Dietary live yeast and increased water temperature influence the gut microbiota of rainbow trout.

AIMS: The objective was to determine the effects of dietary substitution of fishmeal (FM) with live yeast and increasing water temperature on the diversity and composition of gut microbiota in rainbow trout. METHODS AND RESULTS: Fish were fed either FM or yeast (Saccharomyces cerevisiae) and reared in water temperatures of either 11°C (cold) or 18°C (warm) for 6 weeks. Luminal content and mucosa were collected from the distal gut and the load, diversity and species abundance of yeast and bacteria were analysed using agar plating, MALDI-TOF and rRNA gene amplicon sequencing. Yeast in the gut of fish fed FM were represented by S. cerevisiae, Rhodotorula spp. and Debaryomyces hansenii, while fish fed yeast contained 4-5 log higher CFU per g of yeast that were entirely represented by S. cerevisiae. For gut bacteria, sequencing of 16S rRNA gene amplicons using Illumina MiSeq showed lower bacterial diversity and abundance of lactic acid bacteria, especially Lactobacillus, in fish reared in warm rather than cold water. Fish fed yeast had similar bacterial diversity and lower abundance of Leuconostocaceae and Photobacterium compared with fish fed FM. CONCLUSIONS: Feeding live yeast mainly increased yeast load in the gut, while increased water temperature significantly altered the gut microbiota of rainbow trout in terms of bacterial diversity and abundance. SIGNIFICANCE AND IMPACT OF THE STUDY: Live yeast can replace 40% of FM without disrupting bacteria communities in the gut of rainbow trout, while increased water temperature due to seasonal fluctuations and/or climate change may result in a gut dysbiosis that may jeopardize the health of farmed fish.

Growth hormone secretion from the Arctic charr (Salvelinus alpinus) pituitary gland in vitro: effects of somatostatin-14, insulin-like growth factor-I, and nutritional status.

This study investigated the influence of nutritional status on the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis in Arctic charr (Salvelinus alpinus). The objectives were to study the regulation of GH secretion in vitro by somatostatin-14 (SRIF) and hIGF-I, and to determine whether pituitary sensitivity to these factors is dependent upon nutritional status. Arctic charr were fed at three different ration levels (0, 0.35, and 0.70% BWd(-1)), and pituitary glands were harvested at 1, 2, and 5 weeks for in vitro study. Both SRIF and hIGF-I inhibited GH secretion from Arctic charr pituitary tissue in long-term (18 h) static hemipituitary culture, as well as after acute exposure in a pituitary fragment perifusion system. This response appeared to be dose-dependent for SRIF in static culture over the range of 0.01-1 nM, but not for hIGF-I. The acute inhibitory action of hIGF-I on GH release in the perifusion system suggests an action that is initially independent of any effects on GH gene expression or protein synthesis. Nutritional status did not affect the sensitivity of Arctic charr pituitary tissue to either SRIF or hIGF-I in vitro, indicating that changes in abundance of pituitary SRIF or IGF-I receptors may not explain the alterations in plasma GH levels found during dietary restriction.

The efficacy of clove oil as an anesthetic for the zebrafish, Danio rerio (Hamilton).

The anesthetic effects of clove-oil-derived eugenol were studied in the zebrafish, Danio rerio (Hamilton). Acute lethality and the effects of exposures to various dosages of eugenol were measured. The estimated 96-h LC50 for eugenol was 21 ppm. Times to induction and recovery from anesthesia were measured and compared with MS-222 under similar conditions. Eugenol induced anesthesia faster and at lower concentrations when compared to MS-222. The recovery times for fish exposed to eugenol were generally longer compared to similar concentrations of MS-222. Doses of 60-100 ppm eugenol produced rapid anesthesia with an acceptably short time for recovery. These findings suggest that eugenol could be an effective anesthetic for use with this species, and when compared to MS-222, its benefits include a lower cost, lower required dosage, improved safety, and potentially lower mortality rates.

Group-level factors associated with chemotherapeutic treatment regimens in land-based trout farms in Ontario, Canada.

The lowest level at which fish farmers ordinarily make management decisions is the individual holding unit. To identify factors associated with chemotherapeutic treatment initiation at the holding-unit level, we created a unit of measurement called the "farm-tank-lot" (FTL), which allowed the movements and mixing of groups of fish to be followed during an entire production cycle. Each FTL was comprised of fish with a common history housed in a specific holding unit. Our 21-month prospective observational study (conducted on 14 land-based trout farms in Ontario, Canada) showed that the FTL was a biologically meaningful unit of concern and a feasible unit of measurement on land-based trout farms.Multivariable logistic and Poisson regressions revealed that fish size and growth rate both were associated negatively with the probability and frequency of treatment. FTLs that existed for longer periods of time were more likely to be treated and treated more often. There was a significant farm effect. Future field studies of disease in cultured fish should incorporate these factors into their design and analysis.

