Overall, 48 female and 6 male breeders were selected (together with further 10 spare males). Sexual maturity occurred on 21 weeks of age and weekly oviposition rate progressively increased from 1 to 23% in the period October-December 2023.
Selected breeders were organised into 6 familiar lines, as reported in Table 1.1, reared in floor pens with controlled environment. Population management and data recording were performed as described for year 2023. Morphological parameters (average, min, max) recorded from all the breeders are reported in Table 1.2. The oviposition curve (January-July) is shown in Figure 1.1; overall, the total number of laid eggs/hen along the same period (188 days) ranged from 45 (family 6) to 87 (family G), thus showing a marked increase compared to year 2023. Average egg weight was 34.9 g, similar to year 2023, ranging from 21 to 52 g. Artificial incubation parameters are listed in Table 1.3 per family line.
Phenotipic characterisation of Modenese (MO) chickens During 2023, new Modenese (MO) birds were found in fancy farms and new adults (13 females and 7 males, 180 days of age) were housed to the Poultry Center on October 2023. A quarantine period was observed to run sanitary tests, then rearing started in December. The birds were individually marked, weighted, and sampled (feathers). Samples were sent to the Molecular Genetics Laboratory (UniTO) for microsatellite marker genotyping in order to analyse individual variability and kinship. Analyses identified 3 familiar lines (Figure 1.2) and were used to set a mating plan to reduce consanguineity. Data on familiar lines in reproduction from March 2024 are reported in Table 1.4. Each familiar line is reared in a floor pen with controlled environment.
Morphological caracterisation of individual breeders showed omogeneous qualitative traits, consistent with wheaten gold colour: yellow shanks, white ear-lobe, orange/yellow eyes, red simple comb with 5-6 spikes, falling to one side in the hen; average values of quantitative morphological traits are reported in Table 1.5.
Occasional ovoposition was registered during November and December 2023; however, oviposition rate showed a constant increase from January to March and a decrease from April to June, variable among the family lines. General oviposition (January-June) and family line oviposition (March-June) curves are shown in Figure 1.3. Overall, the total number of laid eggs/hen along the same period (98 days) ranged from 11.8 (family 1) to 26.3 (family 2). Average egg weight was 54.2 g, ranging from 40 g and 65 gg.
Eggs set in April were incubated to select new breeders; 186 eggs were incubated and 133 chicks were hatched. Artificial incubation parameters are listed in Table 1.6. Family 1 showed higher fertility and lower embryonic mortality values than other family lines, thus also hatching percentage was remarkably higher.
Chick were reared in a floor pen with controlled environment during the growing period; at 40 days of age birds were sexed and males and females were reared separately from then on. The chicks underwent vaccination programme as follows: a) Marek disease, at 1 day of age; b) Newcastle disease, at 1, 4, 20 weeks of age, and before sexual maturity; c) salmonellosis, at 1, 7, and 18 weeks of age. Body weight was registered from May to December, on hatch and at 4, 8, 12, 18, 24, and 30 weeks of age, to monitor the growth curve; the data are shown in Figure 1.4 for males and females.
Males were sampled (feathers) at about 40 days of age and genotyped in order to select new breeders and set mating plans, according to individual variablity index (see Actions 2 and 4). Female breeders were selected according to kinship and to the numbers of birds avaiable for each family line; overall, 25 female breeders were selected. Family line characteristics, with the numerousness of breeders and their IC, are reported in Table 1.7. Three spare males were also selected and reared, in case of any breeder decease.
Family lines were set in December 2024 and will be reared along the entire reproductive season from January to July 2025, to monitor oviposition and reproductive performances.
Phenotipic characterisation of Nero d’Italia (NI) and Brianzolo (Br) turkeys
Brianzolo breed (BR) – The reproductive period of the three couples of Brianzolo (BR) breed, started during the second year, continued during May-July 2023 in controlled environment. Oviposition was constant in the period March-July 2023, after a period of sexual rest (see Report on the activities of the second year); oviposition rate was low, not higher than 29%, and individual egg production was 25 eggs in 16 oviposition weeks. All breeders were moved to outdoor enclosures from August to December 2023; occasional oviposition was recorded during this period only in September and October. Eggs set during the period April-May 2023 (n=24) were incubated, fertility was 96%, embryonic mortality 21%, and hatchability/incubated eggs 75%, corresponding to 18 hatched poults.
