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\begin{document}

\title{Hacking Techniques Improve Health and Nutritional Status of Nestling
White-tailed Eagles}



\author[1]{Miguel Ferrer}%
\author[2]{Rhian  Evans}%
\author[3]{Joanna Hedley }%
\author[4]{ Simon  Hollamby}%
\author[5]{Anna Meredith}%
\author[6]{Virginia Morandini}%
\author[2]{Owen  Selly}%
\author[2]{Claire  Smith}%
\author[7]{Phil Whitfield}%
\affil[1]{Estación Biológica de Doñana}%
\affil[2]{Royal Society for the Protection of Birds Scotland Headquarters}%
\affil[3]{Royal Veterinary College}%
\affil[4]{Melbourne Veterinary School}%
\affil[5]{University of Edinburgh}%
\affil[6]{Oregon State University}%
\affil[7]{Natural Research Ltd}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Birds of prey frequently feature in reintroductions and the hacking
technique is typically used. Hacking involves removing large nestlings
from donor populations, transferring them to captivity, feeding them ad
libitum. Potentially, via the hacking method, stress of captivity and
disruption of parental feeding may be detrimental. Alternatively,
provision of ad libitum food may be advantageous. Although hacking has
underpinned reintroduction project successes there has been no research
on how the method may affect the health and nutritional status of
translocated birds during captivity. We compared blood chemistry data
from 55 young White-tailed Eagles, translocated from Norway as part of
the species' reintroduction to Scotland, from sampling soon after
arriving in captivity and again ([?] 42 d later) before their release.
Numerous significant differences between first and second samples were
found, but no significant interactions showed that sexes responded
similarly to captivity. According to hematological and biochemical
metrics, individuals showed several changes during captivity, including
in red blood cell parameters, plasma proteins and white cellular
parameters related to the immune system, that indicated improved health
status. Captivity with ad libitum food was associated with decreased
urea and uric acid values: high values can indicate nutritional stress.
Urea values became more normally distributed before release, indicating
that ad libitum food had reduced nutritional differences between early
nestlings in the season and later ones. Despite plentiful food, both
sexes lost body mass before release, suggesting an inherent
physiological mechanism to improve flight performance in fledglings. We
conclude that hacking improved the health and nutritional status of
released eagles which is likely to enable birds to cope with greater
costs of exploratory behavior which they may require in reintroduction
projects. In this context, we note the absence of survival differences
between hacked and wild raptors in previous research.%
\end{abstract}%



\sloppy


\textbf{Abstract}

Birds of prey frequently feature in reintroductions and the hacking
technique is typically used. Hacking involves removing large nestlings
from donor populations, transferring them to captivity, feeding
them\emph{ad libitum} . Potentially, via the hacking method, stress of
captivity and disruption of parental feeding may be detrimental.
Alternatively, provision of \emph{ad libitum} food may be advantageous.
Although hacking has underpinned reintroduction project successes there
has been no research on how the method may affect the health and
nutritional status of translocated birds during captivity. We compared
blood chemistry data from 55 young White-tailed Eagles, translocated
from Norway as part of the species' reintroduction to Scotland, from
sampling soon after arriving in captivity and again ([?] 42 d later)
before their release. Numerous significant differences between first and
second samples were found, but no significant interactions showed that
sexes responded similarly to captivity. According to hematological and
biochemical metrics, individuals showed several changes during
captivity, including in red blood cell parameters, plasma proteins and
white cellular parameters related to the immune system, that indicated
improved health status. Captivity with \emph{ad libitum} food was
associated with decreased urea and uric acid values: high values can
indicate nutritional stress. Urea values became more normally
distributed before release, indicating that \emph{ad libitum} food had
reduced nutritional differences between early nestlings in the season
and later ones. Despite plentiful food, both sexes lost body mass before
release, suggesting an inherent physiological mechanism to improve
flight performance in fledglings. We conclude that hacking improved the
health and nutritional status of released eagles which is likely to
enable birds to cope with greater costs of exploratory behavior which
they may require in reintroduction projects. In this context, we note
the absence of survival differences between hacked and wild raptors in
previous research.

