Pauline Wischhusen
email: pauline.wischhusen@inra.fr

Maroussia Parailloux

Pierre-André Geraert

Mickael Briens

Maïté Bueno

Sandra Mounicou

Brice Bouyssière

Philip Antony

Jesu Prabhu

Sadasivam S Kaushik

Benoit Fauconneau

Brice Bouyssiere

Antony Jesu Prabhu

Sadasivam J Kaushik

Stéphanie Fontagné-Dicharry

Effect of dietary selenium in rainbow trout (Oncorhynchus mykiss) broodstock on antioxidant status, its parental transfer and oxidative status in the progeny

Keywords: Rainbow trout, Broodstock, Progeny, Selenium, Antioxidant

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

While fishmeal based diets naturally contain high amounts of selenium (Se) covering the nutritional requirements [START_REF] Hilton | The requirement and toxicity of selenium in rainbow trout (Salmo gairdneri)[END_REF], plant based diets are generally lower and vary more widely in their Se content [START_REF] Combs | Selenium in global food systems[END_REF]. With the ongoing replacement of fish meal and fish oil in aquafeeds, there is a need to better understand the metabolic role of Se, which is complex and not yet fully explored. The Se requirements in fish are reported to vary between 0.15 and 0.70 mg/kg diet [START_REF] Jesu Prabhu | Mineral requirements of fish: a systematic review[END_REF], depending on species [START_REF] Watanabe | Trace minerals in fish nutrition[END_REF], life stage [START_REF] Bell | Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri)[END_REF] and bioavailability of the Se form [START_REF] Wang | Organic selenium sources, selenomethionine and selenoyeast, have higher bioavailability than an inorganic selenium source, sodium selenite, in diets for channel catfish (Ictalurus punctatus)[END_REF]Penglase et al., 2014a). Thereby, organic Se forms have been described

to have a higher bioavailability compared to inorganic forms, leading to increased tissue Se content [START_REF] Le | Bioavailability of selenium from different dietary sources in yellowtail kingfish (Seriola lalandi)[END_REF][START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF][START_REF] Jesu Prabhu | Mineral requirements of fish: a systematic review[END_REF]. Dietary Se, both in surplus and deficiency, can have severe consequences for the fish. Already a dietary Se level of 3 mg/kg, slightly above the recommended level, can in the long term induce adverse effects [START_REF] Berntssen | Safe limits of selenomethionine and selenite supplementation to plant-based Atlantic salmon feeds[END_REF]. On the other hand, Se deficiency can reduce the expression and activity of selenoproteins [START_REF] Hesketh | Nutrigenomics and selenium: gene expression patterns, physiological targets, and genetics[END_REF][START_REF] Elia | Effects of selenium diets on growth, accumulation and antioxidant response in juvenile carp[END_REF]Penglase et al., 2014a), of which many are involved in the antioxidant protection of the cell [START_REF] Papp | From selenium to selenoproteins: synthesis, identity, and their role in human health[END_REF]. In a previous study, working with low Se levels in rainbow trout (Oncorhynchus mykiss) fry, the positive impact of Se supplementation on Se-dependent GPX activity and gene expression was demonstrated, especially when an organic Se supplement was used [START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF]. Se supplementation was found to increase the expression of selenoprotein P not only in rainbow trout, but also in zebrafish (Danio rerio) (Penglase et al., 2014a;[START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF][START_REF] Pacitti | Selenium supplementation in fish: A combined chemical and biomolecular study to understand Sel-Plex assimilation and impact on selenoproteome expression in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Wang | Expression of selenoprotein genes in muscle is crucial for the growth of rainbow trout (Oncorhynchus mykiss) fed diets supplemented with selenium yeast[END_REF]. Under a dietary Se deficiency, besides the effects at the cellular level, a reduced growth and an increased mortality in fish are also reported [START_REF] Wang | The pathology of selenium deficiency in Cyprinus carpio L[END_REF]. In rainbow trout, the negative impact of Se deficiency on body weight could not be detected [START_REF] Bell | Some effects of selenium deficiency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri)[END_REF], but liver vitamin E levels were significantly reduced and the combined effect of vitamin E and Se deficiency led to a reduced weight gain [START_REF] Bell | Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri)[END_REF]. Most of the studies investigating the effect of dietary Se on the metabolism in fish have been undertaken with juveniles or early stages and there is little information on the effects of Se in broodstock nutrition, except studies investigating the impact of Se-supplementation at high levels [START_REF] Hardy | Effects of dietary selenomethionine on cutthroat trout (Oncorhynchus clarki bouvieri) growth and reproductive performance over a life cycle[END_REF]Penglase et al., 2014aPenglase et al., , 2014b)). However, the reproduction period is substantially different to other life stages as energy is not used for growth, but mobilized to the reproductive organs; especially in females where nutrients are accumulated in the ovarian oocytes during vitellogenesis. Therefore, Se deficiency effects observed at early stages might be different in broodstock fish and might affect spawn quality. Male fertility and reproduction have been shown to be reduced in several animal species fed Se-deficient diets [START_REF] Hosnedlova | A summary of new findings on the biological effects of selenium in selected animal species-a critical review[END_REF] and could be linked to the presence of the selenoprotein GPx4 in male sperm [START_REF] Imai | Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice[END_REF]. A reduced antioxidant capacity of offspring by maternal Se deficiency have already been described in broilers [START_REF] Pappas | Maternal organo-selenium compounds and polyunsaturated fatty acids affect progeny performance and levels of selenium and docosahexaenoic acid in the chick tissues[END_REF][START_REF] Zhang | Different sources of maternal selenium affect selenium retention, antioxidant status, and meat quality of 56-day-old offspring of broiler breeders[END_REF][START_REF] Xiao | The protective effects of different sources of maternal selenium on oxidative stressed chick embryo liver[END_REF]. Furthermore, the dietary supplementation of Se in poultry breeders led to an increased fertility of males and maternal transfer of Se, improving the oxidative status in the progeny [START_REF] Słowińska | Effect of organic and inorganic forms of selenium in diets on turkey semen quality[END_REF][START_REF] Wang | Influence of dietary selenomethionine supplementation on performance and selenium status of broiler breeders and their subsequent progeny[END_REF][START_REF] Zhang | Different sources of maternal selenium affect selenium retention, antioxidant status, and meat quality of 56-day-old offspring of broiler breeders[END_REF]. Se toxicity trials undertaken with zebrafish [START_REF] Thomas | Developmental and persistent toxicities of maternally deposited selenomethionine in zebrafish (Danio rerio)[END_REF] and brown trout (Salmo trutta) [START_REF] Covington | Effects of in situ selenium exposure and maternal transfer on survival and deformities of brown trout (Salmo trutta) fry[END_REF] suggest that a parental transfer of Se in fish exists at toxic levels. The current study was undertaken to address whether similar effects are detected in broodstock fish fed nutritionally relevant dietary levels of Se in different forms.

Material and Methods

Experimental set up

Three-year-old rainbow trout (Oncorhynchus mykiss) females and males of the same genetic origin from the INRA experimental fish farm of Lées-Athas (Pyrénées-Atlantiques, France) were used as broodstock. Rainbow trout broodstock were randomly allocated to three circular 2-m diameter tanks supplied with flow-through (1L per sec) spring water at 8 ± 1 °C with 25 females (initial mean weight: 1.1 ± 0.2 kg, second reproduction season) and 15 males (0.9 ± 0.3 kg) per tank (stocking density: 40 kg/m 3 ). The fish were hand fed once a day to visual satiation for a period of 6 months prior to spawning. Each fish was tagged in the dorsal muscle with a passive integrated transponder (PIT, 12 x 2 mm, ISO 11784/11785, IER, Suresnes, France) and the individual growth was followed until spawning. During the spawning period, fish were regularly checked for ovulation and at spawning, oocytes of each female were collected through stripping. At the first spawning date, one female from the SSgroup and two females from the SO-group were over-mature (with oocytes exhibiting polar condensation of vitellus and lipid goblets) and they were excluded from the trial as well as one NC female during the second spawning date due to low spawning quantity and quality.

Otherwise, two pools of 50 oocytes from each female were lined up to measure total length and afterwards weighed to calculate the average diameter and weight of oocytes, respectively. All other eggs were fertilized with sperm collected on the same day from a pool of males of the same dietary group. Eggs from each female were reared separately in small trays supplied with flow-through spring water at 8 ± 1 °C to follow individual hatching data. The embryonic development was followed until swim-up fry stage. The survival was monitored and dead eggs were removed every two days. Survival rates were calculated at eyed stage (33 dpf), hatching (46 dpf) and swim-up stage (65 dpf).

Sample collection

Broodstock fish were sampled at spawning. First, fish were anaesthetized with benzocaine (30 mg/L) for stripping and afterwards killed by a sharp blow on the head for liver and muscle sampling. Muscle tissue was sampled in duplicate at the dorsal fin as approximately 15g of fillet. The weights of whole fish, spawn (total eggs collected from each female after stripping and removal of coelomic fluid) and liver were recorded to calculate absolute and relative fecundity (number of eggs/female and number of eggs/kg female, respectively), gonadosomatic index (GSI, percentage of spawn weight to weight of female) and hepatosomatic index (HSI, percentage of liver weight to weight of fish). Tissue samples and oocytes (2 x 50 units per female) were immediately frozen in liquid nitrogen and stored at -80 °C until further analysis. The progeny was sampled at swim-up fry stage. Pooled fry, for every female separately, were euthanized with an overdose of benzocaine (60 mg/L), weighed and afterwards subdivided in different sample size according to planned analysis, before freezing as whole-body fry in liquid nitrogen and stored at -80°C until further analysis.

