To understand how male age affects seminal fluid, we conducted a literature search following PRISMA eco-evo guidelines (O’Dea et al., 2021). We used a search string for abstracts, titles, and keywords “(sfp* OR seminal fluid OR seminal plasma) AND (ageing OR age OR aging OR senescence)” to identify studies which test how advancing male age affects seminal fluid, using two search engines: SCOPUS and Web of Science (WoS), on December 14^th 2021, accessed through the University of Oxford server. The searches returned a total of 738 hits from WoS (year range: 1991 to 2021) and 620 from SCOPUS (year range: 1941 to 2021). After duplicate deletion, which was done using Rayyan (Ouzzani et al., 2016), we obtained a total of 970 unique papers. We then screened the abstracts of these papers using pre-defined inclusion and exclusion criteria (see below), before screening the full-texts to obtain a final list of papers from which relevant data was extracted.

To be retained for full-text screening, the paper had to be a research article (not review or meta-analysis), on any animal, and measure a seminal fluid trait for males of different ages, judged from its abstract. We excluded studies during abstract screening if they were on the wrong topic, did not compare males of different ages, did not have clear ageing data, only covered a small proportion of lifespan (e.g., only included young males), did not measure seminal fluid traits, or only measured seminal fluid during maturation of males (i.e., during juvenile or pubertal stages). The initial screening of abstracts produced a total of 94 studies whose full texts were considered in more detail.

When assessing full texts, to be included in our analysis review, a study needed to: compare males of non-overlapping age groups, compare non-sperm components of the seminal fluid (like oxidative stress enzymes, proteins, hormones, lipids, and macro- or micronutrients), report sample sizes of males in each age group and exact ages (or range of ages) to which males in each age group belonged. We excluded studies whose full texts were not available (two studies), or which were not in English (three studies). We additionally conducted a scoping search on Google Scholar to obtain additional papers which might have been missed in our systematic screening and search. This was done by using the keywords “seminal fluid protein + aging + ageing + senescence” for each of the following taxa: “bulls,” “insects,” “pigs,” “rodents,” “humans,” “birds,” “mammals,” “fish,” and searching the first five search result pages for relevant studies.

From all studies which fulfilled our inclusion criteria, we collected information on how male age affected various non-sperm characteristics of the ejaculate, as described in the paper. Additionally, we collected data on factors which could modulate the influence of seminal fluid ageing, such as: male mating history (i.e., whether males were held as virgin or not prior to testing), at which ages males were sampled, what fraction of average lifespan was covered and sampling methodology. The fraction of average lifespan covered is likely to influence whether seminal fluid ageing is detected in a study because ageing trajectories are expected to follow a non-linear pattern, with senescence being more prominent in late-adult life (e.g., Jones et al., 2014; Lemaître et al., 2020b).

Male mating history could influence the ageing of seminal fluid, such that if males are kept virgins, old males would have stored seminal fluid for longer durations, thus have more degraded SFPs and higher accumulation of oxidative damage than mated old males or virgin young males. On the other hand, old virgin males would accumulate higher quantities of SFPs than younger virgin males (Koppik et al., 2018; Sepil et al., 2020). If previously mated males are tested the quantity of seminal fluid produced would depend on the timing of the last mating, number of times the male mated in succession and its rate of replenishment, given that the abundance of SFPs within accessory tissues/glands decreases significantly immediately after a mating event (Hopkins et al., 2019a; Sepil et al., 2019). Furthermore, if mating history is not controlled for, then older males would have mated more times over their life (e.g., Aich et al., 2021), and thus have undergone more rounds of SFP replenishment and thus potentially experienced a higher turnover of the glandular tissue producing the SFP than young males.

Male sampling methodology (if samples are collected longitudinally or cross-sectionally) can also have a large impact on the study outcome. Cross-sectional sampling of males makes age-dependent individual-level deterioration in ejaculate traits with advancing age harder to detect (Nussey et al., 2013), especially if low-quality males selectively disappear (Bouwhuis et al., 2009; Hämäläinen et al., 2014). This non-random age-dependent mortality could lead to biased sampling of males, where younger age classes would have higher variance and might bias estimates of averages in seminal fluid traits compared to old age classes. Thus, cross-sectional studies might underestimate male reproductive senescence, compared to longitudinal sampling measuring the same individuals at different ages.

Overall, we obtained data from 27 papers through our systematic searches, and seven additional papers from Google Scholar (see Supplementary Table S1 for the full list of included studies). Out of these 34 studies, 14 reported how male age affected SFPs (see Table 1), although some of these studies reported changes in total protein content, while others, changes in specific SFPs only. 10 studies reported data on oxidative damage levels or anti-oxidants present in seminal fluid (henceforth collectively called “oxidative stress,” see Table 2). Apart from these two components of the seminal fluid, a smaller fraction of studies assessed the concentration of lipids or lipoproteins (four studies), minerals/vitamins content (four studies), sugar content (two studies), or hormone concentrations (four studies) in the seminal plasma/ejaculate.

