Enhanced Toxicity for Mice of Pertussis Vaccines When Preserved with Merthiolate

Pertussis vaccines preserved with 0.01% Merthiolate are more toxic for mice than unpreserved vaccines prepared from the same parent concentrate and containing the same number of organisms. The toxicities of both Merthiolate (0.01%)-preserved and unpreserved vaccines increased when the number of organisms injected was increased. An increase in mortality was observed when Merthiolate was injected separately, before or after an unpreserved saline suspension of pertussis vaccine.     

Chironomid biomass determination from larval shape

SUMMARY.

• 1 Ash-free dry weights and dry weights of each larval instar were determined for nine chironomid taxa using unpreserved material. Larval weight (wμg) was related to body length measurements (l mm) by the equation In w= In a+b In l.
• 2 For a given taxon. the constants In a and b were dependent on larval size and shape, respectively: b was found to increase with larval length, while taxa with characteristically stout larvae yielded higher values for In a.
• 3 Comparison of measurements of freshly-killed chironomid larvae with measurements taken after preservation in 70% ethanol showed no change in body shape due to preservation. Length measurements from larvae preserved in this way are therefore suitable for use in the calculation of larval biomass when the relationship to the unpreserved weight is known.
• 4 Maximum larval lengths and mean body proportions (length: width ratios) are reported for thirty-two chironomid taxa.

     

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.     

Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies

Stable isotope analysis of carbon (C) and nitrogen (N) in animal tissues is an important approach to investigate the tropic status and habitat of marine species. Some biases due to lipid extractions and preservation can hinder the interpretation of results, yet their effects have not been investigated in squid. In this study, we evaluated the effects of lipid extraction and preservatives (dimethylsulfoxide (DMSO), 70% ethanol, and 10% buffered formaldehyde) on the δ¹³C, δ¹⁵N and C:N ratios in squid muscle. Beaks were placed under the same treatments with the exception of DMSO. Muscle and beak samples remained under treatment for 375 days and 416 days, respectively. Our results indicate that lipid extractions increased the mean values of unpreserved samples by 0.8‰ for δ¹³C and by 0.68‰ for δ¹⁵N. Preservatives also affected the isotopic composition in muscle at different magnitudes. DMSO remarkably reduced and increased the variability for δ¹³C and δ¹⁵N values among samples, formalin mainly reduced δ¹³C values by 1.5‰, whereas ethanol increased both δ¹³C and δ¹⁵N by ≤ 0.8‰. Lipid extractions eliminated the effect of DMSO and ethanol for δ¹³C and δ¹⁵N, and formalin only for δ¹⁵N. In beak, negligible shifts in δ¹³C, δ¹⁵N and C:N ratios were recorded after preservation in ethanol and formalin. Although lipid extractions can be recommended to reduce the effect of preservation, further research is needed to develop correction models for isotopic shifts associated with both lipid extractions in unpreserved and preserved muscle tissues. Lipid extractions per se could introduce a bias that may have important implications for ecological studies.     

Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-dried Sperm in Eggs of Nile Tilapia

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.     

Molecular taphonomy of animal and plant cuticles: selective preservation and diagenesis

The nature of organic material and the environment in which it is deposited exert a major influence on the extent to which biomacromolecules are preserved in the fossil record. The role of these factors is explored with a particular focus on the cuticle of arthropods and leaves. Preservation of the original chemistry of arthropod cuticles is favoured by their thickness and degree of sclerotization, and the presence of biominerals. Decay and burial in terrestrial as opposed to marine, and anoxic rather than oxygenated conditions, likewise appear to enhance preservation. The most important factor in the long-term preservation of the chemistry of both animal and plant cuticles, however, is diagenetic alteration to an aliphatic composition. This occurs even in amber, which encapsulates the fossil, eliminating almost all external factors. Some plants contain an original decay-resistant macromolecular aliphatic component but this is not the case in arthropods. It appears that the aliphatic components of many plant as well as animal fossils may be the result of diagenetic polymerization. Selective preservation as a result of decay resistance may explain the initial survival of organic materials in sediments, but in many cases longer-term preservation relies on chemical changes. Selective preservation is only a partial explanation for the origin of kerogen.     