All procedures were performed in compliance with the European Directive 2010/63/EU for the protection of animals used for scientific purposes and the French Decree no. 2013-118 for animal experimentation.

Experimental diets

Diets were manufactured using a twin-screw extruder (BC 45, Clextral, Firminy, France) at the INRA experimental facilities in Donzacq (Landes, France). The three diets (NC, SS and SO) were based on plant-derived proteins and had similar levels of crude protein (50%) and total lipid (22%) and differed only in Se content (Table 1). The NC-diet containing 0.3 mg Se/kg feed was not supplemented with Se and served as negative control. The SS and SO diets were supplemented with either sodium selenite (SS-diet) or hydroxy-selenomethionine (Selisseo®, SO-diet). In both supplemented diets, the targeted Se concentration was 0.6 mg Se/kg, which remained lower to concentrations usually found in fish meal based diets [START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF], but feed analyses gave a final dietary Se content of 0.8 mg Se/kg in SS diet and 0.7 mg Se/kg in SO diet.

Proximate, mineral and fatty acid analysis

For proximate composition of diets, dry matter (DM) was determined after drying at 105 °C for 24h, protein (N × 6.25) by Kjeldahl method after acid digestion [START_REF] Iso | Water Quality-Determination of Kjeldahl Nitrogen-Method after Mineralization with Selenium[END_REF], ash after 10h incineration at 550 °C and gross energy in an adiabatic bomb calorimeter. Total lipid in diets and broodstock muscle was extracted and quantified gravimetrically following [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF], using dichloromethane instead of chloroform. Fatty acid methyl esters were prepared and analyzed as previously described [START_REF] Fontagné | Lipid peroxidative stress and antioxidant defence status during ontogeny of rainbow trout ( Oncorhynchus mykiss)[END_REF]. Total phosphorus was determined by the molybdate blue/ascorbic acid method at 820 nm after mineralization and acid digestion [START_REF] Afnor | Viandes, préparations de viande et produits à base de viande -Détermination de la teneur en phosphore total[END_REF]. Total Se was measured using inductively coupled plasma mass spectrometry (ICP-MS, Agilent series 7500cx) by Ultra Trace Analyses Aquitaine (UT2A, Pau, France) for diets, broodstock tissues and oocytes according to [START_REF] Vacchina | Total selenium quantification in biological samples by inductively coupled plasma mass spectrometry (ICP-MS)[END_REF] and by the Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les Matériaux (IPREM, Pau, France) for swim-up fry as described by [START_REF] Godin | Influence of dietary selenium species on selenoamino acid levels in rainbow trout[END_REF]. Selenomethionine (SeMet) and selenocysteine (SeCys) were analyzed by liquid chromatography (HPLC, Agilent series 1200) coupled to ICP-MS by UT2A for oocytes according to Vacchina et al. (2018) and by IPREM for swim-up fry according to [START_REF] Godin | Influence of dietary selenium species on selenoamino acid levels in rainbow trout[END_REF].

Antioxidant enzyme activity and mRNA levels

Antioxidant enzyme activity was measured as previously described by [START_REF] Fontagné-Dicharry | Antioxidant defense system is altered by dietary oxidized lipid in first-feeding rainbow trout (Oncorhynchus mykiss)[END_REF] in 0.3 g samples of broodstock liver and pooled whole-body fry. Briefly, all samples were first homogenized with an Ultra-turrax in an eight-time dilution of 20 mMphosphate buffer (pH 7.4) containing 1mM EDTA, afterwards centrifuged at 2000g for 10 min, the supernatant diluted in 0.5% triton solution and incubated for one hour before using the different enzyme assays. The glutathione peroxidase (GPX, EC 1.11.1.9) activity was measured in a solution of 50 mM phosphate buffer (pH 7.4), 1 mM EDTA, 2 mM sodium azide, 2 mM GSH, 0.1 mM NADPH and 0.2 mM glutathione reductase (GR) following the reduction of cumene hydroperoxide (0.2 mM) for Se-dependent GPX (SeGPX) and H2O2 (50 µM) for total GPX (totGPX) at 30 °C and 340 nm. Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured by monitoring the inhibition of nitrotetrazolium reduction at 550 nm, and was expressed as the amount of enzyme required to inhibit the rate of nitrotetrazolium reduction by 50% per mg protein at 37 °C. The catalase (CAT, EC 1.11.1.6) activity was measured by following the reduction of H2O2 in a solution of 67 mM phosphate buffer, 1 mM EDTA and 20 mM H2O2 (pH 7.4) at 30 °C and 240 nm. GR (EC 1.8.1.7) activity was determined measuring the reduction of NADPH in a solution containing 100 mM phosphate buffer (pH 7.5), 1 mM GSSG and 50 µM NADPH at 30 °C and 340 nm. Glutathione-Stransferase (GST, EC 2.5.1.18) activity was measured in a solution of 100 mM phosphate buffer (pH 6.5), 1 mM EDTA, 1 mM GSH and 1 mM CDNB at 30°C and 340nm, using the extinction coefficient of 9.61 mM -1 /cm. One unit of enzyme is defined as the amount needed to catalyze the reaction of 1 µM substrate per minute. The protein concentration was determined according to [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF], using bovine serum albumin (BSA) as a standard.

Total RNA was isolated from 100 mg samples of broodstock liver and a pool of three wholebody swim-up fry by using Trizol reagent (Invitrogen, Cergy-Pontoise, France). For quantitative RT-PCR, complementary DNA was generated from 1 mg total RNA using SuperScript III RT (Invitrogen) and a mix of oligo (dT)15 and random primers (Promega, Charbonnières, France). For each sample, RT was performed in duplicate and quantitative PCR analyses were performed in LightCycler® 480 Instrument II (Roche Diagnostics, Meylan, France) using LightCycler® 480 SYBR Green I Master mix (Roche Diagnostics).

Total reaction volume was 6 μL, with 2 μL of the diluted RT reaction mixture (dilution 40) and 4 μL of master mix added with 0.4 mM of each primer (Table 2). The PCR protocol was initiated at 95 °C for 10 min for initial denaturation of the cDNA and hot-start Taq-polymerase activation, followed by 45 cycles of a three-step amplification program (15 s at 95 °C, 10 s at the melting temperature of 60 °C, 4.8 s at 72 °C). Melting curves were systematically monitored (5 s at 95 °C, 1 min at 65 °C, temperature slope at 0.11 °C/s from 65 to 97 °C) at the end of the last amplification cycle to confirm the specificity of the amplification reaction.

Relative quantification of target gene transcripts was performed using β-actin as the reference gene and NC as the reference group using the ΔΔCt method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

Glutathione and oxidative status in the progeny

Total (GSHt), oxidized (GSSG) and reduced glutathione (GSH) were measured in 0.3 g homogenized liver tissues and 1 g of pooled oocyte and fry samples using Cayman glutathione assay kit (Bertin Pharma, Montigny-le-Bretonneux, France) according to the manufacturer's instructions with protein concentration assessed by the method of [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] using BSA as a standard. Lipid peroxidation was assessed by measuring concentration of 8-isoprostanes (8-isoPT), the product of non-enzymatic peroxidation of arachidonic acid by reactive oxygen species in 0.5 g of pooled fry as described by [START_REF] Fontagné | Effects of dietary oxidized lipid and vitamin A on the early development and antioxidant status of Siberian sturgeon (Acipenser baeri) larvae[END_REF] using Cayman Chemical 8-isoprostane enzyme immunoassay kit (Spi-Bio, Massy, France) according to the manufacturer's instructions. Protein oxidation was assessed using the spectrophotometric determination of protein carbonyls by derivatization with 2,4-dinitrophenylhydrazine (DNPH) at 370 nm according to [START_REF] Armenteros | Analysis of protein carbonyls in meat products by using the DNPH-method, fluorescence spectroscopy and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS)[END_REF] with protein concentration calculated from absorption at 280 nm in the blank sample using bovine serum albumin (BSA) as standard. HPLC was used for determination of vitamin C according to the method described by [START_REF] Maeland | Examination of the qualitative ability of some cold water marine teleosts to synthesise ascorbic acid[END_REF]. Tocopherols were extracted from a pool of six oocytes and three whole-body fry according to [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF] using dichloromethane instead of chloroform. They were analyzed by HPLC on a Phenomenex Luna® PFP(2) column (100 Å, 3 µm, 100 x 2 mm), with an isocratic mobile phase of methanol/water (93:7 v/v) at a flow rate of 0.5 mL/min and column oven temperature of 40 °C using a Waters Alliance 2695 separation module with a Waters 2475 multi wavelength fluorescence detector set at 295/330 nm (excitation/emission) according to [START_REF] Górnaś | An alternative RP-HPLC method for the separation and determination of tocopherol and tocotrienol homologues as butter authenticity markers: A comparative study between two European countries[END_REF]. The concentration of tocopherol per individual was calculated using the average weight of oocyte or whole-body fry.

