Analysis of Minor Proteins Present in Breast Milk by Using WGA Lectin.

Breast milk is a complex and dynamic biological fluid and considered an essential source of nutrition in early life. In its composition, the proteins have a relevant biological activity and are related to the multiple benefits demonstrated when compared with artificial milks derived from cow's milk. Understanding human milk composition provides an important tool for health care providers toward the management of infant feeding and the establishment of breastfeeding. In this work, a new technique was developed to increase the knowledge of human milk, because many of the components remain unknown. To isolate minor proteins present in breast milk by using WGA lectin, breast milk was centrifuged to remove cells and separate the fat phase from the serum phase. The serum obtained was separated into two groups: control (n = 3; whole serum sample from mature milk) and WGA lectin (n = 3; sample processed with WGA lectin to isolate glycosylated proteins). The samples were analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). A total of 84 different proteins were identified from all of the samples. In the WGA lectin group, 55 different proteins were isolated, 77% of which had biological functions related to the immune response. Of these proteins, there were eight WGA lectin group exclusives, and two had not previously been described in breast milk (polyubiquitin-B and POTE ankyrin domain family member F). Isolation by WGA lectin is a useful technique to detect minor proteins in breast milk and to identify proteins that could not be observed in whole serum.

Self-directed learning using computer simulations to study veterinary physiology: Comparing individual and collaborative learning approaches.

BACKGROUND: Advances in technology enable new educational resources geared towards situated learning and leading students to a more active education. Self-directed learning methodologies used along with simulators may represent a good alternative to traditional teaching methods. The aims of this study were to analyse veterinary students' degree of acceptance of self-directed learning using the PhysioEx simulator in physiology, and to evaluate self-directed learning outcomes using different approaches (individual vs. collaborative). METHODS: The study was carried out over three academic years. Students conducted different activities on the PhysioEx simulator, either in an individual or cooperative mode. Once the activities were finished, students voluntarily participated in an opinion survey regarding self-directed learning methodology. Finally, an evaluation of learning outcomes was made through Kahoot!. RESULTS: Students expressed a high degree of satisfaction with this self-directed learning method, with the combination of self-directed learning and gamification being scored the highest. Although students prefer the collaborative method, no differences in learning outcomes were found between the two learning approaches. CONCLUSION: The self-directed learning method in combination with gamification increased the motivation of students, who obtained suitable learning outcomes regardless of whether an individual or collaborative approach was followed.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Spatial and Pregnancy-Related Changes in the Protein, Amino Acid, and Carbohydrate Composition of Bovine Oviduct Fluid.

Knowledge of how the biochemical composition of the bovine oviduct is altered due to the oviduct anatomy or the presence of an embryo is lacking. Thus, the aim of this study was to assess the effect of (І) oviduct anatomy and (ІІ) embryo presence on oviductal fluid (OF) protein, amino acid, and carbohydrate composition. Cross-bred beef heifers (n = 19) were synchronized and those in standing estrus were randomly allocated to a cyclic (non-bred) or pregnant (artificially inseminated) group. All heifers were slaughtered on Day 3 after estrus. The oviducts ipsilateral to the corpus luteum from each animal were isolated, straightened and cut, separating ampulla and isthmus. Each portion was flushed with 500 µl of PBS enabling recovery of the oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (8-cell embryos; pregnant group) were located in the isthmus of the oviduct. Samples of flushing medium from the isthmus and ampulla were used for proteomic (n = 2 per group), amino acid (n = 5), and carbohydrate (n = 5) analysis. For proteomic analysis, total protein from cyclic and pregnant samples were labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3-10, 17 cm, Protean® IEF cell system, Bio Rad). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). Images were obtained with a Typhoon 9410 scanner and analyzed with Progenesis SameSpots software v 4.0. Amino acid content in the OF was determined by high performance liquid chromatography (HPLC). Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. For the proteomic assessment, the results of the image analysis were compared by ANOVA. For both amino acid and carbohydrate analyses, statistical analysis was carried out by 2-way ANOVA with the Holm-Sidak nonparametric post hoc analysis. On Day 3 post-estrus, OF composition varied based on (І) anatomical region, where isthmic metabolites were present in lower (i.e., lactate, glycine, and alanine) or higher (i.e., arginine) concentrations compared to the ampulla; and (ІІ) embryo presence, which was correlated with greater, arginine, phosphoglycerate kinase 1, serum albumin, α-1-antiproteinase and IGL@ protein concentrations. In conclusion, data indicate that the composition of bovine OF is anatomically dynamic and influenced by the presence of an early embryo.

Regulation of boar sperm functionality by the nitric oxide synthase/nitric oxide system.

PURPOSE: Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects. METHODS: Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media. RESULTS: Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes. CONCLUSIONS: These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.

A new device for deep cervical artificial insemination in gilts reduces the number of sperm per dose without impairing final reproductive performance.

