Effect of Dietary Linoleic Acid (18:2n-6) Supplementation on the Growth Performance, Fatty Acid Profile, and Lipid Metabolism Enzyme Activities of Coho Salmon (Oncorhynchus kisutch) Alevins.

A 12-week feeding trial aimed to evaluate the effects of dietary linoleic acid (LA, 18:2n-6) on the growth performance, fatty acid profile, and lipid metabolism enzyme activities of coho salmon (Oncorhynchus kisutch) alevins. Six experimental diets (47% crude protein and 15% crude lipid) were formulated to contain graded LA levels of 0.11%, 0.74%, 1.37%, 2.00%, 2.63%, and 3.26%. Each diet was fed to triplicate groups of 50 alevins with an initial body weight of 0.364 ± 0.002 g, which were randomly assigned to 18 white plastic tanks (0.8 × 0.6 × 0.6 m, 240 L/tank). Fish were reared in a freshwater flow-through rearing system and fed to apparent satiation four times daily. The survival rate was not significantly different among the treatments (p > 0.05). However, the 1.37% LA group significantly improved the final body weight and specific growth rate (SGR) (p < 0.05) of alevins. The feed conversion ratio (FCR) in the 1.37% LA group was significantly lower than those in other groups (p < 0.05). The whole-body lipid content significantly decreased (p < 0.05) with dietary LA levels increasing from 0.74% to 2.00%. The fatty acid composition of the total lipid in muscle was closely correlated with those in the diets. The dietary LA level of 1.37% led to significantly higher activities of liver lipoprotein lipase (LPL) and hepatic lipase (HL) than those of other groups (p < 0.05). Hepatic malate dehydrogenase (MDH) and fatty acid synthase (FAS) decreased with the increase in the dietary LA levels from 0.11% to 1.37%. The lowest MDH and FAS activities were obtained in the 1.37% LA group (p < 0.05). This study indicated that an appropriate amount of dietary LA was beneficial for the growth and lipid metabolism of coho salmon alevins, and the results of the quadratic regression analysis of the SGR and FCR indicated that the optimal dietary LA requirements were 1.25% and 1.23% for coho salmon alevins, respectively.

A rapid classification tool for deformities in Atlantic salmon (Salmo salar) embryos.

Deformities in fish embryos are important for their survival in later life stages. However, a consistent way to refer to and classify salmonid embryo deformities does not exist. Expanding on reports of alevin deformities, we developed a classification tool for distinguishing the deformities observed in a collection of preserved Atlantic salmon (Salmo salar) embryos. Deformities were classified based on the deformed body part and a qualitative subtype. This classification tool uses external morphology, requires minimal equipment and can be applied from the first appearance of optic vesicles to hatch.

A novel gnotobiotic experimental system for Atlantic salmon (Salmo salar L.) reveals a microbial influence on mucosal barrier function and adipose tissue accumulation during the yolk sac stage.

Gnotobiotic models have had a crucial role in studying the effect that commensal microbiota has on the health of their animal hosts. Despite their physiological and ecological diversity, teleost fishes are still underrepresented in gnotobiotic research. Moreover, a better understanding of host-microbe interactions in farmed fish has the potential to contribute to sustainable global food supply. We have developed a novel gnotobiotic experimental system that includes the derivation of fertilized eggs of farmed and wild Atlantic salmon, and gnotobiotic husbandry of fry during the yolk sac stage. We used a microscopy-based approach to estimate the barrier function of the skin mucus layer and used this measurement to select the derivation procedure that minimized adverse effects on the skin mucosa. We also used this method to demonstrate that the mucus barrier was reduced in germ-free fry when compared to fry colonized with two different bacterial communities. This alteration in the mucus barrier was preceded by an increase in the number of cells containing neutral mucosubstances in the anterior segment of the body, but without changes in the number of cells containing acidic substances in any of the other segments studied along the body axis. In addition, we showed how the microbial status of the fry temporarily affected body size and the utilization of internal yolk stores during the yolk sac stage. Finally, we showed that the presence of bacterial communities associated with the fry, as well as their composition, affected the size of adipose tissue. Fry colonized with water from a lake had a larger visceral adipose tissue depot than both conventionally raised and germ-free fry. Together, our results show that this novel gnotobiotic experimental system is a useful tool for the study of host-microbe interactions in this species of aquacultural importance.

