Induction of Gynogenetic and Androgenetic Haploid and Doubled Haploid Development in the Brown Trout (Salmo trutta Linnaeus 1758)

Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation‐induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 ± 2.4% and 16.14 ± 2.90% and the survival rates at the first feeding stage of 10.48 ± 3.48% and 12.78 ± 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiation‐induced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.     

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350 Gy of X‐rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non‐irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non‐irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X‐rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.     

Activation of the Albino Sterlet Acipenser ruthenus Eggs by UV‐Irradiated Bester Hybrid Spermatozoa to Provide Gynogenetic Progeny

Meiotic gynogenesis was induced in the albino form of sterlet Acipenser ruthenus by activation of eggs with UV‐irradiated bester (Huso huso x Acipenser ruthenus) spermatozoa followed by inhibition of the second meiotic division performed by a heat shock. Obtained putative gynogenetic progeny were all albinos. The genetic verification based on three microsatellite DNA markers confirmed the only maternal inheritance of the progeny from the gynogenetic experimental groups. Cytogenetic analysis proved the gynogenetic sterlets were diploids. Application of the albino phenotype together with the molecular and the cytogenetic diagnostic approaches enabled to evaluate the efficiency of the spermatozoa irradiation and application of the heat shock to restore diploid state in the gynogenetic zygotes.     

Induction of Gynogenesis and Androgenesis in Goldfish Carassius auratus (var. oranda)

Chromosome engineering was used to develop lines that expressed the stable phenotypic characteristics of goldfish, Carassius auratus (var. oranda). To obtain androgenetic individuals, carp eggs were irradiated and fertilized with sperm from goldfish bearing telescopic eyes. The fertilized eggs were divided into four groups and three of them were subjected to heat-shock treatment at 42°C for 2.0 min at 34, 37 and 40 min after fertilization. All groups exhibited low viability of embryos and after hatching the embryos were severely deformed on the head, yolk sac and tail and exhibited 100% mortality, within 2 days after hatching. To obtain gynogenetic individuals, a thin layer of carp milt was subjected to ultraviolet irradiation for 26 min to neutralize its genetic material and used to fertilize a mixture of eggs derived from three different variations of C. auratus individuals with characteristics such as round fat body, triple tail, red and black colour and telescopic eyes. The fertilized eggs were divided into six groups and five of them were subjected to heat-shock treatment at 39.5°C for 2.5 min at 15 (group 2), 20 (group 3), 30 (group 5) and 45 (group 6) min after fertilization. The percentage of fertilized eggs that survived ranged from 20 to 50% and the percentage of larval survival in groups 2–5 was 22–28% with lower levels in groups 1 and 6, ranging from 10–12%. However, 40–60% of the larvae exhibited severe deformities on the head, yolk sac and tail, whereas the rest developed normally. Fish with the typical oranda phenotype were observed in all the groups and about 1% of the fish were characterized by a triple tail but there was no fish with telescopic eyes. The results indicate that gynogenetic individual goldfish can be produced but that the induction of androgenesis requires further improvement of the techniques.     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

Early Life Stage Survival and Susceptibility of Brook Trout,Coho Salmon,Rainbow Trout,and Their Reciprocal Hybrids to Infectious Hematopoietic Necrosis Virus

Abstract

Triploid (heat-shocked) and diploid groups of rainbow trout Oncorhynchus mykiss, brook trout Salvelinus fontinalis, coho salmon Oncorhynchus kisutch, and reciprocal hybrids were produced, monitored for early life stage survival, and evaluated for susceptibility to infectious hematopoietic necrosis virus (IHNV). The female rainbow trout × male brook trout triploid hybrids had significantly greater (P < 0.01) survival than the diploid hybrids of this cross. The heat-shocked hybrid group of the female rainbow trout × male coho salmon also exhibited significantly greater survival to the eyed egg stage of development than the untreated group of this hybrid. Studies of the susceptibility of treatment groups to a 1990 IHNV isolate from the Hagerman Valley were conducted by using a standardized immersion exposure procedure at one or two different mean body weights. The diploid brook trout and coho salmon and two triploid hybrids (female rainbow trout × male brook trout or male coho salmon) were significantly less (P < 0.05) susceptible to IHNV than the pure-species diploid and triploid rainbow trout groups.     

