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 x 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 galactosel,3Nacetylgalacyosamine( Gall,3GalNAc)terminating O-glycans and l,2-fucosylated glycans secretory GCs in the duodenum; (3) increase in 2,6-sialoglycans and high-mannose N-linked glycans secretory GCs but reduction in GCs-secreting sulfoglycans in the ileum; (4) increase in Gall,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

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

Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice

Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter “A”. Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics

Atti SISVET 2021

The urinary bladder cavity is lined by a highly specialized epithelium, the urothelium, which prevents permeation of solutes and noxious agents back into the bloodstream and underlying tissues, serving also as a sensor and transducer of physiological and nociceptive stimuli[1]. In addition, in physiological conditions the urothelium can also function as a secretory tissue[2]. A mucous layer protects the urothelium of the urinary bladder from potentially harmful environmental substances, attachment of bacteria and proteolytic enzymes present in the urine[3]. Moreover, the glycan composition of urothelial mucous layer could have a role in the intravesical pharmacological treatments[4]. Despite their considerable importance in urinary bladder physiology, few studies are available about the glycoconjugates expressed in non-human species. In this study the glycoconjugate pattern of urothelium lining the horse urinary bladder was investigated. Tissue fragments from three horse stallions in good health status, aged 2.5-4 years, were fixed in 4% (w/v) PBS-buffered paraformaldehyde, embedded in paraffin wax and stained with a panel of twelve lectins, in combination with saponification and sialidase digestion (Ks). The urinary bladder urothelium has three distinct layers from the basal zone to the lumen consisting of basal, intermediate and superficial cells (umbrella cells). Cytoplasm of basal cells showed glycans ending with Neu5AcGal1-3GalNAc, GlcNAc and with terminal/internal Man (Ks-PNA, GSA II, and Con A II reactivity). A sub-population of intermediate cells also displayed terminal Neu5Ac2-6Gal/GalNAc, NeuNacα2-3Galβ1-4GlcNAc, Gal1-3GalNAc, Gal, terminal and sialic acid-linked GalNAc, internal GlcNAc and Fucα1-2Galβ1-4GlcNAc (MAL II, SNA, PNA, GSA I-B4, SBA, ks-SBA, Ks-WGA, and UEA I reactivity). The cytoplasm of umbrella cell population contained all the above cited sugar residues. Moreover, LTA-reactive fucosylated glycans and Ks-DBA-positive sialoderivatives were found indifferent scattered umbrella cells. These sialoglycans were secreted in the bladder lumen. The bladder luminal surface stained with MAL II, SNA, PNA, Ks-PNA, and GSA I-B4 displaying a coating of sialo- and galactose-terminating glycoconjugates. These findings show that different glycosylation patterns exist along the horse bladder urothelium, and different sub-populations of umbrella cells are present secreting the sialoglycans which constitute the protective gel layer lining the bladder. Compared to results of a similar study carried out onthe donkey bladder urothelium[2], the present research reveals a species-specific glycan pattern and could contribute to a better understanding of the differences between domesticated odd-toed ungulate mammals via comparative glycopattern investigation. [1] Lasič et al. Properties of the urothelium that establish the blood-urine barrier and their implications for drug delivery, Reviews of Physiology, Biochemistry and Pharmacology Vol 168, 2015. [2] Desantis et al. In situ characterization of glycans in the urothelium of donkey bladder: Evidence of secretion of sialomucins, Acta Histochemica, 115:712–718, 2013. [3] Kreft et al. Formation and maintenance of blood–urine barrier in urothelium, Protoplasma, 246:3–14, 2010. [4] Lopedota et al. Spray dried chitosan microparticles for intravesical delivery of celecoxib: preparation and characterization, Pharmaceutical Research, 33:2195–2208, 2016

