Estimation of sperm concentration of wild and reconditioned brown trout, Salmo trutta L.

Sperm concentrations were assessed for milt samples taken from three stocks of brown trout, Salmo trutta L., (wild resident trout, and reconditioned and seareared sea trout) using direct counts, spermatocrit and an optical density of 1:1000 dilutions. There were significant linear relationships between spermatocrit and absorbance, and both spermatocrit and absorbance with sperm concentration. The results gave sperm concentrations from 2.2 × 10⁹ mL^?1 to 26 × 10⁹ mL^?1. Mean concentrations differed significantly between stocks; the reconditioned males had a mean concentration of 9.1 × 10⁹ mL^?1, sea-reared males 18.0 × 10⁹ mL^?1 and 8.0 × 10⁹ mL^?1 in 1994 and 1995, respectively, and wild resident males 13.3 × 10⁹ mL^?1. Sperm concentrations from all three stocks of trout were negatively correlated with length, and therefore, age. It was indicated that sufficient milt was being produced to achieve good rates of fertilization, although some of the larger trout did have low sperm counts, suggesting that fertilizing ability may decrease with male size or age within any one stock.     

Effect of GnRHa injection on milt volume in recently stripped rainbow trout Oncorhynchus mykiss

In this study, the effect of gonadotropin‐releasing hormone agonist (GnRHa) injection on milt production in spent rainbow trout was investigated. On day 0, 25 newly matured male rainbow trout (Oncorhynchus mykiss) were stripped manually, and sperm quantity (vol: mL fish^?1) and quality, spermatocrit (%), sperm count (cell mL^?1), motile sperm percentage and motility duration (s) were evaluated. After stripping, fish were randomly divided into five groups: intact; sham (injected with propylene glycol as a hormone vehicle); and groups receiving 4, 8 or 16 μg kg^?1 BW of [d ‐Ala⁶ Des‐Gly¹⁰] mGnRHa. On day 7, the fish were stripped again and the same sperm characteristics as on day 0 were measured. At the beginning of the experiment, there were no significant differences in any of the sperm quantity characteristics between groups. On day 7, expressible milt volume was significantly reduced compared with day 0 (P<0.05, t‐test) in the intact and sham groups but milt quality remained the same (P>0.05, t‐test). The present study shows that GnRHa injection with a concentration as low as 4 μg kg^?1 BW after first stripping could prevent a significant reduction in milt quantity collected 7 days later without any adverse effects on sperm quality.     

Production and storage of sperm from the black sea bass Centropristis striata L

Black sea bass Centropristis striata L. are protogynous hermaphrodites that develop and spawn as females before changing sex to male. Since all fish eventually become males, determining the relationship between sperm production, sperm quality and seasonal changes in plasma levels of testosterone (T) and 11‐ketotestosterone (11‐KT) could be useful for identifying appropriate males to maintain as broodstock. Milt and blood samples were collected three times during an 8‐week spawning season. Milt volume (3.5±0.76 mL kg^?1), sperm density (3.2 × 10⁸± 0.31 cells mL^?1), sperm production [11 × 10⁸±3.4 cells kg^?1 body weight (BW)] and sperm motility (80±0.6%) were at their highest during the first sampling interval and coincided with the highest 11‐KT levels (1.0± 0.11 ng mL^?1). All of the sperm indices decreased to their lowest levels during the final 3 weeks of the study. Sperm viability was highly correlated (adjusted R²=0.84) with sperm motility. Sperm cryopreserved in modified Mounib's extender (MME) had the highest post‐thaw motility compared with two other extenders. Post‐thaw motility of sperm cryopreserved in MME was not different from fresh after 90 days of storage. There was no difference in fertilization rates between fresh (69±2.4%) and post‐thaw (67±4.1%) sperm samples taken from the same male or among males. These results demonstrate that the quality of black sea bass spermatozoa is higher earlier in the spawning season and that acceptable post‐thaw fertilization rates can be obtained from cryopreserved sperm.     

