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Effects of waterless live transportation on survivability, physiological responses and flesh quality in Chinese farmed sturgeon (Acipenser schrenckii)
Aquaculture 518 (2020) 734834
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Effects of waterless live transportation on survivability, physiological responses and flesh quality in Chinese farmed sturgeon (Acipenser schrenckii)
T
Wensheng Wanga, Yongjun Zhangb, Yan Liuc, Nóra Adányid, Xiaoshuan Zhanga,⁎ a
Beijing Laboratory of Food Quality and Safety, College of Engineering, China Agricultural University, 100083, PR China Shandong Youth University Of Political Science, School of Information Enginerring, Jinan 250103, PR China c Beijing Wuzi University, Beijing 101149, PR China d National Agricultural Research and Innovation Center, Food Science Research Institute, Budapest, Hungary b
ARTICLE INFO
ABSTRACT
Keywords: Waterless live transportation Acipenser schrenckii Physiological response Survivability Flesh quality
Waterless transport is an alternative method to realize the larger volume and higher survival rate of live aquatic products, this paper aims to investigate the effects of transport multi-stressors on the blood physiology and flesh quality, then optimize the setting of ambient parameters during waterless live transportation. Amur sturgeon, Acipenser schrenckii is selected as research objective because it is a representative fish of Chinese farmed sturgeon species. Fish were divided into three groups, Group I, Group II, and Group III. The fish in group I were cultured in the normal living environment (21 °C, with water), and Group II were transported in a lower temperature but normal living environment (4 °C, with water), but fish in Group III were transported in a waterless environment (4 °C, without water). Blood and muscle for assay were sampled at every 8 h intervals (0 h, 8 h, 16 h, 24 h and 32 h). The results indicate that temperature and oxygen are the critical ambient parameters in waterless live transportation, and significantly influence the fish survivability and blood physiology; blood glucose (GLU) and serum cortisol (COR) could be regarded as the representative indexes to assess the physiological stress level. Furthermore, the fish in waterless condition (Group III) exhibit no significant changes in flesh quality, such as total protein and crude fat after transport. These findings provide the theoretical evidence to understand the effects of multi-transport stressors on live fish and apply waterless transport in actual situation.
1. Introduction Live fish are particularly popular in China because it is believed in Chinese culture that freshly killed fish would bring vigorous energy to the body and has more delicious flavour than dead fish (Nie et al., 2018; Zhang et al., 2019, 2018a). Therefore, live aquatic products marketing is regarded as a value-adding process because live aquatic products would obtain substantially higher prices with lower processing costs (Zhang et al., 2019). Nowadays, the sales of live fish, especially those high-value species, have significantly expanded and the sales price has risen 5–10 times, which brought considerable economic benefits (Fabinyi et al., 2016; Yang et al., 2016). However, it is a relatively complicated process to transport a large quantity of live fish, which needs better control of the potential transport stimulus in order to prevent the physiological stress response of fish; otherwise, excessive physiological stress could lead to the decline of fish vitality, even to death (Harmon, 2009; Refaey et al., 2017). With the continuous development of live fish transport technology, there are mainly two transport strategies, including closed system and
⁎
open system (Berka, 1986; Das et al., 2015). Representing by some sealed containers, the closed system contains life-supported units while the open system has many technical variants, ranging from small transport fish-cans, up to transport trucks and tank wagons, which usually includes water-filled containers with appropriate control of ambient condition such as water temperature and salinity (Berka, 1986; Zhang et al., 2019). However, both of those technologies present a necessary to transport a large amount of water, which would inevitably increase energy consumption and transportation costs (Stieglitz et al., 2012). As a novel transport strategy, regulated waterless transportation for live fish is considered as a green and economical solution to realize the high survival rate and significant volume (Refaey et al., 2018; Zhang et al., 2018b). Based on the characteristics of ecological ice temperature of cold-water fish, the waterless transport method can reduce not only the respiratory metabolism but also physical damage caused by the stress response. Existed literatures show that the waterless transport method has been applied in to some cold-water fish species live transportation such as crucian carp, channel catfish Ictalurus punctatus and
Corresponding author at: China Agricultural University, Beijing 100083, PR China. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.aquaculture.2019.734834 Received 24 April 2019; Received in revised form 2 December 2019; Accepted 5 December 2019 Available online 07 December 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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rainbow trout fingerlings (Mi et al., 2012; Shabani et al., 2016; Refaey et al., 2017). In practice, it is crucial to reduce the number of potential stressors as well as to minimize the duration of exposure to these potential stressors in the process of waterless live transportation, because changes in fish homeostasis are associated with the intensity and duration of physiological stress responses (Harmon, 2009; Madaro et al., 2016; Oyoo-Okoth et al., 2011). Moreover, our previous studies showed that the ambient factors could significantly influence the survival rate of Urechis unicinctus, and the corresponding combinatorial optimization, as well as association controlling, were essential to ensure the efficiency of waterless live transportation (Zhang et al., 2018b). Therefore, to better understand the effects of multiambient-stressors on fish physiology and enhance the efficiency of waterless live transportation, it is of vital importance to monitor the fluctuations of the critical ambient parameters and to figure out the mechanism of the physiological changes of cold-water-fish (Jakkhupan et al., 2015; Tian, 2016; Xiao et al., 2017; Zhang et al., 2017). Sturgeon have been cultured in many regions around the world, and the sturgeon aquaculture in China has become popular since 1998 with eleven species and hybrids being cultured (Wang and Chang, 2007; Wei et al., 2011). Amur sturgeon, Acipenser schrenckii, nearly accounts for 15% of total farmed sturgeon production in China, is one of the most popular species in Chinese live aquatic products markets (Ni et al., 2014; Nikoo et al., 2014; Wei et al., 2011). Thus, the paper aimed to understand the affiliation between ambient stressors in waterless transportation and the vitality of fish, and assess the effects of physiological response on typical blood chemistry indexes and flesh quality parameters, so that to realize a better optimization and control of waterless transportation for live fish.
cleanliness of subsequent experiments. 2.1.3. Stage 3 Dormancy processing. Picking out 25 fish randomly and inducing them into a hibernated state by gradient cooling of water at the rate of 1–2 °C until 4 °C in water tanks with a suitable dissolved oxygen level. 2.1.4. Stage 4 Packaging. Catching the dormant fish gently from the water tank and placing them on the tray to weigh and measure length, then packaging them into the plastic bags and filling with pure oxygen. 2.1.5. Stage 5 Waterless live transportation. In this stage, fish was in the same state of dormancy as Stage 4 and was placed on the tray line by line in case of fluctuations and movements during waterless live transportation. Meanwhile, the critical ambient parameters like temperature and humidity values were obtained by the smart sensor system. 2.1.6. Stage 6 Recovery and biochemical analysis. After Stage 5, some part of the fish was selected to recover from dormant state and examine the mortality, while another part of the fish was selected to slaughter for further biochemical analysis. 2.2. Critical ambient parameters collection and survivability analysis To explore the relationship between critical ambient parameters and the survivability of Amur sturgeon (Acipenser schrenckii), each fish of Group III was packaged in a sealed plastic bag that filled with oxygen and equipped with smart sensors made in our laboratory (Zhang et al., 2019); and meanwhile, five sealed plastic bags filled with oxygen and equipped with smart sensors but no fish were set as a basal level to obtain the control group data. Considering the respiratory action of live fish and the potential stress factors in the waterless condition, four ambient parameters: oxygen (O2), carbon oxide (CO2), ambient temperature (T), and ambient relative humidity (RH) were monitored and collected simultaneously by the smart sensors system (Shabani et al., 2016; Stieglitz et al., 2012; Zeng et al., 2014; Zhang et al., 2017). Besides, to calculate the survival rate and do further analysis of waterless live transportation, the number of dead fish needed to count at every time interval. It was calculated by the following eq. (1) to obtain the survival rate of waterless live transportation.