Virulence variations of Flavobacterium columnare in rainbow trout (Oncorhynchus mykiss) eyed eggs and alevin.

Flavobacterium columnare (Fc) is the causative agent for columnaris disease (CD) in several fish species and an emerging problem for rainbow trout aquaculture. We characterize the virulence phenotype of two Fc isolates, CSF-298-10 and MS-FC-4, against trout from two sources, NCCCWA and a production stock (PS), at the eyed egg and alevin life stages. Immersion challenges demonstrated that NCCCWA eyed eggs were susceptible to the Fc isolate MS-FC-4 (>97% mortality) but no mortality was observed against PS eyed eggs. The CSF-298-10 had little effect on any eyed eggs tested and was not highly virulent to any alevin till day six post-hatch, up to 38% for NCCCWA and ~80% PS alevin. The MS-FC-4 strain produced ≥80% mortality any day an immersion challenge occurred post-hatch. Significant difference in CFU counts was recorded between the Fc strains on 2 days post-hatch immersion challenges. Counts for the NCCCWA alevin were 4.4 × 103  CFU/ml-1 and 1.8 × 106  CFU/ml-1 for the CSF-298-10 strain and MS-FC-4 strain, respectively, and for the PS alevin CSF-298-10 measured 9.9 × 101  CFU/ml-1 and 3.8 × 105  CFU/ml-1 for MS-FC-4. These two Fc isolates present stark differences in virulence phenotypes to both eyed eggs and alevin and present an interesting model system for virulence kinetics and potentially alternative pathogenic pathways.

Organization of glomerular territories in the olfactory bulb of post-embryonic wild chinook salmon Oncorhynchus tshawytscha.

The post-embryonic odor imprinting paradigm suggests Chinook salmon (Oncorhynchus tshawytscha) acquire memory to stream-specific amino acid olfactory odors prior to emergence as fry. Because effects of olfactory experience on development can be examined by mapping olfactory sensory neurons extending into distinct territories of glomerular neuropil in the olfactory bulb, glomerular patterning from early yolk-sac larva to fry was documented in wild salmonids, a temporal scale not yet thoroughly explored. Labeling olfactory sensory neurons with anti-keyhole limpet hemocyanin (anti-KLH) revealed seven spatially conserved glomerular territories visible at hatch and well established by the late yolk-sac larva developmental stage. Because of the responsiveness of microvillous olfactory sensory neurons to amino acids, corresponding glomeruli in the lateral bulbar region were mapped using anti-calretinin. The dorsolateral territory, distinct glomeruli of the lateral glomerular territory and the ventromedial glomeruli were immunoreactive to both KLH and calretinin. This study offers a morphological description of glomerular patterning in post-embryonic stages in wild Chinook salmon, a temporal window previously shown to be significant for olfactory imprinting. J. Morphol. 278:464-474, 2017. © 2017 Wiley Periodicals, Inc.

Immunolocalization of morphogen sonic Hedgehog in salmon fry (Salmo salar) / Inmunolocalización del morfógeno sonic Hedhehog en alevines de salmón (Salmo salar)

