Store Viable Sperm Research Articles

Female long-term sperm storage results in viable offspring in the Himalayan Mountain Pitviper, Ovophis monticola.

The ability of females to store sperm for extended periods in their reproductive tracts (termed long-term sperm storage, LTSS) has been reported across a diversity of vertebrate taxa. The evolutionary, ecological, and physiological significance of LTSS is wide-ranging and includes the ability to produce offspring when mates may be temporally scarce by way of decoupling copulation from ovulation, inbreeding avoidance, and the generation and maintenance of genetic diversity in progeny. Among vertebrate lineages, nonavian reptiles exhibit a remarkable capacity for LTSS, with the production of viable offspring reported after periods exceeding 6 years since prior contact with a potential mate. Given that female reptiles are able to store viable sperm for prolonged periods, it is important to disentangle LTSS from that of facultative parthenogenesis (FP), a reproductive trait which appears widespread among all reptile lineages. The implications of this distinction are particularly important in the context of the development and management of captive breeding programs. To accurately determine between the two reproductive strategies, genomic screening is highly recommended. Following a period of isolation for 13 months from a potential male mate, a female Himalayan Mountain Pitviper (Ovophis monticola) produced a clutch of three male offspring. Here, through genome-scale analyses of the female and her progeny, we document the first record of LTSS in this genus and exclude FP as the alternative hypothesis.

The developmental high wire: Balancing resource investment in immunity and reproduction.

The strategic allocation of resources into immunity poses a unique challenge for individuals, where infection at different stages of development may result in unique trade‐offs with concurrent physiological processes or future fitness‐enhancing traits. Here, we experimentally induced an immune challenge in female Gryllus firmus crickets to test whether illness at discrete life stages differentially impacts fitness. We injected heat‐killed Serratia marcescens bacteria into antepenultimate juveniles, penultimate juveniles, sexually immature adults, and sexually mature adults, and then measured body growth, instar duration, mating rate, viability of stored sperm, egg production, oviposition rate, and egg viability. Immune activation significantly impacted reproductive traits, where females that were immune challenged as adults had decreased mating success and decreased egg viability compared to healthy individuals or females that were immune challenged as juveniles. Although there was no effect of an immune challenge on the other traits measured, the stress of handling resulted in reduced mass gain and smaller adult body size in females from the juvenile treatments, and females in the adult treatments suffered from reduced viability of sperm stored within their spermatheca. In summary, we found that an immune challenge does have negative impacts on reproduction, but also that even minor acute stressors can have significant impacts on fitness‐enhancing traits. These findings highlight that the factors affecting fitness can be complex and at times unpredictable, and that the consequences of illness are specific to when during an individual's life an immune challenge is induced.

Sperm Mass Longevity and Sperm Storage in the Female Reproductive Tract ofPlethodon shermani(Amphibia: Plethodontidae)

Abstract: In salamanders in the seven families composing the Salamandroidea, sperm storage allows a female to obtain male sperm months before oviposition. This study reveals the ability of captive female Red-legged Salamanders, Plethodon shermani (Plethodontidae), to store and retain viable sperm for long periods of time. By staging male–female courtship trials, we found that a female’s retention of the male sperm mass is visible in her cloaca for up to 4 d (X = 2.8 d). Given the prolonged breeding season exhibited by this species (>10 wk), it is unlikely that the sperm mass functions efficiently to prevent female remating in that season. In addition, oviposition and egg-rearing in the laboratory revealed that P. shermani females can store viable sperm in their sperm storage organs (spermathecae) for ≥9 mo. Histology and microscopy showed that a subset of females even retained sperm in their spermathecae after oviposition, but significantly less of their lumina were occupied by sperm compared with the...

