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CARACTERÍSTICAS DEL DESARROLLO DE
LA CAPACIDAD DIGESTIVA DURANTE LA
ONTOGENIA LARVARIA Y JUVENIL DEL
ATERINÓPSIDO CHIROSTOMA ESTOR, SIN
INTERFERIR LA MADURACIÓN DIGESTIVA
FEATURES OF DIGESTIVE ENZYME CAPACITY DEVELOPMENT DURING
THE LARVAL AND JUVENILE ONTOGENY OF THE ATHERINOPSID
CHIROSTOMA ESTOR, WITHOUT IMPAIRING DIGESTIVE MATURATION
Elva Mayra Toledo-Cuevas
Universidad Michoacana de San Nicolás de Hidalgo, México
Miguel Ángel Hernández González
Comité Estatal de Sanidad e Inocuidad Acuícola de Michoacán, México
Dariel Tovar-Ramírez
Centro de Investigaciones Biológicas del Noroeste, México
Ana Mauricia Ávalos Sánchez
Telebachillerato Michoacán Plantel 104, México
María Antonia Herrera-Vargas
Universidad Michoacana de San Nicolás de Hidalgo, México
Luis Sergio Villafuerte Herrera
Telebachillerato 55 Epitacio Huerta, México
María Guadalupe Zavala Páramo
Universidad Michoacana de San Nicolás de Hidalgo, México

pág. 10906
DOI: https://doi.org/10.37811/cl_rcm.v9i4.19639
Características del Desarrollo de la Capacidad Digestiva Durante la
Ontogenia Larvaria y Juvenil del Aterinópsido Chirostoma Estor, sin
Interferir la Maduración Digestiva
Elva Mayra Toledo-Cuevas1
mayra.toledo@umich.mx
https://orcid.org/0000-0003-0862-9455
Instituto de Investigaciones Agropecuarias y
Forestales, Universidad Michoacana
de San Nicolá s de Hidalgo
Tarímbaro, México
Miguel Ángel Hernández González
miguelaxo86@gmail.com
https://orcid.org/0009-0003-9234-6171
Comité Estatal de Sanidad
e Inocuidad Acuícola de Michoacán CESAMICH
Morelia, Michoacán, México
Dariel Tovar Ramírez
dtovar04@cibnor.mx
https://orcid.org/0000-0003-1204-9576
Centro de Investigaciones Biológicas del
Noroeste CIBNOR
Lab. de Fisiología Comparada y Genómica
Funcional. La Paz, B.C.S.
México
Ana Mauricia Ávalos Sánchez
ana.avalos@telebachillerato.michoacán.gob.mx
https://orcid.org/0009-0004-5801-8067
Telebachillerato Michoacán Plantel 104
Morelia, Michoacán
México
Maria Antonia Herrera-Vargas
antonia.herrera@umich.mx
https://orcid.org/0000-0002-8537-2561
Instituto de Investigaciones sobre los
Recursos Naturales, Morelia Universidad
Michoacana de San Nicolá s de Hidalgo
México
Luis Sergio Villafuerte Herrera
luis.villafuerte@telebachillerato.michoacan.gob.mx
Telebachillerato 55
Epitacio Huerta, Michoacán
México
María Guadalupe Zavala Páramo
maria.zavala.paramo@umich.mx
https://orcid.org/0000-0002-4545-3519
Centro Multidisciplinario
de Estudios en Biotecnología
Facultad de Medicina Veterinaria y Zootecnia
Tarímbaro, Universidad Michoacana de
San Nicolá s de Hidalgo
México
1 Autor principal
Correspondencia: mayra.toledo@umich.mx

pág. 10907
RESUMEN
El pez blanco Chirostoma estor es una especie con alto potencial de cultivo y calidad nutricional. A
pesar de los avances en su cultivo y alimentación, aún no se cuenta con una alimento balanceado que
permita el buen crecimiento obtenido con el alimento vivo. En este trabajo se estudió el desarrollo de
la actividad digestiva en la especie, desde la eclosión hasta los 150 días, usando solo alimento vivo
para evitar afectar la maduración digestiva. Los análisis de actividad se realizaron con técnicas
fluorómetricas (enzimas pancreáticas) y espectofotométricas (enzimas intestinales) validadas para
evaluar maduración digestiva. El crecimiento observado fue comparable con el obtenido utilizando
luz contínua y microdietas con proteína soluble y consorcio bacteriano. Se evidenció la necesidad de
incluir rotíferos y nauplios de Artemia en los primeros estadíos. Los indicadores de maduración
digestiva evidenciaron que C. estor sigue el modelo descrito para peces gástricos, con las
particularidades de ser tardío -posterior a los 5 meses de vida-, presentar niveles muy elevados de
actividad citosólica intestinal (leucin alanin peptidasa) y mantener esta actividad y la anclada a la
membrana de los enterocitos durante el estadio juvenil. Por consiguiente, se recomienda un destete
posterior al primer mes de vida, induciendo una maduración temprana.
Keywords: agástrico , intestino corto, alimento vivo, maduración digestiva tardía
Artículo recibido 04 Agosto 2025
Aceptado para publicación: 29 Agosto 2025

pág. 10908
Features of Digestive Enzyme Capacity Development During the Larval
and Juvenile Ontogeny of the Atherinopsid Chirostoma Estor, Without
Impairing Digestive Maturation
ABSTRACT
The silverside Chirostoma estor is a fish species with a high culture potential and nutritional quality.
However, despite the advances in its culture, there is not yet a balanced diet that allows a similar
growth obtained from feeding live feed. In this work, C. estor were fed only with live feed to avoid
impairing the digestive maturation. The development of the digestive activity was studied using
fluorometric (pancreatic enzymes) and spectrophotometric (intestinal enzymes) validated methods to
evaluate digestive maturation, from the first until 150 days post-hatching (dph). The obtained growth
was equivalent to that previously reported using continuous light and feeding fish with a microdiet
containing soluble protein and a bacterial consortium. The results show the importance of feeding
with rotifers and Artemia nauplii during early stages. Also, the digestive maturation indexes studied
show that C. estor follows the maturation model already described for gastric fish, with some
particularities: a late maturation, after 150 dph, very high levels of the intestinal cytosolic leucine
alanine peptidase and the maintenance of high levels of this and the brush border enterocyte
membrane digestive enzymes until the juvenile stage. Therefore, weaning after the first month of life
is recommended, enhancing early digestive maturation.
Keywords: agastric, short intestine, live feed, late digestive maturation

