Abstract
Triploid Atlantic salmon are sterile and used in aquaculture to prevent escapees from breeding in the wild. Meanwhile, triploids suffer poor animal welfare in the latter marine growth phase. Previous experiments have mainly tested smaller fish, and physiological differences between triploids and diploids tended to be subtle or non-existing. We therefore hypothesized that triploidy first becomes a disadvantage at larger body sizes where scaling constraints become more magnified in triploids owing to them having larger cells with lower surface to volume ratios. We measured metabolic rates, stress responses, hypoxia tolerance, and critical thermal maximum in big (≈3 kg) triploid and diploid Atlantic salmon. Additionally, we assessed gill histology metrics. Big triploids had higher standard metabolic rates, lower aerobic scopes, and reduced tolerances to hypoxia and thermal stress. Oxygen extraction coefficients were overall lower in triploids, suggesting reduced efficiency in gill oxygen uptake. This was further supported by lower lamellar densities which indicate less gill surface area. In conclusion, big triploid Atlantic salmon were more vulnerable to environmental extremes driven by oxygen supply limitation and higher basal maintenance costs. This provides a mechanistic explanation for why triploids become prone to animal welfare issues in the latter growth phase of marine aquaculture.
三倍体大西洋鲑鱼为无菌型，用于水产养殖以防止逃逸个体在野外繁殖。与此同时，三倍体在后期海洋生长阶段动物福利较差。以往的实验主要测试较小的鱼类，三倍体和二倍体之间的生理差异往往很细微甚至不存在。因此，我们假设三倍体首先在较大体型时成为劣势，因为三倍体细胞更大且表面积比较低，缩放限制更为明显。我们测量了≈大三倍体和二倍体大西洋鲑鱼的代谢率、应激反应、缺氧耐受性和临界热极大值。此外，我们还评估了鳃组织学指标。大三倍体患者标准代谢率更高，有氧内窥镜较低，且对缺氧和热应激的耐受性降低。三倍体的氧气提取系数整体较低，表明鳃吸收氧气效率降低。这一观点还得到了层状密度较低的支持，这表明鳃表面积较小。总之，大型三倍体大西洋鲑鱼更易受到由氧气供应限制和基底维护成本增加驱动的环境极端影响。这为三倍体鱼在海洋水产养殖后期生长阶段容易出现动物福利问题提供了机制解释。
Keywords Aerobic scope, Animal welfare, Cell size, Critical thermal maximum, Gill histology, Hypoxia
toleranceKeywords： Aerobic scope, Animal welfare, Cell size, Critical thermal maximum, Gill histology, Hypoxia
tolerance
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.
基于海笼的大西洋鲑鱼（Salmo salar）养殖的一个主要可持续性问题是野生中逃逸养殖鱼与鲑鱼杂交的情况 1,2.自20世纪70年代以来，养殖鲑鱼经过多代的选择性繁殖，以获得理想的生产性状 3,4驯化过程中的权衡使养殖鲑鱼更不适应野外生存 5,6,7.因此，驯化基因型的引入可能会伤害已经承受其他人为活动压力的野生鲑鱼种群 2,8,9.
通过在水产养殖中使用无菌鱼类可以避免渗入。目前，唯一可靠的商业规模生产不育鱼的方法就是诱导三倍体——即三套完整的染色体，而普通二倍体鱼只有两套10.三倍体鱼类群通过加压受精卵，防止第二极体从雌性配子中挤出，从而保留两套母体和一组父系染色体 10,11.三倍体的后果是细胞体积更大，因为它们含有的DNA比细胞多50%。与此同时，器官的相对大小和身体比例大致保持相似。因此，三倍体鱼相对于同大小的二倍体鱼，细胞数量较少但细胞更大12.
三倍体可能导致生理上的问题，导致水产养殖中的健康状况下降。在全球最大的大西洋鲑鱼生产国挪威，已有记录显示三倍体鲑鱼在海洋生长后期相较二倍体动物福利下降 13,14. 因此，挪威食品安全局在2023年后对三倍体大西洋鲑鱼在海笼养殖中实施了临时禁令15.然而，三倍体目前仍在加拿大和澳大利亚等其他鲑鱼产区使用。
在商业海笼中使用三倍体大西洋鲑鱼时，显著的动物福利问题包括更高的死亡率、伤口和溃疡的发生率增加，以及普遍更容易感染传染病13,14,16,17.海相生长减缓、瘦弱鱼类数量增加以及捕捞时分级质量下降，也使得三倍体大西洋鲑鱼从经济角度看吸引力降低18,19,20,21.
三倍体的潜在生理影响已被广泛研究，以帮助理解水产养殖中的潜在益处与挑战10,22,23.这里可以推测，拥有多余染色体的细胞应导致更高的基础维持成本，从而在静息时提高标准代谢率（SMR）。尽管三倍体细胞数较少，可能会抵消这一效应。体积较大的细胞，表面积与体积比较低，也应限制细胞与细胞内空间之间的氧交换速率容量，可能限制剧烈活动或急性压力下的最大代谢率（MMR）。更高的标准和较低的最大代谢率都会降低支持任何能量消耗活动的有氧范围。有氧范围的缩小导致对环境缺氧的脆弱性增加，这是鲑鱼海笼中普遍存在的问题 24,25.此外，随着变温鱼类能量需求随温度增加而氧溶解度下降，更高的基础维护成本和氧气吸收能力降低，应导致耐热性降低，这在鲑鱼产区夏季热浪预计将愈发严重且频繁时令人担忧 26,27.