Statistical analysis

Results are given as mean ± standard error (SEM). Differences between dietary groups were analyzed in statistical software R (R Development Core Team, 2008) or SigmaStat 3 (SPSS, Chicago, IL, USA) using one-way ANOVA. Tocopherol data were analyzed using two-way ANOVA to compare data from oocytes with data at swim-up fry stage. All data were tested for normal distribution and homogeneity and only in case the assumptions were not respected, the ANOVA was performed instead on ranks. The binary spawning data were analyzed pairwise using χ 2 test. Percentages were arc-sin transformed before analysis. A Newman-Keuls post-hoc test was used to compare means, when a significant difference (P < 0.05) was found.

Results

Growth and reproductive performance in broodstock

All three diets were readily accepted by rainbow trout broodstock. In males and females, no significant difference in initial or final body weight was detected between the three dietary groups (Table 3). In females, no significant difference in daily growth between the control group and the supplemented groups was found. However, the daily growth index was higher in females fed SO compared to SS. In the groups NC and SS, six males and in SO, one female and four males died during the feeding trial before spawning. HSI, muscle lipid content and muscle fatty acid profile (data not shown) were not significantly different between groups for both males and females. At the end of the spawning period in January, the total number of fish spawning was significantly higher in the two Se-supplemented groups SS and SO compared to the control group NC. Also, females fed the organic Sesupplemented diet SO began to spawn significantly earlier compared to females fed the inorganic Se-supplemented diet SS (Fig. 1). No significant difference in reproductive performance, including GSI, fecundity or egg size could be detected (Table 3). During embryonic development, there was no significant difference in survival between NC and the supplemented groups. However, the survival rate was significantly lower in the SS-group than in SO from hatching onwards.

Se and its transfer from broodstock to progeny

Total Se content was significantly higher in muscle of females fed SO, compared to the ones fed NC or SS (Fig. 2A). The same tendency was noticed in female liver, but no significant difference between the three treatments was detected (Fig. 2B). Total Se content in milt was not significantly different between groups (Fig. 2C). On the other hand, total Se content in oocytes was significantly higher for females fed SO and lower in NC group compared to the SS treatment (Fig. 2D).

The majority of Se in oocytes was present in the form of SeMet, which was significantly higher in Se-supplemented groups compared to the control group NC with the highest concentration in the organic Se treatment, while the SeCys content was not significantly different between the groups (Table 4). As for oocytes, the total Se content in swim-up fry before first-feeding was the lowest in NC, followed by SS and highest in SO (Fig. 2E).

However, with smaller sample size for Se speciation analyses (n = 3), no significant difference between the groups SS and SO were found (Table 4). SeCys content was not significantly different between the three dietary groups of swim-up fry. Only swim-up fry from the organic Se-supplemented group SO showed an increase in SeMet concentration compared to the two other groups (Table 4). The total Se content per individual decreased significantly from oocyte to swim-up fry stage in SS (18 ± 2 to 15 ± 1 ng Se per individual) and SO (29 ± 3 to 23 ± 1 ng Se per individual) groups whereas no significant change in Se content was noticed for NC (11 ± 1 ng Se per individual).

Changes in selenoproteins and other antioxidant enzyme activity and mRNA levels in broodstock and progeny

There was no effect of broodstock nutrition on GPX (Fig. 3A and3B) or other antioxidant enzyme activity in liver of female or male (Table 5). In whole-body swim-up fry, totGPX activity was significantly higher in both Se-supplemented treatments with the highest activity in the SO group (Fig. 3C). This was associated with higher SeGPX activity in SO-group compared to the two other groups. The activity of other antioxidant enzymes was not significantly affected by Se supplementation (Table 5).

The mRNA expression of selenoprotein genes including the different GPX genes was not significantly affected by dietary Se supplementation in broodstock female liver except for SelPa and SelPa2 that were higher expressed in liver of broodstock females fed the inorganic Se-supplemented diet SS compared to the two other groups (Fig. 4A). The other antioxidant genes not coding for selenoproteins were not significantly affected by dietary Se supplementation in female liver (Fig. 4B). In male liver, all the three selenoproteins P were found to be higher expressed in the Se-supplemented treatments SS and SO while GPx1a was significantly higher expressed only in the inorganic Se treatment SS and MsrB1 only in the organic Se treatment SO (Fig. 5A). GPX4a2 mRNA levels were significantly higher in liver of broodstock male fed the organic Se-supplemented diet SO than in the inorganic Sesupplemented group SS. Regarding non-selenoprotein genes, CAT, Gclc and MsrB2 mRNA levels were significantly higher in liver of broodstock males fed the two Se-supplemented groups while Keap1 mRNA levels were only higher in male livers from the inorganic Se supplemented treatment SS compared to the control group NC (Fig. 5B). In whole-body swim-up fry, gene expression of the SO group with organic Se supplementation was enhanced not only for the two selenoproteins Pa and Pa2, but also for the glutathione peroxidases GPx1a and GPx1b2 (Fig. 6A). For other non-selenoproteins involved in antioxidant protection, no significant difference in mRNA levels could be detected between the swim-up fry groups except for CAT and MsrB2 that were higher expressed in the organic Se-supplemented group SO (Fig. 6B).

Glutathione levels in rainbow trout broodstock and offspring

GSHt, GSSG and GSH contents in male or female liver were not significantly different between the three dietary groups (Fig. 7A and7B). In whole-body swim-up fry, GSH was significantly lower in the organic Se-supplemented group SO, while GSSG was higher in both Se-supplemented groups compared to the control group NC (Fig. 7C). Glutathione content was also measured in oocytes, but GSSG was not detectable, while no significant difference in total glutathione was detected between the three dietary groups of oocytes (0.68 ± 0.04 µmol/mg protein).

Oxidative status in rainbow trout offspring

At whole-body swim-up fry stage, the GSH/GSSG ratio was significantly lower in the offspring from broodstock fed the Se-supplemented diets SS and SO compared to the control group NC (Table 6). The concentration of 8-isoprostanes was significantly lower in whole-body swim-up fry in the inorganic Se treatment SS compared to the control group NC and organic Se-supplemented group SO. Parental Se-supplementation had no significant effect on the concentration of protein carbonyls in whole-body swim-up fry. However, there was a tendency for lower protein carbonyl content in the organic Se treatment SO, indicative of an improved antioxidant status. This was confirmed with vitamin C that displayed significantly higher amounts in whole-body swim-up fry from the organic Se-supplemented SO treatment compared to the two other groups.

Tocopherol content in rainbow trout offspring

Organic Se supplementation in rainbow trout broodstock diet significantly increased the αtocopherol concentration per individual in the offspring. The concentrations of γ-and δtocopherols were not significantly affected by dietary broodstock Se supplementation. A significant decrease between oocyte and swim-up fry stage was noticed for δ-tocopherol (Table 7).

Discussion

The recent edition of NRC (2011) stated that micronutrient requirements for farmed fish and shrimp are another area where knowledge is lacking; whether dietary micronutrient requirements should be based on minimal levels required to prevent clinical deficiency signs or impaired growth or based on other criteria is an open question. Studies on the determination of nutrient requirements should rely on more than one response criterion to test the effect of increasing levels of a nutrient and eventually estimate minimal dietary inclusion levels for each criterion tested, whenever possible.

Organic and inorganic Se supplementation affects spawning rate and timing

We did not find any positive effect of Se-supplementation to a plant-ingredient based diet on the growth performance of rainbow trout broodstock. It could indicate that the Se levels used in the control diet did not provoke a severe deficiency situation, as was also shown, in an earlier study with rainbow trout, that a Se level of 0.025 mg Se/kg did not significantly affect growth rates [START_REF] Bell | Some effects of selenium deficiency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri)[END_REF]. On the other hand, the growth performance in all treatments of this experiment was low, which can be attributed in part to low water temperatures and the use of plant-based diets or it might be related to the increased demand of energy in reproductive organs during spawning period. These factors might have masked the impact of Se-supplementation on broodstock growth. We did not also find any significant effect of the form of Se-supplementation on reproductive performance parameters, including absolute and relative fecundity, GSI and egg size and weight. These results are in line with observations in zebrafish, where no significant difference in number of eggs and mating success could be found between groups fed deficient (0.09 mg Se/kg) vs. replete (0.65 mg Se /kg) diets (Penglase et al., 2014a). Also, similar to the mentioned zebrafish trial, the survival of the progeny was not significantly different between the control group and fish fed Se supplemented diets, however we found a slightly lower survival in the inorganic supplemented group compared to the control group. But even without a detectable effect on growth, the number of females spawning was significantly higher in both Se-supplemented groups and females fed organic Se in form of hydroxy-selenomethionine spawned significantly earlier compared to females receiving the sodium selenite diet. The low survival in progeny of selenite group can be traced back to given ascending females, spawning at the early spawning dates, while progeny of the majority of females in this group did not show any elevated mortality. The differences in spawning pattern and survival between the groups receiving different forms of dietary Se supplementation could be connected to modified vitellogenesis and blood steroid concentrations as these parameters were described to be affected in rainbow trout females fed dietary SeMet at a high level of 4.54 mg/kg [START_REF] Wiseman | Chronic exposure to dietary selenomethionine increases gonadal steroidogenesis in female rainbow trout[END_REF]. It might have been interesting to also analyze plasma vitellogenin levels in the present study.

The results indicate that a plant-protein based diet with a basal Se level of 0.3 ppm was not sufficiently deficient to affect growth of rainbow trout broodstock, but might negatively affect reproduction by decreasing the number of females spawning, which can be improved by supplementing Se, especially as organic form.

