Proteomic Characterization of Zinc‐Binding Proteins of Canine Seminal Plasma

The zinc‐binding proteins (ZnBPs) of the seminal plasma are implicated in different processes related to sperm–egg fusion. The aim of this study was to characterize the ZnBPs of canine seminal plasma using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and mass spectrometry. The ZnBPs were isolated from the ejaculates of five dogs by affinity chromatography and subjected to 2D‐PAGE analysis. The acquired spots, detected across the gels, were analysed by mass spectrometry. Using 2D‐PAGE analysis, it was shown that canine seminal plasma comprised about 46–57 zinc‐binding polypeptides, with molecular mass ranging from 9.3 to 138.7 kDa and pI at pH 5.2–10.0. It was found that zinc‐binding polypeptides of low molecular masses (9.3–19.0 kDa and pI at pH 6.1–10.0) were predominant in the seminal plasma, and seven polypeptides, with molecular masses ranging from 11.7 to 15.4 kDa and pI at pH 6.8–8.7, were characterized by high optical density values. In addition, analysis with mass spectrometry (LC‐MS‐MS/MS) revealed that the identified seven polypeptides are canine prostate‐specific esterase (CPSE), which is the main proteolytic enzyme of the seminal plasma. The findings of this study indicate an important regulatory role of seminal plasma zinc ions in the functional activity of CPSE, which is of great significance for maintaining the normal function of canine prostate and the spermatozoa functions.     

Dietary resveratrol improves immunity but reduces reproduction of broodstock medaka <Emphasis Type="Italic">Oryzias latipes</Emphasis> (Temminck &amp; Schlegel)

Here, we investigated the effect of dietary resveratrol (20, 40, and 80 µg/g BW/day) on cell-mediated immunity (activity of spleen phagocytes and proliferative response of lymphocytes) and reproductive parameters (egg and sperm quality, i.e. fecundity—total number of eggs produced by individual fish, fertility, embryo survival, and hatching rate) in medaka. Fish fed feed with resveratrol at 40 and 80 µg/g BW/day had significantly higher metabolic activity and intracellular phagocyte killing activity than control. The proliferative lymphocyte activity of the fish from R80 group was greater by more than 20 % in comparison with the control group (P < 0.05). The percentage of macrophages (MO) and their mean fluorescence intensities (MFI) in R40 and R80 groups were significantly higher compared to C and R20 groups (P < 0.05). The differences in MO and MFI values ranged from 52.5 % (±1.5; R0 group) to 65.8 % (±1.6; R80 group) and from 23.2 (±1.4; R0 group) to 38.2 (±2.4; R80 group), respectively. Moreover, resveratrol at 80 µg/g BW/day decreased liver COX activity, i.e. 5.4 in R80 group and 7.9 in R0 group (P < 0.05). The motility parameters of the sperm obtained from the males fed feed supplemented with resveratrol at 80 µg/g BW/day exhibited the highest values except the linearity, which was lower as compared to the control (P < 0.05). The results indicate that diet supplemented with resveratrol at a dosage of 40 µg/g BW/day improves phagocyte killing ability and lymphocyte proliferation in broodstock and accelerates offspring hatch. Also, the results suggest that COX activity influences sperm and oocyte quality in fish; the presence of a COX inhibitor in the dose of 40 µg/g BW/day decreased the embryo survival.     

The effect of feeding the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) on cell-mediated immunity and protection against Yersinia ruckeri in pikeperch (Sander lucioperca)

The present study examined the influence of leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB), a natural product HMB, on nonspecific cell‐mediated defence mechanisms and protection against enteric redmouth disease (ERM) in pikeperch (Sander lucioperca). β‐hydroxy‐β‐methylbutyrate was fed in a pelletted ration at 50 mg kg^?1 of the feed for 8 weeks. The phagocytic ability and potential killing activity of blood and pronephric phagocytes were examined in HMB‐ and control‐fed fish before and after 8 weeks of feeding HMB. Simultaneously, the proliferative response of blood and pronephric lymphocytes stimulated by mitogens concanavaline A and lipopolisaccharide were examined in experimental and control groups. Following 8 weeks of HMB feeding, a challenge test was performed by injecting the fish with live pathogenic bacteria Yersinia ruckeri. β‐hydroxy‐β‐methylbutyrate applied in the diet for 8 weeks prompted a statistically significant (P<0.05) increase in phagocytic ability and potential killing activity of the blood and pronephric phagocytes and the proliferative response of blood and pronephric lymphocytes. The changes in these mechanisms correlated with protection against Y. ruckeri, the ERM pathogen. The results showed that feeding HMB increased the nonspecific cell‐mediated defence mechanisms and protection against ERM by reducing cumulative mortality (30%) following the challenge with pathogenic bacteria. Future studies will include determination of optimal doses and protocols of oral application of HMB to maximize the immunomodulatory effects and protection against viral diseases in intensive pikeperch culture.     

