Aquaculture International (2020) 28:1473–1483

https://doi.org/10.1007/s10499-020-00535-y

Effect of stocking density and water exchange

on performance and stress tolerance to low and high
salinity by Litopenaeus vannamei postlarvae reared
with biofloc in intensive nursery phase

Héctor M. Esparza-Leal 1 & Jesús T. Ponce-Palafox 2 & Píndaro Álvarez-Ruiz 1 &

Ely S. López-Álvarez 1 & Nadia Vázquez-Montoya 1 & Mariel López-Espinoza 1 &
Magnolia Montoya Mejia 1 & Rosa L. Gómez-Peraza 1 & Eusebio Nava-Perez 1

Received: 7 August 2019 / Accepted: 5 March 2020 / Published online: 13 March 2020
# Springer Nature Switzerland AG 2020

Abstract
The effect of stocking density and water exchange was evaluated on growth
performance of Litopenaeus vannamei postlarvae (PL), reared in a biofloc-based
system at the nursery level and exposed to short-term low-high salinity stress. A
bioassay was conducted with four treatments in triplicate: T1 = 8500 postlarvae/m3
without water exchange, T2 = 16,500 postlarvae/m3 without water exchange, T3 =
8500 postlarvae/m 3 with 50% water exchange per week, and T 4 = 16,500
postlarvae/m3 with 50% water exchange per week. At 0 (PL′22), 15 (PL′37), 30
(PL′52), and 45 (PL′67) days of culture, a group of shrimp from each treatment
was exposed to acute stress by low (27 to 2‰) and high (27 to 60‰) salinity.
After 45 days, the water exchange rate and water exchange rate × stocking
density interaction affected shrimp survival, but shrimp growth was affected only
by stocking density. The mean weight of shrimp in the treatments with the lowest
stocking density was higher than that with the highest stocking density, indepen-
dently from the water exchange rate. In the salinity stress tests, shrimp survival
was affected by the interaction between the salinity stress tests and treatments.
Generally, shrimp showed a greater tolerance when subjected to low salinity (27
to 2‰). In the culture with biofloc at the nursery level, it was confirmed that
L. vannamei shrimp improve their tolerance to a sudden low salinity endpoint; a
directly proportional relationship of survival with ontogenetic development in low
and high salinity was determined.

Keywords Litopenaeus vannamei . Salinity stress . Survival . Growth . Density . Water exchange

* Héctor M. Esparza-Leal
hesparza@ipn.mx

Extended author information available on the last page of the article

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1474 Aquaculture International (2020) 28:1473–1483

Introduction

Penaeid shrimps inhabit from postlarvae to juveniles in lagoon-estuarine systems, where

