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Considering the beneficial effects of n-3 polyunsaturated fatty acids (PUFA) for human health (Leskanich and Noble, 1997) several studies have been carried out to enrich animal products with these compounds. Both eggs and poultry meat enriched with n-3 PUFA may be considered as valid n-3 PUFA alternative sources to natural occurring fish products (Meluzzi et al., 2001; Sirri et al., 2002)...

Maintenance or improvement of sperm quality during storage could prevent the loss of fertilizing capacity associated with stored turkey semen. Therefore the optimization of stored turkey semen could be useful to breeder industry since the commercial production of this bird relies almost entirely on artificial insemination. Previous research have shown that He-Ne laser irradiation in mammalian sperm increased the motility (Stato, 1986), decreased the mortality, promoted the acrosome reaction, which have a pivotal role in assisted fecundating programmes as therapy for resolving infertility in domestic animals..........

Semen of the domestic turkey cannot be stored longer than 6 h without a loss of fertilizing capability. The improvement of long-term liquid storage procedures of semen is important since the commercial production of turkey relies almost entirely on artificial insemination. Therefore, studies improving storage regimens would allow longer storage and consequently hen fertility (Iaffaldano and Meluzzi, 2003). Since the search for an optimal extender composition for semen storage is still in progress, the aim of this paper has been to study the effects of different extenders on the quality of turkey semen during the storage for 48 h at 5°C.

Fish production requires great efforts and energies and causes different impacts/effects on the ecosystems. The development of aquaculture is possible, in the medium and long term, only if this is done responsibly, and if the production is reached from both an economic and environmental point of view. In our study we have worked on fry production using 1.5 g sized Dicentrarchus labrax obtained rearing larvae for 120 days after the hatching of the eggs.

IMTA is one of the most innovative and sustainable farming systems, exhibiting the best technique available in rearing aquatic organisms belonging to different positions along the trophic levels. In the literature and in legislation, the environmental benefits of IMTA protocols have been extensively recognized, mainly for its capability to reduce the ecological footprint of intensive aquaculture systems and concretely address the Sustainable Development Goal no. 14 (SDG 14). However, lesser attention is given to the assessments of its role in enhancing the zootechnical performance, animal welfare, and flesh quality of the species involved. To the best of our knowledge, this is the first review that aims to offer a systematic analysis of the existing literature on the main commercial motivations that could draw the attention of stakeholders, including consumers and fish farmers, towards a greater social acceptability and implementation of the IMTA system on a large scale. The findings suggest that, beyond its environmental advantages, IMTA systems can positively influence the productivity, growth, survival, feed efficiency, and animal health and welfare (AH&W), as well as the nutritional quality of the harvested species, thus offering significant economic and market value both in terms of Environmental, Societal and Governance (ESG) parameters and One Health.

Significant changes in the quality and metabolic profile of fresh turkey sperm as a result of both cryopreservation and reproductive age have already been individually confirmed in our previous studies. This new dataset adds a relevant piece to the tangled puzzle of changes in metabolite levels affecting cryopreserved turkey sperm quality, taking into consideration two different reproductive period ages. Fresh semen samples were collected at 44 and 56 weeks of age and exposed to the cryopreservation process. All fresh and frozen-thawed samples were subjected to analysis of mobility, viability and osmotic tolerance as parameters for evaluating the sperm quality, while NMR spectroscopy was used to assess the quantitative changes in water and lipid-soluble metabolites. Our results showed that the cryopreservation process significantly affected all of the measured qualitative parameters both at 44 and 56 weeks. Concerning the metabolic profile, a greater number of quantitative changes for both water and lipid-soluble components were found in frozen semen at 56 weeks than at 44 weeks of age. These data could contribute to identifying new strategies aimed at improving freezing procedures even as reproductive age increases., (© 2024 The Author(s).)

Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese , Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5-16%) and between birds within a breed (BP = 3-7%; BS = 7-31%; PP = 6-22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen's fertilizing ability are required.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca 2+ -binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study aimed to improve postharvest management of flat oysters reared in a longline system in the mid Adriatic Sea, using short-term storage and package in an innovative closed-circuit system. For the trial, 870 oysters were employed, divided into three experimental groups (A, B, and C), N = 270 oysters each group, whereas the remaining 60 oysters were used for the 2 controls. Each group differed in relation to the time spent in the depuration tank and the time of packaging: group A was packed and immediately transferred to the cell; group B was depurated in a tank for 48 h, then packed and transferred to the cell; group C was depurated in a tank for 48 h and then packed, depurated for another 24 h and transferred to a cell. Samples of each group were sampled at different times of permanence in cell (t0) up until 12 days (t12) for biomorphometric, sensorial, nutritional, and microbiological analysis. Although the nutritional and sensorial quality of the oysters was more pronounced in group A, B and C groups also showed good results. In these two groups, thanks to the use of the modern water recirculation system the quality and safety of oysters was improved by reducing the presence of sludge and eliminating fecal contaminants completely than A treatment and seawater control. These results were also confirmed by the tank control, where a more extended depuration period positively influenced the same parameters emphasizing the importance of the adequate depuration processes in oyster production., (© 2023 The Authors. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools ( n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 10 6 per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 10 6 exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher ( p < 0.05) in the mid-range sperm concentrations of 25 × 10 6 (49.9% and 19.7%) and 35 × 10 6 (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration.