pág. 10909
INTRODUCTION
The silverside Chirostoma estor, endemic to Lake Patzcuaro, Mexico, is valued for its regional
socioeconomic importance and nutritional content, particularly its high levels of long-chain omega-3
polyunsaturated fatty acids essential to human health (Martínez-Palacios et al., 2020). A member of
the Atherinopsidae family, C. estor is agastric with a short intestine and lacks anatomical adaptations
for its limited digestive system (Horn et al., 2006; Ross et al., 2006), aside from a well-developed
branchial sieve and pharyngeal teeth suited to its zooplanktivorous habit (Ross et al., 2006; Martínez-
Palacios et al., 2019).
As an ancient marine species, C. estor hatches from small eggs and requires live feed for the first
three months of life (Martínez-Palacios et al., 2008). Early weaning was achieved using a high-protein
microdiet supplemented with Lactobacillus acidophilus and L. plantarum (Martínez-Angeles et al.,
2022), though final growth and specific growth rates did not surpass those from live feed in this or
prior studies (Toledo-Cuevas et al., 2011). Frequent feeding of juveniles improved growth and body
composition and reduced skeletal deformities (Melo et al., 2023). However, the knowledge of the
development of key digestive enzymes during ontogeny is essential for understanding C. estor’s
digestive physiology and designing species-specific diets.
In the first month post-hatching, fish larvae undergo significant anatomical and physiological
changes, including pancreatic maturation and the onset of enzyme secretion. Enterocyte maturation
follows, marked by increased activity of brush border enzymes and decreased cytosolic peptidase
activity. The final stage involves stomach development, with pepsin activation and the start of acid
digestion of proteins and lipids (Lazo et al., 2011; Zambonino-Infante et al., 2008). Understanding this
maturation process is essential for selecting suitable ingredients and formulating digestible, balanced
diets (Moyano et al., 2005).
A preliminary study on the digestive physiology of C. estor detected very high levels of the intestinal
cytosolic leucine-alanine peptidase (leu-ala) activity (Toledo-Cuevas et al., 2011) that may suggest a
functional compensation for an apparently restricted digestive anatomy, since this feature has also
been found in other atherinopsids (Toledo-Cuevas et al., 2024).

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Therefore, this study aimed to characterize the digestive ontogeny (pancreatic and intestinal) of C.
estor during larval and juvenile development, and the use of these enzyme activities as markers to
determine the model and timing of digestive maturation. To prevent the effects of poorly formulated
diets or premature weaning (Hamlin et al., 2000; Zambonino-Infante & Cahu, 2001; Zambonino-
Infante et al., 2008), fish were fed exclusively live feed for five months post-hatching.
MATERIALS AND METHODS
Source of fish and rearing conditions. Fertilized eggs of C. estor were obtained from broodstock at
the pilot farm of the Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana
de San Nicolás de Hidalgo, M éxico. Eggs were incubated at 25 °C in 1 L Zug jars with 7 g/L salinity
and 100 ml/min water flow until hatching. Larvae were collected as they swam up and transferred to
2.5 L plastic tanks placed inside a 45 L tank with a closed recirculating system and a 50 L biological
filter for the first 15 days post-hatching (dph). From day 15 to 45 dph, larvae were reared in the 45 L
tank. Afterwards, they were moved to 90 L plastic raceways with a closed recirculation system and
weekly water replacement. At 100 dph, juveniles were transferred to 1000 L tanks, remaining until the
trial ended at 150 dph. All cultures were maintained in freshwater, under a natural photoperiod, at
23.31 ± 0.35 °C—close to the species' optimal rearing temperature (Martínez-Palacios et al., 2002a).
Water temperature was recorded hourly using sensors (Thermotraker Pro), and physicochemical
parameters were assessed every 10 days. Salinity was evaluated with a refractometer (ATAGO S/Mill-
E), dissolved oxygen with an oximeter (YSI 55/25), pH with a potentiometer (Fisher, Accumet), and
ammonia, nitrites, and nitrates with a photometer (YSI-9500). All parameters were kept within
optimal ranges for the species (Martínez-Palacios et al., 2004).
Fish were fed live feed, ad libitum, three times a day (Martínez-Palacios et al., 2008). From hatching
to 25 dph, they received Brachionus plicatilis rotifers, followed by co-feeding with Artemia
franciscana nauplii from 15 to 60 dph. From 60 to 150 dph, feeding consisted exclusively of A.
franciscana metanauplii.
Sampling. Sampling was conducted in the morning before the first feeding, following a 17-hour fast.
Previous data showed trypsin activity remains stable up to 22 hours of fasting (not shown).

pág. 10911
Four pools of fish of at least 65 mg wet body weight were collected every third day (1, 3, and 5 dph),
then every five days (5 to 25 dph), and finally every 15 days (60 to 150 dph). From 45 dph onward,
when fish exceeded 100 mg, four individual fish were sampled to reduce animal use. Additionally,
12–15 fish per time point were collected in individual tubes for fluorometric analyses. Fish were
euthanized with ice-cold water, rinsed with distilled water, and excess water removed.
Samples consisted of whole fish aged 1 to 45 dph and dissected intestines and hepatopancreas from
individual fish aged 60 to 150 dph. Handling was done on ice-cold metal plates; samples were
weighed, snap-frozen in liquid nitrogen, and stored at -80°C. Wet body and organ weights were
measured using a microbalance (Mettler Toledo MX5) and an analytical scale (Mettler Toledo
AB204-S). To assess wet body weight (WBW; mg), larvae were pooled prior to each sampling: 60–
160 individuals at 1–3 dph and 10 individuals from 5–150 dph. Total length (TL; millimeters) was
measured for 10 larvae using a digital vernier.
Growth data analysis. The following formula was used to calculate the specific growth rate:
SGR = 100 (Ln WBW2- Ln WBWh)/ ∆t
Where WBW2 is the mean weight body mass (mg) at the end of this study (150 dph) or either at 25 or
27 dph, as indicated in the discussion section; WBWh is the mean weight body mass (mg) at hatch; ∆t
age at time 2 minus age at hatching (days).
The mean growth rate (mm/day) was calculated as the increment in TL from hatching to the periods 1
to 45 and 60 to 150 dph, based on previous reports (Alvarez et al., 2021):
GR = (TL2 – TL1)/ ∆t
Where TL2 = total length of fish at time 2 (either 45 or 150 dph); TL1 = total length at time 1 (either 1
or 60 dph), and ∆t = age at time 2 minus age at time 1.
Digestive enzyme activity assays
Fluorometric analysis. Individual fish or digestive organs (hepatopancreas and intestines, from 60-
150 dph; n = 12-15) were used to evaluate the activity of pancreatic enzymes: trypsin and lipase, as
reported before (Rotllant et al., 2008). The homogenate was prepared as indicated by Toledo-Cuevas
et al. (2011).

