Abstract

Introduction
A major sustainability concern of sea cage-based Atlantic salmon (Salmo salar) aquaculture is the occurrence of escaped farm fish interbreeding with salmon in the wild1,2. Cultured salmon have been selectively bred for desirable production traits over multiple generations since the 1970’s3,4, and trade-offs in the domestication process make cultured salmon less adapted to surviving in the wild5,6,7. Introgression of domesticated genotypes may therefore hurt wild salmon populations that already are under pressure from other anthropogenic activities2,8,9.

Introgression can be avoided by using sterile fish in aquaculture productions. Presently, the only reliable method to create sterile fish at a commercial scale is via the induction of triploidy – that is three complete sets of chromosomes as opposed to two sets in normal diploid fish10. A triploid fish group is created by pressurizing fertilized eggs to prevent the extrusion of the second polar body from the female gamete and thereby retaining two maternal chromosome sets along with one paternal set10,11. A consequence of being triploid is that cells become larger as they contain 50% more DNA. Meanwhile, relative organ sizes and body proportions remain roughly similar. A triploid fish will therefore comprise of fewer but larger cells relative to a diploid counterpart of the same size12.

Triploidy may cause physiological disadvantages leading to reduced health in aquaculture. In Norway, the world’s largest producer of Atlantic salmon, it has been documented that triploids suffer reduced animal welfare particularly in the latter part of the marine growth phase relative to diploids13,14. The Norwegian Food Safety Authority therefore imposed a temporary moratorium after 2023 on the use of triploid Atlantic salmon in sea cage-based aquaculture15. However, triploids are presently still being used in other salmon producing countries such as Canada and Australia.

Notable animal welfare issues when using triploid Atlantic salmon in commercial sea cages include higher mortalities, increased occurrences of wounds and ulcers, and generally being more susceptible to infectious diseases13,14,16,17. Reduced growth during the marine phase, higher occurrences of emaciated fish, and lower quality gradation at harvest also make triploid Atlantic salmon less attractive from an economic point of view18,19,20,21.

The underlying physiological implications of being triploid has been extensively studied in salmonids to help understand the potential benefits and challenges in aquaculture10,22,23. It can here be theorized that having cells with an extra set of chromosomes should lead to higher basal maintenance costs, resulting in elevated standard metabolic rates (SMR) when at rest. Although this effect may be offset by triploids consisting of fewer cells. Larger cells with lower surface to volume ratios should also limit exchange rate capacities of oxygen between cells and intracellular spaces, potentially restricting maximum metabolic rates (MMR) during strenuous activities or acute stress. A potentially higher standard and lower maximum metabolic rate would both contribute to a reduced aerobic scope for supporting any energetically costly activity. A reduced aerobic scope leads to higher vulnerability to environmental hypoxia, a prevailing issue in salmon sea cages24,25. Moreover, as energetic demands in ectothermic fish increase with temperature while oxygen solubility in water decreases, a higher basal maintenance cost and a reduced capacity for oxygen uptake should then result in a lower thermal tolerance, which is a concern as summer heatwaves are projected to get worse and more frequent in the future in salmon producing regions26,27.

When investigating these above mentioned theoretical predictions, empirical studies on triploid salmonid physiology have occasionally found support for them, but often also reported no or subtle effects depending on experimental context. For instance, reduced aerobic scope has been implied in triploid brook char (Salvelinus fontinalis) owing to an elevated SMR28, and in triploid chinook salmon (Oncorhynchus tshawytscha) owing to a reduced oxygen carrying capacity of the blood29. Meanwhile triploid Atlantic salmon had a lower aerobic scope at 10.5 °C although it was similar to diploid counterparts at 3 °C30. In contrast, other studies found similar critical swimming speeds30,31,32, as well as similar metabolic rates between triploid and diploid salmonids33,34,35, indicating that triploidy did not impose a substantial physiological disadvantage. With regards to environmental vulnerability, indicators of lower hypoxia tolerance primarily at elevated temperatures has been found in different triploid salmonid species, albeit effects tended to be subtle36,37,38. Additionally, Bowden et al.,35 reported negligible differences in thermal tolerance between triploid and diploid Atlantic salmon while Verhille et al.39 reported impaired tolerance to high temperatures in triploid rainbow trout (Oncorhynchus mykiss).