In situ characterization of glycans in the horse bladder urothelium

The urinary bladder cavity is lined by a highly specialized epithelium, the urothelium, which prevents permeation of solutes and noxious agents back into the bloodstream and underlying tissues, serving also as a sensor and transducer of physiological and nociceptive stimuli [1]. In addition, in physiological conditions the urothelium can also function as a secretory tissue [2]. A mucous layer protects the urothelium of the urinary bladder from potentially harmful environmental substances, attachment of bacteria and proteolytic enzymes present in the urine [3]. Moreover, the glycan composition of urothelial mucous layer could have a role in the intravesical pharmacological treatments [4]. Despite their considerable importance in urinary bladder physiology, few studies are available about the glycoconjugates expressed in non-human species. In this study the glycoconjugate pattern of urothelium lining the horse urinary bladder was investigated. Tissue fragments from three horse stallions in good health status, aged 2.5-4 years, were fixed in 4% (w/v) PBS-buffered paraformaldehyde, embedded in paraffin wax and stained with a panel of twelve lectins, in combination with saponification and sialidase digestion (Ks). The urinary bladder urothelium has three distinct layers from the basal zone to the lumen consisting of basal, intermediate and superficial cells (umbrella cells). Cytoplasm of basal cells showed glycans ending with Neu5AcGal1-3GalNAc, GlcNAc and with terminal/internal Man (Ks-PNA, GSA II, and Con A II reactivity). A sub-population of intermediate cells also displayed terminal Neu5Ac2-6Gal/GalNAc, NeuNacα2-3Galβ1- 4GlcNAc, Gal1-3GalNAc, Gal, terminal and sialic acid-linked GalNAc, internal GlcNAc and Fucα1-2Galβ1-4GlcNAc (MAL II, SNA, PNA, GSA I-B4, SBA, ks-SBA, Ks-WGA, and UEA I reactivity). The cytoplasm of umbrella cell population contained all the above cited sugar residues. Moreover, LTA-reactive fucosylated glycans and Ks-DBA-positive sialoderivatives were found in some scattered umbrella cells. These sialoglycans were secreted in the bladder lumen. The bladder luminal surface stained with MAL II, SNA, PNA, Ks-PNA, and GSA I-B4 displaying a coating of sialo- and galactose-terminating glycoconjugates. These findings show that different glycosylation patterns exist along the horse bladder urothelium, and different sub-populations of umbrella cells are present secreting the sialoglycans which constitute the protective gel layer lining the bladder. Compared to results of a similar study carried out onthe donkey bladder urothelium [2], the present research reveals a species-specific glycan pattern and could contribute to a better understanding of the differences between domesticated odd-toed ungulate mammals via comparative glycopattern investigation. [1] Lasič et al. Properties of the urothelium that establish the blood-urine barrier and their implications for drug delivery, Reviews of Physiology, Biochemistry and Pharmacology Vol 168, 2015. [2] Desantis et al. In situ characterization of glycans in the urothelium of donkey bladder: Evidence of secretion of sialomucins, Acta Histochemica, 115:712–718, 2013. [4] Lopedota et al. Spray dried chitosan microparticles for intravesical delivery of celecoxib: preparation and characterization, Pharmaceutical Research, 33:2195–2208, 2016

The menstrual cycle of the Baboon (Papio hamadryas) evaluated by vaginal cytology and hormonal variations

