Food effects on some reproductive aspects of rainbow trout, Oncorhynchus mykiss, in a Venezuelan fish farm

Samples of rainbow trout, Oncorhynchus mykiss, were fed with three different dried diets, labeled D1, D2 and D3, the first ones were expanded and the last one pelleted type food. Their protein content ranged from 36% to 42%, and only D1 diet presented a pigment in its composition. The gonodasomatic index (GSI) as well as the gonadial development showed a different behavior in males and females for all the samples tested. A continuous sperm production was observed in males fish, while in female fish the GSI showed a tendency to increase through the whole study, and they showed a reproductive rest period on july 92. Fish relative fecundity and egg diameter were similar in all cases. The gonodial maduration age was shorter for the female that were fed with D1 and D2, while the 50% of the trout population that got D3 had a gonodial maduration period two months longer. For all trouts tested the fertility was low with a maximum value of 49%. The hatchery from the tested trouts with D2 intakes had a mortality value of 62%. Meanwhile, those with D1 and D3 intakes showed mortality values of 36% and 43% respectively.     

Does the Rainbow Trout Ovarian Fluid Promote the Spermatozoon on Its Way to the Egg?

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the “turn-and-run” behavior involving asymmetric flagellar beating and Ca²⁺ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)–sperm interaction reflect the specific features of the spawning process in this species.     

Oil content and fatty acid composition of commercially important Turkish fish species

The oil content and fatty acid composition of commercially important Turkish fish species (anchovy,Engraulis encrasicholus; freshwater rainbow trout,Salmo gairdneri; and cultured salmon,S. salar) were determined. Palmitic (16∶0), palmitoleic (16∶1), oleic (18∶1), and docosahexaenoic (22∶6) acids were the most abundant fatty acids in all species. Eicosapentaenoic acid (20∶5) was twice as high in the anchovy oil as in the rainbow trout and salmon oils. Significant quantities of linoleic acid (18∶2) and docosahexaenoic acids (22∶6) were found in both rainbow trout and salmon samples. The individual fatty acid data obtained from rainbow trout and salmon were similar to each other. All three fish species contain high levels of n-3 polyunsaturated fatty acids and would be suitable for inclusion in the formulation of low-fat highly unsaturated diets.     

Are chemical alarm cues conserved within salmonid fishes?

A wide diversity of fishes possess chemical alarm signalling systems. However, it is not known whether the specific chemicals that act as alarm signals are conserved within most taxonomic groups. In this study we tested whether cross-species responses to chemical alarm signals occurred within salmonid fishes. In separate laboratory experiments, we exposed brook charr (Salvelinus fontinalis), brown trout (Salmo trutta), and rainbow trout (Oncorhynchus mykiss) to chemical alarm signals from each of the three salmonid species and from swordtails (Xiphophorus helleri). In each case, the test species responded with appropriate antipredator behavior to all three salmonids alarm cues, but did not react to swordtail cues. These data suggest that chemical alarm cues are partially conserved within the Family Salmonidae. For each species tested, the intensity of the response was stronger to conspecific alarm cues, than to heterospecific alarm cues, indicating that salmonids could distinguish between chemical cues of conspecifics versus heterospecifics. These results suggest that the chemical(s) that act as the alarm cues may be: 1) identical and that there may be other chemical(s) that allow the test fish to distinguish between conspecifics and heterospecifics, or 2) that the cues that act as signals are not identical, but are similar enough to be recognized.     

LC-PUFA Biosynthesis in Rainbow Trout is Substrate Limited: Use of the Whole Body Fatty Acid Balance Method and Different 18:3n-3/18:2n-6 Ratios

Five experimental diets with constant total C₁₈ PUFA and varying 18:3n-3/18:2n-6 ratios were fed to rainbow trout over an entire production cycle. The whole-body fatty acid balance method demonstrated a clear trend of progressively reduced fatty acid bioconversion activity along the n-3 and n-6 pathways, up to the production of 20:5n-3 and 20:4n-6, respectively. This suggests that the pathway exhibits a “funnel like” progression of activity rather than the existence of a single rate limiting step. The production of 22:5n-3 and 22:6n-3 was more active than that of 20:5n-3. However, despite this trend in reduced apparent in vivo net enzyme activity, the efficiency of the various bioconversion steps (measured as % of bioconverted substrate) confirmed an opposing trend. A 3.2-fold higher Δ-6 desaturase affinity towards 18:3n-3 over 18:2n-6 and an 8-fold greater Δ-5 desaturase affinity towards 20:4n-3 over 20:3n-6 were recorded. The main results of the study were that (1) rainbow trout are quite efficient at bioconverting 18:3n-3 to 22:6n-3, and (2) the LC-PUFA biosynthetic pathway is substrate limited. Fillet n-3 LC-PUFA concentrations increased with the increasing dietary supply of 18:3n-3. Despite an almost identical dietary supply of n-3 LC-PUFA, originating from the fish meal fraction of the diets, the fillets of trout fed the diet richest in 18:3n-3 were 2-fold higher in n-3 LC-PUFA than fish fed low 18:3n-3 diets. Nevertheless, fillets of trout fed a fish oil control diet contained more than double the amount of n-3 LC-PUFA compared to fish fed the diets richest in 18:3n-3.     

