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Spotted wolffishAnarhichas minorreproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh sperm to fertilize the eggs that are obtained. In this study, we evaluate the possibility to store spotted wolffish sperm by refrigeration. Spotted wolffish sperm has the particularity that is already motile on stripping, and currently it is not possible to immobilize and reactivate. Thus, sperm refrigeration protocols should focus in extending this motility period that usually lasts up to 2 days. In a first experiment, we evaluated the possibility that the motility period of the sperm was limited by contamination with urine. The urea concentration in the sperm obtained both by stripping (17.10 ± 1.98 mg/dL) and directly from the testis (12.59 ± 2.37 mg/dL) was similar (p > 0.05), which indicate that the sperm collection method used avoid contamination with urine. Afterwards, we tested the possibility that the sperm motility period was limited by energy stores. The ATP concentration (initial value 5.65 ± 0.86 nmol/109cells) remained stable (p = 0.099) during 30 h after sperm collection, and similar values (p = 0.329) were recorded at end of sperm storage in both diluted (3.88 ± 1.35 nmol/109cells) and undiluted samples (4.76 ± 1.08 nmol/109). This indicates that the low intracellular ATP consumption, derived from the slow sperm motility, can probably be compensated rapidly enough by mitochondrial synthesis of ATP in the spotted wolffish sperm. In both experiments, diluted sperm kept higher percentage of motile cells during the storage time.

Trachypithecus, which currently contains 20 species divided into four groups, is the most speciose and geographically dispersed genus among Asian colobines. Despite several morphological and molecular studies, however, its evolutionary history and phylogeography remain poorly understood. Phayre's langur ( Trachypithecus phayrei) is one of the most widespread members of the genus, but details on its actual distribution and intraspecific taxonomy are limited and controversial. Thus, to elucidate the evolutionary history of Trachypithecus and to clarify the intraspecific taxonomy and distribution of T. phayrei, we sequenced 41 mitochondrial genomes from georeferenced fecal samples and museum specimens, including two holotypes. Phylogenetic analyses revealed a robustly supported phylogeny of Trachypithecus, suggesting that the T. pileatus group branched first, followed by the T. francoisi group, and the T. cristatus and T. obscurus groups most recently. The four species groups diverged from each other 4.5-3.1 million years ago (Ma), while speciation events within these groups occurred much more recently (1.6-0.3 Ma). Within T. phayrei, we found three clades that diverged 1.0-0.9 Ma, indicating the existence of three rather than two taxa. Following the phylogenetic species concept and based on genetic, morphological, and ecological differences, we elevate the T. phayrei subspecies to species level, describe a new species from central Myanmar, and refine the distribution of the three taxa. Overall, our study highlights the importance of museum specimens and provides new insights not only into the evolutionary history of T. phayrei but the entire Trachypithecus genus as well.

The aquaculture of meagre (Argyrosomus regius) requires methods for the control of reproduction that enable the production of families from specific individuals for selective breeding programs. We experimentally determined the parameters required for an in vitro fertilisation protocol. A total of 14 females and 5 males (mean ± S.D. weights of 20.45 ± 6.22 and 15.94 ± 2.75 kg, respectively) were used. Selected females had vitellogenic oocytes >550 μm in diameter and males had fluid sperm upon application of abdominal pressure. Both sexes were treated with an injection of 15 μg kg−1 of gonadotropin-releasing hormone agonist (GnRHa) to induce oocyte maturation/ovulation and enhance sperm production. To determine the timing of ovulation and window of high egg viability, females were stripped serially every 2.5 h beginning 35 h after GnRHa treatment. Sperm was obtained 24 h after GnRHa treatment and was diluted 1/4 in modified Leibovitz for storage at 4 °C until use. Sperm quality parameters such as percentage initial spermatozoa motility, duration of motility, velocity and density were determined using computer assisted sperm analysis (CASA). In vitro inseminations were made in duplicate or triplicate batches of eggs from each spawn by mixing 0.5–1 mL of eggs, 20–40 μL diluted sperm (pooled from two males) and 100 mL of seawater. Fertilisation success was examined at spermatozoa (spz): egg ratios between ~2000 and 400,000 spz egg−1. The optimal time for stripping ovulated females was ≤3 h after ovulation, which was the window of optimal egg viability. Ovulation under the conditions of this study was close to 38 h after GnRHa treatment, with a range from 35 to 41 h. Beginning from 3 h after ovulation, egg viability declined probably due to overripening. Sperm diluted in Leibovitz maintained motility and velocity for as long as 7 h after collection. Spermatozoa motility (%) and average path velocity (VAP, μm/s) of sperm samples obtained from males before GnRHa injection declined rapidly after activation compared to the samples obtained 24 h post-injection, with significant decreases respectively after 75 and 45 s. A minimum ratio of 150,000 spermatozoa egg−1 was necessary to ensure high fertilisation success. The acquired knowledge of the present study will aid the aquaculture industry and future research on selective breeding programs for meagre. info:eu-repo/semantics/acceptedVersion