Poults, marked and weigted on hatch, were reared in floor pens with controlled environment up to adult age. Poults underwent vaccination for Newcastle disease at 1, 4, and 19 weeks of age. Body weight was registered on 4, 10, 16, 21, 30, 33, and 35 weeks of age and average registered values are reported in Table 1.8 for males and females. Body weight from hatch to 16 weeks of age was very similar in both sexes, while males showed a higher body weight increase in the following weeks. Birds were moved to outdoor enclosures in the period August-December 2023 and then again to controlled environment in the following months. Mortality rate during the growing period was 11% (2 birds), then 5 females and 11 males reached adult age. Sexual maturity in females occurred on 30-31 weeks of age, in December.
At the beginning of March 2023, familiar lines were organised: 1 group with 3 females hatched in 2022 (TV), and 2 groups with 5 females hatched in 2023 (TG, 2 females in one group and 3 females in the other). It is planned to mate TV females in turn with each of the 3 males hatched in 2022, and TG females with males hatched in 2023, randomly chosen, and changed every third week. All the birds underwent morphological characterisation, by the recording of both qualitative and quantitative parameters; qualitative parameters were homogeneous and consistent to the breed standard, showing reticulated silver bronze livery, orange-red caruncles, dark iris, light brown beak with blackish stripes, pinkish shanks; average quantitative parameters are reported in Table 1.9. Oviposition curve constantly increased from January to March (6% to 80%) for TG females, while TV females oviposition started in March, and the weekly average oviposition was 26%. General oviposition (January-June) and family line oviposition (March-June) curves are shown in Figure 1.5. Altogether, total egg production/hen along the oviposition period (177 days) ranged from 21 eggs (family 1) to 49 eggs (family 3). Average egg weight was 72.5 g, ranging from 58 g to 93 g.
Eggs set in April were incubated to select new breeders; 138 eggs were incubated and 75 poults were hatched. Artificial incubation parameters are summarised in Table 1.10; fertility was high, however embryonic mortality was also very high, thus consequently average hatchability was as low as 54%, ranging from 42% to 70% across the family lines. Poults, marked and weighted on hatch, were reared in floor pens in controlled environment up to adult age, as previously described for the Modenese chicken breed. Brianzolo turkey growth curve, recorded along the period May-December 2024, is shown in Figure 1.6. All the birds were sampled (feathers) for microsatellite marker genotyping and for population structure and kinship analyses (see Action 2). As a whole, 62 birds were characterised.
The analysis of 4 polymorphic markers showed the existence of 13 alleles. Average variability indexes are reported in Table 1.11. Inbreeding rate was estimated through the evaluation of average observed heterozygousis (mean=0.657, median=0.750). Two distinct genetic groups were identified throught the analysis of the population genetic structure, however the family lines are not clearly separated from each other, as shown by birds’ distribution in Figure 1.7.
Nero d’Italia breed (NI) – A new group of Nero d’Italia turkeys (n=10) was housed to the Poultry Center on July 2023, in order to set a nucleus population. A quarantine period was observed to run sanitary tests, then rearing started in September. All the birds were marked, sexed, then males and females were reared separately in outdoor enclosures in the period September-December, and in floor pens in controlled environment in the following months. Body weight was registered on 98, 126, 160, 190, and 220 days of age; growth curve for males (n=3) and females (n=7) is reported in Figure 1.8. Sexual maturity of females occurred on 45 weeks of age in April 2024.
At the beginning of March, 3 family lines were randomly organised, each made of 2-3 females and 1 male, which was changed every third week. Morphological characterisation was performed on all the birds through the evaluation of both qualitative and quantitative parameters; morphological traits were homogeneous and consistent with the breed standard, showing black livery, red caruncles, dark iris, dark beak, dark red to purple shanks; average quantitative parameters are reported in Table 1.12. NI breeder management and reproductive data recording were performed as described for the BR breed. Oviposition results are summarised in Figure 1.9 and artificial incubation parameters are listed in Table 1.13. Altogether, total oviposition/hen along the oviposition period (177 days) ranged from 18 eggs (family 1) to 31 eggs (family 2). Average egg weight was 78.2 g, ranging from 65 g to 92 g. Overall, 84 eggs were incubated and 30 poults were hatched. A high variability across the family lines was observed in fertility, embryonic mortality, and, therefore, hatchability.
