1
1 Effect of increased rearing temperature on digestive function in cobia early juvenile
2 Short title: Effect of temperature on Cobia digestion
3
4 M. Yúfera
a
, M.V. Nguyen
b
, C. Navarro-Guillén
a1
, F.J. Moyano
c
, A-E.O. Jordal
d
, M. Espe
e
, L.E.C.
5 Conceição
f
, S. Engrola
g
, M.H. Le
b
, and I. Rønnestad
d
6
a
Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Campus Universitario Rio San Pedro s/n,
7 11519 Puerto Real, Spain
8
b
Institute of Aquaculture, Nha Trang University, 02 Nguyen Dinh Chieu st, Nha Trang, Vietnam
9
c
Department of Biology and Geology, University of Almería, 04120 Almería, Spain
10
d
Department of Biological Sciences, University of Bergen, NO-5020, Bergen, Norway
11
e
Institute of Marine Research, Bergen, Norway
12
f
Sparos Lda, Olhão, Portugal
13
g
Centre of Marine Sciences of Algarve (CCMAR), University of Algarve, Campus de Gambelas, University
14 of Algarve, 8005-139 Faro, Portugal
15
16
17 Corresponding author: manuel.yufera@icman.csic.es
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19
1
Present address: Centro de Ciências do Mar do Algarve (CCMAR), Universidade do Algarve, Faro,
20 Portugal.
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22
2
23 Abstract
24 The present study is focused to elucidate the main characteristics of the digestive function of this
25 carnivorous fast-growing fish living at high temperatures. With this aim, we have examined the effects
26 of an increased temperature from 30 to 34°C on the daily pattern of gastrointestinal pH, enzymatic
27 proteolytic digestive activity and the feed transit time in early juveniles of cobia (Rachycentron
28 canadum), a fast-growing carnivorous fish species living in tropical and subtropical waters with an
29 increasing aquaculture production. Fish were fed two meals a day. Gastric luminal pH was permanently
30 acidic (mean pH values: 2.76 - 4.74) while the intestinal pH increased from neutral/slightly acidic to
31 slightly alkaline when the digesta was present, with an increasing alkalinity from proximal to distal
32 intestine (mean pH values: 6.05 to 7.69). The temperature did not affect the gastric pH but a slightly
33 higher acidity was induced in the intestine at 34°C.
34 Pepsin activity showed a daily rhythm at 30 °C with maximum in the middle of the light period, while
35 at 34°C some hourly changes coinciding with feed adding without a clear daily trend during the 24-h
36 period were observed. The trypsin activity exhibited a daily rhythm at both temperatures with an
37 increase after morning feeding to reach a maximum several hours later. Average pepsin activity during
38 the daily cycle was slightly higher at 34 °C (6.1 and 7.3 U mg
-1
BW at 30 and 34 °C respectively), but
39 values were significantly different only at 8 and 24 h after the morning meal. Similarly, the trypsin
40 activity was significantly affected by the temperature only at 8 and 16 h after the morning meal, but
41 daily activity averages were similar (1.20 and 1.29 U g
-1
BW at 30 and 34 °C respectively).
42 The partial transit rates of the first meal in the stomach for each period inter-samplings were higher
43 during the first 4-h period and decreased progressively along the rest of the 24-h cycle at both
44 temperatures, but no significant differences were detected at 30 °C. In addition, the transit was notably
45 faster at 34 °C particularly during the first 8 h after feeding, with rates between 100 and 65% of total
46 volume displaced (intake or released) during each 4-h period. In the intestine the transit rate was
47 relatively constant and similar at both temperatures during 12 h after feeding. Then the rates remained
48 very low during the following 12 h.
49 Residence time of the first meal was longer at 30 than at 34 °C, particularly in the stomach (12h:02min
50 vs 4h:54min respectively). In the intestine the difference was not so large (8h:18min vs 6h:24min
51 respectively). In a parallel study with under same conditions, cobia reared at 30 °C grew faster and
52 showed better a more favorable feed conversion ratio than those at elevated temperature (34 °C). The
53 present results indicate that at 34 °C, a subtle increase of proteolytic activity cannot compensate for
54 the faster gut transit rate. Therefore, 30 °C is more appropriate temperature for the early on-growing
3
55 of cobia because at higher temperatures the digestion efficiency decrease being one of the causes for
56 a lower growth.
