Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique

assessment of cortisol and dhea concentrations in griffon vulture gyps fulvus feathers to evaluate its allostatic load

AbstractThe use of a non-invasive approach to collect biological samples from natural populations represents a great means of gathering information while avoiding handling animals. Even if corticosterone is the main glucocorticoid investigated in birds, there has been observed a proportional direct link between corticosterone and cortisol concentrations. Dehydroepiandrosterone (DHEA) can be produced by the adrenal cortex and should have prominent antiglucocorticoid properties also in birds. The aim of this study was to verify if there is any difference in the cortisol and DHEA feather concentrations between clinically normal and physiologically compromised Griffon vultures (Gyps fulvus) through the non-invasive approach of collecting moulted feathers without having to pluck them from the bird. The study was carried out using 8 physiologically compromised (PC) Griffons and 9 clinically normal Griffons considered as the control (CTRL) group that were necropsied or from the wildlife rehabilitation centre. Primary and secondary covert feathers were either collected directly from the birds' cage floors, or, in the case of dead Griffons, they were plucked off the animals. The results, obtained by RIA, revealed that both cortisol (P<0.01) and DHEA (P<0.05) feather concentrations were higher in the PC than in the CTRL group. No difference was observed by comparing the cortisol/DHEA ratio between the two evaluated groups (P=0.15). Pearson's correlation coefficients showed no correlation between feather hormone concentrations in the PC group (r=0.01, P=0.96) while a positive correlation in the CTRL group (r=0.65, P=0.006) was observed. In conclusion, our study reveals that moulted feathers can be a non-invasive and an interesting tool to evaluate the allostatic load of wild birds and they allowed better understanding the relationship between hormones of the hypothalamic–pituitary–adrenal axis and the physiological status of the birds

Sequenced RAPD markers to detect hybridization in the barbary partridge (<i>Alectoris barbara</i>, Phasianidae)

In the Alectoris partridges (Phasianidae), hybridization occurs occasionally as a result of the natural breakdown of isolating mechanisms but more frequently as a result of human activity. No genetic record of hybridization is known for the barbary partridge (A. barbara). This species is distributed mostly in North Africa and, in Europe, on the island of Sardinia (Italy) and on Gibraltar. The risk of hybridization between barbary and red-legged partridge (A. rufa: Iberian Peninsula, France, Italy) is high in Sardinia and in Spain.We developed two random amplified polymorphic DNA (RAPD) markers to detect A. barbara x A. rufa hybrid partridges.We tested themon 125 experimental hybrids, sequenced the relative species-specific bands and found that the bands and their corresponding sequences were reliably transmitted through a number of generations (F1, F2, F3, BC1, BC2). Our markers represent a highly valuable tool for the preservation of the A. barbara genome from the pressing threat of A. rufa pollution

Lack of detection of West Nile virus in an islander population of chelonians during a West Nile virus outbreak.

In 2011, several outbreaks of West Nile disease occurred in Sardinia (Italy). The region hosts several chelonian species. Because of the increasing concern on the potential role that ectotherms may play in the ecology of West Nile virus (WNV), in October 2011 blood samples were collected from 41 endemic Sardinian chelonians and tested for the presence of active WNV infection or neutralizing antibodies by real time polymerase chain reaction (RT-PCR) and serumneutralisation, respectively. Neither WNV neutralising antibodies (0%; 95% CI: 0‑8.4%) nor WNV RNA (0%; 95% CI: 0‑6.8%) were found in the tested samples. According to the results of this screening survey, it is unlikely that chelonians are involved in the epidemiology of the 2011 WNV outbreaks in Sardinia

Identification and characterization of novel <i>Mycoplasma</i> spp. belonging to the hominis group from griffon vultures

Mycoplasmas are commensals and pathogens of various avian species, and are also regularly found in birds of prey, although their significance to birds&#x2019; health remains unclear. Here we describe two novel Mycoplasma isolated from the upper respiratory tract of four Eurasian griffon vultures (Gyps fulvus) housed in a wildlife recovery centre in Sardinia (Italy). By sequencing the 16S rRNA gene and the entire 16S/23S intergenic spacer region, the new strains were classified within the Mycoplasma taxonomy at the group and cluster levels, showing that the two isolates fall into the Mycoplasma synoviae and Mycoplasma hominis clusters of the hominis group, respectively. We combined molecular tools and immunoblotting methods in order to further characterize these isolates, and antigenic analyses overall confirmed the molecular findings. Different levels of pathogenicity and prevalence of these strains might have different implications for the conservation and reintroduction of vultures

Differences in semen freezability and intracellular ATP content between the rooster (<i>Gallus gallus domesticus</i>) and the Barbary partridge (<i>Alectoris barbara</i>)

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P &lt; 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P &lt; 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P &lt; 0.0001), while ATP values significantly decreased after thawing (P &lt; 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P &lt; 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing. In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezabililty between the two species

