Seminal fluid reduces female longevity and stimulates egg production and sperm trigger oviposition in a moth

Previous studies suggest that a number of factors in relation to mating may reduce female longevity and stimulate egg production and oviposition. However, it is still not clear whether these factors act on these parameters independently or in a collective way. Here we carried out a series of experiments including mating trials and seminal fluid injection to determine the factors responsible for reducing female longevity and stimulating egg production and oviposition in relation to mating in the moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Results show that seminal fluid and sperm work collectively to allow females to achieve maximum realized fecundity (number of eggs laid) in E. kuehniella but these factors play different roles in the process and their actions are independent. Seminal fluid signals females to allocate resources to ova, resulting in shorter longevity and greater egg production while eupyrene (not apyrene) sperm in the spermatheca trigger females to lay maximum number of eggs. We suggest that the receptors for seminal fluid signal may be located in the female reproductive tract and haemolymph, and those for sperm signal may be in the spermatheca. Hypotheses that females prolong their longevity by oosorption, physical injuries by males reduce female longevity, and mechanical stimulation by males triggers oviposition, are not substantiated in the present study.     

Seminal plasma affects prostaglandin synthesis in the porcine oviduct

Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF_2α synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE₂ to PGF_2α, expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE₂ to PGF_2α and PGFM to PGF_2α ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE₂ to PGF_2α and PGFM to PGF_2α ratios in the porcine oviduct.     

Sperm viability and sperm competition in insects

Sperm quality plays an important role in vertebrates in determining which male has the advantage when two or more males compete to fertilize a female's ova. In insects, however, the importance of sperm quality has never been considered, despite sperm competition being widespread and well studied in this group. We tested the hypothesis that sperm viability, measured as the proportion of live sperm, covaried with the intensity of sperm competition in insects. In a pairwise comparison of seven closely related species pairs, each comprising a monandrous and a polyandrous species (i.e., with and without sperm competition, respectively), we found that in all cases the polyandrous species had a higher proportion of live sperm in their sperm stores. The distribution of the percentage of live sperm showed considerable inter- and intraspecific variation, suggesting that, all else being equal, males will vary in their ability to fertilize ova on the basis of sperm viability alone. Our results suggest that sperm viability is one of a suite of male adaptations to sperm competition in insects.     

Sperm viability matters in insect sperm competition

Experimental studies in insects have shown how sperm competition can be a potent selective force acting on an array of male reproductive traits . However, the role of sperm quality in determining paternity in insects has been neglected, despite the fact that sperm quality has been shown to influence the outcome of sperm competition in vertebrates . A recent comparative analysis found that males of polyandrous insect species show a higher proportion of live sperm in their stores . Here, we test the hypothesis that sperm viability influences paternity at the within-species level. We use the cricket Teleogryllus oceanicus to conduct sperm competition trials involving prescreened males that differ in the viability of their sperm. We find that paternity success is determined by the proportion of live sperm in a male's ejaculate. Furthermore, we were able to predict the paternity patterns observed on the basis of the males' relative representation of viable sperm in the female's sperm-storage organ. Our findings provide the first experimental evidence for the theory that sperm competition selects for higher sperm quality in insects. Between-male variation in sperm quality needs to be considered in theoretical and experimental studies of insect sperm competition.     

Manganese deficiency in maize affects pollen viability

Maize (Zea mays L. cv. G2) was grown with 0.55 mg L^–1 (sufficient), or 0.0055 mg L^–1 (deficient) manganese in sand. Manganese-deficient plants developed visible deficiency symptoms and showed poor tasseling and delayed anther development. Compared to Mn-sufficient plants, Mn-deficient plants produced fewer and smaller pollen grains with reduced cytoplasmic contents. Manganese deficiency reduced in vitro germination of pollen grains significantly. Ovule fertility was not significantly affected by Mn. But in Mn-deficient plants seed-setting and development was reduced significantly.     

Seminal plasma addition attenuates the dilution effect in bovine sperm

Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 x 10(6) sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 x 10(6) sperm/mL in EYC extender with (+SP) and without (-SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 x 10(6) sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 x 10(6)/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the -SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the -SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR- 14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the -SP samples at a concentration of 10 x 10(6) sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.     