The influence of lipid‐extraction and long‐term DMSO preservation on carbon (δ13C) and nitrogen (δ15N) isotope values in cetacean skin

Stable isotope analysis (SIA) has rapidly become a useful tool to study the ecology of wild animal populations, especially for elusive, wide‐ranging predators like marine mammals. The development of projectile biopsy techniques resulted in the collection of thousands of cetacean tissue samples that were archived in a dimethyl sulfoxide (DMSO) solution for long‐term, multidecadal preservation. Here we examine the influence of DMSO preservation on carbon (δ¹³C) and nitrogen (δ¹⁵N) values by comparing a set of paired delphinid skin samples stored frozen without preservative and in DMSO for up to 22 yr. Treatment of paired frozen and DMSO‐preserved skin in a 2:1 chloroform:methanol solution yielded similar δ¹³C and δ¹⁵N values, revealing that DMSO and lipid contamination have similar isotopic effects on skin, and that these effects can be removed using routine lipid‐extraction methods. Further, amino acid concentrations in DMSO‐preserved and frozen skin tissue were similar, providing independent evidence of minimal protein alteration due to preservation. Access to a rich archive of skin samples preserved in DMSO will expand our ability to examine temporal and spatial variability in the isotope values of cetaceans, which will aid our understanding of how their ecology has been influenced by historical changes in environmental conditions.     

Current status of regulating biotechnology-derived animals in Canada: animal health and food safety considerations

Development of an effective regulatory system for genetically engineered animals and their products has been the subject of increasing discussion among researchers, industry and policy developers, as well as the public. Since transgenesis and cloning are relatively new scientific techniques, transgenic animals are 'novel' organisms for which there is limited information. The issues associated with the regulation of transgenic animals pertain to environmental impact, human food safety, animal health and welfare, trade and ethics. It is a challenge for the developers to prove the safety of the products of biotechnology-derived animals and also for regulators to regulate this increasingly powerful technology with limited background information. In principle, an effective regulatory sieve should permit safe products while forming a formidable barrier for those posing an unacceptable risk. Regulatory initiatives for biotechnology-derived animals and their products should be able to ensure high standards for human and animal health, a sound scientific basis for evaluation; transparency and public involvement, and maintenance of genetic diversity. This review proposes a regulatory regime that is based on scientific risk based assessment and approval of products or by-products of biotechnology-derived animals and its application in context to Canadian regulations.     

Conservation breeding programmes: an important ingredient for species survival

Captive populations for the primary purpose of securing the survival of species through stable self-sustaining populations need to be founded and managed according to sound scientific principles. Given the current rate of habitat fragmentation and its effect on animal populations, species conservation over the long term increasingly will require management to reduce risk of extinction. This may include ex situ populations which can support and interact demographically and genetically with wild populations. This paper presents a review of what can be achieved through a multidisciplinary approach, involving science, interactive management, politics, environmental education, habitat preservation and habitat restoration. Examples of what conservation-oriented breeding programmes have contributed to date, what represents a viable population and what is meant by effective conservation are provided, and various scenarios for the future are discussed.     

Vocal Repertoire and Its Behavioral Contexts in the Pied Tamarin, <Emphasis Type="Italic">Saguinus bicolor</Emphasis>

The study of animal sound signals can be useful in assisting conservation strategies. Understanding the vocal repertoires of endangered species and the behavioral contexts in which they are given is relevant for monitoring protocols, such as those based on automated sound recordings. The pied tamarin (Saguinus bicolor) is at risk of extinction because of deforestation and urban growth in its restricted geographic range. Between 2012 and 2015 we studied the vocal repertoire of the species and the contexts in which different signals are emitted. We made focal recordings of eight free-living groups, two rescued individuals, and one temporarily captive group of pied tamarins in Manaus, central Brazilian Amazonia. From the 766 sounds analyzed we identified 12 distinct signals within the range of 2–11 kHz. Most signals were emitted during resting or locomotion. Less frequently emitted signals were associated with intergroup agonistic interactions, foraging, and infant-exclusive vocalizations. These results increased the known vocal repertoire of the pied tamarin providing more reliable baseline data for monitoring the species by means of automated or focal sound recordings.     

