The cattle tick Rhipicephalus microplus is an ectoparasite with high economic importance to bovine culture, mainly in tropical and subtropical regions. The resistance of the tick from the commercial acaricides has hindered its control, thus motivating the search for new strategies. The purpose of this study was to perform a critical review about the main molecular targets of R. microplus that are useful for the discovery of new acaricides. Bibliographic search was conducted in the databases PubMed, ScienceDirect and CAB Direct, using the following descriptors: ‘Rhipicephalus microplus’, ‘Boophilus microplus’, ‘molecular targets’ and ‘action’, published between 2010 and 2021. Out of the 212 publications identified, 17 articles were selected for study inclusion. This review described 14 molecular targets and among these 4 are targets from commercial acaricides. Most of them are enzymes to catalyse important reactions to tick survival, related to energetic metabolism, mechanisms of biotransformation and neurotransmission. The data will be helpful in the development of new more effective and selective acaricides.

The supplementation of ruminant diets with exogenous cellulolytic enzymes can improve their digestibility and feeding value. The objective of this study was to determine the effect of supplementing roughage (rice straw) and concentrate with inoculants containing four fungal strains (Pleurotus ostreatus, Phanerochaete chrysosporium, Trichoderma reesei and Trichoderma viride) and four bacterial strains (Paenibacillus polymyxa, Bacillus megaterium, Bacillus circulans and Bacillus subtilis), given separately or as a mixture, as a source of exogenous cellulolytic enzymes, on basic rumen parameters in vitro, including digestibility and methane production. A batch culture trial was used to select the best supplements, and a long-term rumen simulation technique (RUSITEC) was used to evaluate the effects of P. chrysosporium, B. subtilis, and a 1 : 1 mixture of these two on dietary component digestibility and fermentation parameters. In the batch culture evaluation, there were significant increases in the organic matter (OM) digestibility, the total gas production expressed as ml/g of dry matter, the OM, the neutral detergent fibre (NDF) and the acid detergent fibre (ADF) of the supplemented rations, as compared to the control, excluding the rations supplemented with T. viride and B. circulans. In the RUSITEC, the ration supplemented with mixed inoculants showed significantly higher digestibility of crude protein, ether extract, NDF and ADF than did the ration supplemented with the P. chrysosporium and B. subtilis inoculants. It can be concluded that the simultaneous use of fungal and bacterial exogenous cellulases on rice straw roughage improves its digestibility, without negative effects on other rumen parameters.

Protein sources from cereals are used in broiler diets, usually in order to reduce feeding costs. However, their efficient use in poultry diets is limited by the level of fiber whose compounds are resistant to digestion in the small intestine; due to this sugars are not digested by endogenous poultry enzymes. The aim of this study was to determine the effect of multi-carbohydrase (MC) and phytase (Phy) on the total retention of nutrients, retention of apparent metabolizable energy corrected for nitrogen (AMEN) (trial 1) and apparent and standardized ileal digestibility of amino acids (trial 2) of rice bran (RB). A total of 245-day-old male broilers (Cobb 500) was distributed at 21-day-old in a completely randomized design in a 2 × 2 + 1 (0 and 200 mg/kg MC; 0 and 50 mg/kg Phy, and basal diet – BD) factorial arrangement of treatments, to give seven replicates and seven birds per replicate. The BD based on corn (trial 1) and cornstarch and casein (trial 2) was used only to determine the coefficients of retention of nutrients and energy, and coefficients of digestibility of amino acids of the RB. The test diets were made by mixing BD and RB 7 : 3 wt/wt basis. There was interaction (P<0.05) between MC × Phy for DM, nitrogen and AMEN, retention and no interaction (P>0.05) for ash, calcium, phosphorous and NDF was observed. Enzymes interacted (P<0.05) on standardized ileal digestibility of arginine, histidine, leucine, methionine, phenylalanine, threonine, valine, aspartic acid, glutamic acid, proline and serine. Dietary combination of MC and Phy resulted in higher (P<0.05) standardized digestibility of arginine, histidine, methionine and threonine relative to single enzyme supplementation or control diet without enzymes. Enzyme isolated inclusions in the diets improved (P<0.05) standardized digestibility of methionine. The supplementation of carbohydrases and Phy in RB will improve the nitrogen, energy and amino acids utilization for broiler chickens.

