Lipid Droplet Analysis Using In Vitro Bovine Oocytes and Embryos

The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post‐mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM‐199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi‐fine toluidine blue‐stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro‐cultured embryos.     

Effects of the needle bladder rust (Chrysomyxa rhododendri) on Norway spruce: implications for subalpine forests

Norway spruce trees in the subalpine forests of the European Alps are frequently attacked by the needle rust Chrysomyxa rhododendri. The obligate parasite undergoes a complex life cycle with a host shift between rhododendrons (Rhododendron sp.) and Norway spruce [Picea abies (L.) Karsten] and causes a yellowing and defoliation of the current-year needles in summer. Infected trees show several anatomical, morphological and physiological modifications, including a decrease in pigment content and net photosynthesis of infected needles, lower biomass production and reduced radial and height growth. The consequences are diminished timber yield and cripple growth. Because of repeated heavy infections in recent years, forest managers report increasing difficulties in both natural regeneration and afforestation at high elevation sites, where rhododendrons occur. This review gives a summary of the present knowledge about the effects of C. rhododendri infections on Norway spruce, including so far unpublished findings and with particular attention to the phenomena of resistant trees. Implications for subalpine forests and counter strategies are discussed.     

A new large‐scale and cost‐effective restoration method for cold‐water coral gardens

1. Gorgonians dwelling on the Mediterranean continental shelf are among the most frequent fishing bycatch taxa. These species display several traits, such as long lifespans and slow growth, which make them very vulnerable to the impacts caused by fishing activities with far‐reaching and long‐lasting effects.
2. Hence, restoration and mitigation actions are crucial to enhance and speed up the natural recovery of damaged cold‐water coral gardens. Given the growing concern to develop effective and affordable restoration actions, the present study aims to propose and technically validate a new large‐scale and cost‐effective restoration method.
3. This technique, named ‘badminton method', consists of attaching bycatch Eunicella cavolini colonies to cobble supports and returning them to the continental shelf by gently throwing the gorgonian transplants directly from a boat.
4. Two consecutive field experiments were conducted in order to find the best cobble type support and gorgonian size to be used: first, to evaluate the landing efficiency of gorgonian transplants at different depths (from 5 to 30 m) and second, to evaluate their capability to maintain a correct position over time.
5. Natural cobbles with large gorgonians attached were the best option. Field results and modelling approaches suggest that the transplants would correctly land on the continental shelf seabed in a predicted area of around 60 m². Moreover, they would successfully maintain an upright position ensuring gorgonian survival over time.
6. The success of this method highlights the feasibility of large‐scale and low‐cost restoration actions with promising results for the conservation and recovery of cold‐water coral gardens.

     

Insights into population genetics,connectivity and demographic history of the longnosed skate Dipturus oxyrinchus (Linnaeus, 1758) in the western Mediterranean Sea

1. Genetic information is crucial for the conservation of Dipturus oxyrinchus (Linnaeus, 1758), a threatened large skate with declining populations over most of its geographical range. The main aim of the present study was to investigate the genetic structure, connectivity and demographic history of the longnosed skate in Sardinia (western Mediterranean Sea).
2. Patterns of population structure were assessed in 175 specimens from six sampling sites. Variation in two mitochondrial genes (cytochrome c oxidase subunit I (COI) and control region) highlighted high genetic diversity and low but significant genetic differentiation among sites, which clustered into three groups corresponding to the north‐west, north‐east and south Sardinian coasts.
3. The observed genetic structuring could presumably depend on a combination of past geological events, contemporary restrictions to dispersal and biological characteristics of the species (e.g. site‐fidelity, no pelagic larval stage, limited dispersal of juveniles and/or adults).
4. Demographic analyses showed signs of past population expansion, but substantial current stability of Sardinian populations. From a conservation perspective, these results are encouraging, and indicate that Sardinian populations are still large and stable, and seem not to have suffered negative side‐effects from the ever‐growing fishing pressure in the region.
5. The occurrence of genetic structuring strongly supported the close monitoring of populations to identify any erosion of their gene pool, and high genetic variability of the Sardinian D. oxyrinchus populations could thus represent priority populations for conservation purposes, providing potential sources for recolonization in cases of local extinctions in other areas of the distribution range of the species.
6. When the sequences from Sardinia were compared with those available from other areas, the data seem to exclude the possibility that the Atlantic and Mediterranean host totally isolated populations or even different species, as recently suggested. However, additional markers and a larger sampling sites are needed to confirm these findings.

     

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200? implanted heifers

Background

Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.

Results

Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.

Conclusions

These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.     

