Case report: An innovative non-invasive technique to manage shell injuries in C. carbonarius.

Shell fractures are one of the most traumatic and recurrent injuries observed in chelonians during clinical practice. The most common causes of fractures are falling, being run over by automobiles, being burned, and wild animal bites. Epoxy, acrylic resin, polyester, fiber-grass blanket, and screw fixation are among the current techniques used to treat fractures. Regarding the difficulty of fracture repair in the carapace, this case report aimed to report a procedure that is effective, less time-consuming, accessible, affordable, and safe for shell fractures in C. carbonarius. During the physical examination, the animal showed two fractures, in the dorsal region of the carapace and right lateral side of the bridge, with subcutaneous tissue exposure and loss of a small piece of dorsocranial carapace. To treat these injuries, the animal was submitted to a resin application. The procedure consists of using ethyl-cyanoacrylate associated with sodium bicarbonate, which produces a more resistant resin that is bactericidal, non-toxic, and easy to apply in a low surgery time compared to the common methods used to fix shell fractures. The resin application was successfully done, and the animal was under care for a month after the fracture reduction. It was observed that the treatment was effective, presenting reduction of the fracture. A month after the procedure, the animal showed no intercurrence. Three years after the procedure, the animal still presents part of the material still fixed to the shell, normal growth, without interference in locomotor capacity. This resin proved to be an innovative and promising alternative way to treat fractures, suggesting the development of new non-invasive approaches for several tissues and different animal species.

Vaginal Microbiota Diversity in Response to Lipopolysaccharide in Gilts Housed Under Three Housing Systems.

The United Kingdom and European Union have banned crates for pregnant sows. However, animals are kept in a restrictive environment for up to four weeks after mating, leading to stress and different responses of the animals' immune system. Here, we used vaginal flushing of gilts to investigate whether housing systems or an experimental inflammatory challenge with lipopolysaccharide (LPS) can modify the gilt vaginal microbiome. Alpha-diversity indices showed differences in the microbiota of gilts housed under different systems (q = 0.04). Shannon alpha-diversity richness was higher in gilts group-housed in pens than in gilts housed in crates (q = 0.035), but not higher than in other groups. The relative abundance of the operational taxonomic unit (OTU) (q < 0.05) revealed specific differences in housing systems before a LPS or saline (SAL control) challenge. We found different abundances in taxa of Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria in gilts housed in the different systems before challenge. After the LPS challenge, significant differences were detected in the relative abundance of OTUs (q < 0.05) for the LPS-challenged group compared with SAL animals for each housing system. The phylum Staphylococcus showed higher abundance among the LPS-challenged gilts than in SAL-challenged animals. Furthermore, Enterobacter was more abundant in the LPS-challenged gilts housed in crates than in SAL-challenged gilts housed in crates. Streptococcus suis, Conchiformibius, Globicatella and Actinobacillus were more abundant in LPS-challenged gilts in indoor group housing than in SAL gilts in the same housing system. Gilts kept outdoors did not show changes in vaginal microbiota after an LPS challenge. Gilts housed in crates showed clinical signs of urogenital infection, whereas gilts housed outdoors and in indoor group housing did not. The relationship between environment, immune response, and microbiota suggested that animals in a poor environments experience difficulties responding to a challenge and their vaginal microbiota is altered as a consequence, with decreased richness of normal vaginal microbiota, and increased opportunistic bacteria. Welfare indicators measured by gilts' responses to housing systems however, do not fully explain mechanisms associated with the unique signature in vaginal microbiota encountered in the different housing systems.

Insights into the influence of canine breed on proteomics of the spermatozoa and seminal plasma.

This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SIGNIFICANCE: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.

Recellularized rat testis scaffolds with embryoid bodies cells: a promising approach for tissue engineering.

Tissue engineering is gaining use to investigate the application of its techniques for infertility treatment. The use of pluripotent embryonic cells for in vitro production of viable spermatozoa in testicular scaffolds is a promising strategy that could solve male infertility. Due to cell-extracellular matrix (ECM) interactions, here we aim to investigate the differentiation of embryoid bodies (EBs) in cultured into decellularized rat testis scaffolds. Decellularized testis (P = 0.019) with a low concentration of gDNA (30.58 mg/ng tissue) was obtained by sodium dodecyl sulfate perfusion. The structural proteins (collagens type I and III) and the adhesive glycoproteins of ECM (laminin and fibronectin) were preserved according to histological and scanning electron microscopy (SEM) analyses. Then, decellularized rat testis were cultured for 7 days with EB, and EB mixed with retinoic acid (RA) in non-adherent plates. By SEM, we observe that embryonic stem cells adhered in the decellularized testis ECM. By immunofluorescence, we verified the positive expression of HSD17B3, GDNF, ACRV-1, and TRIM-36, indicating their differentiation using RA in vitro, reinforcing the possibility of EB in male germ cell differentiation. Finally, recellularized testis ECM may be a promising tool for future new approaches for testicular cell differentiation applied to assisted reproduction techniques and infertility treatment.Abbreviations: ACRV-1: Acrosomal vesicle protein 1; ATB: Penicillin-streptomycin; DAPI: 4,6-Diamidino-2-phenylindole; EB: Embryoid bodies; ECM: Extracellular matrix; ESCs: Pluripotent embryonic stem cells; GAGs: Glycosaminoglycans; gDNA: Genomic DNA; GDNF: Glial cell line-derived neurotrophic factor; H&E: Hematoxylin and eosin; HSD17B3: 17-beta-Hydroxysteroid dehydrogenase type 3; PBS: Phosphate-buffered saline; PGCLCs: Primordial germ-cell-like cells; RA: Retinoic acid; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SSCs: Spermatogonial stem cells; TRIM-36: Tripartite Motif Containing 36.