6 pages, 4 figures, Consecutive treatments with recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) stimulate spermatogenesis and potentiate sperm production in pubescent specimens of the oligospermic Senegalese sole (Solea senegalensis). However, sperm production in response to the hormones is highly variable, and the steroidogenic potential of the testis may be diminished due to sustained hormone supply. Here, we compared the effectiveness of low (9 μg/kg) and high (18 μg/kg) doses of rFsh and rLh to improve sperm production in adult sole during late winter-early spring (onset of the natural spawning period), and in autumn under a controlled temperature of 12 °C (period of testicular recrudescence). Treatment with rFsh over six weeks during spring, followed by a single rLh injection, did not enhance sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by high Lh plasma levels (~17 ng/ml) before rFsh treatment. In contrast, in autumn, when the Lh circulating levels were much lower (~3 ng/ml), the low doses of rFsh and rLh generated a four-times increase in sperm production, whereas the high doses of the hormones were ineffective. However, treatment with rLh, regardless of the effect of rFsh, improved the motility of spermatozoa during both spring and autumn. These data confirm that consecutive rFsh and rLh treatments increase sperm production and quality in adult sole males, although they seem to be highly sensitive to the rFsh dose. The efficiency of recombinant gonadotropins also appears to be season-dependent despite the asynchronous nature of the sole testis, This work was funded by the Spanish Ministry of Science and Innovation (MINECO) (AGL2017-84013-R to F.C. and AGL2013-41196-R to J.C.), and the Spanish National Institute for Agronomic Research (INIA)-European Fund for Economic and Regional Development (FEDER) (RTA2014-00048 to N.D.). Participation of FC, SR and WG was funded, respectively, by a “Ramon y Cajal” contract (MINECO), a predoctoral grant from AGAUR (Government of Catalonia), and a predoctoral grant from the National Board of Science and Technology (CONACYT, Mexico). This work was also supported by the CERCA Programme (Government of Catalonia).

Under intensive captive conditions, wild-caught flathead grey mullet (Mugil cephalus) females remained arrested in previtellogenic stages of gonadal development and no sperm could be obtained from males. With the aim to induce oogenesis from previtellogenesis to oocyte maturation, induce the release of sperm and obtain fertilized eggs, female and male flathead grey mullet were treated with Mugil cephalus single-chain recombinant gonadotropins (rGths), follicle-stimulating (rFsh) and luteinizing (rLh) hormones. In Experiment 1, fish were treated with a weekly dose of rFsh (15 μg kg−1), which in females significantly (P 75% motile spermatozoa) was obtained from males in both experiments, and in Exp. 2 the addition of rLh induced the production of higher quantities of sperm that were used to fertilise the eggs. Although fertilisation was low (0.4%), these fertilized eggs with embryo development produced viable larvae (71% hatching). In comparison, control females remained arrested at previtellogenesis and control males did not produce sperm. The study demonstrated that both rGths are effective to induce the process of oogenesis in female flathead grey mullet and to obtain flowing sperm from males, adding more data to confirm the roles of the Gths in teleost gametogenesis. This is the first report, in a teleost species, of the use of rGths (rFsh and rLh) to induce oogenesis from previtellogenesis through to maturation to obtain eggs and larvae. This advance provides the bases for the development of therapies for the use in the aquaculture of teleost of commercial interest or the conservation of endangered species. info:eu-repo/semantics/acceptedVersion

Background Dysphagia related to intubation affects almost one in five patients in the intensive care unit; it may vary due to fluctuations in cerebral status and fatigue and lead to pneumonia, re-intubation and increased mortality. Taking care of patients' nutritional needs is an integral part of nursing and nurses must therefore be able to assess risk of dysphagia at all hours. However, Danish validated methods for use in the critically ill patient population are lacking. Aim To translate and adapt the Yale Swallow Protocol method for implementation in a Danish intensive care unit setting. Methods Translation and adaption followed WHO's recommendations for translation and adaption of instruments. Face validity of the Danish version was evaluated in four group interviews. These were digitally recorded, transcribed and analysed using deductive content analysis. All interviewees gave informed consent. Findings The translated version of the Yale Swallow Protocol was easy to understand, although nurses needed to have the method explained and needed training in using the method. In addition, the Yale Swallow Protocol must be adapted to local practice in terms of identifying patients at risk of dysphagia and deciding where to document results of the screening. Conclusion The translated version of the Yale Swallow Protocol proved conceptually and culturally sound and acceptable to intensive care unit nurses. Implementation should be followed by theoretical and practical training to build a nursing vocabulary relating to dysphagia and strengthen nurses' sensitivity to clinical manifestations consistent with dysphagia.

Individuals differ in how they cope with stressful situations along a behavioural continuum, being proactive and reactive at the extremes of this continuum. Proactive individuals are usually bold, highly active and take risks, while reactive organisms are generally shy, exhibit low activity and avoid risky situations. Definitions of stress coping styles state that proactive and reactive traits are consistent over time and across contexts. The present study evaluated the individual differences in stress coping style, physiological changes and reproductive status in Senegalese sole juveniles and breeders over three and two-years, respectively. To determine stress coping style, the fish were subjected to three individual (restraining, new environment, confinement) and one group screening test (risk taking). Both groups were tested on three occasions, juveniles were tested each year and adults were tested in the first year and twice (spring and autumn) in the second year. On the third year, a proportion of the juveniles initiated puberty and the reproductive status of all individuals was assessed and compared with their behavioural responses. Results demonstrated individual differences that were consistent with proactive and reactive traits in juveniles and breeders. Significant intra-individual repeatability and consistency of juveniles and breeder's behavioural responses were observed over time and across situations. In addition, glucocorticoid levels (cortisol) were consistent for individuals. Another result to highlight was that juveniles that past puberty and initiated gametogenesis had significant higher activity, risk predisposition and lower plasma cortisol levels compared to fish that remained immature (did not initiate puberty). info:eu-repo/semantics/acceptedVersion