Short and Long‐Term Preservation of Sablefish Sperm from Baja California, Mexico

AbstractSablefish Anoplopoma fimbria is a species with high potential for aquaculture in Mexico. The development of protocols for short‐ and long‐term preservation of Sablefish sperm becomes necessary to store viable sperm that can be used as needed. In October and November of 2011 and 2012, Sablefish broodstock (mean weight, 3.5 kg; SD, 0.02) were captured using deepwater longlines in an area localized between the cities of Tijuana and Ensenada, Baja California, Mexico. The sperm concentration was evaluated by spectrophotometry. We determined the efficiency of two extender solutions in the short‐term storage of Sablefish sperm and evaluated three cryoprotectants (DMSO, methanol, and glycerol) and three cryopreservation protocols. Significant differences in sperm concentration between years and months were found. No differences were found in sperm activation time between both extender solutions. Significant differences were found among storage time, extender, month, year, and sperm motility or membrane integrity. A significant correlation between sperm motility and membrane integrity was found for both extenders in November. Significant differences were found among type and concentration of cryoprotectant, incubation time, and motility. Significant differences among cryopreservation protocols, cryoprotectant, and sperm motility and membrane integrity were registered. Glycerol (10%) registered the best motility (78%) and membrane integrity using the third cryopreservation protocol when samples were placed 5 cm above the liquid nitrogen for 9 min before being plunged in liquid nitrogen. These results will allow us to establish a reproduction program for Sablefish that will be used as a tool to advance the culture of this species in Mexico.

Females suffer a reduction in the viability of stored sperm following an immune challenge

Despite the ubiquitous nature of sperm storage in invertebrates, relatively little is known about its costs, or the impact that immune activation can have on a female's ability to maintain viable sperm stores. We explored the effects of an immune challenge on sperm storage under food-limited and ad libitum conditions in the field cricket, Teleogryllus oceanicus, by injecting mated adult females with either a LD5 dose of live bacteria or a nonpathogenic immune elicitor [lipopolysaccharide (LPS)] and then scoring the viability of their stored sperm. Females that were infected with bacteria showed a reduction in the viability of stored sperm 48h after infection; interestingly, this pattern was not evident when females were injected with LPS. Reduction in sperm viability post-infection may reflect a reproductive trade-off between immune function and sperm store maintenance, as only females injected with bacteria showed an elevated antibacterial immune (lytic) response. Alternatively, bacteria may act directly on sperm quality. Dietary manipulations showed that lytic activity in females is condition dependent, irrespective of their immune challenge treatment. Diet affected the ability of females to maintain the viability of stored sperm, suggesting that sperm storage is condition dependent. That bacterial infection associated with a reduction in stored sperm quality has potentially important implications for the outcomes of sperm competition in T.oceanicus and in other species in which females store sperm between matings.

Ovarian fluid of receptive females enhances sperm velocity

The females of several internal fertilizers are able to store sperm for a long time, reducing the risk of sperm limitation. However, it also means that males can attempt to mate outside females' receptive period, potentially increasing the level of sperm competition and exacerbating sexual conflict over mating. The guppy (Poecilia reticulata), an internally fertilizing fish, is a model system of such competition and conflict. Female guppies accept courtship and mate consensually only during receptive periods of the ovarian cycle but receive approximately one (mostly forced) mating attempt per minute both during and outside their sexually receptive phase. In addition, females can store viable sperm for months. We expected that guppy females would disfavour sperm received during their unreceptive period, possibly by modulating the quality and/or quantity of the components present in the ovarian fluid (OF) over the breeding cycle. Ovarian fluid has been shown to affect sperm velocity, a determinant of sperm competition success in this and other fishes. We found that in vitro sperm velocity is slower in OF collected from unreceptive females than in OF from receptive females. Visual stimulation with a potential partner prior to collection did not significantly affect in vitro sperm velocity. These results suggest that sperm received by unreceptive females may be disfavoured as sperm velocity likely affects the migration process and the number of sperm that reach storage sites.

