Oxidative stress and lateral muscle development in Siberian Sturgeon (Acipenser baeri): preliminary observations.

Embryonic development in bony fish is strongly influenced by environmental factors, mainly by temperature and dissolved oxygen, but little is known about their influence on Sturgeons. Since 1998 the international trade of Sturgeons has been regulated under CITES due to depletion of wild stocks. Sturgeon aquaculture as well as larvae production is, therefore, important because it may contribute to the repopulation of the wild stocks (Bronzi et al., 2011; Di Giancamillo et al., 2012).The aim of this study is to monitor oxidative stress status and muscle development during embryonic and precocious larval phases in Siberian Sturgeon (Acipenser baeri), when subjected to different incubating temperatures (13°, 16° and 19°C).Siberian sturgeon eggs were subjected to different incubation temperatures. Data regarding water quality parameters were recorded, as well as mortalities, embryonic period length, and precocious larvae behavior. Sampling was performed in the same developmental stage for each temperature, at five time points: 48 hours post-fertilization, embryo movements, hatching, schooling and yolk-sac full absorption. The observed hatching rates were between 85.5% and 98.8% with significant differences concerning the different experimental temperatures (T19 vs T13 and T19 vs T16, p<0,05). Histological, histochemical and immunohistochemical analyses will be performed to assess ontogeny of the lateral muscle and stress biomarkers (Lushchak, 2011), and gene expression will be analyzed for muscle development (Johnston, 2006). The obtained results will be compared with those concerning teleosts and will possibly contribute to better rearing conditions of sturgeons.This study has been approved by the ethic committee of the Università degli Studi di Milano, with the following authorisation code: OPBA_20_2016.

Rearing temperature effect on the skeletal muscle fibres of Acipenser baerii yolk-sac larvae

Siberian sturgeon farming is important because it provides an alternative source of caviar and meat, but also for the conservation of the endangered natural stocks. Farmed fish is continuously subjected to stress factors, of which, water temperature is considered a major one (Schram et al., 2006). It has been demonstrated that physiological stress may have serious negative consequences on growth (Wendelaar Bonga, 1997) and that fish larvae appear less tolerant than adults to temperature variations (Stefanovich et al., 2016). The present study aims at investigating the stress response and development in precocious stages of siberian sturgeon when subjected to different rearing temperatures, by analysing ontogeny, growth and stress response of yolk-sac larvae. This study was approved by the Ethic Committee of the University of Milan (OPBA_20_2016). Fertilized Siberian sturgeon eggs were reared at 16°C, 19°C and 22°C until complete yolk-sac absorption. Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. Water parameters and larvae development data were registered. Histological, histochemical and immunohistochemical analyses were performed in order to assess ontogeny and stress biomarkers and whole body cortisol was measured by a specific microtitre radioimmunoassay (RIA). Statistical analysis was performed with SAS software (v. 9.3, Cary Inc., NC). Larvae subjected to the highest water temperature showed a faster yolk-sac absorption and larvae body weight significantly increased from hatching onwards. Structural normal development considering the three different temperatures investigated from hatching until the end of the trial was observed. Significant differences were found between temperatures regarding body weight and cortisol levels (P<0.01). A qualitative stronger expression of stress markers was noticed in larvae subjected to the lower temperature. Even if this study indicates that lower rearing temperatures would appear more suitable for Siberian sturgeon rearing, further studies would be necessary to evaluate the temperature effect on a mid-long term basis

Is there adaptation of the exocrine pancreas in wild animal? The case of the Roe Deer.

International audienceABSTRACT: BACKGROUND: Physiology of the exocrine pancreas has been well studied in domestic and in laboratory animals as well as in humans. However, it remains quite unknown in wildlife mammals. Roe deer and cattle (including calf) belong to different families but have a common ancestor. This work aimed to evaluate in the Roe deer, the adaptation to diet of the exocrine pancreatic functions and regulations related to animal evolution and domestication. RESULTS: Forty bovine were distributed into 2 groups of animals either fed exclusively with a milk formula (monogastric) or fed a dry feed which allowed for rumen function to develop, they were slaughtered at 150 days of age. The 35 Roe deer were wild animals living in the temperate broadleaf and mixed forests, shot during the hunting season and classified in two groups adult and young. Immediately after death, the pancreas was removed for tissue sample collection and then analyzed. When expressed in relation to body weight, pancreas, pancreatic protein weights and enzyme activities measured were higher in Roe deer than in calf. The 1st original feature is that in Roe deer, the very high content in pancreatic enzymes seems to be related to specific digestive products observed (proline-rich proteins largely secreted in saliva) which bind tannins, reducing their deleterious effects on protein digestion. The high chymotrypsin and elastase II quantities could allow recycling of proline-rich proteins. In contrast, domestication and rearing cattle resulted in simplified diet with well digestible components. The 2nd feature is that in wild animal, both receptor subtypes of the CCK/gastrin family peptides were present in the pancreas as in calf, although CCK-2 receptor subtype was previously identified in higher mammals. CONCLUSIONS: Bovine species could have lost some digestive capabilities (no ingestion of great amounts of tannin-rich plants, capabilities to secrete high amounts of proline-rich proteins) compared with Roe deer species. CCK and gastrin could play an important role in the regulation of pancreatic secretion in Roe deer as in calf. This work, to the best of our knowledge is the first study which compared the roe deer adaptation to diet with a domesticated animal largely studied

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection

lmmunolocalization of regulatory peptides and 5-HT in bovine male urogenital apparatus