Initial steps on mapping differentially expressed proteins in bovine preantral follicles and ovarian tissue: An approach using single-follicle MALDI-MS and mass spectrometry imaging (MSI) analysis.

The molecular mechanisms regulating follicular development and ensuring primordial follicle activation remain undefined. To help elucidate these mechanisms, this proteomic study of bovine ovarian tissue identified the differential molecular profiles of preantral follicles together with the spatial distribution of the most abundant molecular components in the tissue. Isolated primordial, primary and secondary follicles were individually placed on a MALDI target plate for mass spectral acquisitions, with detection of different m/z ranges. Ovarian tissue was sectioned and analysed in the m/z 400-2,000 range. Results of the first analysis indicated a similarity pattern in the molecular protein profile among different follicular classes in the m/z ranges of 100-1000 and 25,000-200,000, but in the m/z ranges of 800-4000, 4000-20,000 and 15,000-70,000, primary and secondary follicles shared similar clustering profiles which were different from primordial follicles (p < .05). In the second analysis, it was possible to correlate some intense molecular components in the tissue from global mass spectrum with the ions detected in the first analysis. Molecular components at m/z 11,325 (±230) were also detected in primary and secondary follicles in the experiment with isolated follicles, in addition to ions at m/z 4,029 (±120), 13,799 (±70), 5,547 (±9), 15,313 (±200), 7,018 (±40) and 7,663 (±90) which were also intensely detected in primary and secondary follicles. The present proteomic approaches evaluated different mass ranges of preantral follicles in bovine ovarian tissue and also indicated the spatial distribution of the most abundant molecular components. This study hopes to pave the way for future research identifying and characterizing specific proteins involved in follicle activation in bovine follicles, in order to better understand folliculogenesis and potentially improve mammalian follicle culture systems.

Seminal Plasma Does Not Influence Canine Semen Stored at 5°C for Long-Term Conservation.

Seminal plasma has several components that protect the sperm cells and assist in the fertilization process. In contrast, the exact role carried out by seminal plasma during the cooling of canine semen remains controversial. Moreover, concerning the long estrus period, the possibility to store chilled semen at 5°C for more than 72 hours and maintain good sperm quality for additional inseminations could increase fertilization rates. Thus, this study aimed to evaluate the seminal plasma influence on quality and oxidative stress of the extended canine semen stored at 5°C for 7 days. Three ejaculate pools from eight healthy dogs were collected by digital manipulation of the penis. The sperm kinetics, sperm vitality (eosin/nigrosin stain), integrity of plasma and acrosomal membranes, morphology, superoxide and hydrogen peroxide production, mitochondrial potential, lipid peroxidation, and oxygen reactive species production (induced and spontaneous thiobarbituric acid [thiobarbituric acid reactive substances, TBARS] assay) were evaluated every 48 hours (M0, M48, M96, and M168) until 7 days (168 hours) in cooled extended (TRIS egg yolk) semen of dogs at 5°C with (+SP) or without (-SP) autologous seminal plasma. No statistical difference was found for sperm kinetics in cooled samples with +SP and -SP during the experimental time period, except for the progressive motility of +SP samples that was higher at M48 than M96 (p = 0.023). The seminal plasma did not influence any other evaluated sperm characteristics. Finally, our results demonstrated that the presence or lack of seminal plasma during cooling the semen of dogs does not influence sperm quality at 5°C. Moreover, the components of the semen extender may contribute to maintaining good sperm quality and low reactive oxygen species production during the long period of the dog's semen cooling, even after semen centrifugation.

Uterine Tissue Engineering: Where We Stand and the Challenges Ahead.

