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This study provides a comparative assessment of fresh semen quality and sensitivity to cryopreservation in several Italian chicken breeds. The research involved 145 roosters from 13 breeds. The results showed a wide variability in all the parameters considered among the different breeds, especially in the quantitative variables of fresh semen, such as volume and concentration. For the qualitative characteristics (sperm membrane integrity and motility parameters), the variability across breeds was more pronounced for frozen than fresh semen. Interestingly, apart from total motility in fresh semen, breed had a significant effect on all semen quality parameters in both fresh and thawed ejaculates. Considering the overall qualitative characteristics, the Robusta maculata, Siciliana, and Mericanel della Brianza breeds produced ejaculates with better semen quality compared to other Italian breeds. By evaluating the main parameters of semen quality, our results underline the potential of these traits to influence the reproductive success and genetic conservation. The Bionda piemontese, Bianca di Saluzzo, Livorno bianca, Pepoi, and Siciliana breeds showed better resilience to cryopreservation, suggesting the need for breed-specific protocols to optimise semen quality after thawing. Importantly, the research highlights the central role of semen quality for both immediate fertilisation success and long-term conservation efforts. Future studies integrating OMICS technologies could elucidate molecular markers influencing breed-specific differences, helping to refine cryopreservation techniques and improve conservation strategies for indigenous Italian chicken breeds. This work contributes valuable insights to global efforts aimed at safeguarding poultry genetic diversity and sustainability.

Specific characteristics of the eggs from nucleus populations of Ancona (AN), Bianca di Saluzzo (BS), Bionda Piemontese (BP), Livorno, Mericanel della Brianza (MB), Mugellese (MU), Siciliana (SI) and Valdarnese (VA) chicken breeds kept in University Research Centres across Italy were evaluated. The physical characteristics considered were whole egg weight and shell, yolk and albumen proportion. The chemical characteristics considered were dry matter (DM), ash and protein content of the yolk and albumen, and the yolk fat. Among the breed groups evaluated in this study, BS and BP produce large eggs that are rich in total crude protein and lipids, with abundant egg whites. MB and MU produce small eggs with a high yolk percentage. AN and SI hens produce medium-small-sized eggs. The heaviest yolks were observed in AN eggs, and the highest shell percentage was found in SI eggs. LB and VA produce medium-large-sized eggs with low yolk lipid content.

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.

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.

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.

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 × 106 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 × 106 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 × 106 (49.9% and 19.7%) and 35 × 106 (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.

The Mediterranean brown trout is one of the most endangered freshwater species. A complicated network of climate and human influences has severely harmed its biodiversity. The introduction of alien trout is one of the most serious threats to native populations’ intraspecific diversity. In Molise region (south-Italy) an important conservation program (LIFE Nat.Sal.Mo project) has recently been proposed to preserve the genetic integrity of native Mediterranean trout. This project, alongside safeguarding and re-establishing the habitats’ usefulness aims to restore the genetic integrity of the autochthonous population. This is one of the major goals, and it is accomplished by employing frozen wild breeder semen in conjunction with proper fertilization techniques to carry out artificial reproduction to enhance genetic diversity in the progeny and maintain fitness within self-sustaining populations. In this regard, the implementation of the first European semen cryobank has played a strategic role for conserving extant genomic diversity of native population. The goal of this review is to outline the procedures developed and guidelines established for the creation of a Mediterranean trout sperm cryobank. Here, we specifically provide an overview of some of the main challenges associated with the implementation of semen cryobank, the results achieved, the prospects for restoring genetic integrity in native populations, and lastly, future views for hatchery management to preserve the wild biodiversity of native salmonid species. During the project timeframe 1,683 semen doses, from 150 native breeders were stored inside the cryobank. Our results clearly showed the efficiency of the freezing procedure used, both in vitro and in vivo. In fact, we recorded satisfactory values of post-thaw sperm motility and viability that ranged from 40% to 80%, and excellent fertilization rate in vivo, which ranged from 64% to 81%.

ABSTRACT: 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.

In external fertilizer fish, ovarian fluid (OF) seems to play a key role in fertilization success, improving spermatozoa swimming performance. These OF/sperm interaction mechanisms are frequently species-specific and/or population-specific and could decrease the risk of genetic introgression of wild populations from introduced or escaped zootechnical individuals. The Mediterranean brown trout (Salmo cettii) is threatened by genetic introgression with strains of domestic brown trout (Salmo trutta) that were introduced for recreational purposes. The aim of our study was to test if native S. cettii females, rather than zootechnical S. trutta, produce OF with a greater ability to upregulate the sperm motility of conspecific males. Thus, we compared the sperm swimming performances of males inhabiting the Biferno River (Molise region—Southern Italy) activated in native S. cettii vs. zootechnical S. trutta female’s OFs. In our study, native females’ OFs (20% diluted), compared to spring water, has the ability to significantly boost the sperm performance of the autochthonous males, while zootechnical S. trutta fails. These preliminary results suggest that OF-sperm interactions could potentially influence or direct the hybridization mechanisms involving the native Mediterranean trout inhabiting the Biferno River and the domestic lineage of brown trout introduced in the past.