2. Materials and methods 2.1. Experimental scenario and process design Amur sturgeon, Acipenser schrenckii, with an average weight (±SD) 750 ± 10.5 g and with a total length (±SD) 48.87 ± 4.25 cm, were obtained from the fish products market of Jinan City, Shandong province, China. A total of 75 fish without visible damage were selected and transferred to the laboratory to be acclimated at water temperature 21 °C for two weeks, which is considered as an optimum rearing temperature. And then the 75 fish were randomly and averagely allocated into fifteen separate water tanks and without any feedings for 24 h of temporary holding before waterless live transportation. And each experimental group used five separate tanks containing five fish each. The experimental treatments were arranged as follows: Group I (Control, water transport,21 °C) - The fish were reared in water for 32 h at 21 °C. Group II (Treatment, water transport,4 °C) - The fish were still reared in water as Group I, but the water temperature was decreased to 4 °C at a rate of 1.5–2 °C/h at the same time. Group III (Treatment, waterless transport,4 °C) – After reared as described as Group II, the fish were distributed into plastic bags (30 cm × 90 cm), in which filled with pure oxygen and equipped with one set of micro-ambient sensors (oxygen, carbon dioxide, humidity and temperature) and smart sensor network nodes for wirelessly precise acquisition. The workflow of waterless live transportation for Amur sturgeon was as follows and the entire experiment was shown in Fig. 1.
t
S urvival (0, t ) =
t
Nt
0
0
N
Dt × 100%, t = 0, 8, 16, 24, 32
(1)
t 0
Nt is the sum of the living fish during the transportation There t experiment, including the fish with weak breath; 0 Dt is the sum of the dead fish during waterless live transportation; N is the total number of fish samples. This result of the equation is utilized to couple with the ambient parameters to obtain a better set of ambient data and more consistent workflow for better waterless live transportation application. 2.3. Physiological measurements and analysis
2.1.1. Stage 1 Aquaculture and catching. All fish were put into the aquaculture pond for normal feeding after being purchased from the fish market, and adapting to the new environment and restore vitality. Then the healthy individuals with no visible mechanical damages were caught as the subjects to conduct the experiments.
To better understand the physiological changes in stress level of Amur sturgeon (Acipenser schrenckii) in the process of waterless live transportation, the blood glucose (GLU) and serum cortisol (COR) were selected as reliable markers of stress level; and meanwhile, the alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) were selected as two significant markers to reflect the extent of physiological effects on fish caused by stress response under such cold-acclimated and airexposed waterless condition. The blood of experimental fish in Group I was sampled at 0, (before experiment), 8, 16, 24 and 32 h post the
2.1.2. Stage 2 Temporary holding. All fish were temporarily held in the water tank for 1–2 days until emptied the metabolites to ensure micro-environment 2
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Fig. 1. The procedure of cold anesthetized waterless live transportation for Amur sturgeon (Acipenser schrenckii).
water transportation at 21 °C for acute stress response level; the blood of experimental fish in Group II was sampled at 0, (before experiment), 8, 16, 24 and 32 h post the water transportation at 4 °C for stress response level; the blood of experimental fish in Group III was sampled at 0, (before experiment), 8, 16, 24 and 32 h post the waterless transportation at 4 °C for stress response level. Three fish were randomly selected at each water tank. Blood sample was collected from caudal vein using 1 mL plastic syringe, while the fish was without anaesthetizing for avoiding the influence of the anaesthetic
on physiological response. Besides, considering the influence of light stress and psychological stress, a piece of paper was used to cover the eyes of every fish to avoid new stress responses occur. And then the blood samples were collected in the 1.5 mL centrifuge and kept on ice for <5 min before centrifuged at 3000g at 4 °C for 15 min. Serum was then collected in 2 mL vacutainers and stored at −20 °C in the refrigerator until the serum stress indicators biochemical test were carried out. Blood glucose (GLU), serum cortisol (COR), alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) were all detected 3
4
90 4.1 ± 0.1d 90.2 ± 0.3a 82.48 ± 0.66c 5042.26 ± 73.21a 95 4.2 ± 0.1cd 86.4 ± 0.7b 85.43 ± 0.52c 5032.88 ± 20.59a 95 4.2 ± 0.1c 85.3 ± 0.3b 90.25 ± 0.78b 4022.12 ± 37.69a
16 h 8h 0h
100 4.4 ± 0.1b 79.6 ± 0.5b 95.21 ± 0.70ac 4737.03 ± 357.01a
32 h 24 h
– 4.0 ± 0.1d 32.7 ± 0.9a 98.97 ± 0.36b 4278.93 ± 29.89a – 4.1 ± 0.1c 28.9 ± 0.8b 99.03 ± 0.07a 4266.69 ± 7.51a
24 h 16 h
– 4.3 ± 0.1c 29.4 ± 0.1b 98.43 ± 0.32b 4297.54 ± 47.55a – 4.5 ± 0.1b 28.7 ± 0.1b 99.16 ± 0.03a 4258.73 ± 33.66a
Treatment level (transported with live fish)
100 4.8 ± 0.1a 37.2 ± 0.4b 98.56 ± 0.72ac 281.43 ± 7.28b Survival rate, (%) Temperature, (°C) Humidity, (%) Oxygen,(%) Carbon oxide,(ppm)
Physiological stress analysis, based on the changes of serum blood glucose (GLU), cortisol (COR), alanine aminotransferase (ALT) and lactic
Analyzed parameters
3.2. Effects on physiological parameters
– 5.1 ± 0.1a 28.5 ± 0.1b 99.24 ± 0.03a 294.21 ± 29.69b
From Table.1, it showed the changes in ambient parameters and survival rate during waterless transport for live Amur sturgeon (Acipenser schrenckii). The ambient temperature decreased significantly in all treatments of Group III (p < .05). The relative humidity was significantly (p < .05) lower in the basal level than treatment level. However, the concentration of O2 and CO2 in treatment level (after waterless live transportation) were significantly (p < .05) lower than the basal level (without fish). Moreover, there were hardly any significant changes (p > .05) of the relative humidity in treatment level provided from the Table.1, as well as the concentration of CO2, while the ambient temperature and concentration of O2 showed significant (p < .05) decrease after waterless transportation.
Survival rate, (%) Temperature, (°C) Humidity, (%) Oxygen,(%) Carbon oxide,(ppm)
3.1. Changes in ambient parameters and survival rate
8h
3. Results
0h
All data were analyzed using the SPSS (version 18.0), and the results were reported as mean ± SD (n = 5) unless otherwise stated. Blood glucose, serum cortisol, alanine aminotransferase and lactic dehydrogenase, flesh quality and chemical composition of muscle between groups in each time intervals were all tested for significant differences by Tukey's test, while using the one-way analysis of variance (ANOVA) followed by Duncan's post hoc test to determine the differences among each time intervals within the same group. Statistically, all significant differences were determined at the p ≤ .05 level for all analyses.
Basal level (transported without fish)
2.5. Statistical analysis
Analyzed parameters
Table 1 Fish survival rate changes and the ambient parameters fluctuations during waterless live transportation in Group III (n = 5; Mean ± SD).
32 h
To better understand the flesh quality changes in the process of waterless live transportation, muscle samples of all groups were obtained by removal of two pieces of fillets, paralleled to the lateral line, between the head and the dorsal fin of both sides of the fish, according to the methodology described by Lefevre et al. (2016). By dividing into two parts, one part of the sample was for the detection of pH value, and the other was quickly removed on ice and stored at −20 °C for the detection of other flesh quality indicators. The muscle pH value was measured, in the front part of fish fillet, within 30 min and 1 h postmortem using a pH probe (Testo 205, Germany). The pH-electrode needs cleaning and recalculation after each measurement by using the buffers 4.01 and 7.00. It is vital to ensure good contact between the electrode surface and the flesh and the final value of pH was based on the stabilized number and calculated as the average of the three measurements. The shear force was determined by using the procedure described by Refaey et al. (2017): using the fish fillets with an average of 5 g by the texture analyzer (TA.XT plus), which performed with a knife-edged blade cutting 80% into the muscle samples perpendicular to the muscle fibers, five measurements per sample (test speed 2 mm/s). Followed by the methods of National Food Safety Standard of P.R. of China, the water content of fish muscle was determined by the method of GB-5009.3-2010, while the total protein was by the Kjeldahl method of GB-5009.5-2010, as well as the crude fat was determined by the Soxhlet method of GB/T-5009.5-2010). Moreover, the analysis of muscle glycogen was utilizing the biochemical kit by spectrophotometer. All the detected parameters were calculated as the average of 5 measurements.