\textbf{Keywords: blood parameters, hatching date, plasma biochemistry,
hematology, urea, cholesterol, free-living raptor}

\textbf{INTRODUCTION}

The global loss of biodiversity is well-documented, with increasing
numbers of species at risk of extinction due to direct or indirect
anthropogenic causes (Mora et al., 2013; Seddon et al., 2014).
Management to reduce the risk of species' extinction includes a wide
variety of actions, including reintroduction. Reintroductions are
intentional translocations of species into parts of their historically
known range from which they have been extirpated (IUCN, 2013). Wildlife
reintroductions are becoming increasingly common, being now considered
an important tool for the conservation of endangered or threatened
species (Armstrong and Seddon, 2008; Soorae, 2016).

Birds of prey (raptors and vultures) are frequently involved in
reintroduction programs. Several such programs have been successful and
typically involve a common practical method: `hacking' (Evans et al.,
2009; Ferrer et al., 2018). This method involves taking large nestlings
(typically when around two-thirds toward fledging in growth) from a
robust donor population and transferring them to the incipient novel
population via hacking facilities. Usually, donor nestlings are
translocated to artificial nest(s) inside hacking facilities
(effectively large aviaries) situated geographically in the recipient
area for the new population. During this period translocated birds are
fed \emph{ad libitum} until they are released from aviaries once it is
judged that the birds are capable of free flight (effectively simulating
`natural' fledging age: Muriel et al., 2011).

Reintroductions by means of the hacking method appear especially
effective in birds of prey (Cade, 2000; Evans et al., 2009; Ferrer and
Morandini 2017, Morandini and Ferrer 2017a) and therefore can be used as
potentially useful tools for population restoration with appropriate
planning, development and monitoring (IUCN, 2013; Morandini and Ferrer,
2017b). There is evidence that in some species at least, manipulation
associated with hacking (extraction of the nestlings, ringing and
tagging them, unlimited amounts of food, keeping them in captivity for
some period, absence of parents) seems to have non-harmful effects on
survival, subsequent reproduction, habitat selection or dispersive
behavior (Morandini and Ferrer, 2017a; Ferrer and Morandini 2017;
Morandini et al., 2017, 2019; Muriel et al., 2015, 2016, 2020).
Nevertheless, as far as we know there are no studies on potential
consequences of health and nutritional condition due to the hacking
method, where food is provided \emph{ad libitum} and there is no contact
with the parents after translocation.

Avian blood chemistry and blood cell analyses can detect possible
pathological states (Meredith et al., 2012). An adequate knowledge of
hematological values is recommended for projects involving research and
management of populations since they can be valuable for the assessment
of the nutritional levels and health status of constituent individuals
(Ferrer and Dobado-Berrios, 1998; Meredith et al., 2012; Ferrer et~al.,
2017a, b). Despite the technology to analyze concentrations of blood
constituents being widely available and well understood, studies of
blood parameters in free-living raptors are still few (Vi\selectlanguage{ngerman}ñuela et al.,
1991; Dobado-Berrios and Ferrer, 1997; Ferrer and Dobado-Berrios, 1998;
Meredith et al., 2012; Hernandez and Margalida, 2010; Montolio et al.,
2018; Flo et al., 2019). Hematological values, including chemical
components, are known to be influenced by many factors: physiological
state, age, sex, nutritional condition, circadian rhythm, seasonal
changes, captivity, pollutants, and plasma storing methods (Gee et~al.,
1981; Rehder and Bird, 1983; Ferrer et~al., 1987; Garcia-Rodriguez
et~al., 1987a, b; Viñuela et~al., 1991; Jenni-Eiermann and Jenni, 1992).

Translocations and reintroductions seem likely to be used more
extensively in the future, especially in the face of rapid global
changes and corresponding distributional shifts of certain species, but
also with regard to the favorable socio-ecological conditions that
remain within the former ranges of threatened species. Consequently, it
is important to improve our understanding of the limitations and
applications of associated techniques, sharing results and thus
increasing our expertise in wildlife restoration strategies.

