Effects of GnRHa treatment during vitellogenesis on the reproductive physiology of thermally challenged female Atlantic salmon (Salmo salar)

Fish husbandry and maintenance

Ninety-five maiden and 88 repeat adult females from the Salmon Enterprises of Tasmania (Saltas) spawning stock were held in 200 (maidens) or 50 (repeats) m³ circular tanks at ambient photoperiod and temperature under standard conditions of husbandry at the Saltas Wayatinah hatchery until January 2009. In mid-January, fish were transferred to temperature-controlled 4 m³ Rathbun tanks (maximum 14 fish per tank, numbers varied according to the availability of stock) under simulated ambient photoperiod according to the following 6 treatment groups; maiden 14 °C, repeat 14 °C, maiden 22 °C, maiden GnRH implant 22 °C, repeat 22 °C and repeat GnRH implant 22 °C (Fig. 1). At the time of transfer, tank temperature was set to match ambient water temperature, and the experimental temperatures were then reached by adjustment a rate not exceeding 1 °C per day. Fourteen and 22 °C represent typical cool, and warm Tasmanian summers respectively. Fish were not fed from the time of transfer to the temperature controlled systems in January consistent with hatchery practice for management of this experimental stock of fish. All fish were maintained at the nominated temperature (14 or 22 °C) until late March when all fish were exposed to a gradual temperature ramp down to 8 °C (Fig. 2) to induce final oocyte maturation and ovulation as in King & Pankhurst (2000).

Sampling protocol

At each destructive sample point (all except the last date), six or seven fish were sampled for each treatment, and fish were selected at random from each replicate tank to ensure that stocking density was comparable between tanks. Maiden and repeat fish reared at ambient temperature were sampled in late October 2008 to establish an early-vitellogenic baseline for each group, and on the 22nd January 2009 at the same time that temperature transfer to 14 or 22 °C occurred for the remaining fish (Fig. 2). Subsequent samples were taken from each group during February 25–27, March 26–27, and April 15–16. After the last destructive sampling in April, five (repeat 22 °C GnRH-treated), six (maiden 22 °C, repeat 22 °C, and repeat 14 °C), or seven fish (maiden 14 °C and maiden 22 °C GnRH-treated) were left to proceed through to ovulation and stripping. A subset of maidens at 22 °C and repeats at 22 °C were implanted with an Ovaplant™ (Syndel, Nanaimo, Canada) cholesterol pellet containing 37 µg of the D-Arg⁶, Pro⁹NEt (GnRHa) on 22nd January (during temperature transfer), and again over February 26–27 to give repeat doses of approximately 7–12 µg kg⁻¹. The dose chosen was designed to stimulate or maintain pituitary secretion of Fsh, but remain below the threshold likely to stimulate an Lh surge and possible premature stimulation of oocyte maturation and ovulation (King & Pankhurst, 2004a). Untreated fish did not receive a sham implant, to simulate real industry conditions.

For sampling, fish were netted from the holding tanks, terminally anaesthetised in Aqui-S™ (Crop and Food, New Zealand), weighed, measured and then blood sampled by caudal puncture using pre-heparinised syringes. Blood plasma was centrifuged at 12,000 g, and stored frozen at −20 °C for later measurement of plasma hormones. Ovaries were excised, weighed and portions allocated to 50-ml pots containing teleost saline for fecundity estimation and follicle measurement. Segments of liver were transferred to 1–2 ml of RNA Later™ (Qiagen, Germany), kept at 4 °C overnight then stored at −80 °C.