extreme variations in water quality exist, mainly of salinity, temperature, and oxygen (Dall
1981). The white shrimp, Litopenaeus vannamei, is a euryhaline organism with a high
osmoregulatory capacity of cellular ion concentration and is able to maintain the osmotic
balance between the intracellular and extracellular media (Delgado-Gaytan et al. 2017). In
shrimp postlarvae, improving the osmoregulation ability increases salinity tolerance (Samocha
et al. 1998). In commercial shrimp farms, extreme salinities are recorded due to the origin and
dynamics of the water source, and salinities of 1 to 50‰ or more can be reached (Ponce-
Palafox et al. 1997; Hurtado et al. 2006; Criales et al. 2011; Martínez-Antonio et al. 2019). The
response of intrinsic and extrinsic factors of shrimp to extreme salinity conditions has been
studied by several authors (Palacios et al. 2004; Martínez-Antonio et al. 2019). In addition,
osmotic stress tests have been used to determine the quality of postlarvae and to infer that the
most resistant ones will have a better growth (Álvarez et al. 2004; Palacios and Racotta 2007),
but very few studies have analyzed the certainty of such a predictive relationship. Postlarvae
that have a higher survival to a salinity stress test are considered to be healthier or of better
quality, assuming that a better physiological condition allows a higher tolerance (survival) to
such stress and that this tolerance depends mainly on the age and nutritional status of the
postlarvae (Samocha et al. 1998). However, it has been found that juveniles and adults can
regulate osmotic pressure in a wide range of salinities under hypoosmotic and hyperosmotic
conditions (Castille and Lawrence 1981). Under clear water conditions, most studies on shrimp
postlarvae transfer have been based on low salinity with extreme cases, going suddenly from
30 to 5‰ with high survival (Jayasankar et al. 2009). In contrast, high salinities studies are
scarce, and the response has been investigated at a maximum interval of 30 to 50‰ (Liu et al.
2007).
In intensive shrimp culture, the critical early stage occurs in the nursery system, which
is the intermediate step between the initial postlarvae and early juveniles (Mishra et al.
2008). Previous studies on nursery systems have shown several benefits, such as optimi-
zation of farm land, increased shrimp survival, and adequate growth performance in the
grow-out phase (Sandifer et al. 1991; Samocha et al. 2000; Samocha et al. 2007). High
stocking densities, high water exchange rates, and the use of high-quality artificial feeds
characterize nursery systems (Mishra et al. 2008). Among the latest high-technology
systems for the cultivation of this phase is biofloc technology (BFT) characterized by
high densities of stoking, high aeration and mixing, high input of organic matter, and low
or no exchange of water (Browdy et al. 2001). Different studies with some penaeid species
confirm that the use of nurseries in the biofloc system contributes to the rapid growth of
cultured organisms (Fóes et al. 2011; Emerenciano et al. 2012; Wasielesky Jr et al. 2013).
BFT has been considered for rearing young postlarvae in short (15 days) and large (30–
71 days) duration nurseries (Emerenciano et al. 2011; Wasielesky Jr et al. 2013).
Postlarvae produced in the BFT have a better nutritional condition and a better quality
than those cultivated in clear water (Suita et al. 2015). However, in the first studies on
osmotic stress in shrimp postlarvae produced in the BFT, no relation was found between
the best condition of the postlarvae in this system and their tolerance to changes in salinity
(Emerenciano et al. 2011). On the other hand, in other species, such as catfish (Pangasius
sp.), a greater survival to changes in salinity was found in juveniles (0.08 g) grown in
biofloc than in clear water (Meritha et al. 2018).

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Aquaculture International (2020) 28:1473–1483 1475

In the super-intensive system in the nursery stage in BFT, high stocking densities (Esparza-
Leal et al. 2015) and better water quality conditions (Legarda et al. 2018) are needed to obtain
high-quality organisms resistant to the critical culture conditions. Therefore, it is necessary to
determine the effect of parameters such as density and water exchange, in the BFT system, on
postlarvae tolerance to critical parameters such as osmotic stress. There is no reference in the
literature describing the effect of water exchange and stocking density in a biofloc-based
system at the nursery level on the salinity stress tolerance (low or high salinity) of shrimp
throughout the culture, and their relation with the performance of shrimp postlarvae in the
culture. Therefore, the aim of this work was to evaluate the effect of stocking density and water
exchange on the stress tolerance (to low and high salinity for over a 150-min holding period)
and performance of L. vannamei postlarvae reared with biofloc at an intensive nursery level.

Material and methods

Experimental design

This study was performed in an outdoor tank facility at the CIIDIR-Unidad Sinaloa of the
Instituto Politécnico Nacional in Mexico. The shrimp culture system included 12 circular tanks
of 0.6 m3 with 400 l of seawater per tank (salinity = 27 ± 3‰), supplied with continuous
aeration with a blower of 5 hp (Sweetwater™, Aquatic Eco-System, Inc., Apopka, FL, USA).
Before stocking postlarvae and starting the study, each tank was filled with unfiltered seawater
and supplied with sugar cane molasses (0.2 g/l per day) as a source of organic carbon for
7 days to promote biofloc formation. During the study, different amounts of sugar cane
molasses, according to the reports of Avnimelech (1999), were added to each tank to maintain
the total ammonia values lower than 1 mg/l. In the tank system in which no water renewal was
performed during the study, only the water lost by evaporation was replaced by adding
freshwater.
L. vannamei postlarvae (PL′21, mean initial weight = 0.003 ± 0.001 g) used in this work
were acquired from a commercial hatchery and transferred to the experimental tanks for
acclimation for 24 h. Four treatments were designed: T1 = 8500 postlarvae/m3 without water
exchange, T2 = 16,500 postlarvae/m3 without water exchange, T3 = 8500 postlarvae/m3 with
50% of water exchange per week, and T4 = 16,500 postlarvae/m3 with 50% of water exchange
per week. Three replicates were randomly assigned to each treatment. Shrimp were fed twice a
day (08:00 and 16:00 h) with commercial feed (7–12% of the biomass; 40% protein). Initially,
the feeding rate was established according to Jory et al. (2001) and, subsequently, the feed was
adjusted daily according to its consumption. The study lasted 45 days.

