Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.     

Haematological and biochemical analysis of healthy neonatal puppies during the immediate foetal‐to‐neonatal transition

For the neonatal patient, precocity of diagnosis is crucial for effectiveness of medical approach. However, the newborn has its own physiological peculiarities due to ongoing adaptive mechanism for extrauterine life and deserves special attention in order to underline a specific management or clinical approach. The objective of this work was to verify clinical adaptations and biochemical balance of neonates during immediate period, with special reference to haematological, renal and metabolic functions. Neonatal puppies (n = 51) were physically examined for vitality and rectal temperature at birth, 5 and 60 min post‐birth. Blood was collected at birth and 60 min post‐birth for analysis of glucose, sodium, potassium, chlorine, blood urea nitrogen (BUN), haematocrit and haemoglobin. Neonatal vitality was lower at birth compared with 5 min and 60 min post‐birth. Progressive decline in rectal temperature (36.5 ± 0.3°C, 34.2 ± 0.2°C, 32.3 ± 0.5°C) was observed at birth, 5 min and 60 min post‐birth, respectively. Puppies presented slight hyponatremia (140.7 ± 0.5 mmol/L) at birth and hypopotassemia (3.5 ± 0.1 mmol/L) and blood urea nitrogen (13.1 ± 0.7 mg/dl) during the first hour, and high haematocrit (45.1 ± 1.0%) and haemoglobin (15.3 ± 0.3 g/dl) concentration. In conclusion, puppies had rapid evolution of vitality. Marked decrease in rectal temperature occurred at 5 min post‐birth. Haematological values of neonates immediately after birth reflected mainly the dam's blood status, not being useful for a blood panel at this time point. The peculiar pattern of BUN, sodium and potassium observed during transition period, suggested that specific reference range should be considered for neonatal puppies.     

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.     

Time course of endocrine changes in the hypophysis-gonad axis induced by hypobaric hypoxia in male rats

Chronic hypobaric hypoxia (CHH) induces a decrease in sperm output and spermatogenesis in male rats. The mechanisms that underlie these changes in testicular function are unknown and could involve changes in the hypophysis-gonad axis. We have tested the hypothesis that changes take place in the endocrine status (FSH, follicle stimulating hormone; LH, luteinizing hormone; testosterone) of rats subjected to CHH. Male Wistar rats were maintained under normobaric or hypobaric conditions (428 torr, 4,600 m). On days 0, 5, 15 and 30 post-exposure, 12 rats were anesthetized, their body weights were measured and blood samples were collected. The testicles were fixed in 4% formaldehyde and processed for histological analysis. In this time course, the FSH levels rose by day 5 post-exposure. On subsequent days, the FSH levels decreased in rats subjected to CHH with a tendency to remain higher than the normoxic group. The LH plasma levels decreased in rats exposed to CHH. Consistent with the decrease in LH levels, the plasma testosterone level decreased significantly after 30 days of CHH exposure. Integrated analysis of hormonal changes in rats subjected to CHH and the body dehydration that occurs in HH allows us to conclude that the effects of CHH on spermatogenesis may be partially related to changes in the hypophysis-gonad hormonal axis.     

Effluent from Citrus Industry: Toxic Parameters of Orange Vinasse

Water, Air, & Soil Pollution - Laboratory snow melting experiments were conducted with actual late-winter snow samples, collected just before the final snowmelt, in two similar northern Swedish...     

Evaluation of Turmeric Extract as an Antioxidant for Frozen Streaked Prochilod (Prochilodus lineatus) Fillets

