M. P. Rosato

NMR metabolic profiling of organic and aqueous sea bass extracts: Implications in the discrimination of wild and cultured sea bass

The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.

Effect of chilling temperature on the long-term survival of rabbit spermatozoa held either in a tris-based or a jellified extender.

As the preservation of the fertilizing capacity of rabbit spermatozoa for several days after semen collection remains a major target for the artificial insemination programs of rabbit breeding, a study was conducted to compare the efficacy of 5 or 15°C as holding temperature in lengthening the preservability of rabbit semen quality during 192 h of storage both in a solid (Cunigel) and a liquid (Tris-Citric acid-Glucose; TCG) extender. Six pooled semen samples (two ejaculates/male; two-three males/pool) were taken and made four aliquots: two aliquots were tenfold diluted with the TCG extender, whereas the other two were tenfold diluted with the Cunigel extender. One aliquot per diluent was stored at 5°C and the second one at 15°C. Sperm motility (light microscope), viability (SyBr-PI staining), plasma membrane functional integrity (Hypo-osmotic swelling test) and acrosome integrity (PSA-FITC staining) were recorded at 0, 48, 120 and 192 h of storage. In liquid-stored spermatozoa, mass motility and viability were significantly higher (p ≤ 0.05) in samples stored at 5°C than at 15°C at all the storage times; at 5°C resulted also higher (p ≤ 0.05) the percentages of both forward motility at 48 h and sperm functional integrity at 120 and 192 h of storage, whereas chilling temperature did not affect acrosome integrity. With the Cunigel extender, all the semen qualitative parameters were significantly higher in sample stored at 5 than 15°C over storage time (p ≤ 0.05); only acrosome integrity at 192 h was not different according to the chilling temperatures. In conclusion, 5°C were better than 15°C for the long-term storage of rabbit semen both in the TCG and Cunigel extender.

The post-thaw irradiation of avian spermatozoa with He-Ne laser differently affects chicken, pheasant and turkey sperm quality

The effects of post-thaw Helium-Neon (He-Ne) laser irradiation on mobility and functional integrity of frozen/thawed chicken, pheasant and turkey spermatozoa were investigated. Cytochrome C oxidase (COX) activity was also determined as a measure of the effect of irradiation on mitochondrial bioenergetics. Semen samples from each species were collected, processed and frozen according to the pellet procedure. After thawing, each semen sample was divided into two subsamples: the first one was the control; the second one was irradiated with a single mode continuous He-Ne laser wave (wavelength 632.8 nm; 6 mW; 3.96 J/cm(2)). Then the samples were assessed for sperm mobility (Accudenz(®) swim-down test), viability (SYBR-14/PI staining), osmotic-resistance (HOS test) and COX activity. The irradiation was effective P<0.05 increasing sperm motility in the turkey semen (0.228 ± 0.01 compared with 0.294 ± 0.02). The irradiation also caused an increase (P<0.05) of the COX activity in pheasant (+135 ± 4%) and turkey (+116 ± 4%) sperm, without affecting viability and osmotic-resistance. The COX was positively correlated (P<0.05) with the viability of chicken sperm, however no significant interactions were found between mobility and COX activity in the three avian species. Due to the difference in energetic metabolism among avian species used in this study, the He-Ne laser irradiation has a differential action on bio-stimulation of turkey, chicken and pheasant spermatozoa. The present results are the first to elucidate the possibility for restoration of motility of cryopreserved avian spermatozoa by bio-stimulation provided via He-Ne laser irradiation.

Cryopreservation of rabbit semen using non-permeable cryoprotectants: Effectiveness of different concentrations of low-density lipoproteins (LDL) from egg yolk versus egg yolk or sucrose

