Comparison of four strategies of ram management in a semen collection centre.

Thirty Merino rams were used to determine the effect of four management systems of rams on semen quality during the anoestrous season. Animals were divided into four groups: Artificial-Photoperiod group (AP; n = 8), which were isolated from females and exposed to artificial long days (16 hr/d) from 1 Feb to 15 Mar; Natural-Photoperiod (NP; n = 8), isolated from females and exposed to the natural photoperiod throughout the experiment; Oestrous-Ewe group (EE; n = 7), housed in a pen adjacent to another pen that housed three ewes in oestrus, and Anestric-Ewe group (AE; n = 7), housed adjacent to another pen that housed three ovariectomized ewes. From 20 Mar to the end of May (10 weeks), semen samples were collected weekly, and blood samples were collected to determine plasma testosterone concentrations. Mean plasma testosterone concentrations, ejaculate volume and reaction time were not affected either by treatment or week. There was a significant effect (p < .01) of ram treatment on sperm concentration, and both TM y PM, and their interaction, were significantly affected by group and week (p < .001). Rams exposed to ewes in oestrus presented the largest sperm concentration (p < .05) compared with the other three groups, although they had the lowest total and progressive motilities (p < .01). In conclusion, management strategy in spring affects semen quality of rams, with the presence of ewes in oestrus being the best plan to increase sperm concentration.

Seventy-Two-Hour LRRK2 Kinase Activity Inhibition Increases Lysosomal GBA Expression in H4, a Human Neuroglioma Cell Line.

Mutations in LRRK2 and GBA1 are key contributors to genetic risk of developing Parkinson's disease (PD). To investigate how LRRK2 kinase activity interacts with GBA and contributes to lysosomal dysfunctions associated with the pathology of PD. The activity of the lysosomal enzyme ß-Glucocerebrosidase (GCase) was assessed in a human neuroglioma cell model treated with two selective inhibitors of LRKK2 kinase activity (LRRK2-in-1 and MLi-2) and a GCase irreversible inhibitor, condutirol-beta-epoxide (CBE), under 24 and 72 h experimental conditions. We observed levels of GCase activity comparable to controls in response to 24 and 72 h treatments with LRRK2-in-1 and MLi-2. However, GBA protein levels increased upon 72 h treatment with LRRK2-in-1. Moreover, LC3-II protein levels were increased after both 24 and 72 h treatments with LRRK2-in-1, suggesting an activation of the autophagic pathway. These results highlight a possible regulation of lysosomal function through the LRRK2 kinase domain and suggest an interplay between LRRK2 kinase activity and GBA. Although further investigations are needed, the enhancement of GCase activity might restore the defective protein metabolism seen in PD.

Acute Anisakiasis: Pharmacological Evaluation of Various Drugs in an Animal Model.

BACKGROUND: The accidental ingestion of the third larval stage of Anisakis can cause acute clinical symptoms, which are relieved via extraction of the larvae. Although this is a highly effective technique, it can only be practiced when the larvae are found in accessible areas of the gastrointestinal tract, and therefore instead the condition has often been treated using various different drugs. AIMS: This study evaluates the effectiveness of gastric acid secretion inhibitors (omeprazole and ranitidine), gastric mucosal protectants (sucralfate) and anthelmintics (mebendazole and flubendazole) in treating anisakiasis in Wistar rats. METHODS: Rats were infected with Anisakis-type I larvae and administered the drugs via a gastric probe. Data were recorded regarding the number of live and dead larvae, their location both within the animal and in its feces, and the presence of gastrointestinal lesions. Additionally, gastric pH was measured and histology performed. RESULTS: While rats in all experimental groups exhibited lesions; those treated with ranitidine and mebendazole showed significantly fewer lesions (50% and 35% of rats exhibited lesions, respectively). Histological examination of the gastric lesions revealed infection-induced changes, but no significant differences were observed between the treated and untreated rats. CONCLUSIONS: Mebendazole was found to be most efficacious in preventing gastrointestinal lesions, followed by ranitidine, which was the most effective antacid of those studied. Both these drugs could thus be considered as part of the conservative management of anisakiasis.

