Fish Physiology and Biochemistry

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Thyroid hormones transiently increase during parr-smolt transformation in coho salmon, Oncorhynchus kisutch, and are believed to trigger morphological, physiological, behavioural, and neural changes. The effectiveness of propylthiouracil (PTU) to induce hypothyroidism in smolting coho salmon was determined by immersing coho salmon, Oncorhynchus kisutch, in 30 mg l−1 PTU from May 1, two weeks prior to the consistent annual total thyroxine (TT4) peak in mid-May, until the last sampling date. Plasma was obtained at two sampling dates from control and PTU -treated coho salmon: May 15, during the plasma TT4 peak; and May 26, after the TT4 peak. Radioimmunoassays were used to measure plasma TT4, total triiodothyronine (TT3), free thyroxine (FT4), and salmon growth hormone (sGH). The PTU -treatment inhibited the natural smoltification-related increases in plasma TT4, TT3 and GH levels compared with controls, but PTU-treatment did not affect these hormone levels when they were low. PTU -treatment increased FT4 and decreased TT3 and sGH levels in the May 26 sample. In the May 15 sample, FT4 levels were unaffected by PTU-treatment, whereas TT4 levels were decreased. These data demonstrate the ability of PTU to induce hypothyroidism in salmonids as shown by the decrease in TT4 and TT3. These data demonstrate that PTU treatment by immersion can induce hypothyroidism in salmonids as shown by: (1) the inhibition of the natural increases of TT4 and TT3; (2) the increase in FT4 levels corresponding to the lowered TT3 levels, suggesting an inhibition of thyroxine 5′-monodeiodinase activity. We also show for the first time that PTU treatment can lower plasma GH levels in salmonids. This lowering of plasma GH level is associated with the decrease in TT3 levels and the increase in FT4 levels. The PTU induced lowering in GH levels may contribute to the observed changes in FT4 and TT3, since GH is known to increase thyroxine 5′-monodeiodinase activity.

The growth performance of carnivorous southern catfish (Silurus meridionalis) was lower when fed a diet containing glucose as a carbohydrate source than when fed one containing starch. To test whether this decreased growth performance was partly due to higher metabolic costs induced by the fast assimilation of dietary glucose, the postprandial metabolic responses of southern catfish juvenile (36.7 ± 1.9 g) fed diets containing either raw or pre-cooked corn starch and glucose were investigated at 27.5°C. The peak metabolic rate of fish fed a raw corn starch diet was significantly higher than that of fish fed precooked corn starch and glucose diets (P < 0.05). The specific dynamic action (SDA) coefficients (energy expended on SDA as a percentage of energy content of the meal) of fish fed raw or precooked corn starch diets were significantly higher than that of fish fed a glucose diet (P < 0.05). The postprandial metabolic rate of fish fed a raw starch diet displayed a classic response – increased metabolic rate immediately after feeding, followed by a decreased metabolic rate several hours after feeding – as has been documented earlier in this species. However, the postprandial metabolic rate of fish fed a glucose diet had two alternating cycles of increasing and decreasing metabolic rate, which has not been reported previously. This bimodal pattern of the SDA curve was also found in a subsequent experiment in which purified d-glucose (0.1 g dry mass) was packed into sections of grass carp (Ctenopharyngodon idellus) intestine (0.05 g dry mass). The results suggest that the digestibility and absorption speed of the dietary carbohydrate has a significant effect on postprandial metabolic response in juvenile southern catfish. The results of this study may provide useful data in terms of the utilization of dietary carbohydrates by carnivorous fishes.

One of the most severe impacts of urbanization on aquatic systems is the increasing presence of low oxygen environments caused by anthropogenic sources of pollution. As urbanization increases nationally and globally, it is becoming exceedingly important to understand how hypoxia affects aquatic fauna, especially fish species. In an effort to better understand the impacts of prolonged hypoxia on fishes, largemouth bass were held at 3.0 and 9.0 mg L−1 for 50 days, which has previously shown to be temporally sufficient to impart plastic phenotypic changes. Following the holding period, fish from each group were subjected to a low dissolved oxygen (DO) challenge of 2.0 mg L−1 for 6 h, and their physiological and hematological parameters were compared with control fish held for 6 h with no change in DO. There were no differences in the physiological stress responses between the two holding groups; however, the low oxygen holding group had increased hemoglobin and hematocrit levels following the 6-h low oxygen challenge compared with the high oxygen group. These results suggest largemouth bass exposed to chronic low oxygen conditions, either naturally or anthropogenically, may possess a beneficial advantage of increased oxygen uptake capacity during periods of low oxygen.

