Evaluation of protein hydrolysis in raw sources by digestive proteases of Senegalese sole (Solea senegalensis,Kaup 1858) using a combination of an in vitro assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of products

The hydrolysis of protein in different animal and plant sources by the intestinal proteases of Senegalese sole (Solea senegalensis) was studied using a combination of an in vitro digestibility assay and the evaluation of the protein fractionation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). In vitro hydrolysis was performed for 90 min in a closed reactor maintained at constant pH and temperature. Samples of the reaction mixture at different time intervals were developed in SDS‐PAGE gels to evaluate the progressive hydrolysis of the different protein fractions in each protein source. A numerical value [coefficient of protein degradation (CPD)], which integrates the information obtained after image analysis of the gels, is proposed for comparison among proteins, according to the intensity of the hydrolysis produced by sole proteases. Additionally, the total free amino acid released from proteins was measured during the in vitro assay. Casein, squid meal and soybean concentrate (SBC) proteins showed very similar degradation patterns, with a quick and almost complete proteolysis within the first minutes of the enzymatic reaction. Fish and krill meals were hydrolysed more progressively. On the contrary, pea meal (PM) and corn gluten meal (CGM) showed scarce changes in their protein profile after 90 min of reaction. For animal protein sources, the final CPD values ranged from 77.6% to 87.0%, showing not significant differences. By contrast, PM (30.5%) and CGM (32.3%) presented significantly lower CPD values (P<0.05) as compared with SBC (90.6%). In general, a linear fitting was found between CPD and the release of free amino acids during in vitro protein hydrolysis. The present study provides detailed information, which, combined with the conventional in vitro digestibility studies, may help in the evaluation of different raw sources according to their protein degradation patterns. This information can be applied directly to estimate the protein nutritional quality of ingredients for Senegalese sole feeds.     

Assessment of enzymatic efficiency on protein digestion in the tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The present study develops an experimental procedure aimed to estimate the efficiency of protein digestion in fish by measuring both gut transit rate and total amount of the main intestinal proteases (trypsin and chymotrypsin). The selected species was the Nile tilapia (Oreochromis niloticus). Total time for digestion, calculated through the estimation of gut transit rate using differently colored feeds, was 7.15 h. Mean production of trypsin and chymotrypsin was 15.94 and 24.11 mU in the proximal intestine and much lower (2,39, 4.90 mU) in the distal intestine. The enzyme efficiency, calculated from the average enzyme activity and time of residence of the digesta in each intestinal section, points to the major role of proximal intestine in protein digestion for this species. Results are discussed in relation to the main features characterizing digestion in stomachless fish.     

Recharge in Volcanic Systems: Evidence from Isotope Profiles of Phenocrysts

Strontium isotope ratios measured from core to rim across plagioclase feldspar crystals can be used to monitor changes in the isotope composition of the magma from which they grew. In samples from three magma systems from convergent margin volcanoes, sudden changes in major element composition, petrographic features, and strontium isotope composition were found to correspond to discrete magmatic events, most likely repeated recharge of more mafic magma with lower ratios of strontium-87 to strontium-86 into a crustally contaminated magma.     

Caspase Activation,Hydrogen Peroxide Production and Akt Dephosphorylation Occur During Stallion Sperm Senescence

To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.     

Use of SDS-page in the assessment of protein hydrolysis by fish digestive enzymes

Inthe field of fish nutrition, the preliminary evaluation of feedstuffs usingin vitro techniques may be an alternative to invivo assays. Degradation of the protein fraction in feedstuffs byseabream (Sparus aurata) digestive enzymes was studiedunder different conditions simulating either acid, alkaline, or acid + alkalinedigestion using a modified pH-stat technique. In addition, a sequential sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the releasedproducts was used both to visualise and to quantify the level of proteinhydrolysis. Gels were analysed by optical densitometry and results wereexpressed as a Coefficient of Protein Degradation (CPD). Values of CPD showedclear differences related to the type of protein digested or the steps followedin the digestion. CDP index provides information based on the enzymaticbreakdown of the proteins visualised in SDS-PAGE gels. The pH-stat evaluation(Degree of Hydrolysis) supplies measurement of the total number of peptidebondsbroken down in a given protein. CDP index mainly focuses on the action ofendoproteases, whereas degree of hydrolysis (DH) includes both endo- andexoproteases. CPD and DH are two complementary indexes capable of measuring theprotein hydrolysis by fish enzymes of a given feedstuffs or diet. Both methodswere in agreement with respect to assessing the hydrolysis of protein. Thesignificance of the SDS-PAGE techniques in the assessment of aquafeeds by fishdigestive enzymes is discussed.     

Evaluation of two ELISA kits for the detection of Aujeszky's disease antibodies in pigs.

This work describes the evaluation of two commercial ELISA kits for the detection of gI antibodies against Aujeszky's disease. A collection of experimental sera from infected pigs, field sample sera, and sera from pigs vaccinated with seven different modified gI-negative commercial vaccines were used to evaluate each test. Both ELISA kits showed good reproducibility, and specificity, but differences could be appreciated in sensitivity when sera obtained at early stages of infection was analysed. These results also indicated that both kits could be used in conjunction with the seven vaccines evaluated in this study.     

Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3–5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, μm; r = −0.56, p < 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p < 0.05). The presence and number of colonies of β-haemolytic Streptococcus were negatively correlated (r = −0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.     

Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics

In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen–thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen–thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1–S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1–FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1–S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.     

Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen–Thawed Stallion Spermatozoa

The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E™). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen–thawed semen samples.