Some properties of bream Abramis brama L. sperm and its cryopreservation

Concentration and motility of spermatozoa, total protein content and its electrophoretic profile, glucose content, activity of aspartate aminotransferase (AspAT) and acid phosphatase (AcP) were assessed in 18 samples of semen from common bream Abramis brama L. males, which were hormonally stimulated to spermiation. Also, milt pooled from four donors was cryopreserved as pellets in vapours of liquid nitrogen (?80 °C) using four extenders (each with or without the addition of hen egg yolk). Mean spermatozoa concentration was 11.68 × 10⁹ mL^?1, and mean spermatozoa motility was about 60%. Protein content in seminal plasma was 2.08 mg mL^?1; both PAGE and SDS–PAGE showed considerable heterogeneity of protein fractions. Mean glucose content was over 11 mg%. AspAT and AcP activities were detected in both seminal plasma and spermatozoa extracts. As calculated to 1 × 10⁹ spermatozoa, AcP and AspAT activities were almost sixfold and 46-fold higher in spermatozoa than in seminal plasma respectively. In the best variant, cryopreservation attempts resulted in 66.6% of eyed embryos (compared with control fertilization) obtained after fertilization of eggs with cryopreserved semen.     

Milt quality and spermatozoa morphology of captive Brycon siebenthalae (Eigenmann) broodstock

In order to develop artificial reproduction in freshwater fish for potential species to be developed in South American aquaculture, milt quality and sperm morphology were studied in yamú (Brycon siebenthalae) under captive conditions during the natural middle spermiation period. The volume of milt collected for each male was 1.8±1.2 mL and the sperm concentration was 13.9±4.0 × 10⁹ spermatozoa mL^?1. Spermatocrit (41.5±10.8%) was positively associated (r²=0.30) with sperm density calculated using a corpuscle counting chamber. Sperm motility was 88±9% and the average duration of forward motility was 41±7 s. Fertilization rate was 84±8% and there was no association between this trait and sperm motility (r²=0.009) or with sperm density (r²=0.073). These results suggest that captive B. siebenthalae broodstock can be reproduced successfully.     

Induced spermiation in 3-year-old sterlet, Acipenser ruthenus L.

Spermiation in 3‐year‐old sterlet, Acipenser ruthenus (L.), males maintained under warm water conditions was induced by intramuscular injection of either (i) (D‐Ala⁶)‐GnRH‐ProNHEt (Kobarelin); (ii) mammalian GnRH analogue+metoclopramide (Ovopel); or (iii) human chorionic gonadotropin (Biogonadyl). The volume of milt, sperm concentration and motility were measured. A higher percentage of spermiating males was obtained after Kobarelin or Ovopel treatment (81.8% and 77.7% respectively) in comparison with fish treated with Biogonadyl (40.0%). However, only stimulation with Ovopel guaranteed motile spermatozoa in all spermiating males. Moreover, treatment with Ovopel resulted in the highest average milt volume, sperm concentration and motility. The average total number of motile spermatozoa (milt volume×sperm concentration×sperm motility) per individual was 0.99×10⁹, 5.31×10⁹ and 0.02×10⁹ after stimulation with Kobarelin, Ovopel or Biogonadyl respectively. Our data indicate that Ovopel is a good stimulator of spermation in sturgeons. Moreover, the time of sexual maturation could be significantly reduced in sturgeons maintained in cages under warm water conditions.     

Seasonal changes in sperm production and quality in the red porgy Pagrus pagrus (L.)