The kinetoplastid parasite Trypanosoma brucei causes African trypanosomiasis in both humans and animals. Infections place a significant health and economic burden on developing nations in sub-Saharan Africa, but few effective anti-parasitic treatments are currently available. Hence, there is an urgent need to identify new leads for drug development. The T. brucei neutral sphingomyelinase (TbnSMase) was previously established as essential to parasite survival, consequently being identified as a potential drug target. This enzyme may catalyse the single route to sphingolipid catabolism outside the T. brucei lysosome. To obtain new insight into parasite sphingolipid catabolism, the substrate specificity of TbnSMase was investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Recombinant TbnSMase was shown to degrade sphingomyelin, inositol-phosphoceramide and ethanolamine-phosphoceramide sphingolipid substrates, consistent with the sphingolipid complement of the parasites. TbnSMase also catabolized ceramide-1-phosphate, but was inactive towards sphingosine-1-phosphate. The broad-range specificity of this enzyme towards sphingolipid species is a unique feature of TbnSMase. Additionally, ESI-MS/MS analysis revealed previously uncharacterized activity towards lyso-phosphatidylcholine despite the enzyme's inability to degrade phosphatidylcholine. Collectively, these data underline the enzyme's importance in choline homoeostasis and the turnover of sphingolipids in T. brucei.

Non-starch polysaccharides (NSP) present in wheat and barley can act as anti-nutrients leading to an increase in digesta viscosity and a reduction in nutrient digestibility. Xylanase, an NSP-degrading enzyme, has been shown to increase nutrient digestibility in pigs. The objectives of this study were: (1) to identify the optimum inclusion level of xylanase in grower pig diets by measuring the effect of increasing enzyme levels on growth performance, the concentration of volatile fatty acids (VFA) and peptide YY concentration in portal and peripheral blood of grower pigs and (2) to increase our understanding of the interrelationships between xylanase inclusion, VFA production and peptide YY secretion. A total of 512 grower pigs ((Large White×Landrace)×MAXGRO) were allocated to pens creating 32 replicates of four pigs per pen per treatment. Pigs were allocated to trial weighing 14.2±0.31 kg and remained on trial until ~41.5±3.31 kg. The experiment was a dose response design with four inclusion levels (0, 8000, 16 000 or 32 000 BXU/kg) of xylanase (Econase XT). Diets were cereal-based wheat, barley mix formulated to meet or exceed the nutrient requirements of grower pigs. Body weight and feed intake were recorded to calculate growth performance. Pen faecal samples were collected to estimate DM, organic matter (OM) and crude fibre (CF) apparent total-tract digestibility. At the end of the trial 16 pigs per treatment were euthanised by schedule 1 procedures. Peripheral and portal blood samples were collected for peptide YY and VFA analysis. The addition of xylanase to the diet had no effect on growth performance, DM, OM or CF total-tract digestibility; however, xylanase tended to have a quadratic effect on ileum pH with higher pH values recorded for pigs fed a diet supplemented with 8000 and 16 000 BXU/kg xylanase (P<0.1). Xylanase had no effect on peptide YY levels or VFA concentration. Total VFA concentration was higher in portal compared with peripheral blood (P<0.05). In conclusion, the addition of xylanase had no effect on grower pig performance, nutrient digestibility, VFA concentration or peptide YY concentration when fed up to 32 000 BXU/kg over a 35-day period. Pig performance was good for all treatments throughout the trial suggesting that diet quality was sufficient thus there were no beneficial effects of adding xylanase.