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.

The creation of genetic resource cryobanks provides a crucial link between in situ and ex situ techniques to improve the efficiency of conservation programs. Aim of the present review is to describe all the activities developed for the implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. These activities can be classified into three main topics: (1) identification of species-specific semen freezing/thawing reference procedures; (2) drafting Standard Operative Procedures (SOP) for the implementation of the semen cryobank; (3) storage of semen doses from Italian chicken and turkey breeds to establish the cryobank. Several trials have been developed to identify a specie-specific semen cryopreservation protocol for chickens and turkeys. The major results are reviewed and a final reference protocol described. Taking into consideration the FAO guidelines for cryoconservation of animal genetic resources, SOP were drafted with the aim to provide technical guidance and logistical support on the choice of priority breeds, selection of birds for semen production, infrastructures and storage sites, birds and semen management, cryopreservation process and doses traceability. Lastly, the Italian Semen Cryobank was created. A total of 112 semen doses from 22 cockerels of three breeds, and 74 doses from 12 turkey males of three breeds were stored in the Cryobank. Breed specific semen quality parameters assessed before and after cryopreservation are reported. The described activities provide information and tools useful for the implementation of semen cryobanking in avian species and might be transferred also to other species after appropriate adaptations.HIGHLIGHTS Implementation of the first Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds Drafting Standard Operative Procedures provides technical guidance and logistical support on the design and establishment of the cryobank Semen cryobank is a precious genetic reservoir and could be useful to safeguard genetic variability in small population in vivo conserved

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.

SUMMARY: The Milanino is a heavy Italian chicken breed and represents an important genetic resource for alternative production systems. Due to the absence of standard management guidelines for local chicken breeds, this study aims to determine the space requirement to achieve a good equilibrium between growth performance and stress response in Milanino chickens reared according to a separate-sex free-range program. A total of 140 birds (70M:70F) were reared in outdoor pens from 75 to 235 d of life according to the following experimental groups (35 birds/group): (M2) males in 2 m2/bird; (F2) females in 2 m2/bird; (M10) males in 10 m2/bird; (F10) females in 10 m2/bird. Growth performance and stress condition were recorded. Bird density affected male body weight, that was higher at the lower density of 10 m2/bird. In contrast, females showed a consistent growth rate irrespective of the bird density suggesting lower space requirements. An overall good adaptability of the breed to the separate-sex free-range system was found, irrespective of both sex and bird density. Moreover, the stress markers values assessed at the end of the growing period suggest the ability of Milanino birds to respond well to environmental stressors. The results provided in this report will be implemented into free-range management guidelines for meat production in the Milanino breed: the separate-sex rearing is recommended during the growing period in outdoor pens and the bird density has to be planned according to the sex.

SUMMARY: The Milanino is a heavy Italian chicken breed and represents an important genetic resource for alternative production systems. In order to support its promotion in the market according to consumer expectations on healthy nutrition and animal welfare, this trial aims to study the slaughter performance and the meat quality in male and female Milanino chickens kept at different rearing density in a separate-sex free-range system. A total of 140 birds (70M:70F) were reared in outdoor pens from 75 to 235 d of life according to the following experimental groups (35 birds/group): M2) males in 2 m2/bird; F2) females in 2 m2/bird; M10) males in 10 m2/bird; F10) females in 10 m2/bird. At 235 d of age, 6 birds per group were slaughtered. Slaughter performance and meat quality were assessed. The Milanino chicken is characterized by high carcass weight and carcass yield among local chicken breeds, and a rearing density of 10 m2/bird is recommended for male birds to obtain heavier carcasses. Milanino meat appears bright and intensely colored, and it is characterized by high protein and low fat contents compared with the standard broiler meat. Total lipids of Milanino meat are characterized by a healthy fatty acid composition, corresponding to a high PUFA/SFA ratio. Milanino breast meat is a good supply of nutraceutical PUFA with a positive low n-6/n-3 PUFA ratio. The ability of the Milanino breed either to synthesize or to transfer to tissue a high quantity of PUFA relevant for human health could be a key factor for its economic valorization.

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.