Poults were marked and weighted on hatch, then reared in floor pens in controlled environment up to adult age, as described for the Modenese chicken breed. NI turkeys growth curve, recorded from May to December 2024, is shown in Figure 1.10.
All the growing birds were sampled (feathers) for microsatellite marker genotyping and for population structure and kinship analyses (see Action 2). As a whole, 22 birds were characterised. The analysis of 4 polymorphic markers showed the existence of 10 alleles. Average variability indexes are reported in Table 1.14. Inbreeding rate was estimated through the evaluation of average observed heterozygousis (mean=0.205, median=0.250). The genetic variability of the subjects was investigated throught the analysis of the population genetic structure, which could not clearly identify distinct family lines (Figure 1.11). Moreover, individual variability index and average kinship index were calculated for each bird. Percentage conservation index was calculated for males, to identify subjects with higher index values to be used as breeders.
Phenotipic characterisation of semen in chicken and turkey breeds
Proteome – The functional informatic analysis of shared and exclusive proteins of different chicken breed semen was concluded during the third year. Preliminary results will be presented in a poster at the European Poultry Congress (Valencia, Spain) in June 2024 (Nonnis et al., 2024). The main results were described and discussed in a scientific report, preliminary to the preparation of a paper to be submitted for the publication to a specialised journal of the field.
Lipids – Laboratory activities for the evaluation of lipid components of semen and of seminal plasma were performed in 2023; final data were verified and organised in dedicated files and the most significant results underwent statistical analysis in 2024. The breeds and the numerousnesses of samples/breed are as follows: Bionda Piemontese 9, Bianca di Saluzzo 9, Mericanel della Brianza 5, Pepoi 10, Robusta Maculata 5. Lipid composition of spermatozoa and of seminal plasma are reported respectively in Tables 1.15 and 1.16. Acidic composition of total phospholipids, the main lipids in both spermatozoa and seminal plasma, is reported in Tables 1.17 and 1.18.
The results of lipid analysis of semen were compared to semen qualitative analysis data (from the first and the second years of the project): Robusta Maculata and Bionda Piemontese breeds, which showed different lipid profiles, proved to display respectively the best and the worst sperm cynetic parameters, as well. The results of this study will be presented at the congress of the Animal Production Science Association (ASPA) to be held in Torino in 2025.
Semen production and quality – In 2024, adult Modenese breed male chickens and adult Brianzolo and Nero d’Italia breed male turkeys were reared to set family groups (see Task 1.2 and 1.3). All the birds were reared in controlled environment and underwent morphological evaluation (see task 1.2 and 1.3). After a few weeks’ training, semen collection was routinely performed from all the males (twice weekly, January-March). Adaptability to milking varied across subjects and across breeds; donor males were identified in MO and BR breeds, while no donors were found in NI turkeys. Average values of quali-quantitative parameters recorded in individual ejaculates for MO and BR breeds are reported in Table 1.19.
Characterisation of the breeds:
- Morphological characterisation, according to the FAO guidelines (2012) to update and validate what was collected during the first TuBAvI project for the Mugellese (Mannelli et al., 2023) and Valdarnese bianca breeds (manuscript work in progress; fig. 1.1) and first characterisation of the Valdarno breed (data elaboration in progress);
- Weight gains (for the Mugellese breed shown in Mannelli et al., 2023 and for the Valdarnese bianca breed shown in Fig 1.2), feed consumption and feed conversion index calculation for the three breeds reared in UniFI farm (for the Mugellese breed shown in Mannelli et al., 2023; for Valdarnese bianca and Valdarno breeds the data validation and processing are in progress), evaluation food consumption with reference to the production of eggs in the families formed after the breeding plan (data collection in progress);
- Daily egg production (for the Mugellese breed reported in Mannelli et al., 2023; for the Valdarnese bianca breed data reported in Fig, 1.3; for the Valdarno breed data collection in progress).