pág. 10912
The measurements were carried out on a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific) in
duplicate. All activities were reported as arbitrary fluorescence units per minute per individual
(U/individual) and as specific activity (U/mg of soluble protein). The percentage of secretion was
calculated from 60 dph when the digestive organs (hepatopancreas and intestine) could be obtained
(Zambonino-Infante & Cahu, 2001). In addition, the ratio of trypsin and lipase activities was
calculated by dividing the total activity of each digestive enzyme. This was obtained from 60 days by
summing the total activity levels obtained in the digestive organs.
Spectrophotometric analysis. Two samples from each pool (individuals or intestines) were used to
measure the activity of cytosolic digestive enzymes (leucine alanine peptidase and acid phosphatase),
while in the other two pool samples, the activity of intestinal brush border membrane (BBM)
digestive enzymes (alkaline phosphatase and aminopeptidase N) was measured.
The homogenate for cytosolic analysis was prepared in cold distilled water using a tissue disruptor
(Fisher Scientific Model 150E), with 10-sec pulses at an amplitude level of 10, until complete tissue
homogenization. The tissues were always kept within ice-cold containers to prevent enzyme
denaturation. The homogenates were centrifuged at 15,700 g for 30 min at 4°C, in a refrigerated
microcentrifuge (Eppendorf 5415 R). The supernatants were stored in 0.1 ml aliquots at –20°C until
analysis. Individual aliquots were used for each digestive enzyme determination to avoid the loss of
enzyme activity by frosting/ defrosting cycles. BBM activities were analyzed in purified intestinal
membranes prepared as reported before (Crane et al., 1979) and modified subsequently (Cahu &
Zambonino, 1994), except for the centrifugation speed (see below). Whole individuals (1-45 dph) and
intestines were homogenized in 30 volumes of cold buffer solution containing 50 mM mannitol, 2
mM Tris, pH 7. Subsequently, 0.1 M CaCl2 was added to a final concentration of 10 mM, followed by
a first centrifugation at 9000 g for 10 min at 4°C. The supernatant was recovered and centrifuged at
24,040 g for 20 min at 4°C. The sedimented BBM was homogenized by sonication for 10-15 seconds
in 1 mL of 0.1 M KCl, 1 mM Dithiothreitol, 5 mM Tris-HEPES, pH 7.5, at 4°C.
The activity of leucine alanine-peptidase (leu-ala) was determined as reported by Nicholson & Kim
(1975), while the acid phosphatase (AcP) activity was measured using the methodology of Bergmeyer
et al. (1974).

pág. 10913
The activity of these enzymes was measured at a final point in a spectrophotometer (Cary 50 Varian).
Alkaline phosphatase (AP) activity was analyzed according to Bergmeyer et al. (1974), while
Aminopeptidase N (APN) activity was assayed according to Maroux et al. (1973). The activity of
these enzymes was kinetically recorded. For leu-ala, one unit of activity was defined as 1 nmol of
substrate hydrolyzed per minute at 37°C. For all the other digestive enzymes, one unit of activity was
defined as 1 μmol of hydrolyzed substrate per minute at 37°C. All assays were performed in triplicate,
and the values were averaged for subsequent calculations.
The soluble protein concentration of all homogenates was determined using the Bradford method
(Bradford, 1976), adapted to a microplate, measuring the absorbance in a photometer (ELIREAD,
Kontrolab). Enzyme activity was calculated as total activity (U/individual or mU/individual) and
specific activity (U/mg protein or mU/mg protein). The ratio of BBM and cytosolic activities was
calculated by dividing the total AP and APN activities, respectively, by the total activity of leu-ala or
AcP. For these calculations, the leu-ala activity was expressed in μmoles, as were the AP and APN
activities.
Statistics
Welch’s two-sample t-test was used to compare the slopes of the decimal-log transformed body
weight and total length, and growth rate from 1-45 and 60-150 dph. One-way analysis of variance or
Kruskal-Wallis’s analysis of variance on ranks (α = 0.05) (depending on the normality and equal
variance of the data) was used to evaluate: a) the influence of the different feed types on the slopes of
decimal log wet body weigh at 1-15, 20-25, 30-45, and 60-150 dph, and on the specific activity of
each digestive enzyme from 1-45 dph; b) the differences on total activities of digestive enzymes
throughout the ontogeny; c) on specific activities, from 1 to 45 dph and 60 to 150 dph, for the sample
differences explained above (whole body versus hepatopancreas or intestine); d) the differences on the
trypsin/lipase and intestinal brush border/cytosolic digestive activities ratios along the fish ontogeny;
and e) the differences on trypsin and lipase secretion values from 60-150 dph. The Shapiro-Wilk and
Fligner-Killeen tests were used to test normality and equality of variance, respectively. A post hoc
comparison, Tukey's Multiple Range Test or Dunn's Pairwise Multiple Comparison Procedure, was
used to determine the significance of differences.

