Age effects on the insemination rate ofAnopheles gambiae s.l. in the laboratory

Age effects on the insemination rate of the Galisua strain ofAnopheles gambiae s.s. (Diptera: Culicidae) and of the Nyanza strain ofAn. arabiensis were investigated in the laboratory. Batches of 7-day old males and females were kept together for 24 h with batches of mosquitoes of the opposite sex of ages ranging from 1–7 days. Males and females were also kept together continuously from emergence. The effect of increasing male/female ratios on the insemination rate was investigated as well. The insemination rate ofAn. arabiensis in the laboratory (96%) was similar to that found in nature, whereas that of our strain ofAn. gambiae s.s. was consistently low (maximum 72%), particularly after females had been together with males for a 24 h period only. The optimum age for insemination was 7 days for males and females of both species. The insemination rate of our strain ofAn. arabiensis was significantly higher at all ages than that of our strain ofAn. gambiae s.s.. The latter strain became inseminated at the earliest when 4 days old. An increase in the male/female ratio significantly enhanced the insemination rate in both strains. It was found that motility of spermatozoa inside the spermatheca, once in contact with saline, could be used as a marker for the approximate time of insemination. It is concluded that the relatively low insemination rate of the Galisua strain ofAn. gambiae s.s. is unlikely to be caused by the rearing conditions. Other factors that may be responsible are discussed.     

The storage of spermatozoa in the oviducal glands of western North Atlantic sharks

Synopsis Spermatozoa stored in oviducal glands of sharks sampled off the North American east coast were revealed by viewing stained tissue sections using light microscopy. Of eleven species surveyed, sperm were found in nine:Alopias vulpinus, Lamna nasus, Carcharhinus obscurus, Carcharhinus plumbeus, Galeocerdo cuvieri, Prionace glauca, Rhizoprionodon terraenovae, Sphyrna lewini andSphyrna tiburo. Three insemination patterns are proposed to account for differences noted in these findings: (1) non-storage/immediate insemination for sharks such asLamna nasus; (2) short-term storage/delayed insemination as found in sharks in which ovulation is prolonged over weeks or months such asRhizoprionodon terraenovae, and (3) long-term storage/repeated insemination, a characteristic of nomadic sharks such asPrionace glauca andCarcharhinus obscurus which can store sperm in specialized tubules for months to years.     

Ultrastructural examination of the insemination reaction in Drosophila

The insemination reaction is a swelling of the female vagina caused by the male ejaculate. This postmating phenomenon is common among species in the genus Drosophila. It could act as a plug securing male paternity. It is not clear, however, what benefits it provides to the female. The structure formed in the female vagina is expelled in some species and disappears gradually in others suggesting different phenomena. Based on ultrastructural examination of the vaginal contents of five Drosophila species (D. mettleri, D. nigrospiracula, D. melanogaster, D. mojavensis, and D. hexastigma), we propose three terms to describe these vaginal structures: the sperm sac, the mating plug, and the true insemination reaction. Each term describes a distinct structure associated with a specific female postmating behavior. This study questions the concept of the insemination reaction as a single phenomenon and discusses its possible functions from an evolutionary perspective.     

Transcervical artificial insemination in the domestic ferret (Mustela putorius furo)

The research was undertaken to develop a successful nonsurgical procedure for artificially inseminating ferrets. A fiberoptic endoscope used in conjunction with a specially designed speculum and catheter permitted cervical catheterization and intrauterine insemination. Sperm were collected from the cauda epididymides of 10 discarded breeder males; the number of sperm in diluted samples used for insemination ranged from 4.4–13.6 × 10⁶/100 μl with progressive motility of sperm ranging from 40 to 60%. Sperm collected from each male were diluted with an egg-yolk extender (TEST) and used to inseminate 8–12 females, with deposition of sperm intravaginally or transcervically into the uterine body 0 or 24 hr after an ovulatory injection of human chorionic gonadotropin (hCG). The vaginal inseminations were used as a control, and no pregnancies resulted after insemination of 26 females. Intrauterine inseminations resulted in 4/24 (17%) of the ferrets pregnant when hCG administration was coincident with insemination, and 19/24 (79%) of the ferrets were pregnant when inseminations were done 24 hr after hCG administration. All inseminated females were euthanized on day 20 after insemination to count fetuses. The mean number of fetuses was 3.1 (range, 1–8). The number (millions) of motile sperm inseminated (X) had a significant effect on the percentage of fetuses (Y). Regression analysis indicated a linear relationship between the two variables, with an R² value of 0.99 and a line of best fit described by the equation Y = 0.029 + 0.034 X. This paper is the first report of transcervical artificial insemination in the domestic ferret (Mustela putorius furo). The method can serve as a model for application to ferrets and other mammals, particularly endangered species. Zoo Biol 17:393–404, 1998. © 1998 Wiley-Liss, Inc.     

