Sperm Displacement Research Articles

Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel

IntroductionMammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the studyThe present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methodsSperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. ResultsA significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20–100 μg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 μM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. ConclusionsPJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

Potential Application of Cryogenic Freezer on Turbot Sperm Cryopreservation

BACKGROUND: Cryopreservation is a common practice to preserve fish sperm for prolonged periods. OBJECTIVE: To examine the effect of different freezing protocols on sperm characteristics, fertilization, and hatching rate of turbot. MATERIALS AND METHODS: Milt was obtained from ten 8-year-old turbot (54.3 ± 1.7 cm in length and 3,106 ±283 g in weight) at the peak of spawning season. Six batches of milts with >90% motility was pooled and diluted to 1:3 by adding dimethyl sulfoxide (DMSO, 10%) as cryoprotectant. Then straws filled with semen were subjected to three freezing protocols (cooling rates). Sperm characteristics were assessed using sperm class analyzer before and after cryopreservation. Cryopreserved and fresh sperm were used for artificial fertilization to assess fertilization and hatching rates. RESULTS: Cryopreservation protocol has significantly deleterious effects on total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity, linearity index, straightness index, oscillation index, and amplitude of lateral head displacement of sperm. However, the beat frequency of cryopreserved sperm was found to be similar to control sperm. The fertilization rate of sperm subjected to three freezing protocols were similar, varying between 65.3 % and 75.6 %, and the hatching rates varied from 51.2% to 70.7%. CONCLUSION: The results show the potential application of cryopreservation in fish hatcheries.

Enhancement of sperm quality and fertility-related parameters in Hubbard grandparent rooster fed diets supplemented with soybean lecithin and vitamin E

The purpose of this study was to investigate the effect of dietary supplementation of different levels of soybean lecithin and vitamin E on semen quality parameters and some reproductive hormones in Hubbard grandparent roosters. The experiment was conducted in a 3 × 2 factorial arrangement with 3 levels of soybean lecithin (0, 1, and 2%) and 2 levels of vitamin E (0 and 300 mg/kg). Semen samples were collected on d 0, 20, 40 and 60 of the experiment and analyzed. Adding 1% soybean lecithin and vitamin E into the diet increased semen volume and sperm concentration, membrane integrity and viability (P < 0.05). Supplementing diets with 1 or 2% lecithin in addition to vitamin E significantly improved total motility and progressive motility (P < 0.05). Vitamin E significantly increased the amplitude of lateral head displacement (ALH) of sperm (P < 0.05). Although there was no effect on LH and FSH when diets were supplemented with vitamin E and 1 or 2% lecithin, testosterone concentration was increased (P < 0.05). Malondialdehyde (MDA) concentration was significantly lower in all 3 treatments containing vitamin E (P < 0.05). It can be concluded that supplementation of rooster diets with vitamin E and 1% lecithin can improve fertility related parameters in Hubbard grandparent roosters.

How biases in sperm storage relate to sperm use during oviposition in female yellow dung flies

Abstract Precise mechanisms underlying sperm storage and utilization are largely unknown, and data directly linking stored sperm to paternity remain scarce. We used competitive microsatellite PCR to study the effects of female morphology, copula duration and oviposition on the proportion of stored sperm provided by the second of two copulating males (S2) in Scathophaga stercoraria (Diptera: Scathophagidae), the classic model for sperm competition studies. We genotyped all offspring from potentially mixed-paternity clutches to establish the relationship between a second male’s stored sperm (S2) and paternity success (P2). We found consistent skew in sperm storage across the three female spermathecae, with relatively more second-male sperm stored in the singlet spermatheca than in the doublet spermathecae. S2 generally decreased with increasing spermathecal size, consistent with either heightened first-male storage in larger spermathecae, or less efficient sperm displacement in them. Additionally, copula duration and several two-way interactions influenced S2, highlighting the complexity of postcopulatory processes and sperm storage. Importantly, S2 and P2 were strongly correlated. Manipulation of the timing of oviposition strongly influenced observed sperm-storage patterns, with higher S2 when females laid no eggs before being sacrificed than when they oviposited between copulations, an observation consistent with adaptive plasticity in insemination. Our results identified multiple factors influencing sperm storage, nevertheless suggesting that the proportion of stored sperm is strongly linked to paternity (i.e., a fair raffle). Even more detailed data in this vein are needed to evaluate the general importance of sperm competition relative to cryptic female choice in postcopulatory sexual selection.

Protective effects of ginseng stem-leaf saponins on D-galactose-induced reproductive injury in male mice.

