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.