This study aimed to investigate the efficiency of turmeric (Curcuma longa L.) extract, applied as a marinade or glaze, as an antioxidant for frozen streaked prochilod (Prochilodus lineatus) fillets. The turmeric extract contained 5.5 ± 0.1 mg curcumin/mL, and the experiment was performed using a diluted extract with 100 µg curcumin/mL. Analysis of the antioxidant activity with 2,2-diphenyl-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals confirmed the high antioxidant activity of curcumin. However, under the experimental conditions, curcumin extract presented no effectiveness for inhibiting lipid oxidation in frozen streaked prochilod fillets, as the concentration of thiobarbituric acid reactive substances did not differ (p > 0.05) between treated and control fillets. Sensory evaluation of the fillet appearance indicated that the change toward a more yellow color in treated fillets negatively affected its acceptance. However, the acceptability of the odor of raw and cooked fillets was similar for treated and control samples. These results may be explained by the fact that good in vitro performance of antioxidants is not always replicated in foodstuffs owing to the complexity of food matrices. Additionally, the low lipid content of the fish and the low amount of curcumin applied to the fillets may have contributed to the inefficiency of turmeric extract.     

Effects of aflatoxin B 1 on performance and health of tambaqui fingerlings ( Colossoma macropomum )

The detection of mycotoxins in feeds and their ingredients in aquaculture gained prominence due to losses caused in production and animal health, mainly the occurrence of aflatoxin (AFB1). The aim of this study was to evaluate the effects of AFB1 on the performance of tambaqui fingerlings (Colossoma macropomum). Four hundred tambaqui were used. Four different treatments were evaluated: treatment T1, considered as the control treatment (CT) with 3.84 μg kg−1; treatment T2, treatment T3 and treatment T4 with 500, 1000 and 2000 μg kg−1 of AFB1, respectively. The AFB1 of the samples (muscle, liver and kidney) was detected by high-pressure liquid chromatography. Four fingerlings from each treatment for histological analysis were examined. Moreover, the performance parameters (weight gain, feed conversion and feed intake) were studied. The levels of toxins used in T2, T3 and T4 represent a reduction in the growth of 14%, 35% and 45%, respectively. The T3 and T4 showed the lowest weight gain (78%) and the worst feed conversion. Aflatoxin B1 in muscle (3.28 μg kg−1) and kidneys (8.8 μg kg−1) in the T3, as well as liver (4.4 μg kg−1) and kidney (4.08 μg kg−1) in T4, was detected. Histopathological changes in liver and kidney tissues of fingerlings were more pronounced in T3 and T4. Fingerlings that consume feed contaminated with AFB1 in concentrations higher than 500 μg kg−1 present decreases in growth, reduction in weight gain and feed intake with increased feed conversion. The consumption of feed contaminated with 1000 and 2000 μg kg−1 of AFB1 caused severe deterioration in the hepatic and renal tissues.     

Compensatory growth and digestive enzyme activity of Litopenaeus vannamei submitted to feeding restriction in a biofloc system

Feeding restriction is a strategy in shrimp farming management that may promote compensatory growth after feeding is re‐established. This study aims to evaluate the effects of two feeding restriction regimens on the compensatory growth and digestive enzymes activity of Litopenaeus vannamei reared in biofloc system. Juvenile shrimp (0.46 ± 0.18 g) were stocked (320 individuals/m³) in 310 L tanks. The experiment comprised two phases: (a) Feeding Restriction (30 days) when shrimp were submitted to three feeding regimes, Control (fed daily), R1F1 (repetitively fasted one day and fed one day) and R2F1 (repetitively fasted 2 days and fed 1 day); and (b) Refeeding (28 days) when shrimp were fed daily. In the restriction phase, shrimp growth and digestive enzyme activities were reduced in R2F1 and R1F1. However, during the refeeding phase, enzyme activities and feed conversion improve significantly in R2F1 and R1F1. Control group attained higher final weight, but its final biomass was similar to R1F1. Litopenaeus vannamei exhibited partial compensatory growth, probably due to improved feed conversion efficiency driven by increased enzyme activity. It is possible to reduce feeding by 50% (R1F1) in biofloc systems for 28 days, without compromising the biomass produced at the end of a 30‐day refeeding period.     

Quantitation Overcoming Matrix Effects of Lipophilic Toxins in Mytilus galloprovincialis by Liquid Chromatography-Full Scan High Resolution Mass Spectrometry Analysis (LC-HR-MS)

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.