Effects of GLUBRAN-2 on the Burst Pressure of Jejunal Loops Thermofused With Vessel Sealing Devices.

BACKGROUND: Off-label use of radiofrequency vessel sealing devices for intestinal thermofusion has been reported as an alternate approach for closing the small and large intestines. The study aimed to evaluate if reinforcing the thermofusion line with a modified N-butyl-2-CyanoAcrylate and methacryloxysulpholane produced improved burst pressure values in ex vivo swine jejunal loops. MATERIALS AND METHOD: A suture-less full-thickness jejunal biopsy was performed with different radiofrequency vessel-sealing devices (Ligasure 5 mm: RFVS-1; Atlast 10 mm: RFVS-2; Cayman Maryland: RFVS-3), and reinforcement with modified cyanoacrylate Glubran-2 (G2) at the thermofusion defect was applied. Burst pressure(BP) values were compared with a control group, wherein a cold blade was utilized to obtain the biopsy, followed by the closing of the jejunum with seven Gambee sutures. RESULTS: Seventy (n = 70) jejunal loop samples were distributed into the experimental groups.The RFVS-1 and -2 groups exhibited BP values similar to those of the suture group. The RFVS-3 group showed significantly lower BP values (P < 0.05) than the suture group. Conversely, in the groups wherein G2 was applied, all BP values were comparable to those of the suture group. BP test in the RFVS-3G2 group showed significantly (P < 0.05) higher values in the group using the same instrument without the glue (RFVS-3). CONCLUSIONS: G2 has been shown to improve the BP on the defects created by instruments that are not completely efficient in intestinal thermofusion and sealing. This experimental model showed that the performance of full-thickness biopsies with RFVS devices and reinforcement with G2 provide feasible and promising results.

Fusarium spp. in Loggerhead Sea Turtles (Caretta caretta): From Colonization to Infection.

With the aim of evaluating the presence of Fusarium spp. in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles (Caretta caretta) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp. were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 (Fusarium keratoplasticum), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp. Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

Retrograde flushing collection and freezing of dromedary camel epididymal spermatozoa with seminal plasma.

The objectives of this study were to describe the parameters of dromedary camel epididymal spermatozoa collected by retrograde flushing (RF) technique and to evaluate the freezability of the collected sperm, diluted with and without the supplementation of seminal plasma (SP). Two experiments were conducted: in Experiment 1, ES were recovered within 6-8 h after castration; selected samples were diluted with a Tris-citrate egg-yolk glycerolated buffer and frozen. In Experiment 2, epididymides were stored for 24 h at 4 °C before RF and semen samples were frozen after dilution with a Tris-lactose egg-yolk glycerolated extender with and without 15% SP. In Experiment 1, eight semen samples were obtained from ten epididymides with a mean of 500 × 106 total spermatozoa recovered, per flushed epididymis. Mean post-thaw motility and progressive motility were 75 and 17%, respectively. In Experiment 2, 15 samples were collected, out of the 18 epididymides (mean number of collected spermatozoa: 700 × 106), and 13 of these samples were of excellent quality. Post-thaw parameters were not satisfactory but the supplementation of the freezing medium with 15% SP improved the progressive motility and kinematic parameters of the spermatozoa.

Surface glycan pattern of canine, equine, and ovine bone marrow-derived mesenchymal stem cells.

The use of bone marrow-derived mesenchymal stem cells (MSCs) for clinical and experimental studies is increasing, but full characterization of MSCs in veterinary species is hindered by the variability in species-specific cell surface marker expression and antibody cross reactivity. Recent studies demonstrated that the glycans in the glycocalyx of MSCs are promising candidates as cell biomarkers. In the present study, we analyzed the glycocalyx of canine MSCs (cMSCs), ovine MSCs (oMSCs), and equine MSCs (eMSCs) using a cell microarray procedure in which MSCs were spotted on microarray slides and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. The signal intensity was then detected using a microarray scanner. The lectin-binding signals indicated that the MSC surface of the investigated species contained both N- and O-linked glycan types, with N-glycosylation predominating over O-glycosylation and fucosylation being more abundant than sialylation. Relative quantification revealed an interspecific difference between these glycans. In addition, cMSCs expressed more α2,3-linked sialic acid (MAL II), terminal lactosamine (RCA120 ), and α1,6 and α1,3 fucosylated oligosaccharides (PSA, LTA); oMSCs exhibited more T antigen (Jacalin), GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1 (DBA), chitotriose (succinylated WGA), and α1,2-linked fucose (UEA I); and eMSCs showed a higher density of α2,6 sialic acids (SNA) and high mannose N-glycans (Con A). Using cell microarray methodology, we have for the first time demonstrated differences in the glycosylation profiles of cMSC, oMSC, and eMSC surfaces. These results could be valuable as resources and references for MSC differentiation and molecular remodeling in clinical cell-based therapy and tissue engineering studies. © 2017 International Society for Advancement of Cytometry.