Establishment and Characterization of Seven Continuous Cell Lines from Freshwater Fish

Abstract

Seven continuous cell lines were established from salmonid and nonsalmonid fishes. Salmonid cell lines derived from rainbow trout Oncorhynchus mykiss and chum salmon O. keta were designated RTE and RTE-2 (rainbow trout embryo), RTT (rainbow trout tail), and SEH (“sake” or chum salmon embryo head). Nonsalmonid cell lines derived from pond smelt Hypomesus olidus, chevron snakehead Channa striata, and goldfish Carassius auratus were designated WF-1 (“wakasagi” fin), SHH (snakehead heart), and EPG (epithelioma papulosum of goldfish), respectively. Optimum growth for most of the cell lines was observed in Eagle's minimum essential medium buffered with sodium bicarbonate (26 mM) or a combination of sodium bicarbonate (8.9 mM) and tris (16 mM). Likewise, most of the cell lines showed optimum growth at the lowest NaCl concentration tested (0.116 M). Optimum growth temperatures ranged from 15 to 20°C for the salmonid cell lines and from 15 to 30°C for nonsalmonid cell lines. Except for RTT, the cell lines were heteroploid. Eleven fish viruses were used to test the susceptibility of these cell lines. Cell lines derived from salmonids developed cytopathic effects (CPE) when infected with 10 of the 11 fish viruses tested, except for RTT, which produced CPE with only 8 of the fish viruses. Six fish rhabdoviruses used in this study elicited a pronounced CPE when inoculated into nonsalmonid cell lines EPG, WF-1, and SHH. Among the new cell lines, RTE-2 showed the best potential for the isolation of fish viruses.     

Biological characteristics of fish germ cells and their application to developmental biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.     

Divergent selection for shape of growth curve in Japanese quail. 6. Hatching time,hatchability and embryo mortality

1. Hatching time, hatchability of fertile eggs and embryo mortality under standard egg storage (1 or 5 days at 12?±?1°C and 55% relative humidity) and incubation conditions (37·5?±?0·2°C and 50% relative humidity) were analysed in lines long-term selected for high (HG) and low (LG) relative weight gain between 11 and 28?d of age, respectively, and constant body weight at 49?d of age.

2. Egg storage duration did not have an effect on average hatching time. LG quail, characterised by a fast postnatal growth rate immediately after hatching, hatched earlier than HG quail with a low early growth rate (about 391 vs. 406?h after egg setting, respectively).

3. In contrast to hatching time, the hatchability of fertile eggs was influenced by line as well as egg storage duration. Extended storage decreased hatching success in both lines. However, LG eggs exhibited a higher hatchability than HG eggs (1?d storage: 96·0 vs. 82·5%; 5?d storage: 88·7 vs. 72·7%, respectively).

4. Lower hatchability resulted mostly from a higher frequency of embryo death during early (up to d 7) and late (d 14 and later) phases of incubation.

5. An inadequate nutrient supply to embryos in consequence of developmental delay seems to be a key factor decreasing viability of embryos during incubation.     

Comparing Sex Steroid Levels During the Annual Cycles of Rainbow Trout (Oncorhynchus mykiss) Diploid Female (XX) and Triploid Female (XXX) Genotypic Sex

In this study, the annual cycle of the gonadal steroids testosterone (T), 11‐ketotestosterone (11‐KT), 17β‐estradiol (E2) and 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one (DHP) was determined using radioimmunoassay and then compared for two populations of rainbow trout, XX diploid females (n = 40) and XXX triploid females (n = 15). In females, E2 and DHP levels were found to be significantly related to body weight (r = 0.22513; p < 0.0001 and r = 0.15831; p > 0.001, respectively). In this group, E2 concentrations peaked in November (25.05 ng /ml), while maximum DHP levels, only measurable from October to April, were attained in February (64.14 ng /ml). No significant differences in hormone ranges related to egg output ability were observed. Finally, sex steroid concentrations were low in the triploid female XXX fish compared to the female XX population. Nevertheless, maximum T (33.85 ng /ml) and 11‐KT (32.35 ng /ml) levels were recorded in January, for XXX. The levels for these two hormones are relatively high and are also significantly associated (r = 0.8430; p < 0.0001). Diploid females showed significantly higher levels of E2 than triploids over the 12‐month study period. The female triploid fish produced the lowest steroid hormone levels, such that these would be the most suitable for human consumption.     

