Anesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L − 1 , EUG 30 mg L − 1 and MTMD 4 mg L − 1 . Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60-75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG were used. The degranulation of primary granules proved to be a useful assay for measuring the effects of stress on neutrophil function in fish. Eugenol and metomidate prevented stress-induced decrease of neutrophil function while MS 222 did not. RightsWorks produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted. AbstractAnesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L − 1 , EUG 30 mg L − 1 and MTMD 4 mg L − 1 . Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60-75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG w...

Reproduction of muskellunge Esox masquinongy has failed in many waters that formerly supported self‐sustaining populations. Laboratory experiments were conducted to isolate causes of such failures. Differential mortality occurred among lots of muskellunge eggs incubated in jars of unaceated lake water over substrates of sand, gravel, silt, aquatic macrophytes, wood, tree leaves, polyethylene screen, and bare glass. High and rapid early mortality (days 1–2), attributable to low dissolved oxygen (DO) concentrations (0–0.1 mg/liter), occurred among eggs incubated on leaves and macrophytes. After day 3, Saprolegnia sp. fungus was implicated in high egg mortalities in jars with inorganic substrates and moderate DO concentrations (3.8–4.1 mg/liter). Lowest mortality rates occurred on organic substrates (silt and wood) amidst intermediate DO concentrations (0.4–1.7 mg/liter) and limited fungal infestation. Among eight midwestern lakes and reservoirs, measured DO at the substrate‐water interface in four of them was high (means, 6.0–8.4 mg/liter) and showed little microstratification; these lakes contain self‐sustaining muskellunge populations. The other four lakes showed extreme DO microstratification and virtual anoxia (means, 0.4–2.4 mg/liter) at the substrate‐water interface; muskellunge populations in these lakes are supported almost wholly by stocking. Suitable spawning substrates in these lakes are aerated by annual reservoir drawdown, have inherently low biological oxygen demand, or support dense beds of stonewort Chara sp. Reproductive failure is associated with spawning areas having deep accumulations of organic matter and dense macrophyte growth. Improvements of spawning habitat to prevent or alleviate hypoxia are among the options available to manage this species.

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A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,30,5,50-tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content. Abstract A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3 0 ,5,5 0 -tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content.