Physiological response of the fish,Cyprinus carpio,to formalin exposure—I. Effects of formalin on urine flow,heart rate,respiration

1. Carp were exposed to 280 ppm formalin. Haematocrit and plasma glucose and lactic acid increased. In moribund fish, blood pH was remarkably lower and plasma protein increased.2. When exposed to formalin, heart rate (HR) and respiration increased briefly, and then decreased.3. Some fish increased urine flow (UF) immediately and maintained higher values for a while, followed by gradual decrease, and others decreased UF consistently. UF stopped at 30 min or longer prior to fish death. Urine osmotic pressure was higher at the 1st to 2nd hour and immediately before UF stopped.4. The relationship between UF, HR and respiration was also discussed.     

Physiological response of the fish, Cyprinus carpio, to formalin exposure--I. Effects of formalin on urine flow, heart rate, respiration.

1. Carp were exposed to 280 ppm formalin. Haematocrit and plasma glucose and lactic acid increased. In moribund fish, blood pH was remarkably lower and plasma protein increased. 2. When exposed to formalin, heart rate (HR) and respiration increased briefly, and then decreased. 3. Some fish increased urine flow (UF) immediately and maintained higher values for a while, followed by gradual decrease, and others decreased UF consistently. UF stopped at 30 min or longer prior to fish death. Urine osmotic pressure was higher at the 1st to 2nd hour and immediately before UF stopped. 4. The relationship between UF, HR and respiration was also discussed.     

Accuracy of body mass estimates of formalin-preserved fish – a review

This paper highlights possible effects of physical and chemical mechanisms of formalin fixation and preservation on biological tissue and reviews the consequent potential inaccuracies on estimates of body mass of small fishes fixed and preserved in formalin. Twenty-six papers including 65 independent experiments with 35 species which examine effects of formalin on body mass estimates on small fishes are included. The effect of the formalin on the specimens depends on the salinity of the water used to dilute the commercial formalin (usually 1:9 formalin: water) before being used to fixate and preserve fish. Mean wet body mass of the specimens from the studies using seawater or fresh water diluted formalin deceases by 13% and increases by 7%, respectively, from before to after being immersed in formalin. The same trend is found with condition factor in the few papers that report this parameter. Body length decreases on average by c. 2% in fixated and preserved fish regardless of whether the formalin is diluted in seawater or fresh water.     

Shifts in drag and swimming potential during grayling ontogenesis: relations with habitat use

The drag coefficient (C_d) of grayling Thymallus thymallus was dependent on body surface conditions and rigidity. At comparable flow conditions, C_d values of a fish preserved in formalin (high body rigidity) were 15–30% lower than those obtained on a freshly-killed fish (medium rigidity); the presence of skin mucus on fish could reduce C_d by 10%. The hydrodynamic potential of grayling increased during ontogenesis, because C_d values decreased (except for yolk sac larvae, which had a particular morphology) and the swimming capacities (in terms of relative muscular mass) increased. Grayling morphology evolves towards hydrodynamically efficient shape at high velocities, and there is a relationship between these shifts in hydrodynamic abilities and the different habitats (in terms of current velocity) used by five morphological groups. Therefore, the concept of hydrodynamic potential (i.e. hydrodynamics of shape and swimming capacities) could be a useful tool in fish ecomorphology and predictions of habitat use.     

The effects of formalin and isopropyl alcohol on length and weight measurements of Sarotherodon mossambicus Trewavas

Two groups of 30 Sarotherodon mossambicus were weighed and measured when alive, after 5 days in 10% formalin, and after 5, 10, 15, 20, 25, 30 and 65 days in 37.5% isopropyl alcohol. Fish from two size classes were preserved and all fish initially increased slightly in length and increased greatly in weight. The greatest increases in length and weight were noted after 5 days in formalin solution. After 65 days in isopropyl alcohol, the length of the preserved fish decreased to approach the live length while the weight of the fish stabilized at a higher than live weight value. This study is the first report of slight length increases or no shrinkage in preserved fish. Comments on the use of standard correction factors for lengthweight studies of preserved fish are provided.     

Immunological parameters of Javanese carp Puntius gonionotus (Bleeker) exposed to copper and challenged with Aeromonas hydrophila.

