Effect of protein on the post-ingestive

response of tilapia fed encapsulated

diets Leandro S. Costa
Visit to download the full and correct content document:
https://ebookmass.com/product/effect-of-protein-on-the-post-ingestive-response-of-til
apia-fed-encapsulated-diets-leandro-s-costa/
 Aquaculture 556 (2022) 738289

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Effect of protein on the post-ingestive response of tilapia fed

encapsulated diets
Leandro S. Costa a, Helder O. Guilherme b, *, Raphael N. Bahiense b, Fábio A.C. Santos b,
Pedro G. Gamarano b, Jose F.L. Olmeda c, Paula A.P. Ribeiro b, Ronald K. Luz b
a
Departamento de Zootecnia, Universidade Federal de Viçosa (UFV), 36570-900, Avenida PH Rolfs, s/n, Campus Universitários, Viçosa, Minas Gerais, Brazil
b
Laboratório de Aquacultura, Universidade Federal de Minas Gerais (UFMG), 31270- 901 Belo Horizonte, Minas Gerais, Brazil
c
Department of Physiology, Faculty of Biology, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, 30100 Murcia, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Two experiments were carried out with Nile tilapia (Oreochromis niloticus) to assess whether protein concen­
Protein intake tration influences post-ingestive signaling and the ability of fish to modulate protein intake from a practical diet.
Feeding behavior The animals were fed three types of encapsulated diet: control, cellulose-based diet (CD) = 0% protein; diet with
Physiology
25% crude protein (D25); and diet with 42% crude protein (D42). In the first experiment, 20 fish were divided
Nutrition
into two groups, one being fed CD and D25 and the other fed CD and D42. In the second experiment, 15 tilapia
Fish
were fed D25 and D42. In the first experiment, the two groups of tilapia consumed more capsules containing D25
(62.83%) and D42 (55.54%), and the animals that received D25 noticed the difference between the capsules
more quickly (7 days of the experiment) than did those that received D42 (11 days). In the second experiment,
there was a preference for capsules containing D25, compared to D42, which was demonstrated after 23 days of
the experiment. The animals adjusted both the amount of crude protein ingested daily (31.86%) and the rate of
daily feed consumption to biomass (1.36%). Tilapia can select the diet that best meets their nutritional needs,
adjusting the protein ingested and feed consumed.

1. Introduction “nutritional reward”, at post-ingestion and post-absorption levels (For­

The term “nutritional wisdom” is used to describe the ability of fish
to regulate nutrient intake (Sánchez-Vázquez et al., 1994; Sanchez-
Vázquez et al., 1995; Simpson and Raubenheimer, 2001; Geurden et al.,
2005; Fortes-Silva et al., 2011a; Fortes-Silva et al., 2016; Santos et al.,
2019; Abd El-Wahab et al., 2020). The control of nutrient intake,
including protein, by fish is similar to that described for mammals, being
done via chemical receptors present in the gastrointestinal tract (Rubio
et al., 2003; Almaida-Pagán et al., 2006; Fortes-Silva et al., 2011a,
2011b; Fortes-Silva et al., 2016), liver tissue (Otero-Rodiño et al., 2016)
and brain tissue (Otero-Rodiño et al., 2015; Babaei et al., 2017;
Comesaña et al., 2018; Comesaña et al., 2020), after evaluating the
nutritional constituents of the food. These chemical receptors originate
signals (neural and hormonal activity) that inform brain centers about
the nutritional properties of foods and, thus, modify eating behavior
(Forbes, 2001). With this information, animals learn to regulate the
intake of a particular nutrient, with its metabolic consequences, called
bes, 2001).
Animals have evolved several mechanisms for regulating ingestion
that enable the perception of the energy content, as well as the defi­
ciency of some nutrients, of a given food. This set of mechanisms was
called “specific hunger”: that is, the animal is able to sense nutrients in
the diet and choose a diet with the required elements. (Fortes-Silva et al.,
2016). However, these mechanisms are not unique in the decision to
ingest or not certain foods (Simpson and Raubenheimer, 2001; Fortes-
Silva et al., 2012). The preference for a particular food, and even the
amount consumed, can be influenced by its “palatability”, a fixed effect
of its sensory properties, including taste and texture (Sánchez-Vázquez
et al., 1994; Simpson and Raubenheimer, 2001). However, classical
ingestion control theories suggest that this event entails changes in some
aspect of the animal’s physiology, which eventually becomes strong
enough to inhibit or stimulate feeding (Forbes, 2001).
Studies using self-demand feeders have shown that fish are able to
select macronutrients, such as proteins, lipids and carbohydrates, and

* Corresponding author at: Universidade Federal de Minas Gerais, Escola de Veterinária, Departamento de Zootecnia, Laboratório de Aquacultura – LAQUA, Av.
Antônio Carlos, 6627, CEP 31270–901 Belo Horizonte, MG, Brasil.
E-mail address: helderog@gmail.com (H.O. Guilherme).

https://doi.org/10.1016/j.aquaculture.2022.738289
Received 22 August 2021; Received in revised form 24 January 2022; Accepted 22 April 2022
Available online 27 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
L.S. Costa et al.
thus regulate the amount of each element to be ingested (Vivas et al.,
2006; Geurden et al., 2007; de Mattos et al., 2016; Fortes-Silva et al.,
2017; Luz et al., 2018; Santos et al., 2019). Morais (2017) and Bertucci
et al. (2019) demonstrated the existence of taste receptors and signaling
elements in the gastrointestinal tract of fish, suggesting that, in addition
to oral taste sensations and palatability, the sensory properties of a diet
can also have functional effects. Thus, the methodology of using
encapsulated foods challenges animals to select or self-compose a post-
ingestion diet, without evaluating organoleptic characteristics such as
flavor and texture (Almaida-Pagán et al., 2006; Fortes-Silva et al.,
2011a; Santos et al., 2019; Almeida et al., 2021).
Proteins are extremely important to fish organism (NRC, 2011).
These molecules compose part of muscle and act as hormones, anti­
bodies, enzymes and molecules that store and transport substances (Tu
et al., 2015). In addition, protein and amino acids play a role in con­
trolling hunger and satiety, thus acting on food ingestion (Forbes, 2001).
In aquaculture, the amount of protein supplied to animals must be
carefully evaluated, as the cost of its inclusion in the diet will impact
final production costs (Kabir et al., 2019). The supply of this nutrient
below the animal’s requirement can affect its growth. At the same time,
its excess in the diet leads to the catabolism of amino acids for energy
supply, increasing ammonia excretion and directly impacting water
quality (NRC, 2011).
The objective of this work was to study the post-ingestive response
related to the control of consumption in tilapia fed with encapsulated
diets of low and high protein content.
2. Material and methods
The experiment was performed at the Laboratório de Aquacultura
(Laqua) of the Departamento de Zootecnia of the Universidade Federal
de Viçosa, following the ethical norms approved by the Ethics Com­
mittee on the Use of Animals of the institution (protocol n◦ 0134/2019).
2.1. Animal facilities and maintenance
Juvenile Oreochromis niloticus, phenotypically inverted to males,
were acquired from the 3D Aqua Ltda. (Belo Horizonte, Minas Gerais,
Brazil) and housed in 40-L aquaria, maintained in a recirculation system
with controlled temperature, constant aeration and a 12Light:12Dark
photoperiod (Key West group DNI). The physical-chemical parameters
of the water remained within ideal levels for the species, with the
following mean values: dissolved oxygen 4.75 ± 1.34 mg L− 1, temper­
ature 27.1 ± 0.8 ◦ C, pH 6.6 ± 0.2 (Probe model HI9146, Hanna in­
struments) and total ammonia 0.03 ± 0.02 ppm L− 1 (Labcon Test).
To avoid dominance and territoriality among animals, which could
affect food ingestion, each aquarium contained a single animal (Fortes-
Silva et al., 2011a; Fortes-Silva et al., 2012), adapted to the experi­
mental facilities for 14 days. During this period, feeding training was
carried out, with the supply of encapsulated diet (Fig. 1A) containing
commercial feed with 32% crude protein and 3800 Kcal g− 1 of crude
energy, supplied twice a day (at 09:00 and 17:00 h). The capsules were
offered to the animals and, after 5 min, the leftovers were collected. The
time of 5 min was determined by testing the stability of the capsule in
water to maintain its structural integrity, thus preventing its breakage
and consequent release of contents into the aquarium.
2.2. Formulation and preparation of encapsulated diet
Two isoenergetic diets with ~4165 Kcal g− 1 of gross energy were
formulated based on the nutritional requirements of juvenile tilapia
(NRC, 2011). Diets varied in protein content, the first with 25% crude
protein and the second with 42% (Table 1). The diets were extruded,
with pellets ranging from 6 to 8 mm in diameter (Imbramx40, Imbramaq
Ltda., Ribeirão Preto, São Paulo, Brazil). Oil was sprayed after extrusion
and the diets were dried and stored in a cold chamber (− 20 ◦ C) until use.
2
Aquaculture 556 (2022) 738289
To eliminate the effect of palatability and organoleptic properties of the
food, the diets were supplied inside gelatin capsules, (number 04,
FAGRON – SM Empreendimentos Ltda, Anápolis, Goiás, Brazil). The
feed pellets were ground to reduce particle size and inserted into the
gelatin capsules with the aid of a manual encapsulator (Ideal®, Arujá,
São Paulo, Brazil).
White- and orange-colored capsules were used in the experimental
phase. This procedure allowed capsule recognition by the fish and
facilitated identification when recording leftovers in the aquariums.
After encapsulation of the experimental diets, a sample of 100 capsules
was taken for individual weighing to obtain the average capsule weight
for each diet. This value was used to calculate the consumption of each
diet as well as the daily intake of protein and energy. Capsule mean
weight and standard deviation for each diet were: D25 = 0.1769 ±
0.009 g; D42 = 0.1680 ± 0.007 g, and CD = 0.1245 ± 0.005 g.
2.3. Experiment 1
An experiment was carried out to evaluate the ability of juvenile
O. niloticus to identify the internal content of capsules after ingestion and
determine whether protein content affects the time required for this
identification The experiment had a completely randomized design with
two treatments and ten replications. Twenty fish (average weight 66.41
± 2.85 g) were housed in 20 aquaria (40-L each). Four types of capsules
with different contents were produced:
- Training capsule: yellow capsule, containing commercial feed and
provided only during the training phase of the animals to consume
the diet;
- Negative control capsule (CD): filled only with cellulose;
- Low protein capsule (D25): filled with experimental diet containing
25% crude protein;
- High protein capsule (D42): filled with experimental diet containing
42% crude protein.
For two weeks, the animals were trained to consume capsules filled
with commercial feed (Fig. 1A), after which they were fasted for two
days before the beginning of the first phase of Experiment 1.
The capsules were initially offered to the animals at a total amount of
10% of live weight, the amount corresponding to 5% of each diet. This
amount was periodically adjusted, according to consumption, so that
there was always a surplus of both offered diets. The control diet (CD)
was offered in white capsules to all animals and the experimental diets,
D25 and D42, were supplied in orange capsules, each to 10 different
animals (Fig. 1B). The first phase of the experiment was completed when
the animals demonstrated a statistically significant and persistent con­
sumption preference for one of the capsules (Fortes-Silva et al., 2011a),
which occurred at day 16 of the experiment. At the end of the first phase,
the animals were fasted for two days before the beginning of the second
phase, during which the internal content of the capsules was inverted.
Thus, the experimental diets, D25 or D42, were offered in white capsules
and CD, with only cellulose, in orange capsules. This procedure was
necessary to demonstrate that capsule color does not interfere with the
food preference of the animals. The second phase was completed when
the animals demonstrated a statistically different and constant per­
centage of consumption of one of the capsules (Fortes-Silva et al.,
2011a), which occurred after another 15 days of the experiment.
Experiment 1 had a total duration of 33 days and food preference was
calculated in relation to the percentage of the offered diets consumed.
2.4. Experiment 2
A second experiment was carried out to evaluate the ability of tilapia
to select and adjust dietary intake based on protein level. Fifteen fish
(average weight 81.39 ± 4.49 g) were housed individually in 15
aquariums (40 L each). After capsule consumption training (same
 L.S. Costa et al.
3