The low number of studies dedicated to male age-related changes in the seminal fluid is also reflected in the limited taxonomic breadth, with a strong focus on mammals (see Figure 1). Within mammals, studies were conducted on farm animals, humans, and laboratory rodents (see Supplementary Table S1). For most studies, males were sampled up to around 80% of their average adult lifespan and 50% of their maximum adult lifespan (see Tables 1 and 2 for lifespan sampled by studies; Supplementary Table S2 for sources of lifespan measurements). Another caveat is that non-significant results might go unpublished and it is difficult to estimate this extent, though a number of studies in our set of papers report no changes with age, so we hope the bias is not strong. In the following review, we restricted our discussion to studies that tested for male age-related changes in SFPs and oxidative stress response, as these aspects of the seminal fluid were better represented compared to other ejaculatory components.

We found some heterogeneity between studies in age-related SFP changes. This could be due to studies reporting quantitative changes in a set of proteins only (rather than all the proteins), or due to specific proteins responding differently to age based on their function or tissue-of-origin (Borziak et al., 2016; Sepil et al., 2020). Proteomics techniques that quantify the whole ejaculate are needed to better elucidate these biological patterns and with the advance of molecular approaches and particularly proteomics, this will become ever more feasible for a range of taxa.

Confounding factors could also explain some of the inconsistencies observed between studies. For instance, male mating history could have a large influence on SFP quantity changes as explained in the methods section above. Another caveat in comparing studies is that males are not always sampled up to old age and so an important fraction of the ageing trajectory is missed. This could be a serious bias, potentially compounded by there being stronger selection to maintain functionality earlier in life, meaning realised phenotypes may in part represent compensatory adaptive responses to the onset of seminal fluid ageing. Disentangling age-related changes from compensatory responses would require sampling beyond the 50% average lifespan into older ages, when the latter responses are expected to wane as the strength of selection declines.

While studies in our review rarely directly discuss the functional importance of the changes in observed SFPs, below, we suggest testable hypotheses for how male seminal fluid ageing might have functional consequences. Overall, studies in our review show changes in specific SFP abundances which are known to influence male fertilization success as well as a variety of female responses. For instance, older male D. melanogaster are less able to delay female remating and stimulate egg laying compared to younger males (Koppik and Fricke, 2017; Ruhmann et al., 2018; Sepil et al., 2020). Similarly in Aedes aegypti mosquitoes, older males are less able to prevent female remating (Agudelo et al., 2021). These responses are largely mediated by SFPs in D. melanogaster (Chapman et al., 2003) and the expression of functionally important SFPs declines with age (Koppik and Fricke, 2017). Therefore, it is possible that the decline in SFPs are driving the changes in female post-mating behavior. For instance, Sepil et al. (2020) found a significant age-related increase in SFP abundances in the accessory glands of unmated males, but no change in SFP abundances in the accessory glands of frequently-mated males. Yet, the authors also found that female egg laying behavior and remating affinity changed as a function of male age following matings with spermless males, hence the seminal fluid alone does contribute to the decline in reproductive function with male age. SFP transfer data can partially explain these findings. While there is no age-related decline in SFP abundances in the accessory glands, old unmated males transferred a lower quantity of SFPs to females compared to younger unmated males. There was no age-related change in the quantity of SFPs transferred to females for frequently-mated males, so it is likely that changes in SFP quality rather than quantity explain the decline in reproductive function with age in this group of males.

Apart from affecting male ability to induce female post-mating responses, age-related changes in seminal fluid might also affect sperm traits. For example, in the jungle fowl Gallus gallus, age-related changes in proteins which affect sperm velocity were detected (Borziak et al., 2016). Thus, the decreased ability of older males to gain paternity under sperm competition and fertilize eggs may be driven by changes in SFPs rather than changes in sperm per se.

While it can be difficult to pinpoint the precise changes responsible, it is becoming increasingly possible to manipulate the expression of individual SFPs to better understand how particular SFPs affect female post-mating behavior and sperm competition. For instance, using a combination of proteomics and RNAi, Marshall et al. (2009) identified a single accessory gland-derived ejaculate protein in the ground cricket Allonemobius socius that influences female egg-laying and declines in expression with male age. Hence, this protein is a prime candidate to explain the waning ability of males to induce female egg laying as the male ages. However, the link between seminal fluid expression and female responses is not necessarily straightforward. RNAi knockdown studies in other taxa have demonstrated that suppressing the expression of individual SFPs can have both positive and negative impacts on fitness-relevant traits such as female fecundity (Xu et al., 2013; Weber et al., 2019), though this may in part reflect the difficulty of measuring fitness components under realistic conditions. A further limitation of many such knockdown or knockout studies is that they tend (often of necessity) to consider only one or a few seminal fluid proteins, whereas in reality the seminal fluid proteome is a highly integrated unit whose individual components co-vary in their expression (Mohorianu et al., 2018; Patlar et al., 2019).