The aim of this study was to analyze the potential effect of hacking on
health and nutritional status of White-tailed Eagle
(WTE:\emph{Haliaaetus albicilla} ) nestlings, in over 50 birds
translocated from western Norway to eastern Scotland. Blood samples were
taken at two events and subsequently analyzed using hematological and
biochemical metrics. The first sample was obtained when birds had been
transferred to Scotland soon after translocation, and the second after
birds had been held in captivity and fed \emph{ad libitum} for c. 44 d
shortly before their release from captivity. We used this double
sampling (including biometric data) across the hacking period, to
examine if the period of captivity had caused any deterioration in
health or nutritional status. Alternatively, the hacking method may have
been beneficial with \emph{ad libitum} food improving the health and
nutritional status of translocated birds during captivity.

\textbf{MATERIALS AND METHODS}

\textbf{Study species and area}

WTE is a large bird of prey with females being larger than males. The
species is Eurasian in distribution and ecologically the Old World
equivalent of the New World's Bald Eagle (\emph{Haliaeetus
leucocephalus} ). Our study was involved with the second phase (east
Scotland) of a reintroduction initiative instigated by an initial (1975
-- 1985: 82 birds) reintroduction and later reinforcement translocations
(1993 -- 1995: 59 birds) using birds from western Norway to the west
coast of Scotland (Green et al., 1996; Evans et al., 2009; Whitfield et
al., 2009a, b; Sansom et al., 2016). The core initiative allowing our
study aimed to provide a further nucleus of WTE population growth on the
east coast of Scotland, situated away from the previous centers of
population re-establishment and subsequent expansions on the west coast.

We sampled nestlings extracted from a wild donor WTE population in
Norway: every June between 2007 and 2012 WTE nestlings were collected
from monitored nests in the western counties of Møre og Romsdal,
Hordaland and Sogn og Fjordane. Single birds were extracted from nests
that had two or three chicks which from field observations were deemed
old enough (five to eight weeks old) to be suitable for translocation on
plumage features and physical appearance (Helander, 1981). Extractions
were coordinated with the goal of a balanced sex ratio (based on
biometrics). Birds were uniquely metal banded (BTO: British Trust for
Ornithology) in Norway prior to translocation, for subsequent
identification.

There were extractions of 15 (2007, 2008, 2009), 19 (2010), 16 (2011)
and six (2012) nestlings per annum, resulting in 86 translocated birds
(85 were released as one 2009 bird died in captivity from aspergillosis,
despite veterinary intervention). Of these, 55 were double sampled for
biochemical and hematological parameters 2009 -- 2012 and are the
subject of this study.

After transport from Norway, all birds had a full clinical examination
within 24 h of arrival to identify any potential abnormalities.
Translocated birds were kept at a hacking facility in northeast Fife,
southeast Scotland, in wooden (three sides enclosed) and wire mesh
aviaries measuring approximately 3.6 x 3 x 2.7 m that had `nesting'
platforms covered in bark chips and soft vegetation, and at least two
long perches. Two or three birds were housed in each aviary, with
similarly sized birds being kept together. Birds were supplied
with\emph{ad libitum} food involving the wide range exploited by WTE
(e.g. Whitfield et al., 2013): fish, birds and mammals, with vitamin and
mineral supplements (Nutrobal®, Vertak, UK) added to the food daily.
Contact with humans was minimized by food being provided via a hatch in
the aviaries' back panel. Captive eagles were kept under daily
observation via `spy holes' in aviaries' back panels for surveillance of
their status and any signs of injury or ill-health, with a qualified
veterinarian on call should the need arise: no eagles were injured
because of our activities.

The first sampling was undertaken shortly after all birds were in the
hacking facility. This allowed for greater efficiency in sampling all
birds at the same time after extraction and transportation from Norway
to Scotland (Table 1). The second sampling was taken shortly before
birds' release from captivity when they were also fitted with patagial
wing tags or color-bands and a VHF telemetry tag, when biometric data
(including body mass) were again taken (Table 1). Birds were aged (days
since hatch date) using the method of Løseth et al. (2019): after T.
Nygård pers. comm.) involving measurement of a central tail feather.
This method was preferred to those in Helander (1981) and Helander et
al. (2007) involving other recorded biometrics when like Løseth et al.
(2019) we also found the ``Nygård'' method to produce more realistic and
consistent results.