Gonadosomatic index (GSI) was calculated as (gonad weight/total body weight) ×100, and condition factor (CF) as (body weight/length³) ×100. Fecundity was determined for a 5 g ovarian segment, and total fecundity was determined by correction for total ovarian weight and expressed as relative fecundity kg⁻¹ body weight as in Pankhurst et al. (2011). Using the procedure described in King & Pankhurst (2004a), fish were checked for ovulation by applying gentle pressure to the abdomen, and if ovulation had occurred, ova were stripped and fertilised for measurement of egg size, fertility and survival to the eyed stage at 250 degree-days of incubation. All animal experiments were conducted in accordance with Australian law under ethical approval issued by the Griffith University and University of the Sunshine Coast Animal Ethics Committees (ENV/25/08AEC and AN/A/09/44 respectively).

Plasma hormone and vitellogenin measurement

Plasma Fsh (all months) and Lh (March and April only) measurements were performed using an RIA developed for O. kisutch by Swanson et al. (1989) with the reagents and modifications described in Anderson & Elizur (2012). The cross reactivity of Fsh in the Lh assay and Lh in the Fsh assay were approximately 4.4% and 6%, respectively (Swanson et al., 1989). ANCOVA was performed as part of a previous study which confirmed that parallelism was present between serial dilutions of S. salar plasma and the purified coho standards (Anderson & Elizur, 2012). The limit of detection (LOD) of the Fsh and Lh assays was approximately 0.6 and 0.5 ng ml⁻¹ respectively.

Plasma levels of E2, T and cortisol (F) were determined by radioimmunoassay using the reagents and procedure for E2 and T described in Pankhurst & Carragher (1992), and for F as in Pankhurst et al. (2008) from 100 µl of plasma extracted with 1 ml ethyl acetate. Extraction efficiency was 78%, 79% and 74% for E2, T and F respectively as determined by recovery of ³H-labelled steroid from replicates of a plasma pool. Assay values were corrected accordingly to account for extraction losses. Interassay variability was determined by repeat measurement of a pooled internal standard and was (CV%) 7.4 (n = 3), 13.9 and 12.3 (n = 2) for E2, T and F respectively.

Plasma Vtg levels were measured by enzyme linked immunosorbent assay using the reagents and protocol as described in Watts et al. (2003). Interassay variability was assessed by repeat measurement of a Vtg standard from the central part of the assay curve and was 13.1 (CV%, n = 7). Pooled internal standards were not used here due to the tendency of Vtg to denature following repeated freeze-thaw cycles. The lower LOD for the assay was 80 ng ml⁻¹.

RNA extraction and cDNA synthesis

Total RNA was isolated from 15 mg of hepatic tissue using the Illustra RNAspin Mini kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s protocol. RNA yield and 260/280 purity ratio were determined using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). An RNA integrity number (RIN) was determined for a random sample of hepatic RNA (n = 48) using a 2,100 bioanalyzer (Agilent, Santa Clara, CA, USA) to establish RNA quality. Four hundred nanograms of liver-derived RNA were then used to synthesise cDNA for use in real-time quantitative PCR (qPCR) using the QuantiTect^® reverse transcription kit (Qiagen, Hilden, Germany). This kit includes a DNA elimination step to remove potential contamination of qPCRs by genomic DNA.

Hepatic gene expression

Gene specific primers (Gsps) for target genes previously optimised and validated for qPCR (Pankhurst et al., 2011) were used to amplify the target hepatic transcripts vtg, zpc and zpb (primers detect both zpba and zpbb, collectively referred to as zpb, Table 1), and the reference transcripts TATA binding protein (tbp), hypoxanthine phosphoribosyltransferase 1 (hprt1), beta-tubulin (β-tub) and elongation factor 1 alpha (ef1α, Table 1). The suitability of using candidate reference genes for normalisation was assessed using GeNorm (Vandesompele et al., 2002) and BestKeeper (Pfaffl et al., 2004) through the site RefFinder (Xie et al., 2012) (Table S1), and the output from these algorithms was then independently validated as suggested by Anderson & Elizur (2012) and Setiawan & Lokman (2010). As a result of this analysis, tbp was used for accurate target gene normalisation, and the level of target gene expression relative to a calibrator sample (pool of randomly selected hepatic cDNA) analysed in every run was calculated using Rest©2008 V2.0.7 (Pfaffl, Horgan & Dempfle, 2002).