Three-day-old soybean seedlings were treated with acifluorfen via liquid culture [50 μM in dark, 1 μM in light (200 μE·m–2·s–1)]. Root elongation in the dark was inhibited only slightly (6%) after 96 h. In the light, acifluorfen inhibited root elongation after 48 h; after 96 h, inhibition was 32%. Hypocotyl length was not affected in either the light or dark. Soluble hydroxyphenolic content per axis was unaffected in the dark but was reduced by about 13 to 25% at 24 to 96 h in the light. Extractable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity per axis was unaltered by herbicide in the dark. A brief transient increase in PAL activity (4 to 24 h) in axes of light-grown seedlings in the presence of acifluorfen was followed by reduced enzyme levels compared to light-grown controls at 72 and 96 h. Although light is required for maximal herbicide action, there was no effect on anthocyanin or chlorophyll accumulation or on the chlorophyll a/b ratio in hypocotyls or light-grown seedlings.

The Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni. In the present study, we show that in contrast to the poor immunogenicity of the enzymatically active native form of SmCE derived from a crude preparation (cercarial transformation fluid), immunization of CBA/Ca mice with two enzymatically inactive forms, namely purified native SmCE or a recombinant SmCE fused to recombinant Schistosoma japonicum glutathione S-transferase (rSmCE-SjGST), after adsorption onto aluminum hydroxide adjuvant, induced specific anti-SmCE immunoglobulin G (IgG) in all mice within 2 weeks of the second immunization. The IgG antibody response to rSmCE-SjGST was mainly of the IgG1 subclass. These results suggest that inactive forms of the antigen could be used to obtain the optimum immunogenic effects as a vaccine candidate against schistosomiasis. Mice immunized with the rSmCE-SjGST on alum had smaller mean worm burdens and lower tissue egg counts when compared with adjuvant alone- and recombinant SjGST-injected controls. The native SmCE was antigenically cross-reactive with homologous enzymes of Schistosoma haematobium and Schistosoma margrebowiei.

Fluazifop-P-butyl, a selective graminicide, has been widely used to control annual grass weeds for more than three decades in Taiwan, and a resistant (R) biotype of goosegrass has consequently appeared. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS) was applied to analyze metabolites of fluazifop-P-butyl in a R biotype of goosegrass. Six signals, including mass-to-charge ratios (m/z) 512, 432, 423, 415, 314, and 160, in positive scanning mode, and four signals, including m/z 788, 623, 593, and 162, in negative scanning mode, were found in the metabolites of the R and S biotypes, respectively. All of the signals of these metabolites in the R biotype showed higher intensity than those of the S biotype, except for m/z 162. Based on the molecular weights of the fragments (MS2 signal) from the molecules (MS1 signal) and comparison with the Metabolite Link Metabolomics database, MassBank database, and related references, one reduced form of fluazifop acid, i.e., 2-[4-(5- trifluoromethyl-2-pyridyloxy) phenoxy] propanol (MW 313); two types of intermediates of MW 163, i.e., 5-trifluoromethyl-2-pyridone and 5-trifluoromethyl-2-hydroxypyridine (or 2-hydroxy-5-trifluoromethyl pyridine); and five possible conjugated compounds containing a common core fragment (MW 255) from fluazifop acid were identified. In addition, another compound, likely degraded from one of the five conjugated compounds, was also detected. Accordingly, the metabolic pathway of fluazifop-P-butyl in goosegrass is described in this study. An enzyme kinetic study on glutathione S-transferase showed that the R biotype has higher affinities to the substrates reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene, with S/R Km ratios of 3.0 and 2.4, respectively. No difference in Vmax was found, revealing that the S biotype has a strong ability to bind GSH and herbicide or target molecules and showed susceptibility to fluazifop.