The effects of injection of amphetamine on female insemination in the black blow fly, Phormia regina (Diptera: Calliphoridae)

Abstract. An earlier study on the blowfly Phormia regina (Meigen) demonstrated that the injection of amphetamine (12 μg) depletes biogenic amine levels in the CNS. In the present study, P. regina females were injected with amphetamine (12 μg), each female was placed with three males and insemination success was determined. Amphetamine inhibited female insemination by 43.3% at 2–90 min post-injection and by 70% at 10–60 min post-injection. At 180–270 min post-injection, there was no significant inhibition of female insemination. This study indicates a possible role in insects for the biogenic amines in female insemination.     

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

The effect of DNA microinjection at various times afterin vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p<0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p<0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p<0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.     

Sperm Competition and Optimal Timing of Matings in Microcebus murinus

Female promiscuity is common in mammals and leads to sperm competition: the sperm of ≥2 males compete for ova. Scientists understand the possible role of optimal insemination periods for male reproductive success in many species as well as the impact of monopolization of receptive females. Information from experiments combined with detailed observations from the field that allow determining the relative impacts of the elements in the same species are rare. We studied sperm competition and the role of optimal insemination periods in gray mouse lemurs (Microcebus murinus), a small solitary nocturnal primate from Madagascar. We used controlled matings to identify the relative impact of both contest and scramble competition, which characterize their mating system, on paternity. Fifteen females mated with 3–6 males in quick succession. Our experiments revealed that the optimal insemination period is during early receptivity. Early but not first mating males are more likely to sire offspring. Comparison with our field data indicate that the timing of male monopolization efforts correspond with the optimal insemination period.     

Age‐specific changes in sperm levels among female tsetse (Glossina spp.) with a model for the time course of insemination

The age, insemination and ovulation status of tsetse flies Glossina pallidipes Austen (n = 154369) and Glossina morsitans morsitans Westwood (n = 19659), captured over 11 years in Zimbabwe, are assessed by ovarian dissection. Instantaneous rates of insemination increase exponentially with age in both species; 90% insemination levels are reached after 5 days post‐emergence in G. m. morsitans and 7 days in G. pallidipes, varying little with season. More than 95% of both species have ovulated by the age of 8 days and 99% by 12 days. Older flies that have not ovulated are > 100‐fold more likely to be caught in October and November than in other months. A 500‐fold decrease in trap catches did not result in any detectible decrease in the probability of females being inseminated. The proportion of partially filled spermathecae rises for approximately 6 days then declines, consistent with some flies having mated more than once. For flies caught on electric nets, with wings undamaged during capture, wing‐fray data are used to extend ovarian age estimates up to 11 ovulations. Among these flies, the volume of sperm in the spermathecae declines little in flies that have ovulated up to seven times; thereafter, it declines by approximately 1% per ovulation. The time course of insemination and the mating frequency of females are important considerations in modelling tsetse fly populations, as well as for the dynamics of interventions involving the release of genetically‐modified insects, which should not be seriously compromised by the limited levels of polyandry currently observed.     

Successful artificial insemination in the corn snake,Elaphe gutatta,using fresh and cooled semen

The design and implementation of assisted reproductive technology to improve genetic diversity and augment captive populations is an important but rarely applied research field in reptiles. Using the corn snake (Elaphe gutatta) as a model, the Henry Doorly Zoo recently produced offspring born as a result of artificial insemination using both fresh, diluted semen, and diluted semen stored at refrigeration for 3 days. Semen was collected noninvasively from sexually mature male corn snakes using a gentle massaging technique, extended in medium then inseminated into the oviducts of adult females. Using molecular genetic techniques to confirm or refute the success of the insemination using primers developed for the black rat snake, Elaphe obsolete, all possible parents and offspring genotypes were evaluated. A paternity‐by‐exclusion analysis verified that the offspring were in fact a result of artificial insemination. Zoo Biol 26:363–369, 2007. © 2007 Wiley‐Liss, Inc.     