Cultured red porgy Pagrus pagrus (L.) males (n=6) were sampled every 2 weeks for milt, in order to monitor changes in sperm quality parameters during a whole spawning period. On 11 January 2001, 60% of the fish were spermiating, increasing to 100% in mid‐February and dropping to 30% by mid‐April. Sperm density showed a slight increasing trend, with mean values ranging between 8.6 and 23.7×10⁹ spermatozoa mL^?1. Sperm motility percentage exhibited a significant improvement during the spawning season (analysis of variance (anova ) P=0.0001). The duration of forward motility for the major part of the monitoring period ranged between 2 and 4 min. Red porgy spermatozoa maintained their viability for many days after whole storage of milt at 4°C. During the monitoring period there were significant changes in the mean duration of sperm survival after cold storage, ranging from 5 to 12 days. The total volume of expressible milt was maximal on 28 March, increasing from a mean value of 1.7 mL to 5.3 mL kg^?1. Milt production of captive‐reared red porgy does not appear to be limiting, when compared with the volume of expressible milt produced by other cultured marine fishes.     

Sperm quality of Brazilian flounder Paralichthys orbignyanus throughout the reproductive season

The aim of this study was to evaluate the sperm quality of Brazilian flounder Paralichthys orbignyanus throughout its reproductive season. Sperm was collected at the beginning, middle and end of the breeding period. Spermatozoa density was maximum at the beginning (12.7 ± 0.92 × 10⁹ cells mL^?1) and at the end (11.8 ± 0.39 × 10⁹ cells mL^?1) of the breeding season (P<0.05). Sperm production and the percentage of spermatozoa moving fast forward increased significantly towards the end of the breeding season (P<0.05). The mean duration of progressive motility of spermatozoa was around 10 min. No difference was observed during the reproductive season in the percentage of motile cells, pH, osmolality and K⁺, Cl^? and Mg²⁺ concentrations in seminal plasma. The concentration of Na⁺ increased throughout the breeding season, reaching 174.62 ± 12.68 mmol L^?1 at the end (P<0.05). There was a decline in the concentration of Ca²⁺ (12.31 ± 3.08 mmol L^?1) in the middle of the breeding season, which coincided with the shortest motility duration of spermatozoa. The information reported in this study should help to improve management and optimize the development of protocols for short‐term storage and cryopreservation of Brazilian flounder semen.     

Diversity of siderophore‐producing bacteria isolated from the intestinal tracts of fish along the Japanese coast

This study examined the diversity of siderophore‐producing bacteria in the intestinal tracts of coastal fish in Japan and screened candidate bacterial strains for probiotic use in aquaculture. Of the 2637 bacteria isolated from the 27 fish specimens (13 species) and six environmental samples collected in this study, 266 isolates exhibited the ability to produce siderophores. Siderophore producers were detected in the intestines of 18 of the fish specimens caught (68%) at densities of 2.3 × 10⁴–2.3 × 10⁸ CFU g^?1, in all three seawater samples at 2.0 × 10²–1.3 × 10³ CFU mL^?1 and in all three sand samples at 2.6 × 10¹–2.8 × 10⁴ CFU g^?1. These findings suggest that siderophore‐producing bacteria are widely distributed in the intestinal tracts of coastal fish and their environments. Analysis of 16S rRNA gene sequences revealed that the siderophore producers belonged to 38 species, of which Vibrio splendidus, Vibrio ichthyoenteri and Vibrio crassostreae accounted for 32.7%, 19.5% and 11.3% of the 266 isolates, respectively, suggesting that these bacteria are indigenous to the intestinal tract of coastal fish. Six bacterial species, Enterovibri norvegicus, Photobacterium leiognathi, Photobacterium phosphoreum, Photobacterium rosenbergii, V. crassostrea and Vibrio scophthalmi were identified as possible candidates for use as probionts in fish aquaculture.     

Incubation rate and larvae size of Persian sturgeon (Acipenser persicus Borodin 1897) in relation to sperm restripping

In this study, 11 wild male brood fish were used and their mean ages were 15.08 ± 1.66. The luteinizing hormone‐releasing hormone analogue (LH‐RH‐A₂) was used to stimulate twice in few days interval (3–7 days) and then semen samples were stripped. The results showed that there was significant difference among the percentage of motile spermatozoa, sperm density and spermatocrit during two strippings (P < 0.05). The duration of sperm motility in first and second stripping was 315/83 ± 162.16 and 212.5 ± 110.53 s respectively. Also, sperm pH was 8.41 ± 0.53 and 8.05 ± 0.33 in first and second stripping respectively. There was significant differences between hatching rate, as well as larvae size significantly changed in both hatching and initiation of active feeding (P < 0.05).     