D-532 fertilization solution is generally used to replace the water or ovarian fluid during artificial reproductive practices in salmonids due to its ability to boost sperm motility and increase fertilization rates compared with natural activation media. However, the maintenance of ovarian fluid in a reproductive microenvironment gives it the advantage of protecting the eggs from potential harmful factors from the external environment and simplifying the field operations related to its removal when D-532 is used alone. In light of this, the aim of the present study was to investigate in vitro, for the first time, the effect of ovarian fluid (OF 100%) on post-thaw sperm swimming performance of Mediterranean trout, comparing it with D-532 and a mixed solution of 50% D-532 and 50% ovarian fluid (OF 50%). The percentage of motile spermatozoa and movement duration was significantly increased in OF 100% and OF 50% compared with D-532. Sperm velocity was higher in D-532, but significant differences were recorded only with OF 100%. In conclusion, these results suggest that the presence of ovarian fluid alone or in combination with D-532 in an artificial microenvironment of reproduction represents a key factor in potentially increasing fertilization success when the frozen semen of Mediterranean brown trout is used.

Intensive fish farming is responsible for the occurrence of various stressors that negatively affect the health of cultured animals. Polyphenols are plant-derived compounds with biological activities and positive influences on the performances and immunity of fish. The aim of the present study was to evaluate the effects of dietary inclusion of a polyphenol extract obtained from Nero di Troia red grape on the quality of farmed sea bass (Dicentrarchus labrax) fillets. Three diets were tested: control (n = 90) received a conventional feed, whereas the two experimental groups (n = 90 each) received the control feed supplemented with the red grape polyphenol extract (GPE) at the concentration of 100 (GPE 100) or 200 mg/kg (GPE 200). The two GPE diets lowered (p < 0.05) the red (a *) and yellow (b *) indexes, fillet hardness, and total lipid content. Chewiness, concentration of saturated fatty acids, and the Atherogenic Index were higher (p < 0.05) in the control group; GPE diets increased (p < 0.05) the polyunsaturated fatty acids content. Furthermore, the concentration of malondialdehyde was lower (p < 0.05) in fillets of the GPE groups, thus confirming the antioxidant effect of the red grape extract and its effectiveness in preventing lipid oxidation.

The recovery, safeguarding, and valorization of autochthonous poultry breeds may contribute toward the preservation of animal biodiversity and utilization of marginal lands that otherwise offer little agricultural or industrial value. A key strategy in promoting local breeds involves the characterization of morphological traits and productive performances, which are influenced by the breed's genetic make-up as well as its environment. The Mugellese breed is an Italian local poultry breed originating in the Mugello area of north-east Tuscany. It is characterized by frugality, resilience and resistance to disease, cold, and heat stress. Moreover, these birds are particularly suitable for free-range farming. The Mugellese chicken is described as a dwarf breed with a medium neck, broad shoulders, fairly long and horizontal wings, wide, and well-developed breast (especially in the hen). Over the course of a 1-yr observation and data collection period, involving 23 breeders and 405 adult chickens, the Mugellese breed showed the following performances: 1) a hen-day egg production characterized by 2 major peaks: the first in the spring time (March-April, 65.75%), and the second in the late summer period (August-September, 51.86%); 2) high true fertility values (94.35%) throughout the entire breeding season; 3) a weight gain of 732.44 ± 117.06 g and a feed conversion ratio of 3.94 ± 2.42 at an age of 140 d; 4) a slaughter yield of 77.80% (± 3.91); v) a respective protein, fat, and mineral content in the yolk and albumen were: 27.21 ± 4.21 g, 57.77 ± 1.03 g and 3.47 ± 0.40 g per 100 g of yolk; and 82.50 ± 0.57 g, 0.12 ± 0.01 g and 5.43 ± 0.34 g per 100 g of albumen. More data are needed to validate the data obtained in this trial., Competing Interests: DISCLOSURES The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been previously investigated. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit semen (spermatozoa and extracellular fluid), to examine the effects of freeze-thawing on proteins changes in semen. Comparative analysis and identification of proteins was carried out using 2-dimensional difference in-gel electrophoresis coupled with a matrix-assisted laser desorption/ionization mass spectrometry. Proteomic raw data are available via ProteomeXchange with identifier PXD034832 for spermatozoa and PXD034853 for extracellular fluid. Respectively, 107 and 28 proteins differed in abundance in spermatozoa and extracellular fluid between fresh and frozen groups. Most of these proteins were involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability. These results broaden the understanding of the effects of cryopreservation on rabbit semen and represent a new starting point for the development of improved freezing procedures., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Brown trout ( Salmo trutta ), like many other freshwater species, is threated by the release in its natural environment of alien species and the restocking with allochthonous conspecific stocks. Many conservation projects are ongoing and several morphological and genetic tools have been proposed to support activities aimed to restore genetic integrity status of native populations. Nevertheless, due to the complexity of degree of introgression reached up after many generations of crossing, the use of dichotomous key and molecular markers, such as mtDNA, LDH-C1 * and microsatellites, are often not sufficient to discriminate native and admixed specimens at individual level. Here we propose a reduced panel of ancestry-informative SNP markers (AIMs) to support on field activities for Mediterranean trout management and conservation purpose. Starting from the genotypes data obtained on specimens sampled in the main two Molise's rivers (Central-Southern Italy), a 47 AIMs panel was identified and validated on simulated and real hybrid population datasets, mainly through a Machine Learning approach based on Random Forest classifier. The AIMs panel proposed may represent an interesting and cost-effective tool for monitoring the level of introgression between native and allochthonous trout population for conservation purpose and this methodology could be also applied in other species.