Aquaculture 556 (2022) 738289

Fig. 1. Experimental scheme. A) Training and maintenance of animals, B) Experiment 1, C) Experiment 2.
L.S. Costa et al. Aquaculture 556 (2022) 738289

Table 1 2.5. Data analysis

Composition of experimental diets.
Ingredients Diets DC The results for daily and final consumption percentage of capsules in
the two phases of the two experiments, as well as the average daily
D25 D42
percentage of protein ingested by the animal and the daily food con­
Fish meal 15.50 21.00 – sumption rate to biomass, are presented as mean and standard error
Soybean meal 24.00 31.00 –
Corn 40.00 31.50 –
(SEM). All data were submitted to Levene’s homoscedasticity test and
Sour rice 5.00 5.00 – the Shapiro-Wilk normality test. Data on percentage of daily and final
Albumin 1.00 1.60 – consumption of capsules were submitted to Student’s t-test (p < 0.05).
Soy oil 8.80 6.60 – The data referring to the average daily percentage of protein ingested by
Cellulose 3.00 1.00 100
the animal and the daily food consumption rate to biomass were sub­
Dicalcium phosphate 0.50 0.50 –
a
Vitamin/Mineral Supplement 0.50 0.50 – mitted to ANOVA and, when significant, Scott-Knott test (p < 0.05).
b
BHT 0.02 0.02 – Statistical analyses were performed with InfoStat® (version 2016, Uni­
Salt 0.50 0.50 – versity of Cordoba, Cordoba, Argentina).
Inert 1.18 0.78 –

3. Results
c
Composition analyzed
Dry matter (DM) 94.0 91.5 –
3.1. Experiment 1
Crude protein (%) 25.69 42.05 –
Gross energy (Kcal/Kg) 4127.00 4204.12 –
Ashes (%) 6.78 8.43 – The tilapia did not show a preference for any of the diets provided at
the beginning of Experiment 1. However, as the experiment progressed,
d
Amino acid profile (g/100 g) the animals increased their consumption of capsules containing the
Threonine 10.2 18 – experimental diets (Fig. 2A and B). The animals in the group that
Valine 12.6 21.7 – received D25 and CD diets showed a consistent and higher intake of D25
Methionine 4.9 9.5 –
on the 7th day of the experiment, which continued until the end of the
Isoleucine 11.5 20.3 –
Leucine 21.3 32.9 –
first phase (P < 0.05) (Fig. 2A). Fish in the group that received D42 and
Phenylalanine 11.3 19.8 – CD diets showed higher consumption of D42 capsules only on the 11th
Lysine 16.5 30 – day, which continued until the end of the first phase (p < 0.05) (Fig. 2B).
Histidine 6.3 11.1 – In the second phase of Experiment 1, when there was an inversion of
Arginine 17 31.3
capsule contents, the animals of both groups (D25 vs CD and D42 vs CD)
–
∑
Essential amino acids (EAA) 111.6 194.6 –
∑
Non-essential amino acids (NEAA) 135.2 235.2 – had greater consumption of capsules containing the control diet on the
1st day (p < 0.05). This preference, however, did not continue
∑ ∑
EAA/ NEAA 0.83 0.83 –
a throughout the evaluation period. The group of animals that received
Vitamin and mineral supplement composition: Vit. A. 1.200.000 UI; Vit.
D3.200.000 UI; Vit. E. 12.000 mg; Vit. K. 2.400 mg; Vit. B1. 4.800 mg; Vit. D25 and CD diets (Fig. 2A) did not show a diet preference between the
B2.4.800 mg; Vit. B6. 4.000 mg; Vit. B12. 4.800 mg; Ac. folic. 1.200 mg; Ac. 2nd and 5th day of the second phase, that is, from the 20th to the 23rd
Pantothenic. 3.750 mg; Vit. C. 48.000 mg; Biotin. 48 mg; Hill. 65.000 mg;Niacin. day of the experiment. However, on the 6th day of the second phase, the
24.000 mg; Fe. 10.000 mg; Cu. 6.000 mg; Mn. 4.000 mg; Zn. 6.000 mg;I. 20 mg; animals showed a statistically higher consumption of the D25 diet (p <
Co. 2 mg; Se. 20 mg. 0.05). This preference remained until the end of Experiment 1. A similar
b
Butil-Hidroxi-tolueno (antioxidant). result was observed for the group of animals that received D42 and CD
c
Values analyzed in the laboratory. Expressed on the basis of dry matter.(Dry diets (Fig. 2B). Consistent consumption preference for one of the diets
matter in greenhouse 105◦ KJeldahl method Energy through the calori-metric was only demonstrated ten days after the beginning of the second phase,
pump Ethereal extract through extraction with petroleum ether Mineralmatter
that is, on the 28th day of the experiment, and remained this way until
in muffle at 600◦ .
d the end of the experiment (p < 0.05). Fig. 3 shows the total consumption
Values analyzed in laboratory; (HPLC and enzymatic).
of capsules at the end of the experiment. In both treatments, the animals
consumed significantly more capsules containing the formulated diet
procedure as in Experiment 1), the animals were fasted for two days
when compared to the control diet resulting in D24 (62.83%) vs. CD
prior to the beginning of the first phase of Experiment 2. In this phase,
(37.17%) and D42 (55.54%) vs. CD (44.46%).
the fish were fed twice a day (at 09:00 and 17:00 h), with two types of
capsules being made available (Fig. 1C):
3.2. Experiment 2

