Courtship activity, copulation & insemination success in a mosquito vector fed a herbal aphrodisiac: Implications for sterile insect technology.

BACKGROUND & OBJECTIVES: In sterile insect technology (SIT), mating competitiveness is a pre-condition for the reduction of target pest populations and a crucial parameter for judging efficacy. Still, current SIT trials are being hindered by decreased effectiveness due to reduced sexual performance of released males. Here, we explored the possible role of a herbal aphrodisiac in boosting the mating activity of Aedes aegypti. METHODS: Males were fed one of two diets in this study: experimental extract of Eurycoma longifolia (MSAs) and sugar only (MSOs). Differences in life span, courtship latency, copulation activity and mating success were examined between the two groups. RESULTS: No deaths occurred among MSA and MSO males. Life span of MSOs was similar to that of MSAs. The courtship latency of MSAs was shorter than that of MSOs (P<0.01). MSAs had greater copulation success than MSOs (P<0.001). In all female treatments, MSAs mated more than MSOs, but the differences in rate were significant only in the highest female density (P<0.05). In MSAs, mating success varied significantly with female density (P<0.01), with the 20-female group (P<0.01) having the lowest rate. Single MSA had better mating success at the two lowest female densities. In MSOs, there were no significant differences in mating success rate between the different female densities. INTERPRETATION & CONCLUSIONS: Our results suggested that the herbal aphrodisiac, E. longifolia, stimulated the sexual activity of Ae. aegypti and may be useful for improving the mating competitiveness of sterile males, thus improving SIT programmes.

Ovarian Stimulation, Intrauterine Insemination, Multiple Pregnancy and Major Congenital Malformations: A Systematic Review and Meta- Analysis- The ART_Rev Study.

INTRODUCTION: Multiple pregnancies are a recognized adverse effect of assisted reproductive technologies; nevertheless, there is no consensus on the incremental risk associated with the ovarian stimulation (OS) used alone and intrauterine insemination (IUI). The relationship between OS and IUI and the risk of major congenital malformations (MCM) is unclear. OBJECTIVE: To summarise the literature and evaluate the risk of multiple pregnancy and MCM associated with OS used alone and IUI used with or without OS compared to natural conception (spontaneously conceived infants without any type of fertility treatments). METHODS: We carried out a systematic review to identify published papers between 1966 and 2014 in MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials. We included observational studies and randomized clinical trials related to the risk of multiple pregnancies and MCM conceived following OS alone or IUI compared to natural conception (spontaneously conceived infants without any fertility treatments). The quality of the included studies was evaluated using The Cochrane Collaboration's tool for assessing risk of bias for RCTs and the Newcastle-Ottawa Scale for observational studies. RESULTS: There were 63 studies included in this review. Our systematic review suggests that the use of any OS alone was associated with an increased risk of multiple pregnancy compared to natural conception (pooled RR 8.80, 95% CI 5.09- 15.20; p= 0.000; 9 studies). Similar increases in the risk of multiple pregnancies were observed following clomiphene citrate used without assisted reproductive technologies. Compared to natural conception, the use of IUI with or without OS was associated with an increased risk of multiple pregnancy (pooled RR 9.73, 95% CI 7.52 -12.60; p= 0.000; 6 studies). Compared to natural conception, the use of any OS alone was associated with an increased risk of any MCM (RR pooled 1.18, 95%CI 1.03-1.36; 11 studies), major musculoskeletal malformations (pooled RR 1.48, 95%CI 1.21-1.81; 7 studies), and malformations of the nervous system (pooled RR 1.73, 95%CI 1.15-2.61; 6 studies). Compared to natural conception, the use of IUI was associated with an increased risk of any MCM (pooled RR 1.23, 95%CI 1.10-1.37; 10 studies), major urogenital (pooled RR 1.52, 95%CI 1.04-2.22; 7 studies), and musculoskeletal malformations (pooled RR 1.54, 95%CI 1.20-1.98; 7 studies). The overall quality of the included studies was acceptable. CONCLUSIONS: The increased risk of multiple pregnancy and certain types of MCM associated with the use of less invasive fertility treatments, such as OS and IUI, found in this review, highlights the importance of the practice framing. Heterogeneity in OS protocols, the combination with other fertility agents, the limited number of studies and the methodological quality differences reduce our ability to draw conclusions on specific treatment. More observational studies, assessing the risk of multiple pregnancy or MCM, as a primary outcome, using standardized methodologies, in larger and better clinically defined populations are needed.

Fitness of transgenic mosquito Aedes aegypti males carrying a dominant lethal genetic system.