Post‐mortem recovery, in vitro maturation and fertilization of fallow deer (Dama dama, Linnaeus 1758) oocytes collected during reproductive and no reproductive season

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post‐mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post‐thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post‐thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.Peer reviewe

Feed Preference, Daily Intake, and Laying Performance of Captive-Born Sardinian Partridges (<i>Alectoris barbara barbara</i> Bonnaterre, 1790) Offered Whole Defrosted Mealworms (<i>Tenebrio molitor</i> L., 1758) as Raw Feed Material with Diet

The competence to locate natural feeding sources is one of the main limiting factors for survival in the wild, especially for captive-born birds. Therefore, environmental enrichment through the diet can be strategic before their release into nature. In this research, a feeding trial was undertaken to evaluate the potential use of yellow mealworm (Tenebrio molitor L.) larvae (TM) provisions to captive bred couples of Sardinian partridges (Alectoris barbarabarbara Bonaterre, 1790) during the laying period. Twenty-four couple-caged Sardinian breeding partridges were enrolled during the laying period (April–May 2019) and randomly allotted to two feeding groups of 12 couples each: (a) the control (CON) group was fed a conventional complete pelleted diet for laying quails; (b) the yellow mealworm enriched group was additionally fed 5% whole, defrosted TM larvae (TM5%) on top of the same amount of the control diet. As a prerequisite, partridges were unaccustomed to eating mealworms before the start of the trial. Daily feed intake (DFI), bodyweight (BW), and number of laid eggs (LE) were monitored over five weeks of experimental feeding. Partridges fed the TM5% diet displayed a higher preference for whole mealworms (first choice and complete consumption) than expressed for the CON pelleted feed. Differences in daily dry matter intake (p = 0.028) between CON and TM5% groups were observed (DMI: 42.6 ± 1.73 vs. 43.4 ± 1.62 g, respectively); the final body weights (BW) (p = 0.098) of birds in the CON group was higher than those in the TM5% group (435 ± 36.9 vs. 416 ± 36.3 g, respectively). Differences in daily energy intake relative to BW (p 0.75) (p < 0.001) were observed between groups, but this was not followed by higher BW, probably due to the absence of grit and inaccessible nutrients and energy (larval exoskeleton). No difference in the average LE per week and egg weight was observed between CON and the enriched TM5% groups, though in the last weeks, a statistically lower number of eggs was laid in TM5% group. Our results suggest that whole yellow mealworms can be a promising feed material to broaden the spectrum of competence for natural feeding sources with similar physical form and nutritional characteristics available in the environment

ASSESSMENT OF CORTISOL AND DHEA CONCENTRATIONS IN THE GRIFFON VULTURE (Gyps fulvus) FEATHERS TO EVALUATE ITS HEALTH CONDITION

During their life, birds face several challenges which can threaten the stability of physiological functions. These situations typically trigger a cascade of endocrine secretions involving the hypothalamic–pituitary–adrenal (HPA) axis, and resulting in the release of both cortisol and corticosterone also in birds. The use of a non-invasive approach to collect samples of biological material derived from natural populations represents a greatly combination for an improvement of knowledge avoiding handling animals. Steroid concentrations in feathers are mainly acquired from the capillary around the follicle during the long period of feather grown reflecting their bloodstream concentrations. Accordingly, a chronic HPA axis activation could be assessed using molted feathers cortisol concentrations. Another hormone, dehydroepiandrosterone (DHEA), is secreted by adrenals and it could be assessed in molted feathers. This study was aimed to evaluate the feasibility of using feathers cortisol and DHEA concentrations to provide a retrospective assessment of the activity of the HPA axis in griffon vulture acquired for health monitoring purposes. For this, we introduced two groups of animals with a known health condition: one group composed by griffons that have experienced a long-term physiologically compromised period (PC groupn=8) and the other group composed by griffons physiologically not compromised (CTRL groupn=9). The feathers cortisol and DHEA analyses were carried out using RIA [1,2] adapted for feathers. The results revealed different feather hormone concentrations between the two groups of animals. The feathers cortisol had a median value higher in the PC group than in the CTRL group, with cortisol concentrations showing a central value 1.6 times higher in those animals that suffered a long-term physiological impairment. The PC group showed also (Kruskal- Wallis test) higher feather concentrations of DHEA (P=0.01) than the CTRL group. Pearson’s correlation coefficients were used to examine the associations between cortisol and DHEA, showing no correlation between cortisol and DHEA feather concentrations in the PC group (r=0.18, P=0.34) and a moderate positive correlation in the CTRL group (r=0.51, P=0.011). In conclusion, our study reveals that molted feathers can be an interesting way to evaluate the physiological status of wild animals by using a non-invasive approach. Our analyses reveal that in addition to cortisol also DHEA could be evaluated to better understand the relationships between these hormones and to determine the resilience condition of wild species