– 0.000 0.000 0.121 0.100
P value
2.4. Flesh quality parameters collection and analysis
Different small letters (a, b, c) in the same line indicate significant differences between groups at each time interval (p ≤ .05); Basal Level; Control (the transported plastic bags without fish in Group III)；Treatment Level; Treatment (the transported plastic bags with fish in Group III). P value = ANOVA between time intervals within each group.
P value
with a biochemical kit (Roche Diagnostics GmbH, Switzerland) and analyzed by an automatic biochemical analyzer (ROCHE/E601).
– 0.000 0.000 0.000 0.000
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Fig. 2. The concentrations changes of glucose (A), cortisol (B), ALT (C) and LDH (D) of the Amur sturgeon, (Acipenser schrenckii) sampled at different time intervals in all three groups. Vertical bars indicate standard deviation; means with one asterisk indicate a significant difference between groups in each time interval (p ≤ .05); * indicates a significant difference between Group III and Group I; ** indicates significant difference between Group III and Group II; while different letters indicate significant differences between times intervals within each group (p ≤ .05).
organoleptic indexes and muscular chemical parameters. The pH value, as an important indicator to evaluate the fish meat quality, its changes in all groups were shown in Fig. 3(A). Overall, there was a decreasing trend in all three groups, no matter whether the fish were placed into a waterless condition or not. However, there was a small increasing (p < .05) trend of pH value in Group III between 8 h and 16 h. Compared to Group I and Group II, the pH value in Group III showed no significant changes in each sampling time. However, Fig. 2 (B) demonstrated that the shear force of fish meat in Group III had significant (p < .05) decrease compared to Group I and Group II before 16 h. The changes of other muscular chemical parameters (water content, total protein, crude fat, and muscle glycogen) were presented in Table. 2. Moreover, it showed no significant (p > .05) difference in the muscular water content between Group I and Group III, and the coefficient of the variable was within 6%. However, there was a significantly (p < .05) increase of water content in Group II within 16 h and then had significantly (p < .05) decrease of water content after 16 h. In terms of total protein, the post waterless transport fish muscle in Group III showed significant (p < .05) decrease before 16 h compared to Group I and Group II, with the coefficient of variation, was
dehydrogenase (LDH), was applied to determine the waterless and lowtemperature effects on Amur sturgeon. As showed in Fig. 2(A), compared to Group I, fish in Group II and Group III showed significant (p < .05) increase of blood glucose after being hibernated for several hours; however, the blood glucose in Group III decreased significantly (p < .05) after 8 h of waterless live transportation but the concentration gradually increased as time passed. In terms of serum cortisol, the fish in Group III (Fig. 2 (B)) showed significant (p < .05) increase and the concentration reached the top after around 8 h of waterless live transportation. From Fig. 2(C), it showed that the concentration of ALT in Group III significantly (p < .05) increasing after waterless live transportation. However, there were no significant differences (p > .05) of the concentration of ALT between Group III and Group II. Furthermore, the concentration of LDH in Group III showed (Fig. 2(D)) significant (p < .05) increase after waterless live transportation compared to Group I, but there were hardly any significant differences compared to Group II. 3.3. Changes in flesh quality The flesh quality was measured by the changes of some typical 5
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Group
Group
360
Group
*
* **
pH value
6.8
Group
**
330
b
a
b
a
a a
Group
b **
**
a
a
Group
*
a
a
a
b
b
6.6
b
*
**
a **
a
a
a
Shear Force(g)
7.0
300
a
a
a b
a
b
b c
c
270
c
a
6.4 240 6.2 0h
8h
16h
24h
16h
8h
0h
32h
24h
32h
Transport time/(h)
Transport time/(h)
Fig. 3. The boxplot of changes in pH value (A) and shear force (B) of Amur sturgeon (Acipenser schrenckii); sampled at different time intervals in all three groups. * indicates a significant difference between Group III and Group I; ** indicates a significant difference between Group III and Group II; while different letters indicate significant differences between times intervals within each group (P ≤ .05). Table 2 Nutrition composition of muscle of the Amur sturgeon, Acipenser schrenckii as affected by different transportation stress (n = 5; Mean ± SD). Sampling Time Group I
P value Group II
P value Group III
P value
0h 8h 16 h 24 h 32 h – 0h 8h 16 h 24 h 32 h – 0h 8h 16 h 24 h 32 h –
Water content (%) 77.44 78.62 78.77 78.12 79.69 0.368 81.67 81.15 83.53 82.42 82.32 0.003 83.26 80.99 79.43 81.54 83.90 0.282
a
± ± ± ± ±
3.15 0.45a 1.73a 0.88a 2.74a
± ± ± ± ±
0.96bc 0.97c 0.26a 1.11b 0.58b
± ± ± ± ±
3.90a 2.09a 3.08a 3.47a 3.19a
CV 0.0255 0.0040 0.0137 0.0074 0.0289 – 0.0117 0.0112 0.0031 0.0134 0.0070 – 0.0536 0.0289 0.0434 0.0482 0.0426 –
Total protein (%, wet weight) 19.69 19.63 19.12 19.36 19.66 0.300 19.78 19.69 19.86 19.80 19.56 0.950 19.57 18.91 18.39 18.29 18.18 0.000
a
± ± ± ± ±
0.55 0.12a 0.35a 1.12a 0.44a
± ± ± ± ±
0.25a 0.73a 0.15a 1.07a 0.50a
± ± ± ± ±
0.38a 0.30b 0.42bc 0.37c 0.43c
CV 0.0021 0.0045 0.0137 0.0340 0.0223 – 0.0129 0.0372 0.0077 0.0540 0.0256 – 0.0220 0.0178 0.0254 0.0223 0.0265 –
Crude fat(%, wet weight) 4.85 ± 4.79 ± 4.68 ± 4.68 ± 4.81 ± 0.089 4.87 ± 4.95 ± 4.81 ± 4.74 ± 4.79 ± 0.496 4.82 ± 4.82 ± 4.01 ± 3.61 ± 3.37 ± 0.000
a
0.05 0.02ab 0.32b 0.09b 0.31ab 0.15a 0.15a 0.32a 0.09a 0.16a 0.35a 0.36a 0.25b 0.49bc 0.21c
CV 0.0031 0.0048 0.0387 0.0212 0.0321 – 0.0314 0.0301 0.0658 0.0186 0.0340 – 0.0809 0.0846 0.0707 0.1503 0.0710 –
Muscle glycogen(mg/g) 2.26 ± 2.27 ± 2.27 ± 2.24 ± 2.24 ± 0.920 2.35 ± 2.37 ± 2.30 ± 2.32 ± 2.22 ± 0.154 2.48 ± 2.31 ± 1.71 ± 1.64 ± 1.50 ± 0.000
a
0.02 0.06a 0.09a 0.05a 0.05a 0.08a 0.14a 0.09ab 0.08ab 0.02b 0.31a 0.11a 0.12b 0.12b 0.06b
CV 0.0067 0.0262 0.0381 0.0202 0.0533 – 0.0355 0.0591 0.0400 0.0326 0.0099 – 0.1399 0.0528 0.0776 0.0851 0.0425 –
CV means the coefficient of variation; while different small letters (a,b,c) indicate significant differences between time intervals within each group (P ≤ .05).
within 3%. For the changes of crude fat of fish muscle, the figures in Table. 2 demonstrated a similar decreasing (p < .05) trend of crude fat in Group III. Besides, as transportation time increased, the concentration of crude fat in Group III gradually decreased (p < .05) while the concentration of muscle glycogen showed significant (p < 0,05) decrease before 16 h in waterless condition.