A lectin-based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile.

The high complexity of glycome, the repertoire of glycans expressed in a cell or in an organism, is difficult to analyze and the use of new technologies has accelerated the progress of glycomics analysis. In the last decade, the microarray approaches, and in particular glycan and lectin microarrays, have provided new insights into evaluation of cell glycosylation status. Here we present a cell microarray method based on cell printing on microarray slides for the analysis of the glycosylation pattern of the cell glycocalyx. In order to demonstrate the reliability of the developed method, the glycome profiles of equine native uncultured mural granulosa cells (uGCs) and in vitro cultured mural granulosa cells (cGCs) were determined and compared. The method consists in the isolation of GCs, cell printing into arrays on microarray slide, incubation with a panel of biotinylated lectins, reaction with fluorescent streptavidin and signal intensity detection by a microarray scanner. Cell microarray technology revealed that glycocalyx of both uGCs and cGCs contains N-glycans, sialic acid terminating glycans, N-acetylglucosamine and O-glycans. The comparison of uGCs and cGCs glycan signals indicated an increase in the expression of sialic acids, N-acetylglucosamine, and N-glycans in cGCs. Glycan profiles determined by cell microarray agreed with those revealed by lectin histochemistry. The described cell microarray method represents a simple and sensitive procedure to analyze cell surface glycome in mammalian cells.

Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and ßN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.

Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)ß1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galß1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galß1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells.

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

The abundance and localization of heat shock proteins (HSP)-60, -70, and -90 in the oviductal ampulla of hamadryas baboon (Papio hamadryas) during the menstrual cycle.

The presence of HSPs in female reproductive and their relationship with the steroid hormone fluctuation have been reported in several mammals but not in non-human primates. The present research dealt with the oviductal expression and localization of the more studied HSPs (60, 70, and 90) as well as the morphological changes in the Hamadryas baboon (Papio hamadryas) during the follicular, preovulatory, and luteal phases of the menstrual cycle. Therefore, western blots, histomorphological, and immunohistochemical analyses were carried out. The results of western blot analysis displayed the lowest HSP expression in the luteal phase. The histomorphology showed that the mucosal epithelium consisted of undifferentiated cuboidal cells in follicular and luteal phases and well-distinguishable columnar ciliated and non-ciliated cells during the preovulatory phase. Immunohistochemistry evidenced that the mucosal epithelium contained cytoplasmic and nuclear HSP60, 70, and 90 immunostaining in the follicular and luteal phases. During the preovulatory phase, the non-ciliated cells showed: (i) cytoplasmic HSP60; (ii) nuclear and cytoplasmic HSP90. Ciliated cells showed cytoplasmic and ciliary HSP70 and ciliary HSP90. The stromal cells and myocytes of muscular layer displayed a decreased cytoplasmic HSP60 in the preovulatory phase and nuclear and low cytoplasmic HSP70 throughout the menstrual cycle. Nuclear HSP90 decreased in ampulla stromal cells and the follicular phase myocytes. These findings indicate that the expression pattern of HSP60,70, and 90 is related to the morphofunctional features of the baboon oviductal ampulla during the menstrual cycle and could represent a referent point for further studies in the oviduct of Primates.

Morphology, Glycan Pattern, Heat Shock Proteins, and Sex Steroid Receptors Expression in the Tubal Fimbria Epithelium of the Baboon Papio hamadryas during the Menstrual Cycle.