In vivo induction of vitellogenin (VTG) in response to the administration of 17β-estradiol (E2) was achieved and the protein was isolated by gel filtration column chromatography of plasma samples. Adult female trout were injected with the vitellogenic fraction every ten days from July to November and levels were measured by RIA from September to December. The results showed a significant decrease (p<0.01) in plasma E2 levels in injected females compared with the controls. In December, after finishing the treatment, the plasma E2 concentration increased, in injected females to reach a level similar to that of control females at vitellogenesis. The in vitro study showed that in early vitellogenic oocyte (from September) the presence of the vitellogenic fraction in the incubation medium causes a significant decrease (p<0.01) in the synthesis of E2 by the oocytes. These data suggest that the concentration of the VTG into the oocyte can alter VTG production by the liver, moderating the production of E2 by the ovary.

Transgenic common carp,Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-1 (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

A 2-week research was carried out to assess water salinity (WS) effects including 0, 15, 35, and 50‰ on osmoregulatory mechanisms and stress indices in Asian sea bass (34.4 g) juveniles. Except for fish reared at 50‰, in the other treatments, it gradually decreased to the prescribed WS during a 10-day period (− 5‰ a day). After a 10-day acclimation period, fish were reared at the prescribed WS for 2 weeks. Fish reared at 15 and 35‰ had higher chloride cell (CC) counts in the interlamellar region. The number of CC in the interlamellar region elevated with increment of WS up to 35‰, but they were pronouncedly reduced in 50‰ group. The diameter of CC in the interlamellar region was not affected by WS. The smallest nucleus diameter of CC in the interlamellar region was observed in fish reared at 15‰ (P < 0.05). The largest and the smallest amounts of serum aspartate aminotransferase were observed in fish reared at freshwater and 15‰, respectively. Fish reared at 35‰ had the highest serum sodium and potassium contents. Serum chloride content and total osmolality increased with increment of WS (P < 0.05). Serum cortisol and glucose contents gradually increased with elevation of WS up to 35‰; then, their contents remarkably decreased. The relative expression of insulin like growth factor-1 in the liver of fish reared at 35‰ was strikingly higher than that in the other groups. The relative expression of HSP70 gene in fresh water group was pronouncedly elevated compared to other treatments. The relative expression of interleukin-1β in 15 and 35‰ groups was higher than that in the other groups; however, the relative expression of lysozyme gene in the liver of fish reared at fresh water was pronouncedly lower than that in the other treatments. The results of this study suggested rearing L. calcarifer at 15‰ closer to the isosmotic point and better provide its welfare.

A 60-day feeding trial was conducted with six experimental diets containing 260 g/kg crude protein and 70 g/kg lipid with varying level of de-oiled rice bran (DORB), viz., 33 (T33), 38 (T38), 43 (T43), 48 (T48), 53 (T53) and 58% (T58) to assess the digestive, metabolic and haematological responses in Labeo rohita. One hundred and eighty (180) fingerlings with ten fish per tank having an average weight of 9.8 ± 0.5 g were randomly distributed within six treatments in triplicates following a completely randomised design. The amylase and lipase activities in the whole intestine did not vary significantly (P > 0.05), but protease activity varied significantly with respect to DORB level in the diet. At higher (53 and 58%) inclusion level of DORB, the protease activities were lower. T33, T38 and T43 groups showed significantly (P < 0.05) higher aspartate aminotransferase (AST) activity compared to the other groups, whereas lowest activity was recorded in the group fed with highest inclusion level (58%). The oxidative stress enzyme like superoxide dismutase (SOD), catalase and glutathione-S-transferase exhibited significantly higher activities in liver of T58 group of L. rohita. However, the activity of catalase and SOD showed no significant variation in gill tissue. The haematological parameter values such as red blood cell (RBC), white blood cell (WBC), haemoglobin (Hb) and packed cell volume (PCV) were lower in the highest DORB fed group (T58) as compared to their lower DORB inclusion counterparts. No change in NBT value was recorded irrespective of DORB inclusion level in the diet. Hence, from these results, it can be concluded that the dietary inclusion of DORB up to 43% in the diet of L. rohita at 26% crude protein and 7% lipid level is best for the optimal activity of digestive, metabolic and haematological parameters.

