2.1 道德聲明

這項研究得到了新不倫瑞克大學 （UNB） 動物護理委員會（動物使用協(xié)議 24007）的批準，并遵循了加拿大動物護理委員會的所有適用福利和實驗指南。

2.2 魚

二倍體和三倍體 S. alpinus 胚胎是在眼卵階段從 Valorēs 育種計劃（加拿大 Shippagan，NB）購買的。它們是2023年4月人工產(chǎn)卵的3只母（池卵）和10只父（池白子）的后代，在受精后210°C-min開始，通過65.5 MPa的靜水壓力機處理5 min誘導(dǎo)了大約一半的卵，其余的保留為二倍體對照。三倍體的眼期存活率（發(fā)貨前）為 58 ± 1%，二倍體為 77 ± 1%。隨后，按照標準飼養(yǎng)程序（Jobling 等人，2010 年）在 UNB 水生設(shè)施（加拿大新罕布什爾州弗雷德里克頓）飼養(yǎng)魚類，二倍體和三倍體分別在供應(yīng)脫氯市政水的流通池中飼養(yǎng)。UNB 的死亡率沒有記錄，但很低，倍性之間沒有明顯差異。

2024 年 1 月，在實驗前的幾個月里，所有魚都被轉(zhuǎn)移到六缸循環(huán)水產(chǎn)養(yǎng)殖系統(tǒng) （RAS） 內(nèi)的 314 L 倍性特異性水箱中。該RAS的水質(zhì)參數(shù)保持在推薦范圍內(nèi)（總氨氮和亞硝酸鹽均為<1.0 mg/L，硝酸鹽為0-400 mg/L，堿度為50-300 mg/L，硬度為>100 mg/L，pH 值為 6.5-8.5;Timmons 等人，2018 年），但單日低堿度 （43 ppm） 除外，通過增加 RAS 的補充水立即得到糾正。在整個 RAS 中，包括直到實驗期結(jié)束，溫度和溶解氧的平均溫度和溶解氧平均分別為 10.3°C ± 1.0°C 和 93% ± 5% 的空氣飽和度。放養(yǎng)密度約為 109 kg m^?3在實驗時，個體平均體重約為160克（表1）。用于該實驗的所有魚都來自單個二倍體和單個三倍體水箱。實驗前20天，室內(nèi)季節(jié)性調(diào)整的光周期從12：12增加到13：11 h（明：暗），以模擬初夏的季節(jié)趨勢，以維持在單獨的RAS內(nèi)的親魚，并且這種13：11狀態(tài)在整個實驗期間保持。

表 1. 臨界游動速度 （U_暴擊）和魚到達U后立即測量的參數(shù)_暴擊在幼年二倍體 （2n） 和三倍體 （3n） 中 Salvelinus alpinus（平均值 ± 標準差，每個倍性 n = 12）。

1 INTRODUCTION
The Arctic charr Salvelinus alpinus L. is a cold-water salmonid species native to Arctic, boreal and temperate regions of the northern hemisphere with great aquaculture potential due to its tolerance of high stocking densities, low optimum rearing temperature and high fillet quality and yield (S?ther et al., 2013; Yossa et al., 2019). However, farmed S. alpinus are prone to preharvest sexual maturation, a process which diverts energy allocation from somatic growth to the gonads, thereby decreasing fillet yield and quality, and increases disease susceptibility (Yossa et al., 2019). Additionally, it creates the risk of genetic introgression of the domesticated genome into locally adapted wild populations in the event of farm escapes, as documented for farmed Atlantic salmon Salmo salar (Glover et al., 2017; Wacker et al., 2023; Wringe et al., 2018).

A practical solution to unwanted sexual maturation of farmed fishes is to use functionally sterile triploid populations (Benfey, 2016; Piferrer et al., 2009). However, extensive pilot-scale and commercial trials with triploid S. salar have identified numerous welfare and farm production concerns when compared to diploids, including cataracts, skeletal deformities and overall reduced survival (Fraser et al., 2012; Madaro et al., 2022; O'Flynn et al., 1997). Although there have been fewer such studies of triploid S. alpinus, there is similar evidence of reduced survival (Chiasson et al., 2009; Fraser et al., 2022) and higher incidence of vertebral deformities (Fraser et al., 2022). Many studies have also shown triploid salmonids to have a reduced ability to tolerate environmental stressors such as high temperature and hypoxia (e.g. Benfey & Devlin, 2018; Hansen et al., 2015; Jensen & Benfey, 2022; Sambraus et al., 2017, 2018; Scott et al., 2015), possibly due to reduced aerobic scope at elevated temperatures (Riseth et al., 2020). There are, however, conflicting findings in the literature: at least two studies have failed to find such an effect of triploidy on aerobic scope or maximum metabolic rate at elevated temperatures (Bowden et al., 2018; Sezaki et al., 1991).