Growth response and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection

This study determined the growth performance and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection. Tilapia were challenged with three doses of S. iniae (8.8 × 10³, 8.8 × 10⁴ and 8.8 × 10⁵ CFU fish^?1 for low, medium and high challenges respectively). Groups of non‐injected and tryptic soy broth‐injected fish were maintained as controls. Significantly (P<0.05) higher mortality (45.0%) occurred in the high challenge treatment than in the low challenge treatment group (29.6%). The medium challenge group had mortality (36.3%) that did not differ significantly from the high or low treatment. Few fish died in the non‐injected and broth‐injected treatments (3.4% and 0.8% respectively). The tilapia that survived S. iniae infection used to assess growth performance were selected from survivors without gross clinical signs of disease. These fish were randomly stocked at a rate of 30 fish into each 57 L aquarium in triplicate and fed to apparent satiation for 8 weeks. No significant differences were detected in weight gain, feed intake, feed efficiency ratio or survival between S. iniae‐survived tilapia and the control treatments following the 8‐week growth performance trial. Following the 8‐week feeding study, tilapia were challenged with 1 × 10⁶ CFU fish^?1 of S. iniae to assess acquired immunity. Mean cumulative mortality was significantly higher (P<0.05) in the control treatments (41.7% for the non‐injected and 43.3% for the broth‐injected fish) than in the low, medium and high challenge treatments (7.4%, 3.3% and 8.3% respectively). Serum protein was significantly (P<0.05) elevated in the S. iniae‐survived tilapia that were subsequently challenged when compared with controls challenged for the first time. Agglutinating antibody titre was significantly higher in the fish in the medium and high challenge treatments, compared with the control fish challenged for the first time. The results suggest tilapia that survive S. iniae challenge without showing overt disease signs performed as well as non‐infected tilapia. Further, the S. iniae‐survived tilapia challenged following the 8‐week growth performance trial gained acquired resistance to homologous S. iniae challenge.     

Hormone-induced ovulation, natural spawning and larviculture of Brazilian flounder Paralichthys orbignyanus (Valenciennes, 1839)

Mature Brazilian flounders Paralichthys orbignyanus were captured in coastal southern Brazil and their reproduction in captivity was studied. Brazilian flounder will spawn naturally in captivity when the water temperature is around 23 °C and 14 h of light is provided daily. Females were induced for ovulation and hand stripping using human chorionic gonadotropin, luteinizing hormone‐releasing hormone analogue or carp pituitary extract. There was no need to inject males, as running milt was observed during the spawning season. Fertilization and hatching rates were above 80% independent of the hormone used. Notochord length at hatching was 2.18±0.07 mm for larvae hatching from naturally spawned eggs. Larvae were reared in salt water (30–35 g L^?1) at 24 °C and under continuous illumination. Larviculture was with green water (Tetraselmis tetrathele 50 × 10⁴ cells mL^?1). Rotifers (10–20 ind mL^?1) were offered as first food 3 days after hatching and gradually replaced by Artemia nauplii (0.5–10 ind mL^?1). Larvae settled to the bottom 20 days after hatching and completed metamorphosis within a week after that. The total length for newly metamorphosed juveniles was 12.9±2.2 mm and the mean survival was 44.8%. The results demonstrate the feasibility of producing Brazilian flounder fingerlings for stock enhancement or grow‐out purposes.     