- White capsule, filled with an experimental diet containing 25%

Initially, the animals did not show a consumption preference be­
crude protein;
tween the offered capsules, a behavior that remained until the 8th day of
- Orange capsule, filled with an experimental diet containing 42%
the experiment (Fig. 4). From the 9th to the 13th day of the experiment,
crude protein.
there was greater consumption of D25 than of D42 (p < 0.05). This same
preference was not observed from the 14th to the 18th day, a period in
The first phase lasted 30 days, the time necessary for the animals to
which the animals consumed both capsules (p > 0.05). From the 19th
demonstrate a constant consumption of the diets. The fish were then
day of the experiment until the end of the first experimental phase, the
fasted for two days before the beginning of the second phase, when the
animals returned to show preference for D25 (p < 0.05). In the second
internal content of the capsules was changed, which lasted for 15 days.
phase, after inversion of the color of the capsules, the animals resumed
At the end of the experimental period, the daily and final consumption
the selection of the D25 diet on the 4th day and continued to do so until
percentage of each diet, the average daily percentage of protein ingested
the end.
by the animal, and the daily food consumption rate to biomass, were
The average daily percentage of protein ingested by the animal was
calculated.
calculated and presented in Fig. 5A. Tilapia could adjust protein con­
sumption during the experimental period. From the beginning of the
experiment until the fifth day, the animals consumed the highest pro­
tein, with a daily average of 34.97%. This value decreased, with an

4
L.S. Costa et al.
Fig. 3. Final consumption of capsules (Mean ± SEM) of groups D25 vs DC and
D42 vs DC, expressed as percentage of total intake, during 33 days of the
experiment. Asterisk represents a significant difference in consumption within
the same group (p < 0.05, n = 10).
-CD: Negative control capsule, filled only with cellulose;
-D25: Low protein capsule, filled with experimental diet containing 25% crude
protein;
-D42: High protein capsule, filled with experimental diet containing 42%
crude protein.
average daily consumption of 32.79% between the 6th and 23rd day,
remaining stable between the 24th and 30th day of the experiment with
an average consumption of 31.97% of crude protein in this period. In the
second phase of the experiment, when the color of the diet’s capsules
was alternated, the animals consumed the highest protein content be­
tween the 33rd and 38th days, with an average of 33.66% of crude
protein. However, this value decreased until consumption stabilized
from the 42nd day onwards, remaining at that level until the end of the
experiment with an average consumption of 31.76% of crude protein, a
value like that of the final of the first phase.
Fig. 5B shows the average daily feed consumption rate to the ani­
mal’s live weight. At the beginning of the experiment, there was a higher
diet consumption, and this consumption decreased until it stabilized.
The highest feed consumption occurred from the beginning of the
experiment until the 9th day, with an average of 3.77% of the biomass.
Between the 10th and 23rd days, consumption was 2.86% of the
biomass, reaching stability between the 24th and 30th days of the
5
Aquaculture 556 (2022) 738289
Fig. 2. Daily frequency of feeding, expressed
as percentage of total diet consumption
(Mean ± SEM), during 33 days of the
experiment: G1 = D25 vs CD; G2 = D42 vs
CD. The contents of the capsules were
inverted after 16 days to verify that the color
of the capsule did not influence consump­
tion. Asterisk represents significant differ­
ences by Student’s t-test (p < 0.05, n = 10).
-CD: Negative control capsule, filled only
with cellulose;
-D25: Low protein capsule, filled with
experimental diet containing 25% crude
protein;
-D42: High protein capsule, filled with
experimental diet containing 42% crude
protein.
experiment, staying at that level until the first phase’s end. During this
period, the average feed consumption was 1.24% of the animal’s
biomass. In the first two days of the second phase of the experiment, the
animals increased consumption (2.80% of live weight). However, from
the 3rd day of the second phase, which corresponds to the 35th day of
the experiment, the consumption was adjusted and remained with an
average of 1.35% of the biomass until the end of the experiment.
4. Discussion
Few works in the literature report the feeding behavior of fish using
encapsulated diets, and in most of them, the study was done with pu­
rified diets. Supplying food inside gelatin capsules allows to isolate the
effect of palatability and to evaluate if the animals can choose the food
through pre- and post-ingestion signals. In our study, tilapia assimilated
the color of the capsules as a pre-ingestive signal and the quality of their
internal content as a post-ingestive signal, for, during experiment 1,
significantly higher consumption of D25 and D42 diets was observed to
the detriment of DC, even after changing the color of the capsule in the
second phase of the experiment (Fig. 2). This identification ability was
evidenced in studies with European sea bass (Dicentrarchus labrax)
(Rubio et al., 2003), sharpsnout seabream (Diplodus puntazzo) (Almaida-
Pagán et al., 2006), Nilotic tilapia (Oreochromis niloticus) (Fortes-Silva
et al., 2011a; Fortes-Silva et al., 2012; Almeida et al., 2021), tambaqui
(Colossoma macropomum) (Filho et al., 2018; Pereira et al., 2018); and
pacamã (Lophiosilurus alexandri) (Santos et al., 2019).
Despite the significant influence of palatability, fish also use indirect
factors such as the physiological state to stimulate the consumption of
some foods (Higuera, 2001), which may have driven the consumption of
D25 and D42 diets to the detriment of DC, as the control diet consisted of
cellulose, which does not provide nutrients to the animals, resulting in a
decrease in the supply of nutrients to the organism. In studies where the
effect of palatability was isolated, the post-ingestive signs indicate to the
animal the quality of the ingested diet. Gastrointestinal receptors
initially make identification of protein intake during protein digestion
(Rubio et al., 2003; Almaida-Pagán et al., 2006; Fortes-Silva et al.,
2011a), and the amino acids absorbed after this process are recognized
by receptors present in liver tissue (Bernardis and Bellinger, 1996).
Hepatic receptors communicate with brain centers via neurotransmit­
ters or hormones, informing the nutritional properties of food and
L.S. Costa et al. Aquaculture 556 (2022) 738289

Fig. 4. Daily frequency of feeding, expressed as percentage of total diet consumption (Mean ± SEM), during 47 days of the experiment. Provision of D25 vs D42. The
contents of the capsules were inverted after 30 days to verify that the color of the capsule did not influence consumption. Asterisk represents significant differences by
Student’s t-test (p < 0.05, n = 15).
-CD: Negative control capsule, filled only with cellulose;
-D25: Low protein capsule, filled with experimental diet containing 25% crude protein;
-D42: High protein capsule, filled with experimental diet containing 42% crude protein.

Fig. 5. (A) Average daily percentage of protein ingested by the animal during the 47 days of the experiment. The average daily percentage of protein was calculated
by adding the amounts of protein consumed from each diet. Letters represent the significant difference between points by the Scott-Knott test (p < 0.05, n = 15). (B)
Daily feed consumption rate to biomass during the 47 days of the experiment. The feed consumption rate was calculated as a percentage of feed consumed to
live weight.

modifying the animal’s eating behavior (Forbes, 2001).
Protein and amino acids are vital for the functioning of the animal
organism (Tu et al., 2015). The level of restriction of these nutrients, in
this study, may have influenced the time taken by the animal to identify
the internal content of the capsules since fish that were challenged with
D25 vs. CD were able to distinguish the D25 capsules faster (Fig. 2A)
significantly increasing its consumption compared to treatment D42 vs.
DC (Fig. 2B), which showed a later significant difference in consump­
tion. The D25 diet was formulated with protein deficiency and some
essential amino acids such as histidine, methionine, threonine, and
valine (Fig. 1), as indicated by the NRC (2011). To date, no studies have
been found on the influence of protein concentration on the time of
identification of a diet by animals.
Terrestrial animals show an appetite for specific nutrients (Forbes,
1998). A deficiency of these essential nutrients is known to reduce feed
intake in fish (Kim et al., 1992a; Walton et al., 1986; Choo et al., 1991;
Kim et al., 1992b). When animals are fed deficient diets, two opposite
responses can be induced: when the deficiency is mild, an increase in
feed intake to reach the absolute level of requirement – as observed in
poultry and swine regarding essential amino acids (Boorman, 1979;
Henry, 1985), or an inhibitory response when the deficiency is more
significant (Higuera, 2001). There was no significant difference in total
consumption between the D25 and D42 capsules in our study. However,
when we analyzed the total consumption of capsules within the treat­
ments D24 vs. DC and D42 vs. DC (Fig. 3), we noticed that the animals
consumed significantly more D25 and D42 capsules, thus increasing
nutrient intake to meet their body’s needs.
In the second experiment, after adjusting for consumption, the ani­
mals ingested an average of 55.6% of D25 and 44.4% of D42 (Fig. 4).
The adjustment also occurred to the crude protein intake and the feed
consumption rate, resulting in 31.86% and 1.36%, respectively (Fig. 5A
and B) after stabilization. Other studies have also reported this eating
behavior of stabilizing the consumption of diets or nutrients (Boujard
and Médale, 1994; Fortes-Silva et al., 2012; Santos et al., 2019). The way