A 30-day acclimation trial was conducted using Tor putitora to elucidate its thermal tolerance, oxygen consumption, haemato-biochemical variables and selected enzymatic activities at five acclimation temperatures (AT). Juveniles of T. putitora were randomly distributed among five treatment groups (20, 23, 26, 29 and 32 ± 0.5 °C). There was a significant curvilinear increase in critical thermal maxima (CTmax) (y = −0.0693x 2 + 1.7927x + 34.628, R 2 = 0.996) and lethal thermal maxima (LTmax) (y = −0.1493x 2 + 2.3407x + 35.092, R 2 = 0.991) with increasing AT. The oxygen consumption rate increased significantly with increasing AT. The Q 10 values were 1.16 between 20 and 23 °C, 3.09 between 23 and 26 °C, 1.31 between 26 and 29 °C and 1.76 between 29 and 32 °C of AT. The acclimation response ratios were ranged between 0.37 and 0.59. Catalase, superoxide dismutase and ATPase activities were increased linearly in liver, gill and kidney, while brain acetylcholine esterase activity decreased linearly with increasing AT. Blood glucose remained unchanged up to AT of 26 °C and increased significantly at AT of 29 and 32 °C. Haemoglobin content was increased linearly with increasing AT. The highest WBC count was observed at 20 °C, and no significant changes found till AT of 26 °C and significantly decreased at 32 °C. Total serum protein and globulin were significantly decreased with increasing AT. Highest values were observed at 20 °C and remained consistent till 26 °C, then decreased significantly. There was no significant change in A/G ratio through the AT 20–29 °C and increased significantly at 32 °C. The increase in CTmax, LTmax and oxygen consumption rate with increasing AT may suggest that the thermal tolerance of T. putitora is dependent on its prior thermal exposure history, and it could adapt to higher AT by altering its haemato-biochemical variables.

Hypoxia can affect various fish populations, including yellow perch Perca flavescens, which is an economically and ecologically important species in Lake Erie, a freshwater system that often experiences hypoxia in the hypolimnetic part of the lake. Fish, similarly to mammals, possess molecular oxygen sensor—hypoxia-inducible factor-1 (HIF-1), a transcription factor that can affect expression of many downstream genes related to animal growth and locomotion, protein synthesis, as well as ATP and amino acid metabolism. HIF-1 is a heterodimer, which consists of two subunits: oxygen-sensitive and oxygen-insensitive subunits, α and β, respectively. In this study, we report first on the molecular cloning and sequencing of P. flavescens HIF-1α. The full-length complementary DNA (cDNA) was isolated and submitted to the GenBank with accession number KT783483. It consists of 3529 base pairs (bp) carrying a single open-reading frame that encompasses 2250 bp of the coding region, 247 bp of the 5′ untranslated region (UTR), and 1032 bp of the 3′ UTR. The “de novo” prediction of the 3D structure of HIF-1α protein, which consists of 749 amino acids, is presented, too. We then utilized One-Step Taqman® real-time RT-PCR technology to monitor changes in HIF-1α messenger RNA (mRNA) copies in response to chronic hypoxic stress. An experiment was conducted using 14-day post-swim-up stage yellow perch larvae with uninflated swim bladders. This experiment included three treatment groups: hypoxia, mid-hypoxia, and normoxia, in four replicates (four tanks per treatment) with the following dissolved oxygen levels: 3, 4, and >7 mg O2/L, respectively. At the end (2 weeks) and in the middle (1 week) of the experiment, fish from each tank were sampled for body measurements and molecular biology analysis. The results showed no differences in survival (∼90%) between treatment groups. Oxygen concentration was lowered to 3.02 ± 0.15 (mean ± SE) mg O2/L with no adverse effect on fish survival. The highest growth rate was observed in the normoxic group. A similar trend was observed with fish body length. The growth rate of fish declined with decreasing water-dissolved oxygen. The number of HIF-1α mRNA copies was not significantly different between hypoxic, mid-hypoxic, and normoxic conditions, and this was true for fish obtained in the middle and at the end of the experiment.