Dietary polyunsaturated fatty acids affect volume and metabolism of Drosophila melanogaster sperm

Dietary fatty acids can accumulate in sperm and affect their function in vertebrates. As Drosophila melanogaster shares several pathways of lipid metabolism and shows similar lipid‐dependent phenotypes but lacks some hormones that in vertebrates regulate lipid metabolism, there is currently no clear prediction as to how dietary fatty acids affect the sperm of D. melanogaster. We manipulated the amount and identity of dietary polyunsaturated fatty acids (PUFA) in the food of D. melanogaster males (a treatment known to affect membrane fluidity) and measured changes in sperm parameters. We found that (a) males reared on food containing PUFA‐rich, plant‐derived lipids showed a slower increase in sperm volume over male age compared to males reared on yeast‐derived lipid food which is richer in saturated fatty acids. (b) The resistance of sperm membrane integrity to osmotic stress was not altered by dietary lipid treatment, but (c) food containing yeast‐derived lipids induced a 46% higher in situ rate of production of reactive oxygen species in sperm cells. These findings show that dietary lipids have similar effects on sperm parameters in Drosophila as in vertebrates, affect some, but not all, sperm parameters and modulate male reproductive ageing. In concert with recent findings of sex‐specific seasonal variation of diet choice in the wild, our results suggest a substantial dietary impact on the dynamics of male reproduction in the wild.     

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.     

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L‐amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase‐like proteins, massive up‐regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self‐perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction‐oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.     

Migrant blackbirds,Turdus merula,have higher plasma levels of polyunsaturated fatty acids compared to residents,but not enhanced fatty acid unsaturation index

Birds have been observed to have dietary preferences for unsaturated fatty acids during migration. Polyunsaturated fatty acids (PUFAs) may increase the exercise performance of migrant birds; however, PUFAs are also peroxidation prone and might therefore incur increased costs in terms of enhanced oxidative damage in migratory individuals. To shed light on this potential constraint, we analyzed plasma fatty acid (FA) composition and estimated the unsaturation index as a proxy for susceptibility to lipid peroxidation of migrants and residents of the partially migratory common blackbird (Turdus merula) at a stopover site during autumn migration. As predicted, migrant birds had higher relative and absolute levels of PUFAs compared to resident birds. This included the strictly dietary ω‐3 PUFA α‐linolenic acid, suggesting a dietary and/or storage preference for these FAs in migrants. Interestingly, the FA unsaturation index did not differ between migrants and residents. These findings suggest a mechanism where birds alter their levels of metabolic substrate without simultaneously increasing the susceptibility of the substrate to lipid peroxidation. In summary, our results are in line with the hypothesis that increased exercise performance during migration might be constrained by oxidative stress, which is manifested in changes in the composition of key FAs to retain the unsaturation index constant despite the increased levels of peroxidizable PUFAs.     

Antioxidative activity and protective effect of probiotics against high-fat diet-induced sperm damage in rats

In this study, antioxidant capability and protective effect of probiotics on reproductive damage induced by diet oxidative stress were investigated. Thirty male Sprague-Dawley rats were randomly divided into three groups with 10 rats in each group. The control group consumed a normal standard diet (5% fat, w/w). The other two treatment groups were fed with a high-fat diet (20% fat, w/w), and a high-fat diet supplemented with 2% probiotics (w/w), respectively. At the end of the experimental period, that is, after 6 weeks, rats were killed. Activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), contents of nitric oxide (NO) free radical and malondialdehyde (MDA) in serum and sperm suspension were examined. Sperm parameters including sperm concentration, viability, motility and DNA integrity were analyzed. The results showed that high-fat diet could induce oxidative stress, shown as significant increases in lipid peroxidation, NO free radical, significant decrease in activities of SOD, GSH-Px, significant reduction in sperm concentration, viability and motility, and damage in sperm DNA (P < 0.05), compared with the control group. These alterations were significantly reversed in the probiotics-supplemented group and had no significant difference in antioxidant capability, lipid peroxidation and sperm parameters compared with the control group. The percentage of sperm with DNA damage was significantly lower than the high-fat diet group and still higher than the control group, which means that probiotics could attenuate sperm damage to some extent. The present results indicated that dietary probiotics had antioxidant activity and the protective effect against sperm damage induced by high-fat diet to some extent.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