\textbf{Blood collecting procedures and sex determination}

The first blood samples were drawn from nestlings 41 - 66 d (mean =
52.4, SD = 5.7) after their estimated hatch, corresponding to the
beginning of hacking. The second samples were drawn when nestlings were
between 86 - 108 d old (mean = 97.6, SD = 5.5), just prior to their
release (Table 1). A total of 55 nestlings, 26 males and 29 females,
were double sampled. Intervals between repeat samples of the same
individual were between 42 - 47 d (mean = 44.0, SD = 2.0).

Blood samples were collected between 11:00 AM and 15:00 PM to avoid
variations in blood parameters due to circadian rhythms
(Garcia-Rodriguez et~al. 1987b; Ferrer 1990; Ferrer et~al., 1994). Blood
samples were collected from the cutaneous ulnar (brachial) vein with the
birds cast in dorsal recumbency. Blood was collected in a 10 ml plastic
syringe (Becton Dickinson S.A., Spain) attached to a 23 gauge 1'' needle
(Monoject, USA). Three blood smears were made using a slide-on-slide
technique. One ml of blood was transferred to a di-potassium ethylene
diamine tetra acetate (EDTA) anticoagulant tube (Teklab UK) for `DNA'
sexing. Birds were subsequently sexed at a (`DNA') molecular level
according to methods described by Ogden et al. (2015). An additional 1
ml EDTA tube was filled and submitted for hematological analysis. A 1 ml
tube containing lithium heparin (Teklab, UK) as an anticoagulant was
filled for blood lead analysis. The remaining sample was transferred to
a 6 ml lithium heparin tube (Becton Dickinson S.A., Spain) and submitted
for biochemical analyses. All samples were kept in a cooler box at
approximately 4° C for a maximum of 8 h prior to submission for
laboratory analysis. Samples arrived within 24 h at Greendale Veterinary
Diagnostics, Surrey, UK, where biochemical and hematologic analyses were
performed\emph{.} Blood lead analysis was performed at Veterinary
Laboratory Agencies (VLA) UK. Packed cell volumes and total plasma
protein levels were also examined within three hours of collection at
the Veterinary Centre, University of Edinburgh, UK.

\textbf{Hematological analyses}

Red blood cell (RBC) counts were measured using an impedance counter
with a \selectlanguage{english}floating threshold by a CellTac analyzer (model MEK 5108K, Nihon
Kohden Corporation, Tokyo, Japan). Hemoglobin (Hb) was measured
photometrically with the HemoCue analyzer (HemoCue, Prospect
Diagnostics, Derbyshire, UK) with microcuvettes preloaded with reagent.
Packed cell volume (PCV) was obtained with a hematocentrifuge by using
plain capillary tubes sealed with Critoseal (Krackeler Scienti\selectlanguage{english}fic Inc.,
NY, USA). Samples were centrifuged at 10,000 g for 5 min and read
manually with a hematocrit reader. White blood cell (WBC) counts were
determined manually by mixing 0.38 ml of 1 \% ammonium oxalate with 0.02
ml of EDTA blood and placing this on a rotor for approximately 5 min.
The resultant mixture was used to \selectlanguage{english}fill the counting chamber of an
improved Neubauer hemocytometer and allowed to stand for 5 min in a
moist box to allow the cells to settle. Counts were performed with phase
contrast microscopy with the 340/0.65 phase objective. A total of 4
large squares (64 small squares) was counted, and the total number was
divided by 20 to obtain WBC X 10\textsuperscript{9}/L. Blood smears were
stained with May-Grunwald-Giemsa. The differential count was based on
100 counted leucocytes. At the same time, thrombocytes were counted
manually. For all samples, thrombocytes were estimated to be adequate
and none were assessed as having a thrombocytopenia, and so were not
considered further.

\textbf{Biochemical analyses}

Samples in lithium heparin were centrifuged at 3000~rpm for 10~min on
arrival at the laboratory and the plasma was separated for biochemical
analysis. Plasma was stored frozen at \selectlanguage{english}- 24degC until analysis.
Determinations of 13 parameters were made in sampled birds: Albumin
(Bromocresol green method), Globulin (Biuret method), Urea (Urease--GLDH
method), Glucose (GOD-PAP method), Cholesterol (Chol. esterase chol.
oxidase--Trinder method), Calcium (o-cresolphthalein complexone
reaction), Aspartate aminotransferase AST (IFCC technique), creatinine
kinase CK (IFCC technique), Uric acid (Uricase--Trinder method), Lactate
dehydrogenase LDH (Pyruvate--lactate method), Sodium (Indirect ion
selective electrode), and Potassium (Indirect ion selective electrode).