The aim of this study was to compare the effect of two permeant-cryoprotectants, dimethylacetamide (DMA) and N-methylacetamide (NMA) used at different concentrations (0%, 2%, 4%, 6%) on the quality and fertility of post-thaw rooster semen. Ejaculates were processed in 7 treatments: Lake pre-freezing+0.1 M trehalose (LPF-T) (control treatment), LPF-T+2% DMA, LPF-T+4% DMA, LPF-T+6% DMA, LPF-T+2% NMA, LPF-T+4% NMA, LPF-T+6% NMA. Sperm quality [sperm membrane integrity (SMI), motility and kinetic parameters] was assessed before and after cryopreservation. Fertility and embryo viability were recorded. Increasing both DMA and NMA concentration from 2 to 6% improved SMI, total motile sperm, progressive motile sperm (PMS), VCL, VSL and VAP values. PMS recovery rates were significantly the highest in 6% DMA, 4% NMA and 6% NMA treatments. Semen cryopreserved with DMA produced the best fertility and embryo viability at 6%; progressive lower values were recorded at lower concentrations, with no viable embryos at 2%. Semen cryopreserved with NMA showed the best fertility values at 2% and lower values were recorded at higher concentrations; live embryos were found in all NMA treatments. Finally, NMA and DMA showed a similar positive concentration dependent effect of the quality of cryopreserved semen. NMA, not DMA, provided the highest fertility and embryo viability values at the lowest 2%. Therefore, the use of NMA is recommended in order to reduce the cryoprotectant concentration, with a concomitant reduction in the risk of toxicity, providing at the same time the adequate cryoprotective action to obtain viable embryos after artificial insemination of cryopreserved chicken semen., (Copyright © 2022. Published by Elsevier Inc.)

Duckweed (Lemna minor) meal was included in the formulation of three experimental feeds (L1, L2, L3) for rainbow trout at 10%, 20%, 28% of the protein source, respectively. Increasing the duckweed inclusion, the other protein sources were adjusted to get isonitrogenous (41%) and isolipidic (20%) diets, as the control diet (LC). 540 fish (mean body weight 124.5 ± 0.7 g) were randomly allocated in 12 tanks divided equally among the four different diets. After 90 days, fish were weighed and the most important productive performances, fillet quality and fatty acid profile were determined. The final body weight in L1 (340.53 g) and L2 (339.42 g) was not different from LC (348.80 g); L3 trout significantly (p < 0.05) exhibited the lowest one (302.16 g). Similar trends were found in final mean length, weight gain, specific growth rate, food conversion rate. Somatic indices were affected by duckweed inclusion. Diets had not significant effects on the proximate composition and fatty acids of the fillet in L1, L2, L3 respect to LC. Based on this study, duckweed meal derived from Lemna minor can be included in the feed for the rainbow trout without negative effects on the growth performances at 20% of the protein substitution.

Semen cryopreservation represents the main tool for preservation of biodiversity; however, in avian species, the freezing−thawing process results in a sharp reduction in sperm quality and consequently fertility. Thus, to gain a first insight into the molecular basis of the cryopreservation of turkey sperm, the NMR-assessed metabolite profiles of fresh and frozen−thawed samples were herein investigated and compared with sperm qualitative parameters. Cryopreservation decreased the sperm viability, mobility, and osmotic tolerance of frozen−thawed samples. This decrease in sperm quality was associated with the variation in the levels of some metabolites in both aqueous and lipid sperm extracts, as investigated by NMR analysis. Higher amounts of the amino acids Ala, Ile, Leu, Phe, Tyr, and Val were found in fresh than in frozen−thawed sperm; on the contrary, Gly content increased after cryopreservation. A positive correlation (p < 0.01) between the amino acid levels and all qualitative parameters was found, except in the case of Gly, the levels of which were negatively correlated (p < 0.01) with sperm quality. Other water-soluble compounds, namely formate, lactate, AMP, creatine, and carnitine, turned out to be present at higher concentrations in fresh sperm, whereas cryopreserved samples showed increased levels of citrate and acetyl-carnitine. Frozen−thawed sperm also showed decreases in cholesterol and polyunsaturated fatty acids, whereas saturated fatty acids were found to be higher in cryopreserved than in fresh sperm. Interestingly, lactate, carnitine (p < 0.01), AMP, creatine, cholesterol, and phosphatidylcholine (p < 0.05) levels were positively correlated with all sperm quality parameters, whereas citrate (p < 0.01), fumarate, acetyl-carnitine, and saturated fatty acids (p < 0.05) showed negative correlations. A detailed discussion aimed at explaining these correlations in the sperm cell context is provided, returning a clearer scenario of metabolic changes occurring in turkey sperm cryopreservation.