Effects of sperm conjugation and dissociation on sperm viability in vitro

Sperm conjugation is an unusual variation in sperm behavior where two or more spermatozoa physically unite for motility or transport through the female reproductive tract. Conjugation has frequently been interpreted as sperm cooperation, including reproductive altruism, with some sperm advancing their siblings toward the site of fertilization while ostensibly forfeiting their own ability to fertilize through damage incurred during conjugate break-up. Conversely, conjugation has been proposed to protect sensitive regions of spermatozoa from spermicidal conditions within the female reproductive tract. We investigated the possibility of dissociation-induced sperm mortality and tested for a protective function of conjugation using the paired sperm of the diving beetle, Graphoderus liberus. Sperm conjugates were mechanically dissociated and exposed to potentially damaging tissue extracts of the female reproductive tract and somatic tissue. We found no significant difference in viability between paired sperm and dissociated, single sperm. The results further indicate that the reproductive tract of female G. liberus might not be spermicidal and conjugation is not protective of sperm viability when damaging conditions do exist. Our results support the interpretation that, at least in some taxa, sperm conjugation is neither protective nor damaging to sperm viability.     

Evolutionary modeling predicts a decrease in postcopulatory sperm viability as a response to increasing levels of sperm competition

Sperm competition has been found to have a strong influence on the evolution of many male and female reproductive traits. Theoretical models have shown that, with increasing levels of sperm competition, males are predicted to increase ejaculate investment, and there is ample empirical evidence supporting this prediction. However, most theoretical models concern sperm number, and although the predictions are likely to apply to other sperm traits that will affect the sperm competitive ability of males, substantiated predictions are difficult unless the evolution of specific traits is explicitly modeled. Here I present a novel theoretical model aiming at predicting evolutionarily stable sperm viability in relation to female mating frequency in a mating system with internal fertilization. At odds with verbal arguments, this model demonstrates that sperm viability is expected to decrease with increasing female remating rates and thus to decrease with increasing levels of sperm competition. The major reason for this is that, with increasing female remating rates, the prospects of future fertilization success will decrease, which acts to reduce the benefit of long-lived viable sperm. An additional interesting result is that, as the cost of sperm viability increases, the overall energy investment in ejaculates will decrease. These novel results should have a strong impact on future sperm competition studies and will also have implications for our understanding of the evolution of female polyandry.     

Seminal prostaglandins in men with subnormal sperm motility and therapeutic treatment

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocelle and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE₂, PGI₂ and PGF_2α. There was a significant difference (p < 0, 025; F-test) between the PGI₂ concentrations in patients with impaired motility (5,6 ± 1,4 pg/mg protein) and normal men (8,8 ± 3,7 pg/mg protein). PGE₂ and PGF_2α were significantly different in patients with varicocele (p < 0,025, F-test). Wide ranges of prostaglandins occured in the Kallikrein-group with no significant differences. We conclude that: a) PGI₁ is an additional prostaglandin compound in seminal plasma. b)its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.     

An advantage for young sperm in the house cricket Acheta domesticus

We show that males of the house cricket Acheta domesticus regularly expel sperm packages (spermatophores) independently of copulation and at a rate that is not affected by the presence of females. We then show for the first time that the age of sperm affects their likelihood of being stored by females after copulation; younger sperm were overrepresented in the female sperm storage organ and therefore in the sperm population used for fertilization. Our results suggest that the reproductive success of males may increase if they deliver ejaculates with young sperm, and the results may explain why the males of several species are regularly observed to discard ejaculates. Our results also suggest that phenomena such as female multiple mating, paternity bias, and/or exaggerated ejaculate sizes may be related to the advantage both genders gain by using young sperm.     