Retracted: Study of testis histology and sperm morphology in the Bunnei,Barbus sharpeyi (Gunther, 1874) induced by luteinizing hormone‐releasing hormone analogue and carp pituitary extract

This study was conducted to determine the effects of hormone treatment on testis structure in Barbus sharpeyi, as well as the morphology of sperm examined by scanning electronic microscopy (SEM). Male B. sharpeyi were divided into three groups (three fish per group) and injected with luteinizing hormone – releasing hormone analogue (LHRH–A₂) or carp pituitary extract (CPE). The first and second groups were treated with 10 μg kg^?1 LHRH–A₂ and metoclopramide (MET), and their testis were sampled pre‐ and Poststripping respectively. The third group received 2 mg kg^?1 CPE and were killed pre‐stripping. Based on the histological results obtained, the testicular connective tissue of the lumen was thicker, and seminal vesicles were of a lower volume, in fish injected with CPE in comparison to the other groups. After treatment with LHRH–A₂ and MET, not all spermatozoa within the testis were ejaculated, and only a small amount of sperm was obtained by abdominal stripping. The highest and lowest diameters of connective tissue within lobules were observed in fish receiving CPE and LHRH–A₂ treatments respectively. There was a significant difference (P < 0.05) in lobule space between the fish injected with the CPE and the fish injected with the LHRH–A₂ and MET. The SEM results revealed that the spermatozoa of B. sharpeyi were composed of a spherical to elliptical head, a cylindrical midpiece, and a lengthy flagellum. In conclusion, it was found that injection with LHRH–A₂ and MET improved the spermatogenic process in comparison to injection with CPE.     

Therapeutic and prophylactic immunization against Streptococcus iniae infection in hybrid striped bass (Morone chrysops×Morone saxatilis)

Vaccination strategies have traditionally been used as preventative or prophylactic measures against disease (prophylactic immunization) in uninfected fish. Alternatively, therapeutic or remedial measures, such as antibiotic administration, are commonly employed to treat disease in infected fish. Vaccination as a therapeutic measure (therapeutic immunization), however, has not been adequately explored in sub‐clinically infected fish. Therapeutic and prophylactic immunization with three Streptococcus iniae vaccines, formalin‐killed whole S. iniae cells (FKC vaccine), concentrated S. iniae extracellular products (greater than 2 kDa) (ECP vaccine) and a combination of killed cells and extracellular products (FKC+ECP vaccine), were tested in hybrid striped bass, Morone chrysops×Morone saxatilis, previously naturally infected with S. iniae. Fish (mean weight 10.0 g) were injected intraperitoneally (IP) or intramuscularly (IM) with one of each of the vaccines, tryptic soy broth (TSB‐control) or non‐injected (non‐injected control) to evaluate therapeutic effects (Trial 1). Survivors of the natural infection and ECP and FKC+ECP vaccine immunization and another lot of non‐injected control fish were immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 at 44 days post‐immunization to evaluate vaccine efficacy (Trial 2). Hybrid striped bass (1.0 g) were also IM injected with S. iniae ECP vaccine at an aquaculture facility and immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 12 weeks post‐immunization (Trial 3). The ECP and FKC+ECP vaccines, regardless of injection route, significantly (P<0.001) increased survival in asymptomatic, sub‐clinically infected fish thereby providing therapeutic merit. Hybrid bass immunized IP or IM had mean per cent survival values ranging from 78 to 96 at 44 days post‐immunization (Trial 1) and 69–97 post challenge (Trial 2). Survival of fish injected with TSB or immunized with FKC vaccine was significantly lowered and ranged from 12 to 13 by IP injection and 40 to 50 by IM injection and thus, the FKC vaccine had no therapeutic effect. The survival of hybrid striped bass IM immunized with S. iniae ECP vaccine in field Trial 3 was 91 and the RPS was 83. These results demonstrate that therapeutic immunization using S. iniae ECP and FKC+ECP vaccines can control a natural S. iniae infection. Furthermore, S. iniae ECP or FKC+ECP vaccines can also be used prophylacticly to protect hybrid striped bass against subsequent pathogen challenge.     