The preservation of genetic variability of autochthonous poultry breeds is crucial in global biodiversity. A recent report revealed small breed size and potential risk of extinction of all native Italian poultry breeds; therefore, a correct assessment of their genetic diversity is necessary for a suitable management of their preservation. In this work, we provided an overview of the contribution to poultry biodiversity of some Italian autochthonous breeds reared in conservation centers devoted to local biodiversity preservation. The level of genetic diversity, molecular kinship, inbreeding, contribution to overall genetic diversity, and rate of extinction of each breed were analyzed with a set of 14 microsatellite loci in 17 autochthonous chicken breeds. To evaluate genetic variability, total number (Na), and effective number (Ne) of alleles, observed (Ho) and expected (He) heterozygosity, and F (Wright's inbreeding coefficient) index were surveyed. The contribution of each analyzed breed to genetic diversity of the whole dataset was assessed using MolKin3.0; global genetic diversity and allelic richness contributions were evaluated. All the investigated loci were polymorphic; 209 alleles were identified (94 of which private alleles). The average number of alleles per locus was 3.62, and the effective number of alleles was 2.27. The Ne resulted lower in all breeds due to the presence of low-frequency alleles that can be easily lost by genetic drift, thus reducing the genetic variability of the breeds, and increasing their risk of extinction. The global molecular kinship was 27%, the average breed molecular kinship was 53%, and the mean inbreeding rate 43%, with a self-coancestry of 78%. Wright's statistical analysis showed a 41% excess of homozygous due to breed genetic differences (34%) and to inbreeding within the breed (9%). Genetic variability analysis showed that 11 breeds were in endangered status. The contribution to Italian poultry genetic diversity, estimated as global genetic diversity, and ranged from 30.2 to 98.5%. In conclusion, the investigated breeds maintain a unique genetic pattern and play an important role in global Italian poultry biodiversity, providing a remarkable contribution to genetic variability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soglia, Sartore, Lasagna, Castellini, Cendron, Perini, Cassandro, Marzoni, Iaffaldano, Buccioni, Dabbou, Castillo, Maione, Bianchi, Profiti, Sacchi, Cerolini and Schiavone.)

The sperm of each avian species and breed have unique characteristics that render them more or less susceptible to the freezing-thawing process; therefore, a suitable cryopreservation protocol that is specific for the sperm of each type of bird is needed. In this context, little information about the common pheasant's sperm is available. Therefore, the aim of this study was to test different parameters at each step of the process of freezing into pellets and thawing to detect the least deleterious parameter settings. Sixteen different protocols were tested by studying two levels in each of the four steps (dilution, equilibration at 5 °C, final dimethylacetamide concentration, and dimethylacetamide equilibration time) comprising the freezing process. The pheasant sperm exhibited a high susceptibility to the damage caused by freezing into pellets; however, the survival of the sperm reached 29%, and the greatest recovered mobility was 22%. The mobility of the sperm was affected by the dilution and the dimethylacetamide concentration, and the viability of the sperm was affected by the equilibration at 5 °C and the dimethylacetamide equilibration. The protocols that caused the least damage to the pheasant sperm were found to be those with higher dilution rates, 10 min of equilibration at 5 °C, and 6% dimethylacetamide equilibrated for 1 or 5 min. In the present study, we individualise some applicable parameters for certain critical steps of the freezing-thawing process; however, further investigations are needed in order to improve upon and complete a suitable protocol for the cryopreservation and thawing of pheasant sperm.

Knowledge of the composition of the gut microbiota in freshwater fish living in their natural habitat has taxonomic and ecological importance. Few reports have been produced on the composition of the gut microbiota and on the presence of LAB in the intestines of freshwater fish that inhabit river environments. In this study, we investigated the LAB community that was present in the gastrointestinal tract (GIT) of Mediterranean trout ( Salmo macrostigma ) that colonized the Biferno and Volturno rivers of the Molise region (Italy). The partial 16S rRNA gene sequences of these strains were determined for the species-level taxonomic placement. The phylogenetic analysis revealed that the isolated LABs belonged to seven genera ( Carnobacterium, Enterococcus, Lactobacillus, Lactiplantibacillus, Vagococcus, Lactococcus, and Weissella ). The study of the enzymatic activities showed that these LABs could contribute to the breakdown of polysaccharides, proteins, and lipids. In future studies, a greater understanding of how the LABs act against pathogens and trigger the fish immune response may provide practical means to engineer the indigenous fish microbiome and enhance disease control and fish health.

Mediterranean trout is a freshwater fish of particular interest with economic significance for fishery management, aquaculture and conservation biology. Unfortunately, native trout populations' abundance is significantly threatened by anthropogenic disturbance. The introduction of commercial hatchery strains for recreation activities has compromised the genetic integrity status of native populations. This work assessed the fine-scale genetic structure of Mediterranean trout in the two main rivers of Molise region (Italy) to support conservation actions. In total, 288 specimens were caught in 28 different sites (14 per basins) and genotyped using the Affymetrix 57 K rainbow-trout-derived SNP array. Population differentiation was analyzed using pairwise weighted F ST and overall F-statistic estimated by locus-by-locus analysis of molecular variance. Furthermore, an SNP data set was processed through principal coordinates analysis, discriminant analysis of principal components and admixture Bayesian clustering analysis. Firstly, our results demonstrated that rainbow trout SNP array can be successfully used for Mediterranean trout genotyping. In fact, despite an overwhelming number of loci that resulted as monomorphic in our populations, it must be emphasized that the resulted number of polymorphic loci (i.e., ~900 SNPs) has been sufficient to reveal a fine-scale genetic structure in the investigated populations, which is useful in supporting conservation and management actions. In particular, our findings allowed us to select candidate sites for the collection of adults, needed for the production of genetically pure juvenile trout, and sites to carry out the eradication of alien trout and successive re-introduction of native trout.