Multiple Paternity in the Common Five-lined Skink (Plestiodon fasciatus)

Multiple mating and sperm storage can influence reproductive strategies of males and females. Male reproductive success is often limited by the ability to acquire mates; therefore, males have evolved strategies to mate with multiple females. In contrast, the number of offspring a female produces is generally not limited by the quantity of sexual partners; thus, selection for multiple matings is expected to be weaker than in males. Evidence of polyandry and sperm storage is widespread among lizards. We investigated whether female Common Five- lined Skinks (Plestiodon fasciatus) could store viable sperm between reproductive cycles, estimated the frequency of multiple paternity, and examined the sharing of paternity within clutches. Females were unable to store viable sperm between successive clutches. Most clutches (65%) had multiple sires, but within those clutches, there was unequal sharing of paternity. Although we cannot determine the function of polyandry from our data, we suggest possible causes of polyandry in the mating system of this species. Multiple mating by males and females has been well documented in numerous vertebrate and invertebrate taxa (Birkhead and Moller, 1998). Although widespread, the evolution of polyandry is more difficult to explain based on the existing literature than is polygyny. For males, mating opportunities are the most severe limitation of reproductive success, and accordingly, selection has driven male strategies toward the acquisition of multiple females (Andersson, 1994; Birkhead and Moller, 1998; Birkhead, 2000). In contrast, for females, a single mating typically can fertilize their ova and reproductive success is generally not constrained by the number of partners (Bateman, 1948; Trivers, 1972; but see Madsen et al., 1992).

Molecular genetic evidence for alternative reproductive strategies in North American pitvipers (Serpentes: Viperidae): long-term sperm storage and facultative parthenogenesis

The first documentation of facultative parthenogenesis (FP) in non-avian reptiles (snakes) occurred in 1997, following the application of molecular genetic methods for parentage analysis, and since has been described in only four additional snake species. Here, in two species of live-bearing (viviparous) North American pitviper snakes, using microsatellite DNA fingerprinting, we describe the first record of a virgin birth by FP in the copperhead (Agkistrodon contortrix), and the first genetically confirmed case of long-term sperm storage (LTSS) of exceptional duration (5 years) in the eastern diamond-backed rattlesnake (Crotalus adamanteus). The capacity of female squamate reptiles (snakes, lizards, and amphisbaenians) to store viable sperm for extended periods of time is a competing hypothesis to FP, and it is often inferred when interpreting certain births where adult females have been isolated from adult male mates for prolonged periods. On reviewing the literature of certain cases of LTSS in snakes, we provide compelling support for the prevalence of FP, casting doubt over the widespread acceptance of LTSS. Accordingly, with the discovery of FP in squamates and the results of the present study, we advocate that in order to differentiate between LTSS and FP, especially under natural conditions, rigorous molecular testing will be required. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 934–942.

Factors Affecting Sperm Quality Before and After Mating of Calopterygid Damselflies

Damselflies (Odonata: Zygoptera) have a more complex sperm transfer system than other internally ejaculating insects. Males translocate sperm from the internal reproductive organs to the specific sperm vesicles, a small cavity on the body surface, and then transfer them into the female. To examine how the additional steps of sperm transfer contribute to decreases in sperm quality, we assessed sperm viability (the proportion of live sperm) at each stage of mating and after different storage times in male and female reproductive organs in two damselfly species, Mnais pruinosa and Calopteryx cornelia. Viability of stored sperm in females was lower than that of male stores even just after copulation. Male sperm vesicles were not equipped to maintain sperm quality for longer periods than the internal reproductive organs. However, the sperm vesicles were only used for short-term storage; therefore, this process appeared unlikely to reduce sperm viability when transferred to the female. Males remove rival sperm prior to transfer of their own ejaculate using a peculiar-shaped aedeagus, but sperm removal by males is not always complete. Thus, dilution occurs between newly received sperm and aged sperm already stored in the female, causing lower viability of sperm inside the female than that of sperm transferred by males. If females do not remate, sperm viability gradually decreases with the duration of storage. Frequent mating of females may therefore contribute to the maintenance of high sperm quality.