BACKGROUND: The aim of this study was to evaluate the reproductive performance of a new artificial insemination (AI) device specifically designed for gilts (Deep cervical AI, Dp-CAI) by means of which the sperm is deposited deeply in the cervix (8 cm more cranial than in traditional cervical insemination-CAI). New AI techniques have arisen in recent decades in the porcine industry, such as post-cervical artificial insemination (PCAI), which involves depositing the sperm in the body of the uterus [through a catheter (outer tube)-cannula (inner tube)] rather than by CAI. Although the PCAI method has been successfully applied in farm conditions to reduce sperm doses without impairing the reproductive performance, this technique has limitations in gilts mainly because of the difficulty involved in introducing the inner cannula through the cranial part of the cervix. For this reason, the Dp-CAI method described herein may be considered as an alternative to CAI and PCAI methods in gilts. RESULTS: Gilts were divided in two experimental groups: 1) Dp-CAI: gilts (n = 1166) inseminated using 1.5 × 109 sperm/45 mL; 2) CAI (as a control group): gilts (n = 130) inseminated using 2.5 × 109 sperm/85 mL. The Dp-CAI method was successfully applied in 88.90% of the gilts, with no differences detected between gilts with 1 or 2 previous oestrus cycles, although the catheter could be introduced more deeply in 2 oestrus gilts (P < 0.05). As the length of the insemination device that could not be introduced increased (at the moment of insemination), so the success rate of the Dp-CAI device fell, as did the total number of piglets born. When the reproductive output in CAI and Dp-CAI was compared, none of the parameters analysed [pregnancy and farrowing rates (%), and number of piglets born (total and live)] showed significant differences. CONCLUSIONS: The use of the Dp-CAI technique provides a new AI method as an alternative to CAI and PCAI for pigs. The device, especially designed for gilts, was used with a high degree of success reducing conventional sperm doses without impairing reproductive parameters.

Oviductal epithelial cells selected boar sperm according to their functional characteristics.

The interaction of oviductal epithelial cells (OECs) with the spermatozoa has beneficial effects on the sperm functions. The aim of this study is to evaluate the in vitro fertilizing capacity of incubating spermatozoa previously selected by density gradient in OEC and determinate some sperm characteristics that could explain the results obtained. In this study, we assessed in vitro fertilization (IVF), tyrosine phosphorylation, phosphatidylserine translocation, nuclear DNA fragmentation, and chromatin decondensation. Three experimental sperm groups, previously selected by Percoll gradient, were established according to the origin of the sperm used for IVF: (i) W30 group: spermatozoa were incubated with oocytes in the absence of OEC; (ii) NB group: after sperm incubation in OEC, the unbound spermatozoa were incubated with oocytes, in the absence of OEC; and (iii) B group: after sperm incubation with OEC, the bound spermatozoa were incubated with oocytes in the OEC plates. The results showed that sperm from the NB group led to a lower IVF yield, accompanied by low penetration rates (NB: 19.6%, B: 94.9%, and W30: 62.9%; P < 0.001) and problems of nuclear decondensation. Moreover, higher levels of tyrosine phosphorylation were observed in the NB group compared with the W30 and B groups (NB: 58.7%, B: 2.5%, and W30: 4.5%; P < 0.01). A similar trend was observed in phosphatidylserine translocation (NB: 93.7%, B: 5.7%, and W30: 44.2%; P < 0.01). These results demonstrate that the OEC exerts a rigorous degree of sperm selection, even within an already highly selected population of spermatozoa, and can capture the best functional spermatozoa for fertilization.

The oviductal transcriptome is influenced by a local ovarian effect in the sow.

BACKGROUND: Oviducts participate in fertilization and early embryo development, and they are influenced by systemic and local circulation. Local functional interplay between ovary, oviduct and uterus is important, as deduced from the previously observed differences in hormone concentrations, presence of sperm, or patterns of motility in the oviduct after unilateral ovariectomy (UO). However, the consequences of unilateral ovariectomy on the oviductal transcriptome remain unexplored. In this study, we have investigated the consequences of UO in a higher animal model as the pig. METHODS: The influence of UO was analyzed on the number of ovulations on the contra ovary, which was increased, and on the ipsilateral oviductal transcriptome. Microarray analysis was performed and the results were validated by PCR. Differentially expressed genes (DEGs) with a fold change ≥ 2 and a false discovery rate of 10 % were analyzed by Ingenuity Pathway Analysis (IPA) to identify the main biofunctions affected by UO. RESULTS: Data revealed two principal effects in the ipsilateral oviduct after UO: i) down-regulation of genes involved in the survival of sperm in the oviduct and early embryonic development, and ii) up-regulation of genes involved in others functions as protection against external agents and tumors. CONCLUSIONS: Results showed that unilateral ovariectomy results in an increased number of ovulation points on the contra ovary and changes in the transcriptome of the ipsilateral oviduct with consequences on key biological process that could affect fertility output.