Effect of Vitamin E and Polyunsaturated Fatty Acids on Cryopreserved Sperm Quality in Bos taurus Bulls Under Testicular Heat Stress

Taurine bulls are highly susceptible to heat stress, leading to increased oxidative stress (OS) and impaired sperm viability. Polyunsaturated fatty acids (PUFAs) supplementation can be an alternative to improve semen quality, which also results in more sperm susceptibility to lipid peroxidation. Moreover, this deleterious effect can be exacerbated in animals affected by heat stress. Vitamin E is a key antioxidant that counteracts lipid peroxidation of sperm membrane caused by OS. Thus, combining PUFAs with vitamin E may improve sperm quality. In this context, this study aimed to evaluate the effect of interaction between PUFAs and vitamin E on sperm quality in Bos taurus bulls under testicular heat stress. Sixteen taurine bulls under testicular heat stress were randomly assigned in four groups: Control, Vitamin E, PUFA, and PUFA?+?Vitamin E. All groups lasted for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables (CASA), membrane and acrosome integrity, mitochondrial activity, susceptibility to oxidative stress, DNA integrity, and sperm-binding capacity. Results showed that vitamin E had a beneficial effect on some sperm characteristics, whereas PUFA supplementation had an adverse effect when the two treatments were evaluated separately. Finally, the association between PUFAs and vitamin E did not improve sperm quality.     

Mitochondria: omega-3 in the route of mitochondrial reactive oxygen species

Mitochondria are the main organelles that produce reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage to macromolecules, including lipids, and can damage cellular membrane structure and functions. Mitochondria, the main target of ROS-induced damage, are equipped with a network of antioxidants that control ROS production. Dietary intake of omega-3 polyunsaturated fatty acids (ω3PUFAs) and consequently the increase in ω3PUFA content of membrane lipids may be disadvantageous to the health because ROS-induced oxidative peroxidation of ω3PUFAs within membrane phospholipids can lead to the formation of toxic products. Mitochondrial control of lipid peroxidation is one of the mechanisms that protect cell against oxidative damage. This review discusses the role of mitochondria in ROS generation and the mechanisms by which it regulates ROS production. The susceptibility to peroxidation of PUFAs by ROS raises the question of the adverse effects of ω3PUFA dietary supplementation on embryonic development and prenatal developmental outcomes.     

Roles of antioxidants on prolonged storage of avian spermatozoa in vivo and in vitro

This review focuses on natural and assisted prevention against lipid peroxidation in avian spermatozoa. The presence of high levels of n-6 polyunsaturated fatty acids (PUFAs) in the plasma membrane creates favorable conditions for the formation of peroxidative products, a major cause of membrane damage which may ultimately impair male fertility. However, a complex antioxidant system involving vitamin C, vitamin E and GSH is naturally present in avian semen. Coupled with a battery of enzymatic defenses (e.g., SOD, GSH-Px either Se- or non-Se-dependent), this system acts to prevent or restrict the formation and propagation of peroxides. The presence of specialized sites dedicated to prolonged sperm storage in avian females raises the question of durable protection of sperm membranes against peroxidation. Preliminary observations have revealed the presence of a specific antioxidant system at these sites in which vitamin C could exert a major role. From a practical standpoint, the extensive use of artificial insemination in poultry, along with the emergence in some species of workable techniques to cryopreserve spermatozoa, demand better control of peroxidation occurring in the plasma membrane of spermatozoa before or during storage. Dietary supplementation with vitamin E is effective in limiting lipid peroxidation of sperm plasma membranes, both in chickens and turkeys. In addition, organic Se with or without vitamin E stimulates Se-GSH-Px activity in seminal plasma. Preliminary observations in female chickens have also revealed the effectiveness of dietary supplementation with vitamin E, organic selenium or both to sustain fertility in aging flocks.     

Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated.Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks.In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age.In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.     

No evidence of trade‐offs in the evolution of sperm numbers and sperm size in mammals

Post‐copulatory sexual selection, in the form sperm competition, has influenced the evolution of several male reproductive traits. However, theory predicts that sperm competition would lead to trade‐offs between numbers and size of spermatozoa because increased costs per cell would result in a reduction of sperm number if both traits share the same energetic budget. Theoretical models have proposed that, in large animals, increased sperm size would have minimal fitness advantage compared with increased sperm numbers. Thus, sperm numbers would evolve more rapidly than sperm size under sperm competition pressure. We tested in mammals whether sperm competition maximizes sperm numbers and size, and whether there is a trade‐off between these traits. Our results showed that sperm competition maximizes sperm numbers in eutherian and metatherian mammals. There was no evidence of a trade‐off between sperm numbers and sperm size in any of the two mammalian clades as we did not observe any significant relationship between sperm numbers and sperm size once the effect of sperm competition was taken into account. Maximization of both numbers and size in mammals may occur because each trait is crucial at different stages in sperm's life; for example size‐determined sperm velocity is a key determinant of fertilization success. In addition, numbers and size may also be influenced by diverse energetic budgets required at different stages of sperm formation.     

Sperm morphology and evidence for sperm competition among parrots

Sperm competition is an important component of post‐copulatory sexual selection that has shaped the evolution of sperm morphology. Previous studies have reported that sperm competition has a concurrently directional and stabilizing effect on sperm size. For example, bird species that show higher levels of extrapair paternity and larger testes (proxies for the intensity of sperm competition) have longer sperm and lower coefficients of variation in sperm length, both within and between males. For this reason, these sperm traits have been proposed as indexes to estimate the level of sperm competition in species for which other measures are not available. The relationship between sperm competition and sperm morphology has been explored mostly for bird species that breed in temperate zones, with the main focus on passerine birds. We measured sperm morphology in 62 parrot species that breed mainly in the tropics and related variation in sperm length to life‐history traits potentially indicative of the level of sperm competition. We showed that sperm length negatively correlated with the within‐male coefficient of variation in sperm length and positively with testes mass. We also showed that sperm is longer in sexually dichromatic and in gregarious species. Our results support the general validity of the hypothesis that sperm competition drives variation in sperm morphology. Our analyses suggest that post‐copulatory sexual selection is also important in tropical species, with more intense sperm competition among sexually dichromatic species and among species that breed at higher densities.     

A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation

Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.     

Relationships among lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in dairy bulls

Sperm membranes contain high concentrations of polyunsaturated fatty acids that are highly susceptible to oxidative damage that interferes with fertilization ability. The objective of this study was to determine associations among lipid peroxidation (thiobarbituric-acid-reactive substance concentration), antioxidant enzymatic activities in frozen spermatozoa, and competitive indices. Semen from multiple ejaculates collected in succession from each bull (four Holstein and four Jersey) was pooled. Heterospermic doses (20x10(6)sperm/0.5mL straw) were made to obtain 16 Holstein/Jersey combinations (equal number of sperm from each bull). Cows were inseminated on observed or synchronized estrus. The sire of calves (N=460) was determined; based on the number of calves sired, a competitive index was obtained for each bull. Prior to preparation of the heterospermic doses, a sub-sample of semen from each bull was taken, processed, frozen, and stored concurrent with heterospermic samples. After thawing, these homospermic samples were assessed for lipid peroxidation, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, DNA fragmentation index (DFI), plasma membrane integrity (PMI), and total progressive motility (assessed by CASA). Sperm lipid peroxidation and the competitive index were negatively correlated (r=-0.78; P<0.05), the DFI and sperm lipid peroxidation were positively correlated (r=0.86; P<0.001), and there were negative correlations (P<0.05) for sperm lipid peroxidation and both PMI and total progressive motility (r=-0.78 and -0.83, respectively). There was neither significant association between SOD activity and competitive index, nor between GPx activity and competitive index. In conclusion, bulls with lower sperm lipid peroxidation had higher chances of siring calves; this was attributed to the deleterious effects of lipid peroxidation on sperm plasma membrane integrity and sperm DNA, which may reduce sperm fertilizing potential.     