Aerobic interval exercise improves renal functionality and affects mineral metabolism in obese Zucker rats.

Obesity, metabolic syndrome, and renal injury are considered risk factors for type 2 diabetes, as well as kidney disease. Functional and structural changes in the kidney as consequence of obesity and metabolic syndrome may lead to impaired mineral metabolism in what is known as chronic kidney disease-mineral and bone disorder. Lifestyle interventions such as physical activity are good strategies to manage these pathologies and therefore, prevent the loss of kidney functionality and related complications in mineral metabolism. In this study, we have used 40 male Zucker rats that were randomly allocated into four different experimental groups, two of them (an obese and a lean one) performed an aerobic interval training protocol, and the other two groups were sedentary. At the end of the experimental period (8 wk), urine, plasma, and femur were collected for biochemical and mineral composition analysis, whereas the kidney was processed for histological studies. The obese rats exhibited albuminuria, glomerulosclerosis, and hypertrophy in glomeruli and renal tubule in some areas, together with alterations in mineral content of plasma but not of femur. The training protocol prevented the generation of albuminuria and glomerulosclerosis, showing a significant action on plasma and bone mineral levels. Therefore, the specific training protocol used in this study was able to prevent the development of diabetic nephropathy and affected the metabolism of certain minerals.

Vitrification induces critical subcellular damages in ram spermatozoa.

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 106 spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.

Pushing the frontiers: boron-11 NMR as a method for quantitative boron analysis and its application to determine boric acid in commercial biocides.

Quantitative boron-11 NMR (11B qNMR) spectroscopy has been introduced for the first time as a method to determine boric acid content in commercial biocides. Validation of the method affords a limit of detection of 0.02% w/w and a limit of quantification of 0.04% w/w, which are low enough to determine boric acid in commercial biocides. Other figures of merit such as linearity (R2 > 0.99), recovery (93.6%-106.2%), intra- and inter-day precision (from 0.7 to 2.0%), uncertainty (3.7 to 4.4%) and matrix effects were also evaluated. This method was successfully applied to determine boric acid in five different commercial biocides in a wide range of concentrations (<0.05 to 10% w/w) providing excellent results when they were compared with those obtained using inductively coupled plasma-mass spectrometry (ICP-MS). The suitability of this method for a fast and reliable quantification of boric acid in commercial biocide preparations has been demonstrated. The absence of the matrix effect allows the application of this validated method for the determination of boric acid in other matrices of diverse composition.

Development and Validation of a Multiresidue Method for the Determination of Pesticides in Dry Samples (Rice and Wheat Flour) Using Liquid Chromatography/Triple Quadrupole Tandem Mass Spectrometry.

A rapid and sensitive multiresidue method was developed and validated for the determination of around 100 pesticides in dry samples (rice and wheat flour) by ultra-performance LC coupled to a triple quadrupole mass analyzer working in tandem mode (UPLC/QqQ-MS/MS). The sample preparation step was optimized for both matrixes. Pesticides were extracted from rice samples using aqueous ethyl acetate, while aqueous acetonitrile extraction [modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method] was used for wheat flour matrixes. In both cases the extracts were then cleaned up by dispersive solid phase extraction with MgSO4 and primary secondary amine+C18 sorbents. A further cleanup step with Florisil was necessary to remove fat in wheat flour. The method was validated at two concentration levels (3.6 and 40 µg/kg for most compounds), obtaining recoveries ranging from 70 to 120%, intraday and interday precision values≤20% expressed as RSDs, and expanded uncertainty values≤50%. The LOQ values ranged between 3.6 and 20 µg/kg, although it was set at 3.6 µg/kg for the majority of the pesticides. The method was applied to the analysis of 20 real samples, and no pesticides were detected.

Improvement of the antioxidant and hypolipidaemic effects of cowpea flours (Vigna unguiculata) by fermentation: results of in vitro and in vivo experiments.

