zudem wäre eine Möglichkeit zur Sicherung der genetischen Diversität geschaffen. Im Rahmen des hier vorgestellten Projektes wurde eine solche Methode erarbeitet. In umfangreichen Abkühl- und Einfrierversuchen konnte eine neue Konservierungstechnik entwickelt werden, bei der das Kryoprotektivum mittels Dialyse dem Bienensperma zugesetzt wird. Dieses Verfahren erhält die native Spermaform, in der die Spermien parallel und inaktiv in dicht gepackten Clustern vorliegen, und erzielt bisher unerreichte Besamungserfolge. So konnten durchschnittlich 1,25 Mio. Spermien in den Spermatheken besamter Königinnen gezählt werden und davon waren ungefähr 90% motil. Besonders vielversprechend ist jedoch, dass 79,4% der Brut weiblich waren. Ein so hoher Anteil weiblicher Brut konnte bislang nicht erreicht werden und wäre für züchterische Zwecke ausreichend.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Dorsal rather than ventral visual pathways discriminate freezing status in Parkinson's disease

BACKGROUND: Although visuospatial deficits have been linked with freezing of gait (FOG) in Parkinson's disease (PD), the specific effects of dorsal and ventral visual pathway dysfunction on FOG is not well understood. METHOD: We assessed visuospatial function in FOG using an angle discrimination test (dorsal visual pathway bias) and overlapping figure test (ventral visual pathway bias), and recorded overall response time, mean fixation duration and dwell time. Covariate analysis was conducted controlling for disease duration, motor severity, contrast sensitivity and attention with Bonferroni adjustments for multiple comparisons. RESULTS: Twenty seven people with FOG, 27 people without FOG and 24 controls were assessed. Average fixation duration during angle discrimination distinguished freezing status: [F (1, 43) = 4.77 p < 0.05] (1-way ANCOVA). CONCLUSION: Results indicate a preferential dysfunction of dorsal occipito-parietal pathways in FOG, independent of disease severity, attentional deficit, and contrast sensitivity.

Safe high-pressure freezing of infectious micro-organisms

We describe how high-pressure freezing of infectious biological material can safely be accomplished with the help of membrane carriers. The method described is easy to perform; however, careful manipulations are required. Existing safety regulations must still be followed. However, the procedure reduces the risk of dissemination of infectious material.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.

Fine structure of hippocampal mossy fiber synapses following rapid high-pressure freezing

Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.

A new microbiopsy system enables rapid preparation of tissue for high-pressure freezing

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm x 1.2 mm x 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen.

[Search for Neospora caninum DNA in bull semen using PCR ]

Neospora caninum represents one of the most frequent abortifaciant organisms worldwide. The parasite is diaplacentally transmitted from the pregnant cow to the fetus, where it normally leads to the delivery of a healthy, however persistently infected calf. Abortion thus is a relative rare event. The transmission of bovine neosporosis occurs in more than 90% of the cases vertically due to the endogenous reactivation of a persistently infected mother. Exogenous infections are therefore responsible for less than 10% of the cases.The question arises about which infection sources may be relevant in this context. In Switzerland, the role of dogs as definitive hosts has been shown to be of low significance in that respect. Recently, discussion focused on the potential of infectious bull semen following natural or artificial insemination. Thus, a few years ago a report documented the detectability of N. caninum-DNA in the semen of naturally infected bulls by nested-PCR. As a consequence, we decided to gain own experience by investigating 5 separate semen specimens per animal, originating from 20 N. caninum-seropositive bulls used for artificial insemination in Switzerland. All probes turned out to be negative by nested PCR. Based upon our laboratory experiences, the potential bull semen-associated Neospora-problem seems not to affect the Swiss bull population, thus there is no evidence to include further respective means of control.

Effects of experimental freezing on soil nitrogen dynamics in soils from a net nitrification gradient in a nitrogen-saturated hardwood forest ecosystem

This study examined effects of soil freezing on N dynamics in soil along an N processing gradient within a mixed hardwood dominated watershed at Fernow Experimental Forest, West Virginia. Sites were designated as LN (low rates of N processing), ML (moderately low), MH (moderately high), and HN (high). Soils underwent three 7-day freezing treatments (0, –20, or –80 °C) in the laboratory. Responses varied between temperature treatments and along the gradient. Initial effects differed among freezing treatments for net N mineralization, but not nitrification, in soils across the gradient, generally maintained at LN < ML ≤ MH < HN for all treatments. Net N mineralization potential was higher following freezing at –20 and –80 °C than control; all were higher than at 0 °C. Net nitrification potential exhibited similar patterns. LN was an exception, with net nitrification low regardless of treatment. Freezing response of N mineralization differed greatly from that of nitrification, suggesting that soil freezing may decouple two processes of the soil N cycle that are otherwise tightly linked at our site. Results also suggest that soil freezing at temperatures commonly experienced at this site can further increase net nitrification in soils already exhibiting high nitrification from N saturation.

Effects of in situ freezing on soil net nitrogen mineralization and net nitrification in fertilized grassland of northern China

Effects of soil freezing on nitrogen (N) mineralization have been the subject of increased attention in the ecological literature, though fewer studies have examined N mineralization responses to successive mild freezing, severe freezing and cyclic freeze–thaw events. Even less is known about relationships of responses to soil N status. This study measured soil N mineralization and nitrification in the field along an experimental N gradient in a grassland of northern China during the dormant season (October 2005–April 2006), a period in which freezing naturally occurs. Net N mineralization exhibited great temporal variability, with nitrification being the predominant N transformation process. Soil microbial biomass C and N and extractable NH4 + pools declined by 40, 52, and 56%, respectively, in April 2006, compared with their initial concentrations in October 2005; soil NO3– pools increased by 84%. Temporal patterns of N mineralization were correlated with soil microbial biomass C and N. N mineralization and nitrification increased linearly with added N. Microbial biomass C in treated soils increased by 10% relative to controls, whereas microbial N declined by 9%. Results further suggest that freezing events greatly alter soil N dynamics in the dormant season at this site, with considerable available N accumulating during this period.