6
L.S. Costa et al.
fish regulate the intake and use of macronutrients is essential for
aquaculture, as the animal can adjust the nutrient concentration in the
body through variation in food intake (Atienza et al., 2004).
Feed selection by animals aims to satisfy nutritional requirements,
although palatability also plays an important role (Higuera, 2001). A
balanced nutrient intake is the main objective of feed selection. Animals
can select, from a range of available rations, different proportions of
each to ensure adequate intake of nutrients and energy, essential for
normal bodily functions and growth (Simpson and Raubenheimer,
1999). In higher vertebrates, feed selection does not appear to be
random or purposeless; it seems to aim to achieve a certain nutrient
intake level (Kyriazakis, 1997). A similar fact was observed in our study.
The animals were offered two rations, with different crude protein and
amino acids levels, one deficient and the other with excess. The animal
showed a significant preference for the consumption of D25. However,
part of the diet consumed was composed of D42, which possibly sup­
plied nutrients in low levels on D25.
Diets with a high protein concentration can decrease consumption
and affect growth in some fish species (Gurure et al., 1995; Santinha
et al., 1996). Such adverse effects have also been observed in higher
vertebrates fed diets containing high protein and amino acids levels (Li
and Anderson, 1983). The reduction in feed consumption by animals, in
this situation, may work as a strategy to reduce the possible harmful
effects of excessive nutrient intake (Higuera, 2001). In this study, the
animals adjusted their daily protein intake by consuming different
proportions of each diet and reducing, at the end of the experiment, the
total food consumption. The average percentage of crude protein in the
food ingested by the animals after the selection stabilization period was
approximately 32% (Fig. 5A). This value is close to that recommended
by the NRC (2011), which indicates a level of roughly 34% of crude
protein, and by Santos et al. (2020), who report better performance of
tilapia fed diets containing 31.12% crude protein. It should be empha­
sized that preferences do not need to coincide with a sensible and
balanced diet formulated to provide maximum growth and efficient feed
conversion (Higuera, 2001).
Feed consumption by fish is affected by climate, water temperature,
dissolved oxygen, nutrient levels, and dietary energy (Higuera, 2001;
Zhou et al., 2018; Mallekh et al., 2003). Our work observed that the feed
consumption rate decreased throughout the experiment and stabilized at
1.36% of the biomass. Values similar to those found in our study were
reported by Omasaki et al. (2017) (1.54%) and Tran-Duy et al. (2008),
who reported consumption of 1.54 and 1.24% of the biomass, respec­
tively. We emphasize that our research was carried out in a water
recirculation system, with temperature, water quality, and photoperiod
control, therefore, without these variables interfering in the food con­
sumption rate.
5. Conclusion
Given the above, we conclude that Nilotic tilapia (O. niloticus) can
select a diet that best meets its nutritional needs, and low-protein diets
provide faster response in the selection process. The animals were able
to adjust the consumption of crude protein by 31.86% through the se­
lection of diets, with a consumption rate of 1.36% of the biomass, these
values being recommended in the production of tilapia in a water
recirculation system.
CRediT authorship contribution statement
Leandro S. Costa: Conceptualization, Methodology, Validation,
Formal analysis, Investigation, Writing – original draft, Writing – review
& editing, Visualization. Helder O. Guilherme: Conceptualization,
Methodology, Validation, Formal analysis, Investigation. Raphael N.
Bahiense: Conceptualization, Methodology, Validation, Formal anal­
ysis, Investigation. Fábio A.C. Santos: Conceptualization, Methodol­
ogy, Validation, Formal analysis, Investigation. Pedro G. Gamarano:
7
Aquaculture 556 (2022) 738289
Conceptualization, Methodology, Validation, Formal analysis, Investi­
gation. Jose F.L. Olmeda: Conceptualization, Methodology, Validation,
Formal analysis, Investigation. Paula A.P. Ribeiro: Conceptualization,
Methodology, Validation, Formal analysis, Investigation, Resources,
Data curation, Investigation, Writing – original draft, Writing – review &
editing, Visualization, Supervision, Project administration, Funding
acquisition. Ronald K. Luz: Conceptualization, Methodology, Valida­
tion, Formal analysis, Investigation, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We are grateful for the financial support granted to this research by
CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico
- CNPq 401117 / 2014-6) and by CAPES (Coordenação de Aperfeiçoa­
mento de Pessoal de Nível Superior), as well as the research grants
granted to professors Ronald Kennedy Luz (CNPq n.308547/2018-7)
and Paula Adriane Perez Ribeiro (CNPq n.308684/2017-6). We would
also like to thank Departamento de Zootecnia da Universidade Federal
de Lavras (UFLA), especiallyh Priscila Vieira Rosa, for facilitating access
to the Enzimologia and Biologia Molecular laboratories.
References
Abd El-Wahab, A., Visscher, C., Teitge, F., Steinhagen, D., 2020. Choice preference of
diets with different protein levels depending on water temperature in Nile tilapia.
J. World Aquacult. Soc. 51, 512–526. https://doi.org/10.1111/jwas.12651.
Almaida-Pagán, P.F., Rubio, V.C., Mendiola, P., De Costa, J., Madrid, J.A., 2006.
Macronutrient selection through post-ingestive signals in sharpsnout seabream fed
gelatine capsules and challenged with protein dilution. Physiol. Behav. 88, 550–558.
https://doi.org/10.1016/j.physbeh.2006.05.027.
Almeida, C.A.L., de Almeida, C.K.L., de Fátima Ferreira Martins, E., Bessonart, M.,
Pereira, R.T., Paulino, R.R., Fortes-Silva, R., 2021. Coping with suboptimal water
temperature: modifications in blood parameters, body composition, and
postingestive-driven diet selection in Nile tilapia fed two vegetable oil blends.
Animal 15. https://doi.org/10.1016/j.animal.2020.100092.
Atienza, M.T., Chatzifotis, S., Divanach, P., 2004. Macronutrient selection by sharp snout
seabream (Diplodus puntazzo). Aquaculture 232, 481–491. https://doi.org/10.1016/
S0044-8486(03)00468-X.
Babaei, S., Sáez, A., Caballero-Solares, A., Fernández, F., Baanante, I.V., Metón, I., 2017.
Effect of dietary macronutrients on the expression of cholecystokinin, leptin, ghrelin
and neuropeptide Y in gilthead sea bream (Sparus aurata). Gen. Comp. Endocrinol.
240, 121–128. https://doi.org/10.1016/j.ygcen.2016.10.003.
Bernardis, L.L., Bellinger, L.L., 1996. The lateral hypothalamic area revisited: ingestive
behavior. Neurosci. Biobehav. Rev. 20 (2), 189–287.
Bertucci, J.I., Blanco, A.M., Sundarrajan, L., Rajeswari, J.J., Velasco, C., Unniappan, S.,
2019. Nutrient regulation of endocrine factors influencing feeding and growth in
fish. Front. Endocrinol. (Lausanne). 10, 1–17. https://doi.org/10.3389/
fendo.2019.00083.
Boorman, K.N., 1979. Regulation of protein and amino acid intake. In: Boorman, K.N.,
Freeman, B.M. (Eds.), Food Intake Regulation in Poultry. Longman, Edinburgh,
pp. 87–126.
Boujard, T., Médale, F., 1994. Regulation of voluntary feed intake in juvenile rainbow
trout fed by hand or by self-feeders with diets containing two different protein/
energy ratios. Aquat. Living Resour. 7, 211–215. https://doi.org/10.1051/alr:
1994023.
Choo, P.S., Smith, T.K., Cho, C.Y., Ferguson, H.W., 1991. Dietary excesses of leucine
influence growth and body composition of rainbow trout. J. Nutr. 121, 1932–1939.
https://doi.org/10.1093/jn/121.12.1932.
Comesaña, S., Velasco, C., Ceinos, R.M., López-Patiño, M.A., Míguez, J.M., Morais, S.,
Soengas, J.L., 2018. Evidence for the presence in rainbow trout brain of amino acid-
sensing systems involved in the control of food intake. Am. J. Phys. Regul. Integr.
Comp. Phys. 314, R201–R215. https://doi.org/10.1152/ajpregu.00283.2017.
Comesaña, S., Conde-Sieira, M., Velasco, C., Soengas, J.L., Morais, S., 2020. Oral and pre-
absorptive sensing of amino acids relates to hypothalamic control of food intake in
rainbow trout. J. Exp. Biol. 223 https://doi.org/10.1242/jeb.221721.
de Mattos, B.O., Nascimento-Filho, E.C.T., dos Anjos-Santos, A., Sánchez-Vázquez, F.J.,
Fortes-Silva, R., 2016. Daily self-feeding activity rhythms and dietary self-selection
of pirarucu (Arapaima gigas). Aquaculture 465, 152–157. https://doi.org/10.1016/j.
aquaculture.2016.09.005.
Filho, E.C.T.N., Mattos, B.O., Santos, A.A., Barreto, K.A., Albinati, R.C.B., Oliveira
Vidal, L.V., Fortes-Silva, R., 2018. Geometric approach to evaluate the energy/
L.S. Costa et al.
protein balance in tambaqui (Colossoma macropomum): can fish ensure nutritional
intake targets by postingestion signals? Aquac. Nutr. 24, 741–747. https://doi.org/
10.1111/anu.12602.
Forbes, J.M., 1998. Voluntary Food Intake and Diet Selection in Farm Animals.
International Oxford, Cabi.
Forbes, J.M., 2001. Consequences of feeding for future feeding. Comp. Biochem. Physiol.
- A Mol. Integr. Physiol. 128, 461–468. https://doi.org/10.1016/s1095-6433(00)
00328-7.