qPCRs were conducted on a Rotor-gene 6,000 series thermal cycler (Qiagen, Hilden, Germany) using the Platinum^® SYBR^® Green qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA, USA) master mix and the following cycling conditions: 50 °C for 2 min; 95 °C for 2 min; 40 cycles of 95 °C for 15 s; 60 °C for 15 s, and 72 °C for 20 s (acquiring). At the end of cycle 40, a melt curve analysis was performed to confirm amplification of a single product as follows: 90 s preconditioning step at 72 °C, followed by a temperature gradient up to 95 °C at 1 °C per 5 s. The 10 µl qPCR reaction contained 5 µl SYBR, 200 nM each primer, 3.6 µl PCR grade water and 1 µl cDNA template. For every gene analysed no-template controls and a calibrator were included to detect possible contamination, and control for in-between run variability, respectively. Negative reverse transcription controls were also analysed to detect the presence of any contaminating genomic DNA.

Statistical analysis

Pairwise comparison of means of morphometric and plasma hormone data was made using an independent samples t-test (October and January). For multiple comparisons (following months) one-way ANOVA with post-hoc comparison of means by Tukeys-b was performed using the SPSS (version 15.0) statistical package. Differences in relative gene expression levels were detected non-parametrically using the Kruskal–Wallis test coupled with Bonferroni’s Correction to reduce the risk of type 1 error. The P value for significance was set at 0.05 for all analyses.

Morphometric data

The mean (±SEM) body weight of maiden spawners (4.01 ± 0.07 kg) was consistently lower than that of repeat spawners (7.03 ± 0.15 kg) during the course of the experiment (data not shown). CF was lower in repeats than maidens in October; however, CF was not different among fish from all groups thereafter (data not shown). There was a general trend of increasing GSI and follicle diameter with sample time across all treatment groups (Figs. 3A and 3B). The GSI of fish from all treatments was similar after introduction to the temperature-controlled tanks and hormonal treatment; however, follicle diameter was lower in all fish reared at 22 °C relative to their respective 14 °C control in April, and did not change as a result of treatment with GnRH. Total fecundity (during development) was highest in repeat spawners, but there was no consistent difference in relative fecundity between repeats and maidens, and no discernible effect of temperature regime or hormonal treatment (Figs. 4A and 4B).

Ovulation, egg fertility and embryo survival

Maidens held at 14 °C ovulated first, followed by a cluster of untreated and treated maidens at 22 °C, repeats at 22 °C, and then repeats at 14 °C (Fig. 5). (Repeats at 22 °C treated with GnRH showed markedly delayed ovulation relative to all other groups. Fecundity and relative fecundity at ovulation was unaffected by temperature or hormonal treatment (Figs. 6A and 6B). At 22 °C, repeat fish had slightly higher absolute, but not relative fecundity. Maiden and repeat spawning fish at 22 °C had markedly smaller post-ovulatory egg diameters and volumes (Figs. 6C and 6D) than their respective controls at 14 °C. Egg size was smaller in maiden fish than repeat fish at 14 °C, although egg size was similar between maidens and repeats at 22 °C. Fertility and eyed egg survival was higher in fish reared at 14 °C than at 22 °C; however, the reduction in embryo survival was less marked in repeats than maidens compared to the respective 14 °C control group (Figs. 6E and 6F). Treatment with GnRH did not significantly affect egg fertility or survival in maidens or repeats relative to the appropriate control at 22 °C.

