Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation

Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p < 0.05) of this fatty acid in his sperm plasma membranes.     

Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.     

Gut pH as a limiting factor for digestive proteolysis in cultured juveniles of the gilthead sea bream (Sparus aurata)

After the development of the gastric function in juvenile fish, dietary proteins enter a two-phase digestive process comprising an acidic gastric phase followed by an alkaline intestinal phase. However, the main gastric protease, pepsin, is strictly dependent on the existence of a low-enough environmental pH. In 20-g gilthead sea bream, Sparus aurata, the mean minimal gastric pH is close to 4.5, while the mean pH in the duodenal portion of the intestine was nearly fixed at 6.5. The mean maximal gastric content of HCl was approximately 20 microEq for a low-buffering diet. Gastric proteases were more severely affected than intestinal proteases when assayed at actual sub-optimal pH values, 4.5 and 6.5, respectively. When the gastric proteases of juvenile fish were pre-incubated with a citric acid buffer at pH 6.0, the activity at pH 4.5 was very low, whereas when they were pre-incubated with the same buffer at pH 3.0, the activity at pH 4.5 was significantly increased; this fact suggests a deficient activation of zymogens during the gastric digestion and points to a potential approach to improve protein digestion in juvenile gilthead sea bream.     

Larval fish distribution and retention in the Canary Current system during the weak upwelling season

The spatial distribution of fish larvae was studied in the Canaries‐African Coastal Transition Zone, outside the strong upwelling season. An onshore–offshore transition in the larval fish community structure was observed, from a coastal assemblage dominated by small pelagics (sardine, anchovy, mackerel), bounded by the upwelling front, to an offshore assemblage dominated by mesopelagic species (mainly Myctophidae, Phosichthydae, Gonostomatidae). Distribution of the neritic larvae was deeply influenced by the intense mesoscale activity found in the area, both horizontally (larvae were advected offshore but were always retained within the upwelling area) and vertically (larvae were deepened in the vicinity of two anticyclonic eddies). A combined effect of the upwelling front and a cyclonic–anticyclonic eddy dipole is likely the successful retention mechanism for these larvae. These results support the current belief that retention may be higher than previously thought in upwelling areas. Oceanic larvae were also collected in higher abundances near the front and an anticyclonic eddy. Neritic and oceanic larvae frequently showed a differentiated position in the water column, although they sometimes coexisted. Finally, larval connectivity between Islands within the Canary archipelago is suggested. The present study thus contributes to the understanding of the complex dispersal and retention processes in the Canaries‐African Coastal Transition Zone. However, results also highlight the poor knowledge of this region compared with the other three main Eastern Boundary Upwelling Systems in terms of ichthyoplankton dynamics. The importance of routine monitoring programs of commercial and non‐commercial species in the area is emphasized.     

Effect of phytase supplementation of a plant‐based diet on phosphorus and nitrogen bioavailability in sea bream Sparus aurata

To assess to what extent addition of phytase to a plant‐based diet results in spatio‐temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant‐based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant‐based diets used in fish aquaculture.     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

Pregnancy Diagnosis in Cats Using a Rapid, Bench-top Kit to Detect Relaxin in Urine

Relaxin is a pregnancy-specific hormone in the queen and is produced by the placenta. Both serum and urinary relaxin levels can be used to diagnose and monitor pregnancy in the cat; however, only serum levels are commonly measured in practice. The present study aimed to assess whether urine could be used for the rapid diagnosis of pregnancy at an early stage in domestic cats using a bench-top kit to detect relaxin. Paired serum and urine samples were collected during the first month of gestation in six cats. The samples were tested by applying neat serum, urine or urine diluted in non-pregnant cat serum to the Witness Relaxin kit. Relaxin concentrations in the paired samples were also measured by radioimmunoassay. All undiluted urine samples from pregnant cats tested negative using the bench-top kit; however, the kit was able to detect relaxin in urine after dilution with non-pregnant cat serum. Using this as the test sample, the kit was accurate at diagnosing pregnancy from 28 days after mating and some samples tested positive at 21 days after mating. This preliminary work could lead to the development of a home pregnancy test for cats.     

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.     

Phytase and sodium diformate supplementation in a plant‐based diet improves protein and mineral utilization in rainbow trout (Oncorhynchus mykiss)

A basal isonitrogenous and isoenergetic plant‐based diet (Control) was supplemented with either 10 g kg^?1 sodium diformate (NaDF), 4000 FTU kg^?1 phytase (Phy) or a combination of both additives (NaDF + Phy). Three hundred juvenile rainbow trout with an average weight of 120 g were randomly distributed into 12 fibreglass tanks (300 L). After 65 days of trial, fish fed diets containing phytase, NaDF or the combination of both additives showed a higher growth rate (P < 0.05) compared to fish fed Control diet. NaDF increased feed intake (P = 0.032), while phytase inclusion resulted in a better feed conversion ratio (P < 0.0001) and a higher N retention efficiency (P = 0.02) compared with the Control. Apparent digestibility of P, Ca, Mg and Zn was improved by the use of phytase (P < 0.005) as well as P, Ca and Mg retention efficiency in fish (P < 0.0001). Using 4000 FTU kg^?1 phytase in plant‐based diets resulted in a 13% and 50% reduction in N and P loadings, respectively. The use of NaDF in combination with phytase in a plant‐based diet for rainbow trout resulted in a higher weight gain than that when NaDF was used alone.     

Nitrate,arsenic, cadmium,and lead concentrations in leafy vegetables: expected average values for productive regions of Chile

High arsenic (As), cadmium (Cd), lead (Pb), and nitrate (NO₃^?) concentrations in soil pose a risk for the human population and compromise food safety. The goal of this study was to obtain preliminary approximations for the expected mean values of As, Cd, Pb, and N-NO₃ in three leafy vegetables (lettuce, spinach, and chard) grown in the central farming regions of Chile. Representative samples (n = 148) of these crops were collected from the Coquimbo, Valparaíso, and Metropolitana Regions. Water extraction and an ion-selective electrode were used to determine NO₃^? contents, while total As, Cd, and Pb contents were determined by atomic absorption spectroscopy. The recorded values were 23.8%, 59.2%, and 97.9% below detection limits for As, Cd, and Pb, respectively. Furthermore, As and Cd intake by leafy vegetables reached 0.26 and 2.30% of the daily intake levels estimated by Chilean authorities. The daily NO₃^? intake by leafy vegetables was 0.44 mg per kg of bodyweight per day (kgbw^?1day^?1), or 12% of the WHO-recommended intake. No analyses were performed for Pb due to highly left-censored data. While the recorded NO₃^?, As, and Cd concentrations in lettuce, spinach, and chard do not apparently pose a health risk, further detailed studies are suggested.