The Amount of MUC5B mucin in cervical mucus peaks at midcycle.

The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.

Developmental expression of mucin genes ASGP (rMuc4) and rMuc5ac by the rat ocular surface epithelium.

PURPOSE: To determine site and time of initiation of expression of the membrane-spanning mucin ASGP (rMuc4) and the goblet cell-specific, gel-forming mucin rMuc5AC by the developing rat ocular surface epithelium. METHODS: Newborn Sprague-Dawley rat pups were killed at 1, 7, and 14 days after birth. Adult rats (weight, 200 g) were used as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect ASGP mRNA using beta-actin as an internal control. Competitive RT-PCR was performed to quantitate rMuc5AC mRNA using an rMuc5AC-competitive reference standard (CRS) as an internal control. In situ hybridization was performed to localize ASGP and rMuc5AC mRNA. Goblet cells were detected by staining with periodic acid-Schiff (PAS) reagent. RESULTS: ASGP mRNA was detected by RT-PCR at 1 day after birth. Compared with beta-actin, the amount of ASGP mRNA showed a progressive increase from 1 to 14 days of postnatal development. By in situ hybridization, the expression of ASGP was first clearly detected at 14 days after birth at the lid margin, where the most stratification of epithelium was seen, and along the adjacent palpebral conjunctiva. This pattern was seen in rat eyelids that were not yet open but appeared about to open. In rat eyelids already open at 14 days after birth, ASGP mRNA was diffusely spread in the apical cell layer of both conjunctival and corneal epithelia. The expression of rMuc5AC was detected by RT-PCR in ocular surface epithelium in rat pups 1 day after birth. Quantitative RT-PCR showed a low level of rMuc5AC RNA expression in conjunctiva of 1-, 7-, and 14-day-old rats followed by a large increase in expression between 14 days and adulthood. The expression of rMuc5AC was first detected by in situ hybridization in a few goblet cells at 7 days after birth. One or two labeled cells were present in the fornical area; some were on the palpebral side of the fornix; others were present on the bulbar side. The distribution and time of appearance of rMuc5AC correlated with that of PAS staining of goblet cells. CONCLUSIONS: The developmental expression of the membrane-spanning mucin ASGP (rMuc4) and the gel-forming mucin rMuc5AC are regionally and temporally separated. Expression of the gel-forming mucin begins at the fornix at 7 days after birth and is correlated with the appearance of goblet cells, whereas, expression of the membrane-spanning mucin begins later at the lid margin at day 14. Expression of the membrane-spanning mucin correlates to eyelid opening.

MUC4 and MUC5B transcripts are the prevalent mucin messenger ribonucleic acids of the human endocervix.

Mucins secreted by the endocervical epithelium protect the surfaces of the reproductive tract epithelium from pathogen penetrance and modulate sperm entry into the uterus. Three large gel-forming mucins, MUCs 5AC, 5B, and 6, are expressed by the endocervical epithelium, as is MUC4, a relatively uncharacterized mucin for which only tandem repeat sequence has been reported. We sought to determine the relative abundance of each of these mucin gene transcripts and to relate their expression to blood progesterone and estradiol. Samples were obtained from six subjects at successive stages in the menstrual cycle. Primers to nontandem repeat sequences of MUCs 4, 5AC, 5B, and 6 were used in semiquantitative reverse transcription-polymerase chain reaction to determine relative abundance of each mucin gene in relation to beta2-microglobulin message control. In order to design primers from a nontandem repeat region of MUC4 so that MUC4 message levels could be quantitated, we obtained approximately 2.7-kilobase nontandem repeat sequence 5' to the tandem repeat sequence of a MUC4 genomic clone. The sequence showed lack of cysteine-rich D-domains and was rich in serine and threonine. Semiquantitative polymerase chain reaction analyses indicated that the principal mucin transcripts of human endocervix are MUC4 and MUC5B, with MUC4 predominant in 15 of 21 samples. When correlated with plasma steroid levels, message levels of both MUC4 and MUC5B were inversely related to progesterone levels.

Factors predictive of survival in ampullary carcinoma.