Seminal prostaglandins in men with subnormal sperm motility and therapeutic treatment

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocele and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE2, PGI2 and PGF2 alpha. There was a significant difference (p less than 0.025; F-test) between the PGI2 concentrations in patients with impaired motility (5.6 +/- 1.4 pg/mg protein) and normal men (8.8 +/- 3.7 pg/mg protein). PGE2 and PGF2 alpha were significantly different in patients with varicocele (p less than 0.025, F-test). Wide ranges of prostaglandins occurred in the Kallikrein-group with no significant differences. We conclude that: a) PGI2 is an additional prostaglandin compound in seminal plasma, b) its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.     

Microbial infection affects egg viability and incubation behavior in a tropical passerine

Many avian species initiate incubation before clutch completion,which causes eggs to hatch asynchronously. This influences broodcompetitive dynamics and often results in nestling mortality.The prevailing hypotheses contend that parents incubate earlybecause asynchronous hatching provides fitness benefits to parentsor surviving offspring. An alternative idea is that early incubationis the best of a bad job because of the costs of delaying incubationto the viability of first-laid eggs. To explore this, we examinedthe potential for microbial infection, and the relative effectsof infection and suboptimal development temperatures on theviability of pearly-eyed thrasher (Margarops fuscatus) eggs.We exposed newly laid eggs for 5 days at either end of a tropicalaltitudinal gradient and cleaned shells of half the eggs toreduce microbial growth. Uncleaned eggs were infected more thanwere cleaned eggs, and infection was greater for eggs exposedat the cool, humid site than at the hot, less humid site. Parentallyincubated eggs, however, were not infected, suggesting thatincubation limits infection. The consequence of exposure toinfection and high ambient temperatures was a dramatic reductionin viability; cleaned eggs held at the cool site had the highesthatching success, which was significantly greater than for uncleanedeggs at this site and for cleaned eggs held at the hot site.This provides the first evidence that microbes can infect unincubatedeggs of a wild bird, and that infection and ambient temperatureact independently to reduce hatching success. These factorscould affect avian life-history strategies in diverse habitats.     

Comparative viability of bovine sperm frozen on a cryomicroscope or in straws

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spermatozoa on the cryomicroscope. Semen was diluted in Test-yolk (20%) extender containing 7% glycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitrogen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicroscope. Thawed samples were diluted with Hepes buffered medium containing 0.1% bovine serum albumin (BSA) and stained with either carboxymethylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with propidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (stained with CMFDA or SYBR-14), which were classified as plasma membrane-intact and 2) spermatozoa with intense red fluorescence, (stained with PI), which were classified as plasma membrane-damaged. Samples frozen using the cryomicroscope contained 29 and 26 % plasma membrane-intact (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. Cryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane- intact sperm cells, while spermatozoa frozen in 0.25-ml straws resulted in 34 and 31% PMI sperm cells for CMFDA and SYBR-14, respectively. No significant difference was observed (P > 0.05) for PMI spermatozoa stained with either CMFDA or SYBR-14. In addition, the ability to recover spermatozoa after freezing on the cryomicroscope establishes the Linkam BCS 196 as a useful tool for the study of sperm cell cryopreservation.     

Immune activation decreases sperm viability in both sexes and influences female sperm storage

All animals are under the constant threat of pathogenic infection. However, little is known regarding the influence of acute infection on sperm viability, particularly in female insects. This information is crucial for our understanding of mating and immune system coevolution, considering that females store sperm and serve as the site of sperm competition. Using the fruitfly, Drosophila melanogaster, we examined the influence of infection on sperm viability and storage. Twenty-four hours after haemocoel inoculation with a pathogen mimic (peptidoglycan, PGN) both sexes exhibited reduced sperm viability, indicating that systemic immune activation played a significant role in gamete survival. Surprisingly, sperm death did not appear to result from a reproductive-immune system trade-off, considering that sperm survived 24 h in vitro once removed from their somatic resources. Instead, our results are most consistent with death owing to immune effector collateral damage. We also examined the potential for sexually transmitted pathogens to influence sperm storage. Females mated with 'infected' males (created by dipping genitalia into a PGN solution) exhibited a higher proportion of empty sperm stores 48 h after mating compared to their controls. Remarkably, these data indicate that females may increase their fitness by removing 'infected' ejaculates from storage over time.     

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.     