The morphological, growth and deposition data reported for the Mugellese breed and published in Mannelli et al., 2023 are under validation through further data collection and, in order to consider the two liveries as two distinct populations, the families belonging to the two standard liveries have been separated.
Phenotypic characterization of autochthonous breeds and species During the third year of the project, blood samples and phenotypic characteristics were collected on animals of the following species/breeds:
- Collo Nudo Italiana breed (species: Gallus gallus) (n = 48)
- Oca Padovana breed (species: Anser anser) (n = 53)
The collection of the above information led to the completion of the activity of recording morphometric measurements and collecting biological material (blood) required by the TuBAvI-2 project. Table 1 summarizes the qualitative phenotypic characteristics (shank and skin color) of all species/breeds involved in the project, and Figure 1 depicts the descriptive statistics (mean and standard deviation) of quantitative phenotypic characteristics (body weight, body length, shank length, shank circumference, wingspan, sternum circumference) recorded on the same species/breeds. Phenotypic characteristics are presented for males and females within breed and follow the guidelines of FAO (Phenotypic characterization of animal genetic resources. FAO Animal Production and Health Guidelines, 2012).
The experimental test for the characterisation of the Ancona breed has been completed. In particular, the behavioral trial was carried out during the months of June and July 2023, during the recordings the minimum and maximum temperatures of the day were noted. The test, as for the previous breed, produced a total of 12 hours of outdoor recording and 12 hours of indoor recording carried out using the Noldus XT software. The test was carried out using the same devices, structures and methods used for the characterization of the Leghorn breed. For this reason, individual devices (bibs) were applied to the individual animals, each outdoor areas and internal boxes had UWB Anchors positioned at the antipodes, 1 UWB Tag positioned in the centre, 1 UWB Gateway, 1 computer to collect the raw files and 10 chips placed on the bibs (Figure 1). The software was able to produce a raw report for each animal with a precise recording of the behavior every 2 seconds, detecting its spatial position within the four quadrants of the area and the Cartesian axis (x, y, z), indicating the peck and the distance covered (Figure 2).
Phenotypic characterization of a Siciliana breed conservation nucleus As part of this task, 28 Siciliana (SI) breeders were raised at the Molise farm, which had been previously agreed. The birds were organized into 2 families and raised outdoors with access to night shelters, following standard poultry breeding guidelines.
Two incubations of SI breed were carried out to verify the level of fertility and hatchability; the results recorded are shown in table 1.1.
Phenotypic characterisation of Romagnolo (RO) and Ermellinato di Rovigo (ER) turkeys During 2023, 20 RO-breed and 15 ER-breed turkeys were reared at the agreement farm located in the municipality of San Giovanni Rotondo (FG). The live weight of male and female of both breeds was recorded monthly. The mean weights, expressed as mean ± standard deviation from the first day of life to 180 days, are presented in Table 1.2. Mortality recorded up to 6 months was 40%. Oviposition in the two breeds started in January and peaked in the second half of March in the RO breed and at the end of March in the ER breed. A gradual decrease in oviposition rate was observed from the third week of June. The average number of eggs produced by each turkey hen was 65 for the ER breed and 70 for the RO breed. Adult individuals of both breeds (n = 6, RO; n = 5, ER) were subjected to morphometric measurements according to the FAO 2012 guidelines. The mean values of the quantitative traits recorded are presented in Table 1.3. Two incubations of eggs were performed for each breed to assess the level of fertility and hatchability, and the recorded results are presented in Table 1.4.
Fifteen eggs were characterised for the RO breed and ten for the ER breed; the eggs were collected during the peak period. Table 1.5 shows the data related to the phenotypic characterisation of the eggs from the two examined breeds.