The intensive use of high-performing strains in poultry production has led to the extinction of several autochthonous chicken breeds and, consequently, loss of genetic variability. Interest in saving biodiversity is growing rapidly and has become a major objective worldwide. The aim of this study was to shed light on the production trends of native Italian poultry breeds and the related market. A questionnaire, which asked about the production cycles, the number of animals and table eggs produced per year and their retail prices was completed by 121 breeders across Italy. The surveyed breeders were divided into two categories: breeders conducting an agrozootechnical farm, referred to as 'farmers' (F); and breeders keeping chickens as backyard poultry, referred to as 'fancy breeders' (FB). Analysis of the data acquired indicated that animals were mainly slaughtered between 6 and 12 months of age, with F processing more animals per year. The same production trend was observed for table eggs. The recorded retail prices of native chicken products were higher than those for conventional products, but similar to those reported for valuable niche poultry products, such as the Poulet de Bresse in France and organic eggs. Knowledge about these highly valuable markets should be used to encourage the use of local breeds in alternative poultry farming and help protect biodiversity.

The most reared species of farm animal around the world is the chicken. However, the intensification of livestock systems has led to a gradual increase in the concentration of a limited number of breeds, resulting in substantial erosion to the genetic pool. The initial step of an 'animal conservation program' entails establishing the actual conservation statuses of the breeds concerned in a defined area; in this case, in Italy. To this end, a survey of breeds was performed by means of a census questionnaire divided into two parts. The first part collected information on breeds, breeders, housing facilities, and management aspects, the results of which are presented here. The second part of the questionnaire regarded chicken products and their markets, and these data will be reported in a second paper. The breed status of six chicken breeds was shown to be exceptionally worrying, with total numbers ranging from just 18 to 186 birds. Population sizes exceeding 1000 birds was identified for just four breeds, the maximum being 3400. Some improvements in status were noted in relation to breeds which had been the subject of conservation efforts in the past. The two most common breeds reported are the Bionda Piemontese, a double-purpose breed, and the Livorno egg-laying hen. Collo Nudo Italiano, Millefiori Piemontese, Pollo Trentino, and Tirolese chicken breeds and the Castano Precoce turkey breed were not listed by breeders at all. The most reported turkey breeds are the Bronzato Comune and the Ermellinato di Rovigo. The population sizes of native Italian poultry breeds were shown to be generally poor. Italian poultry farmers and the population at large are largely ignorant about indigenous poultry breeds. Thus, promoting the virtues of Italian breeds would help their conservation by encouraging breeders to rear these birds and consumers to buy their products. The identification of strategies to facilitate access to pure breed birds is essential, and will require the collaboration of university research centers, public entities, and breeders. The results presented in this paper constitute the initial part of a more complex conservation program.

The genomic variability of local Italian chicken breeds, which were monitored under a conservation plan, was studied using single nucleotide polymorphisms (SNPs) to understand their genetic diversity and population structure. A total of 582 samples from 23 local breeds and four commercial stocks were genotyped using the Affymetrix 600 K Chicken SNP Array. In general, the levels of genetic diversity, investigated through different approaches, were lowest in the local chicken breeds compared to those in the commercial stocks. The level of genomic inbreeding, based on runs of homozygosity (F ROH ), was markedly different among the breeds and ranged from 0.121 (Valdarnese) to 0.607 (Siciliana). In all breeds, short runs of homozygosity (ROH) (<4 Mb in length) were more frequent than long segments. The patterns of genetic differentiation, model-based clustering, and neighbor networks showed that most breeds formed non-overlapping clusters and were clearly separate populations, which indicated the presence of gene flow, especially among breeds that originated from the same geographical area. Four genomic regions were identified as hotspots of autozygosity (islands) among the breeds, where the candidate genes are involved in morphological traits, such as body weight and feed conversion ratio. We conclude that the investigated breeds have conserved authentic genetic patterns, and these results can improve conservation strategies; moreover, the conservation of local breeds may play an important role in the local economy as a source of high-quality products for consumers.

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 10 6 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 10 9 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis-CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 10 6 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

In mid Adriatic Sea the common sea snail is the habitual snail eaten, but over the years fishermen started to capture also the long sea snail, a possible alternative for human consumption. This study aims to compare the quality traits of the edible fraction in the common and long sea snails. In this study, common and long sea snail samples were provided by fishermen in November 2018 and March 2019. Total weight (meat and shell), fractions of meat and shell (after having extracted the edible part), fatty acid, elements in meat, and calcium content in shells were determined. Meat quality traits showed high nutritional value without significant differences between the two species. The fatty acid profile showed n3/n6 ratio significantly different both considering the season of sampling (November: 4.1; March: 2.38) and the species of sea snail (common: 4.98; long: 2.86). The long species showed a higher yield in the total body and calcium content concentrations. However, the long sea snail showed 50% lower meat yield compared with the common sea snail. In conclusion, the long sea snail can be used as an alternative to the common sea snail for human consumption.