Panax ginseng is a perennial plant in the Araliaceae family. In this study, we investigated the protective effects of ginseng stem-leaf saponins (GSLS) isolated from P. ginseng against D-galactose-induced reproductive function decline, oxidative stress, and inflammatory response. Reproductive injuries were induced in mice via the subcutaneous injection of D-galactose (300 mg/kg) for six weeks. The mice were then treated with GSLS by intragastric administration. GSLS inhibited markers of oxidative stress and inflammatory cytokines induced by D-galactose in serum, liver and kidney, whereas GSLS increased the activities of antioxidant enzymes. Compared to the mice treated only with D-galactose, GSLS treatment significantly increased the average path velocity, straight line velocity, curvilinear velocity, and amplitude of the lateral head displacement of mouse sperm. Meanwhile, GSLS significantly increased the testosterone level and reduced the cortisol, FSH, and LH levels. Histopathological examination revealed alterations in the number and the arrangement of spermatogenic cells in the seminiferous tubules of the mice in the GSLS group. GSLS treatment suppressed MAPKs pathway activation in testes. These results suggest that GSLS can attenuate D-galactose-induced oxidative stress and inflammatory response in serum, liver and kidney, and ameliorate reproductive damage by inhibiting MAPKs signaling pathway.

Evolutionary insight from a humble fly: sperm competition and the yellow dungfly.

Studies of the yellow dungfly in the 1960s provided one of the first quantitative demonstrations of the costs and benefits associated with male and female reproductive behaviour. These studies advanced appreciation of sexual selection as a significant evolutionary mechanism and contributed to the 1970s paradigm shift toward individual selectionist thinking. Three behaviours in particular led to the realization that sexual selection can continue during and after mating: (i) female receptivity to remating, (ii) sperm displacement and (iii) post-copulatory mate guarding. These behaviours either generate, or are adaptations to sperm competition, cryptic female choice and sexual conflict. Here we review this body of work, and its contribution to the development of post-copulatory sexual selection theory. This article is part of the theme issue 'Fifty years of sperm competition'.

Body weight of the two sexes determines the occurrence of polyandry in a moth

Polyandry has been a central topic in the study of animal behaviour. Yet, it is still not clear how the body weight of each sex affects the probability of female remating, and what each sex gains from polyandry. Here we aimed to shed some light on these questions using the Mediterranean flour moth, Ephestia kuehniella (Lepidoptera: Pyralidae). We found that virgin pairs mated regardless of their weight to ensure fertilization. However, only 58% of once-mated females remated. The probability of female remating increased when the female and/or the second male was heavier. Our findings indicate that fecundity, polygamy and longevity are functions of body weight in each sex. The nature of the last male sperm precedence in this species allows males and nonvirgin females to regulate their mating decisions. We propose that once-mated females would benefit from accepting heavier second males to replace most sperm from the lighter first mates and rejecting lighter second males to avoid displacement of sperm from the first heavier mates. Heavier males are more coercive, probably also preferred by females, and thus more likely to achieve mating with heavier nonvirgin females to replace most sperm from the previous males and dominate paternity. As body weight is heritable, we suggest that the fitness of both sexes rises when they pass on genes for this trait through female encouragement or avoidance of sperm displacement and male coercion of mated females into remating. We conclude that the two sexes play interactive roles in determining polyandrous patterns depending on their body weight.

Functional morphology of the female reproductive system of a crab with highly extensible seminal receptacles and extreme sperm storage capacity.

We studied the functional morphology of the female reproductive system of the purple stone crab Danielethus crenulatus. The most remarkable feature is the relative storage capacity and extensibility of the seminal receptacles. These receptacles are a pair of simple sacs that lack internal structures dividing the internal lumen. Differences in seminal receptacle size and contents are accompanied by conspicuous changes in receptacle lining at a tissue level. Full seminal receptacles contain discrete sperm masses formed by hardened fluid and densely packed spermatophores. Different sperm masses are likely from different mates and their stratified disposition within the seminal receptacles is compatible with rival sperm displacement and last sperm precedence. Additionally, the anatomical structure of the vulva and vagina suggest active female control over copula. We discuss our results in the general context of sperm storage in brachyurans and the implications for the mating system of this species.

A Note on the Fertilization Success of the First Male during Successive Mating in the Japanese Rhinoceros Beetle Trypoxylus dichotomus septentrionalis

It is empirically well known that adults of both sexes of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis (Kono) copulate repeatedly under laboratory conditions. However, details of the fertilization success of respective males involved in multiple mating are unknown. To determine the degree of sperm displacement in successive mating in this beetle species, we conducted a male-swapping crossing experiment under laboratory conditions. A mutant line with white compound eyes in the adult stage, of which the white-eye state shows simple recessive Mendelian inheritance to the wild type regardless of sex, was employed in the experiment. Individual adult female mutants were consecutively mated with a mutant and a wild-type adult male in a different order. Although the sample sizes of our experiment were limited, the eye color of all adults of the next generation was inherited from the first-mated males. Therefore, our results strongly suggest that the degree of sperm displacement is very low or absent, or even that sperm transfer does not occur in secondary matings, in this beetle species.