Phenotypic characterization of Collo nudo italiana chicken breed In the project proposal submitted, it was planned to maintain the Bianca di Saluzzo breed. Later, in agreement with the other PAs, this breed was replaced by the Collo Nudo italiana breed, as there was no conservation nucleus of this breed yet. In October 2023, the request for replacement of the breed was approved by the Ministry, and activities were started to breed a group of Collo nudo italiana chickens. All individuals (n = 40) were raised outdoor at the Apulian farm. Morphometric measurements were carried out on the available adults and the data obtained are presented in Table 1.6.
The body weight of all subjects, both male and female, was monitored monthly. Table 1.7 reports the average weight values, expressed as mean ± standard deviation, recorded from the first day of life up to 180 days of age. Egg laying was monitored during the period from January to September 2024. It began towards the end of January, reaching its peak (80%) in the second week of March (Figure 1.1). The average number of eggs produced per hen during the reference period ranged between 95 and 110. During the first year of laying (2024), 30 eggs were characterized, and the results are shown in Table 1.8. A preliminary artificial incubation test was carried out on 26 eggs in 2024, with a recorded fertility rate of 84.6% and a hatching rate of 76.9%.
Table 1.9 presents the results obtained. The GLM procedure was employed to determine the effects of the considered variables (breed, sex, and their interactions) on the evaluated immune markers. A significant effect of breed and sex was observed only for LYZ. Significantly higher values of LYZ (ng/mL) were found in ER males, whereas in the RO breed, the highest values of this marker were observed in males compared to females (p<0.05). Although higher levels of C3 (µg/mL) were observed in males of both breeds compared to females, no significant differences were noted. IgY concentrations did not show significant differences between breeds and sexes, but the RO breed exhibited higher concentrations (mg/mL) than the ER breed. Understanding the parameters analyzed provides a preliminary insight into the immune status of these breeds, offering a useful framework for assessing their health and responsiveness to pathogens.
Phenotypic characterisation of fresh semen The assessment of fresh semen quality was carried out for the following turkey breeds: RO and ER. The following quanti-qualitative parameters were evaluated: sperm volume and concentration, cellular membrane integrity (by flow cytometry) total sperm motility, progressive motility, and kinetic parameters (by CASA system with SCA software).
The results obtained from the assessment of fresh semen quality of the native turkey breeds considered are reported in Table 1.10.
Lipid profile analysis was performed on the spermatozoa of SI breed roosters, using the Folch’s method to extract total lipids with a commercial kit (Fatty Acid Extraction Kit, Low Standard – Sigma Aldrich). The total lipids were separated into the neutral lipid classes by thin layer chromatography. Total phospholipid, triglyceride, free fatty acid and cholesterol ester classes were subjected to reflux trans-methylation followed by determination of fatty acid composition by gas chromatography. FAMEs were separated on a capillary column (Zebron ZB-FAME 30 m × 0.25 mm × 20 µm). The fatty acids of the samples were compared with the peak retention times of the 37 FAME standard (Supelco). The results obtained are presented in Table 1.11.
Phenotypic characterisation of Livorno, Siciliana, Bianca di Saluzzo, Bionda Piemontese, and Millefiori Piemontese chicken breeds During the period under consideration, Livorno (L) and Siciliana (S) chicken breeds, hatched in April 2021 and raised at the Poultry Breeding facility of the Department of Veterinary Sciences at the University of Pisa, completed their second laying cycle by October-November 2023. All animals were raised on sandy soil in outdoor enclosures with partial roofing, under natural light and temperature conditions.
Monitoring of daily egg production continued until the end of the laying cycle (see figure 1.1.1); the weight of eggs evaluated in the five central months of the reproductive season is shown in figure 1.1.2 and the shape index of these eggs was equal to 74.1 and 68.9 for the Livorno and Siciliana breeds respectively, highlighting also in the second laying a decidedly different shape of the egg in these two breeds: standard oval the first and sharp oval the second. The assessment of food consumption by second-laying hens was completed by June 2023, and the egg FCE was calculated (see table 1.1.1). Replacement subjects born in April 2023 were monitored for growth, mortality, and morphometric characteristics as outlined in the project (figure 1.1.3 and figure 1.1.4).