In addition to changes in the abundance of individual proteins or changes to the composition of the seminal proteome, ageing can potentially impact seminal fluid through alterations to protein quality. A loss of protein homeostasis – proteostasis – is a well-known feature of ageing, characterized by a failure of chaperones, stress-response factors, and protein degradation machinery to respond to stress and prevent protein misfolding (Labbadia and Morimoto, 2014). The role of failing proteostasis in loss of SFP quality in ageing males is currently unclear but has the potential to impact ejaculate function.

Work in D. melanogaster suggests that factors other than SFP quantity may be responsible for the decline in seminal fluid-mediated functions with male age (Sepil et al., 2020). Aged males, that are known to have compromised fertility and reduced seminal fluid function, still appear capable of levels of seminal proteome production and transfer that are similar to young males (Sepil et al., 2020). However, several proteins in aged males show evidence of qualitative changes via mass shifts on Western blots (Sepil et al., 2020). While it remains to be investigated how widespread age-related changes in SFP quality are and what the functional consequences are, it nonetheless raises the possibility that a decline in SFP functionality with age is primarily related to proteostasis loss, rather than diminishing amounts of SFPs.

While our systematic review showed general age-related declines in SFPs, how the somatic tissues which produce SFPs are affected by ageing across taxa still remains unclear. Generally, the size of prostates/accessory glands tends to increase as males grow older (Jin et al., 1996; Atalan et al., 1999; Rezaei et al., 2015; Reyes-Hernández and Pérez-Staples, 2017), but shrinkage with age was also reported in few studies (Mazeed and Mohanny, 2010; Santhosh and Krishna, 2013). However, the overall size of the organ does not necessarily predict protein content, as found in A. ludens (Herrera-Cruz et al., 2018) and D. bipectinata (Santhosh and Krishna, 2013). In humans, the increase in prostate size is known as benign prostatic hyperplasia (Berges and Oelke, 2011; Zhang et al., 2013), however prostate size varies among ethnic groups and so does the rate of change with age (i.e., Bolivian Tsimane, Trumble et al., 2015) or the occurrence of enlarged prostates in older males (Mubenga et al., 2020). Some theory predicts that the enlargement of the prostate is a side-effect of cellular hyperfunction that causes ageing of this tissue (Blagosklonny, 2021). The hyperfunction theory of ageing proposes that suboptimal nutrient-sensing molecular signaling in late-life causes ageing via excessive biosynthesis, as opposed to energy-tradeoffs (Lind et al., 2019).

The studies we reviewed consistently found that antioxidant quantity in the seminal fluid decreases with increasing male age, while oxidative stress markers tend to increase in the seminal fluid as males age. Reactive oxygen species (ROS) are unstable, free radical compounds and are required for vital cellular processes (Finkel and Holbrook, 2000; Hajam et al., 2022), but can also be deleterious to cells. For instance ROS play a role in sperm activation and changing sperm motility, e.g., in humans (Aitken et al., 2022) with the potential to influence male reproduction (Mannucci et al., 2022). However, work in D. melanogaster shows that while older males have higher metabolic rates in their sperm, ROS production is actually lower in these sperm (Turnell and Reinhardt, 2020).

Antioxidants, on the other hand, play a key role in stabilizing free radicals generated as part of cellular processes (Hood et al., 2019), and an imbalance between antioxidants and ROS causes oxidative stress. Oxidative stress has been shown to influence sperm homeostasis and can cause sperm DNA damage thus affecting male fertility (Mannucci et al., 2022) and has been shown to be key in regulating various intracellular pathways related to sperm, and activation of various sperm transcription factors (Aitken and Baker, 2006; Sabeti et al., 2016; Aitken, 2017). Our review suggests that older males have lower antioxidant levels but higher oxidative stress markers in their seminal fluid, and thus may have higher oxidative stress than young males. The decline in antioxidants might indicate a tradeoff where ageing males cannot maintain optimal antioxidant levels if these are energetically costly.

The mechanisms for why older males have higher oxidative stress could be several. For instance, ROS from sperm could “leak” into seminal fluid or somatic cells which produce SF could accumulate more ROS damage over time in old versus young males. This increase in oxidative stress in older males could have severe hypothesized functional consequences, such as higher oxidative damage to sperm, or the fertilized egg, and reduced sperm performance, which can be tested by future studies. More studies are needed to disentangle the origin/cause of age-dependent changes in ROS production in seminal fluid, the consequences of scavenging by SF antioxidants, and the overall effects on sperm, male and female reproduction.