Effective Se transfer from broodstock to progeny

Tissue analysis revealed elevated female liver and muscle tissue Se concentrations in supplemented groups, compared to the control group, and the concentration in fish fed diets with organic Se supplementation were even higher compared to fish fed inorganic Se diets.

The increase of Se in muscle tissue of fish fed Se-supplemented diets was also recently reported in Atlantic salmon (Salmo salar), finding higher Se retention for organic Se supplementation in the form of SeMet compared to inorganic supplementation [START_REF] Sele | Selenium and selenium species in feeds and muscle tissue of Atlantic salmon[END_REF]. The higher retention for organic Se (SeMet), compared to inorganic Se (selenite) was previously described also in rainbow trout fry [START_REF] Rider | Supranutritional dietary intake of selenite and selenium yeast in normal and stressed rainbow trout (Oncorhynchus mykiss): implications on selenium status and health responses[END_REF][START_REF] Godin | Influence of dietary selenium species on selenoamino acid levels in rainbow trout[END_REF].

However, this effect is not systematic, as in some other studies, no effect on tissue Se accumulation was described, when working with similar amounts of Se as studied here [START_REF] Han | The effects of dietary selenium on growth performances, oxidative stress and tissue selenium concentration of gibel carp (Carassius auratus gibelio)[END_REF]. We found elevated Se concentrations in the progeny at oocyte and swim-up fry stage before first feeding in Se-supplemented groups with higher transfer in the group supplemented with hydroxy-selenomethionine. These results are in line with results obtained in hens that describe the parental transfer of Se to the progeny [START_REF] Pappas | Maternal organo-selenium compounds and polyunsaturated fatty acids affect progeny performance and levels of selenium and docosahexaenoic acid in the chick tissues[END_REF][START_REF] Wang | Influence of dietary selenomethionine supplementation on performance and selenium status of broiler breeders and their subsequent progeny[END_REF]. The Se speciation in oocytes and swim-up fry could show that the SeMet can be found in tissues in higher concentrations compared to SeCys. Thereby, Se supplementation increased the SeMet levels, while SeCys was not affected. The elevated levels of SeMet in oocytes of the selenite treatment are surprising, especially as they are not found any more at swim-up fry stage. Indeed selenite in the Se metabolism is known to be metabolized to selenide and not transformed to SeMet [START_REF] Burk | Regulation of Selenium Metabolism and Transport[END_REF]. The elevated status of SeMet in the oocytes of the inorganic Se supplemented group found in this study might be linked to a transfer of SeMet from broodstock to the progeny, a process which could have been limited in the control group compared to the Se-supplemented groups. In a study with rainbow trout fry fed a diet supplemented with sodium selenite or SeMet, SeMet was found to be higher in the SeMet treatment compared to control group and selenite treatment, whereas in comparison to SeMet, the SeCys concentration was not affected by the supplementation at all [START_REF] Godin | Influence of dietary selenium species on selenoamino acid levels in rainbow trout[END_REF]. However, there were huge differences in the amount of Se identified as SeMet and SeCys between the two tissues analyzed in this study, which could be also connected to different extraction methods used. In this case, the absolute concentration values should be considered carefully and preferably trends at each stage regarded. However, it cannot be excluded that other seleno-components are present in fry as other Se-containing peaks were observed in chromatograms.

These results showed that Se-supplementation to diets with low basal levels could increase the tissue Se concentration and that there was a transfer of Se from the broodstock to the progeny. The bioavailability and transfer of Se was stronger in broodstock fed hydroxyselenomethionine compared to selenite.

An altered antioxidant metabolism in deficient Se broodstock and offspring compensated by Se supplementation especially in organic form

In animals, selenoprotein P is a selenoprotein often found to be regulated by Se level [START_REF] Yuan | Regulation of selenoprotein P concentration and expression by different sources of selenium in broiler breeders and their offspring[END_REF]Penglase et al., 2014a;[START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF] and this was confirmed in the present study where the SelPa expression was significantly lower in the control group.

In hepatic tissue of both males and females, the strongest effect was found in the group fed inorganic Se supplemented diets, while in the whole-body swim-up fry, the effect is detectable only in the organic Se supplemented groups. In humans, [START_REF] Steinbrenner | Selenoprotein P protects endothelial cells from oxidative damage by stimulation of glutathione peroxidase expression and activity[END_REF] found that hepatocyte-derived selenoprotein P concentrations can stimulate the GPX1 expression and activity in astrocytes. Similar to selenoprotein P, GPX1 mRNA levels and activity are often described to decrease in mammals under Se deficiency [START_REF] Chow | Response of glutathione peroxidase to dietary selenium in rats[END_REF][START_REF] Sunde | Selenium status highly regulates selenoprotein mRNA levels for only a subset of the selenoproteins in the selenoproteome[END_REF] and have been found to be a good biomarker of Se status in fish [START_REF] Bell | Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri)[END_REF]Penglase et al., 2014a;[START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF][START_REF] Pacitti | Selenium supplementation in fish: A combined chemical and biomolecular study to understand Sel-Plex assimilation and impact on selenoproteome expression in rainbow trout (Oncorhynchus mykiss)[END_REF]. Likewise, the present results showed a lower GPX1a expression in male liver of the control group, compared to the inorganic Se treatment, but there was no effect in female liver or GPX activity in broodstock male or female liver. In whole-body swim-up fry, GPX1a1 and GPX1b2 expression, in correlation with GPX activity was increased in fry coming from broodstock fed the organic Se supplemented diet. In male liver, also GPX4a2 was influenced by dietary Se supplementation with significantly higher levels in the organic Se supplemented treatment, compared to the group fed inorganic Se, while in a study with rainbow trout fry GPX4a1 was found to be the most sensitive isoform in terms of low Se levels [START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF]. Se deficiency was described to lead to decreased levels of MsrB protein activity and mRNA expression in various tissues [START_REF] Moskovitz | Selenium-deficient diet enhances protein oxidation and affects methionine sulfoxide reductase (MsrB) protein level in certain mouse tissues[END_REF]. In the present study, the selenoprotein MrsB1 was higher expressed only in male liver of the group fed organic Se-supplemented diets, while the non-selenoprotein MsrB2 was regulated by dietary Se not only in male liver of both supplemented groups, but also in the progeny of the group fed organic selenium. In comparison to mice, where the selenoprotein MsrB1 is the most dominant Msr in liver and affected by dietary Se levels [START_REF] Novoselov | Regulation of selenoproteins and methionine sulfoxide reductases A and B1 by age, calorie restriction, and dietary selenium in mice[END_REF], these results in rainbow trout could indicate a different MsrB hierarchy in fish as MsrB2 was more sensitive to low dietary Se levels than MsrB1.

These results showed that parental Se nutrition, especially supplementation with hydroxyselenomethionine as an organic source of Se, can enhance not only the expression of proteins involved in antioxidant defense, such as SelP, GPX and MsrB2, but can also increase the activity of GPX in the progeny, while the expression of some hepatic proteins in the broodstock was more strongly affected even by a supplementation with selenite.

Contrasted oxidative status in progeny related to Se broodstock nutrition

As an integral part of several glutathione peroxidases, Se plays an important role in metabolizing hydrogen peroxides and lipid hydroperoxides into water and lipid alcohols [START_REF] Brigelius-Flohé | Tissue-specific functions of individual glutathione peroxidases[END_REF]. This function is complementary to that of vitamin E as a lipid-soluble antioxidant and both are functioning in close link [START_REF] Hamre | Metabolism, interactions, requirements and functions of vitamin E in fish[END_REF]. It has been shown that a combined deficiency of vitamin E and Se can lead to severe consequences, including suppressed growth, muscular dystrophy, exudative diathesis and death in fish [START_REF] Poston | Vitamin E and selenium interrelations in the diet of Atlantic salmon (Salmo salar): gross, histological and biochemical deficiency signs[END_REF][START_REF] Gatlin | Effects of singular and combined dietary deficiencies of selenium and vitamin E on fingerling channel catfish (Ictalurus punctatus)[END_REF][START_REF] Watanabe | Trace minerals in fish nutrition[END_REF], while Se-deficiency symptoms appear less severe, if adequate levels of vitamin E are provided [START_REF] Bell | Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri)[END_REF][START_REF] Bell | Some effects of selenium deficiency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri)[END_REF]. In the present study, vitamin E requirements were assumed to be fulfilled as per the NRC (2011) with more than 50 mg tocopherol per kg diet. As organic Se supplementation led to an increase in αtocopherol levels in the progeny, this possibly indicates a Se deficiency or reduced Se availability in the control group and in the group receiving sodium selenite. Indeed, Sedeficiency has been reported to cause a reduction of vitamin E levels [START_REF] Hamre | Metabolism, interactions, requirements and functions of vitamin E in fish[END_REF]. These results are in line with an earlier study in hens, describing significant increase in yolk vitamin E concentration by the supplementation of hydroxy-selenomethionine [START_REF] Tufarelli | 2-Hydroxy-4-methylselenobutanoic acid as new organic selenium dietary supplement to produce selenium-enriched eggs[END_REF].