57
58 Key words: Temperature, GIT luminal pH, Digestive enzyme, Gut transit time, Rachycentron canadum
59
60 Introduction
61 Water temperature is a key factor affecting metabolic rates in fish and therefore has an evident impact
62 on feed intake, nutrient utilization and growth (Brett, 1979; Buentello et al., 2000). To cope with the
63 wide range of temperatures in the oceans depending on the geographic location and environmental
64 cycles, the various fish species have adapted their feeding behavior and physiology to the temperature
65 conditions of their particular habitat (Brett, 1979; Somero, 2004, 2010). Many studies have examined
66 different perspectives of physiological responses to changes in temperature.
67 Particularly relevant is the way the ingested nutrients are digested before their incorporation into
68 growing tissues. In spite of a large research effort, the effect of temperature on fish digestion is far
69 from being well understood. The digestive function includes different processes from feed capture to
70 assimilation of nutrients that may be affected in different manners by temperature changes. Generally,
71 the feed intake increases with increased temperatures up to levels close to the upper tolerance limits
72 (Fernández-Montero et al., 2018; Pérez-Casanova et al., 2009). Digestive enzyme activity has been
73 traditionally assessed in two ways. On one side, in vitro experiments for the enzyme characterization
74 performed with enzyme extracts show that activity increases with increasing temperature usually up
75 to values exceeding those representative of their natural habitats, and also beyond lethal levels
76 (Alarcón et al., 1998; Fernández et al., 2001; Gelman et al., 2008; Tanji et al., 1988). On the other hand,
77 information about digestive enzyme activities analyzed in live fish at different temperatures is also
78 available (Bowyer et al., 2014; Hani et al., 2018; Mazumder et al., 2018; Miegel et al., 2010; Sharma et
79 al., 2017). However, these studies are based on only one sampling point during the postprandial
80 response and, also report contradictory responses among the different studied species.
81 Gut evacuation rate also increases at increasing temperatures up to a certain limit, leading to lower
82 residence time in the digestive tract (De et al., 2016; Fernández-Montero et al., 2018; Handeland et
83 al., 2008; Temming and Herrmann, 2001). However, the estimation of evacuation rate has usually been
84 performed under unrealistic feeding conditions in which the fish has been refed until satiation after a
85 starvation period. Digestion efficiency will depend on the relation between enzymatic activity and gut
86 transit time that are not short punctual facts but long dynamic cyclic processes usually occurring along
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87 a whole day. Consequently, only experiments performed in routine feeding may provide realistic
88 information.
89 Other species-specific digestive characteristics may also strongly affect the digestion process. That is
90 the case of the gut luminal pH that conditions the activation of proenzymes in the gut, which may vary
91 among fish, particularly within the stomach (Bucking and Wood, 2009; Papastamatiou and Lowe, 2005;
92 Papastamatiou et al., 2007; Secor and Carey, 2016; Yúfera et al., 2004, 2007, 2012; Solovyev et al.,
93 2018). Optimum temperature for growth does not necessarily coincide with maximum feed intake,
94 highest digestion efficiency or optimal feeds utilization. In fact, from the point of view of aquaculture,
95 the aim is to optimize all these factors to obtain the better juvenile quality and weight gain at a
96 reasonable cost and with the lowest environmental impact.
97 Cobia (Rachycentron canadum), is a fast-growing species inhabiting tropical and subtropical waters
98 with a broad geographical distribution over several continents. The species may reach up to 60 Kg and
99 has a high-quality white flesh, being considered an excellent marine fish for aquaculture. It is being
100 produced mainly in Asian-Pacific coast and in a lesser extent in the Gulf of Mexico with a world
101 production above 40.000 tons during the last years (FAO, 2018; Tveteras, 2016). In Vietnam, it is one
102 of the main marine fish in large scale commercial aquaculture (Nhu et al., 2011). In South Vietnam and
103 other Southeast Asian regions water temperature in ponds and tanks ranges between 27 and 30 °C,
104 but may reach up to 36 °C during the daytime in the warmer season. For this reason, it is necessary to
105 understand the responses to increased temperatures, particularly in this region in the scenario of
106 global warming (IPCC 2015). Effect of rearing temperature on growth in cobia juveniles has been
107 examined by Sun and Chen (2009, 2014). In these studies, cobia juveniles were reared in the rage 20
108 to 35 °C and highest growth rates were observed in the range 27-31 °C. An unsolved question is how
109 these temperature changes affect the digestion process.