Individual animals commonly adopt different stress coping styles that have been shown to impact reproductive success and differ between sexes (female/male) and origin (wild/hatchery). Hatchery reared Senegalese sole (Solea senegalensis) exhibit a behavioural reproductive dysfunction and a complete failure to spawn viable eggs. Hence, the present study examined whether 1) reproductive success of Senegalese sole was linked to coping styles and 2) behavioural differences exist in relation to sex or origin. A total of 198 breeders held in two research institutions were submitted to three individual tests (restraining, new environment and confinement) and one grouping test (risk taking). In addition, a blood sample to quantify cortisol, glucose and lactate levels was obtained from each individual after completing the individual tests. Senegalese sole breeders showed individual differences in behaviour across the different behavioural tests that were consistent with proactive and reactive coping styles traits. However, the most striking result was that reproductive success, sex and origin of Senegalese sole was not biased to any specific coping style. Indeed, the behavioural responses were similar and consistent between fish grouped by reproductive success, sex and origin. This study presented information that contrasts with different studies on dominant aggressive species and indicated that social non-aggressive species such as Senegalese sole follow a cooperative strategy that favours equal opportunities between stress coping styles and sexes. Therefore, results suggest that maintaining both coping styles strategies are fundamental for a sustainable breeder population approach. info:eu-repo/semantics/acceptedVersion

Aim of study: The reproductive performance over a five year period in three different sole broodstocks: wild males with cultured females (WM), cultured males with wild females (CM), and a control of cultured males and females (C). Area of study: Cantabria, Northern Spain. Material and methods: The parental contribution was evaluated through the allocation of hatched larvae and a behavioural study was performed using underwater video recordings. Main results: Fertilized natural spawns were only obtained from group WM showing a significantly higher mean annual volume of floating eggs compared with groups CM and C. Over the entire 5-yr a higher number of spawns, total and floating volumes of eggs were obtained compared with groups CM and C. The analysis of four polymorphic microsatellites enabled the identification of the individuals involved in the fertile spawns by parental assignment. The percentage of individuals contributing to spawns ranged between 56.3% and 75% showing fidelity patterns. However between 46.2% and 68.6% of the contribution to production was a result of the crossing of three single couples. According to the behaviour analysis, group WM presented the highest peak of activity at 20:00 h and a higher activity profile during the night, moreover, the courtship behaviours “swim follow” and “swim followed”, were only recorded in group WM. Research highlights: The study has importance for aquaculture as it demonstrated how broodstocks with wild males and cultured females had adequate egg production for a commercial operation to supply eggs and implement single sex breeding programs., Sí

The life cycle of Senegalese sole (Solea senegalensis) is not closed in captivity due to a reproductive dysfunction related to the lack of participation of cultured male breeders in the courtship. To discern a possible solution to this social reproductive dysfunction, the main objective of this study was to determine the effect of the presence of spawning wild Senegalese sole breeders on the courtship and reproduction of cultured breeders. Three experimental groups were formed: Control group (n = 10) formed with only cultured sole; groups M1 and M2 constituted of mixed-origin sole (10 cultured and 8 or 9 wild breeders). All cultured breeders were from the same stock, which had never successfully reproduced, whilst the wild broodstock had spawned viable eggs in captivity. All groups were held in the same captive conditions and spawning, and behaviour were recorded for each group over four spawning seasons. All spawns were collected, and the parameters registered were floating and non-floating eggs, fertilization and hatching rates. In addition, parental analysis was made of larvae from viable spawns. Behaviour was studied with video recordings to compare locomotor activity and courtship behaviours including the "Follow" behaviours where sole swim after each other in a procession. Fertilized spawns were obtained from the mixed-origin groups (M1 and M2) including spawns involving a cultured male. The cultured males also participated actively in the "Follow" behaviours with the courting wild sole and this participation of cultured males increased significantly over the four years of the experiment. Male percentage participation in the "Follow" behaviours was positively correlated (R = 0.81) to participation in spawning to indicate the importance of increasing cultured male participation in the "Follow" behaviours. A total of seven spawns were obtained from a cultured male that fertilized eggs from one cultured female and two wild females. The same cultured female also spawned with a wild male. No viable spawns were obtained from the Control group and locomotor activity and courtship behaviour counts were significantly lower than in the experimental mixedorigin groups. This is the first report of cultured male breeders participating in reproductive behaviour and spawning, which could be associated with social learning processes, mate selection and dominance where cultured males copied the behaviour and spawning of wild Senegalese sole breeders. Spanish National Institute for Agronomic Research (INIA)-FEDER [RTA201100050, RTA2014-00048] FCT - Foundation for Science and TechnologyPortuguese Foundation for Science and Technology [UID/Multi/04326/2019] info:eu-repo/semantics/submittedVersion

Meagre is a relatively new aquaculture species with great potential in large scale European aquaculture. The primary objective of the study was to describe, for the first time, parentage allocation and assign offspring to their parents for an industrial scale production system. A total of 800 meagre fish were sampled from two large cages in January and May 2016, both part of a commercial farm site in Valencia, Spain. All fish originated from the same spawning event obtained from a broodstock of 6 females and 13 males. However, due to differential growth during the juvenile stage the fish were graded into two groups, a group of larger juveniles that was transferred to one cage (batch 1) and a group of smaller juveniles that were transferred to the second cage (batch 2). Total length and weight was measured for all fish that were genotyped with a 10 microsatellite loci multiplex to infer parentage based on parental genotypes. Parentage assignment rate was high (87.5% for batch 1 and 95% for batch 2) and provided evidence that offspring belonged to 20 families. Half of the broodstock was identified as probable parents of the offspring (five females and seven males).Between the two sea-cages, a slight differential composition for the same families was encountered. The fifteen shared families that the offspring were assigned to, were analyzed for statistical significant differences concerning body weight and total body length, differences which were observed in 3 families for both batches. We estimated the heritability for body weight and total body length, as well as the genetic and phenotypic correlations for these two traits. Batch 1 showed higher heritability estimates than batch 2 with the genetic and phenotypic correlation estimates being almost the same for both batches. Certain parents contributed more offspring and exhibited dominance in spawning. Similarly, the growth related traits of body weight and total body length of the dominant parents correlate, putatively, with the statistical important differences that are observed in these three families. info:eu-repo/semantics/acceptedVersion