With the purpose of carrying out a diagnosis of the different pathologies that affect the salmon fry stage (Salmo salar) and analyze the regeneration phases of the organizational centers and subjacent tissue in case of an amputation, we realized a study that allowed identifying the temporary and spatial location of the Sonic Hedgehog (Shh) morphogen in hatched fry stage. Fifteen salmon fry (Salmo salar) were used. They were anesthetized with 5 % benzocaine (BZ-20®, Veterquímica), fixed in 10 % buffered formalin, and embedded in paraffin, Shh polyclonal antibody (Santa Cruz H-160, rabbit) was used diluted at 1/100. They were subsequently rinsed in PBS-1 % Triton and incubated with anti-rabbit conjugated polymer antibody and HRP for 10-15 min. The development was done with DAB (Vector) for 1-5 min. The negative control was incubated without primary antibody. As an internal positive control the notochord was considered. Serial sagittal sections were analyzed consigning tissues and organs marked positively and were described morphologically. The objective of recognizing the spatial and temporal location of Shh was achieved. The notochord, spinal cord neurons and ganglia, the basal layer of the skin and also the lepidotriquias escleroblastos were positively identified for Shh. Finally positivity was also observed in the intestine and renal tubules. The heterogeneity observed in the location of the Shh morphogen suggests its potential use as a marker of regulatory centers in Salmo salar, and a potential advantage in the diagnosis of malformations of salmon fry stage, in addition to a better understanding of tissue regeneration.

Con el fin de llevar a cabo un diagnóstico de las diferentes patologías que afectan a la etapa de alevín de salmón (Salmo salar) y analizar las fases de regeneración de los centros de organización y el tejido subyacente en caso de una amputación, se realizó un estudio que permitió identificar la ubicación temporal y espacial del morfógeno Sonic hedgehog (Shh) en la etapa de alevines eclosionados. Se utilizaron quince alevines de salmón (Salmo salar). Fueron anestesiados con benzocaína al 5% (BZ-20®, Veterquímica), se fijaron en formalina tamponada al 10%, e incluidos en paraplast. Se utilizó Shh anticuerpo policlonal (Santa Cruz H-160, conejo) dilución 1/100. Se enjuagaron posteriormente en PBS-1% Triton y se incubaron con anticuerpo conjugado con polímero anti-conejo y HRP durante 10-15 minutos. Se utilizó como sustrato DAB (Vector) durante 1-5 minutos. El control negativo se incubó sin anticuerpo primario. Como un control positivo interno se consideró la notocorda. Se analizaron secciones sagitales en serie consignando los tejidos y órganos marcados positivamente y se describieron morfológicamente. Se logró el objetivo de reconocer la localización espacial y temporal de Shh. La notocorda, las neuronas de la médula espinal y los ganglios, la capa basal de la piel y también los escleroblastos de las lepidotriquias fueron identificados positivamente para Shh. También se observó positivo el intestino y los túbulos renales. La heterogeneidad observada en la ubicación del morfógeno Shh sugiere su uso potencial como un marcador de los centros de regulación en Salmo salar, y una ventaja potencial en el diagnóstico de las malformaciones de la etapa de alevines de salmón, además de una mejor comprensión de la regeneración de tejidos.

Estudio morfológico de la retina de salmones (Salmo salar) / Morphological study of the retina of salmon (Salmo salar)