In seeking alternative cryoprotectants to glycerol for a reference chicken semen freezing procedure, the aim of the present study was to compare the effect of two concentrations of N-Methylacetamide (MA) and two thawing rates on the quality of frozen-thawed semen. Semen samples were diluted in Lake pre-freezing extender, including 0.1 M trehalose in presence of 6% or 9% MA, loaded into straws, frozen in nitrogen vapors, and stored in liquid nitrogen. The following thawing treatments were used: 5 °C for 100 s and 38 °C for 30 s. Sperm quality (cell membrane integrity, motility and kinetic parameters) was assessed before and after cryopreservation. The decrease of MA concentration from 9 to 6% improved sperm quality after freezing/thawing and this effect was dependent on thawing temperature. Decreasing the MA concentration from 9 to 6% improved the proportion of undamaged membrane, motile, and progressive motile sperm recovered after thawing at 5 °C for 100 s; in contrast, no effect of the MA concentration was observed thawing at 38 °C for 30 s. Therefore, the treatment with 6% MA and thawing at 5 °C for 100 s has given the best cryoprotective action. These results contribute to improve the efficacy of the current chicken semen cryopreservation procedures., Competing Interests: The authors declare no conflict of interest.

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 ( p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank., Competing Interests: The authors declare no conflict of interest.

The aim of our study was to test the effectiveness of a simple semen cryopreservation procedure, developed for cultivated salmonid, on the wild salmonid of the Mediterranean area and to evaluate the effect of different thawing rates and sperm-to-egg ratios. The semen of five individual males was diluted into a final extender concentration of 0.15 M glucose and 7.5% methanol and loaded into 0.25 mL plastic straws, and a final sperm concentration of 3.0 × 10 9 sperm/mL was obtained. After equilibration, the straws were frozen by exposure to liquid nitrogen vapor at 3 cm above the liquid nitrogen level for 5 min. The semen was thawed at 40 °C/5 s or 10 °C/30 s. The sperm cryosurvival was evaluated by examining in vitro the sperm motility parameters using the CASA system, followed by fertilization trials in vivo, using three different sperm-to-egg ratios 6 × 10 5 , 4.5 × 10 5 and 3 × 10 5 :1. The applied cryopreservation procedure resulted in remarkably high (85.6%) post-thaw sperm total motility, when the semen was thawed at 40 °C/5 s, whilst the highest fertilization rate (53.1%) was recorded for a sperm-to-egg ratio of 4.5 × 10 5 :1. According to these outcomes, the cryopreservation procedure that was tested turned out to be effective for the wild population of Mediterranean brown trout and practical for the creation of the first European semen cryobank foreseen as part of our "LIFE" Nat.Sal.Mo. project.

The aim was to compare the effect of dimethylacetamide (DMA) and N-methylacetamide (NMA) concentrations on the quality and fertility of post-thaw chicken semen. Ejaculates were obtained from 30 Hi-Line White roosters and processed according to the following treatments: lake pre-freezing extender + 0.1 M trehalose (LPF-T) + 6% DMA (control treatment), LPF-T + 9% DMA, LPF-T + 6% NMA, and LPF-T + 9% NMA. Sperm quality (viability, motility, and kinetic traits) was assessed before and after cryopreservation. A total of 15 laying hens per treatment were inseminated to assess fertility and embryo viability. Sperm cryopreserved in presence of DMA had significantly better in vitro quality compared to NMA, showing the highest proportion of viable and progressive motile sperm recovered after thawing. Furthermore, proportion of progressive motile sperm and the VCL, LIN, ALH, and WOB mean values were significantly improved in semen samples frozen/thawed with 6% compared to 9% cryoprotectant concentration. However, the best cryoprotective action on sperm quality played by DMA and the lowest cryoprotectant concentration did not translate into a concomitant advantage in in vivo semen fertility that showed no differences between cryoprotectant and cryoprotectant concentration treatments. Finally, the cryoprotectant DMA and NMA showed an opposite effect on embryo viability in comparison with the effect played on in vitro semen quality, being NMA more efficient than DMA on preserving viable embryos. The present results suggest the urgency to further decrease the cryoprotectant concentration in poultry semen freezing procedures and to assess the specific toxic effect of cryoprotectant on sperm integrity, fertility, and embryo development., (© 2019 Poultry Science Association Inc.)

This study was designed to optimize the semen freezing protocol of the native Mediterranean brown trout inhabiting the Molise rivers through two experiments: an in vitro analysis of the effects of two basic extenders combined with three cryoprotectants on post-thaw semen quality; and an in vivo test to assess the fertilization and hatching rate. Semen was diluted at a ratio of 1:3 in a freezing medium composed of a glucose extender (A) or mineral extender (B). Each basic component contained 10% dimethylsulfoxide, dimethylacetamide or methanol. The post-semen quality was evaluated considering motility, duration of motility, viability and DNA integrity. The basic extender and cryoprotectant were shown to have significant effects on these variables, and the best results were obtained using extender A or B combined with dimethylsulfoxide (P < 0.05). These freezing protocols were selected for fertilization trials in vivo. Fertilization and hatching rates were significantly higher in fresh semen. No significant differences were observed in frozen semen using extender A or B, although higher percentages of eyed eggs and hatching rates were recorded using extender A. According to our in vitro and in vivo results, the glucose-based extender and dimethylsulfoxide emerged as the best combination for an effective cryopreservation protocol for semen of this trout.