BACKGROUND: The antioxidant capacity and hypolipidaemic effects of Vigna unguiculata, as well as their potential improvement by different fermentation and thermal processes were studied using in vitro and in vivo methods. RESULTS: Phenolic content and reducing capacity of legume acetone extract were significantly increased by different fermentation processes, and by the thermal treatment of fermented legume flours. TBARS inhibiting capacity was increased by fermentation but not by thermal treatment. A higher ability to decrease Cu(2+)/H2O2-induced electrophoretic mobility of LDL was found in fermented when compared to raw legume extracts, and a higher protective effect on short term metabolic status of HT-29 cells was found for raw and lactobacillus-fermented Vigna followed by naturally fermented Vigna extracts. Significant improvements in plasma antioxidant capacity and hepatic activity of antioxidant enzymes were observed in rats that consumed fermented legume flours when compared to the untreated legume or a casein-methionine control diet. In addition, liver weight and plasma levels of cholesterol and triglycerides were also positively affected by untreated or naturally fermented Vigna. CONCLUSION: V. unguiculata has demonstrated its potential as a functional food with interesting antioxidant and lipid lowering properties, which can be further augmented by fermentation processes associated or not to thermal processing.

Genomic Characterization of Quality Wool Traits in Spanish Merino Sheep.

The native Spanish Merino breed was the founder of all the other Merino and Merino-derived breeds worldwide. Despite the fact that this breed was created and improved to produce the highest quality fine wool, the global wool market crisis led to the wholescale crossing of most of the herds with breeds for meat purposes. Nevertheless, there are still some purebred animals with a high potential for producing quality wool. The objective of this study was to characterize the current wool quality of the breed and identify genes associated with these parameters. To achieve this, over 12,800 records from the most representative animals of the breed (registered in the herd book) were analyzed using the Australian OFDA 2000 system, for parameters such as fiber diameter (FD), standard deviation (SD), coefficient of variation (CV), fibers over 15 microns (>15%), staple length (SL), and comfort factor (CRV). Additionally, animals with the most extreme FD values were whole-genome sequenced using NGS. Genome-wide association studies (GWAS) determined the association of 74 variants with the different traits studied, which were located in 70 different genes. Of these genes, EDN2, COL18A1, and LRP1B, associated with fibers over 15%, and FGF12 and ADAM17, associated with SL, play a key role in hair follicle growth and development. Our study reveals the great potential for recovering this breed for fine wool production, and identifies five candidate genes whose understanding may aid in that selection process.

Cooling rate modifies the location of aquaporin 3 in spermatozoa of sheep and goat.

The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins' (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P < 0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P < 0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P < 0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.

Unified Method for Target and Non-Target Monitoring of Pesticide Residues in Fruits and Fruit Juices by Gas Chromatography-High Resolution Mass Spectrometry.

A new polyvalent wide-scope analytical method, valid for both raw and processed (juices) fruits, combining target and non-target strategies, has been developed and validated to determine low concentrations of 260 pesticides, as well as many potential non-target substances and metabolites. The target approach has been validated according to SANTE Guide requirements. Trueness, precision, linearity, and robustness values were validated in raw fruit (apple) and juice (apple juice) as representative solid and liquid food commodities. Recoveries were between 70-120% and two ranges of linearity were observed: 0.5-20 µg kg-1 (0.5-20 µg L-1 apple juice) and 20-100 µg kg-1 (20-100 µg L-1 apple juice). The limits of quantification (LOQs) reached were lower than 0.2 µg kg-1 in apple (0.2 µg L-1 apple juice) in most cases. The developed method, based on QuEChERS extraction followed by gas chromatography-high resolution mass spectrometry (GC-HRMS), achieves part-per-trillions lower limits, which allowed the detection of 18 pesticides in commercial samples. The non-target approach is based on a retrospective analysis of suspect compounds, which has been optimized to detect up to 25 additional compounds, increasing the scope of the method. This made it possible to confirm the presence of two pesticide metabolites which were not considered in the target screening, phtamlimide and tetrahydrophthalimide.

Colostrum Quality Assessment in Dairy Goats: Use of an On-Farm Optical Refractometer.