Dietary Ascorbyl Monophosphate Depresses Lipid Peroxidation in Rainbow Trout Spermatozoa

Abstract

This study was designed to analyze lipid peroxidation in spermatozoa of rainbow trout Oncorhynchus mykiss by an optimized thiobarbituric acid (TBA) method, and to evaluate the effect of graded levels of dietary antioxidant (ascorbic acid in the form of ascorbyl monophosphate, AP) on TBA values of spermatozoa. Sperm from rainbow trout fed diets supplemented with AP at 0, 110, or 870 mg/kg were sampled during the reproductive season. The group given the unsupplemented diet had the lowest ascorbic acid concentration in seminal plasma during most of the spermiation season compared with groups fed diets with AP While the ascorbic acid concentration in seminal plasma decreased toward the end of the reproductive season, spermatozoa malondialdehyde production, indicative of increasing peroxidation, tended to increase. At the end of the season, significant differences (P < 0.05) were found in peroxidation levels in spermatozoa from fish fed different levels of ascorbic acid. The most abundant polyunsaturated fatty acid in sperm lipids, docosahexaenoic acid (C22:6), was significantly lower in lipids from spermatozoa of the AP-devoid (control) group than in the 870-mg/kg supplement group. By the end of the reproductive season, spermatozoa from the control group had significantly higher peroxidation levels than did spermatozoa from fish given AP (110 and 870 mg/kg). Thus, it is the first evidence in fish that dietary AP supplements can increase seminal plasma ascorbic acid concentrations, and it suggests that peroxidative damage to spermatozoa can be reduced during the reproductive season, which in turn may affect sperm fertilizing ability.     

Reproductive efficiency and maternal-offspring transfer of gossypol in rainbow trout (Oncorhynchus mykiss) fed diets containing cottonseed meal.

In a preceding study, complete substitution of fish meal protein with cottonseed meal (CM) protein did not affect the survival or growth rate of adult rainbow trout over a 6-mo period. Gossypol, a naturally occurring compound in cottonseeds, has an antifertility effect in terrestrial animals, but information regarding salmonid fish is lacking. Female rainbow trout in this experiment were fed diets with either 0, 25, 50, 75, or 100% (diets 1 to 5) of the fish meal protein replaced with CM protein until first maturation and spawning to study long-term effects on growth and reproduction. Feeding diets containing CM over a total period of 10 mo did not result in differences in growth and mortality compared to the control group (P > 0.05). Increased CM incorporation levels resulted in decreased (P < 0.05) blood hemoglobin (10.6 +/- 1.3, 8.4 +/- 1.8, 7.3 +/- 1.1, 6.9 +/- 0.8, and 5.6 +/- 1.4 g/dL) and hematocrit (49.6 +/- 3.9, 38.5 +/- 9.3, 34.4 +/- 3.7, 34.8 +/- 4.9 and 28.0 +/- 6.8%) levels in diets 1 to 5, respectively. The CM incorporation level had no effect (P > 0.05) on the number of eggs produced per female but led to a reduction (P < 0.05) in egg weight. Eyed stage survival of embryos was low in all dietary groups and did not show differences (P > 0.05). However, an increasing CM incorporation level led to a linear increase (P < 0.05) in the number of females that produced no viable embryos (23.1, 37.5, 42.9, 60.0, and 71.4%). Gossypol in the diet was absorbed by the female trout and transferred to the eggs (0, 2.2 +/- 0.5, 6.7 +/- 1.6, 10.6 +/- 4.2, and 20.0 +/- 2.6 micrograms/g in diets 1 to 5, respectively). A high concentration of gossypol remained in the juveniles at the swim-up stage (endogenous yolk-absorbed) (0.6 +/- 0.3, 2.4 +/- 0.3, 3.4 +/- 0.0, and 4.7 +/- 1.0 micrograms/g, diets 2 to 5, respectively). The findings suggest that replacement of the dietary fish meal protein with CM protein has no effect on fish growth and mortality but may lead to a reduction in reproductive performance in female rainbow trout.     