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more, but females influence insemination success by removing spermatangia.

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at −196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing–thawing.

Gonopod mechanics in &lt;em&gt;Centrobolus&lt;/em&gt; Cook (Spirobolida: Trigoniulidae) II. Images &lt;em&gt;&lt;/em&gt;

Gonopod mechanics were described for four species of millipedes in the genus Centrobolus and are now figured using scanning electron microscopy (SEM) with the aim to show the mechanism of sperm competition. Structures of sperm displacement include projections on a moveable telopodite and tips on a distal process (opisthomerite). Three significant contact zones between the male and female genitalia were recognized: (1) distal telopodite of the coleopod and the vulva, (2) phallopod and the bursa, (3) sternite and legs of the female.

Gonopod mechanics in Centrobolus Cook (Spirobolida: Trigoniulidae) II. Images

Gonopod mechanics were described for four species of millipedes in the genus Centrobolus and are now figured using scanning electron microscopy (SEM) with the aim to show the mechanism of sperm competition. Structures of sperm displacement include projections on a moveable telopodite and tips on a distal process (opisthomerite). Three significant contact zones between the male and female genitalia were recognized: (1) distal telopodite of the coleopod and the vulva, (2) phallopod and the bursa, (3) sternite and legs of the female.

Symmetry in ejaculate volumes of Centrobolus inscriptus Attems (Spiroboloidea: Trigoniulidae)

I test for symmetry in ejaculate volumes measured as disintegrations per minute (dpm) deposited by males in ejaculate storage organs of Centrobolus inscriptus in single, double and artificially-terminated matings. Combined data showed symmetry in dpm values (left-right: X ± SE = 48.11 ± 126.1828; n = 21). No significant differences between the left and right ejaculate volumes occured (T = 711.5, n = 59, P = 0.19). Symmetry in ejaculate volumes was consistent with the mechanism of sperm displacement i. e. mixing-self-sperm displacement by phallopods.

Symmetry in ejaculate volumes of &lt;em&gt;Centrobolus inscriptus&lt;/em&gt; Attems (Spiroboloidea: Trigoniulidae)

I test for symmetry in ejaculate volumes measured as disintegrations per minute (dpm) deposited by males in ejaculate storage organs of Centrobolus inscriptus in single, double and artificially-terminated matings. Combined data showed symmetry in dpm values (left-right: X ± SE = 48.11 ± 126.1828; n = 21). No significant differences between the left and right ejaculate volumes occured (T = 711.5, n = 59, P = 0.19). Symmetry in ejaculate volumes was consistent with the mechanism of sperm displacement i. e. mixing-self-sperm displacement by phallopods.

Elaborate gonopods in the myriapod genus Chersastus (Diplopoda: Trigoniulidae)

Two-paired gonopods of Chersastus were examined under light and scanning microscopy. Species-specific structures with complexities are described. These gonopods are test models for elaborate genitalia hypotheses: lock-and-key, pleiotropism, genitalic recognition, mechanical conflict of interests, cryptic female choice. Structures of potential sperm displacement are described.

Sperm displacement in the Drosophila bipectinata species complex: Evidence for interspecific variations

We studied sperm displacement in two members of the Drosophila bipectinata species complex i.e., Drosophila parabipectinata and Drosophila malerkotliana by employing mutant and wild type strains. We found a significant increase in the productivity of remated females than that of once mated ones in all the crosses of the two species. The P2′ values range from 0.67 to 0.70 in D. malerkotliana and 0.52 to 0.56 in D. parabipectinata. These results were compared with that of our earlier finding in D. bipectinata belonging to the same complex. We found that though the three species exhibit differences in the degree of sperm displacement, the pattern is somewhat similar in the three species. However, as far as its genetic variability and population size maintenance is considered, D. malerkotliana and D. bipectinata (P2′ is 0.60–0.67) are at the forefront. Our finding is also supported by the fact that D. parabipectinata is derived from D. bipectinata and has not diverged much since its emergence as a separate species. The differences in the pattern of sperm displacement may be attributed to differences in remating latency, duration of copulation in first mating and sperm usage pattern among the members of this complex.

Do candidate genes mediating conspecific sperm precedence affect sperm competitive ability within species? A test case in Drosophila.