Effects of rainbow trout freshness on n‐3 polyunsaturated fatty acids in fish offal

The effects of rainbow trout cold storage on the quality of offal left after fish processing to fillets with skin were determined. The intact farmed rainbow trout were kept at 2 °C in ice for 0, 4, 7, and 14 days of storage. The offal was, immediately after processing, frozen at ?20 °C and analysed after a month‐long frozen storage; fillets (non‐frozen) were analysed as well. Non‐protein nitrogen, volatile bases, trimethylamine, lipid oxidation (peroxide value, anisidine value, UV‐VIS spectra, and fluorescence) and fatty acid composition were determined. The offal consists in 15% of protein and in about 20% of chloroform/methanol‐extractable lipids, with n‐3 polyunsaturated fatty acids (n‐3 PUFA) accounting for 20.37 ± 1.25% of the fatty acids. The fish storage duration was found to exert a significant (p = 0.05) effect on the changes in lipids and nitrogen compounds. No losses of long‐chain n‐3 PUFA in the offal were detected during the 2 wk of storage in ice plus 1 month at ?20 °C. The rainbow trout offal is a valuable – rich and stable – source of n‐3 PUFA.     

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish.     

Significance of docosahexaenoic acid for rainbow trout (Oncorhynchus mykiss) larvae

N-3 fatty acids are essential for rainbow trout (Oncorhynchus mykiss). Our investigations specify the fatty acid requirements of rainbow trout larvae. One group of larvae was fed with Artemia ssp. rich in linolenic acid (18:3 n-3) and octadecatetraenoic acid (18:4 n-3), while the other group received a commercial fish feed containing high levels of long-chain polyunsaturated fatty acids (PUFA), mainly docosahexaenoic acid (22:6 n-3). After two and four weeks of feeding, the growth and fatty acid pattern of the larvae were determined. The fatty acid composition of the diet is reflected in the triglyceride composition of the fish, but there is no sign of conversion of 18:3 n-3 and 18:4 n-3 into long-chain polyunsaturated fatty acids. This is caused by limited chain elongation rather than by low enzyme activity of desaturase. In the triglycerides of the larvae, high levels of 18:4 n-3 were determined. This fatty acid was not transformed into the corresponding long-chain polyunsaturated fatty acids. Deficiency of 22:6 n-3 resulted in reduced larval growth. Therefore, it can be stated that docosahexaenoic acid is essential for rainbow trout larvae.     

Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone,Haliotis discus hannai

Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.     

Fatty acid composition of 19 species of fish from the black sea and the Marmara Sea

Evidence suggests that differences in fatty acid composition among various fish species may be due to differences in diet or to environmental factors such as temperature, salinity, and depth at which the fish are caught. The beneficial effects of a diet containing fish on cardiovascular or other diseases have been associated with their high content of eicosapentaenoic (20∶5n-3) and docosahexaenoic (22∶6n-3) acids. In this study we analyzed the fatty acid composition of the flesh of 18 different species of marine fish and of cultured rainbow trout. The fish were obtained from the Black and the Marmara Seas, both of which have unique biological and ecological systems as well as eutrophication and pollution. The contents of 20∶5n-3 and 22∶6n-3 in the marine fish ranged from 4.2 to 13.3 wt% of total fatty acids, and from 6.6 to 40.8 wt%, respectively. The most important differences from other studies on oceanic fish were the tendencies toward higher percentages of 16∶0 and 22∶6n-3. The n-3 series of polyunsaturated fatty acids were present as 32.4±1.9% of the total fatty acids. The present study suggests that mature and immature Pomatomus saltator, as well as Engraulis encrasicolus, Mullus surmuletus, Sardina pilchardus, Mugil cephalus, and Sarda sarda may be preferred for the Turkish diet as a result of their high 20∶5 n-3 and 22∶6 n-3 contents. The cultured rainbow trout Oncorhynchus mykiss is not as good a source of n-3 fatty acids as are the marine fish.     