A convincing and consistent physiological explanation for differences between triploid and diploid salmonids has therefore not yet been demonstrated. However, laboratory experiments have primarily utilised smaller fish and typically in freshwater, although the prevailing animal welfare issues first tend to emerge when triploid Atlantic salmon become much larger during the latter marine sea cage production phase13,14. It would therefore be interesting to consider the theoretical implication of physiological scaling effects across body size between diploids and triploids.

Larger-sized fish are generally assumed to have a lower thermal optimum and a lower aerobic scope owing to geometrical scaling effects causing oxygen supply limitation40,41,42. Moreover, this consequently implies that fish species may become smaller as an adaptation to global warming43,44,45. From this perspective, a triploid fish can be considered an experimental model that encompass certain aspects of being a larger-bodied animal due to their larger cell sizes and lower surface to volume ratios46, factors that likely impose comparable geometrical scaling constraints on functionality. An example of this is the growth patterns of muscle cells, where fish generally rely less on hyperplasia and more on hypertrophy of cells as they grow larger, and in adult Atlantic salmon continued muscle growth relies solely on hypertrophy47,48. Interestingly, diploid Atlantic salmon have approximately one-third more muscle fibres per myotome owing to higher rates of fibre recruitment and lower rates of hypertrophic growth than triploid counterparts49 highlighting that triploids indeed may functionally resemble larger animals.

In zebrafish (Danio rerio), triploid models have been established to investigate fundamental effects of different cell and genome sizes (46). Triploid zebrafish larvae have been reported to perform better in colder conditions, while they perform worse at higher temperatures and show slightly worse hypoxia tolerance than diploid counterparts, indicating oxygen supply limitations in more challenging conditions50,51.

Impairments to physiological capacities and environmental tolerance limits in triploids can therefore be theorized to be similar to what will happen as a fish becomes larger. Furthermore, the magnitude of reduced robustness with increasing body size should then be greater in triploid relative to diploid salmon in aquaculture contexts. This would explain why the latter marine growth phase is when triploids are reported to struggle the most13,14. From an applied aquaculture perspective, it would be valuable to investigate whether the welfare issues of triploid Atlantic salmon in the final phase of sea cage production indeed are a consequence of size-dependent rate limitations that become exacerbated by larger cell sizes, making them more vulnerable to various stressors when compared to diploid counterparts. If so, this would also make larger-sized triploids more vulnerable to summer heatwaves and hypoxia events in the sea cage environment.

The purpose of this study was to measure key physiological capacities of larger-sized (≈ 3 kg) triploid Atlantic salmon as compared to diploid counterparts when acclimated to a mid-seawater temperature of 12 °C. First, we performed respirometry trials to measure metabolic rate traits and acute hypoxia tolerance. Then we performed critical thermal maximum (CT max) trials and assessed haematological parameters in fish subjected to this imposed thermal stress. Additionally, we did gill histology analyses on all the fish tested to potentially provide a morphological link to the physiological data.

We hypothesized that the larger-sized triploid Atlantic salmon would have a lower maximum metabolic rate and lower aerobic scopes compared to diploid counterparts of similar sizes, driven by larger cells with higher surface to volume ratios limiting physiological rates at the cellular level. A reduced capacity for oxygen uptake in triploids should also translate into a reduced hypoxia tolerance and a reduced oxygen extraction coefficient. Acute thermal tolerance was also hypothesized to be lower in triploids owing to larger-sized cells making it more difficult to maintain homeostasis. Overall, we hoped to demonstrate an obvious and more consistent difference in physiological capacities and environmental limits between larger-sized diploid and triploid Atlantic salmon when compared to past experiments on smaller-sized fish.

基于海籠的大西洋鮭魚（Salmo salar）養(yǎng)殖的一個(gè)主要可持續(xù)性問題是野生中逃逸養(yǎng)殖魚與鮭魚雜交的情況 1,2.自20世紀(jì)70年代以來，養(yǎng)殖鮭魚經(jīng)過多代的選擇性繁殖，以獲得理想的生產(chǎn)性狀 3,4馴化過程中的權(quán)衡使養(yǎng)殖鮭魚更不適應(yīng)野外生存 5,6,7.因此，馴化基因型的引入可能會傷害已經(jīng)承受其他人為活動壓力的野生鮭魚種群 2,8,9.