Factors related to shell deaths during artificial incubation of ostrich eggs

The ostrich industry experiences a high rate of embryonic mortalities during artificial incubation of eggs. Embryonic deaths were studied from data recorded on 37,740 fertile eggs incubated artificially during the 1998-2005 breeding seasons. Roughly 10,000 eggs that sustained embryonic mortalities were classified according to the stage and nature of death, i.e. before 21 days of incubation, after 21 days of incubation, deaths after pipping and rotten eggs. Although infection may have played a role in approximately 1300 rotten eggs, no detailed knowledge of the pathogens involved was available. The remainder of deaths could not be related to pathogens and the deaths were thus generally referred to as non-infectious. The overall level of embryonic mortality in all the eggs studied was 28.5 %. Overall embryonic mortality was affected by incubator, with higher levels (57.0 %) found in eggs incubated in an African Incubator and also in eggs that were transferred between incubators during incubation (38.1%). Overall embryonic mortality also increased in eggs produced by older females. Eggs produced in the autumn had the highest level of embryonic mortality at 53.6 %, whereas eggs produced in the winter had a marginally higher level of embryonic mortalities of 29.2 % compared with eggs produced during summer (27.4 %). Eggs produced by South African (SA) Black males crossed to Zimbabwean Blue females had high levels of embryonic losses of 45.7 %. The embryonic mortality of eggs produced by SA Blacks or Zimbabwean Blue breeding birds subjected to pure breeding was similar at approximately 33-34 %, but embryonic mortality was improved in eggs produced by Zimbabwean Blue males crossed to SA Black females (27 %). Embryonic mortality was increased in eggs that were set directly (32.0 %) or subjected to longer than 6 days of storage (43.5 %). Embryonic mortality was affected by year. The results that were obtained will assist in determining non-infectious factors that have a negative effect on hatching success. Steps can thus be taken to eliminate such factors that may compromise hatching success.     

Storage of sperm in the uterovaginal junction and its incidence on the numbers of spermatozoa present in the perivitelline layer of hens' eggs

1. The numbers of spermatozoa found in the perivitelline layer (perivitelline spermatozoa) of hens' eggs during a 14-d period after insemination were found to be log-dose dependent (r = 0.99) on the quantities of spermatozoa inseminated intravaginally in these hens (50, 100, 200 or 400 million/female). 2. Highly significant correlations were also observed between the perivitelline spermatozoa and the proportion of uterovaginal sperm-storage tubules containing spermatozoa on day 14 after insemination. 3. These data confirm that the number of perivitelline spermatozoa in eggs laid on day 2 after artificial insemination (AI) are highly correlated with the mean percentages of fertility of its duration over a 14-d or 24-d period. As a consequence, eggs laid by the 10% highest or the 10% lowest females primarily classified on the basis of this variable exhibited on average 99% or 49.7% fertility, respectively, over a two-week period after AI.     

Susceptibility of Four New Salmonid Cell Lines to Infectious Hematopoietic Necrosis Virus

Abstract

Four salmonid cell lines, CoE 45, CoE 115, CoE 345, and RBTE 45, were established from embryonic tissues of coho salmon Oncorhynchus kisutch and rainbow trout O. mykiss. In vitro challenges of the new lines were conducted with four isolates of infectious hematopoietic necrosis virus (IHNV). Two of the IHNV isolates used for the challenges were derived from infected tissues of rainbow trout, one was derived from chinook salmon O. tshawytscha, and the other isolate was derived from coho salmon. To standardize the virus challenges of the new cell lines, several established piscine cell lines (EPC, CHSE 214, CSE-119, RTH-149, RTG, and RTS) were challenged in the same way as the new lines. Each of the lines was challenged with virus at a single low multiplicity of infection (0.01 plaque-forming unit per cell). Virus yields were quantitated by plaque assay on epithelioma papulosum cyprini (EPC) cells on day 3. Results of the challenge experiments revealed different levels of production of virus for each isolate on the various cell lines. Overall, the new cell line derived from rainbow trout, RBTE 45, was quite susceptible to all viruses tested. The three cell lines newly derived from coho salmon embryo were not as resistant to the replication of IHNV as was the established coho salmon cell line, CSE-119. An established cell line, EPC, derived from an epithelial tumor of common carp Cyprinus carpio, remained the most susceptible to all four IHNV isolates tested.     

Effects of Salinity on Yersinia ruckeri Infection of Rainbow Trout and Brown Trout

Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.     