All biochemical analyses were conducted under ILAB 600 (Instrumentation
Laboratory UK, Cheshire, UK) according to manufacturer instructions. For
some samples, there was insuf\selectlanguage{english}ficient sample volume to realize analysis of
all the biochemical metrics. The laboratory's quality control was to run
daily internal quality control material and to peer review the results
with other laboratories by using two external quality assurance schemes:
the Randox International Quality Assessment Scheme and the American
Veterinary Laboratory Association.

\textbf{Statistical analyses}

All data are expressed as mean +- standard deviation (SD). Normality in
distribution of variables was univariate tested and log or square
transformed when necessary to meet normality, after they were
back-transformed for tables and graphics. We used MANOVA and regression
methods to examine our hypotheses on blood hematological and
biochemistry parameters according to sample timing and birds' age and
sex. In analyses, we standardized hatching dates using the earliest
estimated record of hatching each year as day 1 and the difference
between the earliest record and each subsequent hatch date. Statistica
8.0 software statistical package was used to perform statistical
procedures, and we used an alpha value of 0.05 to assess significance of
results.

\textbf{RESULTS}

Mean values of selected blood parameters according to sample timing are
shown in Table 2. Using a MANOVA analysis, significant differences
between first and second samples and between sexes were found, but there
was no significant interactive effect between sample and sex (Table 3).
Significant differences between the two samples were found in most of
the parameters (Table 2). Among hematological parameters, all showed
significant differences early and later in hacking, including red blood
cells, lymphocytes and monocytes. Hemoglobin, PCV and thrombocytes
decreased during captivity, but white blood cells and most related
component metric values increased. All the biochemical parameters, apart
from AST and LDH, showed significant differences (Table 2). Albumin and
globulin increased as did cholesterol, calcium and potassium.
Nitrogenous components, urea or uric acid, decreased together with
glucose and sodium. Weights (body mass) of the nestlings varied
significantly, being lower in the second sample. This decrease in body
mass was similar for both sexes (Fig. 1).

For urea, the form of the distribution also changed from a skewed
(quasi-Poisson) distribution in the sample when entering the hacking
facility (Shapiro-Wills = 0.889, P \textless{} 0.001), to a normal
distribution of values after their stay in the hacking facility
(Shapiro-Wills = 0.965, P = 0.246, Fig. 2). A significant linear
regression between standardized hatching date and urea level was found
only for the first samples (r = 0.396, R\textsuperscript{2} = 0.16, P =
0.010), but not for the second samples, after their stay in captivity (r
= 0.297, R\textsuperscript{2} = 0.08, P = 0.059) (Fig. 3).

\textbf{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 \emph{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 \emph{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 \emph{ad libitum} for more than one month. These results
suggest a process of hemodilution associated with the \emph{ad
libitum}food (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 (\emph{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
(\emph{Buteo jamaicensis} ), CK levels in birds that had not \selectlanguage{english}flown rose
dramatically above baseline in the 24 h after a short period of exercise
compared to a non-signi\selectlanguage{english}ficant rise from a higher baseline in
\selectlanguage{english}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 \selectlanguage{english}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 (Vinuela 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 to\emph{ad
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
Eagles\emph{Aquila 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)\emph{.} For example, early hatched Spanish Imperial Eagle
nestlings were better nourished than later ones (Ferrer, 1994; Muriel et
al. 2015)\emph{.} 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
through\emph{ad 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.

\textbf{DATA AVAILABILITY STATEMENT}

The raw data supporting the conclusions of this article will be made
available at

the CSIC public repository: \textless{}http://hdl.

handle.net/\ldots{}..

\textbf{ETHICS STATEMENT}

Procedures used in this study complied with and were licensed according
to laws, regulations and guidelines for all relevant activities (both in
Norway and Scotland), and permits for transfer of birds between the
countries. All applicable international, national, and institutional
guidelines for the care and use of animals were followed. All sampling
was undertaken by qualified experienced veterinarians.