pág. 10914
The influence of diet (type of live feed) and age on the specific activity of digestive enzymes was
analyzed using ANCOVA, considering age as a continuous covariate and diet as a factor. All statistical
analyses were performed using R 4.2.2 software (R Core Team, 2020).
RESULTS
This is the first study describing the species' growth from hatching to 150 days after hatching. A
potential growth profile of C. estor was observed during the larval and juvenile stages for WBW and a
linear profile for TL, with the following equations: WBW = 0.005dph4.24, R² = 0.98; TL = 3.21dph -
6.18, R² = 0.97 (Fig. S1). When both growth parameters were transformed to decimal base logarithms,
two phases were observed (Fig. 1). The growth slope in WBW and TL was significantly greater in the
period between one to 45 dph (log10 WBW = 0.06dph - 0.26, R² = 0.97; log10 TL = 0.02 dph + 0.75,
R² = 0.92; mg or mm ± standard deviation) than between 60 to 150 (log10 WBW = 0.01dph + 1.46,
R² = 1.00; log10 TL = 0.003dph + 1.22, R² = 1.00) (d.f. = 16 and d.f. = 18, respectively, P < 0.00001).
In addition, the mean growth rate (GR) for the period from 1 to 45 dph was higher (0.54 ± 0.02) than
for 60 to 150 dph (0.29 ± 0.01) (d.f. = 17.39, P < 0.00001). On the other hand, the specific GR for the
larval period (30 dph) was 13.69 g/100 g, while for the entire period it was 5.39 g/100 g.
Figure 1.
Decimal-log wet body weight (mg ± standard deviation, SD, n = 10 (except for 1 to 10 dph, where
pools of 60-160 individuals were needed), black circles) and decimal-log total length (mm ± SD, n =
10, black triangles) from Chirostoma estor during the larval and juvenile development. Growth is

pág. 10915
shown separately with the respective regression lines, with the following equations: 1 to 45 dph:
log10 WBW = 0.06dph - 0.26, R² = 0.97; log10 TL = 0.02 dph + 0.75, R² = 0.92; 60 to 150 dph: log10
WBW = 0.01dph + 1.46, R² = 1.00; log10 TL = 0.003dph + 1.22, R² = 1.00. Horizontal broken lines
indicate the period of the different feeding regimes. WBW: Wet body weight; TL: Total length.
The influence of the different live feeds on the log 10 WBW slope during the development of C. estor
is shown in Table 1. No significant differences were observed in the growth slope when feeding
rotifers alone or when co-feeding rotifers and Artemia nauplii. Nevertheless, the growth slope
decreased when fish were fed on Artemia nauplii and Artemia metanauplii.
Table 1. Effect of the feeding regime on the slope of decimal-log wet body weight at different age
periods of Chirostoma estor.
Age (dph)
1 to 15 20 to 25 30 to 45 60 to 150 Statistics
Daily feeding
regime
Rotifers Rotifers +
Artemia nauplii
Artemia
nauplii
Artemia
metanauplii
d.f. F
Slope 0.07
±0.01a
0.10
±0.01a
0.03
±0.00b
0.01
±0.00c
3 118.97
Different letters indicate significant differences between age periods (slope mean ± standard error, S.E.; n = 10, P <0.00001).
dph: days post-hatching.
Regarding the effect of age on digestive activities, total trypsin and pancreatic lipase activity
increased throughout the development of C. estor (Fig. 2A, C). In contrast, the specific trypsin
activity showed a fluctuating profile during the first 45 dph, with a significant increase at the end of
this period (Fig. 2B). When specific activity was measured in the digestive organs (hepatopancreas
and intestines) from 60 to 150 dph, the profile was high and stable. The specific lipase activity
showed a significant increase on the third day, after which it decreased and remained at low levels
until 45 dph. However, specific activity during the 60 to 150 dph period was high and relatively stable
(Fig. 2D).

pág. 10916
Figure 2
Total and specific activity of pancreatic digestive enzymes during the larval and juvenile development
of Chirostoma estor. A and B: trypsin; C and D: lipase. Statistical analysis of the specific activity was
performed in two periods: 1 to 45 and 60 to 150 dph due to tissue differences (digestive organs from
60 to 150 dph). Each circle represents the mean of twelve to fifteen individuals. Different letters
indicate significant differences (P < 0.05) between days post-hatching. Statistical significance was
based on One-way ANOVA or Kruskal-Wallis Analysis of Variance on Ranks, followed by Tukey's
Multiple Range Test or Dunn's Pairwise Multiple Comparison Procedure, depending on normality and
variance. The internal graph in A shows the levels of trypsin-specific activity during the first 30 dph.
Horizontal broken lines indicate the period of the different feeding regimes. dph: days post-hatching.
The total activity of the cytosolic digestive enzymes, AcP and leu-ala, increased throughout larval and
juvenile development of C. estor (Fig. 3A, C). The AcP specific activity levels were very low and
stable during the first 45 dph. Similarly, the activity recorded between 60 to 150 dph was stable, but
with higher levels since dissected intestines were analyzed in this period (Fig. 3B). In contrast, the
specific activity of leu-ala remained constant during the first 45 days. Subsequently, from 60 to 150
dph, the activity showed a significant increase at 105 dph, with similar activity levels thereafter (Fig.
3D).

pág. 10917
Figure 3.
Total and specific activity of the intestinal cytosolic digestive activities during larval and juvenile
development of Chirostoma estor. A and B: acid phosphatase; C and D: leucine alanine peptidase. The
statistical analysis of the specific activity was performed in two periods: 1 to 45 and 60 to 150 dph,
due to tissue differences. Each circle represents the mean of two pools of individuals or two intestines.
Different letters indicate significant differences (P < 0.05) between days post-hatching. Statistical
significance was based on One-way ANOVA or Kruskal-Wallis Analysis of Variance on Ranks,
followed by Tukey’s Multiple Range Test or Dunn’s Pairwise Multiple Comparison Procedure,
depending on normality and variance. Horizontal broken lines indicate the period of the different
feeding regimes. dph: days post-hatching.
The total activity of the intestinal brush border membrane digestive enzymes, AP and APN, increased
during larval and juvenile development of C. estor, albeit at very low levels (Fig. 4A, C). On the other
hand, the specific AP activity was also low but fluctuated during the first 45 days. In the second
developmental period, the activity significantly rose until 105 dph, falling afterwards (Fig. 4B). APN-
specific activity was low and fluctuating during the first 45 dph, while for the period from 60 to 150
dph, APN activity was analogous to that observed for AP. However, a second increase was observed
after the significant increase at 105 dph (Fig. 4D). Interestingly, the specific activity of all the
intestinal digestive enzymes cytosolic and membrane-bound) showed an abrupt increment at 105 dph.