Understanding pesticide metabolism in plants and microorganisms is necessary for pesticide development, for safe and efficient use, as well as for developing pesticide bioremediation strategies for contaminated soil and water. Pesticide biotransformation may occur via multistep processes known as metabolism or cometabolism. Cometabolism is the biotransformation of an organic compound that is not used as an energy source or as a constitutive element of the organism. Individual reactions of degradation–detoxification pathways include oxidation, reduction, hydrolysis, and conjugation. Metabolic pathway diversity depends on the chemical structure of the xenobiotic compound, the organism, environmental conditions, metabolic factors, and the regulating expression of these biochemical pathways. Knowledge of these enzymatic processes, especially concepts related to pesticide mechanism of action, resistance, selectivity, tolerance, and environmental fate, has advanced our understanding of pesticide science, and of plant and microbial biochemistry and physiology. There are some fundamental similarities and differences between plant and microbial pesticide metabolism. In this review, directed to researchers in weed science, we present concepts that were discussed at a symposium of the American Chemical Society (ACS) in 1999 and in the subsequent book Pesticide Biotransformation in Plants and Microorganism: Similarities and Divergences, edited by J. C. Hall, R. E. Hoagland, and R. M. Zablotowicz, and published by Oxford University Press, 2001.

Enzymatic activity assays, used mainly to show biochemical transformations in humans and mammals, have recently found increasing application in birds. Enzyme activity in birds is affected by numerous factors, including age, sex, species, breed, nutrition, physiological state, and farming techniques. In large-scale poultry breeding, in a flock that may number over ten thousand birds, individual birds may be in different stages of development of a disease process, and may respond differently to stress factors present during rearing. There is also high individual variation among birds, so that results of enzyme activity analyses fall within fairly broad ranges. The aim of this paper is to review experiments on the activity of selected antioxidant, liver and cardiac enzyme profiles in turkey tissues. The results of many years of measurements of the activity of selected enzymes, presented in this study, may be considered physiologically normal for this group of birds. Analyses of the activity of these enzymes are important in determining whether oxidative stress reactions are induced in cells and which cells or organs have been damaged. During oxidative stress, which leads to cell damage or organ dysfunction (of the liver or heart), there is generally an increase in the activity of AST, ALT, ALP, LDH, GGT, SORD, MDH, AC, G6PC, G6PD, Cp, CK and HBDH, and a decrease in that of SOD, GPx, CAT, ACHE, BCHE, SDH, Ca2+ATPase. On the basis of the literature reviewed it can be concluded that the use of feed components and supplements with antioxidant or immunostimulatory properties can mitigate oxidative stress, which is manifested as beneficial changes in the activity of these enzymes.

Exogenous enzymes are commonly added to poultry diets to improve nutrient utilisation, reduce excretion of nutrients into the environment as well as to improve zootechnical performance and reduce the cost of production. A series of four experiments were conducted in broilers to determine the efficacy and tolerance to an enzyme product (Vegpro™) containing protease and xylanase activities in wheat-soybean meal (SBM) diets fed to broilers from 1 to 39 or 42 d of age. Collectively, four experiments tested the following exogenous protease activity levels in wheat-SBM diet at levels of 0, 2,500, 5,000, 10,000, 20,000, 30,000, 40,000 or 100,000 HUT/kg of feed. Analysis of the four experiments together confirmed that Vegpro™ improved average daily gain and feed conversion ratio, especially in birds up to 21 d of age. Overall, inclusion of Vegpro™ to broiler diets at 10,000 HUT protease per kg of feed was shown to provide the maximum performance benefit (i.e. weight gain and feed conversion ratio). An exceptionally high protease activity (100,000 HUT/kg, ten times the manufacturer's recommended inclusion rate) was well tolerated and did not adversely affect the measured health and performance indicators from 1 to 42 d of age.