Supplementing the feed of pikeperch [Sander lucioperca (L.)] juveniles with MacroGard and its influence on nonspecific cellular and humoral defense mechanisms

This study examined the influence of β‐1.3/1.6‐glucan (MacroGard) on the innate immune system in healthy pikeperch (Sander lucioperca) juveniles. MacroGard was fed in a pelleted ration of 1 or 2 g kg^?1 feed for 6 weeks. The control group of fish was fed feed with no supplement. Blood, pronephros and spleen samples for the study of nonspecific cellular and humoral defense mechanisms were collected from 10 fish from each group. No changes were observed in either the behaviour or the health of the fish throughout the rearing period. Fish mortality was not noted in any of the groups. Additionally, the supplementation of feed with MacroGard did not have a significant impact on the fish growth rate as expressed in both absolute and relative values (P>0.05). The condition of the fish from the experimental group did not differ from that of the control group specimens, and the feed conversion ratios were also very similar (P>0.05). The results showed that MacroGard administered in two doses significantly (P<0.05) activated the metabolic activity and potential killing activity of spleen phagocytes, compared with the control‐fed pikeperch. The highest phagocyte activity was observed with a dose of 2 g kg^?1. The proliferative response of pronephros lymphocytes indicated a similar pattern. MacroGard significantly increased proliferative response of lymphocytes stimulated by mitogens (P<0.05). MacroGard also significantly increased the lysozyme activity and total immunoglobulin levels in the serum. In this study neither significantly different ceruloplasmine activity or total protein level in serum were observed. The result showed that MacroGard at the optimal dose of 1 g kg^?1 feed and a twofold higher dose does not have a negative influence on the hepatocytes or increased nonspecific cell‐ and humoral‐mediated immunity in healthy pikeperch.     

Impact of diets with different proportions of linseed and sunflower oils on the growth, liver histology, immunological and chemical blood parameters, and proximate composition of pikeperch Sander lucioperca (L.)

The aim of the study was to determine the impact of applying different proportions of linseed (LO) and sunflower (SFO) oils in pikeperch diets on growth, histological changes in the liver, immunological and blood chemical parameters. The fish were fed isoenergetic and isoprotein feeds containing SFO (group 100SFO) or LO (group 100LO) in quantities of 67 g kg/feed, and a mixture of oils: 47 g SFO and 20 g LO kg/feed (group 70SFO/30LO) and 20 g SFO and 47 g LO kg/feed (group 30SFO/70LO). Dietary ratios of polyunsaturated fatty acids from the n-3 and n-6 series (n3/n6 index) were 0.36–2.15. Pikeperch were reared for 56 days in three replicates for each dietary treatment. Various dietary oils and ratios of n3/n6 did not impact fish growth, feed conversion ratio, viscerosomatic and hepatosomatic index, and size of the hepatocytes. Feeding the fish high quantities of LO and SO oils (groups 100LO and 100SFO) reduced the immunological response of the phagocytes and lymphocytes in the fish. Moreover, this resulted in significant differences among groups in the quantity of linolenic and linoleic acid in whole fish bodies, viscera, fillets, and livers. Various quantities of vegetable oils in the fish diets did not impact the quantity of arachidonic, eicosapentaenoic and docosahexaenoic acid in the fillets and livers. The immunological index and low quantities of linoleic acid in the fillets obtained in group 30SFO/70LO indicate that the n3/n6 dietary ratio of 1.35 was the most advantageous for feeding juvenile pikeperch feeds with vegetable oils.     

The effect of cyclooxygenase (COX) inhibitors on Japanese medaka (Oryzias latipes) reproduction parameters fed with high level of arachidonic acid (20:4 n-6)

Broodstock of mature Japanese medaka (Oryzias latipes Temminck and Schlegel, 1846) (mean weight 0.45 ± 0.03 g; age 6 months) were maintained in glass aquaria (8 males and 8 females in each aquarium). The fish were fed commercial feed (group C) or this feed was supplemented with arachidonic acid ARA (2 % of total fatty acids in feed; group A) and one of the COX inhibitors: resveratrol (group AR) or NS-398 (group ANS) with amount 20 mg kg^?1 BW day^?1 of one of the COX inhibitors. The effect of 2 weeks of feeding experimental diets on reproductive parameters (fecundity, embryo and larval survival, hatching rate, and larvae body weight) was analyzed. Larvae (obtained from experimental dietary treatments) were reared for 2 weeks. The percent of spawning female and fecundity was similar in dietary treatments. COX inhibitors in broodstock diet caused a decrease in embryo survival rate. Eggs of medaka from group ANS and AR revealed the lowest hatchability rate. In contrast, group A revealed a higher hatchability rate at the same time. Larval body weight after hatching was significantly the highest in this group. At the end of the experiment, larvae body weight ranged from 1.62 ± 0.43 mg in group AN to 2.89 ± 0.74 mg in group A. These results show that the COX inhibitors decreased gamete and larval quality regardless of high levels of ARA in broodstock diet.     

Isolation and Characterization of Phenol-Degrading Psychrotolerant Yeasts

Water, Air, & Soil Pollution - To investigate the differential contribution of extracellular polymeric substances (EPS) to polycyclic aromatic hydrocarbon (PAH) degradation by fungi and...     

Induced androgenesis as a biotechnology method for obtaining DH plants in Daucus carota L.

The influence of various factors on the efficiency of obtaining double haploid lines in anther cultures was examined in Daucus carota L. The impact of the genotype, the formation of donor plants and the growth conditions was investigated. The effects of various regeneration media were analysed. Acclimatisation of androgenic plants and their evaluation were also conducted. Androgenesis was induced on B₅ medium containing 2,4-D and NAA at 0.1 g·L^?1. Depending on the genotype, 1.2–305.3 embryos per 100 anthers were obtained. The highest number of embryos (5.5 per 100 anthers) was obtained from donor plants with the first-order shoots containing one umbel. Cultivation of donor plants under controlled conditions improved the number of embryos from 305 in the open field to 1764 in the growth chamber. Regeneration of plants from androgenetic embryos proceeded most effectively on B₅ medium without hormones and amino acids. Regenerated plants adapted in over 60%. Ploidy analysis showed the presence of 92% of plants with a doubled chromosome set and 8% with a tetraploid number of chromosomes. Plants with a doubled chromosome set were 73% homozygous for the glucose phosphate isomerase isoenzyme and 100% homozygous for the aspartate aminotransferase isoenzyme, which confirms their gametic origin.