Elevated vitamin E and Se levels in the progeny of supplemented groups thus should provide them a better protection against oxidative damage. This was supported also by an elevated vitamin C level in organic Se-supplemented group in this study, as was also reported by [START_REF] Kumar | Immuno-protective role of biologically synthesized dietary selenium nanoparticles against multiple stressors in Pangasinodon hypophthalmus[END_REF] in striped catfish. However, protein carbonyls as an indicator of protein oxidation were not significantly affected by Se-supplementation in the present study, even though there was a tendency for lower protein carbonyl levels in the group with organic Se-supplementation. An effect might have been found at the single tissue level as in mice, where fewer protein carbonyls were found in isolated liver, kidney, cerebrum and cerebellum, when fed higher Se levels [START_REF] Moskovitz | Selenium-deficient diet enhances protein oxidation and affects methionine sulfoxide reductase (MsrB) protein level in certain mouse tissues[END_REF]. A closer look on 8isoprostane levels showed a decrease only in the group with sodium selenite supplementation. This result is surprising as [START_REF] Küçükbay | The effects of dietary organic or inorganic selenium in rainbow trout (Oncorhynchus mykiss) under crowding conditions[END_REF] found that, both inorganic and organic Se supplementation led to a linear decrease of both serum 8isoprostane and malondialdehyde (MDA) levels. Similarly, in common carp, selenite supplementation did not even affect MDA levels, which were only decreased by feeding SeMet or nano-Se [START_REF] Saffari | Effects of different dietary selenium sources (sodium selenite, selenomethionine and nanoselenium) on growth performance, muscle composition, blood enzymes and antioxidant status of common carp (Cyprinus carpio)[END_REF]. As 8-Isoprostanes are products of fatty acid (arachidonic acid) peroxidation, a more indirect effect of Se could be suspected in the present study as dietary Se has been shown to affect fatty acid composition in a previous study [START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF]. Also, the GSH/GSSG ratio in the whole-body swimup fry was found to be lower in supplemented groups compared to the control group, which is accompanied by an absence of effect on the mRNA levels of both glutamate-cysteine ligase catalytic subunit (Gclc), needed for the synthesis of glutathione, and glutathione reductase (GR), needed for the reduction of glutathione, while the GPX activity was higher in supplemented groups. In more developed fry fed with different Se level, not only the GPX activity, but also the GSH/GSSG ratio was higher in groups fed Se-supplemented diets [START_REF] Fontagné-Dicharry | Influence of the forms and levels of dietary selenium on antioxidant status and oxidative stress-related parameters in rainbow trout (Oncorhynchus mykiss) fry[END_REF]. The different results might be connected to differences in the glutathione metabolism, which seems to be boosting at swim-up fry stage as levels of total glutathione increased about ten times compared to oocytes and oxidized glutathione levels only became detectable at swim-up fry stage in this study. The indirect effect of parental feeding on glutathione status in the progeny will need further investigation.

The absence of significant effects on some markers of oxidative status, especially linked to glutathione metabolism, may indicate that even with the clear difference in Se transfer from broodstock to the progeny, the Se deficiency in the non-supplemented group was not critical enough to provoke a strong oxidative stress. Otherwise, the Se deficiency in broodstock might induce other metabolic effects leading to changes in oxidative status such as modification of one-carbon metabolism as suggested by [START_REF] Speckmann | Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice[END_REF] in mice.

Conclusion

The present results demonstrate that dietary Se-supplementation in plant-ingredient based diets with low basal selenium levels can enhance the total number of females spawning, while hydroxy-selenomethionine supplementation even led to an earlier spawning. It was demonstrated for the first time a parental transfer of Se in the progeny with the higher effect observed with the better bioavailability of organic Se. In the progeny before first feeding, the high Se levels led to an increase in the activity and mRNA expression of GPX and other proteins involved in antioxidant protection, along with elevated vitamin E and C levels. The impact of Se broodstock nutrition on glutathione metabolism in the progeny is intriguing and requires further investigation. Further work with juvenile fish at later developmental stages is warranted to show, if the parental effects persist after exogenous feeding of the progeny. Total saturates include 12:0, 15:0, 17:0 and 20:0 and total monoenes include 14:1 and 20:1. (kg, n=9-11) 1.20 ± 0.12 1.04 ± 0.09 1.03 ± 0.13 DGI 1 (%, n=9-11) 0.34 ± 0.08 0.28 ± 0.14 0.29 ± 0.12 HSI 1 (%, n=5)

1.1 ± 0.1 1.1 ± 1.1 1.5 ± 0.2 Muscle lipid content (%, n=5)

6.8 ± 0.8 6.7 ± 1.1 6.3 ± 0.6

Female performance

Initial mean body weight (kg, n=25) 1.13 ± 0.05 1.11 ± 0.03 1.10 ± 0.04 Final mean body weight (kg, n=24-25) 1.30 ± 0.07 1.21 ± 0.04 1.32 ± 0.05 DGI 1 (%, n=10-21) 0.27 ± 0.05 ab 0.17 ± 0.04 b 0.32 ± 0.05 a HSI 1 (%, n=10-21)

1.1 ± 0.1 1.0 ± 0.1 1.1 ± 0.2 Muscle lipid content (%, n=8-18) 7.9 ± 0.6 6.2 ± 0.5 6.9 ± 0. 1 DGI, daily growth index = 100 x (final body weight 1/3 -initial body weight 1/3 )/duration (182 days); HSI, hepato-somatic index = 100 x (wet liver weight/fish weight); GSI, gonadosomatic index = 100 x (wet spawn weight/female weight); Spawn weight = total egg weight per female after stripping and removal of coelomic fluid; absolute fecundity = egg number per female; relative fecundity = egg number/kg female. Vitamin C (mg/kg) 29 ± 1 b 30 ± 2 b 37 ± 2 a Within rows means ± SEM (n = 8) not sharing a common superscript letter are significantly different (P < 0.05) according to one-way ANOVA followed by Newman-Kauls post-hoc test. Means not sharing a common superscript letter are significantly different (P < 0.05) according to one-way ANOVA followed by Newman-Keuls test. Fig. 7. Glutathione contents in female liver (A), male liver (B), and whole-body swim-up fry (C) from rainbow trout broodstock fed different Se sources over 6 months at a constant water temperature of 8 ± 1°C. Bars represent means ± SEM (n = 8, except for male liver: n = 5). Difference between total (GSHt) and oxidized glutathione (GSSG) represents reduced glutathione (GSH). Means not sharing a common superscript letter are significantly different (P < 0.05) according to one-way ANOVA followed by Newman-Keuls post-hoc test.

Fig. 1 .

 1 Fig. 1. Total numbers of females spawning in rainbow trout broodstock fed different Se sources over a six months period prior to spawning at a constant water temperature of 8 ± 1°C. For each date, total numbers of females spawning, not sharing the same superscript letter, are significantly different (P < 0.05) by pairwise comparison of the χ 2 .

Table 1 .

 1 Formulation and composition of experimental diets (g/100g dry weight).

	Diet

8

Table 2 .

 2 Oligonucleotide primers used to assay mRNA levels by real-time quantitative RT-

	744
	745	PCR.
	Gene	Accession no.	Forward primer sequence	Reverse primer sequence	Amplicon size
	SelPa SelPa2 SelPb GPX1a GPX1b1 CA357669.1 HF969249.1 MH085054.1 HF969250.2 HE687021 GPX1b2 HE687023 GPX4a1 HE687024 GPX4a2 HE687025 GPX4b CA344428.1 TR HF969247.1 MsrB1 BX313019.3 MsrB2 BX311214.3 MsrB3 CX041708.1 MsrA BT073595.1 SOD1 AF469663.1 SOD2 CA352127.1 CAT BX087110.3 GR HF969248.1 Gclc GSONMT00065033001 GSTπ BX302932.3 Nrf2 CA360709.1 Keap1 GSONMT00034445001 Keap1X GSONMT00019673001 NF-κB BX880658.3 IκBα BT074199.1 AJ438158.1 β-Actin SelP, selenoprotein P; GPX1, glutathione peroxidase 1 ; GPX4, glutathione peroxidase 4; cagccacctggttggagtat cctggagtagggccacca accttgctgagccagaaact cagacgaccacacctgtcat gtgtcgttcctcatcgtgaa ggaacgtcagtctcccacat aatgtggcgtcactctgagg caattctcctgatggccaaa cgagctccatgaacggtacg tgcttcccgttcacatccac tcggacatcaggagaactgc tccttcccattcacatccac gaaaggcttcctgggaaatg ctccaccacactgggatcat agaaatacaggggcgacgtt gcatctccgcaaactgagag ttggaggtcaggagccaggt accctttcccttgggctgtt acaaaatcaaggcgaccaac ggcagagagaacaggtcgtc ctggcctgccttcactgaga ttcccacatcggaccttgaa aggggacagagatgcccttc cccatgagcctctttgaacg tcaggttggccttccttcta cgaggtgagaaccacactga agatggctatgcttgggatg acctgtgcaggtctcttcgt tggtcctgtgaagctgattg ttgtcagctcctgcagtcac tccctgacctgacctacgac ggcctcctccattaaacctc tgatgtcacacaggtgcgta gtgggctcagtgttgttgag ctaagcgcagcgtcatagtg acacccctgtctgacgacat caaccaactggcagacaatg cctttgacaaggggatgaga tcgctgactggacgaaagga cgaaggtcctcaacgccatc tgagctgcagcaatgtctga gttgggcaatgggtagaagc gctacgtgatgtctgcccct ggtacctcatagcggccagt actgggaggttatgacggga cctccgtcctctcatgcttc cagcgtcctaccaggctaaagagat gctgttcgatccatccgcactat agagacagactgcgctccac cggccttcagtagcctctct gatgggccagaaagacagcta tcgtcccagttggtgacgat 746	82 90 87 72
	747	TR, thioredoxin reductase; SOD1, superoxide dismutase 1; SOD2, superoxide dismutase 2;
	748	CAT, catalase; GR, glutathione reductase; Gclc, glutamate-cysteine ligase catalytic subunit;
	749	GSTπ, glutathione S-transferase π; Nrf2, nuclear factor erythroid-2 related factor 2; Keap1,
	750	Kelch-like ECH-associated protein 1; NF-κB, nuclear factor kappa-light chain-enhancer of
	751	activated B cells; IκBα, NF-κB inhibitor α.
	752

Table 3 .