The data concerning the Piedmontese breeds Bianca di Saluzzo (BS), Bionda Piemontese (BP), and Millefiori Piemontese (MP) preserved at the Poultry Centre for the Conservation of Local Genetic Resources (Carmagnola, TO – supervised by Prof. Achille Schiavone) have been collected and are made available as part of the technical consultancy contract between UniPI and UniTO starting from 05/03/2022 (refer to the technical report by the UniTO consultant).
Phenotypic characterisation of Ancona (A) breed chickens Monitoring of daily egg production during the second egg-laying cycle of Ancona hens continued until the end of October 2023 (see figure 1.2.1), while egg weight detection concluded in July (see figure 1.2.2).
Characterisation of laying hens’ adaptation capacity to seasonal environmental stimuli In 2023, the qualitative characterisation of the yolk and albumen of the eggs produced during the second cycle of laying of the L and S breeds was completed in June. The egg components were evaluated (see table 1.3.1), and chemical analyses of the yolk and egg white were conducted (see table 1.3.2).
Genetic characterisation of native chicken breeds through the study of polymorphisms in candidate genes involved in heat stress response The genomic data were returned by LGCgenomics to our research unit, which performed an analysis of the data, shown in Table 1. The total samples were 700 and 21 polymorphisms (SNP) in the Heat Shock Protein codifying genes were analysed for each of them (Table 1). Contrary to the expected, only one SNP in analysis was found polymorphic within animals: Chen2015_A-69G_hsp70_R. The following SNP was presented as normal homozygous (T:T) in 98.43% of the animals, as heterozygous (T:A) in 0.42%, and as homozygous for polymorphism (A:A) in 1.14%. The animals showing a heterozygous profile were three in total belonging to the Mugellese and Bionda Piemontese breeds. The animals showing a homozygous profile for polymorphism were 8 in total and belonging to different breeds, among which: Livorno, Modenese, Siciliana and Polverara (Figure 3).
Genetic characterisation using microsatellite markers Genetic characterisation with microsatellite markers is planned to provide useful tools for the conservation of biodiversity in nucleus populations managed by PAs and in the semen Cryobank. Specifically, the following objectives are considered:
- identifying male candidates for replenishment and semen production;
- characterising new genetic lines in breeds under conservation;
- characterising new chicken breeds.
Characterisation of male candidates – For the characterisation of candidates for semen production, 20 subjects of the Ermellinata breed and 16 subjects of Robusta Lionata were analysed. For replenishment and management plans, candidates present in the conservation nuclei of UniMOL, UniFI, UniTO, and UniMI were characterised. The number and breed of genotyped subjects are listed in Table 2.1.
Total 436 subjects from the Gallus gallus species from conservation centres were analysed.
Post-quality control analyses, were completed for all the species involved in the project, namely Gallus gallus, Anser anser, Numida meleagris, and Anas platyrhynchos. For individuals belonging to these species/breeds, the following genetic diversity indices were estimated: observed heterozygosity (Ho), expected heterozygosity (He), minimum allele frequency (MAF), and Runs of Homozygosity (ROH), defined as DNA segments that are identical on both chromosomes within the population and their respective geographic islands (Table 2).
Selection of males for reproduction and semen production Genotyping data were used to calculate the kinship matrix by evaluating the number of alleles shared by each pair of individuals belonging to the same breed. The average kinship among all breeders (P) and the average kinship with family lines (Pf) were then calculated. Principal Coordinate Analysis (PCoA) was used to highlight the existing genetic variability in the sampled groups. The results were graphically represented (Figure 4.1), in order to make the genetic differences between the subjects evident. As an estimate of inbreeding, average observed heterozygosity was analysed, along with number of samples (N), number of different alleles (Na), number of effective alleles (Ne), Shannon Index (I), effective heterozygosity (He), and fixation index (F).
Individual variability index (H-ind) and genetic distance (DPS) were evaluated to establish male selection plans. Based on the results of the previously reported analyses, and in order to maximise male selection efficacy, the individual variability index (H-ind) and kinship index (P or Pf) were combined to create a new index, the conservation index (IC), which is calculated as the difference between H-ind and P/Pf. The conservation index can range from 1 to -1; higher values mean lower consanguinity and higher genetic variability. Estimated indexes were used to evaluate both utilization priorities of specific subjects for reproduction and the genetic variability of males used to produce semen doses to be stored in TuBAvI Cryobank. Results are described in the genetic analysis reports published in the project website, at the corresponding breeds’ pages.