there are four critical control parameters (CCPs): temperature, oxygen, carbon oxide and stress changes (Zhang et al., 2019). Therefore, in this research, four ambient stressors (temperature, humidity, oxygen & carbon oxide) were monitored by smart sensors to analyze the relationship between ambient parameters changes and the survivability of waterless live transportation. Table.1 shows that the survival rate of Amur sturgeon during the waterless live transportation is decreasing smoothly as time passes. Compared to Group III, the decreasing speed of the ambient temperature is slower, in which maybe the aerobic respiration of live fish that generates much more quantity of energy released to the environment. Furthermore, also because of the aerobic respiration, the ambient humidity changes significantly (p < .05) compared to Group III, and reaches a relatively high level after 16 h of transportation. As it is for the absence of live fish, the oxygen consumption leads to a significant decrease, with average decreasing rate at 51.93%/h in the previous 16 h, while in the last 16 h the rate is 48.56%/h, which is just opposite with ambient carbon oxide changes. The results also indicate that the
4. Discussion 4.1. Key ambient parameters and survivability Responses to stress-related ambient disturbances in fish are often characterized as primary, secondary, and tertiary (Kordon et al., 2018; Hur et al., 2019). Furthermore, if severe enough, acute stressors (e.g., air exposure, temperature change) may lead to the consequence of death, which affects the activeness and survivability of waterless transportation for fish (Pottinger et al., 2016; Pottinger, 2017). In terms of the quality control management for waterless live transportation, 6
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Fig. 4. The interaction between survival rate of Chinese farmed sturgeon (Acipenser schrenckii) and ambient parameters changes during waterless live transportation.
live sturgeon gradually enter a relatively stable status after 8 h of waterless transportation, and could live for >32 h. With 4 °C of temperature control and oxygen-filled packaging, the fish survival time in waterless live transportation was observed >32 h in this research. From Table.1, the oxygen level in Group III decreases
faster than in the Control Group (Basal Level), partly because the waterless circumstance stimulates the respiration of the fish, which leads to the increase of oxygen consumption at the same time. Besides, the change of ambient oxygen is opposite with ambient carbon oxide. By analyzing the interaction between the survival rate and different 7
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ambient parameters, as shown in Fig. 4, the temperature and filled‑oxygen in bag are the most critical ambient stressors which directly influence the survival rate of the waterless live transportation. Thus, the results illustrate that the survival rate of waterless live transport is bound up with the ambient temperature and oxygen concentration in plastic bag.
Due to every individual fish is under a low-temperature and no-water condition; consequently, to provide the nutrients to organism and keep alive, some nutrients in fish meat would be degraded so that causing the denaturation of the muscle proteins, which could be reflected on the reducing the muscle of the muscle SF (Kristoffersen et al., 2006; Refaey et al., 2017). From Table 2, it presents that the nutritional values add up to >100%, and what causes this kind of discrepancies mainly including two aspects of error: one is the systematic error, which mainly due to several batches of tests during data acquisition; while another one is the random error, which mainly due to the outliers when artificially remove them. Additionally, Table 2 shows that the transport stress led to the increase of water content of fish muscle since the waterless live transportation, which perhaps causing a lower hardness of muscle after 16 h of transportation thereby decrease the shear strength. After all, the relationship between water content and shear force is a negative correlation (Dunajski, 1979; Aramli et al., 2016; Refaey et al., 2017).
4.2. Physiological parameters and stress level In previous studies, blood glucose and serum cortisol levels are considered as important indicators of stress response when fish subjected to stressful situations (Long et al., 2019; Mi et al., 2012). From Fig. 2 (A) & (B), the stress level of Amur sturgeon in Group III reaches the peak value after 8 h of waterless live transportation but decreases slowly as time passes. However, the stress level is still high (p < .05) compared to Group I and Group II at the same time, which indicates low-temperature and waterless condition has a significant impact on the physiological stress level. Furthermore, in response to low-temperature, the overexpressed cortisol is able to enhance glycolysis pathway, which results in a maintenance in catabolism and energy metabolism over a period of time, when Amur sturgeon was subjected to the low-temperature and waterless transportation (Nie et al., 2018; Bao et al., 2018; Jia et al., 2018; Kordon et al., 2018; Lermen et al., 2004). Except for the two physiological parameters above, the changes in alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) level indicate the generalized stress reactions and could show the damage degree of the organism in vivo of Amur sturgeon (Bao et al., 2018; Li et al., 2018). As a mark of metabolic adaption in the fish to different physiological demands, ALT could provide preliminary evidence for subtle changes in the process of waterless transportation. From Fig. 2 (D), a significant increase level of LDH in Group III indicates the anaerobic metabolism goes up. It has been noted that some changes of lactate dehydrogenase (LDH) concentration seem to stem from the stress increase induced by air exposure, especially exposed to such high‑oxygen (Patterson et al., 2017; Wen et al., 2017).
5. Conclusions As a novel live fish transportation method, waterless live transportation is more high-efficiency and low-cost compared to traditional live fish transportation. To minimize the aqua- and transport losses, it is essential to understand the physiological mechanism and level of stress response in the process of waterless live transportation. In this paper, Amur sturgeon, Acipenser schrenckii was selected as the research subject, and the effects of waterless live transportation on its survivability, physiological responses and flesh quality were presented in this paper. From this research, the results indicate properly optimizing of critical ambient parameters of waterless live transportation is conductive to achieve >32 h keep-alive status of sturgeon. Furthermore, lowtemperature and air exposure could lead to sustained physiological response, which would promote capabilities of anaerobic metabolism in live sturgeon under a waterless environment and thus consume more basal energy to stable the homeostasis. At the same time, there are no significant changes in flesh quality of fish during the low-temperature and waterless live transportation, which means that this live transport approach can guarantee high transport quality to a certain extent. Therefore, the possibility to prepare for and respond to challenges to realize the high volume of cold-fish waterless live transportation is to cope with the accordance between transport stress response and transport management and how to strengthen the stress response monitoring and do further studies on waterless transport optimization are the next research direction for waterless transportation management.
4.3. Flesh quality and muscular parameters Muscle pH is an important parameter of flesh quality evaluation, and muscle shear force is an indicator of tenderness of muscle (Roth et al., 2005). Besides, scientific literature in meat science has evaluated the relationship between different pH values and flesh quality. For instance, high pH in beef longissimus thoracic reduces muscle fibre transverse shrinkage and light scattering (Hughes et al., 2017). The decrease in muscle pH due to transport stress has a noticeable effect on the physical properties of muscle change, which causing the denaturation of the muscle proteins and fatty acid, which possibly reflected on reducing the muscle shear force (SF) (Kristoffersen et al., 2006; Refaey et al., 2017). In this respect, from Fig. 3(A), there is a decreasing trend in muscle pH value between two groups during waterless live transportation while there are nearly no apparent changes in muscle pH value which means no significant increase or decrease during waterless live transportation, which may due to the physiological regulation of the living body itself. As a crucial factor for overall acceptance and consumers' satisfaction, texture or hardness of fish meat is the response of the tactile senses to physical stimuli that result from contact between some part of the body and the food (Veland and Torrissen, 1999; Zarifi et al., 2017). Of course, the characteristic of the texture varies widely due to the different species like fish, vegetables and fruits which have dominant quality characteristic. Moreover, from certain aspects, the index of the shear force is an indirect parameter to reflect part of the texture, partially indicate the degradation of fish muscle which causes lower shear strength. However, according to the present research, transport stress in waterless live transportation leads to declining in the flesh SF directly, which in turn, it would attribute to the declining of muscle pH value.
Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the final manuscript entitled “Effects of Waterless Live Transportation on Survivability, Physiological Responses and Flesh Quality in Chinese Farmed Sturgeon (Acipenser schrenckii)”. Acknowledgments This research is supported by the project assignment on the crossdisciplinary cooperation of Beijing Science and Technology New Star Program (Project ID Z181100006218123), Beijing Science and Technology Project (Project ID Z181100001018033) and Application of Key R&D Program (Public Welfare Specialized) Projects in Shandong Province in 2019:Research on intelligent ambient quality control technology for aquatic products transportation (Project ID 2019GNC106079). 8
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Effects of waterless live transportation on survivability, physiological responses and flesh quality in Chinese farmed sturgeon (Acipenser schrenckii)
T
Wensheng Wanga, Yongjun Zhangb, Yan Liuc, Nóra Adányid, Xiaoshuan Zhanga,⁎ a
Beijing Laboratory of Food Quality and Safety, College of Engineering, China Agricultural University, 100083, PR China Shandong Youth University Of Political Science, School of Information Enginerring, Jinan 250103, PR China c Beijing Wuzi University, Beijing 101149, PR China d National Agricultural Research and Innovation Center, Food Science Research Institute, Budapest, Hungary b
ARTICLE INFO
ABSTRACT
Keywords: Waterless live transportation Acipenser schrenckii Physiological response Survivability Flesh quality
Waterless transport is an alternative method to realize the larger volume and higher survival rate of live aquatic products, this paper aims to investigate the effects of transport multi-stressors on the blood physiology and flesh quality, then optimize the setting of ambient parameters during waterless live transportation. Amur sturgeon, Acipenser schrenckii is selected as research objective because it is a representative fish of Chinese farmed sturgeon species. Fish were divided into three groups, Group I, Group II, and Group III. The fish in group I were cultured in the normal living environment (21 °C, with water), and Group II were transported in a lower temperature but normal living environment (4 °C, with water), but fish in Group III were transported in a waterless environment (4 °C, without water). Blood and muscle for assay were sampled at every 8 h intervals (0 h, 8 h, 16 h, 24 h and 32 h). The results indicate that temperature and oxygen are the critical ambient parameters in waterless live transportation, and significantly influence the fish survivability and blood physiology; blood glucose (GLU) and serum cortisol (COR) could be regarded as the representative indexes to assess the physiological stress level. Furthermore, the fish in waterless condition (Group III) exhibit no significant changes in flesh quality, such as total protein and crude fat after transport. These findings provide the theoretical evidence to understand the effects of multi-transport stressors on live fish and apply waterless transport in actual situation.
1. Introduction Live fish are particularly popular in China because it is believed in Chinese culture that freshly killed fish would bring vigorous energy to the body and has more delicious flavour than dead fish (Nie et al., 2018; Zhang et al., 2019, 2018a). Therefore, live aquatic products marketing is regarded as a value-adding process because live aquatic products would obtain substantially higher prices with lower processing costs (Zhang et al., 2019). Nowadays, the sales of live fish, especially those high-value species, have significantly expanded and the sales price has risen 5–10 times, which brought considerable economic benefits (Fabinyi et al., 2016; Yang et al., 2016). However, it is a relatively complicated process to transport a large quantity of live fish, which needs better control of the potential transport stimulus in order to prevent the physiological stress response of fish; otherwise, excessive physiological stress could lead to the decline of fish vitality, even to death (Harmon, 2009; Refaey et al., 2017). With the continuous development of live fish transport technology, there are mainly two transport strategies, including closed system and
⁎
open system (Berka, 1986; Das et al., 2015). Representing by some sealed containers, the closed system contains life-supported units while the open system has many technical variants, ranging from small transport fish-cans, up to transport trucks and tank wagons, which usually includes water-filled containers with appropriate control of ambient condition such as water temperature and salinity (Berka, 1986; Zhang et al., 2019). However, both of those technologies present a necessary to transport a large amount of water, which would inevitably increase energy consumption and transportation costs (Stieglitz et al., 2012). As a novel transport strategy, regulated waterless transportation for live fish is considered as a green and economical solution to realize the high survival rate and significant volume (Refaey et al., 2018; Zhang et al., 2018b). Based on the characteristics of ecological ice temperature of cold-water fish, the waterless transport method can reduce not only the respiratory metabolism but also physical damage caused by the stress response. Existed literatures show that the waterless transport method has been applied in to some cold-water fish species live transportation such as crucian carp, channel catfish Ictalurus punctatus and
Corresponding author at: China Agricultural University, Beijing 100083, PR China. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.aquaculture.2019.734834 Received 24 April 2019; Received in revised form 2 December 2019; Accepted 5 December 2019 Available online 07 December 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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rainbow trout fingerlings (Mi et al., 2012; Shabani et al., 2016; Refaey et al., 2017). In practice, it is crucial to reduce the number of potential stressors as well as to minimize the duration of exposure to these potential stressors in the process of waterless live transportation, because changes in fish homeostasis are associated with the intensity and duration of physiological stress responses (Harmon, 2009; Madaro et al., 2016; Oyoo-Okoth et al., 2011). Moreover, our previous studies showed that the ambient factors could significantly influence the survival rate of Urechis unicinctus, and the corresponding combinatorial optimization, as well as association controlling, were essential to ensure the efficiency of waterless live transportation (Zhang et al., 2018b). Therefore, to better understand the effects of multiambient-stressors on fish physiology and enhance the efficiency of waterless live transportation, it is of vital importance to monitor the fluctuations of the critical ambient parameters and to figure out the mechanism of the physiological changes of cold-water-fish (Jakkhupan et al., 2015; Tian, 2016; Xiao et al., 2017; Zhang et al., 2017). Sturgeon have been cultured in many regions around the world, and the sturgeon aquaculture in China has become popular since 1998 with eleven species and hybrids being cultured (Wang and Chang, 2007; Wei et al., 2011). Amur sturgeon, Acipenser schrenckii, nearly accounts for 15% of total farmed sturgeon production in China, is one of the most popular species in Chinese live aquatic products markets (Ni et al., 2014; Nikoo et al., 2014; Wei et al., 2011). Thus, the paper aimed to understand the affiliation between ambient stressors in waterless transportation and the vitality of fish, and assess the effects of physiological response on typical blood chemistry indexes and flesh quality parameters, so that to realize a better optimization and control of waterless transportation for live fish.
cleanliness of subsequent experiments. 2.1.3. Stage 3 Dormancy processing. Picking out 25 fish randomly and inducing them into a hibernated state by gradient cooling of water at the rate of 1–2 °C until 4 °C in water tanks with a suitable dissolved oxygen level. 2.1.4. Stage 4 Packaging. Catching the dormant fish gently from the water tank and placing them on the tray to weigh and measure length, then packaging them into the plastic bags and filling with pure oxygen. 2.1.5. Stage 5 Waterless live transportation. In this stage, fish was in the same state of dormancy as Stage 4 and was placed on the tray line by line in case of fluctuations and movements during waterless live transportation. Meanwhile, the critical ambient parameters like temperature and humidity values were obtained by the smart sensor system. 2.1.6. Stage 6 Recovery and biochemical analysis. After Stage 5, some part of the fish was selected to recover from dormant state and examine the mortality, while another part of the fish was selected to slaughter for further biochemical analysis. 2.2. Critical ambient parameters collection and survivability analysis To explore the relationship between critical ambient parameters and the survivability of Amur sturgeon (Acipenser schrenckii), each fish of Group III was packaged in a sealed plastic bag that filled with oxygen and equipped with smart sensors made in our laboratory (Zhang et al., 2019); and meanwhile, five sealed plastic bags filled with oxygen and equipped with smart sensors but no fish were set as a basal level to obtain the control group data. Considering the respiratory action of live fish and the potential stress factors in the waterless condition, four ambient parameters: oxygen (O2), carbon oxide (CO2), ambient temperature (T), and ambient relative humidity (RH) were monitored and collected simultaneously by the smart sensors system (Shabani et al., 2016; Stieglitz et al., 2012; Zeng et al., 2014; Zhang et al., 2017). Besides, to calculate the survival rate and do further analysis of waterless live transportation, the number of dead fish needed to count at every time interval. It was calculated by the following eq. (1) to obtain the survival rate of waterless live transportation.