The aims of this study were the characterization of the upper olfactory epithelium of cultured and wild Senegalese sole mature males at histological and transcriptomic (using RNA-Seq) level. No significant differences in tissue structure, cell types and cellular distribution pattern (olfactory sensory neurons) were identified between cultured and wild specimens. Deep transcriptomic analysis showed 2387 transcripts were differentially expressed between cultured and wild groups. A detailed analysis identified the differentially expressed transcripts included some olfactory receptors (OR, TAAR and V2R-like) and transcripts related with the control of reproduction such as the brain aromatase cytochrome P450 and tachykinin-3. Also a wide set of genes related with lipid sensing, metabolism and transport were differentially expressed and these transcripts were often down-regulated in cultured fish. Furthermore, cultured males presented a higher expression of genes related with goblet cells and mucin production that modulates innate and adaptive immune responses. All these changes in gene expression could be explained by different nutritional status and diet preference. The different expression of transcripts related to olfaction, reproduction, nutrient sensing and immune system demonstrate distinct differences in functionalities between cultured and wild soles providing new clues about the sexual dysfunction in this species.

A multiple GnRHa injection method for inducing spawning of high fecundity and quality in meagre was optimized. Specifically, the study (a) examined how long females would continue spawning in response to consecutive, weekly GnRHa injections, if maintained at the natural spawning temperature, and (b) evaluated whether males can produce adequate amounts of good quality sperm for the same period, with the assistance of a GnRHa therapy. Combined with stable temperatures (19–20 °C) that are prevalent during the early spawning season (April–May) in the Mediterranean Sea, each weekly GnRHa injection induced ~ 2 consecutive spawns per week for a period of 17 weeks. Fish spawned consistently 2 days after treatment and produced high fecundity and egg quality ( i.e. fertilization, hatching and early larval development). Maximum mean total fecundity (± S.D.) obtained was 1,415,000 ± 149,000 eggs kg − 1 in 32 spawns, being the highest total season fecundity reported for meagre, and significantly higher than the estimated maximum potential fecundity for the species. At the same time, sperm of consistently high quality ( i.e. initial motility, duration of motility and storage survival) was produced in response to treatment with multiple GnRHa implants, though towards the end of the experiment a significant reduction (ANOVA, P ≤ 0.05) in spermatozoa density was observed. This protocol is expected to enhance greatly commercial production of meagre. Statement of relevance The present study reports on an optimized protocol for the induction of consistent and repeated spawning in meagre, based on the use of weekly GnRHa injections combined with stable temperatures that are prevalent at the early spawning season in the Mediterranean Sea. This protocol produced eggs of high fecundity and quality ( i.e. fertilization, hatching and early larval development) for a period of > 4 months, and it is expected to enhance greatly commercial production of meagre.

A non-ossified unilateral subcutaneous fibroma was diagnosed in the distal femoral region of a 5-year-old Nooitgedacht mare. Histopathological examination of the excised mass revealed long interweaving bundles of semi-mature monotonous collagenous connective tissue with fusiform nuclei without mitotic figures. The mare made an uneventful recovery following surgical removal of the neoplasm. Subcutaneous fibromas should be considered in the differential diagnosis of skin swellings associated with the limbs of horses.

Methods are needed to manage the sperm of Senegalese sole (Solea senegalensis), which will enable the industry to use artificial fertilisation to reproduce hatchery raised sole and implement breeding programs. The present study aimed to (a) describe the male reproductive and urinary system, (b) describe the effects of urine contamination on sperm quality and (c) examine the use of extenders for short term chilled storage of sole sperm. Nine males were dissected to describe the male reproductive and urinary system. A total of 49 males were examined and 32 (65.3%) provided adequate sperm samples of the study. Initially the samples were described by appearance (colour, transparency and fluidity) and sub-samples analysed for sperm quality, urea concentration, osmolality, pH and protein concentration. Cell concentration and sperm quality parameters, percentage motility, curvilinear velocity (VCL) and average path velocity (VAP), were measured using ImageJ CASA. Control samples and samples diluted (1:3) in six different extender solutions (modified Leibovitz, Ringer, NAM, Sucrose, Stor Fish® and Marine Freeze®) were stored short-term (4 °C) and tested zero, three, six and 24 h after collection. The close proximity of the reproductive and the urinary systems, especially the sperm ducts being attached to the urinary bladder makes obtaining sperm without urine contamination appear difficult. All the samples appeared to be contaminated with urine. Samples that appeared to be contaminated with urine (yellow colour) had similar spermatozoa cell concentration and urea concentration as samples that appeared not to be contaminated with urine (whitish colour), although motility was significantly lower in yellow samples. Seminal plasma urea concentration was positively correlated with osmolality. Cluster analysis grouped samples with significantly higher sperm quality and pH and significantly lower urea concentration and osmolality to indicate that urine contamination negatively affected sperm quality by increasing osmolality and decreasing pH. Amongst the six extender solutions Leibovitz and Marine Freeze® preserved significantly higher percentage motility 24 h after collection. Ringer, NAM and Stor Fish® were intermediate and Sucrose was similar to control samples that significantly decreased motility 3 h after collection. Taken together all sole sperm samples probably had urine contamination, which is difficult or impossible to avoid especially if all the sperm available needs to be collected. The extenders, Leibovitz and Marine Freeze® were used to maintain sperm quality and mitigate the negative effects of urine contamination. The collection and short term chilled storage in extenders of sole sperm from the majority of males in a broodstock (65.3%) can provide a valid sperm management system for industrial application for artificial fertilisation, however, further work is needed. info:eu-repo/semantics/acceptedVersion