pág. 10918
Figure 4.
Total and specific activity of intestinal border membrane-digestive enzymes during larval and juvenile
development of Chirostoma estor. A, B: alkaline phosphatase; C, D: aminopeptidase N. Statistical
analysis of specific activity was performed in two periods: 1 to 45 and 60 to 150 dph due to tissue
differences. Each circle represents the mean of two pools of individuals or two intestines. Different
letters indicate significant differences (P < 0.05) between days post-hatching. Statistical significance
was based on One-way ANOVA followed by Tukey’s Multiple Range Test. The internal graph in B
shows the levels of alkaline phosphatase specific activity during the first 45 dph. Horizontal broken
lines indicate the period of the different feeding regimes. dph: days post-hatching.
The analysis of the influence of feed regime (feed type) on the specific digestive activity is shown in
Table 2. As is observed, the activity of trypsin increased when fish were fed exclusively with Artemia
nauplii. Although fish were fed on Artemia nauplii from day 30, the sample at this age was taken
before feeding. Therefore, the influence of this feed was noted up to 45 dph. The lipase activity
showed a fluctuating profile, with a tendency to decrease. However, feeding fish with Artemia nauplii
also positively influenced lipase activity. No significant differences were detected in the activity of
intestinal cytosolic digestive enzymes in relation to the feeding regime. Regarding the brush border
membrane enzymes, co-feeding with rotifers and Artemia nauplii had a negative effect on AP and

pág. 10919
APN activities, while their activity increased when fish were fed exclusively on Artemia nauplii
(Table 2).
Table 2. Effect of feeding regime on the specific digestive enzyme activity during the ontogeny of
Chirostoma estor.
Feeding regime Rotifers Rotifers +
Artemia nauplii Artemia nauplii
Age (dph) 1 3 5 10 15 20 25 30 45
Trypsin 27.18
 4.91a
36.04
 5.36a
30.45
 3.96a
44.08
 5.12a
10.83
 1.31a
15.56
 2.54a
2.02
0.19a
13.02
 2.52a
104.93
23.46b
Lipase 2391.88
412.47
a
5502.70
284.52
a
4078.86
321.33
a
1945.61
420.28
a
246.30
26.38
a
100.60
14.64a
140.19
18.74b
397.45
29.55b
268.82
31.79b
Alkaline
phosphatase
29.69
 0.38a
44.49
1.96a
27.13
1.28a
38.32
4.85a
11.84
6.89a
33.75
0.02a
5.37
0.77b
3.18
0.98b
27.77
0.39ab
Aminopeptidase N 332.47
 49.52a
155.64
17.12a
484.77
28.61a
432.03
65.12a
173.25
0.12a
155.50
8.74a
16.97
0.50b
42.54
6.19b
182.15
36.36ac
For the one-way ANOVA analysis, feeding regime was considered from 1 to 20 with rotifers, 25 to 30
with co-feeding on rotifers and Artemia nauplii, and from 45 dph with exclusively Artemia nauplii.
This was because, on days 20 and 30, fish were sampled before feeding and, therefore, before the
change in feed type. Alkaline phosphatase and aminopeptidase N are expressed as mU/mg of protein
 10-3. dph, days post-hatching.
When analyzing the influence of the feeding regime and age of the fish, it was found that the activities
of digestive pancreatic enzymes were significantly swayed by the interaction of both variables (Table
3).
Also, diet and its interaction with age affected the specific activity of leu-ala. Meanwhile, acid
phosphatase activity was influenced by both diet and fish age. Likewise, age and its interaction with
diet influenced AP activity, while specific APN activity was controlled by age, feeding regime, and
their interaction (Table 3).

pág. 10920
Table 3. Results of the ANCOVA model for the effect of diet (feeding regime) and fish age on the
digestive enzyme activities of Chirostoma estor during its larval and juvenile ontogeny. The model
considered age as a covariate and diet as a factor.
Covariate and
factor
Specific activity
d.f. SS F P
Trypsin Age 1 22992 21.53 <0.00001
Diet 2 38337 17.95 <0.00001
Age:Diet 2 53629 25.11 <0.00001
Lipase Age 1 244657268 136.80 <0.00001
Diet 2 46535987 13.01 <0.00001
Age:Diet 2 59980386 16.77 <0.00001
leu-ala Age 1 56 0.05 0.83
Diet 2 13545 5.79 0.01
Age:Diet 2 22922 9.79 0.01
Acid
phosphatase
Age 1 104.96 9.72 0.00
Diet 2 80.17 3.71 0.03
Age:Diet 2 5.74 0.27 0.77
Alkaline
phosphatase
Age 1 0.00 17.45 0.00
Diet 2 0.00 0.68 0.51
Age:Diet 2 0.00 22.23 <0.00001
APN Age 1 0.42 25.93 <0.00001
Diet 2 0.21 6.52 0.00
Age:Diet 2 0.12 3.67 0.03
On the other hand, a significant increase in the percentage of trypsin secretion was observed on day
75, unlike lipase secretion (Fig. 5A, B).

pág. 10921
Figure 5.
Percentage of secretion. A: Trypsin and B: lipase. C: Trypsin/lipase activity ratios. Each bar represents
the mean of twelve to fifteen individuals or organs. Different letters indicate significant differences (P
< 0.05) between days post-hatching. Statistical significance was based on Kruskal-Wallis Analysis of
Variance on Ranks followed by Dunn’s Pairwise Multiple Comparison Procedures. The internal graph
in C shows the levels of the trypsin/lipase activity ratio during the first 30 days. dph: days post-
hatching.
Regarding the trypsin/lipase ratio, three significant increases were observed at 20, 45, and 60 dph (Fig
5C). With respect to the ratios of BBM/cytosolic activities, two common and statistically significant
increases were observed for AP/leu-ala and AP/AcP, at 3 and 105 dph. The increase at 3 dph was also
observed for APN/leu-ala and APN/AcP, but another significant increase was observed at 120 dph for
the latter (Fig. 6).