2. Materials and methods 2.1. Experimental scenario and process design Amur sturgeon, Acipenser schrenckii, with an average weight (±SD) 750 ± 10.5 g and with a total length (±SD) 48.87 ± 4.25 cm, were obtained from the fish products market of Jinan City, Shandong province, China. A total of 75 fish without visible damage were selected and transferred to the laboratory to be acclimated at water temperature 21 °C for two weeks, which is considered as an optimum rearing temperature. And then the 75 fish were randomly and averagely allocated into fifteen separate water tanks and without any feedings for 24 h of temporary holding before waterless live transportation. And each experimental group used five separate tanks containing five fish each. The experimental treatments were arranged as follows: Group I (Control, water transport,21 °C) - The fish were reared in water for 32 h at 21 °C. Group II (Treatment, water transport,4 °C) - The fish were still reared in water as Group I, but the water temperature was decreased to 4 °C at a rate of 1.5–2 °C/h at the same time. Group III (Treatment, waterless transport,4 °C) – After reared as described as Group II, the fish were distributed into plastic bags (30 cm × 90 cm), in which filled with pure oxygen and equipped with one set of micro-ambient sensors (oxygen, carbon dioxide, humidity and temperature) and smart sensor network nodes for wirelessly precise acquisition. The workflow of waterless live transportation for Amur sturgeon was as follows and the entire experiment was shown in Fig. 1.
t
S urvival (0, t ) =
t
Nt
0
0
N
Dt × 100%, t = 0, 8, 16, 24, 32
(1)
t 0
Nt is the sum of the living fish during the transportation There t experiment, including the fish with weak breath; 0 Dt is the sum of the dead fish during waterless live transportation; N is the total number of fish samples. This result of the equation is utilized to couple with the ambient parameters to obtain a better set of ambient data and more consistent workflow for better waterless live transportation application. 2.3. Physiological measurements and analysis
2.1.1. Stage 1 Aquaculture and catching. All fish were put into the aquaculture pond for normal feeding after being purchased from the fish market, and adapting to the new environment and restore vitality. Then the healthy individuals with no visible mechanical damages were caught as the subjects to conduct the experiments.
To better understand the physiological changes in stress level of Amur sturgeon (Acipenser schrenckii) in the process of waterless live transportation, the blood glucose (GLU) and serum cortisol (COR) were selected as reliable markers of stress level; and meanwhile, the alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) were selected as two significant markers to reflect the extent of physiological effects on fish caused by stress response under such cold-acclimated and airexposed waterless condition. The blood of experimental fish in Group I was sampled at 0, (before experiment), 8, 16, 24 and 32 h post the
2.1.2. Stage 2 Temporary holding. All fish were temporarily held in the water tank for 1–2 days until emptied the metabolites to ensure micro-environment 2
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Fig. 1. The procedure of cold anesthetized waterless live transportation for Amur sturgeon (Acipenser schrenckii).
water transportation at 21 °C for acute stress response level; the blood of experimental fish in Group II was sampled at 0, (before experiment), 8, 16, 24 and 32 h post the water transportation at 4 °C for stress response level; the blood of experimental fish in Group III was sampled at 0, (before experiment), 8, 16, 24 and 32 h post the waterless transportation at 4 °C for stress response level. Three fish were randomly selected at each water tank. Blood sample was collected from caudal vein using 1 mL plastic syringe, while the fish was without anaesthetizing for avoiding the influence of the anaesthetic
on physiological response. Besides, considering the influence of light stress and psychological stress, a piece of paper was used to cover the eyes of every fish to avoid new stress responses occur. And then the blood samples were collected in the 1.5 mL centrifuge and kept on ice for <5 min before centrifuged at 3000g at 4 °C for 15 min. Serum was then collected in 2 mL vacutainers and stored at −20 °C in the refrigerator until the serum stress indicators biochemical test were carried out. Blood glucose (GLU), serum cortisol (COR), alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) were all detected 3
4
90 4.1 ± 0.1d 90.2 ± 0.3a 82.48 ± 0.66c 5042.26 ± 73.21a 95 4.2 ± 0.1cd 86.4 ± 0.7b 85.43 ± 0.52c 5032.88 ± 20.59a 95 4.2 ± 0.1c 85.3 ± 0.3b 90.25 ± 0.78b 4022.12 ± 37.69a
16 h 8h 0h
100 4.4 ± 0.1b 79.6 ± 0.5b 95.21 ± 0.70ac 4737.03 ± 357.01a
32 h 24 h
– 4.0 ± 0.1d 32.7 ± 0.9a 98.97 ± 0.36b 4278.93 ± 29.89a – 4.1 ± 0.1c 28.9 ± 0.8b 99.03 ± 0.07a 4266.69 ± 7.51a
24 h 16 h
– 4.3 ± 0.1c 29.4 ± 0.1b 98.43 ± 0.32b 4297.54 ± 47.55a – 4.5 ± 0.1b 28.7 ± 0.1b 99.16 ± 0.03a 4258.73 ± 33.66a
Treatment level (transported with live fish)
100 4.8 ± 0.1a 37.2 ± 0.4b 98.56 ± 0.72ac 281.43 ± 7.28b Survival rate, (%) Temperature, (°C) Humidity, (%) Oxygen,(%) Carbon oxide,(ppm)
Physiological stress analysis, based on the changes of serum blood glucose (GLU), cortisol (COR), alanine aminotransferase (ALT) and lactic
Analyzed parameters
3.2. Effects on physiological parameters
– 5.1 ± 0.1a 28.5 ± 0.1b 99.24 ± 0.03a 294.21 ± 29.69b
From Table.1, it showed the changes in ambient parameters and survival rate during waterless transport for live Amur sturgeon (Acipenser schrenckii). The ambient temperature decreased significantly in all treatments of Group III (p < .05). The relative humidity was significantly (p < .05) lower in the basal level than treatment level. However, the concentration of O2 and CO2 in treatment level (after waterless live transportation) were significantly (p < .05) lower than the basal level (without fish). Moreover, there were hardly any significant changes (p > .05) of the relative humidity in treatment level provided from the Table.1, as well as the concentration of CO2, while the ambient temperature and concentration of O2 showed significant (p < .05) decrease after waterless transportation.
Survival rate, (%) Temperature, (°C) Humidity, (%) Oxygen,(%) Carbon oxide,(ppm)
3.1. Changes in ambient parameters and survival rate
8h
3. Results
0h
All data were analyzed using the SPSS (version 18.0), and the results were reported as mean ± SD (n = 5) unless otherwise stated. Blood glucose, serum cortisol, alanine aminotransferase and lactic dehydrogenase, flesh quality and chemical composition of muscle between groups in each time intervals were all tested for significant differences by Tukey's test, while using the one-way analysis of variance (ANOVA) followed by Duncan's post hoc test to determine the differences among each time intervals within the same group. Statistically, all significant differences were determined at the p ≤ .05 level for all analyses.
Basal level (transported without fish)
2.5. Statistical analysis
Analyzed parameters
Table 1 Fish survival rate changes and the ambient parameters fluctuations during waterless live transportation in Group III (n = 5; Mean ± SD).
32 h
To better understand the flesh quality changes in the process of waterless live transportation, muscle samples of all groups were obtained by removal of two pieces of fillets, paralleled to the lateral line, between the head and the dorsal fin of both sides of the fish, according to the methodology described by Lefevre et al. (2016). By dividing into two parts, one part of the sample was for the detection of pH value, and the other was quickly removed on ice and stored at −20 °C for the detection of other flesh quality indicators. The muscle pH value was measured, in the front part of fish fillet, within 30 min and 1 h postmortem using a pH probe (Testo 205, Germany). The pH-electrode needs cleaning and recalculation after each measurement by using the buffers 4.01 and 7.00. It is vital to ensure good contact between the electrode surface and the flesh and the final value of pH was based on the stabilized number and calculated as the average of the three measurements. The shear force was determined by using the procedure described by Refaey et al. (2017): using the fish fillets with an average of 5 g by the texture analyzer (TA.XT plus), which performed with a knife-edged blade cutting 80% into the muscle samples perpendicular to the muscle fibers, five measurements per sample (test speed 2 mm/s). Followed by the methods of National Food Safety Standard of P.R. of China, the water content of fish muscle was determined by the method of GB-5009.3-2010, while the total protein was by the Kjeldahl method of GB-5009.5-2010, as well as the crude fat was determined by the Soxhlet method of GB/T-5009.5-2010). Moreover, the analysis of muscle glycogen was utilizing the biochemical kit by spectrophotometer. All the detected parameters were calculated as the average of 5 measurements.