La retina de peces teleósteos como pez cebra, se ha transformado en un importante modelo para el estudio de la plasticidad neuronal y la neurogénesis. Se ha demostrado además que la retina experimenta cambios ontogenéticos para adaptarse a distintos medios ambientes durante su vida. Este estudio tiene como objetivo describir el desarrollo ontogenético de la retina del alevín de salmón desde la eclosión hasta la fase de juvenil. Se trabajó con 30 salmones divididos en tres grupos de 10. Grupo I: recién eclosionados, con saco vitelino y 18 mm de longitud. Grupo II: sin saco vitelino y 30 mm de longitud. Grupo III: 100 mm de longitud. Cinco alevinesde cada grupo fueron procesados según el protocolo de Hanken & Wassersug para medir los diámetros dorsoventral y nasal-temporal utilizando el cartílago que protege al globo ocular. Los restantes cinco ejemplares fueron seccionados con micrótomo Microm en forma seriada (5 µm) y procesados con técnica H&E/Azul de Alcián. Se midieron las capas de la retina en un microscopio óptico Zeiss, con cámara Powershot incorporada y con un software Image Tool 3.0. El Grupo 1 presentó grandes ojos pigmentados, con aspecto de copa óptica embrionaria, la retina está estratificada en capas. La Capa Nuclear Interna (CNI) mide 62±10 µm y la capa plexiforme interna (CPI) 10±2 µm. El Grupo 2 presenta cambios en el espesor de ellas. La CNI disminuye su espesor a 45±8 µm y la Plexiforme aumenta a 25±5 µm. En los peces juveniles del Grupo 3, la CNI alcanza el espesor mínimo (15±3 µm), por el contrario, la capa Plexiforme interna aumenta su espesor hasta alcanzar (70±10 µm). En los tres grupos estudiados observamos en la periferia de la retina una zona proliferativa germinativa, que corresponde a un remanente del neuroepitelio embrionario, responsable del crecimiento continuado de la retina. La retina de los salmones puede ser también un importante modelo para el estudio de la ontogenia, la plasticidad neuronal y la neurogénesis. Esta neurogénesis en la retina de peces facilita la reordenación celular a lo largo de la ontogenia, lo que potencialmente permite la optimización del sistema visual a los cambios en las demandas visuales. Este estudio puede ser de utilidad para facilitar el diagnóstico en las patologías de ojo en salmonicultura y también puede contribuir a conocer mejor la regeneración de tejidos. Por otro lado, con estudios posteriores, la neurogénesis de la retina de peces podría extrapolarse al tratamiento de enfermedades humanas con daño a nivel retineal, tales como glaucoma, desprendimiento de retina y retinopatía diabética.

The retina of teleost fish zebrafish, has become an important model for studying neuronal plasticity and neurogenesis. It was further shown that the retina undergoes ontogenetic changes to adapt to different environments during their lifetime. This study aims to describe the ontogenetic development of the retina of juvenile salmon from hatching to the juvenile stage. We worked with 30 salmon divided into three groups of 10. Group I: newly hatched with yolk sac and 18 mm in length. Group II: without yolk sac and 30 mm in length. Group III: 100 mm long. Five fry each group were processed according to the protocol of Hanken & Wassersug to measure dorsoventral and nasal-temporal diameters using the cartilage that protects the eyeball. The remaining five specimens were sectioned with a microtome Microm serially (5 µm) and processed with technical H-E / Alcian blue. The layers of the retina were measured on a Zeiss optical microscope with camera Powershot built and with Image Tool 3.0 software. Group 1 showed large pigmented eyes, looking embryonic optic cup, the retina is stratified in layers. The inner nuclear layer (CNI) measured 62±10 microns and the inner plexiform layer (CPI) 10±2 µm. Group 2 presents changes in the thickness of them. The CNI decreases in thickness to 45±8 µm and the plexiform increased to 25±5 µm. In juvenile fish of group 3, the CNI reaches the minimum thickness (15±3 µm), by contrast, the inner plexiform layer thickness increases up to (70±10 µm). In the three groups observed in the periphery of the retina one proliferative germinative zone, which corresponds to a remnant of the embryonic neural epithelium responsible for the continued growth of the retina. The retina of the salmon can also be an important model for the study of ontogeny, neuronal plasticity and neurogenesis. This retinal neurogenesis fish rearrangement facilitates cell along ontogeny, potentially allowing optimization of the visual system to changes in the visual demands. This study may be useful to help diagnose pathologies in eye salmon and can also contribute to better understand tissue regeneration. On the other hand, with later studies, fish's retinal neurogenesis could be extrapolated to the treatment of human retinal diseases, such us glaucoma, retinal detachment o diabetic retinopathy.

