Basal levels of inorganic elements,genetic damages,and hematological values in captive Falco peregrinus

It is essential to determine the basal pattern of different biomarkers for future evaluation of animal health and biomonitoring studies. Due to their great displacement capacity and to being at the top of their food chains, birds of prey are suitable for monitoring purposes. Furthermore, some birds of prey are adapted to using resources in urban places, providing information about this environment. Thus, this study determined the basal frequency of micronuclei and other nuclear alterations in peripheral blood erythrocytes of Falco peregrinus. Hematological and inorganic elements analysis were also performed. For this purpose, 13 individuals (7 females and 6 males) were sampled in private breeding grounds. Micronucleus, nuclear buds, nucleoplasmic bridges, notched nuclei, binucleated cells and nuclear tails were quantified. Inorganic elements detected included the macro-elements Ca, P, Mg, Na, Cl, S and K as well as the micro-elements Fe, Al and Zn. Our study found similar values compared to previous studies determining the reference ranges of hematologic parameters in falcons. The only different value was observed in the relative number of monocytes. Thus, this study is the first approach to obtaining reference values of cytogenetic damage in this species and could be useful for future comparisons in biomonitoring studies. Keywords: Biological monitoring, basal DNA damage, falcon, reference value, hematology

In recent years, different organisms have provided information about environmental quality (Parmar et al., 2016). Birds of prey due to their great displacement capacity and to being at the top of the food chains are suited for monitoring purposes (Lodenius and Solonen, 2013). Top bird predators have been evaluated regarding exposure to pesticide, metal and other chemical substances (Lodenius and Solonen, 2013; de Wit et al., 2019; Aver et al., 2020; Frixione and Rodríguez-Estrella, 2020). In biomonitoring studies, it is possible to evaluate biomarkers at the molecular, cellular, morphological and physiological levels in different species. Among the available bioassays, there are several that are more invasive or less invasive to animals (Valdes, 2010). A technique that estimates the exposure level in DNA without having to sacrifice the organism exposed is the Erythrocyte Nuclear Abnormalities (ENA) assay. The ENA assay is considered a proper tool for detecting the DNA damage because its analysis includes micronuclei and the other nuclear alterations analyses (Gomes et al., 2015). Most studies about genotoxicity in birds include only micronuclei in analysis, excluding the other nuclear variations, which may be more frequent than micronuclei. Nuclei with smaller or larger evaginations, nuclei with vacuoles and nuclei with a deep slit are alterations that can also be observed (Carrasco et al., 1990; Quero et al., 2016; de Souza et al., 2017; de Faria et al., 2018; Silveira et al., 2022).Some birds of prey, such as Falco peregrinus, have adapted to using resources in urban areas. According to Pollack et al. (2017) these species provide information, especially about the widespread consequences of urbanization, giving an insight into its influence on animal behavior and physiology, as well as guiding investigations in humans. The aim of this study was to determine the frequency of micronuclei and other nuclear alterations in peripheral blood erythrocytes of Falco peregrinus, as a first approach to obtain reference values of cytogenetic damage in this species. Furthermore, knowledge was obtained regarding other physiological parameters, such as hematological analysis and the quantification of inorganic elements.All birds used in this study belong to Criatório Hayabusa - Consultoria Ambiental Ltda., a wildlife center and commercial breeder located in São Francisco de Paula (Rio Grande do Sul, Brazil). The area has approximately 48 ha and is considered to be in an excellent state of conservation, distant from urban areas and areas where crops are cultivated. The individuals are housed in outdoor aviary cages (4 m (width) x 4 m (height) x 4 m (length)) containing a couple of birds each. The birds had been captive for at least four years and were fed daily with Coturnix coturnix and water ad libitum. The sex of the birds was determined through external sexual size dimorphism, wherein the male can carry up to 50% less loads than the female (Mills et al., 2019). In addition, the bird’s age (juvenile/adult) was defined according to information on a metal ring.The procedures involving animals were conducted in compliance with the guidelines approved by the Committee on Ethics in the Use of Animals of the Universidade La Salle (CEUA-UNILASALLE, number 003/2017), and authorized by the Ministry of the Environment (MMA) through the Sistema de Autorização e Informação da Biodiversidade (SISBIO) for scientific activities (number 59921-1).Blood samples of 13 individuals, collected by the center’s veterinarian, were drawn from the ulnar vein of the wing using heparinised syringes. The samples were immediately smeared onto clean glass slides, where two slides were prepared per individual. The slides were sent to the laboratory. Remaining blood samples were also transported to the laboratories at below 8 ºC for the analysis of hematological and inorganic elements. The animals were identified as to sex and age (juvenile/adult). All the birds sampled were apparently healthy, without any signs of illness.In the laboratory, the slides were prepared according to Grisolia and Cordeiro (2000). At least 2,000 erythrocytes for each animal were scored using bright-field optical microscopy at a magnification of ×200-1000. Coded slides were blind-scored by a single observer. The presence of ENA was evaluated according to procedures by Carrasco et al. (1990) and Quero et al. (2016), using mature erythrocytes to estimate the frequency of the following nuclear lesions: (i) micronuclei (MN); (ii) nuclear buds (NBud); (iii) binucleated cell (BN); (iv) nuclear tails (NT); (v) nucleoplasmic (NB); and (vi) Notched (NO).The content of inorganic elements in the blood samples was analyzed by particle-induced X-ray emission (PIXE) (Johansson et al., 1995). As the PIXE system requires the use of solid samples, the blood samples were dried at 60 °C. Once dried, the samples were homogenized and pressed into 2 mm thick pellets before being placed in the target holder inside the reaction chamber (pressure about 10^-5 mbar). A 3 MV Tandetron accelerator provided a 2.0 MeV proton beam with an average current of 3 nA at the target. The X-rays produced in the samples were detected by a Si(Li) detector with an energy resolution of ca. 150 eV at 5.9 keV. The spectra were analyzed with the GUPIX software package and the results were expressed in mg/g (Campbell et al., 2000). The same sample was evaluated in three independent analyses in order to obtain mean and standard deviation.The hematological evaluation was carried out in a commercial laboratory (BLUT’S Centro de Diagnóstico, Produtos e Serviços Veterinários, Porto Alegre-RS, Brazil) according to standard methods. Together with the results of the present study, information about other studies were taken as reference to interpret hematologic parameters.The normality of the variables was evaluated using the Kolmogorov-Smirnov test. To compare the parameters of the study population, Studentt-, and Mann-Whitney U non-parametric tests were used. The critical level for rejection of the null hypothesis was considered to be P < 0.05.The interspecific variations in the spontaneous frequencies of nuclear alterations are probably related to the intrinsic individual factors associated with ingestion, accumulation, metabolism and excretion of the xenobiotics to which the organism is exposed daily. Furthermore, the correct functioning of the DNA repair also could be involved in this interspecific response to DNA damage (Jha, 2004). Information on 13 animals sampled is presented in