A decreased expression of interferon stimulated genes in peri-implantation endometrium of embryo transfer recipient sows could contribute to embryo death.

Pig pregnancy succeeds thanks to a well-coordinated system ruling both maternal immune activation and embryonic antigen tolerance. In physiological pregnancies, the maternal immune system should tolerate the presence of hemi-allogeneic conceptuses from the pre-implantation phase to term, while maintaining maternal defence against pathogens. Allogeneic pregnancies, as after embryo transfer (ET), depict high embryo mortality during the attachment phase, calling for studies of the dynamic modifications in immune processes occurring at the maternal-foetal interface, for instance, of interferon (IFN)-stimulated genes (ISGs). These ISGs are generally activated by IFN secreted by the conceptus during the process of maternal recognition of pregnancy (MRP) and responsible for recruiting immune cells to the site of embryo attachment, thus facilitating cell-antigen presentation and angiogenesis. We performed RNA-Seq analysis in peri-implantation (days 18 and 24) endometrial samples retrieved from artificially inseminated sows (hemi-allogeneic embryos (HAL) group) or sows subjected to ET (allogeneic embryos (AL) group) to monitor alterations of gene expression that could be jeopardising early pregnancy. Our results showed that endometrial gene expression patterns related to immune responses differed between hemi- or allogeneic embryo presence, with allogeneic embryos apparently inducing conspicuous modifications of immune-related genes and pathways. A decreased expression (P < 0.05; FC < -2) of several interferon ISGs, such as CXCL8, CXCL10, IRF1, IRF9, STAT1, and B2M, among others was detected in the endometrium of sows carrying allogeneic embryos on day 24 of pregnancy. This severe downregulation of ISGs in allogeneic pregnancies could represent a failure of ET-embryos to signal IFN to the endometrium to warrant the development of adequate immunotolerance mechanisms to facilitate embryo development, thus contributing to elevated embryo death.

Realidad virtual de bajo coste. Una nueva aplicación para rehabilitación motora de los miembros superiores en patología neurológica: Estudio piloto / Low-cost virtual reality. A new application for upper extremity motor rehabilitation in neurological pathology: Pilot study

Antecedentes y objetivo: El objetivo de este estudio es presentar una nueva aplicación de realidad virtual (RV) de bajo coste basada en el dispositivo Leap Motion Controller (LMC) desarrollada para la rehabilitación motora de miembros superiores tras patología neurológica y mostrar su viabilidad clínica realizando una experiencia piloto. Material y métodos: El LMC permite la interacción con las aplicaciones virtuales mediante la captación de los movimientos de la mano. Se realizó una prueba piloto con cuatro pacientes con afectación de miembros superiores con Upper Extremity Motor Score (UEMS) mayor de 10. Se les valoró con los test Box and Block (BBT) y tarea de escritura del Jebsen-Taylor Hand Function (JTHF) antes y después de la intervención. Resultados: Los pacientes completaron el protocolo de nueve sesiones de 30 minutos de duración dividido en tres sesiones por semana. En el BBT pasaron de 38 (DE 20) cubos antes de la intervención a 44 (DE 21,72) cubos tras finalizarla. En el JTHF pasaron de 28,25 s (DE 8,61) a 26, 75 s (DE 21,72). No hubo diferencias estadísticamente significativas. La usabilidad del dispositivo se valoró con la escala Quebec User Evaluation of Satisfaction with assistive Technology, versión 2.0 (QUEST) siendo la seguridad, efectividad y facilidad de uso los aspectos prioritarios. Conclusión: Se presenta un nuevo desarrollo de RV basado en el dispositivo LMC y se ha comprobado su viabilidad clínica en pacientes neurológicos con afectación de los miembros superiores. Es preciso realizar un estudio clínico con una muestra amplia para valorar su posible efectividad clínica como elemento de tratamiento.(AU)