The nutrient availability in animal feeds can be improved by including exogenous enzymes to the feed, either by helping breakdown anti-nutritional factors or by increasing digestibility of complex ingredients thereby releasing more nutrients for utilisation. This process can improve the efficiency of meat and egg production, increase animal health, decrease feeding costs and reduce nutrients in animal waste. Proteases are protein-digesting enzymes that are used in animal nutrition to break down storage proteins in various plant materials and proteinaceous anti-nutrients in vegetable proteins. The analysis of exogenous proteases in feed additives and after they have been added to feed has proven technically challenging. Accordingly, the purpose of this work was to validate a method for the determination of the activity of protease in animal feed additives and supplemented animal feed. The approach used for the assay was to adapt an assay based on the hydrolysis of haemoglobin. The method validations examined a range of parameters including; linearity & range; uncertainty, sensitivity, accuracy and studies designed to highlight any possible matrix effects on various types of supplemented feed. The assay method described herein is convenient and inexpensive and could be applied to the analysis of proteases in animal feeds during quality control and in investigating fraudulent adulteration of feed to ensure the authenticity and traceability of the product.

The aim of this study was to test the hypothesis of an improved gut environment of post-weaning piglets when administered a blend of essential oils (EO; thymol and cinnamaldehyde) and an enzyme combination (xylanase and β-glucanase (XB)) either alone or in combination. To assess the effect of dietary treatments, faecal nutrient digestibility and microbial counts, as well as ileum histology and gene expression of inflammatory mediators were evaluated. One hundred and ninety-two weaned piglets were allocated into four experimental treatments, and fed the basal diet (CTRL) either without or with EO, XB or their combination (EO+XB) for a 42-day period. The experiment concerning digestibility was designed with two periods (period I: days 15 to 21; period II: days 29 to 35) and the faeces were collected on days 20, 21, 34 and 35. On day 42, six piglets from each treatment were slaughtered. It was found that EO, XB and EO+XB supplementation did not affect (P>0.05) the growth performance of the piglets from days 0 to 42. Moreover, no dietary effect on faecal score was observed. Faecal digestibility of dry matter, organic matter, ash, dietary fibre, lipid, CP and NDF were increased from period I to period II (P<0.01 to P=0.06), while no effect (P>0.05) of EO, XB or their combination on the faecal digestibility was observed at both periods. Compared with the CTRL diet, dietary XB reduced the faecal Lactobacillus and Escherichia coli counts but increased the Lactobacillus to Coliforms ratio on day 42 (P=0.02, 0.03 and 0.03, respectively), and all the additives supplementations decreased the counts of faecal Coliforms on day 42 (P<0.01). XB supplementation increased the villus to crypt ratio (P=0.04) and reduced the mucosal macrophages number (P<0.01) in the ileum compared with the CTRL group, and dietary EO or EO+XB decreased the number of lymphatic follicles (P=0.01 and P<0.01, respectively) and mucosal macrophages (P=0.02 and P<0.01, respectively). In addition, the interleukin (IL)-1α was downregulated in piglets treated with EO+XB compared with the EO group (P=0.02). In conclusion, the administration of EO, XB or their combination was effective in improving ileum histology, and EO+XB supplementation might benefit the modulation of the expression of ileum inflammatory cytokines in piglets.

Published literature on the effect of carbohydrase enzymes (CE) on energy availability, nutrient digestibilities and growth performance of broilers fed corn-soybean meal-based diets is reviewed. Data from experiments involving CE preparations displaying single- to multiple-activities has shown improved metabolisable energy by 3% and digestibility of protein by 5%. Despite the observed variation in absolute values for individual amino acids, the improvement in amino acid digestibility appeared to be 27% of the undigested fraction. The positive effect on nutritive value are consistent with the improvements in body weight gain (0.8 to 10.4%; mean = 4.3%) and feed efficiency (1.3 to 9.3%; mean = 3.7%). Possible mechanisms contributing to these effects and the factors thought to explain the variability in responses has been discussed.

Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.