Fortes-Silva, R., Martínez, F.J., Sánchez-Vázquez, F.J., 2011a. Macronutrient selection in
Nile tilapia fed gelatin capsules and challenged with protein dilution/restriction.
Physiol. Behav. 102, 356–360. https://doi.org/10.1016/j.physbeh.2010.11.036.
Fortes-Silva, R., Sánchez-Vázquez, F.J., Martínez, F.J., 2011b. Effects of pretreating a
plant-based diet with phytase on diet selection and nutrient utilization in European
sea bass. Aquaculture 319, 417–422. https://doi.org/10.1016/j.
aquaculture.2011.07.023.
Fortes-Silva, R., Rosa, P.V., Zamora, S., Sánchez-Vázquez, F.J., 2012. Dietary self-
selection of protein-unbalanced diets supplemented with three essential amino acids
in Nile tilapia. Physiol. Behav. 105, 639–644. https://doi.org/10.1016/j.
physbeh.2011.09.023.
Fortes-Silva, R., Oliveira, I.E., Vieira, V.P., Winkaler, E.U., Guerra-Santos, B.,
Cerqueira, R.B., 2016. Daily rhythms of locomotor activity and the influence of a
light and dark cycle on gut microbiota species in tambaqui (Colossoma macropomum).
Biol. Rhythm. Res. 47, 183–190. https://doi.org/10.1080/09291016.2015.1094972.
Fortes-Silva, R., Costa, L.S., Paulino, R.R., Rodrigues, E.J.D., Sanchez-Vazquez, F.J.,
Rosa, P.V., 2017. Hybrid fish model (Pseudoplatystoma reticulatum♀ × Leiarius
marmoratus♂) to study feeding behaviour: protein source self-selection and demand-
feeding rhythms. Aquac. Res. 48, 3705–3718. https://doi.org/10.1111/are.13196.
Geurden, I., Cuvier, A., Gondouin, E., Olsen, R.E., Ruohonen, K., Kaushik, S., Boujard, T.,
2005. Rainbow trout can discriminate between feeds with different oil sources.
Physiol. Behav. 85, 107–114. https://doi.org/10.1016/j.physbeh.2005.03.010.
Geurden, I., Corraze, G., Boujard, T., 2007. Self-feeding behaviour of rainbow trout,
Oncorhynchus mykiss, offered diets with distinct feed oils. Appl. Anim. Behav. Sci.
108, 313–326. https://doi.org/10.1016/j.applanim.2006.12.006.
Gurure, R.M., Moccia, R.D., Atkinson, J.L., 1995. Optimal protein requirements of young
Arctic charr (Salvelinus alpinus) fed practical diets. Aquac. Nutr. 1, 227–234.
Henry, Y., 1985. Dietary factors involved in feed intake regulation in growing pigs: a
review. Livest. Prod. Sci. 12, 339–354. https://doi.org/10.1016/0301-6226(85)
90133-2.
Higuera, M., 2001. Effects of nutritional factors and feed characteristics on feed intake.
In: Food Intake in Fish, 827, pp. 250–268.
Kabir, K.A., Verdegem, M.C.J., Verreth, J.A.J., Phillips, M.J., Schrama, J.W., 2019. Effect
of dietary protein to energy ratio, stocking density and feeding level on performance
of Nile tilapia in pond aquaculture. Aquaculture 511, 634200. https://doi.org/
10.1016/j.aquaculture.2019.06.014.
Kim, K. Il, Kayes, T.B., Amundson, C.H., 1992a. Requirements for sulfur amino acids and
utilization of D-methionine by rainbow trout (Oncorhynchus mykiss). Aquaculture
101, 95–103. https://doi.org/10.1016/0044-8486(92)90235-D.
Kim, K. Il, Kayes, T.B., Amundson, C.H., 1992b. Requirements for lysine and arginine by
rainbow trout (Oncorhynchus mykiss). Aquaculture 106, 333–344. https://doi.org/
10.1016/0044-8486(92)90265-M.
Kyriazakis, I., 1997. The nutritional choices of farm animals: To eat or what to eat? In:
Forbes, J.M., Lawrence, T.L.J., Rodway, R.G., Varley, M.A. (Eds.), Animal Choices.
British Society of Animal Science, Edinburgh, pp. 55–65.
Li, E.T.S., Anderson, G.H., 1983. Amino acids in the regulation of food intake. In:
Nutrition Abstract and Reviews in Clinical Nutrition, 53, pp. 169–178.
Luz, R.K., Boluda-Navarro, D., Sánchez-Vázquez, F.J., Fortes-Silva, R., Portella, M.C.,
Zamora Navarro, S., Madrid, J.A., 2018. European seabass (Dicentrarchus labrax)
ability to discriminate between diets made with different dietary fat sources. Aquac.
Nutr. 24, 65–73. https://doi.org/10.1111/anu.12534.
Mallekh, R., Lagardère, J.P., Eneau, J.P., Cloutour, C., 2003. An acoustic detector of
turbot feeding activity. Aquaculture 221, 481–489. https://doi.org/10.1016/S0044-
8486(03)00074-7.
Morais, S., 2017. The physiology of taste in fish: potential implications for feeding
stimulation and gut chemical sensing. Rev. Fish. Sci. Aquac. 25, 133–149. https://
doi.org/10.1080/23308249.2016.1249279.
National Research Council (NRC), 2011. Nutrient Requirement of Fish. Committee on
Animal Nutrition, Board on Agriculture, National Academic Press, Washington, DC,
pp. 102–134.
8
Aquaculture 556 (2022) 738289
Omasaki, S.K., Janssen, K., Besson, M., Komen, H., 2017. Economic values of growth
rate, feed intake, feed conversion ratio, mortality and uniformity for Nile tilapia.
Aquaculture 481, 124–132. https://doi.org/10.1016/j.aquaculture.2017.04.013.
Otero-Rodiño, C., Librán-Pérez, M., Velasco, C., López-Patiño, M.A., Míguez, J.M.,
Soengas, J.L., 2015. Evidence for the presence of glucosensor mechanisms not
dependent on glucokinase in hypothalamus and hindbrain of rainbow trout
(Oncorhynchus mykiss). PLoS One 10. https://doi.org/10.1371/journal.
pone.0128603.
Otero-Rodiño, C., Librán-Pérez, M., Velasco, C., Álvarez-Otero, R., López-Patiño, M.A.,
Míguez, J.M., Soengas, J.L., 2016. Glucosensing in liver and Brockmann bodies of
rainbow trout through glucokinase-independent mechanisms. Comp. Biochem.
Physiol. Part - B Biochem. Mol. Biol. 199, 29–42. https://doi.org/10.1016/j.
cbpb.2015.09.008.
Pereira, R.T., Paulino, R.R., de Almeida, C.A.L., Rosa, P.V., Orlando, T.M., Fortes-
Silva, R., 2018. Oil sources administered to tambaqui (Colossoma macropomum):
growth, body composition and effect of masking organoleptic properties and fasting
on diet preference. Appl. Anim. Behav. Sci. 199, 103–110. https://doi.org/10.1016/
j.applanim.2017.10.004.
Rubio, V.C., Sánchez-Vázquez, F.J., Madrid, J.A., 2003. Macronutrient selection through
postingestive signals in sea bass fed on gelatine capsules. Physiol. Behav. 78,
795–803. https://doi.org/10.1016/S0031-9384(03)00082-9.
Sánchez-Vázquez, F.J., Martínez, M., Zamora, S., Madrid, J.A., 1994. Design and
performance of an accurate demand feeder for the study of feeding behaviour in sea
bass, Dicentrarchus labrax L. Physiol. Behav. 56, 789–794. https://doi.org/10.1016/
0031-9384(94)90244-5.
Sanchez-Vázquez, F.J., Madrid, J.A., Zamora, S., 1995. Circadian rhythms of feeding
activity in sea bass, Dicentrarchus labrax L.: dual phasing capacity of diel demand-
feeding pattern. J. Biol. Rhythm. 10, 256–266. https://doi.org/10.1177/
074873049501000308.
Santinha, P.J.M., Gomes, E.F.S., Coimbra, J.O., 1996. Effects of protein level of the diet
on digestibility and growth of gilthead sea bream, Sparus aurata L. Aquac. Nutr. 2,
81–87.
Santos, F.A.C., Fortes-Silva, R., Costa, L.S., Luz, R.K., Guilherme, H.O., Gamarano, P.G.,
Ribeiro, P.A.P., 2019. Regulation of voluntary protein/energy intake based practical
diet composition for the carnivorous neotropical catfish Lophiosilurus alexandri.
Aquaculture 510, 198–205. https://doi.org/10.1016/j.aquaculture.2019.05.038.
Santos, W.M., Costa, L.S., López-Olmeda, J.F., Costa, N.C.S., Santos, F.A.C., Gamarano, P.
G., Ribeiro, P.A.P., 2020. Effects of dietary protein levels on activities of protease
and expression of ingestion and protein digestion-related genes in Nile tilapia
juveniles. Aquac. Res. 51, 2973–2984. https://doi.org/10.1111/are.14635.
Simpson, S.J., Raubenheimer, D., 1999. Assuaging nutritional complexity: a geometrical
approach. Proc. Nutr. Soc. 58, 779–789. https://doi.org/10.1017/
S0029665199001068.
Simpson, S.J., Raubenheimer, D., 2001. A framework for the study of macronutrient
intake in fish. Aquac. Res. 32, 421–432. https://doi.org/10.1046/j.1365-
2109.2001.00593.x.
Tran-Duy, A., Schrama, J.W., van Dam, A.A., Verreth, J.A.J., 2008. Effects of oxygen
concentration and body weight on maximum feed intake, growth and hematological
parameters of Nile tilapia, Oreochromis niloticus. Aquaculture 275, 152–162. https://
doi.org/10.1016/j.aquaculture.2007.12.024.
Tu, Y., Xie, S., Han, D., Yang, Y., Jin, J., Liu, H., Zhu, X., 2015. Growth performance,
digestive enzyme, transaminase and GH-IGF-I axis gene responsiveness to different
dietary protein levels in broodstock allogenogynetic gibel carp (Carassius auratus
gibelio) CAS III. Aquaculture 446, 290–297. https://doi.org/10.1016/j.
aquaculture.2015.05.003.
Vivas, M., Rubio, V.C., Sánchez-Vázquez, F.J., Mena, C., García García, B., Madrid, J.A.,
2006. Dietary self-selection in sharpsnout seabream (Diplodus puntazzo) fed paired
macronutrient feeds and challenged with protein dilution. Aquaculture 251,
430–437. https://doi.org/10.1016/j.aquaculture.2005.06.013.
Walton, M.J., Cowey, C.B., Coloso, R.M., Adron, J.W., 1986. Dietary requirements of
rainbow trout for tryptophan, lysine and arginine determined by growth and
biochemical measurements. Fish Physiol. Biochem. 2, 161–169. https://doi.org/
10.1007/BF02264084.
Zhou, C., Xu, D., Lin, K., Sun, C., Yang, X., 2018. Intelligent feeding control methods in
aquaculture with an emphasis on fish: a review. Rev. Aquac. 10, 975–993. https://
doi.org/10.1111/raq.12218.
Another random document with
no related content on Scribd:
 O thou blossom of eastern silence,
Take thy ancient way untrodden of men.
Go on thy dustless path of mystery,
Reach thou the golden throne of God,
And be our advocate
Before His Silence and His compassionate speechlessness.