Mammalian sperm metabolism: oxygen and sugar, friend and foe

Mammalian spermatozoa expend energy, generated as intracellular ATP, largely on motility. If the sperm cell cannot swim by use of its flagellar motion, it cannot fertilize the egg. Studies of the means by which this energy is generated span a period of six decades. This review gives an overview of these studies, which demonstrate that both mitochondrial oxidative phosphorylation, for which oxygen is friend, and glycolysis, for which sugar is friend, can provide the energy, independent of one another. In mouse sperm, glycolysis appears to be the dominant pathway; in bull sperm, oxidative phosphorylation is the predominant pathway. In the case of bull sperm, the high activity of the glycolytic pathway would maintain the intracellular pH too low to allow sperm capacitation; here sugar is enemy. The cow's oviduct has very low glucose concentration, thus allowing capacitation to go forward. The choice of the pathway of energy generation in vivo is set by the conditions in the oviduct of the conspecific female. The phospholipids of the sperm plasma membrane have a high content of polyunsaturated fatty acids represented in their acyl moieties, rendering them highly susceptible to lipid peroxidation; in this case oxygen is enemy. But the susceptibility of the sperm membrane to lethal damage by lipid peroxidation allows the female oviduct to dispose of sperm that have overstayed their welcome, and so keep in balance sperm access to the egg and sperm removal once this has occurred.     

Sperm head abnormalities are associated with excessive omega‐6 fatty acids in two finch species feeding on sunflower seeds

In a rapidly changing world, it is important to understand how urban environments impact wildlife. For example, supplementary feeding of birds, though well‐intended, might have unexpected negative effects on the health of individual animals. Sunflower seeds are commonly provided in garden bird feeders, but they contain high levels of linoleic acid (LA), an omega‐6 polyunsaturated fatty acid (PUFA). Omega‐6 PUFAs are associated with increased oxidative stress, which can damage cell membranes, and in particular sperm cells. We assessed the level of LA in the blood of two seed‐eating finch species, greenfinches Chloris chloris and hawfinches Coccothraustes coccothraustes, caught in and in environments with direct access to sunflower seed feeders (Norway), and compared these with the level of LA in a smaller number of individuals sampled in in a rural area with low incidence of sunflower seed feeders (Czech Republic). Furthermore, we investigated the relationship between the proportion of LA in the blood (as well as the proportion of 10 other fatty acids) and sperm quality (the frequency of sperm head abnormalities and sperm swimming speed). We found that both finch species, but particularly greenfinches caught near feeders, exhibited levels of LA that were considerably higher than those previously reported for other wild birds. We also found that the proportion of LA was positively correlated with the frequency of abnormal sperm heads (sperm missing the acrosome), while there was no significant effect of fatty acid composition on sperm swimming speed. Our results indicate that the sperm quality of finches may be negatively affected by a high intake of sunflower seeds, adding to a growing body of research showing that supplementary feeding may have detrimental side effects for urban animals. This is particularly relevant for the greenfinch, which is currently affected by disease and population declines.     

Secondary sexual characters and sperm traits in coho salmon Oncorhynchus kisutch

A study was undertaken to examine secondary sexual characters (spawning colouration and overall body size) in relation to sperm metrics in one alternative reproductive tactic of coho salmon Oncorhynchus kisutch : large hooknose males that spawn in dominance-based hierarchies. Males with less intense red spawning colouration had higher sperm velocities than males with darker red spawning colouration. There was no relationship between male body size and sperm metrics. These results suggest that within an alternative reproductive tactic, variation in sperm competition intensity may select for a trade-off between investment in sexual colouration and sperm quality.     

