Patrick Prunet

Exploration of the hypothalamic–pituitary–adrenal function as a tool to evaluate animal welfare

Measuring HPA axis activity is the standard approach to the study of stress and welfare in farm animals. Although the reference technique is the use of blood plasma to measure glucocorticoid hormones (cortisol or corticosterone), several alternative methods such as the measurement of corticosteroids in saliva, urine or faeces have been developed to overcome the stress induced by blood sampling itself. In chronic stress situations, as is frequently the case in studies about farm animal welfare, hormonal secretions are usually unchanged but dynamic testing allows the demonstration of functional changes at several levels of the system, including the sensitization of the adrenal cortex to ACTH and the resistance of the axis to feedback inhibition by corticosteroids (dexamethasone suppression test). Beyond these procedural aspects, the main pitfall in the use of HPA axis activity is in the interpretation of experimental data. The large variability of the system has to be taken into consideration, since corticosteroid hormone secretion is usually pulsatile, follows diurnal and seasonal rhythms, is influenced by feed intake and environmental factors such as temperature and humidity, age and physiological state, just to cite the main sources of variation. The corresponding changes reflect the important role of glucocorticoid hormones in a number of basic physiological processes such as energy metabolism and central nervous system functioning. Furthermore, large differences have been found across species, breeds and individuals, which reflect the contribution of genetic factors and environmental influences, especially during development, in HPA axis functioning. Usually, these results will be integrated with data from behavioral observation, production and pathology records in a comprehensive approach of farm animal welfare.

Smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch): plasma prolactin, growth hormone, thyroid hormones, and cortisol.

The status of circulating growth hormone and prolactin during the parr-smolt transformation and during seawater adaptation of coho salmon (Oncorhynchus kisutch) was investigated in relation to changes in plasma levels of thyroxine, triiodothyronine, and cortisol, and in hypoosmoregulatory ability. Sampling (biweekly or monthly) occurred between early February and October. When peak hypoosmoregulatory ability was achieved (mid-April), one group of fish was acclimated to seawater over a period of 18 hr and was sampled 1, 3, and 7 days after the introduction of fish to seawater and biweekly thereafter. Plasma prolactin levels rose steadily from the first sampling date to a peak of 15 ng/ml in early April, declined rapidly, and remained low until June when a second increase occurred. Prolactin declined to 2 ng/ml within 1 day of the beginning of seawater adaptation. Growth hormone increased twofold from February to late March, and achieved plateau levels of 20 ng/ml in the period from mid-April to July and then gradually declined to 10 ng/ml in September and October. Plasma levels of growth hormone in seawater-acclimated fish were similar to those of freshwater coho, but with larger fluctuations; no increase was apparent during the first week of seawater acclimation. Plasma cortisol and plasma triiodothyronine increased at the same time as plasma growth hormone; increases in plasma thyroxine occurred later. In general, both growth hormone and cortisol levels were elevated when hypoosmoregulatory ability was high. Conversely, prolactin levels generally showed a negative relationship with hypoosmoregulatory ability.

Differential expression of gill Na + ,K + -ATPase - and -subunits, Na + ,K + ,2Cl - cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

SUMMARY This study examines changes in gill Na+,K+-ATPase (NKA) α- and β-subunit isoforms, Na+,K+,2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and after seawater (SW) transfer in May/June. Gill NKA activity increased from February through April, May and June among both strains in freshwater (FW), with peak enzyme activity in the landlocked salmon being 50% below that of the anadromous fish in May and June. Gill NKA-α1b, -α3, -β1 and NKCC mRNA levels in anadromous salmon increased transiently, reaching peak levels in smolts in April/May, whereas no similar smolt-related upregulation of these transcripts occurred in juvenile landlocked salmon. Gill NKA-α1a mRNA decreased significantly in anadromous salmon from February through June, whereas α1a levels in landlocked salmon, after an initial decrease in April, remained significantly higher than those of the anadromous smolts in May and June. Following SW transfer, gill NKA-α1b and NKCC mRNA increased in both strains, whereas NKA-α1a decreased. Both strains exhibited a transient increase in gill NKA α-protein abundance, with peak levels in May. Gill α-protein abundance was lower in SW than corresponding FW values in June. Gill NKCC protein abundance increased transiently in anadromous fish, with peak levels in May, whereas a slight increase was observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-α1a, -α1b and -α3 isoforms may be important for potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.