Plasma hormones and Vtg

There was no difference in the level of plasma Fsh between groups of fish from October to February (Fig. 7). In March, plasma Fsh was elevated in treated and untreated maiden fish reared at 22 °C relative to the untreated group maintained at 14 °C, and Fsh levels were similar among both maiden groups at 22 °C. There was no significant difference among plasma Fsh levels in repeat fish in March. There was no significant difference in plasma Fsh levels between groups of fish in April. There were no differences in the mean (±SEM) plasma Lh levels between any groups of fish in March (0.91 ± 0.08 ng ml⁻¹) or April (0.85 ± 0.037 ng ml⁻¹) (data not shown).

Plasma E2 levels were suppressed at in February in both groups of maiden, but not repeat fish reared at 22 °C relative to their respective control at 14 °C (Fig. 8A). In March, plasma E2 levels in maidens were suppressed in both groups at 22 °C and there was also suppression at 22 °C in repeats treated with GnRH. In April, all 22 °C groups were suppressed relative to maidens held at 14 °C. T levels were not different between groups in October or January, and repeat fish treated with GnRH had higher T levels than the corresponding control group at 22 °C in February (Fig. 8B). Plasma T levels were suppressed at 22 °C in maidens treated with GnRH in March and April relative to maidens at 14 °C, and were suppressed in both groups of repeats held at 22 °C in April relative to the control group at 14 °C. Mean (±SEM) plasma F levels were elevated in maidens (15.7 ± 2.0 ng ml⁻¹) relative to repeats (3.3 ± 1.2 ng ml⁻¹) in October, but there were no differences among groups between January and March (data not shown). In April, F was elevated in repeats at 14 °C (23.1 ± 6.4 ng ml⁻¹) relative to all other groups (range 2.4–8.8 ng ml⁻¹).

Plasma Vtg levels increased through reproductive development and were similar among groups until February when levels were suppressed in maidens at 22 °C relative to 14 °C (Fig. 9A). In February, suppression of plasma Vtg was also observed for repeat fish treated with GnRH, and to some extent the untreated 22 °C group relative to the corresponding control at 14 °C. There was some recovery in plasma Vtg levels by March, with only repeats treated with GnRH showing significant suppression at 22 °C. In April, there were no differences between fish at 14 and 22 °C for either age class; although, maiden fish had higher levels of plasma Vtg than repeat fish at 14 °C.

Hepatic gene expression

Hepatic levels of relative vtg gene expression increased markedly from November to January, and in February, vtg expression was suppressed in repeats at 22 °C but this effect appeared to be offset by treatment with GnRH (Fig. 9B). In maidens, vtg gene expression was unaffected by maintenance at 22 °C or treatment with GnRH in February. In March, vtg expression was suppressed in GnRH-treated maiden fish at 22 °C, compared to the 22 °C maiden control group, but not maidens at 14 °C. There were no differences among repeat GnRH-treated and control fish held at 22 °C. In April, maidens held at 22 °C and treated with GnRH showed suppression relative to maidens at 14 °C, and both groups of repeats held at 22 °C showed suppressed expression relative to repeats at 14 °C.

Hepatic zpb gene expression was down-regulated in repeat fish relative to maidens in October; although there was no difference in gene expression by January between groups (Fig. 10A). In February, zpb expression was significantly reduced in maiden fish at 22 °C receiving GnRH implantation relative to maidens at 14 °C, and both groups of repeats at 22 °C showed suppression relative to repeats at 14 °C. In March, zpb expression was suppressed in both groups of maidens held at 22 °C, and repeats at 22 °C treated with GnRH. In April, there were no significant differences between treatments even though there was a general trend towards lower gene expression at elevated temperature. In October and January, the level of zpc gene expression was not different between maidens and repeats (Fig. 10B). In February, zpc expression was higher in repeats than maidens at 14 °C, and gene expression was higher in maidens at 22 °C than at 14 °C. There was suppressed zpc expression in maidens held at 22 °C treated with GnRH, relative to maidens at 14 °C in March. In April, the same effect was present. There were no significant differences among fish held at 22 °C in April; however, there was a general trend towards lower gene expression levels at elevated temperature.