Self-learning through the PhysioExTM 9.0 simulator as a teaching tool in Veterinary Physiology. The opinion of the students

[EN] Self-learning has been proposed as an active and plausible methodology to promote the capability of students to reach assigned objectives. During the academic year of 2016-2017, the course of Veterinary Physiology (included in the degree of Veterinary Medicine, University of Murcia, Murcia, Spain) was given using a self-learning method through the computer simulator PhysioExTM 9.0. The practice consisted in  solving 6 exercises, performing simulated laboratory actions, knowing the consequences of each of their actions and answering a series of questions that were discussed afterwards with their classmates. The objective of this learning methodology was to teach students to work independently as well as a team member, promoting their skills to solve problems that might appear later in their professional life. After the practice, the students completed a voluntary survey whose results showed a satisfying opinion about using this self-learning methodology, reaching an average score on the proposed statements (a total of 7) between 4.01 and 4.71 on a Likert scale from 1 to 5. Additionally, the students associated the practice with concepts as ‘classmates’, ‘doubts’, ‘dynamic’, ‘better’, ‘help’ and ‘knowledge’. In conclusion, this activity increased the collaborative learning process of students and enhanced dynamism in class.http://ocs.editorial.upv.es/index.php/HEAD/HEAD18Soriano Úbeda, C.; García Vázquez, F. (2018). Self-learning through the PhysioExTM 9.0 simulator as a teaching tool in Veterinary Physiology. The opinion of the students. Editorial Universitat Politècnica de València. 407-414. https://doi.org/10.4995/HEAD18.2018.8005OCS40741

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

BackgroundThe in vivo concentration of bicarbonate (HCO3-), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3- concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased.ResultsOnce exposed to the capacitation medium, the intracellular pH (pH(i)) of spermatozoa increased immediately even at low concentrations of HCO3-, but only extracellular concentrations of and above 15mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15mmol/L of HCO3- stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3- concentration to 15mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%).ConclusionsOptimising HCO3- concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15mmol/L of HCO3- in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the European Regional Development Fund (FEDER), Grants AGL2012-40180-C03-01-02 and AGL2015-66341-R), Fundacion Seneca (20040/GERM/16) and by a grant R01-HD-038082 (to P. E. V.) from the National Institutes of Health (NIH), USA

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

[EN] Background: The in vivo concentration of bicarbonate (HCO3), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3 concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. Results: Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3 stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3 concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Conclusions: Optimising HCO3 concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3 in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcineSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the European Regional Development Fund (FEDER), Grants AGL2012–40180-C03–01-02 and AGL2015–66341-R), Fundación Séneca (20040/GERM/16) and by a grant R01-HD-038082 (to P.E.V.) from the National In- stitutes of Health (NIH), US

The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

[EN] OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the eggSIThe research was supported by the Spanish MINECO (Spain) and FEDER (AGL2012-40180-C03-01-02 and AGL2015-70159-P) and the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 260759 [L.J.]. We thank the Electron Microscopy Service, Image Analysis and Molecular Biology Sections of University of Murcia for their technical assistanc

Reproductive Outcomes and Endocrine Profile in Artificially Inseminated versus Embryo Transferred Cows

[EN] The increasing use of in vitro embryo production (IVP) followed by embryo transfer (ET), alongside with cryopreservation of embryos, has risen concerns regarding the possible altered pregnancy rates, calving or even neonatal mortality. One of the hypotheses for these alterations is the current culture conditions of the IVP. In an attempt to better mimic the physiological milieu, embryos were produced with female reproductive fluids (RF) as supplements to culture medium, and another group of embryos were supplemented with bovine serum albumin (BSA) as in vitro control. Embryos were cryopreserved and transferred while, in parallel, an in vivo control (artificial insemination, AI) with the same bull used for IVP was included. An overview on pregnancy rates, recipients’ hormonal levels, parturition, and resulting calves were recorded. Results show much similarity between groups in terms of pregnancy rates, gestation length and calves’ weight. Nonetheless, several differences on hormonal levels were noted between recipients carrying AI embryos especially when compared to BSA. Some calving issues and neonatal mortality were observed in both IVP groups. In conclusion, most of the parameters studied were similar between both types of IVP derived embryos and the in vivo-derived embryos, suggesting that the IVP technology used was efficient enough for the safe production of calvesSIThis research was funded by European Union, Horizon 2020 Marie Sklodowska-Curie Action, grant number REPBIOTECH675526 and as well as by the Ministry of Economy and Competitiveness (Spain), grants number AGL2015-66341-R & AGL2015-70140-R MINECO-FEDER and Fundación Séneca, grant number 20040/GERM/1

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage

[EN] Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.SIMCIN/AEI/10.13039/501100011033/FEDERUniversity of LeónFPU17/04142PRE2018-086400Junta de Castilla y LeónFSE PO 14-2

The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment

[EN] Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300 μM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescence microscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage.SIMCIN/AEI/10.13039/501100011033/FEDERUniversity of LeónMINECOJunta de Castilla y Leó

Bos taurus and Cervus elaphus as Non-Seasonal/Seasonal Models for the Role of Melatonin Receptors in the Spermatozoon

[EN] Melatonin is crucial in reproduction due its antioxidant, hormonal, and paracrine action. Melatonin membrane receptors (MT1/MT2) have been confirmed on spermatozoa from several species, but functionality studies are scarce. To clarify their role in ruminants as reproductive models, bull (Bos taurus, non-seasonal) and red deer (Cervus elaphus, highly seasonal) spermatozoa were analyzed after 4 h of incubation (38 °C, capacitating media) in 10 nM melatonin, MT1/MT2 agonists (phenylmelatonin and 8M-PDOT), and antagonists (luzindole and 4P-PDOT). Motility and functionality (flow cytometry: Viability, intracellular calcium, capacitation status, reactive oxygen species (ROS) production, and acrosomal and mitochondrial status) were assessed. In bull, MT1 was related to sperm viability preservation, whereas MT2 could modulate cell functionality to prevent excess ROS produced by the mitochondria; this action could have a role in modulating sperm capacitation. Deer spermatozoa showed resistance to melatonin and receptor activation, possibly because the samples were of epididymal origin and collected at the breeding season’s peak, with high circulating melatonin. However, receptors could be involved in mitochondrial protection. Therefore, melatonin receptors are functional in the spermatozoa from bull and deer, with different activities. These species offer models differing from traditional laboratory experimental animals on the role of melatonin in sperm biologySIThis research was funded by the Ministry of Economy, Industry and Competitivenes MINECO (Spain), grant number AGL2013-43328

Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM

Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits. Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.MINECO, FEDER, Fundación Senec