The prayer frightened the eagles, unaccustomed to human voices.

But ere they were excited to fury our little ritual was over and we hid
ourselves under the stunted pine. The eagles, left without any
breakfast, looked out and scanned the sky for a sign of their parent.
They gazed below where flocks of parrots and jays flew, small as
humming birds. Wild geese came trailing across the snowy peaks
where they had spent the night on their journey southward. Soon
they too grew small as beetles and melted into space. Hour after
hour passed, yet no sight of the big eagle! The full-fledged eaglets
felt hungrier and hungrier, and began to fret in their nest. We heard a
quarrel going on in the interior of the eyrie which grew in intensity
and noise till one of them left home in disgust and began to climb the
cliff. He went higher and higher. Up and up he walked without using
his wings. By now it was past midday; we had luncheon, yet still
there was no sight of the parent birds. We judged that the eagle left
in the nest was the sister, for she looked smaller than the other
eaglet. She sat facing the wind, peering into the distance, but she
too grew down-hearted. Strange though it may sound, I have yet to
see a Himalayan eagle which does not sit facing the wind from the
time of its birth until it learns to fly, as a sailor boy might sit looking at
the sea until he takes to navigating it. About two in the afternoon that
eagle grew tired of waiting in the nest. She set out in quest of her
brother who was now perching on the top of a cliff far above. He too
was facing the wind. As his sister came up his eyes brightened. He
was glad not to be alone and the sight of her saved him from the
melancholy thought of flying for food. No eagle-child have I seen
being taught to fly by its parents. That is why younglings will not
open their wings until driven by hunger. The parent eagles know this
very well and that is why when their babies grow up, and the time
has come, they leave them and go away.
The little sister laboriously climbed till she reached her brother's side
but alas, there was no room for two. Instead of balancing themselves
on their perch, the sister's weight knocked her brother almost over.
Instantly he opened his wings wide. The wind bore him up. He
stretched out his talons, but too late to reach the ground. He was at
least two feet up in the air already, so he flapped his wings and rose
a little higher. He dipped his tail—which acted like a rudder and
swung him sideways, east, south, east. He swung over us and we
could hear the wind crooning in his wings. Just at that moment a
solemn silence fell on everything; the noises of insects stopped;
rabbits, if there were rabbits, hid in their holes. Even the leaves
seemed to listen in silence to the wing-beats of this new monarch of
the air as he sailed higher and higher. And he had to go away up, for
only by going very far could he find what he sought. Sometimes
eighteen hundred to three thousand feet below him, an eagle sees a
hare hopping about on the ground. Then he folds his wings and
roars down the air like lightning. The terrible sound of his coming
almost hypnotizes the poor creature and holds him bound to the spot
listening to his enemy's thunderous approach, and then the eagle's
talons pierce him.
Seeing her brother go off in this way, and being afraid of loneliness,
the sister suddenly spread her wings too. The wind blowing from
under threw her up. She also floated in the air and tacked her flight
by her tail toward her comrade and in a few minutes both were lost
to sight. Now it was our turn to depart from those hills in search of
our pigeon. He might have gone to Dentam. But it behoved us to
search every Lamasery and baronial castle which had served Gay-
Neck as a landmark in his past flights.
CHAPTER V
 ON GAY-NECK'S TRACK
s we descended into the bleak oblivion of the
gorges below, we suddenly found ourselves in a
world of deepening dark, though it was hardly three
in the afternoon. It was due to the long shadows of
the tall summits under which we moved. We
hastened our pace, and the cold air goaded us on.
As soon as we had descended about a thousand
feet and more it grew warmer by comparison, but as night came on
apace the temperature dropped anew and drove us to seek shelter in
a friendly Lamasery. We reached that particular serai where the
Lamas, Buddhist monks, most generously offered us hospitality. They
spoke to us only as they had occasion in serving us with supper and
in escorting us to our rooms. They spend their evenings in meditation.
We had three small cells cut out of the side of a hill, in front of which
was a patch of grassy lawn railed off at the outer edges. By the light
of the lanthorns we carried, we found that we had only straw
mattresses in our stone cells. However, the night passed quickly, for
we were so tired that we slept like children in their mothers' arms.
About four o'clock next morning I heard many footsteps that roused
me completely from sleep. I got out of bed and went in their direction,
and soon I discerned bright lights. By climbing down and then up a
series of high steps, I reached the central chapel of the Lamasery—a
vast cave under an overhanging rock, and open on three sides.
There, before me stood eight Lamas with lanthorns which they quietly
put away as they then sat down to meditate, their legs crossed under
them. The dim light fell on their tawny faces and blue robes, and
revealed on their countenances only peace and love.
Presently their leader said to me in Hindusthani: "It has been our
practice for centuries to pray for all who sleep. At this hour of the
night even the insomnia-stricken person finds oblivion and since men
when they sleep can not possess their conscious thoughts, we pray
that Eternal Compassion may purify them, so that when they awake
in the morning they will begin their day with thoughts that are pure,
kind and brave. Will you meditate with us?"
I agreed readily. We sat praying for compassion for all mankind. Even
to this day when I awake early I think of those Buddhist monks in the
Himalayas praying for the cleansing of the thoughts of all men and
women still asleep.
The day broke soon enough. I found that we were sitting in a cleft of a
mountain, and at our feet lay a precipice sheer and stark. The tinkling
of silver bells rose softly in the sunlit air, bells upon bells, silver and
golden, chimed gently and filled the air with their sweet music: it was
the monks' greeting to the harbinger of light. The sun rose as a
clarion cry of triumph—of Light over darkness, and of Life over Death.
Below I met Radja and Ghond at breakfast. It was then that a monk
who served us said: "Your pigeon came here for shelter yesterday."
He gave a description of Gay-Neck, accurate even to the nature of
his nose wattle, its size and colour. Ghond asked: "How do you know
we seek a pigeon?" The flat-faced Lama, without even turning an
eyelash, said in a matter of fact tone: "I can read your thoughts."
Radja questioned with eagerness: "How can you read our thoughts?"
The monk answered: "If you pray to Eternal Compassion for four
hours a day for the happiness of all that live, in the course of a dozen
years He gives you the power to read some people's thoughts,
especially the thoughts of those who come here.... Your pigeon we
fed and healed of his fear when he took shelter with us."
"Healed of fear, my Lord!" I exclaimed.
The Lama affirmed most simply: "Yes, he was deeply frightened. So I
took him in my hands and stroked his head and told him not to be
afraid, then yestermorn I let him go. No harm will come to him."
"Can you give your reason for saying so, my Lord?" asked Ghond
politely.
The man of God replied to him thus: "You must know, O Jewel