pág. 10922
Figure 6.
Intestinal brush border and cytosolic activity ratios. A and B: AP/leu-ala and AP/AcP activities ratios,
respectively. C and D: APN/leu-ala and APN/AcP activities ratios, respectively. Each bar represents
the mean of two pools of individuals or digestive organs. Different letters indicate significant
differences (P < 0.05) between days post-hatching. Statistical significance was based on One-way
ANOVA or Kruskal-Wallis Analysis of Variance on Ranks, followed by Tukey’s Multiple Range Test
or Dunn’s Pairwise Multiple Comparison Procedure, depending on normality and variance. dph: days
post-hatching.
DISCUSSION
Growth performance and the impact of nutrients and weaning time
The growth of C. estor, in terms of body weight and total length, in this study resembles that found in
other studies for the species, under similar conditions (Martínez-Palacios et al., 2002b; Navarrete-
Ramírez et al., 2011), although this is the first time that the growth profile has been analyzed from the
larval and juvenile stages up to 150 dph. The specific growth rate found for the larval period is also
analogous to previous studies (Toledo-Cuevas et al., 2011; Martínez-Chávez et al., 2014; Juárez -

pág. 10923
Gutiérrez et al., 2021), suggesting a common and normal development for the species in the present
study.
On the other hand, a higher decimal-log wet body weight and mean growth rate were found for the
first period (1-45 dph), compared to the second period (60-150 dph). A similar performance has been
reported in the anchovy Engraulis encrasicolus and the sardine Sardine pilchardus. These species
showed higher growth rates than mackerel Scomber scombrus, horse mackerel Trachurus trachurus
and hake Merluccius merluccius, suggesting that, for the cupleoids, swimming ability is more
important than the development of a large mouth (Alvarez et al., 2021). Also, for the thick lip grey
mullet Chelon labrosus, improving locomotor function is critical for food capture and predator
avoidance (Gilannejad et al., 2020). Constant movement and a functional mouth have been described
in C. estor one-day post-hatch larvae (Martínez-Palacios et al., 2006), and food has been found in
their digestive tract at two days of age (Martínez-Angeles et al., 2022), suggesting a fast-growing
larval stage in C. estor, which supports our findings. Similar fast larval growth has been described in
two other atherinopsids, Odontesthes bonariensis and O. hatcheri (Toledo-Cuevas et al., 2024).
Although different growth phases are related to the morphophysiological changes occurring during
fish development, these can also be influenced by the composition of the live feed. The SGR obtained
in this study (at 25 days) is higher than that obtained by Juárez -Gutiérrez et al. (2021) at 27 days
(16.47 vs 14.43) and by Martínez-Angeles et al. (2022) (13.69 vs 10.87, at 30 days), suggesting a
better response of C. estor larvae to co-feeding on rotifers and Artemia rather than feeding exclusively
on rotifers. Similar results were obtained for the pikeperch Sander lucioperca fed on a combination of
rotifers and Artemia nauplii (Imentai et al., 2020; Yanes-Roca et al., 2018). A higher average protein
and free amino acids content in Artemia nauplii than in rotifers can result in higher larval growth
(Hamre et al., 2002; Carvalho et al., 2003; Conceição et al., 2010; Hamre, 2016).
On the other hand, the reduction in the slope of the decimal-log body weight between 60 to 150 dph
may be attributed to the different nutrient and energy content of Artemia nauplii compared to Artemia
metanauplii (Sorgeloos et al., 2001; Guevara & Lodeiros, 2003), which could cause an imbalance in
the nutritional requirement for the juvenile stage. Alternatively, the growth slowdown could also be
caused by the difficulty of Artemia in meeting the energy and nutrient requirements of fish after 60

pág. 10924
days, related to the small size of Artemia metanauplii and the large amount of live feed required by
larger fish, as suggested in other studies (Hamre et al., 2002).
Fish are known to maximize their energy gain by ingesting larger, higher-calorie prey (Sorgeloos et
al., 2001; Hernández -Rubio et al., 2006; Bittar et al., 2012).
It is important to note that feeding only live feed for this study was performed to seek the maturation
of the digestive system of the species, under cultured conditions, avoiding the introduction of a
balanced diet, which could impair the maturation process (Zambonino-Infante & Cahu, 2001;
Zambonino-Infante et al., 2008). Previous studies have determined that the digestive maturation in C.
estor did not occur during the larval stage, that is, the 30 days post-hatching (Toledo-Cuevas et al.,
2011). This may be the reason of why the best SGR obtained before (Martínez-Angeles et al., 2022) at
30 dph (13.79 %/day) is barely comparable with the one obtained in this study (13.69 at 30 dph) when
it should have been much higher in the former study due to various factors that should have improved
growth: 1) the fish were fed on a microdiet containing 278 g of soluble protein/kg and a mixture of
Lactobacillus (0.5% L. acidophilus and 0.5% L. plantarum). The very high levels of the cytosolic
(lysosomal) leu-ala activity found in C. estor (Toledo-Cuevas et al., 2011) suggests its great capacity
to digest soluble proteins that would enter the intestine by pinocytosis, already demonstrated in
juveniles of the species (Toledo-Cuevas et al., unpublished); 2) the inclusion of probiotics in feeds
accelerates digestive maturation (Tovar et al., 2002; Ringø et al., 2020), and 3) the use of continuous
illumination (24L) in the culture of C. estor also increases growth (Martínez-Chávez et al., 2014;
Corona-Herrera et al., 2022 ). However, in the study of Martínez-Angeles et al. (2022), larvae were
fed only on rotifers, and weaning occurred at 10 dph. Better results would undoubtedly have been
obtained if the fish had been fed on rotifers and Artemia, and especially if weaning were performed at
a later stage of development, since, as discussed later, the maturation of the C. estor digestive system
seemed to occur after 105 dph. The nutrigenomics study conducted on C. estor weaned at 10 dph
clearly shows that, at this early stage, larvae are not ready to be fed on microdiets (Juárez -Gutiérrez et
al., 2021).
A similar negative impact on larval growth of weaning at an early stage has previously been reported
in other fish species (Chen et al., 2022).