Oviductal Transcriptome Is Modified after Insemination during Spontaneous Ovulation in the Sow.

Gene Expression Microarray technology was used to compare oviduct transcriptome between inseminated and non-inseminated pigs during spontaneous oestrus. We used an in vivo model approaching the study from a physiological point of view in which no hormonal treatment (animals were in natural oestrus) and no artificial sperm selection (selection was performed within the female genital) were imposed. It is therefore emphasised that no surgical introduction of spermatozoa and no insemination at a site other than the physiological one were used. This approach revealed 17 genes that were two-fold or more up-regulated in oviducts exposed to spermatozoa and/or developing embryos and 9 genes that were two-fold or more down-regulated. Functional analysis of the genes revealed that the top canonical pathways affected by insemination were related to the inflammatory response and immune system (Network 1) to molecular transport, protein trafficking and developmental disorder (Network 2) and to cell-to-cell signalling and interaction (Network 3). Some of the genes in network 1 had been previously detected in the oviduct of human and animals, where they were over-expressed in the presence of spermatozoa or pre-implantation embryos (C3, IGHG1, ITIH4, TNF and SERPINE1) whereas others were not previously reported (SAA2, ALOX12, CD1D and SPP1). Genes in Network 2 included RAB1B and TOR3A, the latter being described for the first time in the oviduct and clearly expressed in the epithelial cells of the mucosa layer. Network 3 integrated the genes with the highest down-regulation level (CYP51, PTH1R and TMOD3). Data in the present study indicate a change in gene expression during gamete encounter at the site of fertilization after a natural sperm selection within the female genital tract. These changes would indicate a modification of the environment preparing the oviduct for a successful fertilization and for an adequate embryo early development.

A new class of marine Euryarchaeota group II from the Mediterranean deep chlorophyll maximum.

We have analyzed metagenomic fosmid clones from the deep chlorophyll maximum (DCM), which, by genomic parameters, correspond to the 16S ribosomal RNA (rRNA)-defined marine Euryarchaeota group IIB (MGIIB). The fosmid collections associated with this group add up to 4 Mb and correspond to at least two species within this group. From the proposed essential genes contained in the collections, we infer that large sections of the conserved regions of the genomes of these microbes have been recovered. The genomes indicate a photoheterotrophic lifestyle, similar to that of the available genome of MGIIA (assembled from an estuarine metagenome in Puget Sound, Washington Pacific coast), with a proton-pumping rhodopsin of the same kind. Several genomic features support an aerobic metabolism with diversified substrate degradation capabilities that include xenobiotics and agar. On the other hand, these MGIIB representatives are non-motile and possess similar genome size to the MGIIA-assembled genome, but with a lower GC content. The large phylogenomic gap with other known archaea indicates that this is a new class of marine Euryarchaeota for which we suggest the name Thalassoarchaea. The analysis of recruitment from available metagenomes indicates that the representatives of group IIB described here are largely found at the DCM (ca. 50 m deep), in which they are abundant (up to 0.5% of the reads), and at the surface mostly during the winter mixing, which explains formerly described 16S rRNA distribution patterns. Their uneven representation in environmental samples that are close in space and time might indicate sporadic blooms.

Boar sperm tyrosine phosphorylation patterns in the presence of oviductal epithelial cells: in vitro, ex vivo, and in vivo models.

Spermatozoa transport through the oviduct is a controlled process that regulates sperm capacitation. A crucial event involved in capacitation is protein tyrosine phosphorylation (TP). This study was undertaken to determine whether similarities exist in protein TP distribution between spermatozoa bound or unbound to oviductal epithelial cells (OEC) in three different conditions: i) in vitro, spermatozoa coincubated with OEC cultures; ii) ex vivo, spermatozoa deposited in porcine oviductal explants from slaughtered animals; iii) in vivo, in which sows were inseminated and the oviduct was recovered. The localization of phosphotyrosine protein was determined using indirect immunofluorescence. The distribution of protein TP was significantly (P<0.05) different between bound and unbound cell populations in all experiments. In sows inseminated close to ovulation, spermatozoa were found mainly in the utero-tubal junction, where spermatozoa exhibited higher proportion of flagellum phosphorylation. Spermatozoa not bound to OEC exhibited high levels of protein phosphorylation (phosphorylated equatorial subsegment and acrosome and/or phosphorylated flagellum) in the ex vivo and in vivo experiments (P<0.05). However, unbound spermatozoa coincubated with OEC in in vitro conditions tended to show intermediate levels of TP (equatorial subsegment with or without phosphorylated flagellum). In spermatozoa bound to OEC, protein TP was located in the equatorial subsegment or presented no phosphorylation (P<0.05). Although sperm capacitation conditions in vivo were not reproducible in vitro in our experimental conditions, sperm and OEC binding seemed to be a mechanism for selecting spermatozoa with a low level of TP in in vivo, ex vivo, and in vitro experiments.