When females mate to multiple males, the last male to mate fathers the majority of progeny. When males of different species inseminate a female, the sperm of the male conspecific to the female is favored in fertilization in a process known as conspecific sperm precedence (CSP). A large number of studies in Drosophila have assayed the genetic basis of sperm competition, with a main focus on D. melanogaster and accessory gland protein genes. Only a few studies have attempted to disentangle the genetic basis of CSP between related species of Drosophila. Although there is no a priori reason to believe that genes influencing intraspecific sperm competitive ability might also mediate conspecific sperm precedence, no study has addressed the question. Here, we test a group of candidate CSP genes between D. simulans and D. mauritiana for their effect on sperm competition in D. melanogaster. The use of P-element insertion lines identified CG14891 gene disruption as the only one causing a significant decrease in second male paternity success relative to wild-type and ebony tester males. The gene disruption affected both sperm displacement and the sperm fertilizing ability. Out of five genes tested using RNA interference, only gene knockdown of CG6864 (Mst89B) significantly reduced the male’s ability to father progeny when second to mate. Our results suggest that CG14891 and CG6864 might have been co-opted from an intraspecies gene function (i.e., sperm competition) into an interspecies avoidance phenotype (i.e., CSP). Alternatively, the dual role of these genes could be a consequence of their pleiotropic roles.

Fluorescent sperm in a transparent worm: validation of a GFP marker to study sexual selection.

BackgroundSexual selection has initially been thought to occur exclusively at the precopulatory stage in terms of contests among males and female mate choice, but research over the last four decades revealed that it often continues after copulation through sperm competition and cryptic female choice. However, studying these postcopulatory processes remains challenging because they occur internally and therefore are often difficult to observe. In the transparent free-living flatworm Macrostomum lignano, a recently established transgenic line that expresses green fluorescent protein (GFP) in all cell types, including sperm, offers a unique opportunity to non-invasively visualise and quantify the sperm of a GFP-expressing donor inside the reproductive tract of wild-type recipients in vivo. We here test several aspects of the reproductive performance of the transgenic individuals and the accuracy of the techniques involved in assessing the GFP-expressing worms and their sperm. We then show the usefulness of these methods in a study on sperm displacement.ResultsGFP-expressing worms do not differ from wild-type worms in terms of morphology, mating rate and reproductive success. In addition, we show that the GFP signal is reliably and unequivocally expressed by all GFP-expressing individuals observed under epifluorescence illumination. However, the intensity of the GFP signal emitted by sperm of GFP expressing donors can vary (which we show to be at least in part due to sperm ageing) and the GFP marker is inherited according to Mendel’s laws in most, but not all, of the individuals. Nevertheless, we argue these two issues can be addressed with an appropriate experimental design. Finally, we demonstrate the value of the GFP-techniques by comparing the number of GFP-expressing sperm in a wild-type recipient before and after mating with a competing sperm donor, providing clear experimental evidence for sperm displacement in M. lignano. This result suggests that sperm donors can displace previously stored sperm and replace it with their own.ConclusionThe availability of the GFP-techniques in a transparent organism provide unique opportunities to visualise and quantify internal processes in the female reproductive tract after mating, which opens new avenues in the study of sexual selection.

Isometric patterns for male genital allometry in four damselfly species

Recent studies have found that insect genitalic traits show negative allometry, i.e., are relatively small in relation to body size. One interpretation of this is that males use their genitalia to stimulate females. Thus, given the nature of damselfly copulation in which males physically reach the rival sperm that females have stored from previous matings, male genitalic traits are not expected to show negative allometry. To test this idea, we assessed (a) the rival sperm displacement function by the mating male and (b) allometry of aedeagal length of four damselfly species (Argia anceps, Argia tezpi, Argia extranea, and Enallagma praevarum). Sperm displacement was assessed by inspecting whether the aedeagus reached the rival sperm during copulation in mating pairs for the four species. To have a standard for comparing allometric patterns, allometry of aedeagal was compared to that of two non-genital traits, tibial, and fourth abdominal segment length. In all cases, the aedeagus was found to reach the rival sperm which supports the idea that stimulation is not the mechanism for sperm displacement but physical displacement. Aedeagal length was isometric, and its slope was lower in general compared to that of tibial length and fourth abdominal segment. Given that this isometric pattern is not common for other odonate species, our interpretation of these varying aedeagal scaling patterns in this insect order is that males’ and females’ sexual interests are in conflict (males are evolving an elongated aedeagus to reach rival sperm while females are evolving unreachable sperm storage organs to prevent displacement of stored sperm). This sexual conflict scenario would favor varying scaling patterns for aedeagal length in odonates. A final interpretation is that the risk of interspecific matings in damselflies, may also explain different species-specific, aedeagal allometries.