Activity of the enzyme β-glucuronidase (EC 3.2.1.31) is found in milk from ruminants with mastitis. However, the use of this enzymic activity as an indicator of mastitis has gained little attention possibly because of its low activity when compared with other mastitis indicators. The determination may therefore be less precise and the analytical procedure very time consuming and labour intensive. The present study optimized the fluorometric determination of the β-glucuronidase activity with respect to substrate concentration, pH, incubation time etc., validated the assay, and developed it into large scale analyses. The assay performance is satisfactory regarding precision, linearity etc., and it appears comparable to analogous fluorometric assays for mastitis indicators in milk. From a local dairy herd, 825 milk samples were analysed for potential mastitis indicators, i.e. β-glucuronidase, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and N-acetyl-β-d-glucosaminidase (NAGase) activity, and for somatic cell counts (SCC) and the variables were compared. Activity of β-glucuronidase was moderately but significantly correlated to SCC (r=0·21; n=768) as well as the other mentioned variables (r=0·25–0·43; n=825). Simple indices based on β-glucuronidase and LDH or NAGase activity were tested as indicators of mastitis (SCC), but were not found to improve the diagnostic value. Future studies may further verify whether β-glucuronidase can compete with well-established indicators of mastitis in cows such as LDH or NAGase as well as determine whether β-glucuronidase activity, in combination with other indicators of mastitis, has an advantage. Nineteen milk samples from subclinical and latent cases of mastitis (individual quarters) were identified for specific pathogens (PCR method) and measured for β-glucuronidase activity. The activity was tested at four different pH levels (5·5, 6·0, 6·5 and 7·0) in order to investigate the possibility of discrimination between pathogens. However, all milk samples (strains of pathogens) had the same pH optimum for β-glucuronidase activity; this may indicate that enzymic activity from mammary tissue and leucocytes dominates over enzyme activity from bacterial cells.

Endosperms of fenugreek and carob which have been leached following their isolation exhibit increased activity of endo-β-mannanase, part of which is released into the surrounding incubation medium. This activity was suppressed by the addition to the endosperms of abscisic acid or endosperm/seed coat leachate. The leachate from carob inhibited the increase of endo-β-mannanase activity in fenugreek and that from fenugreek inhibited enzyme activity in carob but more weakly; neither of these leachates inhibited the production of α-amylase in wheat endosperm. Polyethylene glycol-induced stress on non-leached endosperms inhibited endo-β-mannanase production, but not when the stress was applied following endosperm leaching. However, the stress did reduce the amount of enzyme released into the surrounding incubation medium. Several isoenzymes of mannanase could be detected on isoelectric focusing activity gels in the endosperms of both fenugreek and carob, but their pis differed between the two species. When activity was suppressed by ABA or leachate, all pl forms declined equally.