This study was designed to identify the most effective non-permeable cryoprotectant (CPA) for the cryopreservation of rabbit semen by comparing the effects of different concentrations of low-density lipoproteins (LDL) on post-thaw sperm quality with those of whole egg yolk or sucrose. In a second experiment, the performance of the non-permeable CPAs identified as most effective was assessed in vivo by determining reproductive performances. Pooled semen samples were diluted to a ratio of 1:1 (v:v) in freezing extender (Tris-citrate-glucose and 16% dimethylsulfoxide as permeable CPA) containing as non-permeable CPAs 6, 8, 10 or 15% LDL from egg yolk, 0.1M sucrose, or 15% egg yolk. The semen was loaded in 0.25mL straws and frozen in liquid nitrogen vapor. After thawing, we determined sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Our results clearly revealed a significant effect of LDL concentration on semen quality. Also, at an optimal concentration of 10%, motility and acrosome integrity were improved over the values recorded for egg yolk (P<0.05). Based on the in vitro data, 3 groups of does (n=30 each) were inseminated with fresh semen or semen frozen using sucrose or 10% LDL. Sucrose led to a significantly higher conception rate than LDL and reproductive performance was similar to that observed for fresh semen. Our findings indicate the markedly better performance of sucrose in vivo as a non-permeable CPA for the cryopreservation of rabbit semen.

Effects of lycopene on in vitro quality and lipid peroxidation in refrigerated and cryopreserved turkey spermatozoa

1. The effects of lycopene-enriched extenders on the in vitro quality of turkey semen including lipid peroxidation were examined after chilled and frozen storage. 2. Five pools of semen diluted in extenders containing 0, 0·05 or 0·1 mg/ml of lycopene were stored at 5°C for 48 h or cryopreserved as pellets and the following variables determined in fresh samples and samples stored chilled or frozen: sperm motility, viability, osmotic resistance, DNA integrity and lipid peroxidation (as malonaldehyde production). 3. Semen quality was generally compromised after storage, especially post-freezing. However, in the presence of the highest dose of lycopene, both the viability and osmotic-resistance of chilled spermatozoa and the DNA integrity of frozen spermatozoa were similar to those of fresh spermatozoa. 4. Greater lipid peroxidation was detected in refrigerated compared to fresh or cryopreserved spermatozoa. However, spermatozoa chilled in lycopene-enriched extenders showed significantly lower malonaldehyde levels than those chilled without lycopene, while the addition of lycopene to the freezing medium served to maintain the lipid peroxidation levels observed in fresh semen. 5. In conclusion, the presence of lycopene in the extender improved the survival of turkey spermatozoa after liquid-storage and protected DNA integrity against cryodamage. The beneficial effects of lycopene observed could be related to its capacity to diminish sperm lipid peroxidation during refrigeration or cryopreservation.

The preservability of turkey semen quality during liquid storage in relation to strain and age of males

It is difficult to maintain turkey semen quality after in vitro liquid storage and the problem is worsened by animal aging. Little is currently known about the effects of both reproductive period and strain on the preservability of qualitative characteristics of turkey semen during liquid storage. The purpose of this study was to evaluate the effect of the reproductive period of two commercial turkey strains on semen quality changes during in vitro storage for upto 48 h at 5 degrees C. Two different periods were considered: first period from 32 to 40 weeks of age and the second one from 44 to 52 weeks. Turkey males from either British United Turkeys (BUT) Big-6 line and Hybrid Large White line (Hybrid) were used. Semen pools of each tom strain were diluted with Beltsville Poultry Semen Extender (BPSE) and the motility, viability and membrane integrity of sperm were evaluated at 3, 24 and 48 h of liquid storage at 5 degrees C. The sperm concentration was significantly affected by period (P<0.01) and strain (P<0.05), with best values in first period and in the Hybrid semen. Besides also the motility, viability and membrane integrity during 48 h of storage were better (P<0.05) in the first period compared to the second one for both strains, particularly in Hybrid semen. During storage it was clearly shown in the first period that Hybrid sperm worsened more than the BUT one: in spite of the motility and viability values were at first (3h) higher (P<0.05) in Hybrid semen, after 48 h of storage the motility did not show any significant difference between strains while the viability resulted even better (P<0.05) in BUT semen. In the second period, although the semen quality decreased during the storage with a similar trend for both strains, better (P<0.05) values were found in BUT semen. Our results indicated that the reproductive period affected the quality of turkey semen in a different manner according to the strain. Moreover BUT semen showed a better in vitro storage ability compared to the Hybrid one.

