Genetic Diversity of 17 Autochthonous Italian Chicken Breeds and Their Extinction Risk Status.

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.

Antimicrobial Effects of Black Soldier Fly and Yellow Mealworm Fats and Their Impact on Gut Microbiota of Growing Rabbits.

This study aimed to evaluate the in vitro antimicrobial activities of two types of insect fats extracted from black soldier fly larvae (HI, Hermetia illucens L.) and yellow mealworm larvae (TM, Tenebrio molitor L.) and their effects as dietary replacement of soybean oil (S) on cecal fermentation pattern, and fecal and cecal microbiota in rabbits. A total of 120 weaned rabbits were randomly allotted to three dietary treatments (40 rabbits/group) -a control diet (C diet) containing 1.5% of S and two experimental diets (HI diet (HID) and TM diet (TMD)), where S was totally substituted by HI or TM fats during the whole trial that lasted 41 days. Regarding the in vitro antimicrobial activities, HI and TM fats did not show any effects on Salmonella growth. Yersinia enterocolitica showed significantly lower growth when challenged with HI fats than the controls. The insect fat supplementation in rabbit diets increased the contents of the cecal volatile fatty acids when compared to the control group. A metataxonomic approach was adopted to investigate the shift in the microbial composition as a function of the dietary insect fat supplementation. The microbiota did not show a clear separation as a function of the inclusion, even if a specific microbial signature was observed. Indeed, HI and TM fat supplementation enriched the presence of Akkermansia that was found to be correlated with NH3-N concentration. An increase in Ruminococcus, which can improve the immune response of the host, was also observed. This study confirms the potential of HI and TM fats as antibacterial feed ingredients with a positive influence on the rabbit cecal microbiota, thus supporting the possibility of including HI and TM fats in rabbit diets.

Growth Performance Analysis of Two Italian Slow-Growing Chicken Breeds: Bianca di Saluzzo and Bionda Piemontese.

Bianca di Saluzzo (BS) and Bionda Piemontese (BP) are two Italian chicken breeds, mainly reared for meat production, primarily in antibiotic-free farming. However, technical information on their growth pattern is still missing. At hatching, 150 unsexed chicks of each breed were weighed, labeled, and reared in indoor pens up to 8 w of age. At 8 w of age, the chicks were separated by sex and randomly transferred to growing pens with access to an external paddock (15 birds/pen; 4 pens/sex for each breed). The body weight (BW) was recorded biweekly for each bird, from hatching to 32 w of age. In order to identify an improvement strategy, the objectives of our study were to analyze the growth pattern of these birds using the Gompertz mathematical model and compare results with other chicken breeds. Polymorphism of the PAX7 gene was also analyzed to test its association with growth traits. Both BS and BP are close to unselected native breeds and, among the Italian local poultry, they are confirmed to be slow-growing birds with an intermediate size between heavy and light chicken breeds. Regarding the PAX7 gene, two alleles were found, F and G, and showed an association with the actual BW in the BP females from 14 w of age onwards. The G allele always exhibited a more favorable effect than the F allele. In small size poultry population, a delicate balance between preservation of biodiversity and performance improvement should be considered. Consequently, the most proper way could be an approach based on a mating scheme to keep inbreeding under control, increase growth rate, and improve commercial maturity.

Polymorphism Analysis of Ch1 and Ch2 Genes in the Siberian Cat.

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders' statements. The sequence of the two genes, namely Ch1 and Ch2, that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats.

Distinguishing industrial meat from that of indigenous chickens with molecular markers.

The aim of investigation was to evaluate a traceability system to detect industrial chicken meat among indigenous products, considering issues that could affect assignment accuracy. The dataset included 2 Italian indigenous meat breeds, namely Bionda Piemontese (2 ecotypes) and Bianca di Saluzzo, one broiler line, and 3 layer lines. Assignment tests were performed using a standard panel of 28 microsatellite loci. To evaluate effects of inbreeding and substructure on assignment accuracy, a simulated dataset was prepared. Broilers and layers belong to homogeneous populations and never enter the clusters of indigenous breeds. Ambiguity or misallocation are expected between the Bionda ecotypes and between the 2 indigenous breeds, but it is unlikely that niche products provided by Bionda and Bianca will compete with one another. Non-random mating reduces accuracy, but only populations having weak genetic differentiation are involved, namely those that are less interesting to discriminate. The dataset can be used as a reference population to distinguish commercial meat from indigenous meat with great accuracy. Misallocations increase as number of loci decreases, but only within or between the indigenous breeds. A subpanel of the most resolving 14 loci keeps sufficient informative content to provide accuracy and to correctly allocate additional test samples within the reference population. This analytical tool is economically sustainable as a method to detect fraud or mislabeling. Adoption of a monitoring system should increase the value of typical products because the additional burden of molecular analyses would improve commercial grade and perception of quality.

Genetic epidemiology of Sarcoptes scabiei in the Iberian wolf in Asturias, Spain.