Experimental evidence that winning or losing a fight does not affect sperm quality in a field cricket

Fighting is a powerful social experience that can affect male reproductive behavior, including ejaculatory strategies. Whereas winners may monopolize females, losers may instead perceive high sperm competition and limited future mating opportunities, and accordingly enhance ejaculate quality to maximize their reproductive success. In male field crickets Gryllus bimaculatus that fight aggressively for control of breeding territories, winners are known to possess sperm of lower quality (viability) compared to losers, but it remains unclear whether this is due to short‐term fighting consequences. To test if the fighting experience per se (winning or losing) affects male adjustment of sperm viability, we subjected males to winning and losing experiences by staging fights against size‐matched rivals of known fighting ability. These rivals were males that previously won or lost a fight and, due to “winner‐loser effects” kept winning or losing subsequent contests. We sampled sperm prior and after the fight and twice in control males with no fighting experience and found no differences in sperm viability across measures. We conclude that males do not tailor their ejaculate quality following a single fight, or based on its outcome. Intrinsic differences in other attributes between winners and loser phenotypes may explain differences in sperm quality previously described in this system.     

Female genotype affects male success in sperm competition

The central question addressed by most studies of sperm competition is: ''what determines which male''s sperm are used at fertilization?'' Empirical and theoretical studies that address this question have traditionally focused on adaptations which enhance male fertilization success while treating the female as a receptacle in which sperm competition is played out. Here we provide evidence which suggests that female genotype strongly influences the outcome of sperm competition. When the sperm of two males are in competition the proportion of offspring fathered by the second male to mate (P₂) was found to be highly repeatable only if the male pair were mated to three different, but genetically similar females (full-sisters to each other; unrelated to either of the males). In contrast, if a male pair were mated to three females that were unrelated then P₂ was either non-repeatable or marginally repeatable. We also show that male success in sperm competition is determined, to a large extent, by gamete and/or male–female compatibility. This conclusion is derived from the observation that P₂ was repeatable among full-sisters mated to different, yet genetically similar male pairings, whilst P₂ was non-repeatable among full-sisters mated to different, genetically distinct male pairings.     

Assessment of boar sperm viability using a combination of two fluorophores

A combination of the fluorophore probes, calcein acetylmethyl ester (CAM) and ethidium homodimer (EH), were used to assess viability of ejaculated boar spermatozoa. Both CAM and EH have been used as indicators of biosynthetic activity and membrane integrity in monolayer cell cultures, with CAM shown to permeate and undergo enzymatic cleavage in viable monolayer cells giving the cell a green fluorescence, and EH penetrating only membrane damaged cells giving cells a red fluorescence. To determine if these fluorophores can be used to assess boar sperm viability, ejaculates from 10 boars were divided into 3 test groups (cytotoxic-treated, swim-up and washed), utilizing a split-ejaculate technique; each group consisted of both a probe-treated and control sample. Sample viability was ascertained in the control groups by visual estimation of the percentage motile spermatozoa, whereas the number of spermatozoa showing green (CAM = viable) or red (EH = non-viable) fluorescence were quantitated for each of the probe-treated groups using a fluorsecein or rhodamine filter, respectively. All spermatozoa exposed to the combined probes had an uptake of one or both fluorophores. The cytotoxic-treated group exhibited 0% gross motility, with 100% of the sperm heads showing red fluorescence. In the swim-up group, no difference was detected (P > 0.05) between control gross motility and the percentage of completely green fluorescing spermatozoa (85% vs. 86.6%, respectively). In the washed group, a significant difference (P = 0.039) was detected between gross motility estimates and the percentage of calcein-green fluorescent spermatozoa (57% vs. 60%, respectively). This study demonstrated that 1) CAM fluoresces only viable sperm, giving off a green fluorescence, 2) EH fluoresces in only non-viable sperm, giving off a red fluorescence, 3) visual estimation of motile sperm can approximate a semen sample's viability, but is not as precise as fluorophore determination, and 4) sperm incubation with the fluorophore combination CAM and EH provided an accurate technique for the objective assessment of boar sperm viability via their distinct fluorescent patterns in boar sperm.