– 0.000 0.000 0.121 0.100
P value
2.4. Flesh quality parameters collection and analysis
Different small letters (a, b, c) in the same line indicate significant differences between groups at each time interval (p ≤ .05); Basal Level; Control (the transported plastic bags without fish in Group III)；Treatment Level; Treatment (the transported plastic bags with fish in Group III). P value = ANOVA between time intervals within each group.
P value
with a biochemical kit (Roche Diagnostics GmbH, Switzerland) and analyzed by an automatic biochemical analyzer (ROCHE/E601).
– 0.000 0.000 0.000 0.000
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Fig. 2. The concentrations changes of glucose (A), cortisol (B), ALT (C) and LDH (D) of the Amur sturgeon, (Acipenser schrenckii) sampled at different time intervals in all three groups. Vertical bars indicate standard deviation; means with one asterisk indicate a significant difference between groups in each time interval (p ≤ .05); * indicates a significant difference between Group III and Group I; ** indicates significant difference between Group III and Group II; while different letters indicate significant differences between times intervals within each group (p ≤ .05).
organoleptic indexes and muscular chemical parameters. The pH value, as an important indicator to evaluate the fish meat quality, its changes in all groups were shown in Fig. 3(A). Overall, there was a decreasing trend in all three groups, no matter whether the fish were placed into a waterless condition or not. However, there was a small increasing (p < .05) trend of pH value in Group III between 8 h and 16 h. Compared to Group I and Group II, the pH value in Group III showed no significant changes in each sampling time. However, Fig. 2 (B) demonstrated that the shear force of fish meat in Group III had significant (p < .05) decrease compared to Group I and Group II before 16 h. The changes of other muscular chemical parameters (water content, total protein, crude fat, and muscle glycogen) were presented in Table. 2. Moreover, it showed no significant (p > .05) difference in the muscular water content between Group I and Group III, and the coefficient of the variable was within 6%. However, there was a significantly (p < .05) increase of water content in Group II within 16 h and then had significantly (p < .05) decrease of water content after 16 h. In terms of total protein, the post waterless transport fish muscle in Group III showed significant (p < .05) decrease before 16 h compared to Group I and Group II, with the coefficient of variation, was
dehydrogenase (LDH), was applied to determine the waterless and lowtemperature effects on Amur sturgeon. As showed in Fig. 2(A), compared to Group I, fish in Group II and Group III showed significant (p < .05) increase of blood glucose after being hibernated for several hours; however, the blood glucose in Group III decreased significantly (p < .05) after 8 h of waterless live transportation but the concentration gradually increased as time passed. In terms of serum cortisol, the fish in Group III (Fig. 2 (B)) showed significant (p < .05) increase and the concentration reached the top after around 8 h of waterless live transportation. From Fig. 2(C), it showed that the concentration of ALT in Group III significantly (p < .05) increasing after waterless live transportation. However, there were no significant differences (p > .05) of the concentration of ALT between Group III and Group II. Furthermore, the concentration of LDH in Group III showed (Fig. 2(D)) significant (p < .05) increase after waterless live transportation compared to Group I, but there were hardly any significant differences compared to Group II. 3.3. Changes in flesh quality The flesh quality was measured by the changes of some typical 5
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Group
Group
360
Group
*
* **
pH value
6.8
Group
**
330
b
a
b
a
a a
Group
b **
**
a
a
Group
*
a
a
a
b
b
6.6
b
*
**
a **
a
a
a
Shear Force(g)
7.0
300
a
a
a b
a
b
b c
c
270
c
a
6.4 240 6.2 0h
8h
16h
24h
16h
8h
0h
32h
24h
32h
Transport time/(h)
Transport time/(h)
Fig. 3. The boxplot of changes in pH value (A) and shear force (B) of Amur sturgeon (Acipenser schrenckii); sampled at different time intervals in all three groups. * indicates a significant difference between Group III and Group I; ** indicates a significant difference between Group III and Group II; while different letters indicate significant differences between times intervals within each group (P ≤ .05). Table 2 Nutrition composition of muscle of the Amur sturgeon, Acipenser schrenckii as affected by different transportation stress (n = 5; Mean ± SD). Sampling Time Group I
P value Group II
P value Group III
P value
0h 8h 16 h 24 h 32 h – 0h 8h 16 h 24 h 32 h – 0h 8h 16 h 24 h 32 h –
Water content (%) 77.44 78.62 78.77 78.12 79.69 0.368 81.67 81.15 83.53 82.42 82.32 0.003 83.26 80.99 79.43 81.54 83.90 0.282
a
± ± ± ± ±
3.15 0.45a 1.73a 0.88a 2.74a
± ± ± ± ±
0.96bc 0.97c 0.26a 1.11b 0.58b
± ± ± ± ±
3.90a 2.09a 3.08a 3.47a 3.19a
CV 0.0255 0.0040 0.0137 0.0074 0.0289 – 0.0117 0.0112 0.0031 0.0134 0.0070 – 0.0536 0.0289 0.0434 0.0482 0.0426 –
Total protein (%, wet weight) 19.69 19.63 19.12 19.36 19.66 0.300 19.78 19.69 19.86 19.80 19.56 0.950 19.57 18.91 18.39 18.29 18.18 0.000
a
± ± ± ± ±
0.55 0.12a 0.35a 1.12a 0.44a
± ± ± ± ±
0.25a 0.73a 0.15a 1.07a 0.50a
± ± ± ± ±
0.38a 0.30b 0.42bc 0.37c 0.43c
CV 0.0021 0.0045 0.0137 0.0340 0.0223 – 0.0129 0.0372 0.0077 0.0540 0.0256 – 0.0220 0.0178 0.0254 0.0223 0.0265 –
Crude fat(%, wet weight) 4.85 ± 4.79 ± 4.68 ± 4.68 ± 4.81 ± 0.089 4.87 ± 4.95 ± 4.81 ± 4.74 ± 4.79 ± 0.496 4.82 ± 4.82 ± 4.01 ± 3.61 ± 3.37 ± 0.000
a
0.05 0.02ab 0.32b 0.09b 0.31ab 0.15a 0.15a 0.32a 0.09a 0.16a 0.35a 0.36a 0.25b 0.49bc 0.21c
CV 0.0031 0.0048 0.0387 0.0212 0.0321 – 0.0314 0.0301 0.0658 0.0186 0.0340 – 0.0809 0.0846 0.0707 0.1503 0.0710 –
Muscle glycogen(mg/g) 2.26 ± 2.27 ± 2.27 ± 2.24 ± 2.24 ± 0.920 2.35 ± 2.37 ± 2.30 ± 2.32 ± 2.22 ± 0.154 2.48 ± 2.31 ± 1.71 ± 1.64 ± 1.50 ± 0.000
a
0.02 0.06a 0.09a 0.05a 0.05a 0.08a 0.14a 0.09ab 0.08ab 0.02b 0.31a 0.11a 0.12b 0.12b 0.06b
CV 0.0067 0.0262 0.0381 0.0202 0.0533 – 0.0355 0.0591 0.0400 0.0326 0.0099 – 0.1399 0.0528 0.0776 0.0851 0.0425 –
CV means the coefficient of variation; while different small letters (a,b,c) indicate significant differences between time intervals within each group (P ≤ .05).
within 3%. For the changes of crude fat of fish muscle, the figures in Table. 2 demonstrated a similar decreasing (p < .05) trend of crude fat in Group III. Besides, as transportation time increased, the concentration of crude fat in Group III gradually decreased (p < .05) while the concentration of muscle glycogen showed significant (p < 0,05) decrease before 16 h in waterless condition.