 3 Growth, spawning parameters and offspring performance of rainbow trout broodstock fed different Se sources over 6 months at a constant water temperature of 8 ± 1°C.

	Diet

Table 4 .

 4 Total Se, SeMet and SeCys concentrations (µg Se/kg wet weight), ratio of SeMet 765 and SeCys over total Se, SeMet to SeCys ratio and percentage of total Se in oocytes and 766 whole-body swim-up fry 767

		Setot	SeMet	SeCys	SeMet/Setot SeCys/Setot SeMet/SeCys %Setot
	Oocytes
	NC	214 ± 12 c	118 ± 18 c	89 ± 8	0.55 ± 0.06	0.42 ± 0.04 a	1.35 ± 0.25 b	97 ± 3
	SS	321 ± 14 b 220 ± 24 b 102 ± 14 0.69 ± 0.06 0.32 ± 0.03 ab 2.24 ± 0.38 ab 100 ± 3
	SO	521 ± 19 a	373 ± 5 a	116 ± 19 0.72 ± 0.02	0.22 ± 0.03 b	3.40 ± 0.56 a	94 ± 1
	Swim-up fry
	NC	197 ± 17 b	47 ± 9 b	13 ± 2	0.24 ± 0.06	0.07 ± 0.01	3.68 ± 0.77 b	31 ± 6
	SS	276 ± 11 a	40 ± 5 b	13 ± 1	0.15 ± 0.03	0.05 ± 0.01	2.95 ± 0.03 b	19 ± 4
	SO	312 ± 10 a	84 ± 6 a	12 ± 2	0.27 ± 0.01	0.04 ± 0.00	6.89 ± 0.78 a	31 ± 1
	768	Values are mean ± SEM (n = 3). Within tissue and column values not sharing a common
	769	superscript letter are significantly different (P < 0.05) according to one-way ANOVA followed
	770	by Newman-Keuls post-hoc test.
	771

Table 5 .

 5 Antioxidant enzyme activity in liver and whole-body swim-up fry from rainbow trout 772 fed different Se sources over 6 months at a constant water temperature of 8 ± 1°C. 773

			Female liver			Male liver			Swim-up fry
		NC	SS	SO	NC	SS	SO	NC	SS	SO
	Enzyme activity
	SOD 1	14 ± 1	14 ± 1	14 ± 1	19 ± 2	24 ± 2	18 ± 2	5.0 ± 0.2 4.9 ± 0.2 5.1 ± 0.3
	CAT 1	409 ± 32 373 ± 45 391 ± 50 502 ± 60 574 ± 52 424 ± 49	11 ± 1	11 ± 1	11 ± 1
	GR 2	8 ± 1	9 ± 1	9 ± 1	12 ± 2	7 ± 1	11 ± 1	8 ± 1	8 ± 1	8 ± 1
	GST 2	145 ± 10 154 ± 22 145 ± 17 232 ± 19 229 ± 35 220 ± 33	54 ± 4	57 ± 3	55 ± 4
	774	Mean values ± SEM (n = 8, except for male liver: n = 5).. Within rows and for each tissue,
	775	means not sharing a common superscript letter are significant different (P < 0.05) according
	776	to one-way ANOVA followed by Newman-Keuls post-hoc test.
	777	1 Expressed in U/mg protein
	778	2 Expressed in mU/mg protein

Table 6 .

 6 Oxidative status assessed by GSH/GSSG ratio, 8-Isoprostanes, protein carbonyls content and vitamin C in whole-body swim-up fry from rainbow trout broodstock fed different Se sources over 6 months at a constant temperature of 8 ± 1°C.

	NC	SS	SO

Table 7 .

 7 Tocopherol content in offspring (oocyte and whole-body swim-up fry) from rainbow 785 trout broodstock fed different Se sources over 6 months at a constant temperature of 8 ± .66 6.31 ± 0.36 a 4.02 ± 0.60 b 0.967 <0.001 0.613 Values expressed in ng tocopherol per individual are means ± SEM calculated from samples 788 of offspring (n = 8 per dietary group and stage). Within rows and within dietary groups (n = 789 16) and developmental stages (n = 24), means not sharing a common superscript letter are 790 significantly different (P < 0.05) according to two-way ANOVA followed by a Newman-Keuls

	786
	787	1°C.
				Dietary group		Stage	p-value
			NC	SS	SO	oocyte	swim-up fry	Diet Stage DxS
	α-tocopherol 1815 ± 146 b 2056 ± 176 ab 2472 ± 168 a 2157 ± 154	2071 ± 132 0.043 0.773 0.794
	γ-tocopherol	254 ± 23	249 ± 26	254 ± 15	263 ± 17	241 ± 18	0.348 0.426 0.400
	δ-tocopherol 5.15 ± 0.68 5.67 ± 0.69 4.68 ± 0791
	792	test.
	793

Acknowledgements

The authors have no conflict of interest to declare for this work that was labelled by the "Institut Carnot France Futur Elevage (IC-F2E)". This present study was supported by I-site E2S from the University of Pau and Pays de l'Adour (P.W., PhD fellowship) and IMR (P.A.J.P., ParSel Project no. 15329). The authors wish to thank P. Maunas and N. Turonnet for the care of fish and F. Terrier, F. Sandres and A. Lanuque for the preparation of diets.

The authors are also very grateful to L. Larroquet, V. Véron and C. Héraud for technical assistance in lipid, enzyme activity and tocopherol analysis. The help of M. Sérusier and M.

Cluzeaud in sampling and training was also greatly appreciated.


MD Marta Silvestri

MD Antoine Coignac

MD Julien Delicque

MD, PhD Astrid Herrero

Frederic Borie

MD, PhD Boris Guiu

MD, PhD Jean-Michel Fabre

MD, PhD Regis Souche
email: frsouche@gmail.com

MD, PhD Fréderic Borie

postoperative pancreatic fistula after distal pancreatectomy: A retrospective case-control study of 157 patients with non-pancreatic ductal adenocarcinoma lesions

Keywords: Pancreas, pancreatectomy, postoperative, pancreatic fistula

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Distal pancreatectomy is pancreatic resection at the left of the mesentericoportal axis and is used to treat both benign and malignant diseases of the body and tail of the pancreas. Postoperative pancreatic fistula (POPF) remains the leading cause of morbidity after distal pancreatectomy, frequency being between 13% and 64% [START_REF] Lillemoe | Distal pancreatectomy: indications and outcomes in 235 patients[END_REF][START_REF] Diener | Efficacy of stapler versus hand-sewn closure after distal pancreatectomy (DISPACT): a randomised, controlled multicentre trial[END_REF][START_REF] Rodriguez | Implications and cost of pancreatic leak following distal pancreatic resection[END_REF][START_REF] Kleeff | Distal pancreatectomy: risk factors for surgical failure in 302 consecutive cases[END_REF]. The definition of POPF was updated by the International Study Group for Pancreatic Fistula (ISGPF) in 2016: 1. Biochemical leak (BL) applying to pancreatic fluid leakage without clinically relevant consequences (POPF grade A according to the former definition) is no longer defined as POPF [START_REF] Bassi | The 2016 update of the International Study Group (ISGPS) definition and grading of postoperative pancreatic fistula: 11 Years After[END_REF]. 2. A drain in situ at discharge is only scored now as POPF grade B when the surgical drain remains in place for more than 21 days after surgery. 3. Intensive care admission for POPF grade C criteria has changed to patient requirement of relaparotomy and/or admission to intensive care unit due to organ failure, and/or pancreatic fistula, leading to death.

The factors predisposing to POPF after distal pancreatectomy remain poorly recognized. The recent meta-analysis by Peng et al. including 20 studies, with a total of 2070 patients (ranging from 33-352 patients per study), indicates that soft pancreatic texture, higher BMI, blood transfusion, massive intraoperative blood loss and prolonged operative time were associated with increased POPF incidence [START_REF] Peng | Risk factors of postoperative pancreatic fistula in patients after distal pancreatectomy: a systematic review and meta-analysis[END_REF]. Many groups have attempted different pancreatic stump closure and/or transection techniques, but unfortunately the data does not show a clear consensus regarding the optimal technique for gland closure.

In current practice, radical anterior modular pancreaticosplenectomy (RAMPS) remains the standard procedure for patients with distal pancreatic ductal adenocarcinoma (PDAC). Personalized parenchyma-sparing distal pancreatectomy should be favored when feasible for benign or borderline lesions in order to prevent new-onset pancreatogenic diabetes mellitus (5% to 42% frequency after distal pancreatectomy) and exocrine insufficiency [START_REF] Maeda | Pancreatogenic diabetes after pancreatic resection[END_REF].