Background and objectives: The aim of this study is to present a new virtual reality (VR) low cost application based on Leap Motion Controller (LMC) device for upper extremity motor rehabilitation after neurological pathology and to demonstrate its clinical feasibility by carrying out a pilot experience. Material and methods: The LMC allows the interaction with virtual applications by capturing the patient's hand movements. A pilot study was carried out with 4 patients with upper limb impairment reflected with Upper Extremity Motor Score (UEMS) greater than 10. They were assessed using the Box and Block (BBT) and the writing task within the Jebsen-Taylor Hand Function (JTHF) before and after the intervention. Results: All patients completed the 9-session, 30-min protocol divided into 3 sessions per week. They went from an average result of 38 (SD 20) blocks in BBT before the intervention to 44 (SD 21.72) after it. They went from 28.25 s (SD 8.61) to 26.75 s (SD 21.72) in the JTHF. Statistically significant differences were no found. The device usability was assessed by the QUEST scale, being the security, effectiveness and ease to use the aspects that patients considered to be a priority. Conclusión: A new VR development based on the LMC device is presented and the clinical feasibility of its application in neurological patients with upper limb involvement has been proven. A clinical study with a large sample size is needed to assess its potential clinical effectiveness as a treatment element.(AU)

[Low-cost virtual reality. A new application for upper extremity motor rehabilitation in neurological pathology: Pilot study]. / Realidad virtual de bajo coste. Una nueva aplicación para rehabilitación motora de los miembros superiores en patología neurológica: Estudio piloto.

BACKGROUND AND OBJECTIVES: The aim of this study is to present a new virtual reality (VR) low cost application based on Leap Motion Controller (LMC) device for upper extremity motor rehabilitation after neurological pathology and to demonstrate its clinical feasibility by carrying out a pilot experience. MATERIAL AND METHODS: The LMC allows the interaction with virtual applications by capturing the patient's hand movements. A pilot study was carried out with 4 patients with upper limb impairment reflected with Upper Extremity Motor Score (UEMS) greater than 10. They were assessed using the Box and Block (BBT) and the writing task within the Jebsen-Taylor Hand Function (JTHF) before and after the intervention. RESULTS: All patients completed the 9-session, 30-min protocol divided into 3 sessions per week. They went from an average result of 38 (SD 20) blocks in BBT before the intervention to 44 (SD 21.72) after it. They went from 28.25 s (SD 8.61) to 26.75 s (SD 21.72) in the JTHF. Statistically significant differences were no found. The device usability was assessed by the QUEST scale, being the security, effectiveness and ease to use the aspects that patients considered to be a priority. CONCLUSIóN: A new VR development based on the LMC device is presented and the clinical feasibility of its application in neurological patients with upper limb involvement has been proven. A clinical study with a large sample size is needed to assess its potential clinical effectiveness as a treatment element.

Discapacidad intelectual y medidas adoptadas durante la pandemia de COVID-19: efectos comportamentales / Intellectual disability and adopted measures during the COVID-19 pandemic: behavioral effects

Actualmente, y en el contexto del estadode emergencia sanitaria, se han implantadouna serie de medidas de prevención y contenciónaplicables a toda la población. Enparticular, los centros sociosanitarios hantenido que adaptarse, diseñando medidasespeciales y aplicando planes de contingencia,que han provocado modificaciones en elentorno habitual de los residentes. Con elfin de observar los efectos de la pandemiasobre la conducta de personas con discapacidadintelectual residentes en un centrosociosanitario, se aplicó el Inventario parala Planificación de Servicios y ProgramaciónIndividual (ICAP). Los resultados, comparadoscon evaluaciones previas a la pandemiapor COVID-19, mostraron que el funcionamientoadaptativo era similar al anterior aeste periodo, pero existía un déficit en destrezasmotoras y en las destrezas de la vidaen comunidad. Así mismo, las alteracionesdel comportamiento habían aumentado, tantoen la puntuación general como en cadauno de sus índices específicos. Además, seencontró una relación inversa entre la edady la conducta adaptativa y una correlación positiva entre la edad y las alteraciones de la conducta. (AU)