Spermatozoon ultrastructure and sperm cryopreservation of the Brazilian dry season spawner fish pirapitinga,Brycon nattereri

The aims of this study were to describe the fresh spermatozoon ultrastructure using scanning and transmission electron microscopy and to improve the sperm cryopreservation methodology for the freshwater fish pirapitinga Brycon nattereri. Extenders (BTS^? and NaCl), straw volumes (0.5 and 4.0 mL), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) were tested. Methylglycol was used as a cryoprotectant agent and sperm was frozen in nitrogen vapour (dry‐shipper). Post‐thawed sperm motility rate, motility quality (score 0=no movement; 5=rapidly swimming spermatozoa), duration of motility and spermatozoon morphology were evaluated. Fresh spermatozoon was 35.06 μm long, the head was ovoid (2.00 × 1.22 μm) with no acrosome, the midpiece was 2.15 μm long and the flagellum was 30.90 μm long with the typical 9+2 axoneme arrangement. Post‐thawed sperm motility rate (70–79% motile sperm), motility quality (score 3.1–3.7) and morphology (9.3–11.6% abnormal spermatozoa) were not affected by any of the parameters tested. The duration of sperm motility was longer when triggered in 1% NaHCO₃ (392–1031 s) compared with 0.29% NaCl (144–338 s). Brycon nattereri sperm cryopreserved under the conditions described above yields over 70% motility and should last long enough to fertilize oocytes, even after 2 years of freezing.     

Spermiation time affect the milt quality indices of the Russian sturgeon,Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833

In this study, the effects of spermiation time are investigated on milt quality of Russian sturgeon over the course of the spawning season. The milt samples were collected from three broodstock batches at three time points including: the beginning, middle and at the end of the spawning season. According to the results, the milt quality parameters including pH, sperm density, spermatocrit, duration of sperm motility and percentage of sperm motility were significantly low in the beginning and end of season than middle of season. The values of milt quality parameters in the middle of season were as follows: (motility percentage: 69.6 ± 3.5, motility duration: 460.3 ± 37.2 s, sperm density: 8.7 ± 0.4 × 10⁹, milt volume: 86.3 ± 8.1 and milt pH: 8.3 ± 0.15). Significant positive correlations were also found between milt pH and sperm motility as well as between sperm density and spermatocrit. In conclusion, our study showed that the middle of season is the best time for collection of milt with appropriate quality in Russian sturgeon. Selection of milt with good quality is necessary aim to cryopreservation of spermatozoa in endangered fish species including Russian sturgeon.     

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

Haemorrhagic septicaemia in the hybrid surubim (Pseudoplatystoma corruscans×Pseudoplatystoma fasciatum) caused by Aeromonas hydrophila

Intensive culture of the hybrid surubim (Pseudoplatystoma corruscans × Pseudoplatystoma fasciatum) in Brazil is responsible for the occurrence of diseases and consequent economic losses. However, the causative agents are not well known. The objective of this study was to isolate and to characterize the pathogenic agent responsible for mortalities in cultured surubim and to demonstrate its virulence. Ten fish from a fish farm located in the Mato Grosso do Sul State (Brazil) were collected and 14 haemolytic bacteria characterized as Aeromonas hydrophila were isolated from the kidneys (eight) and brain (six). As an experimental challenge, fish weighing 98.1±23.6 g were injected with 1 mL of saline solution and 2 × 10², 2 × 10⁴, 2 × 10⁶ and 2 × 10⁸ CFU A. hydrophila mL^?1. Fish infected with 2 × 10⁸ CFU showed increased external and internal symptoms and mortality of 50±12.5% after 96 h. Increased A. hydrophila concentration was responsible for a decrease in haematocrit percentage and erythrocyte number, lymphocytes and eosinophils, as well as an increase in monocytes, neutrophils, serum agglutination titre and serum antimicrobial activity. It was concluded that A. hydrophila was responsible for characteristic symptoms of bacterial haemorrhagic septicaemia as well as important haematological and immunological alterations, which led to surubim mortality.     

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10⁵ plaque‐forming units (PFU) mL^?1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory‐reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10³ PFU mL^?1) than among groups that survived exposure to VHSV (1.0–2.9 × 10² PFU mL^?1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39–47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.