Failure of passive immunity transfer is one of the main causes of increased susceptibility to infectious agents in newborn kids. To ensure successful transfer of passive immunity, kids need to be fed high-quality colostrum, containing an adequate concentration of IgG. This work evaluated the quality of colostrum obtained in the first 3 days postpartum from Malagueña dairy goats. The IgG concentration in colostrum was measured using an ELISA as a reference method, and it was estimated by optical refractometer. Colostrum composition in terms of fat and protein was also determined. The mean concentration of IgG was 36.6 ± 2.3 mg/mL, 22.4 ± 1.5 mg/mL and 8.4 ± 1.0 mg/mL on days 1, 2 and 3 after parturition, respectively. Brix values obtained using the optical refractometer were 23.2%, 18.6% and 14.1% for days 1, 2 and 3, respectively. In this population, 89% of goats produced high-quality colostrum with IgG concentrations of >20 mg/mL on the day of parturition, but this percentage declined dramatically over the following 2 days. The quality of the fresh colostrum estimated with the optical refractometer was positively correlated with those obtained using ELISA (r = 0.607, p = 0.001). This study highlights the importance of feeding first-day colostrum to newborn kids and demonstrates that the optical Brix refractometer is suitable for the on-farm estimation of IgG content in colostrum.

Novel insights and mechanisms of diet-induced obesity: Mid-term versus long-term effects on hepatic transcriptome and antioxidant capacity in Sprague-Dawley rats.

AIMS: The study of molecular mechanisms related to obesity and associated pathologies like type 2-diabetes and non-alcoholic fatty liver disease requires animal experimental models in which the type of obesogenic diet and length of the experimental period to induce obesity deeply affect the metabolic alterations. Therefore, this study aimed to test the influence of aging along a rat model of diet-induced obesity in gene expression of the hepatic transcriptome. MAIN METHODS: A high-fat/high-fructose diet to induce obesity was used. Mid- (13 weeks) and long-term (21 weeks) periods were established. Caloric intake, bodyweight, hepatic fat, fatty acid profile, histological changes, antioxidant activity, and complete transcriptome were analyzed. KEY FINDINGS: Excess bodyweight, hepatic steatosis and altered lipid histology, modifications in liver antioxidant activity, and dysregulated expression of transcripts related to cell structure, glucose & lipid metabolism, antioxidant & detoxifying capacity were found. Modifications in obese and control rats were accounted for by the different lengths of the experimental period studied. SIGNIFICANCE: Main mechanisms of hepatic fat accumulation were de novo lipogenesis or altered fatty acid catabolism for mid- or long-term study, respectively. Therefore, the choice of obesity-induction length is a key factor in the model of obesity used as a control for each specific experimental design.

Genomic Population Structure of the Main Historical Genetic Lines of Spanish Merino Sheep.

According to historiographical documentation, the Romans first began to select Merino sheep in the Iberian Peninsula during the first century, with the aim of obtaining a breed appreciated for the quality of its wool. This process continued locally during the Middle Ages, when Spanish sheep were protected, and their export to foreign countries was banned. It was during the 16th century when individual Merino sheep were allowed to spread around the world to be used to improve the wool quality of local breeds. However, the wool crisis of the 1960s shifted the selection criteria of the Merino breed towards meat production at the expenses of wool. Consequently, individuals that display the genetic and phenotypic characteristics of those sheep originally bred in the kingdom of Spain in the Middle Ages are extremely difficult to find in commercial herds. In this study, we characterized the genetic basis of 403 individuals from the main historical Spanish Merino genetic lines (Granda, Hidalgo, Lopez-Montenegro, Maeso, Donoso and Egea), which were bred in isolation over the last 200 years, using a genomic approach based on genotyping data from the Axiom™ Ovine 50K SNP Genotyping Array. Our analysis included measuring population structure, genomic differentiation indexes, runs of homozygosity (ROH) patterns, and an analysis of molecular variance (AMOVA). The results showed large genetic differences between the historical lines, even though they belong to the same breed. In addition, ROH analysis showed differences due to increased inbreeding among the ancient generations compared with the modern Merino lines, confirming the breed's ancestral and closed origin. However, our results also showed a high variability and richness within the Spanish historical Merino lines from a genetic viewpoint. This fact, together with their great ability to produce high-quality wool, suggests that ancestral Merino lines from Spain should be considered a valuable genetic population to be maintained as a resource for the improvement of wool-producing sheep breeds all around the world.