Hamadryas baboons (Papio hamadryas) form part of a highly successful branch of the primate family (Cercopithecidae), commonly referred to as savannah baboons. They are broadly distributed over the African continent, and the Arabian Peninsula inhabiting the semiarid regions [1]. Baboons are menstruating primates, the average length of a normal reproductive cycles is 32 (24-38) days with close similarity to women's [2]; so that the baboon is considered a valuable model for increasing knowledge to overcome human reproductive disorders. This study aims s to acquire knowledge on the modifications of vaginal cytology and Fallopian tubes during the menstrual cycle of females living in captivity. The stage of the menstrual cycle of 14 healthy adult females of different ages (8.5 yrs) and weights (10.5 kg) on the day of laparoscopic salpingectomy has been evaluated. The baboons breed with high fertility rates in captivity so that laparoscopic salpingectomy was employed as irreversible contraceptive surgical therapy for an extensive control birth program in the Safari Zoo (Fasano - BR). The clinical activity was authorized with written informed consent by the Zoo’s property (Lion 3000 S.p.a.) and obtained the favorable opinion of the ethics committee of DETO (05/2020). The stage of the reproductive cycle was analyzed by 1) vaginal cytology (Harris-Shorr’s and Diff-Quik staining), 2) endocrine changes measuring 17β-estradiol (E2) and progesterone (P4) concentrations in peripheral plasma by ELISA, 3) histological uterine tubes morphology. The stage of the cycle can be also approximated by external observation of the perineum so that the perineal turgescence, characteristic of the follicular phase, was checked during the observation of external genitalia. Laparoscopic evaluations monitored the presence of corpora lutea and the Graafian follicle and when ovulation had occurred, the increased vascularized fimbriae too. Changes in the type of vaginal cells (basal and parabasal, small and large intermediate, anuclear keratinized, erythrocytes, neutrophils) found during the follicular and luteal phases (early and late) of the ovarian cycle were analyzed. Based on hormonal evaluations and cytological observations we found 9/14 subjects in the follicular phase (mean E2 concentration 150±73 pg/ml; mean P4 concentration 0.3±0.1 ng/ml; presence of small and large intermediate, anuclear keratinized cells) and 5/14 females in the luteal phase (mean E2 concentration 50±20 pg/ml; mean P4 concentration 4±0.5 ng/ml; presence of basal and parabasal cells, neutrophils) of their reproductive cycle. Histological investigations revealed morphological and morphometric changes in the uterine tube segments related to the stage of the menstrual cycle. In addition, a different degree of hyperemia was observed in the infundibulum between follicular and luteal stages. The acquired knowledge could be the starting point to propose a hormonal contraceptive strategy for baboons living in captivity with little effect on social interactions such as grooming relationships, aggression, affiliation and sexual behavior while reducing their reproductive success. [1] Newman TK, Jolly CJ, Rogers J. Mitochondrial phylogeny and systematics of baboons (Papio). American Journal of Physical Anthropology, 124: 17-27, 2004. [2] Stevens VC, Sparks SJ, and Powell JE. Levels of estrogens, progestogens, and luteinizing hormone during the menstrual cycle of the baboon. Endocrinology, 87: 658, 1970

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), alpha 2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Gal beta l,3GalNAc (T antigen) (KsPNA) and terminal N-acet-ylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acet-ylglucosamine (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), alpha 2,6-and alpha 2,3-linked sialic acid (MAL II), alpha 1,2-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 alpha 1,3-linked fucose (LTA), whereas some other cells displayed intraluminal secretion of mucin-type glycans terminating with GalNAc alpha 1,3(LFuc alpha 1,2)Gal beta 1,3/4GlcNAc beta 1 (DBA). The luminal surface expressed the most complex glycan pattern in the urothelium because only alpha 1,3-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.(c) 2022 Elsevier GmbH. All rights reserved

Effects of the Multistrain Probiotic Slab51® (SivoMixx®) on the Intestinal Morphology and Microbiota composition of farmed Guinea Fowls (Numida meleagris)

Probiotics are beneficial bacteria with positive effects on health and growth efficiency of host animals by influencing gut microbiota or modifying immune status, as well as by stimulating digestive processes [1]. To understand the effectiveness of the probiotic mixture Slab51® (SivoMixx®, Ormendes SA, Jouxtens-Mezery, CH) on intestinal morphology, mucus layer composition and caecal microbiota diversity, forty 10-days old Guinea fowls (Numida meleagris) were assigned to two groups: control group (C), receiving drinking water, and treated group (P), receiving water plus the multistrains probiotic (2x1011 UFC/L). Both groups were housed in two adjacent sheds (12 m2 each), with litter on the bottom, under controlled photoperiod and natural aeration. Through all the trial, both the groups received ad libitum the same commercial pellet feed (Cruciani, Montappone, MC, Italy), as starter followed growing feed, that changed in proximate composition in relation to the age of the animals. At the end of the normal growth process, animals were slaughtered by electrical stunning and bleeding at 120 days of age, and intestines were collected. Samples from duodenum, ileum and caecum were processed for morphological and morphometric studies, and conventional glycohistochemistry. Caecal samples were also used to assess the microbiota by 16S metataxonomic approach. Group P showed a modification of intestinal morphology characterized by significant increase of villus height, villus width, depth of crypts, and goblet cells per villus in all investigated tracts. Caecal microbiota of birds varied considerably and comparing the relative abundance of the main OTUs a positive enrichment of several beneficial taxa like Oscillospira, Eubacterium, Prevotella and members of the Ruminococcaceae was observed. High levels of diversity 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), that are a vital energy source for enterocytes, improve glucose metabolism and exert an overall anti-inflammatory effect. Probiotic administration enriches 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 a probiotic supplementation positively affects the morphology and microbiota diversity of Guinea fowl intestin

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