Diclofenac, a non-steroidal anti-inflammatory pharmaceutical agent was responsible for the death of millions of Gyps vulture's in the Indian sub-region with the safety of the other non-steroidal anti-inflammatory drugs (NSAIDs) being questionable. With preclinical safety testing not well established for avian species unlike for mammalian and environmental toxicity, we ask the question if a preclinical model could have predicted the toxic effect of the drug. For this study, we test an Organisation for Economic Co-operation and Development (OECD) guideline 223 for assessing the acute toxic potential of pesticides in birds by exposing three avian species to the drug. Exposed Japanese quails (Coturnix japonica) and Muscovy ducks (Cairina moschata) demonstrated clinical signs and pathology similar to those previously reported in vultures viz. hyperuricemia, depression, death, visceral gout and nephrosis. However, exposed domestic pigeons (Columba livia domestica) were insensitive. Following a pharmacokinetic analysis, the drug was well absorbed and distributed in the pigeons with a half-life below 6 h. A toxicokinetic evaluation in quails showed poisoning was due to metabolic constraint, with a half-life and mean residence time above 6 h and 8 h respectively resulting in death. Toxicity seen in the ducks was however not related to metabolic constraint but hyperuricemia as metabolism was rapid [half-life (1-2 h) and mean residence time (2-3 h)] irrespective of survival or death. Despite succumbing to diclofenac, the established oral median lethal dose (LD50) of 405.42 mg/kg and 189.92 mg/kg in Japanese quails and Muscovy ducks respectively from this study were substantially higher than those reported for Gyps vultures (0.098 mg/kg) which is as a result of the rapid elimination of the drug from the body in the former species. More importantly, it suggests that these species are not suitable as surrogates for non-steroidal anti-inflammatory drug toxicity testing and that the toxicity of diclofenac in vultures is idiosyncratic most likely as a result of species specific metabolism.

Mycoplasma gallisepticum (MG) is the primary cause of chronic respiratory disease in poultry. We investigated the protective efficacy of the live-attenuated ts-11 and 6/85 MG vaccines against a local MG strain and, in order to enhance signs and mimic a typical field situation, we co-infected birds with a virulent strain of QX-like infectious bronchitis virus (IBV). Both vaccines showed similar ability to protect infected chickens from clinical signs, although ts-11 performed slightly better. Despite the lower protection against clinical disease, 6/85-vaccinated birds had significantly (P ≤ 0.05) lower tracheal lesion scores and mucosal thickness at day 28 post-vaccination (7 days post-challenge [dpc] with MG, 2 dpc IBV) and day 31 post-vaccination (10 dpc MG challenge, 5 dpc IBV) compared to ts-11 vaccinated birds, but these difference was not significant at day 33 (12 dpc MG, 7 dpc IBV). Pathogen infection and replication was assessed by qPCR, and the 6/85 vaccine produced a more significant (P ≤ 0.05) reduction in MG replication in the lungs, kidneys and livers but enhanced late replication in bursae and caecal tonsils. In contrast, the ts-11 vaccine had a more pronounced reductive effect on replication in tracheas, air sacs, bursae and heart at days 28 and 31, yet increased replication in lungs. Interestingly, both vaccines provided non-specific protection against IBV challenge. The co-challenge model provided useful data on vaccine efficacy, especially on days 31 and 33, and tracheas, lungs, air sacs, kidneys, liver and caecal tonsils were the best organs to assess.

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID) was responsible for the death of millions of vultures on the Asian subcontinent, following the consumption of diclofenac contaminated carcasses. The aim of this research was to establish if liver slices could serve as an alternate means of predicting the toxicity of NSAIDs in Gyps vultures. The Cape vulture liver slices was prepared and incubated with four NSAIDs for 6 h. A percent clearance of 1.0 ± 0.253, 0.58 ± 0.153, 0.961 ± 0.312 and 1.242 ± 0.406 (%/h*g) was attained for diclofenac, carprofen, ketoprofen and meloxicam respectively. Both meloxicam and diclofenac exerted toxic effects on the hepatic cells. Protein content indicated that the vulture tissue had lower enzyme levels than expected for an animal of its size. The poor distinction between the ex vivo hepatic percent clearance of meloxicam and diclofenac indicates that liver slices is not an ideal model to investigate NSAIDs toxicity in Cape vulture.