Tissue engineering is an innovative approach to develop allogeneic tissues and organs. The uterus is a very sensitive and complex organ, which requires refined techniques to properly regenerate and even, to rebuild itself. Many therapies were developed in 20th century to solve reproductive issues related to uterus failure and, more recently, tissue engineering techniques provided a significant evolution in this issue. Herein we aim to provide a broad overview and highlights of the general concepts involved in bioengineering to reconstruct the uterus and its tissues, focusing on strategies for tissue repair, production of uterine scaffolds, biomaterials and reproductive animal models, highlighting the most recent and effective tissue engineering protocols in literature and their application in regenerative medicine. In addition, we provide a discussion about what was achieved in uterine tissue engineering, the main limitations, the challenges to overcome, and future perspectives in this research field. Impact Statement This review presents the applications of tissue engineering in uterine reconstruction and in regenerative therapies of uterine wall injuries.

Anatomo-histological evaluation of male reproductive system of the juvenile Caiman crocodilus yacare / Evaluación anátomo-histológica del sistema reproductor del Caiman crocodilus yacare macho juvenil

SUMMARY: The Caiman crocodilus yacare was once close to extinction. Studies about the male reproductive tract may aid in their reproduction and conservation. In this work, after sedation and euthanasia, seven young male C. yacare were submitted to necropsy, and macroscopic evaluation of the reproductive system, while the three others were admitted for histological study. The histological sections were stained with Hematoxylin & Eosin and Masson's Trichrome. After opening the pleuroperitoneal cavity it was possible to identify that the testicles were disposed in pairs and attached to its dorsal surface. The epididymis showed elongated and convoluted shapes and were located at the cranial margin of the testicles, following its medial portion, which was the same portion that the "vas deferens" stems from until the opening of the cloaca. The crocodile phallus presented a tubular shape, with conical appearance, displaying little resistance while maintaining its flexibility, compatible with a fibrocartilaginous tissue. On light microscopic analysis it was possible to observe that the testis was delimited by the tunica albuginea. The seminiferous tubules were contorted, and the interstitial space was filled with interstitial tissue and Leydig cells. The epididymal ductus were covered with non-ciliated pseudostratified epithelium with cells varying between cuboidal and prismatic shapes. The ductus deferens were characterized by a narrow girth shrouded with non- ciliated pseudostratified prismatic epithelium. The phallus of the crocodile was covered with a non-keratinized squamous epithelium surrounded by connective tissue. The findings support anatomic and histologic knowledge Alligatoridae reproductive system, enabling further research in the C. yacare reproduction and conservation.

RESUMEN: El Caiman crocodilus yacare ha estado en peligro de extinción. Los estudios sobre el aparato reproductor del macho pueden ser de ayuda en su reproducción y conservación. En este trabajo, fueron sometidos a necropsia y evaluación macroscópica del aparato reproductor, siete machos jóvenes de C. yacare, mientras que otros tres fueron utilizados para su estudio histológico. Las secciones histológicas se tiñeron con hematoxilina y eosina y tricrómico de Masson. Después de examinar la cavidad pleuroperitoneal se pudo identificar que los testículos estaban dispuestos en pares y adheridos a su superficie dorsal. El epidídimo presentaba formas alargadas y contorneadas y se ubicaba en el margen craneal de los testículos, siguiendo su porción medial, que era la misma porción de donde parten los conductos deferentes hasta la apertura de la cloaca. El pene del cocodrilo presentaba forma tubular, de apariencia cónica, mostrando poca resistencia manteniendo su flexibilidad, compatible con un tejido fibrocartilaginoso. En el análisis microscópico óptico se pudo observar que el testículo estaba delimitado por la túnica albugínea. Los túbulos seminíferos estaban contorsionados y el espacio intersticial estaba lleno de tejido intersticial y células intersticiales (células de Leydig). El epidídimo estaba cubierto con epitelio pseudoestratificado no ciliado con células que variaban entre formas cuboideas y prismáticas. Los conductos deferentes se caracterizaron por una circunferencia estrecha envuelta en un epitelio prismático pseudoestratificado no ciliado. El pene del cocodrilo estaba cubierto con un epitelio escamoso no queratinizado rodeado de tejido conectivo. Los hallazgos corroboran el conocimiento de la anatomía del sistema reproductivo de Alligatoridae, lo que permite una mayor investigación sobre la reproducción y conservación de C. yacare.

Proteomic data of seminal plasma and spermatozoa of four purebred dogs.

Semen contains several proteins that are important to fertilization and to identify reproductive failures. There are proteins that are specie-specific expressed, although differs among several breeds. This article provides experimental data describing the protein profile of seminal plasma and spermatozoa of four healthy purebred dogs: Golden Retriever (n=3), Bernese Mountain Dog (n=4), Great Dane (n=3), and Maremmano-Abruzzese Sheepdog (n=3), housed at São Paulo state, Brazil. Semen samples were collected by manual stimulation of the penis in a presence of a teaser bitch, when possible. The seminal plasma and sperm cells were separated by centrifugation and prepared for mass spectrometry. The gene ontology annotation of the proteins found is described. This is the first time that proteomic profile of the semen of purebred dogs is described. These data are a valuable resource to improve the biotechnologies of reproduction applied to canid species.