Stefano Cecchini

Dynamics of collagen indicating amino acids, in embryos and larvae of sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata), originated from broodstocks fed with different vitamin C content in the diet

The dynamics of hydroxyproline–proline (HYP–PRO) and hydroxylysine (HYL), as amino acids indicators of collagen, has been studied in fertilised eggs, embryos and fasting larvae of sea bass (Dicentrarchus labrax L.) and gilthead sea bream (Sparus aurata L.). Broodstocks of the two species were fed every second day with two types of diets: one containing sufficient ascorbate for a normal growth and the other with an extra addition of a very high dose (2000 mg kg−1 feed) of coated l-ascorbic acid (AA). The collagen hydroxylation expressed as the HYP-PRO ratio, hydroxylysine (HYL1 and HYL2) and total ascorbate (TAA) concentrations were analysed in several embryonic and larvae developmental stages. TAA concentrations in the newly fertilised eggs and larvae resulted significantly higher in offspring of broodstocks fed an additional dose of 2000 mg AA/kg feed than in those fed a normal diet (P<0.05 or P<0.01). The differences in HYP–PRO ratio values between the groups fed AA-supplemented and unsupplemented diets, were significant for all the analysed stages, being higher in the supplemented groups. The values of HYL were higher, although not always significantly, for all the analysed stages, in offspring of the supplemented group, compared to the offspring of the unsupplemented one. In conclusion, a vitamin C dose of 2000 mg kg−1 feed, delivered every second day to sea bass and gilthead sea bream broodstock, increases the collagen synthesis in embryos and fasting larvae, in comparison with a diet containing vitamin C at the recommended concentration for growth.

Ascorbate dynamics in embryos and larvae of sea bass and sea bream, originating from broodstocks fed supplements of ascorbic acid

The transfer of L-ascorbic acid (AA) from broodstock to the fertilised eggs and its dynamics in embryos and fasting larvae, has been studied in sea bass (Dicentrarchus labrax L.) and sea bream (Sparus aurata L.). Two types of diets were fed to the broodstocks: one containing sufficient ascorbate for normal growth and the other with an extra addition of a very high dose (2,000 mg/kg feed) of ethyl-cellulose-coated L-ascorbic acid.The concentration of AA and total ascorbate (L-ascorbic acid plus dehydroascorbic acid) was detected through several embryo and larval development stages, including larvae just before feeding. In sea bass and sea bream, the mean (SD) concentration of total ascorbate in fertilised eggs originating from broodstock fed AA supplemented diet, was 218.5 (17.7) and 122.4 (5.1) μg/g wet weight respectively. This was significantly higher (P < 0.01) than the unsupplemented groups, which contained only 155.9 (6.9) and 103.9 (3.5) μg/g wet weight respectively. A diet with a vitamin C content adequate for normal growth, may not be sufficient for broodstock when the goal is transfer of TAA to embryos.

Ontogenetic profile of innate immune related genes and their tissue-specific expression in brown trout, Salmo trutta (Linnaeus, 1758)

The innate immune system is a fundamental defense weapon of fish, especially during early stages of development when acquired immunity is still far from being completely developed. The present study aims at looking into ontogeny of innate immune system in the brown trout, Salmo trutta, using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs and hatchlings at 0, 1 h and 1, 2, 3, 4, 5, 6, 7 weeks post-fertilization was subjected to RT-PCR using self-designed primers to amplify some innate immune relevant genes (TNF-α, IL-1β, TGF-β and lysozyme c-type). The constitutive expression of β-actin was detected in all developmental stages. IL-1β and TNF-α transcripts were detected from 4 week post-fertilization onwards, whereas TGF-β transcript was detected only from 7 week post-fertilization onwards. Lysozyme c-type transcript was detected early from unfertilized egg stage onwards. Similarly, tissues such as muscle, ovary, heart, brain, gill, testis, liver, intestine, spleen, skin, posterior kidney, anterior kidney and blood collected from adult brown trout were subjected to detection of all selected genes by RT-PCR. TNF-α and lysozyme c-type transcripts were expressed in all tissues. IL-1β and TGF-β transcripts were expressed in all tissues except for the brain and liver, respectively. Taken together, our results show a spatial-temporal expression of some key innate immune-related genes, improving the basic knowledge of the function of innate immune system at early stage of brown trout.

Serum disposition of bovine lactoferrin after oral and anal administration and its proteolytic cleavage by gastric transit in rainbow trout (Oncorhynchus mykiss W.).