pág. 10925

pág. 10926
Impact of age and nutrients on digestive enzyme activities
The feeding regime influenced the digestive activity of most of the analyzed enzymes. Although the
nutritional content of the live feed used in this study was not assessed, differences have been reported
between rotifers and different life stages of Artemia (Hamre et al., 2002; Carvalho et al., 2003;
Guevara & Lodeiros, 2003; Guermazi et al., 2008; Conceição et al., 2010; Hamre, 2016; Ringø et al.,
2020). The protein content of B. plicatilis and Artemia nauplii varies depending on several factors
(Guevara & Lodeiros, 2003; Guermazi et al., 2008; Øie et al., 2011; Peykaran Mana et al., 2014),
although higher protein levels have occasionally been reported in the latter (Hamre et al., 2002;
Conceição et al., 2010; Kotani et al., 2017; Peykaran Mana et al., 2014). This could explain the
increase in trypsin activity from 45 dph. Higher trypsin activity was also reported in S. lucioperca
when feeding on rotifers, followed by Artemia (Imentai et al., 2022). Furthermore, C. estor larvae
could acquire more protein when feeding on Artemia nauplii since their larger size (compared with
rotifers) makes them easier to capture (Guevara & Lodeiros, 2003; Conceição et al., 2010), which
would increase trypsin activity since it is stimulated by its substrate (Cahu et al., 2004). On the other
hand, although a higher lipid content is sometimes reported in Artemia nauplii than in rotifers (Hamre
et al., 2002; Conceição et al., 2010; Peykaran Mana et al., 2014; Hamre, 2016), which may explain
the gradual increase in lipase activity at 30 and 45 dph, the opposite trend of trypsin and lipase
activities during the first 45 days is also related to the progressive shift from the use of lipids as the
main nutrient to proteins, as reported for this and other species (Cara et al., 2003; Toledo-Cuevas et
al., 2011). The higher phospholipid levels reported in rotifers than in Artemia nauplii (Øie et al., 2011;
Dhont et al., 2013) could explain the decrease in alkaline phosphatase during the larval stage of C.
estor, since AP activity is stimulated by nutrients containing organic phosphate, such as phospholipids
(Lallès, 2020). However, AP activity may also be positively influenced by a possible higher
consumption of Artemia nauplii by C. estor larvae, whose larger size than rotifers would facilitate it.
AP activity is also significantly stimulated by food intake (Lallès, 2020). Higher AP levels were
observed in S. lucioperca fed on Artemia or in combination with rotifers (Imentai et al., 2022).
Finally, the different protein content between rotifers and Artemia nauplii could explain the
differences in APN activity.

pág. 10927
An increase in APN activity was found to be related to the shift of rotifers to Artemia in G. morhua
larvae (Kvåle et al., 2007) and S. lucioperca (Imentai et al., 2022). APN activity is influenced by the
level and source of dietary protein (Nicholson et al., 1974; Zambonino Infante & Cahu, 1994; Kvåle
et al., 2007). However, APN is present in other organs besides the BBM of the intestine (Tang et al.,
2016), which may influence the profile found for APN in C. estor larvae since their whole body was
used until 45 dph. Future studies should include the evaluation of the nutritional content of the live
feed to clarify all these diet-related results of digestive enzyme activity.
On the other hand, age (throughout larval and juvenile stages) and the type of live feed were also
observed to affect all the digestive enzymes studied in C. estor. Changes in the digestive enzyme
activity during fish development have been widely described in other species, as has the influence of
the nutrient content and their form (Zambonino-Infante et al., 2008; Zambonino-Infante & Cahu,
2010). In addition to the above-mentioned differences in the nutritional content between rotifers and
Artemia, nutrients also differ across the different Artemia stages. The nutritional content of Artemia is
influenced by abiotic and biotic factors, feeding strategy, food bioavailability, and development stage,
especially since Artemia feeding begins at the metanauplii stage (Guevara & Lodeiros, 2003;
Guermazi et al., 2008; Peykaran Mana et al., 2014).
Digestive enzyme activity features and maturation markers in C. estor
The maturation process of the digestive system can be monitored through the profile and activity
levels of the different digestive enzymes synthesized in their respective organs, which has led to the
discovery of biochemical markers of maturation in fish. Nevertheless, all these studies have been
conducted mainly in commercial species that develop functional stomachs (Zambonino-Infante et al.,
2008). Most studies on agastric fish have not been designed to investigate the profile of digestive
enzyme activities during fish ontogeny, or they did not assess intestinal enzyme activities
(Zambonino-Infante et al., 2008; Gisbert et al., 2013; Le et al., 2019). However, some studies of this
kind have been carried out on South American silversides (known as pejerreyes) and on C. estor, the
silverside of the Lake of Patzcuaro, to elucidate the model and timing of their digestive maturation
(Pérez-Sirkin et al., 2020; Toledo-Cuevas et al., 2011, 2024). All these atherinopsid species are not
only agastric but also have a short intestine (Toda et al., 1998; Ross et al., 2006; Toledo-Cuevas et al.,