Water quality analysis

During the experiment, pH (monitored with a Hanna 213 pH meter, Hanna Instruments,
Woonsocket, RI, USA), temperature (°C), and dissolved oxygen (DO; mg/l) (both monitored
using a YSI 55 digital oxygen meter with an integrated thermometer, Yellow Springs, OH,
USA) were measured in each experimental tank twice a day (≈ 08:00 and 16:00 h). Water
salinity (‰) was monitored weekly with an Atago refractometer (Novatech International,
Houston, TX, USA). Total ammonia-N (TAN; mg/l), nitrite-N (NO2–N; mg/l), nitrate-N
(NO3–N; mg/l), phosphate-P (PO4−3; mg/l), and total suspended solids (TSS; mg/l) were

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1476 Aquaculture International (2020) 28:1473–1483

analyzed weekly using the methods described by Strickland and Parsons (1972) and APHA
(American Public Health Association) (1998).

Growth performance

During the study, biometric variables were assessed weekly, weighing 100 shrimp from each
experimental tank using a digital balance (precision 0.01 g; Ohaus Corporation, Parsippany,
NJ, USA). Shrimp were returned to their original tanks after weighing. At the end of the study,
all the shrimp that survived in each experimental tank were weighed and counted to evaluate
their growth and survival. In addition, specific growth rates (SGR), biomass, and feed
conversion ratios (FCR) were calculated as follows: SGR = 100 × (Ln final weight − Ln initial
weight day) and FCR = feed offered (g) weight gain (g). To calculate the final shrimp
survival rate, the organisms of each replicate of the treatments that were taken for the salinity
stress tests were counted as undead organisms.

Stress tolerance

At 0 (PL′22), 15 (PL′37), 30 (PL′52), and 45 (PL′67) days of culture, a group of shrimp

(intermolt stage) of the three replicates of each of the four treatments (T1, T2, T3, and T4; 90
shrimp per treatment per stress test) were exposed to acute stress tests, at low (27 to 2‰) and
high (27 to 60‰) salinity for 150 min.
Hyper-saline seawater (≈ 90‰) brought from a natural lagoon was diluted with freshwater
to reach 2 and 60‰ salinity. Plastic bottles (0.5 l) were filled with water at different salinities
and used as experimental units. The conditions under stress tests were 28.6 ± 0.5 °C and pH
8.2 ± 0.1. Abruptly and suddenly, shrimp in the intermolt stage (30 postlarvae of each replicate
per stress test) were placed in the plastic bottles (10 postlarvae per unit) containing a salinity
level of 2 or 60‰ depending on the stress test. Shrimp survival was monitored for 5 min each.
Shrimp that did not respond to mechanical stimuli were considered dead.

Statistical analysis

Data of water quality, shrimp performance, and salinity stress tolerance were analyzed by two-
way analysis of variance using the SAS/STAT® software (SAS Institute Inc., Cary, NC, USA).
The statistical model included data of the four treatments using water exchange rate (without
water exchange and with 50% water exchange per week) and stocking density (8250 and
16,500 postlarvae/m3) as main effects and interaction term. When differences were found
between main effects, comparisons among treatments were performed using Tukey HSD tests.
Effects of treatments were considered significant at P < 0.05. Survival (%) data were trans-
formed (arcsine of the square root) before their analysis (Zar 1996).

Results

Water quality

The values of temperature, DO, salinity, and pH did not differ significantly among treatments
(P > 0.05). During the study, the mean values of DO, temperature, salinity, and pH fluctuated

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Aquaculture International (2020) 28:1473–1483 1477

between 6.2 and 6.4 mg/l, 28.6 and 28.8 °C, 26 and 27 g/l, and 8.2 and 8.3, respectively
(Table 1). The highest concentrations of nutrients and TSS (P < 0.05) were found in the
treatment with higher density and without water exchange (T2). On the other hand, the lowest
concentrations (P < 0.05) were determined in the treatment with the lower density and with
water exchange (T3).