Risk of Salmonella transmission via cryopreserved semen in turkey flocks

To investigate the possibility to carry pathogen bacteria in turkey flocks via cryopreserved semen, research was carried out 1) to investigate the microbial contamination of fresh and frozen thawed turkey semen and 2) to evaluate the effect of the freezing-thawing process on the survival of 3 serovars of Salmonella spp. experimentally inoculated in turkey semen. Five pools of semen diluted 4-fold were cooled, added with 8% of dimethylacetamide as a cryoprotectant, and aliquots of 80 muL were directly plunged into liquid nitrogen to form frozen pellets. Mesophilic viable counts, total and fecal coliforms, Enterobacteriaceae, enterococci, Campylobacter spp., and Salmonella spp. were investigated on fresh and thawed samples. Further, 5 pools of diluted semen were each divided into 3 subsamples, inoculated with 7.8 +/- 0.2 log cfu.mL(-1) of Salmonella Liverpool, Salmonella Montevideo, and Salmonella Braenderup, respectively, and cryopreserved before to assess the postthaw viability of Salmonella spp. strains. Fresh semen was highly contaminated by all of the saprophytic bacteria investigated and the cryopreservation process reduced the amount of mesophilic viable count and total coliforms (P < 0.05) and fecal coliforms, Enterobacteriaceae, and enterococci (P < 0.01) by about 1 log cfu.mL(-1). Conversely, neither Campylobacter spp. nor Salmonella spp. were found as endogenous bacteria in semen. In the inoculated semen, both Salmonella Liverpool, Salmonella Montevideo, and Salmonella Braenderup colonies were recovered postthaw, showing a significant reduction of 2.03 +/- 0.28, 3.08 +/- 0.22, and 2.72 +/- 0.23 log cfu.mL(-1), respectively, compared with the fresh semen (P < 0.001). In conclusion, the cryopreservation process allowed us to obtain a low reduction of microbial count both in endogenous saprophytic bacteria and artificially inoculated Salmonella spp. strains; therefore, the possibility of Samonella spp. transmission to flocks through the use of infected cryopreserved semen does exist.

Comparison of different extenders on the quality characteristics of turkey semen during storage

Riassunto Confronto di differenti diluenti sulle caratteristiche qualitative del seme conservato di tacchino. È stato valutato l’effetto di tre differenti diluenti: BPSE, Lake e IGGKPh sulla qualità del seme di tacchino durante la conservazione per 48 h a 5°C. Ogni pool di seme è stato diviso in 3 aliquote le quali sono state diluite rispettivamente con i tre medium e determinate in vitro la motilità (procedura Accudenz®), la vitalità (SyBr-PI) e l’integrità di membrana previo stress ipoosmotico. La qualità del seme di tacchino peggiora (P<0,01) durante la conservazione per 48 h, tuttavia i valori di vitalità, mobilità ed integrità di membrana risultano più alti (P<0,01) dopo 24 h e 48 h con BPSE rispetto ai diluenti IGGKPh e Lake.

The He-Ne laser irradiation improves the quality of stored rabbit semen

Abstract The aim of the study was to investigate the effect of different energy doses of Helium-Neon (He-Ne) laser irradiation on rabbit semen quality during 48 h of storage. From 30 commercial hybrid bucks semen was collected by artificial vagina. The ejaculates of bucks were pooled (3-4 ejaculates/pool) and in total 8 semen pools were used. Each pool was divided into four aliquots. One sample was not irradiated (control), the other ones were irradiated with a single mode continuous He-Ne laser wave (wavelenght 632,8) and exposed to the following energy doses: 3.96, 6.12 and 9 J/cm2 respectively. Samples were kept at 15° C during laser irradiation. They were subsequently stored again at the same temperature for a further 48 h. Samples were evaluated for sperm motility (Accudenz procedure), sperm viability (SyBr-PI procedure) and acrosome integrity (PSA-FITC procedure) at 3,24 and 48h of storage. Laser irradiation at all energy doses utilized significantly improved motility (P>0.01), viability (P>0.01) and acrosome integrity (P>0.05) than in control either at 24 and 48 h of storage. Moreover, the energy dose of 6.12J/cm2 recorded a better semen quality during storage compared to the 3,96 and 9J/cm2 ones respectively. No significant differences for measured seminal quality parameters were found at 3h; this might be due to the fact that irradiation treatment requires a longer period of time to determine modification on semen quality. The present study shows that laser irradiation, as shown in a previous research for turkey semen, seems to improve the quality of rabbit semen stored for up 48h even if higher energy doses were utilized. Therefore, the irradiation laser might be a useful technique to enhance quality of stored rabbit semen, although further studies are need.