A pharmacological and endocrinological study of female insemination inPhormia regina (Diptera: Calliphoridae)

Injections of octopamine, dopamine, and the octopaminergic agonists, clonidine and naphazoline, into the thoracic hemocoel enhanced female insemination in sugar-fed (sexually unreceptive)Phormia regina. Topical applications of the juvenile hormone (JH) analogue, methoprene, also enhanced female insemination in sugar-fed (sexually unreceptive)P. regina. Since JH plays a role in receptivity in protein-fed females, it was originally hypothesized that one agonist, clonidine, enhanced female insemination by acting on the corpus allatum (CA) to increase JH biosynthesis. Two or three doses of the antiallatal agent, precocene II, prior to administration of clonidine, did not inhibit clonidine-enhanced female insemination. Removal of the corpus allatum also did not have a significant effect on clonidine-enhanced female insemination. Measurement of juvenile hormone (JH) biosynthesis/release in corpora allata, which were removed at 1, 3, 5, and 7 h postinjection, revealed that clonidine does not affect JH biosynthesis/release. Our study reveals a possible role for the biogenic amines in female insemination in insects. We suggest that the octopaminergic agonist, clonidine, acts downstream from the corpus allatum on the regulatory mechanisms involved in female insemination.     

Biochemical genetics of mosquitofish III. Incidence and significance of multiple insemination

Evidence for multiple insemination in the mosquitofish, Gambusia affinis, was detected by statistical analysis of the enzyme phenotypes in mother-offspring combinations for three loci. The probability of nondetection of multiple insemination events for diallelic loci with two or more males mating with a female was presented. The frequency of multiple insemination for G. affinis is at least 56% but is probably nearer to 100%. The high incidence of multiple insemination together with sperm storage and delayed fertilization could have important effects on allele frequencies through time and space. These effects are due to the increase in effective population size and overlap among generations.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

Obtaining an Interspecific Hybrid of Cranes by Artificial Insemination with Frozen–thawed Semen

Studies of artificial insemination of cranes and cryoconservation of their semen have been carried out in the nursery of rare species at the Oka Biosphere Reserve for many years. The criterion of successful cryoconservation of the semen is the obtaining of fertilized eggs after artificial insemination by the thawed semen. An experiment is described on artificial insemination of females of the white-naped crane Grus vipio by the frozen–thawed semen of the Siberian white crane G. leucogeranus after one-year storage of semen in liquid nitrogen. As a result, an interspecific hybrid of cranes was obtained, which confirmed the possibility of producing a bank of cryoconserved crane semen. The use of the white-naped crane females was due to the absence of conspecific males and unavailability of Siberian white crane females. Problems of artificial insemination and cryoconservation of semen of rare crane species are discussed.     

Obtaining an Interspecific Hybrid of Cranes by Artificial Insemination with Frozen–thawed Semen

Studies of artificial insemination of cranes and cryoconservation of their semen have been carried out in the nursery of rare species at the Oka Biosphere Reserve for many years. The criterion of successful cryoconservation of the semen is the obtaining of fertilized eggs after artificial insemination by the thawed semen. An experiment is described on artificial insemination of females of the white-naped crane Grus vipio by the frozen–thawed semen of the Siberian white crane G. leucogeranusafter one-year storage of semen in liquid nitrogen. As a result, an interspecific hybrid of cranes was obtained, which confirmed the possibility of producing a bank of cryoconserved crane semen. The use of the white-naped crane females was due to the absence of conspecific males and unavailability of Siberian white crane females. Problems of artificial insemination and cryoconservation of semen of rare crane species are discussed.     

Can small male shrimps achieve copulation in the presence of larger ones?