Currently, and due to the existing healthemergency context, prevention and containmentmeasures applicable to the entirepopulation have been implemented. In particular,social and health centers have hadto adapt, designing a package of specialmeasures and applying guidelines and contingencyplans that have caused changes inthe habitual environment of the residents.In order to observe the effects of the pandemicon people with intellectual disabilitiesin social health centers, the Inventoryfor Client and Agency Planning (ICAP) wasapplied. The results, compared to previousevaluation, showed that adaptive functioningwas similar to that before this period,but there was a decrease in motor and socialskills. Furthermore, behavioral problems hadincreased, in the general index and in each of its specific index. In addition, an inverse relationship was found between age and adaptive behavior and a positive correlation between age and behavior disorders. (AU)

Instrucciones previas: breve guía para un ejercicio de autonomía / Advance instructions: short guide to an exercise of autonomy

Las instrucciones previas, también denominadas decisiones o voluntades anticipadas,o testamento vital, constituyen una manifestación de la voluntad de los pacientes y, por tanto, son expresión de la autonomía de la persona. Se definen como el proceso en el que una persona planifica los cuidados sanitarios y la asistencia que desea recibir en el futuro, en el momento en que no sea capaz de tomar decisiones por sí misma.La planificación anticipada de cuidados y decisiones (PACD), que incluye elaborar esos documentos de voluntades anticipadas, es un modo de relación asistencial sobre la que todavía hay poca experiencia en nuestro país.La PACD se define como un "proceso voluntario de comunicación y deliberación entre una persona capaz y profesionales implicados en su atención, acerca de los valores, deseosy preferencias que quiere que se tengan encuenta en la atención sanitaria que recibirácomo paciente, fundamentalmente en losmomentos finales de la vida" (1).El objetivo es que la persona, junto conlos profesionales, delibere y establezca cómoquiere ser tratada durante el proceso terapéutico.Incluye los valores relevantes delpaciente, las preferencias sanitarias de caráctergeneral, las indicaciones sanitariasconcretas (rechazo o solicitud de intervenciones,donación de órganos...), expresiónde sentimientos hacia otros ("lo siento","gracias", "te perdono", etc), y/o designarun representante.A través del documento de instruccionesprevias que confirma que se ha dado el procesode reflexión, comunicación y descripciónde esas voluntades anticipadas, las personaspueden decidir acerca de los tratamientos ycuidados de salud que desean o no recibircuando se encuentren en una circunstanciaen la que no puedan manifestar su voluntadpersonalmente.Su fundamento ético es:Respetar y promover la autonomía del paciente.Asegurar que se valoren las situaciones decompetencias limitadas o inexistentes. (AU)

Advance instructions, also called decisionsor advance directives, or living wills,constitute a manifestation of the will of the patients and, therefore, are an expression of the autonomy of the person. They are defined as the process in which a person plans the health care and assistance they want to receive in the future, at the time when they are not able to make decisions for themselves. Advance Care Planning and Decisions(ACPD) which includes developing these advance directives documents. It is something that the law does not collect but does not prevent, and there is very little experience and research on it. They define it as a "voluntary process of communication and deliberation between a capable person and professionals involved in their care, about the values, desires and preferences that they want to betaken into account in the health care thatthey will receive as a patient, fundamentallyin the moments end of life" (1).The objective is for the person, togetherwith the professionals, to establish how theywant to be treated during the therapeuticprocess. It includes the relevant values ofthe patient, general health preferences, specifichealth indications (refusal or requestfor interventions, organ donation ...), expressionof feelings towards others ("I'm sorry","thank you", "I forgive you", etc), and/or appoint a representative.Through the advance directive documentthat confirms that the process of reflection,communication and description of these advancedirectives has been given, people candecide about the treatments and health carethey want or not to receive when they are ina circumstance in the one that cannot expresstheir will personally.Its ethical foundation is:Respect and promote the autonomy of thepatient.Preserve that value in situations of limited or nonexistent skills. (AU)

The role of semen and seminal plasma in inducing large-scale genomic changes in the female porcine peri-ovulatory tract.