Sperm freezing is of interest not only for animal breeding. The ability to use sperm in frozen form for AI is a key factor in ensuring the long-term preservation of genetic diversity through the creation of a sperm cryobank. The most widely used freezing method involves using straws as packaging sperm, then freezing them on liquid nitrogen (N2) vapor and finally immersing them in N2. The cryosurvival of sperm cells also varies among different animal species and has been correlated with the freezing rate, i.e., the distance of the straws above the N2 level. Identifying a freezing procedure that will report a fertilization rate as close as possible to that of fresh semen appears to be a challenge. Thus, there is a clear need to standardize the entire freezing process. In this research, a new device for semen cryopreservation is presented. The cryopreservation prototype allows management of the freezing rate by varying the heights of the support straws above the N2 level. A stainless steel box with a suitable floating frame equipped with a support for the straws was designed and fabricated. The holder is connected to the floating frame by means of two threaded rods that allow for height adjustment by screwing in appropriate guides. Two K-type thermocouples were used, one to measure the temperature of the nitrogen vapor at the height of the support and the other to measure the temperature of the semen inside the straw. In this way, different freezing protocols can be compared. By managing the freezing rate, the sperm freezing procedure, which is specific to animal species, could be standardized and thus variability in results could be minimized. In addition, improved cryosurvival and post-freezing sperm fertilization rates are expected.

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.

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.

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.

The aim of this study was to evaluate the effects of different cold-storage time intervals between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice from 1 to 6 h, until freezing. Fresh and post-thaw sperm motility was evaluated by a Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency (BCF) was recorded, whilst the amplitude of lateral displacement of the spermatozoon head (ALH) underwent a significant increase. In frozen semen, no significant difference was observed for all the motility parameters evaluated, except for the total motility between 1 and 6 h of storage and the duration of sperm movement between 1 and 5 h. Cold-storage time did not significantly affect the percentage of live embryos following the use of frozen semen. In conclusion, our results showed that, if necessary, the Mediterranean brown trout semen can be frozen even until 6 h post-collection without losing its fertilizing ability.

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 FST 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.

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).

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 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.

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 × 106 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 × 109 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 × 106 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.

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 (FROH), was markedly different among the breeds and ranged from 0.121 (Valdarnese) to 0.607 (Siciliana). In all breeds, short runs of homozygosity (ROH) (

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.

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.

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 × 109 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 × 105, 4.5 × 105 and 3 × 105:1. 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 × 105:1. 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.

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.

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.

This study was designed to compare Cortalap® extender with tris-citric acid-glucose (TCG), Lepus® and Merk III®, to assess the in vitro preservability of rabbit spermatozoa stored for 72 h at 5°C. The best extender identified in vitro was then compared with fresh semen to determine fertility and prolificacy rates in vivo. Eight semen pools were split into four subsamples and each of them was diluted to a ratio of 1:10 (volume:volume) with four different extenders (Lepus®, Cortalap®, TCG and Merk III®) and stored at 5°C for 72 h. Sperm motility, viability, sperm membrane and acrosome integrity were evaluated in both fresh and chilled semen at 4, 24, 48 and 72 h of storage. The results showed that Cortalap® was the best extender to preserve the quality of rabbit semen in vitro over 72 h in comparison with the other extenders (P® was used for an artificial insemination (AI) trial to be compared with fresh semen. Two groups of does (n=30 each) were inseminated with fresh and chilled semen. The fertility and prolificacy rates for the does inseminated with chilled semen were significantly lower compared with those inseminated with fresh semen (P® proved to be the best extender to preserve semen quality in vitro after 72 h at 5°C, the in vivo results showed that its use is not recommended in AI programmes.

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.

Neuroglia has been historically considered the “glue” of the nervous system, as the ancient Greek name suggests, being simply referred as non-neuronal cells, with supporting functions for neurons in the CNS of mammalian and lower vertebrates. All around the world, approximately 283 cell lines were obtained from fish, yet none of these was from the brain of Sparus aurata, neither in cell lines nor as primary culture. Here we describe a novel in vitro reproducible neuroglial marine model for establishing primary neuroglial cell cultures, by dissociating the whole brain of seabream juveniles. We showed that proliferating neural stem cells produced alongside three generating lineages, such as neuronal precursor cells, astroglial precursor cells and oligodendroglia precursor cells, which developed respectively neurons, astrocytes and oligodendrocytes. The radial glia, finely described by morphological studies and immunochemical antigen expression, showed a peculiar spatial distribution, giving rise simultaneously both to astrocytes and neuronal precursors within a highly proliferative assemblate. Radial glia cells were assessed by glial fibrillary acidic protein (GFAP) and vimentin reactivity, astrocytes by GFAP, neurons by the neuron-specific markers for ubiquitin carboxy-terminal hydrolase 1 (UCHL1) and intermediate filament associated protein (NF), whereas myelinating oligodendrocytes were immunostained with anti-myelin basic protein (MBP) and anti-O4. Our findings suggest that seabream neuroglial cells gain in 3-4 weeks of culturing proliferation, neuroglial differentiation, and oligodendrocyte maturation with myelination, thus disclosing on the possibility that mixed neuroglial cultures can accelerate the maturation of oligodendrocytes and the regeneration of CNS injury in fish.

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.