Periovulatory Hormonal Profiles after Estrus Induction and Conception Rate by Fixed-Time AI in Payoya Goats during the Anestrous Season.

Sexual activity in domestic goats is positively influenced by reducing the photoperiod. Various protocols have therefore been developed in goats for the induction and synchronization of estrus during those months in which their sexual activity is reduced. The present observational study evaluates the periovulatory hormonal profile in Payoya goats (n = 24), during a non-favorable photoperiod (i.e., spring), being treated for estrus induction. The treatment comprised the vaginal insertion of sponges impregnated with progestogen (fluorogestone acetate, FGA), together with cloprostenol and equine chorionic gonadotrophin (eCG), 48 h before the end of the treatment. When the treatment ended, the plasma concentrations of the LH, FSH, progesterone and estradiol were determined. The goats were inseminated 46 h after the sponge withdrawal, and a pregnancy diagnosis was carried out 40-45 days after the insemination. Various parameters were monitored, such as the peaks of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol, and their respective intervals, in reference to the time of the sponge withdrawal. The conception rate was 62.5%, and the kidding rate was 50%. The results record the hormonal release pattern after the estrus synchronization treatment based on the FGA, and the differences between the pregnant and non-pregnant goats. The findings suggest that the LH peak produced after the estrus synchronization treatment, both in terms of the amplitude and the time of increment, is involved in the reproductive failure detected.

Assessment of wastewater pollution by gas chromatography and high resolution Orbitrap mass spectrometry.

As a novelty, the combination of headspace solid phase microextraction and gas chromatography coupled to an Orbitrap mass analyzer in full scan mode (HS-SPME-GC-Orbitrap-MS) was evaluated for the monitoring of organic pollutants in wastewaters. The developed methodology showed good linearity (R2 > 0.999), sensitivity as well as suitable relative recoveries (89-115%) and precision values (RSD = 1-16%) for 15 polycyclic aromatic hydrocarbons (PAHs) selected as target compounds. Naphthalene, acenapthene and phenanthrene were found in the analyzed samples (influent and effluent wastewaters). Naphthalene was present in 62% of them, ranging from 1.33 to 24.32 ng L-1. Acenapthene was observed in 1 single sample (4.17 ng L-1) while phenanthrene was found in 7 samples (1.51-8.67 ng L-1). In addition, in order to identify other pollutants in the samples, retrospective analyses were addressed through target and non-target screenings. An in-house database containing close to 1,000 pollutants including, among others, polychlorinated biphenyls (PCBs), brominated diphenyl ethers (BDEs) and pesticides, was applied in the post-target analysis. For the non-target screening, after a deconvolution process, high resolution filtering (HRF) and Kovats retention index (KI) were used for tentative analyte identification. Thus, 51 additional pollutants were tentatively identified in the wastewaters, most of them used as flavoring agents and household product ingredients, highlighting the presence of linear alkyl benzenes (LABs).

A combined healthy strategy for successful weight loss, weight maintenance and improvement of hepatic lipid metabolism.