The oviductal fimbria is the first extraovarian anatomical structure that the cumulus-oocyte complex (COC) encounters, and is sensitive to sex hormone changes. The morphology, glycan pattern, expression of heat shock proteins (HSPs), estradiol receptor (ER), and progesterone receptor (PR) were investigated in the oviductal fimbria epithelium of the baboon (Papio hamadryas) during the menstrual cycle. The morphology was investigated by light and scanning electron microscopy; the glycopattern was characterized using conventional and lectin histochemistry; HSPs (60, -70, -90), ER, and PR were localized immunohistochemically. Well-differentiated ciliated and nonciliated cells were present only during the preovulatory phase. The nonciliated cells contained small apical protrusions and thin microvilli. During the preovulatory phase (1) the luminal surface of the fimbria displayed acidic glycans, complex N-glycans containing fucose, and oligolactosamine residues; (2) nonciliated cells expressed HSP60 and HSP90 in the apical blebs, HSP70 in the nucleus and cytoplasm, as well as nuclear ERα and PR; (3) ciliated cells showed HSP70 in the nucleus, cytoplasm, and cilia that also expressed HSP90 and PR. These results are related to the function of the fimbria where the early COC-oviduct crosstalk occurs and may represent a benchmark for translational studies of other primates.

Altered glycosylation in secreting cells of the gastric glands of aquaporin-4-deficient mice.

Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin-4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4-deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4-deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5-µm thick, were analyzed by histochemical methods (Periodic acid-Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA-I, AAA, LTA). Immunohistochemical methods such as anti-Muc6 for neck cells and anti- ß- H+/K+-ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions. RESEARCH HIGHLIGHTS: Glycopatterns in gastric glands were compared between wild type (WT) and AQP4-deficient knockout (KO) mice by histochemical and lectin-binding methods. In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed. In the parietal cells lower fucosylation also resulted. AQP4-deficiency affects glycosylation and could result in altered functionality and pathological conditions.

Response of laying hens fed diet supplemented with a mixture of olive, laurel, and rosemary leaf powders: Metabolic profile, oxidative status, intestinal histomorphology, and egg quality.

This study aimed to evaluate the effects of a mixture of olive, laurel, and rosemary leaf powders, on the oxidative state, biochemical, immune, intestinal morphophysiological parameters, and egg quality of laying hens. One hundred Lohmann Brown hens (28 weeks old) were equally assigned to two groups (n. 50) corresponding to a basal control diet (CON) or the diet supplemented with 6 g/kg feed of leaf powder mixture (LPM) containing olive, laurel, and rosemary leaves (1:1:1), for 60 days. Oxidative status, biochemical indices, immune response, cecal short chain fatty acids (SCFAs), intestinal morphological characteristics, and some egg traits were evaluated at the end of the experiment. The results indicated that LPM improved (P < 0.05) the oxidative status (TOS, ROMs), the immune system (IL-6, IL-1ß, and TNF-α), the total protein and HDL cholesterol content, whereas it decreased (P < 0.05) total cholesterol and LDL cholesterol. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), and alanine aminotransferase were significantly (P < 0.05) lower in the LPM than in the CON group. A significant increase (P < 0.05) in SCFA content in the caecum, as well as in villi height and crypt depth in both duodenum and ileum of LPM-treated hens, was observed. Egg quality parameters were not influenced (P > 0.05) by LPM. These findings indicate that LPM can be considered a candidate as an antioxidant ingredient for functional food in laying hens.

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species.

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Morphology of the syrinx of three species of birds from Brazilian cerrado (Psittacara leucophthalmus, Rhynchotus rufescens and Cariama cristata): Gross anatomy and light microscopy study.

The aim of this study was to describe the morphology of the trachea and syrinx at macroscopic and light microscopy levels of three species of birds from different orders that inhabit the Brazilian cerrado. For that, five adult specimens (three males and two females of each species) of white-eyed parakeet (Psittacara leucophthalmus), red-winged tinamou (Rhynchotus rufescens) and red-legged seriema (Cariama cristata) were used. The trachea and syrinx of the birds were collected and destined for anatomical and histological studies. The trachea of the studied birds presented an elongated path and originated in the larynx and extended caudally to the syrinx. No sexual dimorphism was observed in the syrinx of the studied species, probably because it is associated with their song, which is very similar between males and females of these species. The findings of this study allowed us to classify the syrinx as tracheal in the white-eyed parakeet and tracheobronchial in the red-winged tinamou and red-legged seriema. In general, the morphological features of the trachea and syrinx were similar to those described for other species of birds, such as the presence of intrinsic and extrinsic syringeal muscles, and the lateral and medial tympaniform membranes, which would represent important anatomical structures in sound production through vibration during expiration and eventual inspiration. The morphological structure of the syrinx in the three avian species of the Brazilian cerrado is consistent with the ability of these avian species to perform a potential vocalization, especially the red-legged seriema that emits characteristic sounds very loud and can carry several kilometres.