Studies identified in our systematic literature review included only a few factors such as proportional lifespan sampled, male mating history and sampling of males to explain differences in seminal fluid ageing.

Besides these, other factors could be predicted to influence SF ageing. For instance, evidence for reproductive ageing has been shown to be stronger in laboratory and captive animals compared to wild ones (Nussey et al., 2013; Zajitschek et al., 2020; Kappeler et al., 2022). Additionally, domestic animals, which were used in a majority of studies found in our systematic review, are kept in semi-controlled conditions and are killed off prior to reaching a senescent age (i.e., post their “prime”). Thus, evidence for reproductive senescence in the seminal fluid may be weaker in domestic animals, although in our review, we found evidence for seminal fluid senescence in both lab and domestic animals.

Other abiotic and biotic factors could influence seminal fluid ageing, such as a male’s social environment. Both sperm and seminal fluid are highly plastic in their expression (reviewed in Perry et al., 2013; and Ramm, 2020). Males are known to invest more in seminal fluid production under more competitive environments, such as under high sperm competition (Hopkins et al., 2019b), possibly at the cost of reduced later-life investment in reproduction (Lemaître et al., 2020a). The costs of ejaculate plasticity have been discussed before (see, e.g., Ramm, 2020), but to our knowledge have not been tested, and whether these costs differ for old versus young males can be investigated in the future. Knowledge of costs could be one factor predicting ageing trajectories of seminal fluid. If seminal fluid production is costly and its continued production causes damage, then strong selection on early reproduction might be favoured and we would expect rapid ageing as a consequence. However, if seminal fluid production is cheap then factors such a sperm competition, male dominance and/ or female preferences might have more scope to shape ageing patterns. For example if old males are socially dominant and preferred by females they might face little competition and selection on seminal fluid is relaxed and thus ageing might arise. Conversely, if older males are more likely to experience sperm competition or female ejaculate rejection then there might be relatively high selection for seminal fluid competency late in life. Additionally, sperm production patterns, i.e., continuously versus one bout early in life, and whether sperm is the limiting factor might be important too, because supply and demand needs to be balanced between these different components through reproductive lifespan. To test these ideas knowledge of ejaculate ageing across a broad range of taxa with different reproductive patterns is necessary.

Mating systems can also influence ageing of seminal fluid. Ageing effects are expected to be more pronounced in polyandrous species where males are likely to invest more in their ejaculates (Veltsos et al., 2022), due to facing a higher risk of sperm competition. However, the influence of sperm competition on male reproductive senescence likely depends on the life-history of a species. For example, in some species, males may preferentially invest resources in producing more SFPs early in life, and suffer faster rates of reproductive senescence later in life (see also Lemaître et al., 2020a). Older males in such species are often inferior in both pre- and postcopulatory competition (Johnson and Gemmell, 2012; Gasparini et al., 2019) and are discriminated against by females (Velando et al., 2011; Rezaei et al., 2015). Alternatively, species with increased levels of sperm competition may evolve increased investment in SF (Immler et al., 2011; Lüpold et al., 2020), which may reduce the rate of senescence in these ejaculate traits (Delbarco-Trillo et al., 2018).

Abiotic factors such as nutrition could also impact the trajectory of ageing of the seminal fluid. Studies on male rats showed that both over- and undernutrition during pregnancy seem to lead to premature male reproductive ageing (reviewed in Zambrano et al., 2021). This is because, at least in mammals, the early stages of development have an overall impact on health and quality of life during adulthood (developmental programming) with endocrine disruptors and maternal nutrition impacting developmental programming.

The impact of male age is not limited to his own and his mates’ reproductive success, but potentially extends to offspring fitness as well. Males are known to influence the fitness of their offspring through mechanisms other than the transmission of DNA (Curley et al., 2011; Crean and Bonduriansky, 2014). Advanced paternal age has been shown to shorten offspring lifespan, exacerbate ageing-related pathology and to alter offspring social behavior (Kong et al., 2012; Brenman-Suttner et al., 2018; Xie et al., 2018). Classically, these impacts were believed to be due to the accumulation of de novo mutations in ageing germ cells. However, recent work suggests that non-genetic mechanisms, such as changes in methylation patterns or small non-coding RNA populations, are more likely to drive the intergenerational effects of ageing (Xie et al., 2018). Importantly, it was recently suggested that seminal fluid might be an under-appreciated mediator of paternal effects (Simmons, 2011; Watkins et al., 2018; Evans et al., 2019; Simmons and Lovegrove, 2019, 2020; Kekäläinen et al., 2020), yet this has not yet been tested in a paternal ageing context.