Reproductive biology and mating conflict in the simultaneously hermaphroditic land snail Arianta arbustorum*

This review summarizes the present knowledge on the reproductive biology, mating system, sperm competition, sex allocation, and mating conflict in the simultaneously hermaphroditic land snail Arianta arbustorum (Linnaeus, 1758) (Helicidae). Field studies and controlled laboratory experiments indicate that mating is random with respect to shell size. However, subtle effects of inbreeding (reduced hatching success of eggs and viability of juveniles) and outbreeding were found. Individuals of A. arbustorum mate repeatedly with different partners and store viable sperm for more than one year. Spermatophore transfer is highly reciprocal, but the number of sperm they contain (800,000–4,000,000) is not necessarily equal. Snails need 3–4 weeks to replenish their autosperm reserves after a successful copulation. Sperm are monomorphic. However, there is considerable among-population—and to a minor extent—within-population variation in total sperm length. Sperm utilization patterns in double-mated individuals of A. arbustorum revealed striking differences among individuals. There is a huge variation in the structure of the spermatheca, which consists of 2–9 blind tubules. Different lines of evidence suggest that the snails might be able to store and expel sperm stored in single tubules and thus promote a selective fertilization of eggs (cryptic female choice). Maternal investment in eggs is considerable. Snails mated 1, 2, or 3 times showed that irrespective of the number of matings the individuals devoted >95% of the resources into the female function.

Sperm storage and low incidence of multiple paternity in the European pond turtle, Emys orbicularis: A secure but costly strategy?

The freshwater pond turtle, Emys orbicularis, has recently suffered from population declines throughout its range, mainly due to habitat destruction. The mating strategies of this species were studied using genetic data from successive clutches within and between years. To test for the occurrence and frequency of multiple paternity and sperm storage, genetic paternity at six microsatellite markers was assessed in 114 embryos and hatchlings from single and subsequent clutches of 11 females (including clutches from the same or consecutive years). Multiple paternity was rare and only found in two out of 20 clutches from 11 females. All annual successive clutches and 58% of the clutches in the next year, were fertilized with sperm from the same male. The use of stored sperm is thus a frequent strategy in E. orbicularis. However, hatching rate, hatchling mass, and hatchling length decreased in clutches fertilized by stored sperm, suggesting sperm depletion or deterioration through time. The occurrence of stored sperm despite an associated reduced reproductive output indicated that mating and/or the fertilization process is costly to females. The low incidence of multiple paternity may simply be the residual consequence of the capacity to store viable sperm. These results provide important and innovative insights for the conservation of E. orbicularis. In threatened populations, management strategies may aim to enhance effective copulations in order to increase the reproductive output of females.

THE EVOLUTION OF MALE TRAITS IN SOCIAL INSECTS

Pair formation in social insects mostly happens early in adult life and away from the social colony context, which precludes promiscuity in the usual sense. Termite males have continuous sperm production, but males of social Hymenoptera have fixed complements of sperm, except for a few species that mate before female dispersal and show male-fighting and lifelong sperm production. We develop an evolutionary framework for testing sexual selection and sperm competition theory across the advanced eusocial insects (ants, wasps, bees, termites) and highlight two areas related to premating sexual selection (sexual dimorphism and male mate number) that have remained understudied and in which considerable progress can be achieved with relatively simple approaches. We also infer that mating plugs may be relatively common, and we review further possibilities for postmating sexual selection, which gradually become less likely in termite evolution, but for which eusocial Hymenoptera provide unusual opportunities because they have clonal ejaculates and store viable sperm for up to several decades.