Semen modifies the expression of genes related to immune function along the porcine female internal genital tract. Whether other pathways are induced by the deposition of spermatozoa and/or seminal plasma (SP), is yet undocumented. Here, to determine their relative impact on the uterine and tubal transcriptomes, microarray analyses were performed on the endocervix, endometrium and endosalpinx collected from pre-ovulatory sows 24 h after either mating or artificial insemination (AI) with specific ejaculate fractions containing spermatozoa or sperm-free SP. After enrichment analysis, we found an overrepresentation of genes and pathways associated with sperm transport and binding, oxidative stress and cell-to-cell recognition, such as PI3K-Akt, FoxO signaling, glycosaminoglycan biosynthesis and cAMP-related transcripts, among others. Although semen (either after mating or AI) seemed to have the highest impact along the entire genital tract, our results demonstrate that the SP itself also modifies the transcriptome. The detected modifications of the molecular profiles of the pre/peri-ovulatory endometrium and endosalpinx suggest an interplay for the survival, transport and binding of spermatozoa through, for instance the up-regulation of the Estrogen signaling pathway associated with attachment and release from the oviductal reservoir.

[Injury of the anterior branch of the posterior interosseous nerve]. / Lesión de la rama anterior del nervio interóseo posterior.

INTRODUCTION: Posterior interosseous nerve syndrome, a branch of the radial nerve at the level of the forearm, is characterized by the motor function loss of some or all of the muscles innervated distally. CLINICAL CASE: A 26-year-old male with a history of proximal radius fracture associated to radial nerve injury, treated with osteosynthesis 7 years earlier, with full recovery, who currently presented intense pain 4 cm distal to the radial head, accompanied by paralysis of Extensor pollicis longus, Extesnor pollicis brevis and Abductor pollicis longus, with paresis of the Extensor indicis propius, in which a diagnosis of entrapment syndrome of the anterior descending branch of the posterior interosseous nerve (SNIP) was performed. DISCUSSION: The conservative management of SNIP is indicated during the first 8-12 weeks, if no improvement is found, the indication for surgical exploration is indicated, and the removal of osteosynthesis material is controversial.

INTRODUCCIÓN: El síndrome del nervio interóseo posterior, rama del nervio radial a nivel del antebrazo se caracteriza por la pérdida de función motora de algunos o todos los músculos inervados distalmente. CASO CLÍNICO: Masculino de 26 años con antecedente de fractura de radio proximal manejado con osteosíntesis que cursó con lesión del nervio radial siete años antes con recuperación completa, acude con dolor intenso a 4 cm distal a cabeza radial, acompañado de parálisis del extensor largo y corto del pulgar y del abductor del pulgar, con paresia del extensor propio del índice, en el que se efectúa diagnóstico de síndrome de atrapamiento de la rama anterior descendente del nervio interóseo posterior (SNIP). DISCUSIÓN: El manejo conservador del SNIP está indicado durante las primeras ocho a 12 semanas, de no mostrar mejoría la indicación de exploración quirúrgica está indicada, siendo el retiro de material de osteosíntesis controvertido.

Lesión de la rama anterior del nervio interóseo posterior / Injury of the anterior branch of the posterior interosseous nerve

Resumen: Introducción: El síndrome del nervio interóseo posterior, rama del nervio radial a nivel del antebrazo se caracteriza por la pérdida de función motora de algunos o todos los músculos inervados distalmente. Caso clínico: Masculino de 26 años con antecedente de fractura de radio proximal manejado con osteosíntesis que cursó con lesión del nervio radial siete años antes con recuperación completa, acude con dolor intenso a 4 cm distal a cabeza radial, acompañado de parálisis del extensor largo y corto del pulgar y del abductor del pulgar, con paresia del extensor propio del índice, en el que se efectúa diagnóstico de síndrome de atrapamiento de la rama anterior descendente del nervio interóseo posterior (SNIP). Discusión: El manejo conservador del SNIP está indicado durante las primeras ocho a 12 semanas, de no mostrar mejoría la indicación de exploración quirúrgica está indicada, siendo el retiro de material de osteosíntesis controvertido.