Spillover effects of a Mediterranean marine protected area on the European spiny lobster Palinurus elephas (Fabricius, 1787) resource

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Isoëtes sabatina (Isoëtaceae,Lycopodiopsida): Taxonomic distinctness and preliminary ecological insights

1. Isoëtes sabatina is the rarest aquatic quillwort in Europe. Although recently found (2013) in Lake Bracciano (central Italy), the species is just one step away from extinction with an estimated population not exceeding 400 individuals and a spatial range of a few hundred square metres.
2. Lake Bracciano is a deep, oligo-mesotrophic Mediterranean volcanic lake that has been subjected to human activities. From January to October 2017, the lake experienced a dramatic water level decrease (up to −1.50 m), which significantly affected the littoral zone and the habitat of I. sabatina.
3. To improve the chances of survival of I. sabatina, the first eco-taxonomic investigation on this species was carried out to describe its genetic distinctness, physical and chemical requirements and companion species.
4. The phylogenetic position of I. sabatina was investigated by applying standard DNA barcoding methods. Simultaneously, during summer 2019, the physical and chemical features of water and sediments of the I. sabatina population and five small Alpine lakes colonized by Isoëtes echinospora – a supposed close relative – were characterized. These data were then compared with the available data on the trophic requirements of the target obligate aquatic Isoëtes, together with Isoëtes lacustris and Isoëtes malinverniana.
5. The present survey confirmed the taxonomic and ecological distinctness of I. sabatina – providing the first evidence of genetic differentiation from I. echinospora. Isoëtes sabatina grows in waters with temperature, conductivity and total alkalinity up to 30°C, 561 μS cm⁻¹ and 3.45 meq L⁻¹, respectively.
6. The edaphic requirements of I. sabatina confirm its outstanding conservation value, and this study offers a basic understanding of how to prevent its extinction. Now, all possible actions must be taken immediately to save this species.

     

Stewardship and management of freshwater ecosystems: From Leopold's land ethic to a freshwater ethic

1. In 1949, Aldo Leopold formalized the concept of the ‘land ethic’, in what emerged as a foundational and transformational way of thinking about natural resource management, biodiversity conservation, and stewardship in terrestrial systems. Yet, the land ethic has inherent linkages to aquatic ecosystems; Leopold himself conducted research on rivers and lakes, and freshwater ecosystems figured widely in his writing.
2. We reflect on the land ethic and other aspects of Leopold's scholarship to identify key messages that provide insight into the stewardship and management of freshwater ecosystems around the globe. We also frame what we call the ‘freshwater ethic’ around Leopold's legacy. Although Leopold could not have envisaged the stressors affecting modern aquatic ecosystems, his core principles remain salient. These apply not only to ecosystem protection, but also to the ethics of modern conservation economics, sustainability, and the protection of natural capital, in which lakes, rivers, and wetlands now figure prominently.
3. We identify key ‘Aldo-inspired’ recommendations for protecting and restoring freshwater ecosystems in the Anthropocene that emanate directly from his writings (e.g. adopt an ecosystem approach, identify win–win–win scenarios, recognize the irreplaceability of wild waters, and strive for freshwater optimism).
4. In an epoch where links between people and nature are becoming more explicit in environmental management, policy, and governance, we suggest that Aldo Leopold's work illustrates how inspirational, seminal thinkers have offered leadership in this domain. We contend that today there is still much that can be learned from Leopold, especially by the next generation of environmental practitioners, to ensure the effective stewardship of our aquatic ecosystems.
5. We submit that the adoption of a freshwater ethic in parallel with Leopold's land ethic will enhance the stewardship of the world's increasingly threatened fresh waters by raising the profile of the plight of fresh waters and identifying enduring actions that, if embraced, will help conserve and restore biodiversity.

     

Using fishers' anecdotes, naturalists' observations and grey literature to reassess marine species at risk: the case of the Gulf grouper in the Gulf of California, Mexico

Designing fishing policies without knowledge of past levels of target species abundance is a dangerous omission for fisheries management. However, as fisheries monitoring started long after exploitation of many species began, this is a difficult issue to address. Here we show how the ‘shifting baseline’ syndrome can affect the stock assessment of a vulnerable species by masking real population trends and thereby put marine animals at serious risk. Current fishery data suggest that landings of the large Gulf grouper (Mycteroperca jordani, Serranidae) are increasing in the Gulf of California. However, reviews of historical evidence, naturalists’ observations and a systematic documentation of fishers’ perceptions of trends in the abundance of this species indicate that it has dramatically declined. The heyday for the Gulf grouper fishery occurred prior to the 1970s, after which abundance dropped rapidly, probably falling to a few percent of former numbers. This decline happened long before fishery statistics were formally developed. We use the case of the Gulf grouper to illustrate how other vulnerable tropical and semi‐tropical fish and shellfish species around the world may be facing the same fate as the Gulf grouper. In accordance with other recent studies, we recommend using historical tools as part of a broad data‐gathering approach to assess the conservation status of marine species that are vulnerable to over‐exploitation.