amongst hunters, that no animal, nor any man, is attacked and killed
by an enemy until the latter succeeds in frightening him. I have seen
even rabbits escape hounds and foxes when they kept themselves
free of fear. Fear clouds one's wits and paralyses one's nerve. He
who allows himself to be frightened lets himself be killed."
"But how do you heal a bird of fear, my Lord?"
To that question of Radja's the holy one answered: "If you are without
fear and you keep not only your thoughts pure but also your sleep
untainted of any fear-laden dreams for months, then whatever you
touch will become utterly fearless. Your pigeon now is without fear for
I who held him in my hand have not been afraid in thought, deed and
dream for nearly twenty years. At present your pet bird is safe: no
harm will come to him."
By the calm conviction in his words, spoken without emphasis, I felt
that in truth Gay-Neck was safe and in order to lose no more time, I
said farewell to the devotees of Buddha and started south. Let me
say that I firmly believe that the Lamas were right. If you pray for
other people every morning you can enable them to begin their day
with thoughts of purity, courage and love.
Now we dropped rapidly towards Dentam. Our journey lay through
places that grew hotter and more familiar. No more did we see the
rhododendrons. The autumn that further up had touched the leaves of
trees with crimson, gold, cerise, and copper was not so advanced
here. The cherry trees still bore their fruits; the moss had grown on
trees thickly, and the wind had blown on them the pollen of orchids,
large as the palm of your hand, blossoming in purple and scarlet.
Many white daturas perspired with dewdrops in the steaming heat of
the sun. The trees began to appear taller and terrible. Bamboos
soared upwards like sky-piercing minarets. Creepers thick as pythons
beset our path. The buzzing of the cicada grew insistent and
unbearable, and jays jabbered in the woods. Now and then a flock of
green parrots flung their emerald glory in the face of the sun, then
vanished. Insects multiplied. Mammoth butterflies, velvety black,
swarmed from blossom to blossom, and innumerable small birds
preyed on numberless buzzing flies. We were stung with the sharpest
stings of worms, and now and then we had to wait to let pass a
serpent that crossed our path. Were it not for the practised eyes of
Ghond who knew which way the animals came and went, we would
have been killed ten times over by a snake or a bison. Sometimes
Ghond would put his ear to the ground and listen. After several
minutes he would say: "Ahead of us bisons are coming. Let us wait till
they pass." And soon enough we would hear their sharp hoofs
moving through the undergrowth with a sinister noise as if a vast
scythe were cutting, cutting, cutting the very ground from under our
feet. Yet we pressed on, stopping for half an hour for lunch. At last we
reached the borders of Sikkim, whose small valley glimmered with
ripening red millet, green oranges and golden bananas, set against
hillsides glittering with marigolds above which softly shone the violets.
Just then we came to a sight that I shall never forget. At our feet on
the narrow caravan road the air burnt in iridescence: the heat was so
great that it vibrated with colours. Hardly had we gone a few yards
when like a thunderclap rose a vast flock of Himalayan pheasants;
then they flew into the jungle, their wings burning like peacocks'
plumes in the warm air. We kept on moving. In another couple of
minutes flew up another flock, but these were mud-coloured birds. In
my perplexity I asked Ghond for an explanation.
He said: "Do you not see, O beloved of felicity, the caravan that
passed here was loaded with millet? One of their sacks had a hole in
it. A few handfuls of millet leaked out on the road before the sack was
sewn up. Later on arrived these birds and fed themselves here. We
came upon them suddenly and frightened them to flight."
"But, O Diadem of Wisdom," I asked, "why do the males look so
gorgeous and why are the females mud-coloured? Is nature always
partial to the male?"
Ghond made the following explanation. "It is said that mother Nature
has given all birds the colours that hide them from their enemies. But
do you not see that those pheasants are so full of splendour that they
can be sighted and killed even by a blind man?"
Radja exclaimed: "Can they?"
Ghond answered: "O, wary beyond thy years, no! The real reason is
that they live on trees and do not come down before the earth is very
hot. In this hot India of ours the air two inches above the ground is so
burning that it quivers with a thousand colours; and the plumage of
the pheasant is similar. When we look at them we do not see birds,
but the many-coloured air which camouflages them completely. We
almost walked on them a few moments ago, thinking them but a part
of the road at our feet."
"That I comprehend," resumed Radja reverently. "But why did the
female look mud-coloured and why did they not fly away with the
male?"
Ghond answered without hesitation. "When the enemy approaches
and takes them by surprise, the male flies up to face the enemy's
bullets though without thought of chivalry. The females' wings are not
so good. Besides she, being of the colour of the earth, opens her
wings to shelter her babies under them, then lies flat on the ground,
completely melting away her identity into that colour scheme. After
the enemy goes away in quest of the corpses of their already
slaughtered husbands, the females run away with their babies into
the nearest thicket.... And if it is not too late in the year, and if their
grown-up babies are not with them, the mother birds singly flop to the
ground and lie there, making the gesture of protecting their young.
Self-sacrifice becomes a habit with them, and habitually they put forth
their wings whether they have any young ones with them or not. That
is what they were doing when we came upon them, then suddenly
they realized that they were without anyone to protect, and as we still
kept on coming down upon them they took to flight, poor fliers though
they are."
With the approach of dusk we took shelter in the house of a
Sikkimese nobleman whose son was a friend of ours. There we found
further traces of Gay-Neck, who had been to their house many times
before, and so when he reached the familiar place on his latest visit
he had eaten millet seeds, drunk water and taken his bath. Also, he
had preened his wings and left two small azure feathers which my
friend had preserved for the sake of their colouring. When I saw them
my heart leapt in joy, and that night I slept in utter peace and
contentment. There was another reason for sleeping well, for Ghond
had told us to rest deeply, as after the following day's march, we were
to spend the night in the jungle.
The next night when we sat on that tree top in the deep jungle, often
did I think of the home of my Sikkimese friend and its comforts.
Imagine yourself marching all day, then spending the night on a vast
banyan tree in the very heart of a dangerous forest! It took us a little
over half an hour to find that tree, for banyans do not often grow in
high altitudes, and also the same reason that made us choose the
banyan made us look for a very large one of its kind—it would be of
no use to us if it were slender enough for an elephant to break down
by walking backwards against it! That is how the pachyderm destroys
some very stout trees. We looked for something tall and so stout that
no elephant could reach its upper branches with his trunk and not
even two tuskers could break it by pushing against it with their double
weight.
 With enormously long reach he almost touched the top of the
tree.