BACKGROUND: During the last decades, attempts have been made to understand the molecular epidemiology of Sarcoptes scabiei, and to detect and clarify the differences between isolates from different hosts and geographic regions. Two main phenomena have been described: (i) host-taxon derived-Sarcoptes mite infection in European wild animals (revealing the presence of three separate clusters, namely herbivore-, carnivore- and omnivore-derived Sarcoptes populations in Europe) and (ii) prey-to-predator Sarcoptes mite infection in the Masai Mara ecosystem. RESULTS: Using one multiplex of 9 microsatellite markers and Sarcoptes mite samples from sympatric Pyrenean chamois, red deer, red fox and Iberian wolf, different population structure analyses revealed concordance with the host-taxon law described for wild animals in Europe, with two main host-derived Sarcoptes mite populations, herbivore- and carnivore-derived. Surprisingly, Iberian wolf derived Sarcoptes populations had the highest genetic diversity among the other populations, including two different subpopulations: one similar to the herbivore-derived Sarcoptes populations, and another similar to carnivore (fox)-derived Sarcoptes mite population. CONCLUSIONS: The host-taxon effect in wild animals is still supported with the maintenance of carnivore- and herbivore-derived Sarcoptes clusters' separation in analyzed mites. However, this phenomenon could be modified with the inclusion of a large predator as wolf in the present work, revealing prey-to-predator Sarcoptes mite infection between the studied host-taxa and suggesting the importance of wolf's immune system for explaining the high variability reported in C. lupus derived mites. Further studies of host diet, behavior and movement, and regarding the role played by its immune system, would be of great help to clarify interactions between the two hypotheses, host-taxon and prey-to-predator.

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits.

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sertãozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.

Applicability of molecular markers to determine parasitic infection origins in the animal trade: a case study from Sarcoptes mites in wildebeest.

The development of non-manipulative molecular tools to determine the origin of parasite infections in the animal trade (if infected before their export or import) is of great interest worldwide for both the animal trade industry and for animal welfare. Molecular tools have a wide range of applications, including forensic identification, wildlife preservation and conservation, veterinary public health protection, and food safety. Nonetheless, genetic markers were not reported to detect the source of infection in the animal trade. In this study we tested the applicability of molecular tools to detect the origin of Sarcoptes mite infection of wildebeest imported by the United Arab Emirate (UAE) from Tanzania. Using one multiplex of seven microsatellite markers and control samples from UAE, Kenya and Italy, we demonstrated the usefulness of the multiplex STR-typing as a molecular tool of pivotal interest to help commercialist, authorities, and conservationists, to identify the geographical origin of parasitic infections.

Two simple techniques for the safe Sarcoptes collection and individual mite DNA extraction.

Availability of mites is a recognized limiting factor of biological and genetic investigations of the genus Sarcoptes. Current methods of deoxyribonucleic acid (DNA) extraction from individual mites also need substantial improvement in efficiency and operator friendliness. We have first developed a technique for efficient and safe extraction of living mites from scabietic skin samples (crusts or deep skin scrapings). Its core device is a large plastic syringe connected with a 1.5-ml Eppendorf tube. The source material is introduced in the syringe and the device in a shoe box with the tip half of the tube emerging. Mites migrate towards a heat source during a minimum of 36 h. Then, the tube is detached and the mites utilized without risks for the operators. A second technique allows operator-friendly manipulation of individual mites for DNA extraction. Fixed mites are isolated by adhesion to a small strip of polyvinyl chloride (PVC) adhesive tape operated with tweezers. Then, mite and strip are plunged in the lyses buffer and the sample twice submitted to thermal shock for disruption of the chitinous exoskeleton. Data show that the tape does not interfere with successive DNA extraction with a commercial kit. The corresponding protocol, that we briefly name "PVC adhesive tape + thermal shock + kit DNA extraction," compares favorably with the available ones.

Effectiveness of the postponed isolation (post-frozen isolation) method for PCR-quality Sarcoptes mite gDNA.

The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin ('postponed isolation' method) are suitable sources of PCR-quality genomic DNA, and to test the effectiveness of this method in comparison with the 'direct isolation' method, often used through force of habit. Hundreds of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a approximately 450 bp product (ITS-2) and multi-locus 10x genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era.

A non-invasive test for sex identification in short-toed Eagle (Circaetus gallicus).

Knowledge of the sex of individuals in natural populations greatly facilitates evolutionary ecology, breeding systems and genetics. Therefore, the development of a simple, not stressing and objective sexing test would facilitate conservation of the Short-toed Eagle (Circaetus gallicus), an endangered Accipitridae species living mainly in southern Europe and Asia. A PCR test was used employing primers that amplify two homologous fragments of both the CHD-W gene, unique of females, and the CHD-Z, occurring in the two sexes. The analysis of the PCR products obtained from blood DNA showed a band of about 380 bp, apparently unique in all individuals. The alignment of the sequences of the two fragments revealed that CHD-W is only 9 bp longer than CHD-Z (387 vs. 378 bp) while CHD-Z lacks the restriction site for Asp700I. After digestion male PCR products showed a unique band of 378 bp while fragments belonging to females resolved into three bands (378, 280 and 107 bp). Using feathers as DNA sources, the individual patterns obtained were identical with the corresponding blood DNA samples. This sexing technique is objective and non-invasive and could be useful for verifying the sex ratio theories and improving the management.