Smoltification and seawater adaptation in Atlantic salmon (Salmo salar): plasma prolactin, growth hormone, and thyroid hormones.

To obtain more information on the role of prolactin and growth hormone during the parr-smolt transformation of Atlantic salmon, a population of fish in fresh water was sampled from January to June during two consecutive years. Gill Na+,K+-ATPase activity increased steadily during smoltification and a plasma thyroxine peak was observed 2-3 weeks before the gill Na+,K+-ATPase peak. On the basis of these two parameters, smoltification was considered complete in our populations in April 1985 and May 1986. Two peaks in plasma growth hormone levels occurred in 1986, one in mid-April and the second in mid-May. In both cases, these peaks coincided with a peak in plasma triiodothyronine and preceded the thyroxine peak by 1-2 weeks. Moreover, the second peak which lasted for 1 month coincided with maximal gill Na+,K+-ATPase activity. A decrease in plasma prolactin levels was observed during smoltification of Atlantic salmon in 2 consecutive years. During this period of decreasing and low plasma prolactin levels, gill Na+,K+-ATPase activity increased to its highest values. Atlantic salmon smolts were also directly transferred into seawater. After 2 days or more in seawater, plasma prolactin levels were not significantly different from those on Day 0, whereas in fresh water they showed large fluctuations. All these data indicate that growth hormone may play an important role in the development of hypoosmoregulatory activity. Increased hypoosmoregulatory ability also appears to be associated with low prolactin levels.

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

BackgroundAlthough bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available.DescriptionIn the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster.ConclusionA publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism.

Growth hormone and thyroid hormones during Atlantic salmon, Salmo salar L., smolting, and after transfer to seawater

Abstract Gill (Na + K + )-ATPase activity of juvenile salmon, Salmo salar L., increased “classically” from February to the end of April in experiments in two different years, and its level before transfer to seawater was correlated directly with seawater performance (survival, plsma osmotic pressure and plasma chloride levels). A thyroxine (T4) surge occurred 2–3 weeks before the peak of gill enzyme levels. After the T4 peak the fish were clearly euryhaline, but T4 levels were not correlated with seawater adaptability. They remained able to adapt rapidly to seawater for at least 1 month after the surge. Two significant increases of tri-iodothyronine (T3) occurred while the fish were in freshwater: transfer to seawater either decreased T3 and T4 or had no effect. In freshwater, plasma growth hormone (GH) levels rose sharply concomitant with the T3 peak, 2 weeks before peak gill ATPase activity. After transfer to seawater GH increased significantly, remaining high for 7–10 days, and returning to base levels after 14 days. GH appeared to play a major role in smolting and seawater adaptation.

Measurements of gill (Na+K+)-ATPase activity and plasma thyroid hormones during smoltification in Atlantic salmon (Salmo salar L.)

Abstract Gill (Na + K + )-ATPase activity, triiodothyronine (T3) and thyroxine (T4) circulating blood plasma levels were monitored in 1983 on yearling Atlantic salmon reared in a hatchery in Brittany. The level of enzyme activity increased between February and April to reach a high plateau between mid-April and mid-May. The same fish presented an acute surge of T4 in mid-April and two small increases of T3 in February and May. The gill enzyme activity and the T4 blood plasma level follow a similar pattern, reaching high values at the same moment. The T4 peak occurs at the new moon and does not seem to be correlated with an increase of temperature. Possible relationships between blood plasma T3, T4 and gill (Na + K + )-ATPase activity are discussed.