Increased erythrocyte size is often suggested as the limiting factor to the respiratory capacity of triploids because this results in reduced surface area relative to volume compared to the smaller erythrocytes of diploids (Benfey, 1999; Benfey & Devlin, 2018). Triploid salmonids also frequently exhibit a higher incidence of erythrocyte nuclear segmentation (ENS) (Clark et al., 2025; Dorafshan et al., 2008; Wang et al., 2010; Wlasow et al., 2014, 2004), a cellular deformity of unknown origin that is defined as the medial division of the nucleus (Yokote, 1982). This deformity has yet to be reported in S. alpinus.

Routine aquaculture operations such as vaccination and grading require fish to be crowded for short periods of time, often in combination with capture (by netting) and removal from the water. Triploid salmonids generally respond well to this in terms of their primary and secondary stress response (Benfey & Biron, 2000; Biron & Benfey, 1994; Hyndman et al., 2003a; Madaro et al., 2024; Preston et al., 2017; Sadler et al., 2000) although triploids may exhibit high mortality if pushed to exhaustion at high temperature (Hyndman et al., 2003b).

Critical swimming velocity (Ucrit) is a simple test frequently used to assess physiological responses to exhaustive exercise in fishes. It is positively correlated with haematocrit and blood haemoglobin levels (Pearson & Stevens, 1991), cardiac output (Clark & Seymour, 2006) and metabolic rate (Horodysky et al., 2011; Norin & Clark, 2016), and overall reflects maximum aerobic capacity (Kolok, 1999; Norin & Clark, 2016; Plaut, 2001). While previous studies have shown that Ucrit is not affected by triploidy in several salmonid species (Bernier et al., 2004; Lijalad & Powell, 2009; Riseth et al., 2020; Scott et al., 2015; Small & Randall, 1989; Stillwell & Benfey, 1997), this has not been examined in S. alpinus.

In this study, we examine the effects of triploidy on the response of this polar species to exhaustive exercise through a Ucrit test with post-exercise blood sampling. Based on previous studies in other salmonids, we hypothesized that Ucrit would not differ between diploids and triploids. We sought to supplement this information with measurements of secondary stress response (blood glucose, lactate and haematocrit), and to investigate the presence, and potential causes, of ENS in S. alpinus. Such information can assist in developing species-specific farm management practices for this relatively understudied salmonid aquaculture species.

2 MATERIALS AND METHODS
2.1 Ethics statement
This research was approved by the University of New Brunswick (UNB) Animal Care Committee (Animal Use Protocol 24007) and followed all applicable welfare and experimental guidelines of the Canadian Council on Animal Care.

2.2 Fish
Diploid and triploid S. alpinus embryos were purchased at the eyed-egg stage from the Valorēs breeding program (Shippagan, NB, Canada). They were progeny of three dams (pooled eggs) and 10 sires (pooled milt) artificially spawned in April 2023, with triploidy induced in approximately half of the eggs via hydrostatic pressure treatment of 5?min at 65.5?MPa beginning 210°C-min post-fertilization and the remainder retained as diploid controls. Survival to the eyed stage (prior to shipping) was 58?±?1% for triploids and 77?±?1% for diploids. Fish were subsequently reared at the UNB aquatic facility (Fredericton, NB, Canada) following standard husbandry procedures (Jobling et al., 2010), with diploids and triploids reared separately in flow-through tanks supplied with dechlorinated municipal water. Mortality at UNB was not recorded but was low and with no obvious difference between ploidies.

In January 2024, all fish were moved to 314-L ploidy-specific tanks within a six-tank recirculating aquaculture system (RAS) for the months preceding experimentation. Water quality parameters in this RAS were maintained within recommended ranges (total ammonia nitrogen and nitrite both <1.0?mg/L, nitrate 0–400?mg/L, alkalinity 50–300?mg/L, hardness >100?mg/L and pH?6.5–8.5; Timmons et al., 2018) with the exception of low alkalinity (43?ppm) on a single day that was immediately corrected by increasing makeup water to the RAS. Temperature and dissolved oxygen averaged 10.3°C?±?1.0°C and 93%?±?5% of air saturation, respectively, for the entire time in the RAS, including through to the end of the experimental period. Stocking density was approximately 109?kg?m?3 at the time of experimentation, with individuals weighing approximately 160?g on average (Table 1). All fish used for this experiment came from a single diploid and single triploid tank. Twenty days prior to experimentation, the seasonally adjusted photoperiod in the room was increased to 13:11?h (light:dark) from 12:12 to mimic the seasonal trends of early summer for the sake of broodstock maintained within a separate RAS, and this 13:11 regime was maintained throughout the experimental period.

TABLE 1. Critical swimming velocity (Ucrit) and parameters measured immediately after fish reached Ucrit in juvenile diploid (2n) and triploid (3n) Salvelinus alpinus (mean?±?standard deviation, n?=?12 per ploidy).