110 Therefore, in this study we have examined the effects of temperature on the digestive function from
111 a global perspective in order to advance in the physiological basis and mechanisms behind digestive
112 efficiency and the corresponding effects on growth in cobia juveniles. Specifically, the aim of this study
113 was to elucidate whether a temperature increase from 30 to 34 °C affects gastrointestinal pH,
114 enzymatic proteolytic digestive activity and feed transit over the whole day period, in early juveniles
115 of this fast growing fish.
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117 Material and Methods
118 Fish rearing and sampling
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119 This study was part of a larger experiment examining growth performance in juveniles fed three
120 different diets (Nguyen et al., 2019). We used the tanks from control treatment for the present study.
121 Cobia juveniles were obtained from a local hatchery in Nha Trang, Vietnam, and acclimatized to final
122 experimental temperatures in two indoor fiberglass 5000-L tanks at Nha Trang University facilities
123 during one week. During the period of acclimatization, water temperature in one tank increased at a
124 rate of 1°C per day up to 34°C, while temperature in the other tank was kept constant at 30°.
125 Acclimatized juveniles with 3.7 ± 0.4 g wet body weight, were randomly distributed to 6 experimental
126 200-L tanks (60 fish tank
-1
) and reared under a light/dark cycle at two temperatures (30 and 34 °C,
127 three tanks for each temperature) in recirculation systems. Water salinity was 29.0 ± 3.1 g L
-1
, pH 7.8–
128 8.3, oxygen level 4.6 ± 0.5 mg L
-1
and NH
3
<0.03 mg L
-1
. Fish were maintained with a 12:00 h illumination
129 period from 6:00 to 18:00 h and fed twice a day (8:00 and 16:00 h local time) according to the most
130 common procedure in the local hatcheries (Nguyen, 2013; 2014). with an The experimental diet was
131 produced at SPAROS Lda (Olhão, Portugal) containing 47% protein and 10% lipid (Table 1). The
132 experimental diet and was formulated based on previous results in cobia (Nguyen et al., 2014). The
133 fish were fed to nearly satiety (until most of the fish losing their appetite) by hand. Eventual uneaten
134 feed and removed fish were recorded for the calculation of daily feed intake and feed conversion ratio,
135 parameters considered in the companion study (Nguyen et al., 2019). After 2 weeks under these
136 conditions, 3 fish per tank (wet body weight: ca. 6-8 g range) were sampled every 4 hours during 24
137 hours, for gut pH, digestive enzyme activity and feed transit determinations. Dissected gut were freeze-
138 dried and sent to Spain for the analyses of enzyme activities at the University of Almeria and the gut
139 transit at the ICMAN-CSIC. All experimental procedures complied with the Guidelines of the European
140 Union Council (2010/63/EU) for the use and experimentation of laboratory animals and were reviewed
141 and approved by the Spanish National Research Council (CSIC) bioethical committee.
142 Measurements of gut pH
143 The gastrointestinal pH was measured in nine individuals at each sampling point and for each
144 temperature condition immediately after sampling using a pH microelectrode (Thermo Orion, Thermo
145 Fisher Scientific Inc) following the procedure described in Yúfera et al. (2012). In short, the fish were
146 dissected to make the digestive track accessible. Next, the tip of the microelectrode (diameter 1.7 mm)
147 was inserted in small slits made in the stomach, anterior intestine, medium intestine and posterior
148 intestine (Fig. 1).
149 Digestive enzyme activity analyses
150 The complete digestive tract of three individuals of each sampling point and temperature were
151 dissected, immediately frozen at -80 °C and later freeze-dried. Enzyme extracts were prepared for