The capacity for insemination and competitive ability of small males of the partially protandrous alpheid shrimpAthanas kominatoensis Kubo which lives in symbiosis with the Japanese purple sea urchinAnthocidaris crassispina (A. Agassiz) were examined. Although male shrimps 1.5 mm in carapace length or larger produced sperm, it was not until they grew to 2.4 mm that they became functional. Males lost their insemination ability when their endopodite (or the whole) of the 2nd pleopods were amputated. The amputation of the 5th pleopods did not affect the insemination ability. The males also lost insemination ability after being infected by 2 species of Isopod ectoparasites, probably because of the transformation of the 2nd pleopods. Small males were shown to be able to achieve copulation in cohabitation experiments with amputated or parasitized large males. The total parasite infection rate in the field was highest in December and lowest in May. From August to October, when the small and large males coexisted, on the average 15.9% of large males were infected and transformed by the parasites. The presence of parasites offers the small males an opportunity for reproduction and is thought to play an important role in the evolution of protandry in this shrimp.     

Adaptive Topography in Fertility-Viability Selection Models: The Haplodiploid Case

A linear combination of partial changes of mean fitnesses from one generation to the next one is shown to be approximately equal to the additive genetic variance in fitness after enough generations and away from equilibrium in random mating haplodiploid populations under arbitrary weak frequency-dependent selection on sex-differentiated viability of individuals and sex-differentiated fertility of matings controlled at a single multiallelic locus. The result can be applied to X-linked locus models in diploid populations. The result is used to deduce approximate adaptive topographies far frequency-independent selection models in the cases of nonsex-differentiated fertilities and multiplicative sex-differentiated fertilities and for kin selection models in family-structured populations under the assumptions of single insemination and multiple insemination of females. Multiple insemination creates frequency-dependent selection regimes.     

Sperm loss at copulation in Culicoides melleus

The loss of sperm at copulation in Culicoides melleus was investigated with respect to the previous sexual experience of the female and the interval between her copulations. Approximately 40% of ejaculated sperm were lost if a male copulated with a female within 16 hours of her previous insemination by another male. By 96 hours after the first insemination, sperm transfer at a second copulation was virtually back to the initial 100% level.     

Insemination patterns in HawaiianDrosophila species (Diptera: Drosophilidae) correlate with ovarian development

In 35 Hawaiian Drosophilaspecies collected from natural populations, all reproductively mature females were inseminated. Onset of receptivity to insemination broadly correlated with the vitellogenic stage of ovarian development, but species and species groups varied in the exact stage of vitellogenesis at which earliest insemination took place. A striking exception was observed in two picture-winged species of the adiastolasubgroup, which were precociously inseminated in previtellogenesis, close to the time of adult eclosion. For a given species, the range of ovarian stages from earliest receptivity to insemination of all females, termed the insemination period, also varied among species. This variability may be due to physiological variation between females in hormonal levels, low population densities, and lek behavior of males, or it may reflect variation in degree of female discrimination toward male courtship attempts. Consideration of the quantities of sperm observed stored in the female sperm storage organs, relative to the numbers of eggs produced, indicates obligatory multiple insemination for some species, notably the fungus breeders. In other species groups, there is no apparent necessity for remating, although it may occur.This paper is No. IV in the series Studies of Oogenesis in Natural Populations of Drosophilidae.     

Sperm mortality, insemination and fertilization in the damselfly Ischnura senegalensis: comparisons between wild and inbred populations

Inbreeding can have deleterious effects on individual or population fitness. To avoid fitness reduction, individuals may adopt behavioral or physiological mechanisms to reduce their investment in the production of offspring with genetically similar mates. We examined whether insemination by inbred males introduced more dead sperm than insemination by wild males by counting sperm in female Ischnura senegalensis (Rambur) sperm storage organs. If inbred males inseminated fewer or lower-quality sperm, females would avoid inferior sperm. Our results revealed three features of damselfly inbreeding: insemination failed in a larger proportion of inbred pairs than in wild pairs, inbred pairs showed significantly reduced fertility, and the numbers of live and dead sperm in an inbred female’s sperm storage organs did not differ from those in wild females. These results suggested that neither sperm quantity nor sperm quality was responsible for low fertility to a significant extent, but some kind of female quality, such as sperm usage or storing ability, was. Although inbred pairs had lower fertility, there were no significant differences between inbred and wild pairs in the total numbers of live or dead sperm. It thus seemed that female choice at the insemination stage was responsible for low fertility rather than sperm quantity or quality measured by live-to-dead ratio.