通過在水產(chǎn)養(yǎng)殖中使用無菌魚類可以避免滲入。目前，唯一可靠的商業(yè)規(guī)模生產(chǎn)不育魚的方法就是誘導(dǎo)三倍體——即三套完整的染色體，而普通二倍體魚只有兩套10.三倍體魚類群通過加壓受精卵，防止第二極體從雌性配子中擠出，從而保留兩套母體和一組父系染色體 10,11.三倍體的后果是細(xì)胞體積更大，因?yàn)樗鼈兒械腄NA比細(xì)胞多50%。與此同時(shí)，器官的相對大小和身體比例大致保持相似。因此，三倍體魚相對于同大小的二倍體魚，細(xì)胞數(shù)量較少但細(xì)胞更大12.

三倍體可能導(dǎo)致生理上的問題，導(dǎo)致水產(chǎn)養(yǎng)殖中的健康狀況下降。在全球最大的大西洋鮭魚生產(chǎn)國挪威，已有記錄顯示三倍體鮭魚在海洋生長后期相較二倍體動物福利下降 13,14. 因此，挪威食品安全局在2023年后對三倍體大西洋鮭魚在海籠養(yǎng)殖中實(shí)施了臨時(shí)禁令15.然而，三倍體目前仍在加拿大和澳大利亞等其他鮭魚產(chǎn)區(qū)使用。

在商業(yè)海籠中使用三倍體大西洋鮭魚時(shí)，顯著的動物福利問題包括更高的死亡率、傷口和潰瘍的發(fā)生率增加，以及普遍更容易感染傳染病13,14,16,17.海相生長減緩、瘦弱魚類數(shù)量增加以及捕撈時(shí)分級質(zhì)量下降，也使得三倍體大西洋鮭魚從經(jīng)濟(jì)角度看吸引力降低18,19,20,21.

三倍體的潛在生理影響已被廣泛研究，以幫助理解水產(chǎn)養(yǎng)殖中的潛在益處與挑戰(zhàn)10,22,23.這里可以推測，擁有多余染色體的細(xì)胞應(yīng)導(dǎo)致更高的基礎(chǔ)維持成本，從而在靜息時(shí)提高標(biāo)準(zhǔn)代謝率（SMR）。盡管三倍體細(xì)胞數(shù)較少，可能會抵消這一效應(yīng)。體積較大的細(xì)胞，表面積與體積比較低，也應(yīng)限制細(xì)胞與細(xì)胞內(nèi)空間之間的氧交換速率容量，可能限制劇烈活動或急性壓力下的最大代謝率（MMR）。更高的標(biāo)準(zhǔn)和較低的最大代謝率都會降低支持任何能量消耗活動的有氧范圍。有氧范圍的縮小導(dǎo)致對環(huán)境缺氧的脆弱性增加，這是鮭魚海籠中普遍存在的問題 24,25.此外，隨著變溫魚類能量需求隨溫度增加而氧溶解度下降，更高的基礎(chǔ)維護(hù)成本和氧氣吸收能力降低，應(yīng)導(dǎo)致耐熱性降低，這在鮭魚產(chǎn)區(qū)夏季熱浪預(yù)計(jì)將愈發(fā)嚴(yán)重且頻繁時(shí)令人擔(dān)憂 26,27.

在研究上述理論預(yù)測時(shí)，三倍體鮭魚類生理的實(shí)證研究偶爾支持它們，但根據(jù)實(shí)驗(yàn)情境，也常報(bào)告無效應(yīng)或微弱效應(yīng)。例如，三倍體溪魚（Salvelinus fontinalis）因SMR升高，被暗示有氧范圍減小28以及三倍體奇努克鮭（Oncorhynchus tshawytscha）中由于血液氧氣攜帶能力降低而出現(xiàn)29.而三倍體大西洋鮭魚的有氧范圍較低，僅為10.5°C，盡管其氧氣范圍與二倍體鮭魚相似，3°C。30. 相比之下，其他研究發(fā)現(xiàn)了類似的關(guān)鍵游泳速度30,31,32以及三倍體和二倍體鮭魚類的代謝率相似33,34,35表明三倍體并未帶來顯著的生理劣勢。關(guān)于環(huán)境脆弱性，在不同三倍體鮭科物種中主要在高溫下發(fā)現(xiàn)了較低的缺氧耐受性指標(biāo)，盡管影響較為微妙36,37,38. 此外，Bowden 等人，35報(bào)告稱三倍體和二倍體大西洋鮭魚在耐熱性上幾乎沒有差異，而Verhille等人則在39報(bào)告三倍體虹鱒（Oncorhynchus mykiss）對高溫耐受性受損。