Shrimp performance in the culture

The variance analysis indicated a significant effect of stocking density only for shrimp growth
(P < 0.01), and the water exchange rate × stocking density interaction was not significant for
this parameter (P > 0.58). Contrarily, shrimp survival was affected by both the water exchange
rate (P < 0.02) and water exchange rate × stocking density interaction (P < 0.00). Initial weight
and FCR (Table 2) showed no significant differences among treatments (P > 0.05). Mean final
weights and SGR after 45 days of culture differed significantly between the stocking densities
of 8500 (T1 and T3) and 16,500 postlarvae/m3 (T2 and T4), independently from water exchange
(P < 0.05; Fig. 1). Shrimp survival in treatments T2, T3, and T4 was significantly lower than that
in T1 (P < 0.05).

Salinity stress tolerance

The variance analysis indicated that in no salinity stress test did the water exchange rate (0 and
50% per week), stocking density (8500 and 16,500 postlarvae/m3), and interaction of water
exchange rate × stocking density affect shrimp survival (Fig. 2). However, the salinity stress ×
treatments (T1, T2, T3, and T4) interaction was significant for shrimp survival at 15, 30, and
45 days of culture (Fig. 2).
In the stress tests, mortality began promptly reaching 100% in 40 min in all treatments at
both salinities (Fig. 2). At 15 days of culture (PL′37), the lowest shrimp survival in the stress
test at low salinity, after 80 min, reached 0% for T1–T2, and, after 100 min, 0% for T3–T4.
Whereas after 40 min under high salinity stress, shrimp survival was 0% for T1–T2; at 60 min,
it was 0% for T1–T4; and at 70 min, it was 0% for T3. At 30 days of culture (PL′52), the low
salinity stress resulted in > 80% shrimp survival in T2, T3–T4, and 70 to 65% after 100 to
150 min in T1; the high salinity stress test reached 55% shrimp survival at 60 min in T1, T2–T4,

Table 1 Mean (± SD) water quality parameters during the experimental period

Parameter/treatments T1 T2 T3 T4

Temperature (°C) 28.6 ± 1.2a 28.7 ± 1.1a 28.8 ± 1.3a 28.8 ± 1.0a
OD (mg/l) 6.4 ± 0.5a 6.3 ± 1.9a 6.3 ± 0.9a 6.2 ± 1.5a
pH 8.2 ± 0.6a 8.3 ± 0.8a 8.2 ± 0.5a 8.3 ± 0.9a
TAN (mg/l) 0.36 ± 0.03b 0.81 ± 0.04a 0.04 ± 0.01c 0.39 ± 0.03b
NO2–N (mg/l) 0.03 ± 0.01b 0.05 ± 0.01a 0.02 ± 0.00d 0.03 ± 0.01c
NO3–N (mg/l) 0.33 ± 0.08b 0.44 ± 0.11a 0.09 ± 0.07c 0.17 ± 0.05c
PO4−3 (mg/l) 1.07 ± 0.12 c 2.70 ± 0.20a 0.25 ± 0.05d 1.69 ± 0.11b
TSS (mg/l) 177 ± 16b 256 ± 13a 149 ± 10d 165 ± 9c

T1 = 8250 postlarvae/m3 without water exchange, T2 = 16,500 postlarvae/m3 without water exchange, T3 = 8250
postlarvae/m3 with 50% water exchange per week, and T4 = 16,500 postlarvae/m3 with 50% water exchange per
week. Different superscript letters within the same column indicate significant differences (P < 0.05)
SD standard deviation, TSS total suspended solids, TAN total ammonia-N

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1478 Aquaculture International (2020) 28:1473–1483

Table 2 Growth performance parameters of Litopenaeus vannamei during the experimental period