\textbf{AUTHOR CONTRIBUTIONS}

CS, RE and OS were RSPB (Scotland) project managers for the WTE east
coast reinforcement project, and as part of their many duties CS and RE
coordinated and assisted in the collection of nestlings in Norway,
organized their transport, supervised and monitored the birds' time in
the hacking facility, and collected/collated essential supporting data.
CS led in data collation on background data for this study. Along with
CS and RE, SH, JH and AM supervised submission, the scope of samples,
and collation of/annual reports on laboratory results. MF conducted data
analyses and led on an initial draft manuscript using some earlier
material by JH, and directly assisted in subsequent revisions by VM and
DPW. DPW undertook analyses on birds' age and coordinated the submitted
MS to which all co-authors contributed and agreed on.

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\textbf{Table 1} . Summary by year of the median date (range) when birds
were collected from Norway, the sexes of the collected birds, the dates
when first and second samples were taken in Scotland, and median date
(range) when birds were released from the hacking aviaries. See main
text for details on samples taken on first and second dates, and sexing
method.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Year} & \textbf{Median collection date (range)} & \textbf{Male}
& \textbf{Female} & \textbf{First sample date} & \textbf{Second sample
date} & \textbf{Median release date (range)}\tabularnewline
\midrule
\endhead
2009 & 19 Jun (13 -- 25 Jun) & 7 & 7 & 26 Jun & 7 Aug & 12 Aug (11 -- 25
Aug)\tabularnewline
2010 & 18 Jun (10 -- 24 Jun) & 7 & 12 & 25 Jun & 11 Aug & 17 Aug (16 --
19 Aug)\tabularnewline
2011 & 18 Jun (13 -- 22 Jun) & 10 & 6 & 24 Jun & 5 Aug & 11 Aug (8 -- 19
Aug)\tabularnewline
2012 & 20 Jun (14 -- 22 Jun) & 2 & 4 & 22 Jun & 10 Aug & 23 Aug (20 --
23 Aug)\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2} . Results of MANOVA analysis of body weight, blood
chemistry and hematological parameters for WTE nestlings, when entering
the hacking facility (52 \emph{+} 5 days of age), and after 44 \emph{+}2
days with \emph{ad libitum} food, when they were 97 \emph{+} 5 days old,
a few days before their release from the hacking facility.

\textbf{Entering hacking Prior to release}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Parameter} & \textbf{n} & \textbf{Mean} & \textbf{SD} &
\textbf{Mean} & \textbf{SD} & \textbf{F} & \textbf{P}\tabularnewline
Hemoglobin (g/dl) & 55 & 13.56 & 0.18 & 10.43 & 0.78 & 82.19 &
\textless{} 0.001\tabularnewline
PCV (1/L) & 55 & 38.66 & 13.14 & 29.73 & 5.34 & 27.76 & \textless{}
0.001\tabularnewline
Red blood cells (10\textsuperscript{12}/L) & 55 & 2.55 & 2.53 & 2.59 &
0.93 & 1.88 & 0.139\tabularnewline
White blood cells (10\textsuperscript{9}/L) & 55 & 12.33 & 15.12 & 15.52
& 6.48 & 6.77 & \textless{} 0.001\tabularnewline
Heterophil (10\textsuperscript{9}/L) & 55 & 6.22 & 3.09 & 9.73 & 5.15 &
9.42 & \textless{} 0.001\tabularnewline
Lymphocyte (10\textsuperscript{9}/L) & 55 & 4.14 & 0.85 & 3.26 & 1.92 &
0.26 & 0.854\tabularnewline
Eosinophil (10\textsuperscript{9}/L) & 55 & 1.08 & 0.54 & 1.42 & 0.92 &
3.47 & 0.020\tabularnewline
Monocyte (10\textsuperscript{9}/L) & 55 & 0.64 & 0.24 & 0.71 & 0.47 &
1.60 & 0.195\tabularnewline
Basophil (10\textsuperscript{9}/L) & 55 & 0.24 & 11.94 & 0.39 & 0.38 &
2.84 & 0.044\tabularnewline
Thrombocytes (10\textsuperscript{9}/L) & 53 & 22.06 & 3.11 & 30.15 &
15.24 & 3.86 & 0.013\tabularnewline
Albumin (g/L) & 55 & 13.18 & 0.50 & 14.32 & 1.22 & 20.03 & \textless{}
0.001\tabularnewline
Globulin (g/L) & 55 & 17.17 & 1.32 & 18.63 & 3.41 & 6.30 & \textless{}
0.001\tabularnewline
Urea (mmol/L) & 41 & 1.55 & 0.77 & 0.72 & 0.35 & 36.02 & \textless{}
0.001\tabularnewline
Glucose (mmol/L) & 41 & 16.04 & 0.15 & 14.55 & 0.94 & 12.25 &
\textless{} 0.001\tabularnewline
Cholesterol (mmol/L) & 41 & 5.49 & 60.57 & 6.73 & 1.73 & 5.33 &
0.002\tabularnewline
Calcium (mmol/L) & 55 & 2.58 & 574.93 & 2.69 & 0.12 & 15.86 &
\textless{} 0.001\tabularnewline
AST (U/L) & 55 & 230.07 & 359.90 & 218.85 & 58.77 & 1.51 &
0.219\tabularnewline
CK (U/L) & 55 & 1086.57 & 188.46 & 1334.14 & 320.60 & 3.81 &
0.013\tabularnewline
Uric acid (\selectlanguage{greek}µ\selectlanguage{english}mol/L) & 55 & 696.18 & 1.46 & 322.76 & 104.44 & 10.29 &
\textless{} 0.001\tabularnewline
LDH (U/L) & 55 & 717.31 & 1.18 & 749.36 & 168.36 & 1.89 &
0.137\tabularnewline
Sodium (mmol/L) & 55 & 155.92 & 0.67 & 154.15 & 2.52 & 3.43 &
0.022\tabularnewline
Potassium (mmol/L) & 55 & 2.35 & 1.13 & 3.07 & 0.62 & 4.28 &
0.008\tabularnewline
Body weight (kg) & 53 & 5.13 & 0.92 & 3.92 & 0.68 & 74.53 & \textless{}
0.001\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3.} MANOVA analyses, effect sizes and power results of
blood hematological and biochemistry parameters as dependent variables
and sample, sex and their (*) interaction as fixed factors. Both sample
(1 or 2, dependent on timing during hacking) and sex had a significant
effect but the interaction did not.