Several studies have shown an immunomodulatory effect of orally administered bovine lactoferrin (LF) in fish, but the process of digestion was not characterized. In the present study, we investigated the fate of bovine LF after oral and anal administration, and studied the appearance of intact LF in the bloodstream and its proteolytic attack during the gastric transit in rainbow trout (Oncorhynchus mykiss) held at 9 degrees C and 18 degrees C. Data obtained showed the presence of intact bovine LF in the bloodstream only after anal administration in fish held at 18 degrees C and the presence of several peptides derived from bovine LF in the gastric content. Immunoblotting analysis showed that only a part of bovine LF-derived peptides reacted with the applied anti-bovine LF antibody. The concentration of intact bovine LF, after 30 min of administration, in the gastric content of fish reared at 18 degrees C, being extremely low, if any, led us to suspect that the immunoregulatory effect of dietary bovine LF shown in fish by several authors is not due to the intact form but to bioactive fragments, originated by the proteolytic attack during the gastric transit, as demonstrated in higher vertebrates.

Dehydration is more effective for the control of embryonic development and larval hatching of Diplectanum aequans (Monogenea, Diplectanidae) than formalin and trichlorphon

Embryonic development and larval hatching of the monogenean Diplectanum aequans, gill parasite of sea bass Dicentrarchus labrax, was studied in relation to different prophylactic treatments. Groups of eggs of D. aequans were submitted to different in vitro treatments: formalin (300 and 100 μL L−1 per 1 hour), Neguvon® (trichlorphon 0.2 mg L−1 per 48 hours) and dehydration for 4 and 8 hours. Percentages of hatched larvae, aborted larvae and undeveloped embryos were estimated in comparison with the control group. Results showed that 300 μL L−1 formalin and dehydration treatments were able to reduce larval hatching significantly, while Neguvon® and 100 μL L−1 formalin treatments had no effect.

Morphometric Evaluation of Interrenal Gland and Kidney Macrophages Aggregates in Normal Healthy Rainbow Trout (Oncorhynchus mykiss) and after Bacterial Challenge with Yersinia ruckeri

Macrophage aggregates (MA) occur in various organs of fish as discrete aggregations of pigmented macrophages. The study presented herein investigates the quantitative modifications from normal anatomical condition, of interrenal gland (IG) and kidney MA in six treatment groups of adult rainbow trout submitted to either specific or aspecific immune stimulation and subsequently challenged with Yersinia ruckeri. Routinely stained tissue sections from both IG and kidney were analysed. The percentage of tissues occupied by MA and the MA density (number/mm(2)) were calculated on at least 10 randomly selected nonoverlapping fields taken from each tissue section. MA morphometric findings from challenged fish were compared to those from a control group. Results showed that fish from control group displayed a statistically significant (P < 0.05) higher percentage of tissue occupied by MA and MA density. Among different treatment groups, anti-Yersinia ruckeri immunized fish, which did not show clinical signs of disease after bacterial challenge, displayed higher values of morphometric parameters compared with symptomatic fish from other groups. Our study demonstrates that the quantification of the area occupied by MA might be an efficient parameter to evaluate the general condition of a salmonid population since it positively correlates with the health status and negatively with stress factor such as the acute bacterial infection.

Antioxidant Potential of the Polyherbal Formulation “ImmuPlus”: A Nutritional Supplement for Horses

In order to counteract harmful effects of oxidative stress due to pathological conditions or physical exercise, horses are often administered dietary supplements having supposed high antioxidant activities. The aim of the present study was to identify the in vitro antioxidant potential of “ImmuPlus”, a polyherbal formulation (Global Herbs LTD, Chichester, West Sussex, Great Britain), containing three medicinal plants (Withania somnifera, Tinospora cordifolia, and Emblica officinalis), known in Ayurveda for their use in human disease treatment. Extracts obtained by different solvents (water, methanol, ethanol, acetone, and hexane) were tested for total antioxidant capacity, total reducing power, scavenging activity against DPPH radical, and total polyphenol and flavonoid contents. Our results showed that, except as regards hexane, all the used solvents are able to extract compounds having high antioxidant activity, even when compared to ascorbic acid. Regression analysis showed significant correlations between antioxidant properties and polyphenol/flavonoid contents, indicating the latter, known for their beneficial effects on health of human and animal beings, as major components responsible for the strong antioxidant capacities. Moreover, obtained results suggest the effective role of the polyherbal mixture as good source of antioxidants in horses.

Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers

Owing to the progressive decline of sperm motility during storage there is a need to find substances capable of enhancing sperm energy metabolism and motility and/or preserving it from oxidative damage. The aim of this study was to evaluate in frozen/thawed bovine spermatozoa the effect of several compounds, such as myo‐inositol, pentoxifylline, penicillamine + hypotaurine + epinephrine mixture (PHE), caffeine and coenzyme Q10+ zinc + d‐aspartate mixture (CZA), on either kinetic or metabolic parameters. Sperm kinetics was evaluated by Sperm Class Analyser whereas specific fluorochromes were used to evaluated mitochondrial membrane potential (MMP), intracellular pH, intracellular calcium concentration and lipid peroxidation. Lipid peroxidation was also evaluated by TBARS analysis. Treatments significantly affected total and progressive motility with different dynamics in relation to the incubation time. After the first hour of incubation, CZA treatment produced the best performance in total and progressive sperm motility as well as in curvilinear velocity, average path velocity and amplitude of head displacement, whereas pentoxifylline stimulated the highest straight‐line velocity. MMP showed higher values (p < 0.01) after treatment with pentoxifylline and PHE. Intracytoplasmic calcium concentration and lipid peroxidation were significantly (p < 0.05) affected by the incubation time rather than the treatments. Intracellular pH varied significantly (p < 0.01) in relation to either the incubation time or treatments. In particular, it showed a progressive increase throughout incubation with values in control group significantly higher than in myo‐inositol, PHE, caffeine, pentoxifylline and CZA groups (7.37 ± 0.03 vs. 7.29 ± 0.03, 7.28 ± 0.03, 7.26 ± 0.03, 7.22 ± 0.03 and 7.00 ± 0.03, respectively; p < 0.01).; however, among treatments, CZA displayed the lowest values. Significant correlations were found between sperm kinetic and metabolic parameters. These findings provide new comparative information on the effects of putative metabolic enhancers on kinetics and metabolic activities of bovine spermatozoa. In this study, a rapid methodological approach for evaluating sperm quality is proposed.

Evaluation of the effects of dexamethasone-induced stress on levels of natural antibodies in immunized laying hens.

Natural antibodies (NAb) are an important humoral component of innate immunity, playing a pivotal role as first line of defence against pathogens even without prior antigen-specific activation or antigen-driven selection. The levels of NAb in plasma of young laying hens were explored in more detail and identified 2,4,6-trinitrophenyl bovine serum albumin (TNP-BSA), as the non-self antigen showing the highest levels of IgΥ- and IgM-NAb. Subsequently, the relation between specific antibody (SpAb) levels and NAb levels, and the effect of dexamethasone (DEX)-induced stress on the acquired Ab response and on NAb levels were examined. According to obtained results, the affinity of NAb and SpAb, measured using the thiocyanate elution method, resulted higher in SpAb than in NAb. After stress induction, IgM-NAb and SpAb levels showed a transient decrease, whereas the levels of IgΥ-NAb were not changed. Moreover, statistical analysis showed positive correlations between IgΥ- and IgM-NAb levels and between IgM-NAb and SpAb levels that are lost as stress has been induced, whereas no correlation was observed between IgΥ-NAb and SpAb levels, neither before nor after the DEX-administration. This indicates that IgM-NAb assessment could be a valid tool to estimate the potential of the acquired Ab response and that the dexamethasone-induced stress condition causes depression of IgM-NAb levels and the acquired Ab response, but it has no evaluable effects on IgΥ-NAb levels.

Seminal plasma of brown trout, Salmo trutta fario (L.) contains a factor able to retain iron at acid pH, typical feature of lactoferrin.

Blood and seminal plasma of brown trout Salmo trutta fario were analyzed for their iron binding potential adopting two different methods. Seminal plasma showed an iron binding capacity that was retained even if samples were exposed at acid pH, similarly to mammalian lactoferrin that binds ferric iron also at acid pH. This suggests that the iron binding capacity is determined by a factor having a lactoferrin-like activity. Moreover, trout seminal plasma proteins were also analyzed in their pattern by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. When seminal plasma was subjected to immunoblotting using goat anti-bovine lactoferrin antibodies as a probe, only a single band having an apparent molecular weight of around 80 kDa was specifically detected, showing that this protein has homology with bovine lactoferrin.