Fluid preservation causes minimal reduction of parasite detectability in fish specimens: A new approach for reconstructing parasite communities of the past?

Long‐term datasets are needed to evaluate temporal patterns in wildlife disease burdens, but historical data on parasite abundance are extremely rare. For more than a century, natural history collections have been accumulating fluid‐preserved specimens, which should contain the parasites infecting the host at the time of its preservation. However, before this unique data source can be exploited, we must identify the artifacts that are introduced by the preservation process. Here, we experimentally address whether the preservation process alters the degree to which metazoan parasites are detectable in fluid‐preserved fish specimens when using visual parasite detection techniques. We randomly assigned fish of three species (Gadus chalcogrammus, Thaleichthys pacificus, and Parophrys vetulus) to two treatments. In the first treatment, fish were preserved according to the standard procedures used in ichthyological collections. Immediately after the fluid‐preservation process was complete, we performed parasitological dissection on those specimens. The second treatment was a control, in which fish were dissected without being subjected to the fluid‐preservation process. We compared parasite abundance between the two treatments. Across 298 fish individuals and 59 host–parasite pairs, we found few differences between treatments, with 24 of 27 host–parasite pairs equally abundant between the two treatments. Of these, one pair was significantly more abundant in the preservation treatment than in the control group, and two pairs were significantly less abundant in the preservation treatment than in the control group. Our data suggest that the fluid‐preservation process does not have a substantial effect on the detectability of metazoan parasites. This study addresses only the effects of the fixation and preservation process; long‐term experiments are needed to address whether parasite detectability remains unchanged in the months, years, and decades of storage following preservation. If so, ecologists will be able to reconstruct novel, long‐term datasets on parasite diversity and abundance over the past century or more using fluid‐preserved specimens from natural history collections.     

Observations on the size, dry weight and energy content of the eggs of some demersal fish species from British marine waters

The diameters, dry weights and calorific value of the eggs of seven gadoid species (cod, Gadus morhua , haddock, Melanogrammus aeglefinus , whiting, Merlangius merlangus , Norway pout, Trisopterus esmarkii , lythe. Pollachius pollachius , saithe, P. virens , ling. Molva molva ) and one pleuronectid (plaice, Pleuronectes platessa ) were measured. Ling eggs contain an oil globule; the eggs of the other species do not.
Preservation in formaldehyde solution caused a small (<4%) reduction in egg diameter but a large (15–30%) reduction in dry weight. There was no significant difference between the dry weights of unpreserved eggs weighed after (a) oven-drying at 60° C or (b) freeze-drying. Equations relating the dry weight of unpreserved eggs to unpreserved diameter and to preserved diameter are given for six species, and general equations that may apply to all North Sea gadoids whose eggs lack an oil globule are calculated. The calorific values of the dried, unpreserved eggs of all species except ling were similar (mean 23.19 kJ g⁻¹, S.D. 1.50) but the value for ling was higher (mean 26.92 kJ g⁻¹, S.D. 2.29). The estimated energy content (kJ 1000 eggs⁻¹) of eggs of each species are tabulated.     

New EU legislation for risk assessment of GM food: no scientific justification for mandatory animal feeding trials

This commentary focuses on the potential added value of and need for (sub)‐chronic testing of whole genetically modified (GM) foods in rodents to assess their safety. Such routine testing should not be required since, due to apparent weaknesses in the approach, it does not add to current risk assessment of GM foods. Moreover, the demand for routine testing using animals is in conflict with the European Union (EU) Commission's efforts to reduce animal experimentation. Regulating agencies in the EU are invited to respect the sound scientific principles applied to the risk assessment of foods derived from GM plants and not to interfere in the risk assessment by introducing extra requirements based on pseudo‐scientific or political considerations.     

