The Use of Antifreeze Proteins in the Cryopreservation of Gametes and Embryos

[EN] The cryopreservation of gametes and embryos is a technique widely used in reproductive biology. This technology helps in the reproductive management of domesticated animals, and it is an important tool for gene banking and for human-assisted reproductive technologies. Antifreeze proteins are naturally present in several organisms exposed to subzero temperatures. The ability for these proteins to inhibit ice recrystallization together with their ability to interact with biological membranes makes them interesting molecules to be used in cryopreservation protocols. This mini-review provides a general overview about the use of antifreeze proteins to improve the short and long term storage of gametes and embryos.S

Non-coding RNA regulation in reproduction: their potential use as biomarkers

[EN] Non-coding RNAs (ncRNAs) are crucial regulatory elements in most biological processes and reproduction is also controlled by them. The different types of ncRNAs, as well as the high complexity of these regulatory pathways, present a complex scenario; however, recent studies have shed some light on these questions, discovering the regulatory function of specific ncRNAs on concrete reproductive biology processes. This mini review will focus on the role of ncRNAs in spermatogenesis and oogenesis, and their potential use as biomarkers for reproductive diseases or for reproduction successSIThe authors would like to acknowledge project AGL2015-68330-C2-1-R (MINECO/FEDER) PTA2016-11987-I contract (MINECO/FEDER) and AQUA-CIBUS international net 318RT0549 funded by CYTE

Probiotics reduce anxiety-related behavior in zebrafish

[EN] There is increasing evidence that gut microbiome could have effects on neurological processes and on behavior. In this study we used the novel tank test (NTT) to analyze zebrafish exploring behavior after four months’ supplementation with probiotics with probed antioxidant and anti-inflammatory properties. Results showed that prolonged ingestion of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 significantly alters the swimming pattern and mean swimming speed in the zebrafish model. After treatment, zebrafish strongly reduced their bottom-dwelling geotactic behavior when placed in a new tank, which could be correlated to a lower state of anxiety.S

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

P. 122-130.e2eCryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage

Long Exposure to a Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics Improves Sperm Quality and Progeny Survival in the Zebrafish Model

This article belongs to the Special Issue Relevant Biomolecules for Germ Cells and Fertilization[EN] The aim of the present experiment is to study the effects of oral ingestion of a mixture of two probiotic bacteria on sperm quality and progenies. Three homogeneous groups of juvenile zebrafish were created. Once having reached adulthood (3 months postfertilization; mpf), each group received different feeding regimens: a standard diet (control), a maltodextrin-supplemented diet (vehicle control), or a probiotic-supplemented diet (a mixture (1:1) of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347). The feeding regime lasted 4.5 months. Growth parameters (weight and length) were determined at 3, 5, and 7.5 mpf. Sperm motility was evaluated using computer-assisted sperm analysis at 5 and 7.5 mpf. Progeny survival, hatching rate, and malformation rate were also evaluated. Results showed that probiotic-supplemented diet improved growth parameters compared with the standard diet. The highest percentage of motile spermatozoa was reported in the probiotic-fed group. Concomitantly, the percentage of fast sperm subpopulation was significantly lower in samples derived from control males. Furthermore, there was a significant difference in progeny survival between the probiotic-fed group and the control group at three developmental times (24 hours postfertilization (hpf), 5 days postfertilization (dpf) and 7 dpf). In conclusion, in zebrafish, prolonged ingestion of a mixture of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 has positive effects on growth, sperm quality, and progeny survivalSIThis work was supported by project AGL2015 68330-C2-1-R (MINECO/FEDER)The authors acknowledge contract PTA2016-11987-I (MINECO/FEDER), Biopolis S.L. (Valencia, Spain), AQUA-CIBUS International Net 318RT0549 (CYTED), and the staff from Planta de Cultivos El Bocal (IEO), in particular, Guadalupe Santos Veiga, Serafín Gago Martínez, Jesús Cociña, Diego Vida

Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics Improves Sperm Quality after Only One Spermatogenic Cycle in Zebrafish Model