因此，尚未有令人信服且一致的生理學(xué)解釋來解釋三倍體鮭魚與二倍體鮭魚的差異。然而，實(shí)驗(yàn)室實(shí)驗(yàn)主要使用較小的魚類，通常是淡水魚類，盡管當(dāng)三倍體大西洋鮭魚在后期海洋籠養(yǎng)生產(chǎn)階段變得更大時(shí)，動物福利問題通常首先浮現(xiàn) 13,14.因此，探討二倍體和三倍體之間體型變化的生理尺度效應(yīng)的理論意義將非常有趣。

由于幾何尺度變化導(dǎo)致氧氣供應(yīng)受限，體型較大的魚通常被認(rèn)為具有較低的熱最優(yōu)和較低的有氧范圍40,41,42.此外，這也意味著魚類物種可能因適應(yīng)全球變暖而變小43,44,45.從這個(gè)角度看，三倍體魚類可以被視為一種實(shí)驗(yàn)?zāi)Ｐ停w了由于細(xì)胞體積較大和表面積比較低而成為體型較大動物的某些方面46這些因素很可能對功能施加了類似的幾何縮放約束。例如肌肉細(xì)胞的生長模式，魚類通常較少依賴細(xì)胞增生，而更多依賴細(xì)胞增生，而成年大西洋鮭魚的肌肉持續(xù)生長完全依賴肥大 47,48.有趣的是，二倍體大西洋鮭魚每個(gè)肌節(jié)的肌纖維數(shù)量約多三分之一，這得益于纖維招募率更高且肥厚生長速率低于三倍體鮭魚49這凸顯了三倍體確實(shí)可能在功能上類似于更大的動物。

在斑馬魚（Danio rerio）中，已建立三倍體模型以研究不同細(xì)胞和基因組大小的基本效應(yīng)（46）。三倍體斑馬魚幼體在寒冷條件下表現(xiàn)更好，但在較高溫度下表現(xiàn)較差，缺氧耐受性略遜于二倍體幼蟲，表明它們在更具挑戰(zhàn)性的環(huán)境中存在氧氣供應(yīng)限制 50,51.

因此，三倍體中生理能力和環(huán)境耐受極限的損害可以被理論化為類似于魚類變大時(shí)發(fā)生的情況。此外，在水產(chǎn)養(yǎng)殖環(huán)境中，體型增大時(shí)三倍體鮭魚的堅(jiān)韌性降低幅度應(yīng)當(dāng)更大。這也解釋了為什么后期的海洋生長階段是三倍體生物最為掙扎的時(shí)期 13,14.從應(yīng)用水產(chǎn)養(yǎng)殖的角度來看，探討三倍體大西洋鮭魚在海籠生產(chǎn)最后階段的福利問題是否確實(shí)源于尺寸依賴的速率限制，而這些限制因細(xì)胞體積較大而加劇，使其相比二倍體鮭魚更容易受到各種壓力源的影響，將是有價(jià)值的。如果是這樣，這也將使體型較大的三倍體在海籠環(huán)境中更容易受到夏季熱浪和缺氧事件的影響。

本研究旨在測量體型較大（≈3公斤）大西洋鮭魚在適應(yīng)12°C中海水溫度后，相較于二倍體鮭魚的關(guān)鍵生理能力。 首先，我們進(jìn)行了呼吸測試，以測量代謝率特征和急性缺氧耐受。隨后，我們進(jìn)行了臨界熱最大值（CT max）試驗(yàn)，并評估了受該熱應(yīng)力影響的魚類的血液學(xué)參數(shù)。此外，我們對所有檢測魚類進(jìn)行了鰓組織學(xué)分析，以可能提供與生理數(shù)據(jù)的形態(tài)聯(lián)系。

我們假設(shè)體型較大的三倍體大西洋鮭魚相比，其最大代謝率和需氧范圍會低于同尺寸的二倍體鮭魚，這主要是由于細(xì)胞體積較大、表面積比較高，限制了細(xì)胞層面的生理速率。三倍體氧氣攝取能力降低也應(yīng)轉(zhuǎn)化為缺氧耐受性降低和氧氣提取系數(shù)降低。三倍體的急性耐熱性也被假設(shè)較低，因?yàn)榧?xì)胞體積更大，使得維持穩(wěn)態(tài)更為困難。總體而言，我們希望展示較大二倍體和三倍體大西洋鮭魚在生理能力和環(huán)境限制上與以往小型魚類實(shí)驗(yàn)相比，存在更明顯且更一致的差異。