This study was to determine the median lethal concentration (LC50) of copper to Javenese carp, Puntius gonionotus (Bleeker), and the immune response after the fish were exposed to sublethal levels of copper and challenged with formalin killed Aeromonas hydrophila. The LC50 of copper on P. gonionotus at 24, 48, 72, 96 and 120 h were estimated as 2.17, 0.91, 0.57, 0.53 and 0.42 mg l(-1), respectively. To determine the effect of copper on the immune system, fish were exposed for 66 days to 0.05, 0.10 and 0.15 mg Cu l(-1). After 56 days of initial exposure to copper, fish were challenged with 0.1 ml of 4.5 x 10(5) cfu ml(-1) formalin killed A. hydrophila and maintained in the same concentration of copper. After the challenge, the immune response was monitored for 2 weeks using haematological and serological assays. During the initial phase of exposure to copper, significant changes were noted in the white blood cell, lysozyme, potential killing activity, total plasma protein, total immunoglobulin and haematocrit levels between the control and treated fish. One week after challenge with A. hydrophila, there was a significant increase in the values of white blood cells, total protein and total immunoglobulin compared to the values before the challenge. However, these values were not significantly different (P>0.05) between the control and the treated fish. In contrast, NBT and lysozyme assays exhibited a significant difference (P<0.05) in fish exposed to 0.10 mg Cu l(-1) (0.525 +/- 0.17; 24.42 +/- 3.35 x 10(2) micromg ml(-1)) and 0.15 mg Cu 1(-1) (0.536 +/- 0.19; 21.78 +/- 1.29 x 10(2) micromg ml(-1)) compared to the control (0.746 +/- 0.31; 30.73 +/- 5.42 x 10(2) micromg ml(-1)) after the bacterial challenge (day 61). There was however no significant difference (P>0.05) in NBT and lysozyme levels in fish exposed to lower level of copper (0.05 mg Cu l(-1)), suggesting the absence of immunosuppressive effects at lower level of exposure.     

秀丽隐杆线虫经福尔马林处理后上调表达基因的分析

秀丽隐杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前已经被广泛应用到生物对各种驱虫剂抗性机制的研究中。福尔马林被普遍用于鱼类寄生虫病的防治中,但是由于长期的应用,许多寄生虫对它产生了一定的抗药性。研究以秀丽隐杆线虫为研究对象,分析了它在福尔马林刺激下基因表达的改变情况。结果显示,经福尔马林处理后差异表达的有676个克隆,通过斑点杂交技术进一步筛选,对其中差异显著的161个克隆进行了测序分析。测序结果经BLAST分析发现:(1)细胞凋亡相关基因的表达发生上调,这些基因编码包括线粒体呼吸链相关蛋白、含TPR序列的蛋白SGT-1、热休克蛋白、氧化应激相关蛋白、胞吞过程相关蛋白、DNA复制和修复相关蛋白以及其他一些重要的凋亡相关蛋白;(2)编码重要的转录调节因子和信号转导相关蛋白如转录激活因子FosB/c-Fos、G蛋白、细胞周期蛋白B、钙结合蛋白、核小体装配蛋白NAP-1等的基因的表达发生上调;(3)能量代谢和蛋白质、脂肪、氨基酸代谢途径中的有些基因的表达也发生上调;(4)除了以上已知功能的基因外,还有一些未命名蛋白质基因。这说明福尔马林对秀丽隐杆线虫的影响除了诱导细胞凋亡之外,还影响其他细胞代谢活动的改变。实验为进一步研究生物体对福尔马林的抗性机制奠定了一定的基础。       

Shrinkage of Gobiocypris rarus, Procypris rabaudi, and Sinilabeo rendahli preserved in formalin

Length measurements of preserved fishes are necessary in many types of fish surveys because logistics often do not allow for fish measurement immediately after catch. If the fixative causes significant shrinkage, then the preserved lengths cannot be directly used to indicate accurate live lengths. The objective of this study was to determine how preservation in formalin affects standard length of Gobiocypris rarus larvae (24‐day‐old and newly hatched), larval Procypris rabaudi (4‐day‐old), and larval Sinilabeo rendahli (12‐day‐old). Fishes were measured (to nearest 0.01 mm) and individually fixed in the appropriate formalin solution (2.5% or 5.0% formalin), then re‐measured at 0.5, 1, 3, 7, 14, 30, 45 and 75 days after preservation to follow the time course of shrinkage. Most of the shrinkage occurred within the first half day after preservation. The 5.0% formalin caused a higher relative shrinkage rate than did the 2.5% solution; however, the difference was not statistically significant. In G. rarus, initial shrinkage of newly hatched larvae was higher than that of 24‐day‐old larvae.     