Desarrollo de la aleta caudal del salmón (Salmo salar) / Salmon caudal fin development (Salmo salar)

Las patologías y traumas de la aleta caudal afectan la natación, dificultan la alimentación y la eficiencia de escape de los peces, además aumentan la susceptibilidad a las infecciones bacterianas y fúngicas. Los salmones adultos pueden regenerar rápida y completamente su aleta si esta es amputada. Sin embargo, se han reportado en el sur de Chile, alevines que expresan defectos anatómicos en la aleta caudal asociados a un alto índice de mortalidad donde no ocurre regeneración. Existen múltiples estudios sobre la aleta caudal de peces adultos pero esta descripción no concuerda con la morfología de la fase de alevín. Nuestro objetivo es describir la anatomía e histología de la aleta caudal del salmón de 15 mm, 30 mm y 60 mm para facilitar el diagnóstico de las patologías tempranas de la aleta caudal. Se trabajó con 60 salmones divididos en tres grupos de 20 en etapas de 15, 30 y 60 mm. Diez salmones de cada grupo fueron procesados con técnicas anatómicas de Hanken & Wassersug . Otros 10 alevines de cada grupo fueron procesados mediante técnicas H&E/azul de Alcian pH 2,5: para glicosaminoglicanos y técnica Histoquímica Picrosirius de Junqueira para colágeno I y III. Al momento de la eclosión de los peces (grupo 1) la aleta caudal no tiene su forma definitiva pero ha iniciado la formación de lepidotriquias. En el grupo 2, la aleta caudal comprende entre 19-20 lepidotriquias y se constituyen dos lóbulos uno dorsal y otro ventral, ambos bajo la notocorda. Los rayos de cada lóbulo crecen más rápido que los rayos que se encuentran entre los lóbulos y se forma un surco entre ellos. En el grupo 3 se observa claramente la aleta bilobulada, se mantienen 19 lepidotriquias que ahora están en proceso de calcificación. Cada lepidotriquia crece distalmente mediante la formación de articulaciones y segmentos. En el grupo 2 se consignó un promedio de 4­5 articulaciones por lepidotriquia y en el grupo 3 han aumentado a 6­10 articulaciones. Esta descripción de la aleta del alevín normal facilita el diagnóstico de la aleta deformada y aporta conocimientos para comparar el desarrollo ontogenético con las fases de la regeneración después de la amputación de la aleta caudal.

Caudal fin pathologies and traumas can affect swimming, impede food and exhaust efficiency, and also increase susceptibility to bacterial and fungal infections. Adult salmon can regenerate their fin quickly and completely if it is amputated. However, yolk sac fry expressing anatomical defects in the caudal fin have been reported in southern Chile and are associated to a high mortality rate where regeneration does not occur. There are many studies on adult salmon but this description does not match the morphology of the juvenile phase. We describe the anatomy and histology of the caudal fin in salmon 15mm, 30 mm and 60 mm to facilitate the early diagnosis of diseases of the caudal fin. We worked with 60 salmon divided into three groups of 20 in steps of 15, 30 and 60 mm. 10 salmon from each group were processed with Hanken & Wassersug anatomical techniques. Another 10 fry from each group were processed using H&E/Alcian blue pH 2.5 techniques: for glycosaminoglycans and technical histochemistry Picrosirius Junqueira for collagen I and III. Upon hatching of fish (group 1) the caudal fin has no definitive form but has commenced training ray or lepidotriquias. In group 2, the caudal fin comprises from 19 to 20 lepidotriquias and two lobes one dorsal and one ventral, both are constituted under the notochord. Each lobe ray grows faster than the rays that lie between the lobes and a groove is formed between them. In group 3clearly shows the bilobed flap, 19 lepidotriquias that are now in the process of calcification are maintained. Each lepidotriquia grows distally by forming joints and segments. In group 2 an average of 4­5 lepidotriquia joints were recorded and in group 3 there was an increase at 6­10 joints. This description of normal fry flap facilitates comparative study of the deformed fin.