To assess the improvement of genetic resources and monitor the inbreeding levels in the species Gallus gallus, Anser anser, Numida meleagris, and Anas platyrhynchos, specific indices (FHOM and FROH) were estimated for each individual, based on the excess of homozygosity within the genome of the breeds. The results, presented as breed averages, are shown in Table 2. The values, ranging from 0 to 1, indicate a low level of inbreeding and a high level of genetic diversity for the two Gallus gallus breeds. However, for the other species/breeds, the indices suggest a high level of inbreeding and, consequently, a low level of genetic diversity.
Biodiversity Genetic characterisation was performed on two additional chicken populations, Modenese and Valdarno, and on new subject from Mericanel della Brianza, Livorno, and Ancona breeds, finalised to the identification of new bloodlines. The complete dataset was used to calculate each breed’s contribution to biodiversity, through the estimate of genetic distances from the other breeds considered and studied in both TuBAvI and TuBAvI-2 project. The genetic distance matrix, calculated with Nei’s method for all the analysed breeds, is shown in Figure 5.1; lower values indicate a smaller genetic distance, thus similarity among populations, while higher values indicate a bigger distance. The principal component analysis highlights the differences among populations, as shown in Figure 5.2. Modenese clusterises with Livorno (various colours), and shows the highest genetic distance with Robusta Lionata, Ermellinata di Rovigo, Siciliana, and with breeds from Northern Italy; Valdarno is closer to northern breeds, but it is distant from Robusta Lionata, Ermellinata di Rovigo, and Livorno (various colours).
The data from the TuBAvI-2 project for the species Gallus gallus were integrated with those from the previous TuBAvI project for the same species. This allowed the estimation of phylogenetic relationships using Reynolds genetic distances and provided insights into the genetic proximity among breeds within the Italian local poultry heritage (Figure 2). To further enhance the analysis of local poultry bioidiversity, the entire dataset of breeds distributed across the national territory was expanded with genotypes from 167 breeds from around the world. This integration enabled the evaluation of genetic distances between Italian breeds and those from other countries on a global scale, as well as the measurement of biodiversity levels across nations. The resutls indicate that the Italian local poultry heritage exhibits high genetic diversity (Figure 3 and Figure 4).
Genetic characterisation data, described in Action 2, were completed and organised into a dataset, which was used to calculate genetic indexes for the analysed breeds/populations. The results were compared to those from the previous TuBAvI project to evaluate inbreeding increase along generations. The results are summarised in Table 6.1, which compares the following parameters, considered as an estimate of the genetic variability: allelic richness (Ne), observed heterozygosis (Ho), fixation index (F), and molecular kinship (P). Values from project TuBAvI-2 relate to populations genotyped in 2023 and 2024.
Regarding breeds under protection at UniFI, Mugellese and Valdarnese bianca were sampled at the end of 2021 and DNA was extracted from the collected samples, sequenced by third parties and processed to highlight any differences in the microbiota (fig. 7.1).
In this action, in collaboration with the PA UniPI, in 2022, the PA UniFI has evaluated the resilience and resistance to pathologies and environmental stresses of the Mugellese, Valdarnese bianca, Livorno and Bianca di Saluzzo breeds in terms of animal welfare and resistance to pathogens, using the response of the intestinal microbiota to environmental stimuli as an information marker. Subjects belonging to these breeds were bred in free-range and fed with the same feed (Monge) at our UniFI headquarters and at the UniPI headquarters, according to the same scheme. At the end of the test, the caecal feces of each test subject were taken from both sites. From the collected samples, DNA was extracted and sequenced by third parties and processed to highlight any differences in the microbiota (for the Bianca di Saluzzo breed the data is shown in Fig. 7.2).
In the following year 2023, the same experimental design was repeated but with Pepoi, Robusta lionata, Ermellinata di Rovigo and Ancona breeds. DNA sequenced by third parties was extracted from the collected samples. We are awaiting for the sequencing results.