Functional evidence for differences in sperm competition in humans and chimpanzees

Sperm competition occurs when the gametes of or more males compete for opportunities to fertilize a given set of ova. Previous studies have demonstrated that certain morphological characteristics are affected by sperm competition intensity (e.g. relative testes size and sperm midpiece volume). This study examined whether aspects of sperm energetics may also be affected by sexual selection. We compared the membrane potential of mitochondria in live sperm between H. sapiens (single partner mating system) and P. troglodytes (multiple partner mating system). Flow cytometry of sperm stained with the carbocyanine fluorescent dye JC-1 (an assay for mitochondrial membrane potential) revealed marked differences in red fluorescence intensity. P. troglodytes sperm showed significantly higher mitochondrial membrane potential. Mitochondria provide a substantial part of the energy required for sperm motility. A higher mitochondrial loading may therefore be associated with enhanced sperm motility and/or longevity. Additionally, examination of JC-1 red fluorescence levels before and after in vitro capacitation revealed further differences. Whereas chimpanzee sperm showed maintenance of membrane potential after capacitation (in some cases even an increase), sperm from humans consistently showed reduction in membrane potential. These results indicate that the sperm of human beings and chimpanzees exhibit marked differences in mitochondrial function, which are affected by selection pressures relating to sperm competition and that these pressures differ significantly between humans and chimpanzees.     

The effects of male social environment on sperm phenotype and genome integrity

Sperm function and quality are primary determinants of male reproductive performance and hence fitness. The presence of rival males has been shown to affect ejaculate and sperm traits in a wide range of taxa. However, male physiological conditions may not only affect sperm phenotypic traits but also their genetic and epigenetic signatures, affecting the fitness of the resulting offspring. We investigated the effects of male‐male competition on sperm quality using TUNEL assays and geometric morphometrics in the zebrafish, Danio rerio. We found that the sperm produced by males exposed to high male–male competition had smaller heads but larger midpiece and flagellum than sperm produced by males under low competition. Head and flagella also appeared less sensitive to the osmotic stress induced by activation with water. In addition, more sperm showed signals of DNA damage in ejaculates of males under high competition. These findings suggest that the presence of a rival male may have positive effects on sperm phenotypic traits but negative effects on sperm DNA integrity. Overall, males facing the presence of rival males may produce faster swimming and more competitive sperm but this may come at a cost for the next generation.     

Male body size and condition affects sperm number and production rates in mosquitofish,Gambusia holbrooki

Sperm number is an important predictor of paternity when there is sperm competition. Sperm number is often measured as maximum sperm reserves, but in species where mating is frequent, males will often be replenishing their reserves. Thus, variation in how quickly males can produce sperm is likely to be important in determining male success in sperm competition. Despite this, little is known about how male size, body condition or diet affects sperm production rates. We counted sperm number in large and small Gambusia holbrooki (eastern mosquitofish) after 3 weeks on either a high or low food diet. Sperm number was significantly higher in both larger males and in well‐fed males. We then stripped ejaculates again either 1, 2, 3, 4 or 5 days later to investigate subsequent sperm production. The rate of sperm replenishment was influenced by an interaction between size and diet. Large, well‐fed males had consistently high levels of sperm available over the 5 days (i.e. rapid replenishment), whereas small poorly fed males showed consistently low levels of sperm availability over the 5 days (i.e. slow replenishment). In contrast, large, poorly fed and small, well‐fed males increased their sperm numbers over the first 3 days (i.e. intermediate replenishment). Our study highlights that when mating is frequent and sperm competition is high, size and condition dependence of maximal sperm number and of sperm production rate might both contribute to variation in male reproductive success.