\textbf{Multivariate Tests of Significance, Effect Sizes and Powers}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
& Test & Value & F & Effect df & Error df & P & Partial eta-squared &
Observed power (alpha=0.05)\tabularnewline
\textbf{Intercept} & Wilks & 0.0001 & 158671.6 & 30 & 46 & \textless{}
0.001 & 0.999 & 1.00\tabularnewline
\textbf{SAMPLE} & Wilks & 0.0590 & 24.4 & 30 & 46 & \textless{} 0.001 &
0.941 & 1.00\tabularnewline
\textbf{Sex} & Wilks & 0.1662 & 7.7 & 30 & 46 & \textless{} 0.001 &
0.834 & 1.00\tabularnewline
\textbf{SAMPLE*Sex} & Wilks & 0.5553 & 1.2 & 30 & 46 & 0.261 & 0.445 &
0.81\tabularnewline
\bottomrule
\end{longtable}

\textbf{FIGURE 1} Weight recession of young eagles during hacking
documented by the two sampling events. Both sexes lost mass in a similar
way (female, upper: male, lower). Circles (female) and squares (males)
indicate means and vertical bars denote 0.95 confidence intervals.

\textbf{FIGURE 2} Distributions of urea values of sample 1 (soon after
entering hacking) and sample 2 (just before the release from hacking).
Sample 1 showed a non-normal distribution (Shapiro-Wills = 0.889, P
\textless{} 0.001) that changed to a normal distribution in sample 2
(Shapiro-Wills = 0.965, P = 0.246), after more than one month
with\emph{ad libitum} food in the hacking facility.

\textbf{FIGURE 3} Linear regression between standardized hatching date
and urea level in sample 1 (soon after entering hacking) and sample 2
(just before the release from hacking). Sample 1 showed a significant
relationship with higher urea levels in later hatched birds (r = 0.397,
R\textsuperscript{2} = 0.16, P = 0.010). This relationship disappeared
in sample 2 (r = 0.297, R\textsuperscript{2} = 0.08, P = 0.059), after
more than one month with \emph{ad libitum} food in the hacking facility.

\selectlanguage{english}
\FloatBarrier
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