Modification of Morphology and Glycan Pattern of the Oviductal Epithelium of Baboon Papio hamadryas during the Menstrual Cycle.

The mammalian oviduct is a highly specialized structure where fertilization and early embryonic development occur. Its mucosal epithelium is involved in maintaining and modulating a dynamic intraluminal fluid. The oviductal epithelium consists of ciliated and non-ciliated (secretory) cells whose differentiation and activity are sex hormone-dependent. In this study, we investigated for the first time both the morphology and the glycan composition of baboon oviductal epithelium during the menstrual cycle. Oviducts were laparoscopically removed from 14 healthy adult female Papio hamadryas whose menstrual cycle phase was assessed based on the sex hormone levels and the vaginal cytology features. Histological investigations were carried out on fimbriae, infundibulum, ampulla, and isthmus separately fixed in 4% (v/v) paraformaldehyde, embedded in paraffin wax, and stained with hematoxylin-eosin for morphological analyses and using a panel of nine fluorescent lectins for glycoconjugate characterization. The histomorphological analysis revealed that in the entire oviduct (i) the ciliated and non-ciliated cells were indistinguishable during the follicular and luteal phases, whereas they were highly differentiated during the preovulatory phase when the non-ciliated cells exhibited apical protrusions, (ii) the epithelium height was significantly higher in the preovulatory phase compared to other menstrual phases, and (iii) the number of ciliated cells significantly (p ≤ 0.05) increased from the fimbriae to the infundibulum and progressively reduced in the other oviductal segments with the lower presence of ciliated cells in the isthmus. The glycan characterization revealed a complex and region-specific composition during the different phases of the menstrual cycle. It can be summarized as follows: (i) high-mannosylated N-linked glycans (Con A reactivity) were present throughout the oviductal epithelium during the entire menstrual cycle and characteristically in the apical protrusions of non-ciliated cells of the ampulla during the preovulatory phase; (ii) sialoglycans with α2,3-linked sialic acids (MAL II binding) were expressed along the entire oviductal surface only during the preovulatory phase, whereas α2,6-linked ones (SNA affinity) were also detected in the surface of the luteal phase, although during the preovulatory phase they were characteristically found in the glycocalyx of the isthmus cilia, and O-linked sialoglycans with sialic acids linked to Galßl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA) were found in the entire oviductal surface during all phases of the menstrual cycle; (iii) GalNAc terminating O-linked glycans (HPA staining) were mainly expressed in the entire oviducts of the luteal and preovulatory phases, and characteristically in the apical protrusions of the isthmus non-ciliated cells of the preovulatory phase; and (iv) fucosylated glycans with α1,2-linked fucose (LTA reactivity) occurred in the apical surface of fimbriae during the luteal phase, whereas α1,3/4-linked fucose (UEA I binders) were present in the apical protrusions of the ampulla non-ciliated cells and in the apical surface of isthmus during the preovulatory phase as well as in the isthmus apical surface of follicular-phase oviducts. These results demonstrate for the first time that morphological and glycan changes occur in the baboon oviductal epithelium during the menstrual cycle. Particularly, the sex hormone fluctuation affects the glycan pattern in a region-specific manner, probably related to the function of the oviductal segments. The findings add new data concerning baboons which, due to their anatomical similarity to humans, make an excellent model for female reproduction studies.

Differential expression of glycans in the urothelial layers of horse urinary bladder.