there are four critical control parameters (CCPs): temperature, oxygen, carbon oxide and stress changes (Zhang et al., 2019). Therefore, in this research, four ambient stressors (temperature, humidity, oxygen & carbon oxide) were monitored by smart sensors to analyze the relationship between ambient parameters changes and the survivability of waterless live transportation. Table.1 shows that the survival rate of Amur sturgeon during the waterless live transportation is decreasing smoothly as time passes. Compared to Group III, the decreasing speed of the ambient temperature is slower, in which maybe the aerobic respiration of live fish that generates much more quantity of energy released to the environment. Furthermore, also because of the aerobic respiration, the ambient humidity changes significantly (p < .05) compared to Group III, and reaches a relatively high level after 16 h of transportation. As it is for the absence of live fish, the oxygen consumption leads to a significant decrease, with average decreasing rate at 51.93%/h in the previous 16 h, while in the last 16 h the rate is 48.56%/h, which is just opposite with ambient carbon oxide changes. The results also indicate that the
4. Discussion 4.1. Key ambient parameters and survivability Responses to stress-related ambient disturbances in fish are often characterized as primary, secondary, and tertiary (Kordon et al., 2018; Hur et al., 2019). Furthermore, if severe enough, acute stressors (e.g., air exposure, temperature change) may lead to the consequence of death, which affects the activeness and survivability of waterless transportation for fish (Pottinger et al., 2016; Pottinger, 2017). In terms of the quality control management for waterless live transportation, 6
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Fig. 4. The interaction between survival rate of Chinese farmed sturgeon (Acipenser schrenckii) and ambient parameters changes during waterless live transportation.
live sturgeon gradually enter a relatively stable status after 8 h of waterless transportation, and could live for >32 h. With 4 °C of temperature control and oxygen-filled packaging, the fish survival time in waterless live transportation was observed >32 h in this research. From Table.1, the oxygen level in Group III decreases
faster than in the Control Group (Basal Level), partly because the waterless circumstance stimulates the respiration of the fish, which leads to the increase of oxygen consumption at the same time. Besides, the change of ambient oxygen is opposite with ambient carbon oxide. By analyzing the interaction between the survival rate and different 7
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ambient parameters, as shown in Fig. 4, the temperature and filled‑oxygen in bag are the most critical ambient stressors which directly influence the survival rate of the waterless live transportation. Thus, the results illustrate that the survival rate of waterless live transport is bound up with the ambient temperature and oxygen concentration in plastic bag.
Due to every individual fish is under a low-temperature and no-water condition; consequently, to provide the nutrients to organism and keep alive, some nutrients in fish meat would be degraded so that causing the denaturation of the muscle proteins, which could be reflected on the reducing the muscle of the muscle SF (Kristoffersen et al., 2006; Refaey et al., 2017). From Table 2, it presents that the nutritional values add up to >100%, and what causes this kind of discrepancies mainly including two aspects of error: one is the systematic error, which mainly due to several batches of tests during data acquisition; while another one is the random error, which mainly due to the outliers when artificially remove them. Additionally, Table 2 shows that the transport stress led to the increase of water content of fish muscle since the waterless live transportation, which perhaps causing a lower hardness of muscle after 16 h of transportation thereby decrease the shear strength. After all, the relationship between water content and shear force is a negative correlation (Dunajski, 1979; Aramli et al., 2016; Refaey et al., 2017).
4.2. Physiological parameters and stress level In previous studies, blood glucose and serum cortisol levels are considered as important indicators of stress response when fish subjected to stressful situations (Long et al., 2019; Mi et al., 2012). From Fig. 2 (A) & (B), the stress level of Amur sturgeon in Group III reaches the peak value after 8 h of waterless live transportation but decreases slowly as time passes. However, the stress level is still high (p < .05) compared to Group I and Group II at the same time, which indicates low-temperature and waterless condition has a significant impact on the physiological stress level. Furthermore, in response to low-temperature, the overexpressed cortisol is able to enhance glycolysis pathway, which results in a maintenance in catabolism and energy metabolism over a period of time, when Amur sturgeon was subjected to the low-temperature and waterless transportation (Nie et al., 2018; Bao et al., 2018; Jia et al., 2018; Kordon et al., 2018; Lermen et al., 2004). Except for the two physiological parameters above, the changes in alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) level indicate the generalized stress reactions and could show the damage degree of the organism in vivo of Amur sturgeon (Bao et al., 2018; Li et al., 2018). As a mark of metabolic adaption in the fish to different physiological demands, ALT could provide preliminary evidence for subtle changes in the process of waterless transportation. From Fig. 2 (D), a significant increase level of LDH in Group III indicates the anaerobic metabolism goes up. It has been noted that some changes of lactate dehydrogenase (LDH) concentration seem to stem from the stress increase induced by air exposure, especially exposed to such high‑oxygen (Patterson et al., 2017; Wen et al., 2017).
5. Conclusions As a novel live fish transportation method, waterless live transportation is more high-efficiency and low-cost compared to traditional live fish transportation. To minimize the aqua- and transport losses, it is essential to understand the physiological mechanism and level of stress response in the process of waterless live transportation. In this paper, Amur sturgeon, Acipenser schrenckii was selected as the research subject, and the effects of waterless live transportation on its survivability, physiological responses and flesh quality were presented in this paper. From this research, the results indicate properly optimizing of critical ambient parameters of waterless live transportation is conductive to achieve >32 h keep-alive status of sturgeon. Furthermore, lowtemperature and air exposure could lead to sustained physiological response, which would promote capabilities of anaerobic metabolism in live sturgeon under a waterless environment and thus consume more basal energy to stable the homeostasis. At the same time, there are no significant changes in flesh quality of fish during the low-temperature and waterless live transportation, which means that this live transport approach can guarantee high transport quality to a certain extent. Therefore, the possibility to prepare for and respond to challenges to realize the high volume of cold-fish waterless live transportation is to cope with the accordance between transport stress response and transport management and how to strengthen the stress response monitoring and do further studies on waterless transport optimization are the next research direction for waterless transportation management.
4.3. Flesh quality and muscular parameters Muscle pH is an important parameter of flesh quality evaluation, and muscle shear force is an indicator of tenderness of muscle (Roth et al., 2005). Besides, scientific literature in meat science has evaluated the relationship between different pH values and flesh quality. For instance, high pH in beef longissimus thoracic reduces muscle fibre transverse shrinkage and light scattering (Hughes et al., 2017). The decrease in muscle pH due to transport stress has a noticeable effect on the physical properties of muscle change, which causing the denaturation of the muscle proteins and fatty acid, which possibly reflected on reducing the muscle shear force (SF) (Kristoffersen et al., 2006; Refaey et al., 2017). In this respect, from Fig. 3(A), there is a decreasing trend in muscle pH value between two groups during waterless live transportation while there are nearly no apparent changes in muscle pH value which means no significant increase or decrease during waterless live transportation, which may due to the physiological regulation of the living body itself. As a crucial factor for overall acceptance and consumers' satisfaction, texture or hardness of fish meat is the response of the tactile senses to physical stimuli that result from contact between some part of the body and the food (Veland and Torrissen, 1999; Zarifi et al., 2017). Of course, the characteristic of the texture varies widely due to the different species like fish, vegetables and fruits which have dominant quality characteristic. Moreover, from certain aspects, the index of the shear force is an indirect parameter to reflect part of the texture, partially indicate the degradation of fish muscle which causes lower shear strength. However, according to the present research, transport stress in waterless live transportation leads to declining in the flesh SF directly, which in turn, it would attribute to the declining of muscle pH value.
Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the final manuscript entitled “Effects of Waterless Live Transportation on Survivability, Physiological Responses and Flesh Quality in Chinese Farmed Sturgeon (Acipenser schrenckii)”. Acknowledgments This research is supported by the project assignment on the crossdisciplinary cooperation of Beijing Science and Technology New Star Program (Project ID Z181100006218123), Beijing Science and Technology Project (Project ID Z181100001018033) and Application of Key R&D Program (Public Welfare Specialized) Projects in Shandong Province in 2019:Research on intelligent ambient quality control technology for aquatic products transportation (Project ID 2019GNC106079). 8
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