Parameter/treatments T1 T2 T3 T4

Initial weight (g) 0.003 ± 0.00a 0.003 ± 0.00a 0.003 ± 0.00a 0.003 ± 0.00a
Final weight (g) 0.102 ± 0.03a 0.088 ± 0.02b 0.107 ± 0.02a 0.077 ± 0.02b
Specific growth ratio (%/day) 7.1 ± 0.6a 6.9 ± 0.5b 7.3 ± 0.3a 6.6 ± 0.5b
Feed conversion ratio 1.1 ± 0.3a 1.2 ± 0.0a 1.0 ± 0.1a 1.1 ± 0.1a
Biomass (kg/m3) 0.19 ± 0.01b 0.25 ± 0.02a 0.13 ± 0.02b 0.21 ± 0.01a
Survival (%) 73.3 ± 15.5a 62.3 ± 11.5b 61 ± 2.0b 65 ± 6.2b

T1 = 8250 postlarvae/m3 without water exchange, T2 = 16,500 postlarvae/m3 without water exchange, T3 = 8250
postlarvae/m3 with 50% water exchange per week, and T4 = 16,500 postlarvae/m3 with 50% water exchange per
week. Different superscript letters within the same column indicate significant differences (P < 0.05)

and, at 150 min, T1 reached 45%, T2 reached 20%, and T3–T4 reached 5%. At 45 days of
culture (PL′67), in the low salinity stress test, shrimp survival was up to 85% in all treatments
after 150 min, and in the same period, in the high salinity stress test, shrimp survival was 60%
for T1, 55% for T2, 25% for T4, and 17% for T3.

Discussion

The white shrimp has a high degree of euryhalinity, which enables it to tolerate sudden salinity
changes according to its ontogenetic development (Dall 1981; Castille and Lawrence 1981).
Shrimp in the BFT system have better growth and survival (Ray et al. 2017) than those
cultivated in clear water and are more resistant to osmotic stress (Castille et al. 1993). In the
BFT system, growth and survival of shrimp are not directly related to salinity change tolerance
(Criales et al. 2011). The white shrimp, L. vannamei, is an organism capable of performing
hyper- and hypo-osmoregulatory functions (Castille and Lawrence 1981); thus, extreme
sudden changes in salinity cause stress that is increased by poor water quality and inadequate
handling (Brock and Main 1994). Among the parameters that modify water quality in an
intensive system are water exchange rate and density (Cohen et al. 2005).
In this work, the water quality parameters showed values within those recommended for
penaeid shrimp (Van Wyk and Scarpa 1999; Frías-Espericueta et al. 2000; Lin and Chen
2001). Results suggest that concentrations of TAN, NO2–N, NO3–N, PO4−3, and TSS were

Fig. 1 Shrimp growth during 45 days of culture under different water exchange and stocking density in a biofloc-
based system: T1 = 8250 postlarvae/m3 without water exchange, T2 = 16,500 postlarvae/m3 without water
exchange, T3 = 8250 postlarvae/m3 with 50% water exchange per week, and T4 = 16,500 postlarvae/m3 with
50% water exchange per week. Letters that differ between lines indicate significant differences (P < 0.05)

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Aquaculture International (2020) 28:1473–1483 1479

Fig. 2 Survival of white shrimp at four different postlarval ages (PL′22, PL′37, PL′52, and PL′57) during 150-
min trials subjected to the effect of acute salinities (2‰ and 60‰) to which the postlarvae were introduced
abruptly. Plots a–d (left) track survival of postlarvae to sudden change from 27 to 2‰ and plots e–h track
survival of postlarvae when changed from 27 to 60‰

affected by stocking density and feed as they were higher in treatments (T2 and T4) with a
higher shrimp density (16,500 orgs/m3).
Postlarval growth was affected significantly only by the stocking density (P < 0.05),
showing the best values in shrimp reared in the treatments with the lowest density (8500

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1480 Aquaculture International (2020) 28:1473–1483