Abstract: Introduction: Posterior interosseous nerve syndrome, a branch of the radial nerve at the level of the forearm, is characterized by the motor function loss of some or all of the muscles innervated distally. Clinical case: A 26-year-old male with a history of proximal radius fracture associated to radial nerve injury, treated with osteosynthesis 7 years earlier, with full recovery, who currently presented intense pain 4 cm distal to the radial head, accompanied by paralysis of Extensor pollicis longus , Extesnor pollicis brevis and Abductor pollicis longus, with paresis of the Extensor indicis propius , in which a diagnosis of entrapment syndrome of the anterior descending branch of the posterior interosseous nerve (SNIP) was performed. Discussion: The conservative management of SNIP is indicated during the first 8-12 weeks, if no improvement is found, the indication for surgical exploration is indicated, and the removal of osteosynthesis material is controversial.

Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa.

Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at -20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.

Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm.

Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-µL aliquots and layered on 1mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9±4.2%, compared to 70% (35.4%±3.0, p<0.001) and 90% (8.2%±3.0, P=0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7±1% vs Control: 60.3±0.7%, P<0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5%±1.9 vs. 24.3%±1.2 Control; P<0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa.

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.

Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm.

The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples with this colloid showed a better resistance to freezing compared with the control sample not only for motility but also for viability.

The ubiquitous hyaluronan: Functionally implicated in the oviduct?

Hyaluronan (hyaluronic acid) is a simple, nonantigenic, nonsulfated glycosaminoglycan present everywhere in the extracellular compartments of the body. Noteworthy, it is highly conserved phylogenetically, from sauropsida to mammals; and plays a plethora of roles from embryonic/fetal development to adult physiological and pathological events, including tumor development. In reproduction, hyaluronan has proven related to initial events as sperm survival, buildup of the sperm reservoir in the oviduct, regulation of sperm capacitation, and prefertilization to later participate in embryo, fetal, and placental development. Synthesis, binding (via the CD44 membrane receptor), and degradation of hyaluronan occur in male and female genital organs, the oviduct being no exception. This review discusses our current knowledge on roles of this ubiquitous glycosaminoglycan on the survival of immunologically foreign spermatozoa in the pig oviduct, a relevant event for fertility. During preovulatory storage in the functional tubal sperm reservoir, spermatozoa are entrapped in a mucus-like tubal fluid. This fluid contains fluctuating levels of hyaluronan, which is synthesized by the lining epithelium by hyaluronan synthase 3. Both hyaluronan and its CD44 receptor are particularly evident in the deep mucosal furrows of the sperm reservoir, in which most spermatozoa are embedded in; kept alive, uncapacitated but also undetected by the immune system of the female. Hyaluronan is also present in the seminal plasma, and evidence points toward an involvement of hyaluronan and its receptor in the local (tubal and possibly uterine) production of antiinflammatory cytokines, such as interleukin-10, pertaining maternal immune tolerance of these foreign cells.

The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa.

Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% L-α-phosphatidylcholine, and Type B: 14-23% L-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.

The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation.

The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 µg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 µg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 µg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 µg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 µg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and "apoptotic-like" changes).

Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants.

Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mM or 0.1 mM of each antioxidant, including oxidative stress (Fe(2+)/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.

Diagnóstico prenatal de una trisomia parcial 5p asociada a hiperecogeneicidad intestinal / Prenatal diagnosis of partial trisomy 5p associated with hyperechogenic fetal bowel

Paciente de 30 años de edad, a la que a las 20 semanas de gestación se le realiza una amniocentesis por presentar feto con pies equinovaros, hiperecogeneicidad intestinal y quiste de los plexos coroideos. En el estudio citogenético se diagnosticó una trisomía parcial del brazo corto del cromosoma 5, de novo. Se analizan los hallazgos necrópsicos y el "síndrome 5p", asi como la relación entre hiperecogenicidad intestinal y la aparición de cromosomopatías (AU)

We report the case of a 30-year-old woman who underwent amniocentesis at 20 weeks of gestation since the fetus showed equinovarus feet, hyperechogenic fetal bowel and choroid plexus cyst. Cytogenetic study identified de novo partial trisomy of the short arm of chromosome 5. We analyze the autopsy findings and "5p syndrome" as well as the association between intestinal hyperechogenicity and the development of abnormal chromosomes (AU)

Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours.

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe(2+) /ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.

Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample.

Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).

In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.