At last we found a tree to our liking. Radja stood on Ghond's shoulder

and I on Radja's, until we reached branches as thick as a man's
torso. I climbed and sat on one of them and from it let down our rope
ladder which we always carried in the jungle for emergencies such as
the present one. Radja climbed up and sat near me, then Ghond
ascended the branch and sat between us. Now we saw that below us
where Ghond had stood it had not only grown dark as the heart of a
coal mine but there glowed two green lights set very close to one
another. We knew too well whose they were. Ghond exclaimed
jovially: "Had I been delayed down there two extra minutes the striped
fellow would have killed me."
Seeing that his prey had escaped him, the tiger gave a thunderous
yell, scourging the air like a curse. At once a tense silence fell and
smothered all the noises of insects and little beasts and it descended
further and deeper until it sank into the earth and seemed to grip the
very roots of the trees.
We made ourselves secure on our perch and Ghond passed the
extremely flexible rope ladder around his waist, then Radja's, then
mine, fastening the rest of it around the main trunk of the tree. We
tested it by letting it bear the weight of one of us at a time. This
precaution was taken for the purpose of preventing a sleep-stricken
member of our group from slipping down to the floor of the jungle, for
after all, in sleep the body relaxes so that it falls like a stone. Finally,
Ghond arranged his arms for pillows for our heads when slumber
came.
Now that we had taken all the necessary precautions we
concentrated our attention on what was happening below. The tiger
had vanished from under our tree. The insects had resumed their
song, which was again and again stilled for a few seconds as huge
shapes fell from far off trees with soft thuds. Those were leopards
and panthers who had slept on the trees all day and were now
leaping down to hunt at night.
When they had gone the frogs croaked, insects buzzed continually
and owls hooted. Noise, like a diamond, opened its million facets.
Sounds leapt at one's hearing like the dart of sunlight into
unprotected eyes. A boar passed, cracking and breaking all before
him. Soon the frogs stopped croaking, and way down on the floor of
the jungle we heard the tall grass and other undergrowth rise like a
hay-cock, then with a sigh fall back. That soft sinister sigh like the
curling up of spindrift drew nearer and nearer, then ... it slowly passed
our tree. Oh! what a relief. It was a constrictor going to the water hole.
We stayed on our tree-top as still as its bark—Ghond was afraid that
our breathing might betray our position to the terrible python.
A few minutes later we heard one or two snappings of small twigs
almost as faint as a man cracking his fingers. It was a stag whose
antlers had got caught in some vines and he was snapping them to
get himself freed. Hardly had he passed when the jungle grew very
tense with expectation. Sounds began to die down. Out of the ten or
a dozen different noises that we had heard all at once, there now
remained only three: the insects' tick tack tock, the short wail of the
stag—no doubt the constrictor was strangling him near the water hole
—and the wind overhead. Now the elephants were coming. Hatis
(elephants), about fifty in a herd, came and played around the place
below us. The squeal of the females, the grunt of the males, and the
run run run of the babies filled the air.
I do not remember what happened next, for I had dozed off into a sort
of waking sleep, and in that condition I heard myself talking pigeon-
language to Gay-Neck. I was experiencing a deep confusion of sleep
and dream. Someone shook and roused me. To my utter amazement
Ghond whispered "I cannot hold you any more. Wake up! Mischief is
abroad. A mad elephant has been left behind. The straggler is bent
on harm. We are not high enough to be altogether out of the reach of
his trunk, and if he raises it far enough he will scent our presence.
Wild elephants hate and fear man and once he gets our odour he
may stay about here all day in order to find out where we are. Rouse
thy vigilance, lad. Draw the blade of alertness before the enemy
strikes."
There was no mistake about that elephant. In the pale light of early
dawn I could discern a sort of hillock darkly moving about under our
tree. He was going from tree to tree and snapping off a few succulent
twigs that autumn had not yet blighted. He seemed greedy and bent
on gorging himself with those delicacies, rare for the time of the year.
In about half an hour he performed a strange trick, putting his fore
feet on the bole of a thick tree, and swinging up his trunk. It gave him
the appearance of a far-spreading mammoth; with that enormously
long reach he almost touched the top of the tree and twisted the most
delectable branches off its boughs. After having denuded it of its
good twigs he came to a tree next to ours, and there did the same
thing. Now he found a slender tree which he pulled down with his
trunk, and placed his forefeet on the poor bent thing and broke it with
a crash under his own weight. He ate all that he could of that one.
While he was breakfasting thus, his rampage frightened the birds and
monkeys, who flew in the air or ran from tree to tree jabbering in
terror. Then the elephant put his feet on the stump of the broken tree
and reached up into ours until he touched the branch on which we
sat. Hardly had he done so when he squealed, for the odour of man
all beasts fear, and swiftly withdrew his trunk. After grunting and
complaining to himself he put up his trunk again very near Ghond's
face. Just then Ghond sneezed almost into the elephant's nostrils.
That struck panic into the latter's heart; he felt beleaguered by men.
Trumpeting and squealing like a frightened fiend, he dashed through
the jungle, breaking and smashing everything before him. Again the
parrots, thick as green sails, flew in the sky. Monkeys screamed and
raced from tree to tree. Boars and stags stampeded on the floors of
the jungle. For a while the din and tumult reigned unchecked. We had
to wait some time before we dared to descend from our perch in
order to resume our homeward journey.
Late that day we reached home after being carried on horseback by a
caravan which we were fortunate enough to meet. All three of us
were dead tired, but we forgot our fatigue when we beheld Gay-Neck
in his nest in our house at Dentam. Oh, what joy! That evening before
I went to sleep I thought of the calm quiet assurance of the Lama who
said: "Your bird is safe."
CHAPTER VI
 GAY-NECK'S TRUANCY
ut the day after our return Gay-Neck flew away
again, in the morning, and failed to put in an
appearance later. We waited for him most
anxiously during four successive days, and then,
unable to bear the suspense any longer, Ghond
and I set out in search of him, determined to find
him, dead or alive. This time we hired two ponies to
take us as far as Sikkim. We had made sure of our path by asking
people about Gay-Neck in each village that we had to pass through.
Most of them had seen the bird and some of them gave an accurate
description of him: one hunter had seen him in a Lamasery nesting
next to a swift under the eaves of the house; another, a Buddhist
monk, said that he had seen him near their monastery in Sikkim on a
river bank where wild ducks had their nest, and, in the latest village
that we passed through on the second afternoon we were told that
he was seen in the company of a flock of swifts.
Led by such good accounts we reached the highest table-land of
Sikkim and were forced to bivouac there the third night. Our ponies
were sleepy, and so were we, but after what seemed like an hour's
sleep, I was roused by a tenseness that had fallen upon everything. I
found the two beasts of burden standing stiff; in the light of the fire
and that of the risen half moon I saw that their ears were raised
tensely in the act of listening carefully. Even their tails did not move. I
too listened intently. There was no doubt that the silence of the night
was more than mere stillness; stillness is empty, but the silence that
beset us was full of meaning, as if a God, shod with moonlight, was
walking so close that if I were to put out my hand I could touch his
garment.
Just then the horses moved their ears as if to catch the echo of a
sound that had moved imperceptibly through the silence. The great
deity had gone already; now a queer sensation of easing the tense
atmosphere set in. One could feel even the faintest shiver of the
grass, but that too was momentary; the ponies now listened for a
new sound from the north. They were straining every nerve in the
effort. At last even I could hear it. Something like a child yawning in
his sleep became audible. Stillness again followed. Then a sighing
sound long drawn out, ran through the air; which sank lower and
lower like a thick green leaf slowly sinking through calm water. Then
rose a murmur on the horizon as if someone were praying against
the sky line. About a minute later the horses relaxed their ears and
switched their tails and I, too, felt myself at ease. Lo! thousands of
geese were flying through the upper air. They were at least four
thousand feet above us, but all the same the ponies heard their
coming long before I did.
The flight of the geese told us that dawn was at hand, and I sat up
and watched. The stars set one by one. The ponies began to graze. I
gave them more rope; now that the night had passed they did not
need to be tied so close to the fire.
In another ten minutes the intense stillness of the dawn held all
things in its grip, and that had its effect on our two beasts. This time I
could see clearly both of them lifting their heads and listening. What
sounds were they trying to catch? I did not have long to wait. In a
tree not far off a bird shook itself, then another did the same thing,
on another bough. One of them sang. It was a song sparrow and its
cry roused all nature. Other song sparrows trilled; then other birds,
and still others! By now shapes and colours were coming to light with
blinding rapidity. Thus passed the short tropical twilight, and Ghond
got up to say his prayers.
That day our wanderings brought us to the Lamasery near Singalele
of which I have spoken before. The Lamas were glad to give us all
the news of Gay-Neck. They informed me that the previous
afternoon Gay-Neck and the flock of swifts who nested under the
eaves of the monastery had flown southwards.
Again with the blessings of the Lamas we said farewell to their
hospitable serai, and set out on Gay-Neck's trail. The mountains
burnt like torches behind us as we bestowed on them our last look.
Before us lay the autumn tinted woods glimmering in gold, purple,
green, and cerise.
CHAPTER VII
 GAY-NECK'S STORY
n the preceding chapter I made scanty references
to the places and incidents through which Gay-
Neck was recovered. Ghond found his track with
certainty the first day of our ten days' search for
him, but in order to see those things clearly and
continuously, it would be better to let Gay-Neck tell
his own Odyssey. It is not hard for us to understand
him if we use the grammar of fancy and the dictionary of imagination.
The October noon when we boarded the train at Darjeeling for our
return journey to town, Gay-Neck sat in his cage, and commenced
the story of his recent truancy from Dentam to Singalele and back.
"O, Master of many tongues, O wizard of all languages human and
animal, listen to my tale. Listen to the stammering wandering
narrative of a poor bird. Since the river has its roots in the hill, so
springs my story from the mountains.
"When near the eagle's nest I heard and beheld the wicked hawk's
talons tear my mother to pieces, I was so distressed that I decided to
die, but not by the claws of those treacherous birds. If I was to be
served up for a meal let it be to the king of the air; so I went and sat
on the ledge near the eagle's nest, but they would do me no harm.
Their house was in mourning. Their father had been trapped and
killed, and their mother was away hunting for pheasants and hares.
Since up to now the younglings had only eaten what had been killed
for them, they dared not attack and finish poor me who was alive. I
do not know yet why no eagle has harmed me; during the past days I
have seen many.
"Then you came to catch and cage me. As I was in no mood for
human company, I flew away, taking my chances as I went, but I
remembered places and persons who were your friends and I stayed