Inhibition of gonadotropin-stimulated ovarian steroid production by polyunsaturated fatty acids in teleost fish

The effects of the polyynsaturated fatty acids (PUFAs)—eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA)—onin vitro steroid production by full-grown prematurational ovarian follicles from goldfish and rainbow trout were investigated. EPA and DHA inhibited gonadotropin-stimulated testosterone production in a dose-related manner, but AA was inhibitory only at the highest dose tested (400 μM). AA alone stimulated testosterone production by increasing cAMP production, but the effects of other PUFAs alone were marginal. The inhibitory actions by PUFAs were not restricted to long-chain PUFAs, as linoleic and linolenic acids had similar actions in the goldfish. The inhibitory action of EPA on testosterone production was reversible upon removal of the PUFA from medium. Testosterone production stimulated by the addition of the cAMP analogues, dibutyryl cAMP, and 8-bromo cAMP, was attenuated by PUFAs, suggesting that they act at a site distal to cAMP formation. A post-cAMP site regulating cholesterol availability may be involved as testosterone production induced by addition of 25OH-cholesterol was not affected by the PUFAs in either fish species. Together, these findings underscore the importance of lipids in ovarian physiology and suggest that PUFAs may participate in the regulation of ovarian steroidogenesis in teleost fish.     

Changes in Tissue Lipid and Fatty Acid Composition of Farmed Rainbow Trout in Response to Dietary Camelina Oil as a Replacement of Fish Oil

Camelina oil (CO) replaced 50 and 100 % of fish oil (FO) in diets for farmed rainbow trout (initial weight 44 ± 3 g fish^?1). The oilseed is particularly unique due to its high lipid content (40 %) and high amount of 18:3n‐3 (α‐linolenic acid, ALA) (30 %). Replacing 100 % of fish oil with camelina oil did not negatively affect growth of rainbow trout after a 12‐week feeding trial (FO = 168 ± 32 g fish^?1; CO = 184 ± 35 g fish^?1). Lipid and fatty acid profiles of muscle, viscera and skin were significantly affected by the addition of CO after 12 weeks of feeding. However, final 22:6n‐3 [docosahexaenoic acid (DHA)] and 20:5n‐3 [eicosapentaenoic acid (EPA)] amounts (563 mg) in a 75 g fillet (1 serving) were enough to satisfy daily DHA and EPA requirements (250 mg) set by the World Health Organization. Other health benefits include lower SFA and higher MUFA in filets fed CO versus FO. Compound‐specific stable isotope analysis (CSIA) confirmed that the δ¹³C isotopic signature of DHA in CO fed trout shifted significantly compared to DHA in FO fed trout. The shift in DHA δ¹³C indicates mixing of a terrestrial isotopic signature compared to the isotopic signature of DHA in fish oil‐fed tissue. These results suggest that ~27 % of DHA was synthesized from the terrestrial and isotopically lighter ALA in the CO diet rather than incorporation of DHA from fish meal in the CO diet. This was the first study to use CSIA in a feeding experiment to demonstrate synthesis of DHA in fish.     

Desaturation and chain elongation of essential fatty acids in isolated liver cells from rat and rainbow trout

Isolated hepatocytes from rainbow trout and rat were incubated with¹⁴C-labeled linoleic acid, linolenic acid, dihomogammalinolenic acid or eicosapentaenoic acid. The most striking difference in the desaturase activity was the lower level of Δ5 desaturase in trout than in rat. No Δ4 desaturation of 22∶4(n−6) to 22∶5(n−6) was observed in either of the two species, while the conversion of 22∶5(n−3) to 22∶6(n−3) was significant in both groups and highest in rainbow trout. The chain-elongating activity was remarkably similar in the two species, except for the “dead-end” elongation which was distinctly more important in fish.     

Interactive effects of dietary palm oil concentration and water temperature on lipid digestibility in rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

An experiment was conducted to evaluate the interactive effects of dietary crude palm oil (CPO) concentration and water temperature on lipid and FA digestibility in rainbow trout. Four isolipidic diets with 0, 5, 10, or 20% (w/w) CPO, at the expense of fish oil, were formulated and fed to groups of trout maintained at water temperatures of 7, 10, or 15 degrees C. The apparent digestibility (AD) of the FA, measured using yttrium oxide as an inert marker, decreased with increasing chain length and increased with increasing unsaturation within each temperature regimen irrespective of CPO level fed to the fish. PUFA of the n-3 series were preferentially absorbed compared to n-6 PUFA in all diet and temperature treatments. Except for a few minor FA, a significant (P < 0.05) interaction between diet and temperature effects on FA digestibility was found. Increasing dietary levels of CPO lead to significant reductions in the AD of saturates and, to a lesser extent, also of the other FA. Lowering water temperature reduced total saturated FA digestibility in trout regardless of CPO level. Based on the lipid class composition of trout feces, this reduction in AD of saturates was due in part to the increasing resistance of dietary TAG to digestion. Increasing CPO level and decreasing water temperature significantly increased TAG content in trout fecal lipids, with saturates constituting more than 60% of the FA composition. Total monoene and PUFA digestibilities were not significantly affected by water temperature in fish fed up to 10% CPO in their diet. The potential impact of reduced lipid and FA digestibility in cold-water fish fed diets supplemented with high levels of CPO on fish growth performance requires further research.     