Antimicrobial peptides (AMPs), components of innate immunity, play an important role in protecting fish. In this study we report the molecular cloning of full open reading frames and characterization of expression of three AMP genes (β-defensin (defb), hepcidin (hep2), piscidin (pisc) in meagre (Argyrosomus regius). A phylogenetic analysis of the expressed sequences obtained shows the defensin isoform forms a clade with the other members of the beta class of this family, hepcidin corresponds to hepcidin 2, and piscidin corresponds to class I of its respective family. Gene expression profiles of AMPs was investigated, by means of quantification of mRNA in nine development stages, from 8 days post-hatching (dph) to accomplishment of juvenile form (120 dph). During development it was demonstrated defb, hep2, pisc were expressed in all stages of larval development and in juvenile tissues (kidney, spleen gut and gill). Moreover, expression patterns suggest the expression levels of theses AMPs are influenced by live prey (rotifer, Artemia) and first intake of commercial diet. Induction experiments in vivo (24 h) and in vitro (4, 12, 24 h) with PAMPs (LPS, poly (I:C), β-glucan) revealed significant changes in gene expression of the three AMP genes, in kidney, spleen, gut and gill. However, expression profiles differed in magnitude and time course response. defb expression shows a similar trend in vivo and in vitro in kidney at 24 h after LPS and β-glucan stimulation. The hep2 expression levels were up-regulated upon β-glucan challenge in vivo, more in gut and gills than kidney, while in vitro hep2 expression was up-regulated in kidney cells by LPS, poly (I:C), β-glucan (4 h). pisc expression was up-regulated in kidney cells, splenocytes by β-glucan, but in gill cells by poly (I:C) and β-glucan in vivo. However, pisc expression was upregulated in kidney cells by β-glucan and gill cells by LPS at 4 post-stimulation in vitro. These data suggest that AMPs play an important role in defense against pathogens, with each AMP having differing efficacies against specific types of microorganisms, although follow-up studies focusing on the biological activities in fish are needed. info:eu-repo/semantics/acceptedVersion

Meagre (Argyrosomus regius), a teleost fish of the family Sciaenidae, is part of a group of marine fish species considered new for Mediterranean aquaculture representing the larger fish cultured in the region. Meagre aquaculture started ~ 25 years ago in West Mediterranean, and the supply of juveniles has been dominated by few hatcheries. This fact has raised concerns on possible inbreeding, urging the need for genetic information on the species and for an assessment of the polymorphisms found in the genome. To that end we characterized the muscle and liver transcriptome of a pool of meagre individuals, from different families and phenotypic size, to obtain a backbone that can support future studies regarding physiology, immunology and genetics of the species. The assembled transcripts were assigned to a wide range of biological processes including growth, reproduction, metabolism, development, stress and behavior. Then, to infer its genetic diversity and provide a catalogue of markers for future use, we scanned the reconstructed transcripts for polymorphic genetic markers. Our search revealed a total of 42,933 high quality SNP and 20,581 STR markers. We found a relatively low rate of polymorphism in the transcriptome that may indicate that inbreeding has taken place. This study has led to a catalogue of genetic markers at the expressed part of the genome and has set the ground for understanding growth and other traits of interest in meagre. info:eu-repo/semantics/acceptedVersion

Disabled people are under-represented in higher education (HE), especially in senior posts. This article reports research on the perspectives of career progression into leadership held by disabled staff in one post-1992 university. Findings indicated that some participants were already engaged in leadership or aspired to such roles, and reported positive experiences. However, participants also experienced barriers to progression, including a lack of awareness of equality and diversity among managers and colleagues, inadequate professional development opportunities and the competitive organisational culture of management that could impact on their health and work–life balance. We conclude that disabled staff in HE do not yet have equal opportunities for progression into leadership roles compared with their non-disabled peers. There is a need for realignment of the culture of leadership and management to make it more compatible with the full inclusion of disabled staff in HE such that their unique contributions are valued.

27 pages, 10 figures, supporting information https://doi.org/10.1371/journal.pone.0174387.s001 https://doi.org/10.1371/journal.pone.0174387.s002, Captive flatfishes, such as the Senegalese sole, typically produce very low volumes of sperm. This situation is particularly prevalent in the first generation (F1) of reared sole males, which limits the development of artificial fertilization methods and the implementation of selective breeding programs. In this study, we investigated whether combined treatments with homologous recombinant follicle-stimulating (rFsh) and luteinizing (rLh) hormones, produced in a mammalian host system, could stimulate spermatogenesis and enhance sperm production in Senegalese sole F1 males. In an initial autumn/winter experiment, weekly intramuscular injections with increasing doses of rFsh over 9 weeks resulted in the stimulation of gonad weight, androgen release, germ cell proliferation and entry into meiosis, and the expression of different spermatogenesis-related genes, whereas a subsequent single rLh injection potentiated spermatozoa differentiation. In a second late winter/spring trial corresponding to the sole’s natural prespawning and spawning periods, we tested the effect of repeated rLh injections on the amount and quality of sperm produced by males previously treated with rFsh for 4, 6, 8 or 10 weeks. These latter results showed that the combination of rFsh and rLh treatments could increase sperm production up to 7 times, and slightly improve the motility of the spermatozoa, although a high variability in the response was found. However, sustained administration of rFsh during spawning markedly diminished Leydig cell survival and the steroidogenic potential of the testis. These data suggest that in vivo application of rFsh and rLh is effective at stimulating spermatogenesis and sperm production in Senegalese sole F1 males, setting the basis for the future establishment of recombinant gonadotropin-based hormone therapies to ameliorate reproductive dysfunctions of this species, This work was supported by the CERCA Programme (Government of Catalonia), and partially funded by the Spanish Ministry of Economy and Competitivity (MINECO) (Grant no. AGL2013-41196-R to JC), and the Spanish National Institute for Agronomic Research (INIA)-European Fund for Economic and Regional Development (FEDER) (Grant no. RTA2014-00048 to ND). Participation of FC and WG was funded, respectively, by a >Ramon y Cajal> contract (MINECO) and a predoctoral grant from the National Board of Science and Technology (CONACYT, Mexico)