Conservation of the red-winged grasshopper, Oedipoda germanica (Latr.): the influence of reproductive behaviour

Reproductive isolation can function as a mechanism to maintain locally adapted gene complexes while decreasing the heterozygosity in distinct populations. As a result, reproductive behaviour should be considered a fundamental factor influencing reproductive isolation. This is of interest to conservation biology when one desires to regulate gene flow between two populations either by creating opportunities for increased dispersal, by relocation of individuals, or by re-introduction of a species to its natural habitat. Reproductive behaviour can also influence the effective population size and the actual population size through the Allee effect. We investigated the reproductive behaviour of individuals from two isolated populations of the red winged grasshopper, Oedipoda germanica, an endangered species in Central Europe. We detail several methods to show how several aspects of the reproductive behaviour of this species interact with the conservation of this species. Foreign males were not disfavoured in mate choice and male body size was also unimportant in mating success. Heterogamic matings were as productive as homogamic matings in terms of total number of eggs per female, egg hatching rate, or nymph survival. Therefore, we suggest that cross-matings of individuals from different populations do not positively influence population size by heterosis effects nor act they negatively in the form of outbreeding depression. We found that female O. germanica were able to store viable sperm for extended periods but egg pods showed a decrease in hatching rate when these females were deprived of additional mating opportunities. Multiple mated females laid more eggs than once-mated females. Hence, females are capable of founding new populations even after only one mating but their reproductive output may be reduced. Present-day gene flow between populations of O. germanica probably does not occur. Consequently, there is an urgent need to pursue habitat management and release programmes that maintain current population sizes of this species. With regard to the reproductive behaviour of the red-winged grasshopper, we suggest that relocation programmes release males and female together and at an early adult stage. Also, because female reproductive output increases with mating activity, the initial release should involve excess females. Because males have a shorter lifespan than females a subsequent release of males at a later time may enhance the overall success of local conservation efforts by resulting in more offspring per female.

Genetic markers substantiate long-term storage and utilization of sperm by female painted turtles.

Most studies of genetic parentage in natural populations have been limited to a single breeding season or reproductive episode and, thus, provide only a snapshot of individuals' mating behaviours. Female turtles can store viable sperm in their reproductive tracts for as long as several years, but the extent to which this capacity is utilized in nature has remained unknown. Here, we employ microsatellite markers to assess genetic paternity in successive clutches of individually marked, free-ranging female painted turtles (Chrysemys picta) over a four year period. The genetic data from 113 clutches from this natural population demonstrate that most females (80.5%) remated each year and that each female generally used a single male's sperm to fertilize all clutches laid within a year. However, sperm usage among females varied considerably, and some females apparently used sperm that had been stored for up to three years to fertilize some or all eggs laid in consecutive nesting seasons. Thus, remating by females is not necessary for continued offspring production from a given sire. Furthermore, 13.2% of all clutches examined showed evidence of multiple paternity, and the genetic paternity patterns across years suggest a 'last in, first out' operation of the females' sperm storage tubules.

Post-copulatory storage of sperm in the seminal bursa of the acoel flatworm Otocelis luteola

Summary Copulation in the acoel Otocelis luteola may result in mutual or one-sided insemination. Fifty specimens cultured with a species of Navicula were allowed to mate in pairs for a period of 6 h, 50 were allowed to mate in pairs for 24 h, and nine were allowed to mate freely for 24 h. All wows were then isolated in separate containers that were examined at frequent intervals in order to record the number of eggs and young. The number of progeny produced by worms that had been inseminated ranged from 1 to 65, and one worm had progeny as late as 55 days after mating. This study shows that O. luteola is able to store viable sperm in its seminal bursa for several weeks.

Parental Role-Reversed Polyandry and Paternity

Parental Role-Reversed Polyandry and Paternity

Anatomical evidence for a countercurrent heat exchanger associated with dolphin testes.