Desarrollo de la médula espinal de salmón (Salmo salar) durante el período post-eclosional / Development of spinal cord of the salmon (Salmo salar) during the period post eclosional

En este estudio se describe el desarrollo post-eclosional de la médula espinal del salmón. Salmo salar. Se utilizaron 200 alevines recién eclosionados, los que fueron cultivados en el Centro de Estudios Acuícolas de la Universidad de Chile. Las condiciones ambientales de cultivo fueron de un 90% de saturación de oxigeno. La temperatura ambiental se mantuvo en 7°C. A los días 1, 3, 5 7 ds post-eclosión, 50 alevines por grupo etario fueron anestesiados y sacrificados por exposición a 5% Benzocaina diluida en agua (Kalmagin 20®, Farquímica). Posteriormente fueron fijados en formalina tamponada al 10% y procesados mediante técnica histológica. Para cada alevín se tomaron a nivel de la aleta dorsal un total de 40 cortes coronales seriados de 5µm de grosor, los que fueron procesados de acuerdo a las técnicas Cresil violeta. La cuantificación neuronal se realizó sobre imágenes microscópicas mediante el método del disector. Los resultados obtenidos se sometieron a una prueba de Coeficiente de Kurtosis con el propósito de analizar el grado de concentración que presentan los valores alrededor de la zona central de la distribución. La médula espinal de los alevines de 1 día es poco diferenciada. En los alevines de 3, 5 y 7 días se diferencian gradualmente las neuronas de la sustancia gris, pero no presenta la distribución característica en forma de "Y" invertida del salmón adulto. El número de neuronas aumenta desde 67+1.7 en el día 1 hasta 88+2.1 en el día 7. Esta observación se puede relacionar con la ausencia de movimientos natatorios de los peces durante los primeros días ya que estos caen sobre la gravilla al fondo de las bateas. Un factor determinante en la adquisición de la morfología de la médula espinal es el inicio de los movimientos natatorios, lo que ocurre aproximadamente al quinto día post-eclosión. La actividad motriz activa permite que las neuronas de la médula espinal sean reclutadas y se formen y activen las redes neurales, permaneciendo finalmente los circuitos más eficientes. El aumento del número de neuronas se puede explicar por neurogénesis post-eclosión, como ocurre en otros teleósteos. Este estudio indica que al momento de la eclosión, el sistema nervioso está muy indiferenciando, y que durante las primeras semanas de vida del alevín ocurre la diferenciación de las neuronas y neurogénesis. Este conocimiento es muy importante debido a que en las pisciculturas se cuidan las ovas, y se descuida la fase del alevinaje en la creencia que los tejidos están constituidos.

We describe the development of the spinal cord during the post eclosion period of the salmon (Salmo salar).We used a total of 200 newly hatched fry grown in the Aquaculture Research Center of the Universidad de Chile. Environmental conditions were of 90% oxygen saturation. Ambient temperature was maintained at 7° C. At days, 1, 3, 5 and 7, post-hatching, 50 fry were anesthetized and sacrificed by exposure to 5% benzocaine diluted in water, (Kalmagin 20 ®, Farquímica). They were then fixed in 10% buffered formalin and processed by histological technique. For each juvenile a total of 40 serial coronal sections of 5µm were taken at the level of the dorsal fin, which were then processed according to cresyl violet techniques.Neuronal quantitation was performed on microscopic images by dissector method. The results obtained were subjected to coefficient Kurtosis test in order to analyze the degree of concentration of values around the central distribution area.The spinal cord of the one-day fry is poorly differentiated. In fry of 3, 5 and 7 days neurons are gradually differentiated, they do not however present the characteristic neuronal distribution inverted "Y" of the adult salmon. The number of neurons increases from 67±1.7 on day one, to 88±2.1 on day 7.This observation may be related to the absence of fish swimming movements during days one and three as these fall on the gravel at the bottom of the trays. A determining factor in the acquisition of the morphology of the spinal cord is the start of swimming movements, which occur at around the fifth day post-hatching.Active motor activity allows spinal cord neurons to be recruited and form to activate neural networks, to remain finally in the most efficient circuits. Increasing the number of neurons can be explained by post-hatching neurogenesis as in other teleosts.This study indicates that at the time of hatching, the nervous system is very undifferentiated and that neuron differentiation and neurogenesis occur during the first weeks of life. This knowledge is very important as fish farms take care of eggs, neglecting the nursery stage in the belief that tissues are formed.