In vitro qualitative characteristics of rabbit spermatozoa after 120 h of solid storage at 5 °C and 15 °C

The effects of different prefreezing semen concentrations on post-thawing quality of turkey semen cryopreserved with the pellet method were investigated. Ten pooled semen samples were each divided into 6 subsamples and diluted with Tselutin extender to obtain a final concentration of 0.5, 1, 2, 3, 4 and 5x109/mL respectively. Subsamples were cooled, added with 8% of dimethylaceta- mide as cryoprotectant and, after 5 minutes of equilibration, seminal aliquots of 80 μL were directly dropped into a liquid nitrogen bath to form frozen pellets. Thawing was performed in few seconds at 75°C. Sperm motility (Accudenz® swim-down test), viability (SyBr-Propidium Iodide staining) and sperm susceptibility to osmotic stress (Hyposmotic-water test) were assessed. Cryopreservation caused an overall loss of sperm quality, however differences in seminal parameters due to the different sperm concentration were observed in turkey spermatozoa after thawing: spermatozoa diluted to 4x109/mL showed significant higher values in mobility, viable and osmotic resistant spermatozoa compared to the other concentrations. This study showed that the post-thaw quality of turkey semen cryopreserved by pellets method was affected differently in relation to prefreezing sperm concentration

effects of afb1 on liver phase i and phase ii enzymes induced in vitro on sparus aurata hepatocytes primary culture

Among all know mycotoxins, aflatoxin B1 is one of the most studied for its hepatotoxic, carcinogenic, mutagenic, teratogenic, and immunosuppressant effects. This work aimed to study the effects of AFB1 acute and chronic exposure on CYP1A and GST enzymes induced in vitro on S. aurata hepatocytes by immunoblot analysis, thus relating the cytotoxic effects leading to cell death by apoptotic studies. Immunofluorescence analysis revealed that cell damage was not recoverable but permanent, as the cellular repair systems were unable to recover the induced toxic insult. Our results showed detection of several CYP1A bands, enlightening an indirect correlation between induction of CYP1A with dose and time of exposure. The decreased expression of CYP1A over prolonged exposure times, along with high toxic concentration, could be related with lethal damage observed on hepatocytes by contrast phase and immunofluorescence analysis. A particular pattern of expression was found for GST isoforms upon AFB1 exposure, identifying each isoform profile two different kind of toxic insult. The 65 KDa and the 49 KDa bands being suggestive for markers of acute and chronic response respectively. Interestingly, apoptosis induction, considered an early lesion to DNA, was found associated with the chronic damage along with the low toxic concentration. The new cell model from S. aurata has been proven to be a useful and valid tool to further investigate the modulated response of liver phase I and II enzymes to AFB1

Effect of different sperm concentrations on the post-thaw viability and motility of turkey spermatozoa cryopreserved by the pellet method

The effects of different prefreezing semen concentrations on post-thawing quality of turkey semen cryopreserved with the pellet method were investigated. Ten pooled semen samples were each divided into 6 subsamples and diluted with Tselutin extender to obtain a final concentration of 0.5, 1, 2, 3, 4 and 5x109/mL respectively. Subsamples were cooled, added with 8% of dimethylaceta- mide as cryoprotectant and, after 5 minutes of equilibration, seminal aliquots of 80 μL were directly dropped into a liquid nitrogen bath to form frozen pellets. Thawing was performed in few seconds at 75°C. Sperm motility (Accudenz® swim-down test), viability (SyBr-Propidium Iodide staining) and sperm susceptibility to osmotic stress (Hyposmotic-water test) were assessed. Cryopreservation caused an overall loss of sperm quality, however differences in seminal parameters due to the different sperm concentration were observed in turkey spermatozoa after thawing: spermatozoa diluted to 4x109/mL showed significant higher values in mobility, viable and osmotic resistant spermatozoa compared to the other concentrations. This study showed that the post-thaw quality of turkey semen cryopreserved by pellets method was affected differently in relation to prefreezing sperm concentration

Age-dependent changes in metabolic profile of turkey spermatozoa as assessed by NMR analysis

<div><p>Metabolic profile of fresh turkey spermatozoa at three different reproductive period ages, namely 32, 44 and 56 weeks, was monitored by Nuclear Magnetic Resonance (NMR) spectroscopy and correlated to sperm quality parameters. The age-related decrease in sperm quality as indicated by reduction of sperm concentration, sperm mobility and osmotic tolerance was associated to variation in the level of specific water-soluble and liposoluble metabolites. In particular, the highest levels of isoleucine, phenylalanine, leucine, tyrosine and valine were found at 32 weeks of age, whereas aspartate, lactate, creatine, carnitine, acetylcarnitine levels increased during the ageing. Lipid composition also changed during the ageing: diunsaturated fatty acids level increased from 32 to 56 weeks of age, whereas a reduction of polyunsaturated fatty acids content was observed at 56 weeks. The untargeted approach attempts to give a wider picture of metabolic changes occurring in ageing suggesting that the reduction of sperm quality could be due to a progressive deficiency in mitochondrial energy producing systems, as also prompted by the negative correlation found between sperm mobility and the increase in certain mitochondrial metabolites.</p></div

Metabolites identified by NMR in fresh spermatozoa from 32, 44 and 56 weeks old turkey male.

<p>Mean values ± SE.</p

Age-dependent changes in metabolic profile of turkey spermatozoa as assessed by NMR analysis - Fig 2

<p>^<b>1</b><b>H NMR spectra of aqueous (A) and organic extracts (B) of turkey spermatozoa at 32 weeks of age.</b> Assignments: 1, Ala; 2, Asp; 3, Gln; 4, Glu; 5, Gly; 6, Ile; 7, Leu; 8, Phe; 9, Tyr; 10, Val; 11, Acetate; 12, Citrate; 13, Formate; 14, Fumarate; 15, Lactate; 16, Ac-carnitine; 17, AMP; 18, Carnitine; 19, Creatine; 20, Glucose; 21, Myo-inositol; 22, CHO; 23, DUFA; 24, UFA; 25, PUFA; 26, PC; 27, PE; 28, SMN. For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194219#sec002" target="_blank">Methods</a> section.</p