The uncertainty concerning the level of pancreatic division is highlighted in this former case in which the compromise between quality of resection, parenchyma-sparing and complication risk is a major concern. Several studies have suggested that the level of pancreatic division has an impact on POPF risk, especially for the cases of body or tail division [START_REF] Pannegeon | Pancreatic fistula after distal pancreatectomy: predictive risk factors and value of conservative treatment[END_REF][START_REF] Sell | The influence of transection site on the development of pancreatic fistula in patients undergoing distal pancreatectomy: A review of 294 consecutive cases[END_REF]. To date, very little attention has been addressed to the possibility that the level of pancreatic division during distal pancreatectomy could be a risk factor for POPF occurrence.

Therefore, the aim of this study was to investigate if the level of division (Neck versus Body-Tail) during distal pancreatectomy influenced POPF occurrence in a homogeneous cohort of pancreatectomies for non-pancreatic ductal adenocarcinoma lesions (non-PDAC) in the era of parenchyma-sparing resection.

PATIENTS AND METHODS

Study design and population

From January 1998 to December 2017, data from all consecutive patients requiring distal pancreatectomy in our public tertiary hospital were prospectively collected and analyzed retrospectively. Since 1998, the laparoscopic approach has been routinely proposed for distal pancreatectomy, initially for selected patients with non-PDAC lesions, and then later also for PDAC. We aimed to generate an adequate sample of patients in which the level of pancreatic division was defined preoperatively by the surgeon according to the localization of the lesion. Therefore, patients with pancreatic ductal adenocarcinoma were excluded, and only patients with pancreatic division using a linear stapler were included in this case-control comparative study.

Anatomic determination between neck and body/tail is illustrated in figure 1. Outcome of patients who underwent either distal pancreatectomy using a body-tail division without neck dissection (Body-Tail group) or neck dissection and division above the mesentericoportal axis (Neck group) were compared (figure 2). The decision on when to perform a distal pancreatectomy was taken by expert senior surgeons and was discussed in the context of a multidisciplinary institutional meeting.

Our primary outcome was POPF occurrence, defined and classified according to the 2016 ISGPF definition [START_REF] Bassi | The 2016 update of the International Study Group (ISGPS) definition and grading of postoperative pancreatic fistula: 11 Years After[END_REF]. The secondary outcomes were 90-days postoperative outcomes and new-onset pancreatogenic diabetes mellitus (PDM) occurrence at 6 months after surgery.

The Institutional Review Board approved this study. Data has been reported in line with STROCSS criteria [START_REF] Agha | The STROCSS statement: Strengthening the Reporting of Cohort Studies in Surgery[END_REF].

Surgical procedure

More than 60 pancreatic resections are performed each year on average in our department. All distal pancreatectomies were done by experienced pancreatic surgeons with significant experience in laparoscopic distal pancreatectomy, as previously described [START_REF] Souche | Robotic versus laparoscopic distal pancreatectomy: a French prospective single-center experience and cost-effectiveness analysis[END_REF][START_REF] Mehta | Laparoscopic versus open distal pancreatectomy: a single-institution case-control study[END_REF]. Spleen preservation was planned whenever possible. Splenic vessels were conserved as described by Kimura et al. [START_REF] Kimura | Spleen-preserving distal pancreatectomy with conservation of the splenic artery and vein: techniques and its significance[END_REF]. Splenic conservation while sacrificing the splenic vessels (Warshaw technique [START_REF] Warshaw | Conservation of the spleen with distal pancreatectomy[END_REF]) was not performed in this patient cohort.

In the case of non-PDAC lesion, the level of pancreatic division was planned before the procedure and confirmed by intraoperative ultrasound for medial lesions. Regarding pancreatic sparing strategy, we decided whenever possible not to dissect the neck of the pancreas and to begin the dissection by deporting on the left of the mesenteric root. In all patients, the pancreas was transected with an Endo-GIA™ stapler (Covidien, Medtronic), without staple line reinforcement. The distal transected pancreas was gently lifted and a medial-to-lateral dissection started. In the case of spleen preservation, the splenic vein and artery were skeletonized toward the hilum of the spleen. This allowed both a lymphadenectomy and a step-by-step division of all the branches coming from the splenic vessels. For distal pancreatectomy with splenectomy, first the splenic artery, and then the vein, were divided after pancreatic transection. This was followed by a medial-to-lateral en bloc dissection posterior to the splenic vein along the retroperitoneal plane. A closed non-aspirating drain was placed at the end of the procedure.

Outcome and data source

Demographic, clinical and surgical data (including neck dissection/no neck dissection), were collected using a standardized case report form for pancreatic surgery and analyzed retrospectively.

Main pancreatic duct diameter at the level of transection was measured on preoperative imaging and pancreatic texture was defined by the surgeon during the specimen examination. Operation duration was defined as the time between incision and closure. After surgery, a physician examined all patients daily until hospital discharge. Thoraco-abdomino-pelvic computed tomography (CT scan) with intravenous contrast injection was performed selectively in patients with suspected abdominal or thoracic complications. POPF was defined and classified according to the 2016 update of ISGPF [START_REF] Bassi | The 2016 update of the International Study Group (ISGPS) definition and grading of postoperative pancreatic fistula: 11 Years After[END_REF]. Drain amylase values were systematically measured at postoperative day 3, and the drain was removed for cases of negative values. All patients with amylase-rich drain fluid after postoperative day 3 received octreotide (standardized post-distal pancreatectomy pathway) regardless of POPF severity. Only clinically relevant grade B/C leak was considered as POPF. A fluid collection was identified through CT scan or ultrasound (fluid presence > 5cm in diameter) with or without clinical relevance. Early postoperative hemorrhage was defined according to the ISGPS [START_REF] Wente | Postpancreatectomy hemorrhage (PPH): an International Study Group of Pancreatic Surgery (ISGPS) definition[END_REF].

Patients were followed at 1 month, and then every 3 months, with systematic clinical and blood examinations (standardized post-distal pancreatectomy pathway, including assessment of fasting plasma glucose and hemoglobin A1c (HbA1c) levels). Postoperative complications were stratified according to Clavien-Dindo (CD) classification, defining major complications by a score of III or above [START_REF] Dindo | Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey[END_REF]. Complications, readmissions and operative mortality were considered as those occurring within 90 days of surgery [START_REF] Mise | After Pancreatectomy, the "90 Days from Surgery" Definition Is Superior to the "30 Days from Discharge" Definition for Capture of Clinically Relevant Readmissions[END_REF].

New-onset PDM was defined according to diagnosis and classification by the American Diabetes Association [START_REF]American Diabetes A: (2) Classification and diagnosis of diabetes[END_REF]: (a) fasting plasma glucose level of 126 mg/dL or greater, (b) hemoglobin A1c (HbA1c) of 6.5% or higher at 6 months after surgery for patients without history of diabetes or use of antidiabetic drugs [START_REF] Maeda | Pancreatogenic diabetes after pancreatic resection[END_REF]19].

Statistical analysis

Baseline characteristics followed by clinical outcomes were analyzed using standard univariate analysis. The median with range is reported for continuous variables, whereas absolute/relative frequencies are reported for categorical variables. Continuous variables were compared using nonparametric tests (Mann-Whitney U). Categorical data were compared with Chi-square tests or Fisher's exact tests depending on size. A P value < 0.05 was considered as statistically significant.

Statistical analyses were performed using SPSS Statistics (Version 20.0) for Macintosh (IBM Corp, Armonk, NY) and SAS software (SAS Institute, Cary, North Carolina).

RESULTS

Patients and surgical data

Out of the 983 pancreatectomies performed in our center, 217 patients required a distal pancreatectomy. According to inclusion/exclusion criteria (PDAC (n=54), technique of division other than stapler (n=6)), we analyzed data from 157 patients who underwent distal pancreatectomy for non-PDAC lesions (F: 91/M: 66). In particular, 53 patients underwent distal pancreatectomy with body-tail division (Body-Tail group), and 104 with neck division (Neck group). Body-Tail and Neck groups were comparable concerning demographic data, period of treatment, BMI, ASA score, comorbidities, type of lesion, median lesion size, pancreatic characteristics, laparoscopic or open approach and spleen conservation indications. Table 1 summarizes the clinicopathologic characteristics of the two groups.

The median length of surgery was similar for both different strategies: 180 min for Body-Tail group and 196 min for Neck group (p = 0.625). No difference was found concerning operative time regarding spleen conservation between Body-Tail and Neck groups. The surgical approach did not differ in the two groups with similar rates of laparoscopy (43 vs. 51%, ref), robot assisted laparoscopy (8 vs. 7 %, p = 0.189) and laparotomy (49 vs. 42%, p = 0.388). Conversion rates to open procedure were similar in both groups (4 vs. 3%, p = 1.000). Spleen preservation was planned and feasible in 29/29 patients and 51/51 patients in Body-Tail and Neck groups respectively (p = 0.501). Median intraoperative blood loss was 95 ml (50-480ml) in Body-Tail group and 110 ml (50-2700ml) in Neck group (p = 0.334). Intraoperative transfusion was not necessary in Body-Tail group, contrary to the Neck group (0 vs. 7%; p = 0.096). Surgical data are detailed in Table 2.