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C ( p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post-thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris-citrate-glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post-thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms., (© 2018 Japanese Society of Animal Science.)

The study aimed to characterize meat quality traits of Milanino chickens reared according to a specific free-range farming program. A total of 120 birds was reared straight-run in outdoor pens (8 m2/bird) from 35 d of life and fed ad libitum a low (16%) protein diet. At 180 d of age, 20 birds (10 birds/sex) were slaughtered, and carcass weight data were recorded. After processing, carcasses were refrigerated at 4°C for 24 hours. Then, the right breast and thigh with skin were collected and color parameters, pH, water-holding capacity (WHC), and chemical composition were determined. The left breast and thigh were stored at -20°C until cooking loss and tenderness evaluation. Milanino was confirmed to be a heavy breed with a sexual dimorphism in relation to adult body weight. A high general carcass yield was recorded. Milanino meat was characterized by high protein and low fat contents compared with the standard broiler meat. Differences in meat composition were recorded according to the sex: females presented higher values of dry matter (breast and thigh), protein (breast), and fat (breast and thigh) contents. The meat with skin presented an intense luminosity, and this trait was higher in the females. The muscle color was characterized by high redness and yellowness indices with differences according to the sex: Higher yellowness index was observed in female carcasses, while higher redness index was detected in male breast samples. The pH muscle values were similar to those reported in other autochthonous breeds. WHC values did not show variation between sexes. In contrast, cooking loss values recorded in thigh samples were lower in males compared to females. The degree of tenderness of Milanino meat was not affected by the sex. However, the potential loss of water and the toughness in Milanino meat were low compared to other local chicken breed meat. The present results support the breeding of Milanino chickens for meat production according to its specific straight-run free-range system.

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.

There is need for standardization of freezing-thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post-thaw motility and to analyse combined effect of the best permeating cryoprotectant (P-CPA) with one of four non-permeating cryoprotectants (N-CPA) on post-thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N-methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N-CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing-thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen-thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post-thaw motility. Moreover, ficoll addition to EG-based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N-CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank., (© 2017 Blackwell Verlag GmbH.)

1. Aim of this study was the development of an optimised cryopreservation pellet procedure for chicken semen and the assessment of DNA and membrane integrity in frozen/thawed spermatozoa in a Hubbard F15 meat type selected strain. 2. The following semen processing conditions were studied: spermatozoa working concentration (SWC), 1.5 vs 2 × 10 9 cells/ml in pre-freezing extender; equilibration of diluted semen at 5°C, 20 vs 40 min; dimethylacetamide concentration, 6% vs 9%; dimethylacetamide equilibration time at 5°C, 1 vs 30 min; thawing at 60°C for 10 vs 50°C for 30 sec. Spermatozoa viability (EtBr exclusion procedure - stress test), mobility (Accudenz® swim-down test) and subjective motility were assessed in fresh and frozen-thawed semen. 3. The lower SWC (1.5 × 10 9 cells/ml) and the higher dimethylacetamide concentration (9%) had positive significant effects on the recovery rate of motile (22% vs 16%) and viable spermatozoa (39 vs 34%), respectively. 4.Membrane (SYBR14-PI staining) and DNA integrity (comet assay) were assessed before and after freezing/thawing according to the optimised protocol. 5. Recovery rates of spermatozoa with undamaged plasma membrane and DNA were 41% and 76%, respectively. The distribution of spermatozoa in classes of DNA damage was also analysed and discussed. 6. It was concluded that pellet cryopreservation was a damaging process mainly for plasma membrane rather than nuclear DNA in chicken spermatozoa.

The aim of this study was to assess the combined effect of dimethylacetamide (DMA) and two non-permeating cryoprotective agents, trehalose and sucrose, on the quality of post-thaw chicken semen. Adult Lohmann roosters (n = 27) were used. Semen was processed according to the following treatments: Lake pre-freezing extender +6% DMA (LPF, control treatment), LPF + 0.1 M trehalose (LPF-T treatment), LPF + 0.1 M sucrose (LPF-S treatment) and LPF + 0.1 M trehalose + 0.1 M sucrose (LPF-TS treatment). Semen was loaded into straws and frozen in nitrogen vapour. Sperm quality (viability, mobility and kinetic parameters) was assessed immediately after thawing (T0) and at 5 (T5), 10 (T10) and 15 min (T15) thereafter. The different cryodiluent combinations significantly affected the kinetic parameters. The presence of trehalose, alone or with sucrose, combined with DMA improved the quality of motion in cryopreserved sperm in comparison to DMA alone (LPF) and DMA with sucrose (LPF-S). In particular, the highest values in linearity (LIN) and wobble (WOB) were measured in the treatment LPF-T. The treatments significantly affected the recovery rate of progressive motile sperm that presented the best value soon after thawing in the LPF-T treatment; moreover, the presence of trehalose, alone (LPF-T) or with sucrose (LPF-TS), significantly improved the recovery rate of progressive motile sperm also at T5 and T10 compared to LPF and LPF-S. The present results show a positive synergic action of DMA and trehalose on motile function of thawed chicken sperm., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This data article contains supporting information regarding the research article entitled "Combined effect of permeant and non-permeant cryoprotectants on the quality of frozen/thawed chicken sperm"(Mosca et. al., 2016) [1]. The combined effect of the permeant cryoprotectants agent dimethylacetamide and the non-permeant cryoprotectants agent trehalose on the quality of frozen-thawed chicken semen was assessed. In particular, the quantitative dimethylacetamide/trehalose ratio was investigated freezing semen samples according to the following treatments: trehalose 0.1 M+0% dimethylacetamide (DMA-0), trehalose 0.1 M+3% dimethylacetamide (DMA-3), trehalose 0.1 M+6% dimethylacetamide (DMA-6).