Preservation effects on wet weight, dry weight, and ash-free dry weight biomass estimates of four common estuarine macro-invertebrates: no difference between ethanol and formalin

Two preservative treatments traditionally used in aquatic sciences, formalin (4%) and ethanol (70%), were compared for their effects on biomass estimations. The effects of both preservatives on wet weight, dry weight, and ash-free dry weight were determined for samples preserved for 10, 21, and 90 days. The effects were studied in four different macrofauna species commonly found in German estuaries: Heteromastus filiformis (Capitellidae, Polychaeta), Hediste diversicolor (Nereididae, Polychaeta), Corophium sp. (Amphipoda, Crustaceae), and Gammarus spp. (Amphipoda, Crustacea). The biomass estimates of preserved samples were compared with those of unpreserved samples. In all four species the loss in wet weight, dry weight, and ash-free dry weight was most pronounced within the first 10 days, and an additional weight loss was recorded between days 10 and 21. However, there was no further loss in weight for samples kept for as long as 90 days in the preservatives. In general, crustaceans exhibited higher weight loss than polychaetes, and smaller species (H. filiformis and Corophium sp.) showed higher weight loss and a higher variability than larger species. As our main result, significant differences between the two preservative treatments did never occur. Our results contradict some earlier investigations on this matter where formalin has been reported to be superior to alcoholic preservatives because weight loss was less pronounced than in ethanol. Factors affecting biomass estimates are discussed and we conclude that, for the macrofauna groups tested, the use of the toxic formalin solution is not justified when the major intent is biomass estimation.     

Bacteriological Quality of Raw Materials Used in Finnish Mink Feed

Mink feed raw materials were analyzed for total bacterial count, the number of faecal streptococci, the coliform count, the number of haemolytic bacteria and the number of sulphite reducing bacteria. The investigation comprised samples from the following raw materials: four slaughter-house offal products, preserved and unpreserved slaughter blood, Baltic herring, cod filletting offal, fish silage, blood meal, fish meal, meat-bone meal, protein concentrate, brewer’s yeast and cereal feed. The slaughter-house offals and unpreserved slaughter blood had the poorest quality, in terms of all the bacterial types for which the samples were analyzed. There were statistically significant differences in bacterial contents between slaughter-house offals from different sources. The preserved slaughter blood had significantly lower bacterial contents as compared to the unpreserved slaughter blood. Single samples of the cod filletting offal, Baltic herring and the blood meal had relatively high total bacterial counts, but the specified mean bacterial counts were relatively low. The bacterial counts for the rest of the investigated raw materials were relatively low.     

Structural features of manganese precipitating bacteria

Studies of biological communities of the past (and their associated activities) are usually dependent upon preservation of fossil material. With bacteria this rarely occurs because of the absence of sufficient fossilizable cellular material. However, some bacteria deposit metabolic products that can, conditions allowing, be preserved indefinitely. In particular, manganese and iron depositing bacteria have the capacity to form preservable microfossils. In order to better understand these microfossils of the past, we have examined present day morphologies of manganese oxidizing bacteria. These bacteria are highly pleomorphic, depending on the growth medium, the age of the culture, and the extent of manganese oxidation. Transmission electron microscopy indicates that manganese may be deposited either intra-or extra-cellularly. The prognosis of the use of morphological information for the interpretation of ancient and modern manganese deposits is discussed.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

Epidermal anatomy of Barthelopteris germarii from the Upper Carboniferous and Lower Permian of France and Germany

The epidermal anatomy of Barthelopteris germarii, a late Paleozoic seed fern, is described on the basis of material from the Upper Stephanian of Central France. A number of features are described for the first time for this species. Some have never been reported for late Paleozoic pteridosperms. Although our material is in most respects very similar to previously described material of B. germarii from Germany, there are also some striking differences, especially the peltate glandular trichomes that are very common in the French but completely absent in the German material. Their presence is most probably an ecological adaptation as in many modern plants. This and other epidermal and gross-morphological features (e.g., the presence of papillae on subsidiary cells of the stomata, anastomosing venation) indicate that B. germarii was well adapted to stressed conditions. Of more general interest is the preservation of the material. There appears to exist a clear relationship between the preservation of anticlinal walls and the thickness of the parenchymatic mesophyll; anticlinal walls are very well preserved where parenchyma was thin. Therefore, the absence of anticlinal walls in fossil cuticles, which has often been used as a taxonomic character, is not necessarily a primary feature.     