Comparison of postoperative outcome

Median follow-up of patients was 49 months (range 3-216). Table 3 details postoperative results for both groups. A clinically relevant POPF was diagnosed in 5.5 and 12.5% (p = 0.388) in Body-Tail and Neck groups respectively. Median postoperative hospital stay was similar in both groups (9 vs. 10 days, p = 0.741). A complicated postoperative course occurred in 17% of Body-Tail group and in 30% of Neck group (p = 0.505). The rate of complication grade ≥ III was significantly lower in Body-Tail group than in Neck group (0 vs. 10.5%; p = 0.016). Complication grade ≥ III (n=11/104) in Neck group was related to (i) POPF grade B (6 percutaneous drainage, of which 2 required general anesthesia), (ii) POPF grade C (reoperations with intensive care unit transfer (n = 2)) and (iii) other causes (colic fistula requiring a colostomy (n = 1) and death (n = 2) related to an acute ischemic stroke and a pulmonary embolism without arguments of underlying POPF).

There were no significant differences in peripancreatic fluid collection rates, postoperative transfusion rates, splenic infarction rates, pulmonary complication rates, reoperation rates and readmission rates between the Body-Tail and Neck groups. There was no significant difference in new-onset PDM 6 months after surgery between the two groups (22.5 vs. 20%; p = 0.439).

DISCUSSION

Several studies have suggested that the body/tail level for pancreatic division has an impact on POPF occurrence that remains a major concern for pancreatic surgeons. In the era of minimally invasive and parenchyma-sparing pancreatectomy for patients with non-PDAC lesions, the purpose of this study was to investigate the impact of pancreatic division level on POPF after distal pancreatectomy. To our knowledge, this is the first study focusing on pancreatic division level in a homogeneous cohort of 157 consecutive distal pancreatectomies for non-PDAC lesions using a linear stapler. Overall, we did not find a higher risk of POPF with division at the body-tail level compared to the neck level. Despite similar POPF rates, we found different patterns of POPF between the two groups. The rate of major complications after pancreatic division at the neck was significantly higher. In parallel, our present study confronts previous results published on postoperative diabetes occurrence, finding no difference in new-onset PDM rates between Body-Tail division and Neck division groups.

Our results highlight that distal pancreatectomy is a major surgical procedure, associated with an overall postoperative morbidity rate of 1.9% and a clinically relevant pancreatic fistula rate of 10 %.

Major complications defined according to the Clavien-Dindo (CD) classification ≥ III accounted for 7% of cases. Given that all the procedures were performed for benign or borderline neoplasms, a soft pancreatic texture and a systematic drainage close to the pancreatic stump could have critically contributed to POPF rate. In terms of major complications and clinically relevant POPF, our results are comparable with previous studies reporting a major complication rate of 11 %, and a clinically relevant POPF rate of 10 % [START_REF] Rosales-Velderrain | National trends in resection of the distal pancreas[END_REF]. In the present study, the rate of major complications after neck division was significantly higher, but CD complication ≥ III was not systematically related to POPF. However these results could suggest that pancreatic fistula, when it occurs, tends to be more severe at this division level in close contact with the mesentericoportal and coeliac axis vascular sutures and collaterals. A peripancreatic fluid collection at this level could be more complex to drain using a conservative approach.

Pannegeon et al found that division of the pancreas at the body rather than the neck level was a risk factor for POPF [START_REF] Pannegeon | Pancreatic fistula after distal pancreatectomy: predictive risk factors and value of conservative treatment[END_REF]. Hashimoto et al suggested that a larger residual gland might generate a greater intraductal pressure, and this increased pressure could overcome the duct/gland ligation [START_REF] Dindo | Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey[END_REF].

Sell et al. were the first team to explore a potential correlation between the level of pancreatic division and the development of pancreatic fistula. They compared distal pancreatectomy with neck versus body-tail division, however without any focus on new-onset postoperative diabetes [START_REF] Sell | The influence of transection site on the development of pancreatic fistula in patients undergoing distal pancreatectomy: A review of 294 consecutive cases[END_REF]. This study concluded that POPF occurs more often when the body-tail is transected, although the majority were biochemical leaks and of minimal clinical significance. Grade B/C pancreatic fistula occurred at equivalent rates regardless of transection site. Nevertheless, 2 major confounding factors were present; inclusion of pancreatic ductal adenocarcinoma and no uniform technique for gland transection over the study period. Obviously, they did not defend that all distal pancreatectomies had to be performed with neck division over the superior mesenteric vein axis given that the majority of pancreatic fistula developed were biochemical leaks and did not change clinical management or outcomes. The authors suggested that transection site should be considered as a variable in future studies of POPF. In addition, and in line with our findings, Malleo et al.

reported that specimen length was not related to POPF [START_REF] Malleo | Assessment of a complication risk score and study of complication profile in laparoscopic distal pancreatectomy[END_REF]. Surprisingly, our analysis from a non-PDAC cohort of patients did not find any difference in newonset PDM rates between Body/Tail and Neck groups. The overall new-onset PDM rate at 6 months was 21%. It is likely that the rate of new-onset PDM after pancreatic resection is frequently underestimated as retrospective reviews have reported variable PDM rates of between 5% and 42% after distal pancreatectomy [START_REF] Maeda | Pancreatogenic diabetes after pancreatic resection[END_REF]. On the other hand, rates may be inflated in studies that rarely consider the incidence of preexisting diabetes before pancreatectomy, with all procedures and neoplasms confounded [19,[START_REF] Maignan | Risk factors of exocrine and endocrine pancreatic insufficiency after pancreatic resection: A multi-center prospective study[END_REF]. In studies dedicated to distal pancreatectomy, a resection of more than 25-44% of the pancreatic volume in non-diabetic patients was shown to be an independent risk factor for new-onset PDM 3 months after distal pancreatectomy [START_REF] Kang | Endocrine Function Impairment After Distal Pancreatectomy: Incidence and Related Factors[END_REF][START_REF] Shirakawa | Pancreatic volumetric assessment as a predictor of new-onset diabetes following distal pancreatectomy[END_REF].

Despite the interesting outcomes of this present study, and the fact that the findings were obtained from a homogeneous cohort of consecutive patients, reflecting real life in a minimally invasive pancreatic surgery team, there are several study limitations. Firstly, its design was retrospective.

Secondly, the relatively limited sample size and thus potential inherent risk of type II errors should be considered. Sample size calculation was difficult due to the lack of data available on the impact of pancreatic division level on POPF rate. Nevertheless, this present study remains useful in the design of further prospective studies.

In conclusion, this study suggests that POPF and new-onset pancreatogenic diabetes mellitus occur at equivalent rates regardless of the level of pancreatic division during distal pancreatectomy in homogeneous patient cohorts. The intention to prevent POPF or diabetes should not influence the surgeon's decision on pancreatic transection site. We recommend a personalized distal pancreatectomy for patients with non-PDAC lesions, mixing laparoscopic approach [START_REF] Venkat | Laparoscopic distal pancreatectomy is associated with significantly less overall morbidity compared to the open technique: a systematic review and meta-analysis[END_REF][START_REF] Yi | A Systematic Review and Meta-Analysis of Laparoscopic and Open Distal Pancreatectomy of Nonductal Adenocarcinomatous Pancreatic Tumor (NDACPT) in the Pancreatic Body and Tail[END_REF], spleen conservation [START_REF] Kleeff | Distal pancreatectomy: risk factors for surgical failure in 302 consecutive cases[END_REF][START_REF] Shoup | The value of splenic preservation with distal pancreatectomy[END_REF][START_REF] Goh | Critical appraisal of 232 consecutive distal pancreatectomies with emphasis on risk factors, outcome, and management of the postoperative pancreatic fistula: a 21-year experience at a single institution[END_REF][START_REF] Collard | Is Routine Splenectomy Justified for All Left-Sided Pancreatic Cancers? Histological Reappraisal of Splenic Hilar Lymphadenectomy[END_REF] and pancreatic parenchyma-sparing with body/tail division when feasible.

Further comprehensive studies are needed to explore the potential correlations between the level of pancreatic transection, volume of remnant/resected pancreas, pancreatic blood supply and risks of POPF/new-onset PDM.

Provenance and peer review

Not commissioned, externally peer-reviewed

FIGURE AND TABLES

Figure 1 -Figure 2 -

 12 Figure 1 -Anatomic determination between neck and body/tail for pancreatic level of division. PV, portal vein; SMV, superior mesenteric vein; CT, celiac trunk; SMA, Superior mesenteric artery

Table 1 -

 1 Clinicopathologic characteristics of patients

Table 2 -

 2 Operative features

Table 3 -

 3 Postoperative results

Table 1 -

 1 Clinicopathologic characteristics of patients

	Body-Tail
	division, n
	(%)	Neck division, n (%)	p value

Table 2 -

 2 Operative features

	Body-Tail
	division	Neck division
	n (%)	n (%)	p value

Table 3 -

 3 Postoperative results

	Body-Tail	Neck division
	division n (%)	n (%)	p value

Acknowledgements:

We thank the University of Montpellier for their support. We are grateful to Dr Jacqueline Butterworth for proofreading of the manuscript. We thank the residents, fellows and nurses of our department for the care they provided to these patients.

The authors have no source of funding to disclose for this study.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.