Obesity is critically related with the development of metabolic and pathophysiological alterations among which non-alcoholic fatty liver disease (NAFLD) is of especial relevance. Although there are numerous strategies to successfully treat obesity, the prevention of weight regain still remains challenging for individuals who have undergone weight loss programs. In such context, diet and physical activity are considered essential for the regulation of body weight and lipid metabolism. In this study, rats were fed a high-fat diet (HFD) to induce obesity and alterations in hepatic lipid metabolism. Obese rats were then treated with single or combined strategies of caloric restriction, physical exercise, and/or pharmacological treatment with an appetite suppressant, to lose weight, reverse the obesity-related alterations in hepatic morphology and lipid metabolism and maintain the beneficial effects of the interventions used. HFD induced excess body weight, hepatic steatosis, altered fatty acid profile, dysregulated gene expression of lipogenic and lipolytic enzymes, as well as plasma markers of liver damage, and modifications in liver antioxidant enzyme activity. Such alterations were ameliorated by caloric restriction in combination with a mixed training protocol and/or food-intake inhibitor administration during a weight loss intervention period of 3 weeks, and the beneficial effects remained after 6 weeks of weight maintenance, with some interesting interactions observed. In conclusion, weight loss strategies assayed were efficient at correcting the obesogenic action of a HFD and related alterations in hepatic functionality through different molecular mechanisms. The beneficial effects were also evident along the post-intervention maintenance period to avoid body weight regain.

1H NMR and multi-technique data fusion as metabolomic tool for the classification of golden rums by multivariate statistical analysis.

1H NMR spectroscopy combined with chemometrics was applied for the first time for golden rum classification based on several factors as fermentation barrel, raw material, distillation method and aging. Principal component analysis (PCA) was used to assess the overall structure, and partial least square discriminant analysis (PLS-DA) was carried out for the analytical discrimination of rums. Additionally, data-fusion of 1H NMR and chromatographic techniques (gas and liquid chromatography) coupled to mass spectrometry was applied to provide more accurate knowledge about rums. This approach provided a classification of samples with lower error rate than the one obtained by the use of a single technique (spectroscopic or chromatographic). The results showed that 1H NMR spectroscopy is an appropriate technique for the suitable classification of >95.5% of the samples. When data fusion methodology of spectroscopic and spectrometric data was performed, the prediction efficiency can reach 100% of the samples.

On-line purge and trap GC-MS for monitoring 1,3-dichloropropene in agricultural water and soil samples.

A simple and reliable method using on-line purge and trap gas chromatography mass spectrometry has been developed for the determination of the fumigant 1,3-dichloropropene (1,3-DCP) in agricultural water and soil samples. The proposed analytical methodology was validated in the target environmental matrices by the analysis of spiked blank matrix samples. Limit of detection values of 0.05 microg/L for water and 0.005 microg/Kg for soils were obtained, while limits of quantitation were of 0.1 microg/L for water and 0.01 mg/kg for soils. Good recoveries (93-104%) and precision values (< 6%) were obtained for the target compound in the studied matrices. This methodology has been successfully applied to the analysis of incurred groundwater samples from an agricultural area, The Campo de Dalías (Almería, South Spain), although 1,3-DCP was not detected. The method was also applied to soil samples from greenhouse treated with a soil fumigant containing 1,3-DCP.

Frozen dog spermatozoa are negatively affected during storage at -80, -21 and -8 ºC.

Cryopreservation in liquid nitrogen (LN2) allows for semen to be stored for long periods of time while there is sustaining of sperm viability. In this study, there was assessment of effects induced by different storage temperatures on cryopreserved dog spermatozoa. After cryopreservation at -196 °C, sperm samples were transferred to storage conditions of -80, 21 or -8 °C. Sperm motility, morphology, viability, acrosome integrity, mitochondrial membrane potential and DNA fragmentation were determined in samples stored at -196 °C (evaluation time =0 h), and then after 12 h and 1, 4, 7 and 15 d of storage at 80, -21 and -8 °C. In samples stored at -80 °C, sperm morphology, viability, acrosome integrity, mitochondrial membrane potential and DNA fragmentation did not differ at successive evaluation times. Progressive motility was less (P < 0.05) after 12 h and total motility after 4 d of storage at -80 ºC as compared with that of the 0 h sample. With storage at the other temperatures (-21 and -8 ºC), there was a reduction of mean values for sperm total and progressive motility, viability and mitochondrial membrane potential after 12 h of storage at these temperatures. Results, therefore, indicate the use of ultra-freezers at -80 ºC to store frozen dog semen allows for maintenance of sperm characteristics for at least 15 d but motility is sustained for only 1 d. Neither of the -21 or -8 ºC storage temperatures were effective for storing of frozen dog sperm and retaining viability.