Acidic Lugol’s solution as an effective fixative for flounder eggs

Fixatives used for preservation of marine fish eggs must maintain the structural integrity of eggs in order to secure determination of developmental stages. Formalin is commonly used as a fixative, however, this compound is associated with health issues, and fish eggs fixated in formalin are often not stageable after longer storage. The present study shows that 5% acidic Lugol’s solution is an efficient fixative for ichtyoplankton, and superior to formalin fixation. Flounder eggs retained their structure and staging was easy even after 1 year of storage in acidic Lugol’s solution.     

Fish gut content from biological collections as a tool for long-term environmental impact studies

Most of the world’s fish fauna is suffering from different types of human impacts and new conservation tools are required. The fish diet analysis is a tool that has been used to evaluate degradation processes of aquatic environments, however, few long-term studies are performed by several reasons (e.g., lack of funding, opportunity). Our aim was to test whether the fish gut content from biological collections can be used for comparisons with current data and, consequently, be used as a tool for long-term environmental impact studies. We compared the gut content of fish preserved for fifteen years in a biological collection with recently sampled fish, considering the factors size of the specimen, preservation time and preservation form. We did not find differences in the gut content percentage of preservation between fish size classes and preservation time. However, we found differences between preservation form, in which the fish fixed in formalin kept the digestive content preserved while the fish preserved directly in alcohol did not. Thus, we encourage the use of fish gut content from biological collections fixed in formalin for long-term ecological studies. Our findings may help elucidate some long-term effects of human impacts on fish fauna.     

The effect of various Aeromonas bestiarum vaccines on non-specific immune parameters and protection of carp (Cyprinus carpio L.)

Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish. Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland. Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination. Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days. The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group. Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI). The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control. Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish.     

Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

Length‐weight relationships of fishes in the Mira basin,Colombia

This study estimated the a and b parameters of the length–weight relationships of 15 fishes from the Mira and Caunapí rivers, San Andrés de Tumaco, Nariño, Colombia. The specimens were captured during four samplings periods between 2015 and 2016 using a different a combination of fishing gillnets and electrofishing. The a values ranged from 0.0002 to 0.04 and b values ranged from 2.86 to 3.6. The data presented here are tentative estimates because the fish have been weighted and measured after fixation in formalin and ethanol, however, these data are references for weight‐length relationship for 15 species, the maximum weight for the same 15, and new maximum lengths for 3 species.     

Fluid‐preserved fishes are one solution for assessing historical change in fish trophic level

1. There are few resources available for assessing historical change in fish trophic dynamics, but specimens held in natural history collections could serve as this resource. In contemporary trophic ecology studies, trophic and source information can be obtained from compound‐specific stable isotope analysis of amino acids of nitrogen (CSIA‐AA‐N).
2. We subjected whole Sebastes ruberrimus and Clupea pallasii to formalin fixation and 70% ethanol preservation. We extracted tissue samples from each fish pre‐fixation, after each chemical change, and then in doubling time for 32–64 days once placed in the final preservative. All samples were subjected to CSIA‐AA‐N, and their glutamic acid and phenylalanine profiles and associated trophic position were examined for differences over time by species.
3. Glutamic acid and phenylalanine values were inconsistent in direction and magnitude, particularly during formalin fixation, but stabilized similarly (in 70% ethanol) among conspecifics. In some cases, the amino acid values of our final samples were significantly different than our initial pre‐preservation samples. Nonetheless, significant differences in glutamic acid, phenylalanine, and estimated trophic position were not detected among samples that were in 70% ethanol for >24 hr.
4. Our results suggest that the relative trophic position of fluid‐preserved specimens can be estimated using CSIA‐AA‐N, and CSIA‐AA‐N estimates for fluid‐preserved specimens should only be reported as relative differences. Timelines of trophic position change can be developed by comparing specimens collected at different points in time, revealing trophic information of the past and cryptic ecosystem responses.