OBJECTIVE: To review the recent Memorial Sloan-Kettering Cancer Center experience with adenocarcinoma of the ampulla of Vater and to identify clinicopathologic factors that have an impact on patient survival. SUMMARY BACKGROUND DATA: The prognosis for patients with tumors of the ampulla of Vater is improved relative to other periampullary neoplasms. Identification of independent prognostic factors in ampullary tumors has been limited by small numbers of tumors and a lack of pathologic review. METHODS: Data were collected prospectively for patients presenting with periampullary carcinomas to the Memorial Sloan-Kettering Cancer Center between October 15, 1983 and June 30, 1995. The correlation between clinicopathologic variables and survival of ampullary carcinoma was tested by the Kaplan-Meier method and log-rank test, and Cox proportional hazards regression. Survival of patients with periampullary adenocarcinomas was compared by the Kaplan-Meier method. RESULTS: In 123 patients presenting with ampullary carcinoma, 101 tumors (82.1%) were resected. Factors significantly correlated with improved survival were resection (p < 0.01), and in resected tumors, negative nodes (p = 0.04) and margins (p = 0.02) independently predicted for improved survival. In periampullary tumors, the highest rates of resection and overall survival (median, 43.6 months) were found in ampullary carcinomas. CONCLUSIONS: Factors predictive of improved survival in ampullary carcinoma include resection, negative margins, and negative nodes. Improved overall survival in ampullary relative to periampullary adenocarcinoma is due in part to a significantly higher rate of resection.

Management of adenocarcinoma of the body and tail of the pancreas.

OBJECTIVE: The authors examined the resectability, operative morbidity mortality, and survival of patients with pancreatic adenocarcinoma of the body and tail compared with lesions in the head. SUMMARY BACKGROUND DATA: Adenocarcinoma of the body and tail of the pancreas is characteristically thought of as a disease that presents late and rarely is operable or resectable. METHODS: In an 11-year period, 1981 patients were admitted and entered into a prospective database at Memorial Sloan-Kettering Cancer Center with a diagnosis of peripancreatic cancer, 1363 of whom had adenocarcinoma of the pancreas, 75% with lesions in the head and 25% with lesions in the body and tail. RESULTS: Of 271 patients resected, 237 (23%) had lesions in the head and 34 (10%) had body and tail lesions. Perioperative mortality was 4% for patients with pancreatic lesions in the head and 0% for patients with pancreatic lesions in the body and tail. Five-year actuarial survival for body and tail lesions was projected at 14% for 5 years. Actual survival was 19%, with three patients alive for more than 5 years. CONCLUSIONS: Adenocarcinoma of the body and tail of the pancreas, although less likely to be resectable at presentation than lesions in the pancreatic head, have similar postresection survival.

Relationship between the fertilization of rainbow trout (Salmo gairdneri) eggs and the motility of spermatozoa.

This investigation evaluated the relationship between the motility of rainbow trout (Salmo gairdneri) spermatozoa and egg fertilization. When sperm:egg ratios were supraoptimal (i.e., >200,000 sperm per egg), neither spermatozoan motility, sperm density or spermatocrit were major factors in determining the percentage of eggs reaching the stage of eye-up. At spermatozoan concentrations near the critical ratio of spermatozoa per egg (i.e., 200,000/egg), there was a significant correlation between fertilization rates and subjective motility estimates. Samples exhibiting better motility required fewer spermatozoa to ensure high fertilization rates, obtaining rates near 90% with as few as 100,000 spermatozoa per egg. Late in the reproductive season, there was a significant correlation between initial sperm density and fertilization rate.

A quantitative assessment of thyroid histopathology of herring gulls (Larus argentatus) from the Great Lakes and a hypothesis on the causal role of environmental contaminants.

Thyroids from 213 adult herring gulls of both sexes were collected during incubation from nine colonies in the Great Lakes basin of eastern North America between 1974 and 1983, and from a single colony in the Bay of Fundy from 1977 to 1982. Qualitative and quantitative histological assessment revealed that the majority of the gulls from the Great Lakes basin suffered from goiter. These thyroids had a greater mass than those from the Bay of Fundy, and were microfollicular and frequently hyperplastic. The histopathology was similar to that previously observed in Pacific salmon from the Great Lakes. These findings are consistent wit a forage fish-borne goitrogenic etiology other than, or in addition to, iodine deficiency. Temporal and spatial differences in the severity of thyroid dysfunction are consistent with the hypothesis that polyhalogenated hydrocarbons are responsible for the goiter development and thyrotoxic effects observed in herring gulls from the Great Lakes area.

Polycystic kidney disease in goldfish (Carassius auratus) from Hamilton Harbour, Lake Ontario, Canada.

Severe kidney enlargement was observed in 6.3% (5 of 80) of goldfish collected from a heavily polluted industrial basin. Externally the fish had generalized swelling and abdominal distension. The kidneys contained numerous large, clear, fluid-filled cysts (polycystic) that ranged in size from microscopic to 4 cm in diameter. Affected kidneys had a wide range of histological changes-including the presence of large multiple cysts that caused severe distension and compression of normal renal tissue, multifocal granulomas, and signs of early, proliferative glomerulonephritis. The histology of affected kidneys is compared with other goldfish from Hamilton Harbour, and with goldfish collected from an alternate site (a population apparently free of polycystic kidney enlargements). This afflication is rare in feral fish populations, and its occurrence in a deteriorated environment such as Hamilton Harbour may provide further evidence of a link between fish health and environmental quality.