This study was designed to determine: (i) the in vitro effects of different freezing rates on post-thaw semen quality of Mediterranean brown trout (Salmo trutta macrostigma) from the Biferno river; and (ii) the in vivo fertilization and hatching percentage of freezing rate giving rise to the best post-thaw semen quality. Pooled semen samples were diluted 1:3 (v:v) in a freezing extender composed of 300 mM glucose, 10% egg yolk and 10% dimethyl sulfoxide (DMSO). The extended semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen surface (1, 5 or 10 cm) for 10 min to give three different freezing rates. Semen samples were thawed at 30°C for 10 s. The variables assessed after thawing were sperm motility, duration of motility and viability. Our results clearly indicate a significant effect of freezing rate on post-thaw semen quality. Semen frozen 5 cm above the liquid nitrogen surface showed the best quality after freezing/thawing. Based on these in vitro data, 2 groups of 200 eggs were fertilized with fresh semen or semen frozen 5 cm above the liquid nitrogen surface. Fertilization and hatching rates recorded for eggs fertilized with frozen semen were significantly lower (25.4% and 22.5%, respectively) than the ones obtained using fresh semen (87.8% and 75.5%, respectively). An effective freezing protocol will allow for the creation of a sperm cryobank to recover the original population of Mediterranean brown trout in the Biferno river.

This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality. 2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN2) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined. 3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found. 4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN2 surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.

The aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study was designed to improve current freezing protocols for rabbit sperm by examining: (1) the toxicity of different permeable cryoprotectants (CPAs) used for standard vapor freezing (conventional freezing); (2) the feasibility of ultrarapid nonequilibrium freezing (vitrification) of sperm in the absence of permeating CPAs; and (3), the addition of bovine serum albumin (BSA), alone or with sucrose or trehalose as osmoprotectants. First, we evaluated the effects on sperm motility of the incubation time (5 to 60 minutes) with different final concentrations (5% to 20%) of glycerol, N-N-dimethylacetamide, dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, and methanol. N-N-dimethylacetamide (5%) and DMSO (5% and 10%) showed the least toxic effects; the use of 10% DMSO producing the best postthaw sperm motility and membrane integrity results (P < 0.05) after conventional freezing. For vitrification, semen was diluted in the absence of permeable CPAs and frozen by dropping semen directly in liquid nitrogen. However, this led to the low or null cryosurvival of sperm postvitrification (0.16 ± 0.4%, 1.8 ± 1.6%, and 94.5 ± 1.4% of motile, membrane-, and DNA-intact sperm cells, respectively). To assess the effects of albumin and osmoprotectants on sperm cryosurvival, sperm was conventionally frozen with 10% DMSO or vitrified in the absence of permeable CPAs without or with 0.5% BSA alone or combined with sucrose or trehalose (range, 0-0.25 M). In the conventional freezing procedure, the addition of BSA alone failed to improve sperm cryosurvival, however, in the presence of BSA plus either sucrose or trehalose, the postthaw motility (using 0.1 M sucrose or trehalose) and DNA integrity (using all additive concentrations) of sperm were significantly better (P < 0.05) than control. Higher numbers of motile and membrane-intact cells were observed when semen was vitrified with BSA alone or with BSA and sucrose (0.1 and 0.25 M) or BSA and trehalose (0.25 M) and a best recovery of DNA-intact sperm was recorded for BSA plus sucrose compared with semen vitrified without osmoprotectants (P < 0.05). Finally, the cryodiluent combinations BSA/sucrose and BSA/trehalose were compared in an insemination trial. Rabbit does were inseminated with fresh semen (N = 56), semen conventionally cryopreserved in the BSA-based cryodiluents containing 0.1 M sucrose or trehalose (N = 56 per group), or semen vitrified in the presence of 0.25 M sucrose or trehalose (N = 8 per group). Fertility rates and live born kids were similar for semen cryopreserved with BSA/sucrose (77% and 7.6) compared with fresh semen (84% and 8.1) and significantly higher than the figures recorded for the conventionally frozen semen in the BSA/trehalose group (52% and 6.1; P ≤ 0.05). In contrast, only one doe inseminated with semen vitrified in the presence of BSA/sucrose became pregnant, though no kids were delivered. The conclusions to be drawn from our study are: (1) incubation times and concentration toxicities established for the main permeable CPAs used for conventional freezing of rabbit sperm indicated that DMSO 10% was the least damaging; (2) CPA-free vitrification of rabbit semen led to a low or null sperm cryosurvival; and (3) enriching the freezing medium with BSA plus adequate amounts of sucrose or trehalose can improve the cryosurvival of rabbit sperm after conventional freezing or vitrification. In our working conditions, BSA/sucrose was more effective than BSA/trehalose at preserving the in vivo fertilization capacity of rabbit sperm cryopreserved using the standard procedure., (Copyright © 2013 Elsevier Inc. All rights reserved.)