Senegalese sole (Solea senegalensis) have a high potential for aquaculture that is hampered by reproductive behavioural problems. These problems result in limited breeder participation in spawning. The present study provided an ethogram and described mate selection and spawning of captive wild Senegalese sole. Two tanks of breeders were studied that had 29 and 25 breeders (mean weight = 1.6 ± 0.1 kg). The behaviour was studied during 20 periods of 24 hours: 10 periods where spawning events were recorded and 10 control periods without spawning events. Periods where spawning occurred had three times more locomotor activity than periods without spawning. Two distinct behaviours, termed the “following” behaviour and the “coupled swim”, were only observed during periods with spawning. The courtship sequence (n=12) began with males predominantly involved in “following” behaviours, whilst females remained mainly stationary on the bottom of the tank. Males rested on the females and encouraged the females to begin swimming. When the female began to swim the male swam under the female and the pair made a “coupled swim” to the surface to release gametes. Gamete release was strictly in pairs of one male with one female. Failed “coupled swims” without gamete release were 5.6 times more frequent than successful “coupled swims”. Mate selection was evident as the sole engaged in: paired spawning, males displayed to females, males encouraged females to spawn and females accepted or rejected the male’s advances. The mate selection process provided the opportunity for fish to dominate the spawning and also demonstrated how fish were excluded from spawning. info:eu-repo/semantics/publishedVersion

9 pages, 7 figures, Chemical communication is better understood in freshwater fish than marine fish. The Senegalese sole (Solea senegalensis) is a marine flatfish wherein one of the problems in aquaculture is the poor reproductive performance of hatchery-bred males. Is chemical communication involved in the reproduction of this species Urine, intestinal fluid and mucus samples were taken from adult fish (wildcaught and hatchery-bred) over the spawning season (March-May), and assessed for olfactory potency using the electro-olfactogram (EOG). The effect of stimulation of the olfactory system with adult female urine on circulating luteinizing hormone (LH) levels was also tested in males. Intestinal fluid and urine were potent olfactory stimuli for both juvenile and adult conspecifics, evoking large-amplitude, concentration-dependent EOG responses, with thresholds of detection at approximately 1:106. However, the amplitude of the response to urine depended on the sex and state of maturity of both the donor and the receiver. Most olfactory activity could be extracted by C18 solid-phase cartridges. Urine from mature females evoked a slight, but significant, increase in circulating LH levels in mature males 30 min after exposure. Furthermore, the olfactory potency of urine differed between wild-caught and hatchery-bred fish; however, contrary to expectations, urine from wild-caught females was less potent than that from hatchery-bred females. Taken together, these results strongly suggest that faeces-and urine-released odorants are involved in reproduction in the Senegalese sole, and establish a basis for further investigation into pheromonal communication in marine teleosts, The study was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria and the EU through FEDER 2014-2020 ‘Programa Operativo de Crecimiento Inteligente’ projects RTA2011-00050 and RTA2014-00048 coordinated by N.J.D. Partially funded by the Ministerio de Economía y Competitividad (MINECO; grant AGL2013-41196-R to J.C., RTA2013-00023-C02-01 to M.M.). E.F. was supported by a PhD FPI-INIA grant awarded by MINECO, Spain. This study also received Portuguese national funds from the Fundação para a Ciência e a Tecnologia (Foundation for Science and Technology, Portugal) through project UID/Multi/04326/2013

Dominance is defined as the preferential access to limited resources. The present study aimed to characterise dominance in a non-aggressive flatfish species, the Senegalese sole (Solea senegalensis) by 1) identifying dominance categories and associated behaviours and 2) linking dominance categories (dominant and subordinate) with the abundance of selected mRNA transcripts in the brain. Early juveniles (n= 74, 37 pairs) were subjected to a dyadic dominance test, related to feeding, and once behavioural phenotypes had been described the abundance of ten selected mRNAs related to dominance and aggressiveness was measured in the brain. Late juveniles were subjected to two dyadic dominance tests (n= 34, 17 pairs), related to feeding and territoriality and one group test (n= 24, 4 groups of 6 fish). Sole feeding first were categorized as dominant and sole feeding second or not feeding as subordinate. Three social behaviours (i. “Resting the head” on another fish, ii. “Approaching” another fish, iii. “Swimming above another” fish) were associated with dominance of feeding. Two other variables (i. Total time occupying the preferred area during the last 2 hours of the 24 h test, ii. Organisms occupying the preferred area when the test ended) were representative of dominance in the place preference test. In all tests, dominant fish compared to subordinate fish displayed a significantly higher number of the behaviours “Rest the head” and “Approaches”. Moreover, dominant sole dominated the sand at the end of the test, and in the group test dominated the area close to the feed delivery point before feed was delivered. The mRNA abundance of the selected mRNAs related to neurogenesis (nrd2) and neuroplasticity (c-fos) in dominant sole compared to subordinate were significantly different. This is the first study to characterise dominance categories with associated behaviours and mRNA abundance in Senegalese sole and provides tools to study dominance related problems in feeding and reproduction in aquaculture.