Palaeoredox geochemistry and bioturbation levels of the exceptionally preserved early Cambrian Indian Springs biota,Nevada, USA

The early Cambrian Indian Springs biota of western Nevada, USA, exhibits Burgess Shale‐type (BST) preservation of a diverse array of animal phyla, including the earliest definitive echinoderms. It therefore provides an important window on animal life during the Cambrian radiation. The objective of this study was to analyse the trace metal palaeoredox geochemistry and bioturbation levels of this BST deposit in order to characterize the palaeoenvironmental conditions in which these animals lived and their remains preserved. A total of 28 rock samples were collected from outcrops at three previously reported intervals of exceptional preservation at the Indian Springs locality, as well as from one interval not exhibiting such preservation. An additional 20 random samples were collected from talus for comparison. In the laboratory, the samples were analysed for trace metal palaeoredox indices (V/Cr and V/(V + Ni) ratios). Bioturbation levels were assessed through X‐radiography and petrographic thin sections using the ichnofabric index (ii) method. Additional samples from coeval strata of the Poleta Formation in the White‐Inyo Mountains, CA, that lack BST preservation were also analysed with the same methodology. Results indicate that oxic bottom water conditions dominated during deposition of these strata, despite consistently low bioturbation levels. This pattern holds for intervals with BST preservation and those without. Although ephemeral incursions of low‐oxygen waters may have taken place, there is no evidence for persistent oxygen restriction in these palaeoenvironments. The low levels of bioturbation indicate limited mixed layer development and a redox boundary near the sediment–water interface, likely allowing post‐burial BST preservation to occur even in this setting dominated by oxic bottom waters. Palaeoecological reconstructions and taphonomic hypotheses relating to the Indian Springs Lagerstätte must consider the palaeoredox conditions revealed in this study. With the dispensing of anoxic bottom waters as a requirement for BST preservation, other models proposing a role for clay minerals, the presence of hypersaline brines and the actions of Fe‐reducing bacteria as mechanisms for exceptional preservation warrant renewed consideration.     

Effect of preservation period on the viscoelastic material properties of soft tissues with implications for liver transplantation

The liver harvested from a donor must be preserved and transported to a suitable recipient immediately for a successful liver transplantation. In this process, the preservation period is the most critical, since it is the longest and most tissue damage occurs during this period due to the reduced blood supply to the harvested liver and the change in its temperature. We investigate the effect of preservation period on the dynamic material properties of bovine liver using a viscoelastic model derived from both impact and ramp and hold experiments. First, we measure the storage and loss moduli of bovine liver as a function of excitation frequency using an impact hammer. Second, its time-dependent relaxation modulus is measured separately through ramp and hold experiments performed by a compression device. Third, a Maxwell solid model that successfully imitates the frequency- and time-dependent dynamic responses of bovine liver is developed to estimate the optimum viscoelastic material coefficients by minimizing the error between the experimental data and the corresponding values generated by the model. Finally, the variation in the viscoelastic material coefficients of bovine liver are investigated as a function of preservation period for the liver samples tested 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h after harvesting. The results of our experiments performed with three animals show that the liver tissue becomes stiffer and more viscous as it spends more time in the preservation cycle.     

Viability and sporulating capability of Coelomycetes preserved under a range of different storage regimes

The viability and sporulating capability of 45 Coelomycetes strains were evaluated. Strain subcultures were maintained under mineral oil, in soil and on agar slant for different periods of time lasting as long as 50 years, 39 years and 2 years, respectively. Of the 34 strains preserved under mineral oil, 20 maintained their viability but lost the sporulating capability with exception of one strain of Pestalotiopsis guepinii. Of the 16 strains also preserved in soil only one was viable and it was not able to sporulate. All 12 endophytic strains, 11 preserved on agar slant and one under mineral oil remained viable; however, the strain preserved under mineral oil lost its sporulating capability, while the strains on agar slant were only able to sporulate after culturing on sterilized alfalfa twigs. The results demonstrate that routine monitoring, and the use of different preservation methods, specially with the addition of sterilized plant tissue on the culture media for promoting conidiomata formation, is necessary for the success of the Coelomycetes long-term preservation.