pág. 10928
2024). The study of some digestive maturation markers for C. estor describes that this process does
not occur before the first 3 months of life (Toledo-Cuevas et al., 2011), and digestive maturation
markers are not yet detected in pejerreyes up to 10 weeks post-hatching (Pérez-Sirkin et al., 2020).
Among the known markers of digestive maturation, cytosolic (lysosomal) intestinal digestive activity
is known to be relevant during early stages of life when the digestive system has not fully matured.
During the process of maturation, this lysosomal activity decreases, coinciding with a pronounced
increase in the activity of digestive enzymes linked to the brush border (Zambonino-Infante & Cahu,
2001; Zambonino-Infante et al., 2008). In C. estor, the present study shows that the cytosolic activity
levels of leu-ala and AcP do not decrease during the first 45 dph but significantly increase at 105 dph.
Since the role of these enzymes has been described in the digestion of nutrients acquired by
pinocytosis (Henning, 1987; Zambonino-Infante & Cahu, 2001; Lazo et al., 2011), and because
pinocytosis has been demonstrated in juveniles of C. estor (Toledo-Cuevas et al., unpublished), it
seems that the species maintains the activity of these enzymes as a functional compensation for its
lack of a stomach and its possession of a thin and short intestine. This digestive strategy appears to be
shared by other aquatic organisms that possess a “primitive” digestive system, such as the pejerreyes
Odontesthes bonariensis and O. hatcheri (Toledo-Cuevas et al., 2024) and the sea cucumber
Isostichopus badionotus, which does not possess a functional stomach (Martínez-Milián et al., 2021).
Although there are reports of leu-ala activity that persists beyond the first month of life in some fish
species, such as the cod Gadus morhua and the Atlantic halibut Hippoglossus hippoglossus, up to 75
and 117 dph, respectively (Kvåle et al., 2007), the activity levels are much lower than those found in
C. estor. Thus, not only is the prolonged presence of leu-ala and AcP a characteristic of this type of
digestive system, but also the very high levels of leu-ala, several times higher than in other agastric
fish with a long intestine and in gastric fish (Toledo-Cuevas et al., 2024). Specialized enterocytes in
zebrafish internalize dietary protein for intracellular digestion throughout their life and are essential
for the growth and survival of fish larvae and mice (Park et al., 2019).
The activity of BBM enzymes, APN and AP, was detected on the first day post-hatching of C. estor
(just like the activity of all the cytosolic and pancreatic digestive enzymes herein studied), and like the
cytosolic digestive activities, their activity increases with age.

pág. 10929
The BBM activity is, however, much lower than that previously reported for C. estor (Toledo-Cuevas
et al., 2011). This, even though in the present study, BBM were purified, suggesting difficulties in the
analytical methods. Notwithstanding this, a significant increase in the activity of both enzymes was
detected at 105 dph and constant levels thereafter. BBM enzymes have been reported as markers of
digestive maturation (Kvåle et al., 2007; Zambonino-Infante & Cahu, 2011), but their specific
activities decline a few weeks after hatching in several fish species (Zambonino-Infante et al., 2008;
Solovyev et al., 2016; Koven et al., 2019; Mozanzadeh et al., 2021). The high and constant levels of
BBM activities found for C. estor suggest that this species maintains all the digestive activity it
possesses (cytosolic and BBM-linked) as a digestive strategy due to its digestive anatomical
limitations: absence of a stomach and a thin and short intestine.
Findings of digestive maturation were observed in C. estor. The ratios of trypsin/lipase activities
suggest the early acquisition of pancreatic function, between 20-45 dph, since at those ages, lipase
activity decreases in relation to protein digestion by trypsin, as also described for the white seam
bream Diplodus sargus (Cara et al., 2003), a profile that resembles previous studies in C. estor
(Toledo-Cuevas et al., 2011). On the other hand, the secretion function of pancreatic enzymes appears
to be acquired between 60-75 dph. It has been suggested that the pancreatic function is completed
shortly after hatching, while secretion begins later in the development, followed by intestinal
maturation (Zambonino-Infante et al., 2008; Lazo et al., 2011). In C. estor, the BBM/cytosolic ratios
showed significant increases at 105, 120 and 150 dph, suggesting a late maturation of intestinal
function (Zambonino Infante & Cahu, 1994), as previously reported (Toledo-Cuevas et al., 2011).
However, it is important to highlight that these ratios were not found in O. bonariensis (Pérez-Sirkin
et al., 2020), suggesting that either the high and prolonged levels of cytosolic enzymes impaired the
finding of this maturation index or that intestinal maturation occurs quite late in development, as
observed for C. estor (Toledo-Cuevas et al., unpublished). Interestingly, at 105 dph, almost all the
analyzed digestive enzyme activities peaked, suggesting that some changes in the microanatomy of
the digestive tract might occur at this age. This should be clarified with future histological studies.

pág. 10930
CONCLUSIONS AND IMPLICATIONS
This study suggests that the combined feeding on rotifers and Artemia nauplii is relevant for optimal
growth of C. estor larvae. It was also found that C. estor digestive system maturation occurs around
105 dph, indicating that weaning should occur after the third month post-hatching. Furthermore, the
maturation indexes described for gastric fish (pancreatic secretion, trypsin/lipase and the
BBM/cytosolic intestinal digestive ratios) are present in C. estor, despite its absence of a stomach.
Nevertheless, some of the compensations for its “limited” digestive system are the prolonged presence
of both cytosolic and brush-border digestive enzymes, at least until 150 dph, in addition to the
unusually high levels of leu-ala activity.
Acknowledgements and conflict of interest
We thank Jesús López García for his essential help rearing C. estor for this study. This work was
funded by CONACyT (Consejo Nacional de Ciencia y Tecnología)(Grant numbers CB-2006-61167,
CB-2007-83920, 104194 and 94136), COECYT (Consejo Estatal de Ciencia y Tecnología de
Michoacán), and Coordinación de la Investigación Científica -UMNSH (Universidad Michoacana de
San Nicolás de Hidalgo). Authors MAHG and AMAS received fellowships during their master’s
degree studies, and LSVH for his bachelor's thesis from CONACyT. The authors declare no conflict
of interest.
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ANEXO
Supplementary material
Fig. S1.
Growth profile of Chirostoma estor during its larval and juvenile development, until 150 dph. Wet
body weight (WBW, mg ± standard deviation, SD, n = 10 (except for 1 and 5 dph, where pools of 60-
130 individuals were used), black circles) showed a potential profile with the equation: WBW =
0.005dph4.237, R² = 0.983. On the other side, Total length (TL; mm ± SD, n = 10, black triangles) show
a linear profile described by the equation: TL = 3.206dph - 6.176, R² = 0.968. Horizontal broken lines
indicate the period of the different feeding regimes. dph. Days post-hatching.