DISCUSSION
Our main objective was to determine if manipulation associated with hacking could have harmful consequences on health and nutritional condition of released young, after an average of 44 d of ad libitum food and no contact with parents. We found no harmful effects across many hematological and biochemical parameters. Alternatively, our results demonstrated important positive changes in health and nutritional parameters.
Body mass of nestlings varied significantly between samples, being lower in the second sample. This decrease in mass was similar for both sexes. The mass of nestling birds increases with increasing age, peaks several days prior to fledging, and then decreases until the day of fledging (Ricklefs, 1968; Morbey et al., 1999; Gray and Hamer, 2001; Sprague and Breuner, 2010). One possible function of such pre-fledging mass recession may be to induce fledging i.e. nestlings lose mass because parents reduce provisioning rates to induce fledging when it is no longer beneficial for young to remain in the nest (Morbey et al., 1999). Another possible function of pre-fledging mass recession is that it helps optimize wing loading for newly fledged young (Ricklefs, 1968; Shultz and Sydeman, 1997; Morbey et al., 1999). Our results support the second hypothesis since food was provided ad libitum throughout the hacking period, without parental influence and any variation during the pre-release (‘pre-fledgling’) period. Consequently, our results suggest a physiological mechanism, probably reducing the total amount of water in the body, as the major cause of weight recession before first flight (Ricklefs, 1968).
Hemoglobin and PCV showed a significant decrease during hacking, which indicates a diminution in the concentration of red blood cells after being fed ad libitum for more than one month. These results suggest a process of hemodilution associated with the ad libitumfood (Garcia-Rodriguez et al., 1987a), supported by the significant increase in blood proteins, globulin and albumin. Blood proteins are essential to exert a colloidal osmotic pressure in order to aid in the preservation of blood volume and blood pH within a narrow range (Strukie, 1976). Consequently, an increase in blood proteins cause an increase in blood osmotic pressure, which would cause an elevation of plasma liquid, generating a temporal hemodilution effect, which induces a higher transitory blood volume (Garcia-Rodriguez et al., 1987a).
Results showed a general increase in cells related with the immune system: total white blood cells, heterophils, eosinophils and basophils. These results indicate an improvement of the immune system of young eagles after the hacking period, suggesting a general health improvement.
Results also showed that glucose levels, and glucose values decreased during hacking, which is in accordance with results for other raptor species (Garcia-Rodriguez et al., 1987a). The physiological explanation for this decrease could be that in a situation of long-term undernutrition, the glucose regulation by insulin would be relaxed. In this case, the utilization of glucose by the cells could decrease and, therefore, produce an increase in the plasma glucose (Groscolas and Rodriguez, 1981).
Cholesterol is associated with fat metabolism (Garcia-Rodriguez et al. 1987a; Alonso-Alvarez et al., 2003) and levels increased after the hacking period. Cholesterol concentrations increased in other eagles when birds were adequately fed, as also in owls, vultures and kites (Ferrer et al., 1987). These increases in cholesterol may be merely caused by the increased lipid ingestion and absorption, and subsequent cholesterol synthesis (Ferrer and Dobado-Berrios, 1998). In Yellow-legged Gulls (Larus michahellis ), cholesterol level was the strongest correlated parameter with cumulative body-mass loss during fasting (Alonso-Alvarez and Ferrer, 2001; Alonso-Alvarez et al., 2002).
Creatinine kinase CK is related to muscular activity (Alonso-Alvarez et al., 2003). Levels of plasma CK were higher towards the end of hacking. CK is found primarily in skeletal muscle, heart muscle, and brain tissue and is often used as a diagnostic indicator of muscle damage (Meredith et al., 2012). Muscle cell disruption and enzyme leakage damage can occur when muscle strength and elasticity is exceeded by exercise effort. With training, muscle cells’ hypertrophy and neuromuscular coordination improves; thus, less cellular disruption occurs and trained individuals have lower CK. In Red-tailed Hawks (Buteo jamaicensis ), CK levels in birds that had not flown rose dramatically above baseline in the 24 h after a short period of exercise compared to a non-significant rise from a higher baseline in flight-trained hawks (Knuth and Chaplin, 1994). Consequently, the rise in CK plasma levels in our WTE could be related to increasing flight exercise as they mature: increased wing flapping was observed in hacked birds in days prior to (and conditional on) their release. At the age of the first sample, the eagles in this study had never flown and, thus, it could be hypothesized that their “untrained” muscles might be more susceptible to enzyme leakage (Meredith et al., 2012).
Calcium levels vary according to ossification process (Viñuela et al., 1991; Dobado-Berrios and Ferrer, 1997) and calcium concentration significantly increased after hacking period. This increase is to be expected in the growth of nestlings and we do not have comparable data on what may be expected for wild nestlings across the same time period.
Urea and uric acid showed a highly significant decrease after the hacking period, suggesting a decrease in catabolism rates due toad libitum food. High values of urea have proven to be good indicators of undernourishment (Garcia-Rodriguez et al., 1987a; Ferrer et al., 1987; Polo, 1995). Urea is a minor pathway for protein degradation in birds but the activity of liver arginase (the enzyme on which urea production in birds depends) increases after a prolonged fast, and hence the rise of urea during protein catabolism may be explained by a greater arginine availability (Garcia-Rodriguez et al., 1987a). Consequently, our results on urea and uric acid levels indicate improved nutritional conditions of young WTE due to hacking.
Interestingly, not only was there a change in urea concentration during hacking, but a change in the form of the distribution at the two sampling events. The first samples showed a quasi-Poisson distribution, with a right tail of elevated urea concentrations. Contrarily, second samples showed a normal distribution of urea values. In conclusion, the hacking period not only decreased the mean urea value but decreased the dispersion and homogenized the nutritional levels of young eagles. This effect could have interesting consequences on subsequent juvenile dispersal distances, as demonstrated in Spanish Imperial EaglesAquila adalberti (Ferrer and Morandini, 2017). It has been frequently found that females reproducing earlier in the season produced larger broods with offspring in better physical condition (Klomp, 1970; Newton & Marquiss, 1984; Ferrer, 1994; Moreno et al., 1997; Casado et al. 2002). For example, early hatched Spanish Imperial Eagle nestlings were better nourished than later ones (Ferrer, 1994; Muriel et al. 2015). In our study, that standardized hatching dates were only strongly correlated with the first sample but not with the second gives support to hacking improving nutritional conditions throughad libitum food, dissipating nutritional differences among donor birds from different territories.
In summary, it has already been established that hacking techniques, when properly applied, should not affect the subsequent dispersal behavior of released young (Morandini and Ferrer, 2017a; Ferrer and Morandini, 2017), whose survival probabilities are equal or slightly higher than wild birds (Evans et al., 2009; Muriel et al., 2020), and whose productivity in their later breeding life is equal or higher than wild birds (Morandini et al., 2017, 2019; Murgatroyd et al., 2018). Our novel study demonstrated that certain health indicators, especially those related with the immune system and nutritional parameters, also showed a significant improvement after the hacking period. Consequently, our study is consistent with others showing no harmful effects in hacking techniques that should prevent their use. This is relevant because wildlife reintroductions are a potentially important tool for conservation of endangered or threatened species, and their use is likely to increase in the future.