     

The effects of four preservation methods on length, weight and condition factor of the clupeid Sardinella aurita Val. 1847

Formalin preservation, freezing, and salted and non‐salted smoking are often used to preserve fishes. Body proportions of fish show changes upon preservation when applying different preserving techniques; most authors report a decrease in length and some authors report changes in weight and condition factor (k). Four groups of round sardinella (Sardinella aurita) were subjected to the above treatments. Initial fork length (cm) and weight (g) ranged from 8.7 to 10.8 cm and 6.74 to 11.4 g, respectively. All fish decreased in both length and weight, with highly significant (P < 0.05) differences. Condition factor decreased in all treatments except formalin. Percentage reductions in length, weight, and condition factor were 11.0, 68.0 and 54.8% with salted smoking; 13.0, 69.3 and 53.3% with non‐salted smoking; 3.0, 14.1 and 6.7% with freezing; and 7.1, 13.3 and 108.1% with formalin preservation, respectively. Shrinkage was the least with freezing, followed by formalin, salted smoking, and non‐salted smoking preservation.     

Inhibition of DNA amplification in marine fish larvae preserved in formalin

Recent advances in molecular biology open the possibility touse formalin-preserved specimens stored in ichthyoplankton collectionsfor population genetics studies. Nine DNA extraction techniqueswere tested on Engraulis mordax larvae preserved in bufferedformalin. However, none of the DNA extracts resulted in thepositive amplification of mitochondrial DNA (mtDNA) (NADH1,16srRNA, 12srRNA and control region fractions). An experimentwith different length-time exposure to formalin done with Cynoscionparvipinnis larvae allowed us to confirm the difficulty of amplifyingmtDNA from larvae preserved in formalin for long time periodsand the possibility of DNA extraction and amplification fromshort-term (less than 48 h) formalin-fixed marine fish larvaepreserved in ethanol (70%). We discuss the possible influenceof physical–chemical complexes associated with the durationof preservation to inhibition of amplification reactions.     

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

Preserving young‐of‐the‐year Perca fluviatilis in ethanol,formalin, or in a frozen state and the consequences for measuring morphometrics

As it is often not possible to immediately analyse individuals sampled in the field, captured fish are preserved and stored for later investigation. The objective of this study was to assess the effects of various preservation methods on subsequent changes in morphometric parameters while also providing correction factors to re‐calculate the original body dimensions when sampled fish are measured at a later date. In this study, juvenile perch (Perca fluviatilis, 66.5 ± 10.2 mm total length) were measured directly after capture, then either frozen at ?20°C, preserved in 70% ethanol, or in 4% formalin. They were again measured after 24 h, 3 days, 7 days, and thereafter on a weekly basis for 8 weeks. Ethanol‐preserved perch greatly decreased length and weight; formalin preservation also led to a comparable length reduction, but increased the weight of the perch. In contrast, frozen perch showed less shrinkage and only moderate weight loss. Of the three preservation methods, freezing clearly caused the fewest distortions. Hence, freezing is recommend as the most preferable preservation method, especially in multi‐disciplinary studies on fish ecology; for all other preservation methods the correction factor over time will have to be pre‐determined for each species and size class.     

Shrinkage correction and length conversion equations for Theragra chalcogramma, Mallotus villosus and Thaleichthys pacificus

Preservation of walleye pollock Theragra chalcogramma, capelin Mallotus villosus and eulachon Thaleichthys pacificus by freezing decreased fork length ( L _F) up to 1·8, 5·6 and 2·7% and reduced mass by up to 8·4, 3·5 and 1·1%, respectively. Shrinkage of walleye pollock standard length ( L _S) was greater for fish in 95% ethanol v . 5% formalin and for fish in 10% formalin v . frozen. Equations describing the shrinkage and loss in mass for these species are presented as well as conversions between different length measurements ( L _S, L _F and total length, L _T) for fishes that were frozen.