[EN] This article reviews the use of hormonal treatments to enhance sperm production in aquaculture fish and the methods available for evaluating sperm quality. The different types of testis development are examined and a brief review is presented of the endocrine regulation of spermatogenesis in fishes, including the increasing evidence of the existence of spermatozoa subpopulations. Hormonal manipulations are employed to induce spermatogenesis in species such as the freshwater eels, to synchronize maximal sperm volume to ovulation for in vitro fertilization and to enhance sperm production in species with poor spermiation. The hormones that are employed include gonadotropins (GtHs) of piscine or mammalian origin, and gonadotropin-releasing hormone agonists (GnRHa) administered by injections or controlled-release delivery systems, with or without dopaminergic inhibitors. Pheromones in the culture water and hormones added to the sperm in vitro have also been employed to enhance spermiation and sperm quality, respectively, in some fishes. Hormonal therapies usually do not affect sperm quality parameters, except in cases where fish fail to spermiate naturally or produce very small volumes of high-density sperm. Different parameters have been used to evaluate fish sperm quality, including sperm volume and density, spermatozoa motility and morphometry, and seminal plasma composition. The development of Computer-Assisted Sperm Analysis (CASA) systems made possible the estimation of a higher number of sperm motion parameters using an objective, sensitive and accurate technique. The development of Assisted Sperm Morphology Analysis (ASMA) software has introduced a new approach for sperm evaluation studies, demonstrating changes in the spermatozoa related to reproductive season, hormonal treatments or the cryopreservation processes, and how these may be related to changes in sperm motility and fertilization capacity. The article concludes with a few practical protocols for the enhancement of sperm production in aquaculture species., We would like to thank the COST Action FA1205 (AQUAGAMETE) titled “Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources - The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry” for initiating the production of this special issue and funding partially this article. JFA has been partly funded by the Spanish Ministry of Economy and Competitiveness (REPRO-TEMP; AGL2013-41646-R) and the European Training Network IMPRESS (Marie SklodowskaCurie Actions; Grant agreement n°: 642893). ND has been funded partly by the INIA-FEDER project RTA2011-00050, and CCM and ND have been supported also by the EU project DIVERSIFY (7FP-KBBE-2013-GA 602131).

Senegalese sole was one of the earliest identified candidate species with high potential for aquaculture diversification in the south of Europe.íts culture has been possible, and commercially attempted, for several decades, but intensive production has been slow to take off. This has been explained mostly by serious disease problems, high mortality at weaning, variable growth and poor juvenile quality. However, a strong and sustained research investment that started in the eighties has led to a better understanding of the requirements and particularities of this species. More recently, better management and technical improvements have been introduced, which have led to important progress in productivity and given a new impetus to the cultivation of Senegalese sole. As a result, the last 5 years have marked a probable turning point in the culture of sole towards the development of a knowledge-driven, competitive and sustainable industry. This review will focus on the main technical improvements and advances in the state of knowledge that have been made in the last decade in areas as diverse as reproductive biology, behaviour, physiology, nutritional requirements, modulation of the immune system in response to environmental parameters and stress, and characterization and mitigation of the main disease threats.ít is now clear that Senegalese sole has important particularities that differentiate it from other current and candidate marine aquaculture species, which bring about important challenges, some still unsolved, but also notable opportunities (e.g. a nutritional physiology that is better adapted to dietary vegetable ingredients), as will be discussed here. © 2014 Wiley Publishing Asia Pty Ltd., SM holds a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO) and is supported by the European Commission Marie Curie Actions (FP7-PEOPLE-2010-RG, Project No. 274184). BC and SE are supported by Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BPD/77210/2011 and SFRH/BPD/49051/2008, respectively).

The assembly, or disassembly, of ecological communities is thought to be driven by competition, environmental filtering, and dispersal limitation. These processes leave patterns in the functional, phylogenetic, and taxonomic diversity of communities. Bat communities in the tropics tend to have many species that are niche specialists with varying dispersal propensities. We investigated the effects of fragmentation on bat communities in an isolated forest fragment and a nearby larger forest preserve in Belize. Over four field seasons (2014–2017), we captured over 1,480 individuals from 32 species using mist nets and harp traps. The community in the fragment was a nested subset of species (20) compared to the preserve (30), and species richness was relatively stable over time. Functional richness was higher in the preserve than in the fragment, and species in the preserve were more closely related phylogenetically than expected by chance. Closely related species and species with different diet guilds co-occurred at both sites more often than distant relatives and those with the same diet guild. Bat species with flexible roost use had higher abundance in the fragment than the preserve, while closely related roost-specialist species had higher abundance in the preserve. Local extirpation and decreased dispersal are the most likely mechanisms of community disassembly in this system, and variation in roosting habits results in nonrandom community composition. These results have significant implications for the effects of ongoing deforestation and habitat fragmentation in Belize and adjacent dry forest areas.

9 pages, 4 figures, 1 table, The intensive culture of the Senegalese sole (Solea senegalensis) is hampered by the low or null fertilization rates exhibited by the first generation (F1) of reared males. To investigate the regulation of the reproductive processes in this species by the pituitary gonadotropins follicle-stimulating and luteinizing hormones (Fsh and Lh, respectively), we developed a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for Lh measurements. Quantification of the Fsh and Lh plasma levels in cultured sole using the Lh ELISA developed here, and a previously developed ELISA for Fsh, indicated that in both males and females circulating Fsh steadily increased during autumn and winter and prior to the major spawning in spring, whereas an Lh surge occurred specifically during spawning. The increase in Fsh was associated with a rise of plasma levels of the steroid hormones testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β (E2), but that of Lh was concomitant with a strong decline of the levels of E2 in females and of 11-KT in males, possibly reflecting a rapid steroidogenic shift promoting the final maturation of gametes. Comparison of the plasma levels of gonadotropins and steroids between wild and F1 fish during autumn and spring revealed that F1 males showed significantly lower plasma Lh titres compared to wild males, whereas the levels of T and 11-KT were similar or more elevated in the F1 fish. These data suggest that an impaired Lh secretion during spawning, and perhaps altered Lh-mediated mechanisms in the testis, may be underlying causes for the low reproductive performance of Senegalese sole F1 males. © 2015 Elsevier Inc, This work was funded by the Spanish Fundación Ramón Areces (to J.C.), and partially supported by the Spanish Ministry of Science and Innovation (MICINN) Aquagenomics project (CSD2007-00002 to S.Z.) and the Spanish National Institute for Agronomic Research (INIA) project RTD2011-00050 (to N.D). Participation of E.F. was financed by a predoctoral fellowship from INIA