在研究上述理论预测时，三倍体鲑鱼类生理的实证研究偶尔支持它们，但根据实验情境，也常报告无效应或微弱效应。例如，三倍体溪鱼（Salvelinus fontinalis）因SMR升高，被暗示有氧范围减小28以及三倍体奇努克鲑（Oncorhynchus tshawytscha）中由于血液氧气携带能力降低而出现29.而三倍体大西洋鲑鱼的有氧范围较低，仅为10.5°C，尽管其氧气范围与二倍体鲑鱼相似，3°C。30. 相比之下，其他研究发现了类似的关键游泳速度30,31,32以及三倍体和二倍体鲑鱼类的代谢率相似33,34,35表明三倍体并未带来显著的生理劣势。关于环境脆弱性，在不同三倍体鲑科物种中主要在高温下发现了较低的缺氧耐受性指标，尽管影响较为微妙36,37,38. 此外，Bowden 等人，35报告称三倍体和二倍体大西洋鲑鱼在耐热性上几乎没有差异，而Verhille等人则在39报告三倍体虹鳟（Oncorhynchus mykiss）对高温耐受性受损。
因此，尚未有令人信服且一致的生理学解释来解释三倍体鲑鱼与二倍体鲑鱼的差异。然而，实验室实验主要使用较小的鱼类，通常是淡水鱼类，尽管当三倍体大西洋鲑鱼在后期海洋笼养生产阶段变得更大时，动物福利问题通常首先浮现 13,14.因此，探讨二倍体和三倍体之间体型变化的生理尺度效应的理论意义将非常有趣。
由于几何尺度变化导致氧气供应受限，体型较大的鱼通常被认为具有较低的热最优和较低的有氧范围40,41,42.此外，这也意味着鱼类物种可能因适应全球变暖而变小43,44,45.从这个角度看，三倍体鱼类可以被视为一种实验模型，涵盖了由于细胞体积较大和表面积比较低而成为体型较大动物的某些方面46这些因素很可能对功能施加了类似的几何缩放约束。例如肌肉细胞的生长模式，鱼类通常较少依赖细胞增生，而更多依赖细胞增生，而成年大西洋鲑鱼的肌肉持续生长完全依赖肥大 47,48.有趣的是，二倍体大西洋鲑鱼每个肌节的肌纤维数量约多三分之一，这得益于纤维招募率更高且肥厚生长速率低于三倍体鲑鱼49这凸显了三倍体确实可能在功能上类似于更大的动物。
在斑马鱼（Danio rerio）中，已建立三倍体模型以研究不同细胞和基因组大小的基本效应（46）。三倍体斑马鱼幼体在寒冷条件下表现更好，但在较高温度下表现较差，缺氧耐受性略逊于二倍体幼虫，表明它们在更具挑战性的环境中存在氧气供应限制 50,51.
因此，三倍体中生理能力和环境耐受极限的损害可以被理论化为类似于鱼类变大时发生的情况。此外，在水产养殖环境中，体型增大时三倍体鲑鱼的坚韧性降低幅度应当更大。这也解释了为什么后期的海洋生长阶段是三倍体生物最为挣扎的时期 13,14.从应用水产养殖的角度来看，探讨三倍体大西洋鲑鱼在海笼生产最后阶段的福利问题是否确实源于尺寸依赖的速率限制，而这些限制因细胞体积较大而加剧，使其相比二倍体鲑鱼更容易受到各种压力源的影响，将是有价值的。如果是这样，这也将使体型较大的三倍体在海笼环境中更容易受到夏季热浪和缺氧事件的影响。
本研究旨在测量体型较大（≈3公斤）大西洋鲑鱼在适应12°C中海水温度后，相较于二倍体鲑鱼的关键生理能力。 首先，我们进行了呼吸测试，以测量代谢率特征和急性缺氧耐受。随后，我们进行了临界热最大值（CT max）试验，并评估了受该热应力影响的鱼类的血液学参数。此外，我们对所有检测鱼类进行了鳃组织学分析，以可能提供与生理数据的形态联系。
我们假设体型较大的三倍体大西洋鲑鱼相比，其最大代谢率和需氧范围会低于同尺寸的二倍体鲑鱼，这主要是由于细胞体积较大、表面积比较高，限制了细胞层面的生理速率。三倍体氧气摄取能力降低也应转化为缺氧耐受性降低和氧气提取系数降低。三倍体的急性耐热性也被假设较低，因为细胞体积更大，使得维持稳态更为困难。总体而言，我们希望展示较大二倍体和三倍体大西洋鲑鱼在生理能力和环境限制上与以往小型鱼类实验相比，存在更明显且更一致的差异。