Reproduction and survival of rainbow trout (Salmo gairdneri) fed linolenic acid as the only source of essential fatty acids

A semipurified test diet containing 1% linolenate as the sole dietary essential fatty acid was fed to a group of rainbow trout (Salmo gairdneri) for 34 months. The fish matured and the eggs produced were hatched. The second generation fry were fed our laboratory diet for 3 months. The growth of these fry was normal. Histologic examinations revealed no abnormality in liver, heart and kidney tissues of the fry during the three month period. Technical Paper No. 5011, Oregon Agricultural Experiment Station, Oregon State University, Corvallis, Oregon 97331.     

Cryopreservation of Gametes and Embryos and Their Molecular Changes

The process of freezing cells or tissues and depositing them in liquid nitrogen at –196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.     

The Phospholipid Composition of Kangaroo Spermatozoa Verified by Mass Spectrometric Lipid Analysis

Cryopreservation of kangaroo sperm has not been successful so far, and yet there is no promising cryopreservation protocol for these cells available. However, conservation of gametes is extremely important, particularly in the context of preserving endangered species. As spermatozoa are comprised of different membrane systems, the composition of these membranes might account for difficulties in cryopreservation. Lipids, as the main components, affect the physical properties of biological membranes and play a major role in sperm maturation. Therefore, knowledge of the lipid composition is crucial for any further step toward the preservation of the species. We used MALDI‐TOF, ESI‐IT, tandem mass spectrometry, and thin layer chromatography to investigate the lipid composition of epididymal spermatozoa of four different kangaroo species. Spectra of these species were very similar with respect to the identified lipid species. Tremendous changes in the lipid composition during the transit of sperm from caput to cauda epididymis could be seen, specifically an increase in poly‐unsaturated fatty acids, ether lipids, and plasmalogens, as well as a reduction in mono‐ and di‐unsaturated fatty acids. Additionally, phosphatidylcholines containing docosatrienoic acid (22:3), a heretofore unknown fatty acid for sperm membranes, showed the highest abundance in kangaroo sperm.     

The effects of tricaine methanesulfonate (MS-222) on plasma nonesterified fatty acids in rainbow trout,Oncorhynchus mykiss

The effects of tricaine methanesulfonate (MS-222), a commonly used fish anesthetic, on plasma nonesterified fatty acid levels (NEFA) were examined in rainbow trout,Oncorhynchus mykiss. Total NEFA levels declined with increasing duration of exposure to MS-222. Most of the decline in total NEFA was due to decreases in saturated fatty acids (14∶0, 16∶0 and 18∶0). The fatty acid displaying the most rapid response to exposure to MS-222 was 20∶5n−3. The lower plasma NEFA levels in anesthetized fish may be explained by depressed lipolysis in the presence of the anesthetic.     

Fatty Acid-Specific Alterations in Leptin,PPARα, and CPT-1 Gene Expression in the Rainbow Trout

It is known that fatty acids (FA) regulate lipid metabolism by modulating the expression of numerous genes. In order to gain a better understanding of the effect of individual FA on lipid metabolism related genes in rainbow trout (Oncorhynchus mykiss), an in vitro time‐course study was implemented where twelve individual FA (butyric 4:0; caprylic 8:0; palmitic (PAM) 16:0; stearic (STA) 18:0; palmitoleic16:1n‐7; oleic 18:1n‐9; 11‐cis‐eicosenoic 20:1n‐9; linoleic (LNA) 18:2n‐6; α‐linolenic (ALA) 18:3n‐3; eicosapentenoic (EPA) 20:5n‐3; docosahexaenoic (DHA) 22:6n‐3; arachidonic (ARA) 20:4n‐6) were incubated in rainbow trout liver slices. The effect of FA administration over time was evaluated on the expression of leptin, PPARα and CPT‐1 (lipid oxidative related genes). Leptin mRNA expression was down regulated by saturated fatty acids (SFA) and LNA, and was up regulated by monounsaturated fatty acids (MUFA) and long chain PUFA, whilst STA and ALA had no effect. PPARα and CPT‐1mRNA expression were up regulated by SFA, MUFA, ALA, ARA and DHA; and down regulated by LNA and EPA. These results suggest that there are individual and specific FA induced modifications of leptin, PPARα and CPT‐1 gene expression in rainbow trout, and it is envisaged that such results may provide highly valuable information for future practical applications in fish nutrition.