Specimens of testis, excurrent duct including the accessory genital glands and urethra throughout its extension were investigated in adult bovines, in order to imm~~nohistochemicalllyo calize both the peptidergic innervation and the epithelial cell types belonging to [he diffuse endocrine system (DES). Immunoreactivities to GRP, met- and leu-enkephalins, CGRP, NPY, substance P, VIP, somatostatin, l3-endorphin and 5-HT antisera were tested by means of a labelled streptavidin-biotin (LSAB) method. Such regulatory substances were found in components of the peripheral nervous system (nerve fibers in the connective and muscular tissues, sub- and intraepithelial nerve terminals, nerve cell bodies and fibers in intramural ganglia), and in epithelia1 endocrine/ paracrine cells. Bovine urogenital apparatus is supplied by many peptide-containing nerves, which contain in many localizations GRP and enkephalins, and to a lesser extent substance P, CGRP, NPY and VIP. A thin network of peptidergic nerves distributes to the musculature of the canalicular organs and accessory glands. The prostatic complex was especially rich in peptidergic innervation, and also contained somatostatin- and 5-HTsecreting endocrine cells. In addition, 5-HT-immunoreactive endocrine cells were found in the bulbourethral gland and urethral epithelium. CGRP-ir nerves were present contacting striated muscle fibers of urethra (motor end plates). The testis was devoid of any immunoreactivity. These data are compared with those obtained in a companion study carried out the same organs in two species of Equidae (Equus caballus and Equus asinus). Different patterns of immunoreactivities can be outlined in these domestic ungulates

Gut glycoconjugates in Sparus aurata L. (Pisces, Teleostei). A comparative histochemical study in larval and adult ages

This study examined the gut of the euryaline fish Sparus aurata, from the pharynx to the rectum. The specimens were collected from adult animals, both sexes, and several larval and juvenile stages, from 4 to 135 days of age. Histochemical methods to distinguish neutral and acidic glycoconjugates, as well as specific techniques to identify acidic glycoconjugates which contained 0-acylated sialic acids were used. The presence and distribution of sugar residues in the oligosaccharide side chain of glycoconjugates were investigated with the use of biotinylated lectins. The pharynx and oesophagus of adult fishes showed the presence of abundant secretory cells which synthesized a large quantity of neutral, as well as sulphated and sialylated glycoconjugates, with different cellular combinations of them in the proximal and distal tract. This may be related to the complex functions carried out by this end of the gut in a marine euryaline fish. Epithelia1 secretory cells were found in the developing oesophagus during larval life (14 days) earlier than in the stomach and intestine (34 days). The simple columnar epithelium that lined the gastric mucosa of adult fish synthesized a mixture of neutral and acidic glycoconjugates, whereas during larval life it was shown to contain neutral glycoconjugates only. The intestinal goblet cells were shown to secrete both neutral and acidic glycoconjugates, especially sulphated forms. The adherent mucus gel of the gastric and intestinal mucosa contained many sugar residues, as revealed by lectin histochemistry. This work clearly demonstrates that the quality of gut mucosubstances varies in different ages and in regions of the fish alimentary canal. This is possibly caused by changes in environmental conditions and may in turn sustain functional alterations of the digestive apparatus

Different putative neuromodulators are present in the nerves which distribute to the teleost skeletal muscle

The presence of putative neuromodulators in the nerve fibres was investigated in white skeletal muscle of two teleost fish not taxonomically correlated and showing different patterns of innervation (multiple versus focal innervation). Cryostat sections of epaxial, hypaxial and adductor mandibulae (AM) muscles of Sparus aurata and Anguilla anguilla were stained histochemically for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Other sections were used for indirect immunohistochemistry (streptavidin-biotin and rhodamine immunofluorescence methods), employing antibodies specific for putative excitatory or inhibitory peptides, including CGRP, substance P, met-enkephalin, bombesin, and VIP. In addition, ultrastructural observations were performed in order to describe the morphology of the motor endplates. A strong immunoreactivity for CGRP and substance P was found in many nerve terminals. Met-enkephalin, bombesin and VIP immunoreactivities were less frequently observed. No immunoreactivity was observed to CCK, NPY or 5-HT. NADPH-diaphorase was identified in nerve fibres of the AM complex only of A. anguilla. Electron microscopy observations evidenced more than one type of synaptic vesicle in motor endplates. Some differences in putative neuromodulator distributions were observed in the two species and muscle complexes, which may be related to the different taxonomical position as well as the different pattern of innervation of white muscle fibres

Ultrastructural features of the gut in the white sturgeon, Acipenser transmontanus

Electron-microscopic examinations of the sturgeon gut were performed. Oesophageal goblet cells were abundant in the stratified epithelium. The ultrastructural features of the secretory granules of the oesophageal and intestinal goblet cells were quite similar to those of other vertebrates. Lobules of multilocular adipose tissue were observed in the deep tunica propriasubmucosa of the oesophagus, in close association with vasculature and large fibre bundles of myelinated and unmyelinated axons. Similarly composed nerve fibre bundles were observed in the cardiac stomach. too. The presence of myelinated axons is an unusual feature in the vertebrate enteric nervous system. Cardiac and fundic zones of the stomach showed an epithelium with columnar ciliated and non-ciliated cells, the latter equipped with fuzzy microvilli. Cells lining the tubular gastric proper glands were markedly granulated. Intestinal superficial epithelium was columnar and contained ciliated, as well as non-ciliated and goblet cells. In the tunica propria all over the intestine, the presence and ultrastructure of granulated cells was in addition described. Intraepithelial granulated leukocytes were seen throughout the alimentary canal. Various types of endocrine cells were seen both in the stomach and in the intestine, the size of their granules was measured and their ultrastructure described and compared to that of mammalian cell types