Advances in the cryopreservation of salmonid semen and suitability for a production-scale artificial fertilization program.

During the past decade, semen cryopreservation techniques in salmonid fish have progressed to where fertilization rates obtained with preserved sperm approach those produced with fresh sperm. Recent advances in the collection, dilution and storage of salmonid spermatozoa are reviewed. The problems encountered during the development and examination of artificial insemination programs in warm-blooded animals are contrasted with those encountered in fish. This paper discusses the advantages of artificial fertilization, and the manner in which advances in cryopreservation techniques could aid in the development and expansion of concentrated, production-scale artificial fertilization programs in salmonid fish.

An epizootic of diffuse glomerulonephritis in white perch (Roccus americanus) from a heavily polluted industrial basin in Lake Ontario.

Examination of 43 white perch from a heavily polluted water basin showed all had severe diffuse glomerulonephritis. There were fibrinoid necrosis of tuft vessels, especially the afferent and efferent arterioles, and thickened basement membranes which ultrastructurally contained dense deposits in a subendothelial location. None of these lesions was found in white bass, a closely related species, from the same source nor in white perch from other relatively nonpolluted sites in Lake Ontario or Lake Erie. This is a dramatic example of a rare lesion in fish and possible represents an immune-complex mediated response to an environmental pollutant.

The quantitative clearance of bacteria from the bloodstream of rainbow trout (Salmo gairdneri).

The blood clearance of 51Cr-labelled heat-killed Salmonella pullorum was biphasic and exponential for each phase. This pattern was the same for all three concentrations of bacteria used. The initial more rapid phase lasted for approximately 15 minutes when roughly 90% of the bacteria had been cleared. The second slower phase started 25 to 30 minutes post-inoculation and lasted for approximately 25 to 30 minutes. The tissue distribution of bacteria showed that in both absolute and relative terms, the kidney was the most important organ, accounting for more than 70% uptake of the bacteria. These kinetics demonstrate that rainbow trout at 8 degrees C are able to clear their bloodstream of Salmonella very efficiently, in a fashion very similar to mammals. It is not know, however, which of the various possible clearance mechanisms dominates each phase.

Quantitative interlake comparison of thyroid pathology in Great Lakes coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tschawytscha) salmon.

Coho salmon (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tshawytscha) from Lakes Ontario, Michigan, Erie, or Huron were found to suffer epizootics of thyroid hyperplasia and goiters which appeared to have an environmental etiology. There were 13-fold differences in goiter prevalence within the Great Lakes, and the differences in goiter frequency were correlated with the degree of thyroid hyperplasia. A means of assessing the degree of thyroid hyperplasia (thyroid index) is described, and the derived index was used to facilitate statistical interlake and interspecies comparisons. Despite the hyperplastic (or goitered) condition in all prespawning or spawning Great Lakes salmon, serum thyroid hormone levels were generally higher than in prespawning coho salmon from the Fraser River, British Columbia, indicating that the Great Lakes fish were not necessarily hypothyroid. The hyperplastic lesions appear to undergo progressive changes: (a) large follicles, partly colloid depleted, surrounded by cuboidal epithelial cells; (b) small follicles, largely colloid depleted, surrounded by columnar epithelial cells (in this form, the follicles commonly assume a trabeculate arrangement); (c) "microfollicles" with greatly enlarged columnar epithelial cells encompassing very small follicles; (d) apparently afollicular lesions with little or no colloid in evidence. There was some evidence of benign invasiveness, although the lesions generally resembled simple hyperplastic parenchymatous goiters seen in humans.

Disseminated hexamitiasis in Siamese fighting fish.

One hundred forty Siamese fighting fish became inappetent and lethargic and developed swollen abdomens. One hundred fish died, many with perforated stomachs. Hexamitid parasites were found in large numbers in the abdominal cavity and in the mesentery and the parenchyma of the liver, spleen, and kidney, where they were closely associated with a chronic inflammatory response. Species of bacteria recovered from the peritoneal cavity were believed to be secondary invaders. The 40 surviving fish, including some sick ones, were treated with nifurpirinol at the rate of 0.2 mg/L and 5 mg/kg of body weight, by means of a bath and a feed medication, respectively. The sick fish recovered, and no more died.

Calcific pancreatitis in a patient with type 5 hyperlipoproteinemia.

The history of a 27-year-old woman with 10 years of episodic abdominal pain and the development of calcific pancreatitis secondary to hyperlipoproteinemia is presented. The relationship between familial and alcoholic hyperlipidemia and pancreatitis is discussed as well as the mechanism of injury to the pancreas.