Cetaceans possess cryptic testes that lie within the abdominal cavity, that are surrounded by primary locomotor muscles, and that are presumably exposed to core or above core body temperatures. It has remained a question as to how cetaceans produce and store viable sperm at these high temperatures. We offer anatomical evidence for a two layer arterio-venous countercurrent heat exchanger at the cetacean testis. Subcutaneous veins from the peripheral surfaces of the dorsal fin and flukes carry cool blood from the fins to the lumbo-caudal venous plexus. The lumbo-caudal venous plexus is juxtaposed to the spermatic arterial plexus, which supplies the testis. Venous plexus flow is form the ventro-lateral margins of the visceral cavity towards the vena cava. Arterial plexus flow is from the aorta towards the ventro-lateral margins of the visceral cavity and into the testis. The existence of a countercurrent heat exchanger suggests that cetaceans potentially compensate for detrimental effects of core temperatures on sperm viability and storage by regulating the temperature of blood flow to the testis.

Forced Copulation in Waterfowl

Abstract 1. Although almost all waterfowl (ducks, geese and swans comprizing the family Anatidae) have basically monogamous mating systems, males of 39 species have been observed to perform or attempt forced copulation (FC) (previously called "rape"). 2. Suggestions that FC is an artifact of crowding in urban mallards, an outlet for thwarted male sex drive, a component of territory defence, or an alternative reproductive strategy of unpaired males are not supported by available evidence. 3. In the case of the mallard, HEINROTH'S (1911) interpretation of FC as a secondary insemination method whereby paired males increase their reproductive success by fertilizing some of the eggs laid by females other than their mate is supported by evidence of various kinds. FCs are performed by paired males; they occur during periods in the breeding season when eggs are being fertilized; they are directed mainly at females in prelaying and laying condition; paired males usually defend their mates against males attempting FC (suggesting defence of genetic paternity); paired males have been seen to copulate forcibly with their own mates after the latter have been subjected to FC (suggesting an antidote insemination strategy). 4. Experiments on captive mallards have shown that eggs can be fertilized by sperm delivered during FC and, since females can store viable sperm for up to 17 days, sperm competition must be taking place. Artificial insemination experiments have shown that (a) the second of 2 competing inseminations 6 hours apart overlays the former insemination and is 70% more potent, (b) there is an insemination "window" within 1 hour of oviposition when the next egg in the clutch is fertilized. The extent to which males time their copulations (FCs, pair copulations, forced pair copulations) to take advantage of these patterns of sperm competition is not known. 5. Studies of the lesser snow goose, northern pintail and lesser scaup indicate that FC is a secondary reproductive strategy of paired males in these species also. Observations on 13 species of dabbling duck provide additional evidence for the selection of fertile females as FC targets and the development of male tactics for achieving and combatting FC. 6. It is difficult to rule out the possibility that females could derive certain benefits from FC inseminations (e.g. genetic diversity of offspring) but the costs and risks of being involved in FC assaults can be high and the females apparently try to avoid FC. Female dabbling ducks can be damaged, or even killed, during assaults and female escape behaviour (high flights, diving, hiding in cover) entails energetic costs and wastage of time that are presumably detrimental to females during the period when they are producing eggs. 7. As HEINROTH suggested, FC probably does not occur in swans, most geese and shelducks because it is incompatible with the major roles that paired males play in defence of breeding territories, mates, nest-sites and broods in these groups. The occurrence of FC in snow and Ross goose may be facilitated by colonial nesting.. 8. Among dabbling ducks, male territoriality is associated with strong mate-guarding and these characteristics appear to conflict with male FC activities. Involvement of paired males in extrapair courtship, polygyny and brood-care may produce additional conflicts with FC behaviour in certain species. 9. In at least 4 species of waterfowl (mallard, northern pintail, lesser snow goose, lesser scaup) behavioural evidence indicates that FC is part of a mixed male reproductive strategy of the type postulated by TRIVERS (1972). Rigorous testing of the hypothesis will require studies to (a) assess the incidence of multiple paternity in wild clutches, (b) distinguish species (or populations within a species) in which paired males actively pursue FC tactics from those in which they merely capitalize on incidental opportunities for FC.