BACKGROUND: Urothelium is a multilayer epithelium covering the inner surface of the urinary bladder that acts as a blood-urine barrier and is involved in maintaining the wellbeing of the whole organism. Glycans serve in the maturation and differentiation of cells and thus play a key role in the morphology and function of the multilayered epithelium. The aim of the present study was to examine the glycoprotein pattern of the horse urinary bladder urothelium by lectin histochemistry. METHODS: The study involved urinary bladders from four horse stallions. Tissue sections were stained with a panel of eleven lectins, in combination with saponification and sialidase digestion (Ks). RESULTS: Basal cells displayed high-mannose N-glycans (Con A), α2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Galßl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acetylglucosamine (GlcNAc) (GSA II), galactose (GSA I-B4), T- and Tn antigens (PNA, SBA). The mature intermediate cells showed additional high-mannose N-glycans, O-linked sialoglycans (sialyl-T antigen, sialyl-Tn antigen), α2,6- and α2,3-linked sialic acid (MAL II), α1,2-linked fucose (UEA I), and GlcNAc (KsWGA). The latter residue marked the boundary with the overlying surface layer. Few Con A positive intermediate cells were seen to cross the entire urothelium thickness. The surface cells showed additional glycans such as T antigen and sialic acids linked to GalNAc binding DBA (KsDBA). Few surface cells contained α1,3-linked fucose (LTA), whereas some other cells displayed intraluminal secretion of mucin-type glycans terminating with GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1 (DBA). The luminal surface expressed the most complex glycan pattern in the urothelium because only α1,3-linked fucose lacked among the demonstrated glycans. CONCLUSIONS: This study showed that the glycan pattern becomes more complex from the basal to surface layer of the urothelium and that surface cells could modify the composition of urine via the secretion of glycoproteins.

Age-Related Changes in the Primary Motor Cortex of Newborn to Adult Domestic Pig Sus scrofa domesticus.

The pig has been increasingly used as a suitable animal model in translational neuroscience. However, several features of the fast-growing, immediately motor-competent cerebral cortex of this species have been adequately described. This study analyzes the cytoarchitecture of the primary motor cortex (M1) of newborn, young and adult pigs (Sus scrofa domesticus). Moreover, we investigated the distribution of the neural cells expressing the calcium-binding proteins (CaBPs) (calretinin, CR; parvalbumin, PV) throughout M1. The primary motor cortex of newborn piglets was characterized by a dense neuronal arrangement that made the discrimination of the cell layers difficult, except for layer one. The absence of a clearly recognizable layer four, typical of the agranular cortex, was noted in young and adult pigs. The morphometric and immunohistochemical analyses revealed age-associated changes characterized by (1) thickness increase and neuronal density (number of cells/mm2 of M1) reduction during the first year of life; (2) morphological changes of CR-immunoreactive neurons in the first months of life; (3) higher density of CR- and PV-immunopositive neurons in newborns when compared to young and adult pigs. Since most of the present findings match with those of the human M1, this study strengthens the growing evidence that the brain of the pig can be used as a potentially valuable translational animal model during growth and development.

Modulation of Morphology and Glycan Composition of Mucins in Farmed Guinea Fowl (Numida meleagris) Intestine by the Multi-Strain Probiotic Slab51®.

Probiotics have become highly recognized as supplements for poultry.Since gut health can be considered synonymous withanimal health, the effects of probiotic Slab51® on the morphology and the glycan composition of guineafowlintestine were examined. The probiotics were added in drinking water (2 × 1011 UFC/L) throughout the grow-out cycle.Birds were individually weighed andslaughtered after four months. Samples from the duodenum, ileum and caecum were collected and processed for morphological, morphometric, conventional and lectin glycohistochemical studies.The results were analyzed for statistical significance by Student's t test. Compared with control samples, probiotic group revealed (1) significant increase in villus height (p < 0.001 in duodenum and ileum; p < 0.05 in caecum), crypt depth (p < 0.001 in duodenum and caecum; p < 0.05 in ileum) and goblet cells (GCs) per villus (p < 0.001) in all investigated tracts; (2) increase in galactoseßl,3N-acetylgalacyosamine(Galßl,3GalNAc)terminating O-glycans and αl,2-fucosylated glycans secretory GCs in the duodenum; (3) increase in α2,6-sialoglycans and high-mannose N-linked glycans secretory GCs but reduction in GCs-secreting sulfoglycans in the ileum; (4) increase in Galßl,3GalNAc and high-mannose N-linked glycans secretory GCs and decrease in GCs-producing sulfomucins in the caecum; (5) increase in the numbers of crypt cells containing sulfate and non-sulfated acidic glycans. Overall, dietary Slab51® induces morphological and region-specific changes in glycoprotein composition of guinea fowl intestine, promoting gut health.

Seminal plasma Alters surface Glycoprofile of dromedary camel cryopreserved epididymal spermatozoa.