In vitro hatching and activation ofTaenia taeniaeformis oncospheres

Previously described techniques for hatching eggs ofTaenia taeniaeformis were found to give inconsistent and generally ineffective results, even the degree of disaggregation of the embryophore varying with the strain of parasite. Furthermore, the hatched and activated oncospheres did not survive in the hatching fluid.Following a series of studies on the composition of the hatching fluids, a more reliable procedure was developed.Pretreatment with hypochlorite, at 0.67% w/w available chlorine, caused disaggregation of the embryophoral blocks of virtually all the eggs. When this was followed by exposure to a solution containing 10 mg.ml^–1 trypsin, 10% ox bile and 10% heat-inactivated foetal calf serum in modified RPMI with HEPES buffer and L-glutamine, about 50% of the viable oncospheres were activated and escaped from the oncospheral membrane. Most of the activated oncospheres survived in this hatching fluid for at least three hours.     

Assessment of serum protein fractions in rainbow trout using automated electrophoresis and densitometry

Background: Although rainbow trout (Oncorhynchus mykiss, Walbaum) are one of the most‐studied fish, electrophoretic techniques and classification of serum protein fractions have not been standardized, such that clinically useful values are lacking. Objective: The aim of the present study was to evaluate preliminarily the serum protein fractions of rainbow trout using automated cellulose acetate electrophoresis and densitometry. Methods: Serum samples from 25 rainbow trout (Oncorhynchus mykiss, Walbaum) were electrophoresed on cellulose acetate plates and quantified using densitometry. Results: A maximum of 6 fractions were identified and numbered, in order of decreasing mobility, as I, II, III, IV, V, and VI. In 3 of 25 (12%) samples, 6 fractions were identified; in 18 (72%) samples, 5 fractions were identified; and in 4 (16%) samples, 4 fractions were identified. Fractions I, V, and VI were always clearly identifiable, whereas fractions II and IV were frequently fused and indistinguishable from fraction III. The pattern with 5 fractions was the most probable type (χ², P<.01). The mean (±SEM) protein concentrations of the 6 fractions were I, 0.8±0.1 g/dL; II, 0.3±0.0 g/dL; III, 1.6±0.1 g/dL; IV, 0.3±0.1 g/dL; V, 0.6±0.0 g/dL; and VI, 0.2±0.0 g/dL. Based on comparison of serum and plasma electrophoretic patterns from 8 fish, fibrinogen was found in fraction V. Conclusion: Automated cellulose acetate electrophoresis and densitometry appear to be a practical method for estimation of serum protein fractions in rainbow trout.     

Virulence and serum-resistance in different clonal groups and serotypes of Yersinia ruckeri.

The virulence in rainbow trout (Oncorhynchus mykiss) of 32 isolates of Yersinia ruckeri, representing a range of biotypes, serotypes, and OMP-types, was examined. Virulence was assayed in fish of average weight 7.7 g by bath challenge for 1 h with approximately 5 x 10(7) cells per ml. Two of the six serotype O1 clonal groups of Y. ruckeri, clones 2 and 5, were virulent, whereas the other four clonal groups, clones 1, 3, 4 and 6, as well as all serotype O2, O5, O6 and O7 isolates examined, were avirulent. Analysis of susceptibility to the bactericidal effect of non-immune rainbow trout serum demonstrated an association between virulence and serum resistance. The virulent serotype O1 clonal groups were serum resistant, whereas the avirulent serotype O1 clonal groups and other serotypes were, with some exceptions, serum sensitive. The fact that some serum resistant isolates were avirulent suggested that other factors may be required for the full expression of virulence. The study also demonstrated that rainbow trout and brook trout (Salvelinus fontinalis) differ in their susceptibility to Y. ruckeri.     

Communications: Agglutination of Salmonid Spermatozoa by Renibacterium salmoninarum

Abstract

Renibacterium salmoninarum (American Type Culture Collection: ATCC 33209) agglutinated spermatozoa of brook trout Salvelinus fontinalis, rainbow trout Oncorhynchus mykiss, chinook salmon O. tshawytscha, white sucker Catostomus commersoni, and goldfish Carassius auratus, but not that of walleye Stizostedion vitreum or bulls Bos taurus. When examined microscopically, the bacteria were seen to be binding to the tails but not the heads of the sperm. The sperm agglutinin may be the previously reported renibacterial hemagglutinin.