Evaluation of resilience for growth under environmental stimuli In September 2023, the growth test for the Ermellinata di Rovigo (ER), Ancona (ANC), Pepoi (PP), and Robusta Lionata (RL) breeds concluded. This test began with hatching at the end of February and was conducted at the UniPI and UniFI facilities. Figure 7.1.1 shows the body weights and growth rates of the Pisa animals during the first 150 days of life; consumption and ICA data are currently being processed. Total mortality rates (1-150 days of age) were 68.4%, 38.1%, 27.3%, and 25.0% for RL, PP, ER, and ANC, respectively, primarily due to colibacillosis, which manifested itself on the 14th day of the animals’ lives. Figure 7.1.2 displays the average body weights of 120-day-old chickens raised at the two sites.
Evaluation of resistance/resilience to bacterial and parasitic infections/infestations and resilience of intestinal microbiota Table 7.2.1 shows final results of the qualitative and quantitative parasitological analyses to detect coccidia (Eimeria spp.), nematodes (roundworms, capillaries, Heterakis spp.), protozoa (Giarda spp. and Cryptosporidium spp.) and Histomonas meleagridis in stool samples collected from the growing chickens monitored in the previous task.
At the age of 120 days, four individual cloacal swabs were taken from the same chickens at both sites to identify pathogens such as Salmonella spp., Clostridium perfringens, Campylobacter jejuni, and commensal bacteria to obtain isolates for evaluating the antibiotic resistance profile and the selection of strains with favourable probiotic characteristics. All samples from the UniPI site tested negative for pathogens. Table 7.2.2 shows the percentage of animals, by breed and location, in the antibiotic susceptibility classes found in the Lactobacilli isolated from them. Isolates PP57E17M and AN03E15M, from Pepoi and Ancona breed respectively, were susceptible to all the antibiotics tested and identified to belong to Ligilactobacillus salivarius species. These isolates were the only ones to show anti-Salmonella activity, in particular good inhibitory activity especially against S. Typhimurium and S. Newport.
At the end of the monitoring period, individual cloacal faecal sampling was conducted to characterise the intestinal microbiota of the chickens, performed by PA UniFI as part of its research program.
Collection of germplasm for the Semen Cryobank The Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds was implemented and semen collected from the breeds described in Action 1 task 1.4 was stored in liquid nitrogen. Semen doses were prepared according to the cryopreservation procedure for Gallus gallus and Meleagris Gallopavo semen described in the Cryobank SOPs. Semen donors were chosen from reared populations on the basis of quantititative and qualitative characteristics of the semen. Due to the small volume of MO chicken and BR turkey ejaculates, semen doses were produced with breed semen pools. Overall, 149 doses of MO breed semen and 26 doses of BR breed semen were stored. NI breed turkeys did not produce semen, therefore it was not possible to prepare semen doses for the Cryobank. Numerousnesses of semen donors and doses are listed in Table 1.8. Semen doses are conserved in liquid nitrogen in the Cryobank.
Some doses were thawed at 5°C (Cryobank SOPs) to evaluate semen quality after freezing/thawing processing. Average sperm quality parameters of cryoconserved semen from the considered breeds are reported in Table 8.2.
Germplasm collection in the Semen Cryobank of Italian Avian Breeds During the reference period the Cryobank of the Semen of Italian Poultry Breeds was expanded with semen doses of the turkey breeds whose semen was evaluated in Action 1. The seminal material was frozen using the procedure described in the cryobank SOP for the Meleagris gallopavo species. The number of donors and semen doses stored for each breed is reported in the table 8.1.
Using the genotypes obtained from the TuBAvI-2 project and the previous TuBAvI project for the species Gallus gallus, an investigation was conducted to identify genes involved in the pigmentation of eggshells and shank coloration (Table 3).
Additionally, with the genotypes of animals belonging to the species Anas platyrinchos (breeds: Mignon and Germanata Veneta), a genomic-wide association study was performed. The Mignon and Germanata Veneta had divergent morphometric characteristics, as evaluated during the implementation of Action 1. Therefore, the genome-wide association analysis enabled the identification of candidate genes associated with the distinct developmental traits of the two breeds (Table 4).
