Fish are ectotherms, and their body temperature control depends on external factors [1]. Water temperature is one of the main environmental cues used by fish to indicate when to reproduce [2,3]. For fertilization, most species release their sperm and eggs into the water during external reproduction [3]. Fish spawning and reproduction are often stimulated by increased water temperatures within a range appropriate for the species [3,4]. Climate change is causing an increasing number of abrupt and frequent changes in water temperature [5], which can lead to fish being exposed to water temperatures outside the optimal thermal ranges for their species [6]. As a result, both in hatcheries and in the wild, sperm may be exposed to variable ambient temperatures on their way to the egg. Consequently, fish physiology will be adversely affected by global warming [7], including gametogenesis and the capacity of fish to successfully reproduce [2].

Generally, spermatozoa of both freshwater and marine fish are immotile in the testis, in seminal fluid and in solutions with osmolality similar to that of seminal fluid [[8], [9], [10], [11]]. The environment controls the motility of teleost spermatozoon [12]. Spermatozoon motility is considered to reflect several metabolic pathways and regulatory mechanisms [3,13], and abnormalities in these factors can cause poor sperm motility. However, less is known regarding how the kinematic parameters and motility patterns of fish change in response to changes in water temperature. Research by Lahnsteiner & Mansour [12], Beirão et al. [14], and Dadras et al. [3] have shown that sperm motility rate and swimming velocity are influenced by temperature. According to several studies [12,[15], [16], [17]], there is a trade-off between increased sperm velocity and decreased motility duration at higher temperatures. The temperature of the water influences the viability of gametes produced by thermally challenged salmonids, as reported by Fenques et al. [18]. The direct or indirect effect of temperature on sperm functionality seems to be highly species-specific, as it has been described [13] that the lower and upper limits of temperature ranges, as well as the optimal temperature for different fish species, differ widely. Short-term storage induces a significant decrease in salmon sperm function parameters [19].

Gamete handling is one of several husbandry procedures that are key for the success in in vitro fertilization. Sperm collection is the very first step of the in vitro fertilization process, and it plays an important role in sperm quality for subsequent stages [20]. In general, fish sperm can be collected either by abdominal massage or by removal of the testes by dissection [21]. In addition, conditions of storage and fish sperm activation are prerequisites for their handling in artificial reproduction [22]. Previous studies in Atlantic salmon (Salmo salar) have shown that short-term storage induces a significant decrease in sperm function parameters in salmon [19], considering standard parameters such as viability and plasma membrane integrity, mitochondrial membrane potential, DNA fragmentation and superoxide anion level (O2−). So far as we know, this is the first study to analyze various sperm functions in Salmo salar semen collected by different methods and subjected to cold storage.

Sperm motility characteristics, such as swimming velocity and motility duration, are significant variables in determining whether artificial reproduction techniques in fish-farming are successful [23,24]. After release into the external medium, fish spermatozoa have a brief period of rapid motility, followed by slow progressive motility (from 30 s to several minutes) [25,26]. During this brief period (the life span of the spermatozoa), the sperm must find an egg and successfully fertilize it [27]. In fact, changes in sperm function are influenced by the ambient temperature during the brief time when sperm motility is sustained [28], and temperature stress has been linked to increased production of reactive oxygen species (ROS), oxidative stress [29], and viability problems [30]. To our knowledge, no study has investigated if different temperatures of the activation media effects sperm motility patterns after short-term cold storage.

The mitochondrion is an organelle crucial for normal sperm functions, and mitochondrial activity is considered a good parameter of sperm quality [31]. Mitochondria are the principal source of ROS within the cell [32,33]. Furthermore, the mitochondrion is the most sensitive structure in the sperm to the effects of cryopreservation [34]. Sperm cryopreservation has been linked to oxidative stress (OS) damage due to an increase in ROS [35]. This causes a reduction in the mitochondrial membrane potential, leading to loss of ATP, impaired motility and viability, and the induction of apoptosis in spermatozoa [36,37]. Due to this sensitivity, it is therefore crucial to obtain a better understanding and more detailed knowledge of the mitochondrial physiology of sperm, and to improve milt handling and storage.

According to Alavi & Cosson [23], fish spermatozoa exposed to various physiological conditions can be objectively compared using computer-assisted sperm analysis (CASA), which classifies sperm motility based on swimming patterns (linear, non-linear, and circular). Thus, CASA can specify their swimming potential of the spermatozoa as well as their ability to reproduce under artificial conditions [38].

The aim of the present study was therefore to assess plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), superoxide anion production levels (cytoplasmatic and mitochondrial), and DNA integrity of sperm from Atlantic salmon as a function of collection methods and storage time at 4 °C, and, secondly, to assess the effect of activation medium temperature on the sperm motility pattern after short-term storage at 4 °C.