Plasma prolactin and cortisol concentrations of stressed coho salmon, Oncorhynchus kisutch, in fresh water or salt water ☆

Juvenile coho salmon, Oncorhynchus kisutch, adapted to fresh water or seawater were either acutely handled or continuously stressed by severe confinement. Chronic stress, independent of external salinity, caused a gradual increase in the concentration of circulating prolactin that persisted for 1 to 5 days but lagged behind the cortisol response which peaked much more rapidly and remained elevated. Acutely stressed fish showed a rapid, more transient increase in plasma cortisol titer with no apparent effect on prolactin. Confinement appeared to be more stressful to fish in salt water than to those in fresh water, as judged by their sodium regulatory ability, hormone profiles, and mortality. Stress always elevated plasma prolactin concentrations, regardless of medium or developmental stage.

Aspects of parr-smolt transformation in anadromous and resident forms of brown trout (Salmo trutta) in comparison with Atlantic salmon (Salmo salar)

This study established the existence of a parr-smolt transformation in the anadromous form of brown trout but no such change in the resident form when reared under ambient photoperiod and temperature. Smolting occurred in March–April in the larger trout of the anadromous form and in April–May in salmon. Silvering, a peak of gill Na+,K+-ATPase activity and a simultaneous decrease in plasma prolactin were observed in both salmon and the anadromous form of trout. Bimodality in length frequency and a surge in growth hormone were observed only in Atlantic salmon. No physiological or morphological changes were observed in smaller (<13 cm) trout of the anadromous form or in the resident form. Hypo-osmoregulatory ability was greatest at the time of peak gill Na+,K+-ATPase activity and increased in spring in all trout, irrespective of smolt status. It is concluded that smolting is not as well developed in brown trout as in Atlantic salmon, and is not necessary for seawater adaptation of brown trout. It is also dependent on both stock origin and fish size.

Hydration of rainbow trout oocyte during meiotic maturation and in vitro regulation by 17,20{beta}-dihydroxy-4-pregnen-3-one and cortisol.

SUMMARY Although oocytes of many teleost fish, especially marine species, are subjected to a hydration process during meiotic maturation, which leads to an important volume increase, no noticeable hydration of the preovulatory oocyte has ever been reported in rainbow trout (Oncorhynchus mykiss). In the present study, oocyte water content and dry mass were monitored using consecutive samples taken in vivo from the same female rainbow trout, from 4–5 days prior to ovulation to up to 7 days post-ovulation. In addition, yolk protein electrophoretic patterns were compared between oocytes sampled prior to germinal vesicle breakdown (GVBD) and unfertilized eggs. Furthermore, the effect of the maturation-inducing steroid (17,20β-dihydroxy-4-pregnen-3-one, 17,20β-P), cortisol and 11-deoxycorticosterone (DOC) on oocyte dry and wet masses, as well as GVBD occurrence was assessed in vitro. Finally, mRNA expression profiles of glucocorticoid and mineralocorticoid receptors as well as 11β-hydroxysteroid dehydrogenase (11β-HSD) were monitored in the periovulatory ovary by real-time PCR. Both in vivo and in vitro data showed, for the first time in rainbow trout, that a significant oocyte hydration occurs during oocyte maturation. In addition, an intra-oocyte dry matter increase was reported in vivo during the periovulatory period. However, yolk protein migration patterns were similar in preGVBD oocytes and unfertilized eggs, suggesting that no or little yolk proteolysis occurs during oocyte maturation. We also showed that oocyte hydration can be induced in vitro by 17,20β-P and cortisol but not by DOC. In contrast, GVBD was only observed after 17,20β-P stimulation. Finally, real-time PCR analysis showed an up-regulation of 11β-HSD and glucocorticoid receptor 2 transcripts in the ovary at the time of oocyte maturation. Together, these results suggest that cortisol could participate in the control of oocyte hydration and possibly in other periovulatory ovarian functions.