postlarvae/m3; T1 and T3). These results agree with those reported by Williams et al. (1996),
who found that growth in L. vannamei is stocking density–dependent. Furthermore, this effect
has also been reported in other penaeid species (Emmerson and Andrews 1981; Ray and Chien
1992; Williams et al. 1996). The mean weight of the organisms (0.08–0.11 g) was lower than
that reported by other authors, who found that, at stocking densities > 3300 and < 17,600
postlarvae/m3, L. vannamei postlarvae can grow between 0.23 and 1.12 g (Cohen et al. 2005;
Wasielesky Jr et al. 2013; Correia et al. 2014). However, in these studies, the density-
dependent growth was similar, although there were differences in shrimp growth. These
differences may be due to factors like environmental conditions, origin, size of organisms,
experimental unit size, study periods, and culture systems.
For the culture of L. vannamei, an inverse relationship between stocking density and
survival has been reported (Williams et al. 1996; Krummenauer et al. 2011); under our
experimental conditions, a significant effect of water exchange rate and water exchange rate
× stocking density interaction was observed in shrimp survival. At the lowest stocking density
without water exchange (T1), shrimp showed the highest survival (73.3%) as compared with all
other treatments (62.8%).
Salinity stress tests showed that survival improved progressively from PL′37 to PL′67
under hypotonic conditions (salinity of 2‰) after 150 min until reaching above 80% for all
treatments; whereas, under hypertonic conditions (salinity of 60‰), survival improved slowly
(PL′37 to PL′67) until reaching less than 60% and 30% in treatments T1–T2 and T3–T4,
respectively, after 150 min. This showed that the degree of euryhalinity improved with age,
but in hypotonic conditions, recovery was better than in hypertonic conditions (Fig. 2). This
survival response of tolerance to salinity in the nursery state has been reported for this and
other penaeid species, mainly at low salinity (Bray et al. 1994; McGraw et al. 2002; Tsuzuki
et al. 2000).
Survival results in salinity ranges of 27 to 2‰ and 27 to 60‰ reveal that the homeostatic
range of L. vannamei is very broad and larger than in other commercial shrimp species, which
tolerate salinities that vary in a range of ± 20‰ (Criales et al. 2011), which is lower than that
found in this work for the white shrimp (± 33‰).
The high mortality encountered during the first 40 h agrees with those reports indicating
that at an early age, the osmoregulation ability of shrimp postlarvae is poor, because the
structures that perform this function (gills) are not fully branched and the exchange area is not
enough to offset an osmotic shock, or cannot yet achieve an efficient ion exchange (Rees et al.
1994; Burbano-Gallardo et al. 2015). Stress tests agree with those obtained by Criales et al.
(2011), who indicate that Farfantepenaeus duorarum postlarvae reach the maximum osmo-
regulatory capacity between PL′35 and PL′55 (mean weight ≈ 0.020–0.045 g). In both salinity
stress tests, the postlarvae that had a higher percentage of survival until the end of the
experiment were those obtained from lower density and without water exchange (T1), and
the lowest survival corresponded to the treatments that had 50% of water exchange (T3 and T4)
under hypertonic conditions. Results indicate that the white shrimp has a better capacity to be a
hyper-osmoregulator than a hypo-osmoregulator organism in the nursery stage.
In summary, under the experimental conditions of the present study, the lowest survival
rates in the salinity stress tests were found in treatments with water exchange, regardless of the
stocking density (T3 and T4). No effect of water exchange on growth was observed when
shrimp postlarvae were reared with biofloc during 45 days. We conclude that, at the nursery
level, growth and survival in the BFT system without water exchange had a positive effect on
stress test results in shrimps stocked at 8500 postlarvae/m3, which functioned mainly as hyper-

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Aquaculture International (2020) 28:1473–1483 1481

osmoregulatory organisms. However, the other treatments with low survival in the salinity
stress test do not necessarily mean that postlarvae were of poor quality.

Funding information This study is a funded project of the Instituto Politécnico Nacional (SIP-20130586).

Compliance with ethical standards

This article does not contain any studies with animals performed by any of the authors.

Conflict of interest The authors declare that they have no conflict of interest.

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Affiliations

Héctor M. Esparza-Leal 1 & Jesús T. Ponce-Palafox 2 & Píndaro Álvarez-Ruiz 1 & Ely S.
López-Álvarez 1 & Nadia Vázquez-Montoya 1 & Mariel López-Espinoza 1 & Magnolia
Montoya Mejia 1 & Rosa L. Gómez-Peraza 1 & Eusebio Nava-Perez 1
1
Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa, Instituto
Politécnico Nacional, Boulevard Juan de Dios Bátiz Paredes # 250, 81101 Guasave, Sinaloa, Mexico
2
Escuela Nacional de Ingeniería Pesquera, Universidad Autónoma de Nayarit, Ciudad de la Cultura Amado
Nervo, 62155 Tepic, Nayarit, Mexico

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