[EN] Infertility is a medical concern worldwide and could also have economic consequences in farmed animals. Developing an efficient diet supplement with immediate effects on sperm quality is a promising tool for human reproduction and for domesticated animal species. This study aims at elucidating the effect of a short-time probiotic supplementation consisting of a mixture of two probiotic bacteria with proven antioxidant and anti-inflammatory activities on zebrafish sperm quality and male behavior. For this purpose, three homogeneous groups of males in terms of motility (<60%) were established. The control group was fed with a normal standard diet. The other received supplements: One group (vehicle control) was fed with maltodextrin and the other received a probiotic preparation based on a mixture (1:1) of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347. The feeding regime was 21 days corresponding with a single spermatogenesis in zebrafish. The preparation did not modify animal weight, positively affected the number of fluent males, increased sperm concentration, total motility, progressive motility, and fast spermatozoa subpopulations. Moreover, the animals fed with the supplement showed different behavior patterns compared to control groups. Our results suggest a diet-related modulation on the exploration activity indicating a lower stress-like conduct. The studied formulation described here should be considered as advantageous in male reproductive biotechnologySIThis work was supported by project AGL2015 68330-C2-1-R (MINECO/FEDER)The authors acknowledge contract PTA2016-11987-I (MINECO/FEDER), Biopolis S.L. (Valencia, Spain), AQUA-CIBUS International Net 318RT0549 (CYTED), and the staff from Planta de Cultivos El Bocal (IEO

Effect of low sperm quality on progeny: a study on zebrafish as model species

[EN] Nowadays a decrease tendency in human sperm quality has been reported mainly in developed countries. Reproductive technologies have been very valuable in achieving successful pregnancies with low quality sperm samples. However, considering that spermatozoa molecular contribution is increasingly important in recent studies, it is crucial to study whether fertilization with low sperm quality could leave a molecular mark on progeny. This study explores the consequences that fertilization with low sperm quality may have on progeny, using zebrafish as a model. Good and bad breeders were established attending to sperm quality analyses and were individually tracked. Significant differences in fertilization and malformation rates were obtained in progenies between high and low quality sperm samples. Moreover an altered miR profile was found in the progenies of bad zebrafish breeders (upregulation of miR-141 and miR -122 in 24 hpf embryos) and as a consequence, some of their targets involved in male sex development such as dmrt1, suffered downregulation. Our results indicate that fertilizing with high sperm quality samples becomes relevant from a new perspective: to avoid molecular alterations in the progeny that could remain masked and therefore produce unexpected consequences in it.SIThis work was supported by AGL2015 68330-C2-1-R project (MINECO/FEDER), PTA2016-11987-I contract (MINECO/FEDER) and AQUA-CIBUS international net 318RT0549 funded by CYTED (Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo

Impact of different levels of handling on Solea senegalensis culture: effects on growth and molecular markers of stress

[EN] Aquaculture routine practices may cause stress induction on the fish and compromise their welfare affecting the production. This experiment aimed to evaluate the potential links between handling during culture with stress responses and growth on Senegalese sole (Solea senegalensis). We worked with two fish cohorts in terms of initial body weight and culture stage: Trial 1 included specimens in the fattening stage (226 ± 4.96 g) and Trial 2 animals in the pre-fattening stage (27.20 ± 0.44 g). The tested culture protocol, which lasted 6 and 4 months for Trial 1 and 2, respectively, mainly reduced handling-derived stressors in the experimental tanks via lowering routine samplings to a minimum. This decrease of the handling-derived stress was reflected in both trials with lower concentration of circulating cortisol in blood plasma from the experimental fish when compared to controls. Moreover, the proposed protocol promoted higher growth in the fish cultured in the less disturbing protocol in Trial 2. Higher specific growth rates and mean body weight and length were reported. In order to further explore the potential beneficial effects of our protocol, we studied the musculoskeletal from Trial 2 gene expression of key genes regulating glucocorticoid signaling pathway and apoptosis: glucocorticoid receptors 1 and 2 (gr1, gr2), heat shock protein 90 AA (hsp90aa), and caspase 6 (casp6). In line with the cortisol reduced level in this trial, gr1, hsp90aa, and casp6 genes showed lower expression in the samples coming from the experimental group. The findings of this study provide valuable information to the aquaculture industry for the management of Solea senegalensis stress and welfare.SIPublicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL

Evaluación por parte de estudiantes de biotecnología de la actividad docente dirigida a adquirir la competencia "uso del cuaderno de laboratorio"

El uso correcto del cuaderno de laboratorio como herramienta de trabajo es una competencia muy demandada por las empresas biotecnológicas y los laboratorios públicos y privados. Presentamos una experiencia docente centrada en el aprendizaje del uso del cuaderno de laboratorio, dentro de la asignatura "Técnicas en Biología Celular", impartida en 5º curso de la Licenciatura de Biotecnología en la Universidad de León. Estos estudiantes no habían cursado otras asignaturas en las cuales el cuaderno de laboratorio fuese una competencia a adquirir. La asignatura es eminentemente práctica, proporcionando el entorno perfecto para presentar el cuaderno de laboratorio. En primer lugar, los docentes proporcionamos a los estudiantes normas y ejemplos para que ellos pudiesen comprender la función y propósito de un cuaderno de laboratorio, así como su función central en la investigación científica. Se insistió en que se debía demostrar capacidad para reflejar de manera honesta la metodología, los resultados obtenidos, su interpretación y las incidencias ocurridas. Tras cada sesión, los docentes revisábamos los cuadernos, discutiendo con los estudiantes los aspectos que podrían ser mejorados. Al finalizar las sesiones se evaluaron los cuadernos, añadiendo anotaciones sobre la evaluación, y se devolvieron a los estudiantes. Posteriormente, se invitó a los estudiantes a participar en una encuesta online y anónima (Moodle) para valorar el proceso de adquisición de la competencia de uso del cuaderno de laboratorio, que fue contestada por 20 de 27 estudiantes. Al 65% le pareció una actividad interesante y útil para su actividad futura, y al 15% a que consideraba que era interesante pero poco relacionada con su actividad futura. También se preguntó sobre su actividad futura, distribuyéndose: 25% en investigación, sector público; 30% investigación, sector privado; 15% otras, sector público; 30% otras, sector privado. Todos los estudiantes que escogieron investigación en el sector público consideraron la actividad útil, mientras que en las otras categorías sólo la mitad lo consideró útil. La revisión diaria del cuaderno le pareció útil al 70%, aunque el 25% consideró que era demasiado exhaustiva. Las indicaciones tras la revisión final fueron útiles para el 60%. El mayor obstáculo para los estudiantes fue la falta de experiencia previa en el uso del cuaderno de laboratorio, aunque este fue también uno de los motivos por el que esta actividad fue considerada como interesante

Cryobiology of cephalopod (Illex coindetii) spermatophores

P. 288-294Cephalopod culture is expected to increase in the near future and sperm cryopreservation would be a valuable tool to guarantee sperm availability throughout the year and to improve artificial insemination programs. We have studied the tolerance of spermatophores from the oceanic squid Illex coindetii to several cryoprotectants, in two toxicity experiments and a cryopreservation test. Five permeating cryoprotectants were tested: Dimethyl sulfoxide (Me2SO), methanol, glycerol, propylene glycol and ethylene glycol. In the first experiment, spermatophores were exposed to the five cryoprotectants at 5% (v/v) and 15% (v/v) at 4 °C for 5 min. In the second experiment, spermatophores were exposed to the cryoprotectants at 15% using different exposure times: 5, 15 and 30 min. In a third experiment, we tested two cryopreservation protocols: LN2 vapor or −80 °C freezer, using a 15% cryoprotectant and 15 or 30 min of exposure. Viability and mitochondrial activity were assessed using Mitotracker deep red, YOPRO1 and Hoechst 33342, by flow cytometry. Spermatozoa in this species remain viable after cryoprotectant exposure but their quality decreased considerably after cryopreservation, only 5–10% of spermatozoa being motile. Flow cytometry demonstrated that Me2SO may be the most appropriate cryoprotectant for I. coindetii spermatozoa, and shows a first approach on cephalopod sperm cryopreservation, opening new possibilities for the research and culture of this group of molluscs