The high viscosity of Camelidae semen continues to present a major impediment for its application in assisted reproduction technology. The exposure of epididymal spermatozoa (ES) to seminal plasma (SP) may provide an approach to enhance the development of assisted reproductive techniques in these important domestic species. Since the sperm glycocalyx plays a key role in reproduction we aimed to evaluate whether SP exposure modifies the surface glycosylation patterns of cryopreserved dromedary ES. Epididymal sperm was collected through retrograde flushing of the cauda epididymidis that were obtained from orchidectomized mature dromedary bulls. The collected samples were then cryopreserved after dilution with a tris citrate clarified egg yolk extender, with and without the supplementation of 15% SP. Post-thaw carbohydrate surface profiles of both control and SP-treated spermatozoa were analyzed using 15 fluorescent lectins. Morpho-functional properties were also investigated via computer assisted sperm analysis. Lectin-binding analysis of the glycocalyx in control sperm revealed the presence of (1) N-glycans terminating with lactosamine (Con A, PHA-L, and RCA120), in both acrosomal and tail regions. Whilst (2) α2,3-/α2,6-linked sialic acids (MALII, SNA), and O-linked glycans terminating with a single N-acetylgalactosamine residue (Tn antigen) (HPA, SBA) along with galactoseß1,3N-acetylgalactosamine (T antigen) (PNA) were observed in the acrosomal cap. The expression of both N-acetylglucosamine (sWGA and GSA II) and terminalαgalactose (GSA I-B4) residues was also noted in the acrosomal cap region of control sperm. Compared with controls, SP treated samples displayed: 1) the appearance of bisected di-triantennary complex-type N-glycans (PHA-E), terminating with lactosamine, as well as an increase of O-glycans terminating with Tn and T antigens in both the acrosomal and tail regions; 2) an increase in glycans containing α2,6-linked sialic acid, N-acetylglucosamine, and αgalactose in the tail region. The cytoplasmic droplets of both control and seminal plasma-treated sperm bound Con A, PHA-E, PHA-L, RCA120, HPA, PNA, sWGA, GSA I-B4, and GSA II. These results indicate that SP treatment affects the glycan composition of the dromedary camel ES glycocalyx. More comprehensive studies are required in order to evaluate the fertilization capacity of SP-treated ES in order to facilitate its application in dromedary camel assisted reproduction technology.

Positive Influence of a Probiotic Mixture on the Intestinal Morphology and Microbiota of Farmed Guinea Fowls (Numida meleagris).

To understand the effectiveness of a probiotic mixture on intestinal morphology, mucus layer composition, and cecal microbiota diversity, 40 10-day-old Guinea fowls (Numida meleagris) were assigned to two groups: the control group (C), receiving drinking water, and the treated group (P), receiving water plus a commercial multi-strain probiotic (Slab51®, 2 × 1011 CFU/L). Birds were slaughtered after 4 months, and the intestines were collected. Samples from the duodenum, ileum, and cecum were processed for morphological and morphometric studies, and conventional glycohistochemistry. Cecal samples were also used to assess the microbiota by 16S metataxonomic approach. Group P showed significant increase in the villus height (p < 0.001 in the duodenum and p < 0.05 in the ileum and cecum), villus width (p < 0.05 in all investigated tracts), depth of crypts (p < 0.001 in the duodenum and cecum; p < 0.05 in the ileum), and goblet cells per villus (p < 0.001 in all investigated tracts) compared with group C. Cecal microbiota of the birds varied considerably and comparing the relative abundance of the main observational taxonomic units (OTUs), a positive enrichment of several beneficial taxa, such as Oscillospira, Eubacterium, Prevotella, and members of the Ruminococcaceae, was observed. The enrichment of those taxa can improve microbiota stability and resilience facing environmental stresses, enhancing its resistance against invading pathogens. Ruminococcaceae, which represent the most important taxon in both groups, and Prevotella have a key role in the gut physiology due to the production of short-chain fatty acids (SCFAs), which are a vital energy source for enterocytes, improve glucose metabolism, and exert an overall anti-inflammatory effect. Probiotic administration enriches the presence of Coprococcus, Oscillospira, and Eubacterium taxa that produce butyrate, which exerts a beneficial effect on growth performance, structure of villi, and pathogen control and has anti-inflammatory properties too. This study indicates that Slab51® supplementation positively affects the morphology and microbiota diversity of the guinea fowl intestine.