One performance and one ileal apparent digestibility (IAD) trial were conducted to investigate the performance, nutrient digestibility and intestinal disaccharidase activity of weaner/grower pigs given diets containing Chinese stored brown rice (CSBR) extruded and with exogenous enzyme supplements. In experiment 1, 96 crossbred (Duroc X Landrace X Large White) piglets weaned at 24 days of age were randomly divided into four groups according to a 2 X 2 factorial design. The animals were given four diets: (1) CSBR without enzyme supplementation, (2) CSBR supplemented with 625 mg a-amylase (2509 enzyme activity units) and 200 mg glucoamylase per kg (20018 enzyme activity units) per kg diet, (3) a normal temperature extruded CSBR (NTECSBR) without enzyme supplementation, and (4) NTECSBR supplemented with 625 mg a-amylase (2508 enzyme activity units) and 200 mg glucoamylase (20010 enzyme activity units) per kg diet. Growth, food consumption and specific activity of disaccharides in sections of the digestive tract were measured. In experiment 2, six male grower pigs with a mean initial body weight 21 kg, fitted with a simple ‘T’ cannula at the terminal ileum were used in a 6X6 Latin-square design. In addition to CSBR and NTECSBR diets with and without enzymes, a low temperature extruded diet LTECSBR with and without enzymes (as for other diets) was introduced. IAD of nutrients was measured. Extrusion resulted in a poorer (P < 0.05) food/gain for weaned pigs. Extrusion and enzyme supplementation had no influence (P > 0.05) on average daily food intake and average daily gain. Extrusion tended to improve (P = 0.075) IAD of starch but IAD of energy, dry matter, crude protein and amino acids were not influenced (P > 0.05). Enzyme supplementation had no effect on IAD of amino acids (P > 0.05), except for increasing IAD of lysine, threonine and isoleucine (P < 0.05). Extrusion and enzyme supplementation resulted in a higher (P < 0.05) maltase activity in the duodenum at day 14 of the trial. However, extrusion decreased (P < 0.05) the activity of maltase, isomaltase, sucrase and lactase in the jejunum at day 28. Enzyme supplementation increased (P < 0.05) the activities of maltase, isomaltase, trehalase and lactase in the jejunum at day 28. There was a positive extrusion X enzyme interaction (P < 0.05) for the activity of maltase and trehalase in the duodenum at day 14; a negative interaction was observed for the activity of lactase, maltase, isomaltase and trehalase in the jejunum at day 28. The results suggest that neither extrusion nor enzyme supplementation are necessary for CSBR to be used in pig diets.

Following the break-up of Gondwana, the drift of Antarctica to its present position and the establishment of the Antarctic Convergence, fish evolution was characterized by adaptation to progressive cooling of the environment. The decrease of erythrocyte number and haemoglobin concentration in the blood of Antarctic teleosts raises several questions concerning the physiology of respiration and the enzymatic role of erythrocytes. Our study of the molecular basis of cold adaptation includes the relationship between molecular structure and biological function of haemoglobins. Species of the suborder Notothenioidei, largely confined within the Convergence, have only one major haemoglobin, which displays the Root effect in oxygen binding; on the other hand, Zoarcidae (a family found at all latitudes) have four or five haemoglobins, only one of which displays the Root effect. In addition, our data indicate that the physiological relevance of erythrocyte-like cells, present in very small number in the blood of haemoglobinless Channichthyidae, may be linked to higher content of enzymes, such as glucose-6-phosphate dehydrogenase, in comparison with erythrocytes of red-blooded fishes.

This study summarizes optimization of techniques for common carp artificial propagation including improvements of activation solution (AS), the process of insemination, and elimination of egg stickiness. The optimum gamete ratio for good fertilization and hatching rate ranged from 8490 to 23672 spermatozoa per egg, when dechlorinated tap water was used. Optimal ratio between eggs (weight in g) and AS (in ml) was defined as 1:1 to 1:2. Different concentrations of AS such as NaCl from 0 to 34 mM (0–68 mOsmol kg–1) did not change fertilization and hatching rates. An AS adopted for carp spermatozoa (45 mM NaCl, 5 mM KCl, 30 mM Tris–HCl, pH 8) was compared with other saline AS; only the 51 mM (102 mOsmol kg–1) NaCl solution decreased fertilization and hatching rate. The AS containing 20 mM Tris–HCl at pH 9 increased fertilization and hatching rates compared to dechlorinated tap water of pH 7 or to AS of pH 6 and 7. Adhesiveness from the eggs was successfully removed by incubation in Alcalase DX (PLN 04715) using two successive steps with different enzyme concentrations. The first step with an enzyme concentration of 2 ml l–1 was applied from 8 to 20 min after fertilization. Later in a second step, the best time for application of alcalase enzyme at a concentration of 20 ml l–1 was for 45 and 60 s at 20 min after fertilization leading to fertilization and hatching rates of 80–87%. The α-Chymotrypsin (EC 3.4.21.1. Merck) was also found effective for elimination of stickiness. Results withα-Chymotrypsin enzyme indicate that the response to success in elimination of stickiness is highly variable mainly due to differences in the environment, quality of water and carp strains.