OX513A is a transgenic strain of Aedes aegypti engineered to carry a dominant, non-sex-specific, late-acting lethal genetic system that is repressed in the presence of tetracycline. It was designed for use in a sterile-insect (SIT) pest control system called RIDL® (Release of Insects carrying a Dominant Lethal gene) by which transgenic males are released in the field to mate with wild females; in the absence of tetracycline, the progeny from such matings will not survive. We investigated the mating fitness of OX513A in the laboratory. Male OX513A were as effective as Rockefeller (ROCK) males at inducing refractoriness to further mating in wild type females and there was no reduction in their ability to inseminate multiple females. They had a lower mating success but yielded more progeny than the wild-type comparator strain (ROCK) when one male of each strain was caged with a ROCK female. Mating success and fertility of groups of 10 males-with different ratios of RIDL to ROCK-competing for five ROCK females was similar, but the median longevity of RIDL males was somewhat (18%) lower. We conclude that the fitness under laboratory conditions of OX513A males carrying a tetracycline repressible lethal gene is comparable to that of males of the wild-type comparator strain.

Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost.

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

Love is blind: indiscriminate female mating responses to male courtship pheromones in newts (Salamandridae).

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.

Treatment with the GnRH antagonist ganirelix prevents premature LH rises and luteinization in stimulated intrauterine insemination: results of a double-blind, placebo-controlled, multicentre trial.

BACKGROUND: This study was designed to assess whether the use of ganirelix in women undergoing stimulated IUI could prevent the occurrence of premature LH rises and luteinization (LH+progesterone rises). METHODS: Women of infertile couples, diagnosed with unexplained or male factor infertility, were randomized to receive either ganirelix (n=103) or placebo (n=100) in a double-blind design. All women were treated with an individualized, low-dose rFSH regimen started on day 2-3 of cycle. Ganirelix (0.25 mg/day) was started if one or more follicles>or=14 mm were visualized. Ovulation was triggered by HCG injection when at least one follicle>or=18 mm was observed and a single IUI was performed 34-42 h later. The primary efficacy outcome was the incidence of premature LH rises (+/-progesterone rise). RESULTS: In the ganirelix group, four subjects had a premature LH rise (value>or=10 IU/l), one LH rise prior to the start of ganirelix and three LH rises during ganirelix treatment, whereas in the placebo group 28 subjects had a premature LH rise, six subjects prior to the start of placebo and 22 subjects during placebo treatment. The incidence of LH rises was significantly lower in ganirelix cycles compared to placebo cycles (3.9 versus 28.0%; P=0.003 for ITT analysis). When excluding subjects with an LH value>or=10 IU/l before the start of ganirelix/placebo the incidence of LH rises was also significantly lower in ganirelix cycles compared to placebo cycles (2.9 versus 23.4%; P=0.003 for ITT analysis). Premature luteinization (LH rise with concomitant progesterone rise>or=1 ng/ml) was observed in one subject in the ganirelix group and in 17 subjects in the placebo group of which three subjects had a premature spontaneous ovulation. Ongoing pregnancy rates per attempt were 12.6 and 12.0% for the ganirelix and placebo groups respectively. CONCLUSIONS: Treatment with ganirelix effectively prevents premature LH rises, luteinization in subjects undergoing stimulated IUI. Low-dose rFSH regimen combined with a GnRH antagonist may be an alternative treatment option for subjects with previous proven luteinization or in subjects who would otherwise require insemination when staff are not working.

Administration of minute quantities of 17beta-estradiol on the nasal area terminates early pregnancy in inseminated female mice.

It is well established that chemical emissions of novel males disrupt intrauterine implantation of fertilized ova in inseminated female mice, but the specific nature of these chemicals is not known. Given that novel males excrete androgens and estrogens in their urine and feces, the current experiments were designed to determine whether nasal application of these steroids could disrupt pregnancy. Nasal application of testosterone propionate to females during early pregnancy had no impact on gestation. However, nasal application of 17beta-estradiol terminated all pregnancies in females at all doses greater than or equal to approximately 1 microg/day. Nasal application of 17beta-estradiol benzoate similarly terminated all pregnancies in females at very low doses. In subcutaneous administration, 17beta-estradiol is also the most potent steroid in disrupting pregnancy compared to other estrogens and androgens. These data suggest the possibility that males' emission of estrogens is among factors mediating the Bruce effect.

Microtubule organization and chromatin configurations in hamster oocytes during fertilization and parthenogenetic activation, and after insemination with human sperm.

The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.

Absence of sperm from the seminal receptacle of female Onchocerca volvulus following multiple doses of ivermectin.

It is already known that multiple doses of ivermectin have a profound effect on embryonic development in Onchocerca volvulus and that this appears to operate mainly at the single cell stage. To investigate this further, we examined adult female O. volvulus originating from patients treated either with placebo or one, four or five doses of ivermectin. The reproductive organs were dissected out of the worm and examined for evidence of oogenesis and for the quantity of sperm and oocytes in the seminal receptacles. A single dose of ivermectin had no measurable effect on oogenesis or on the amount of sperm and oocytes compared to placebo. However after multiple doses of ivermectin a significantly lower proportion of seminal receptacles contained sperm and there was a significantly higher proportion of ovaries with impaired oogenesis compared to placebo. It is concluded that the reduction in the number of multicellular embryonic stages from worms exposed to multiple doses of ivermectin is due, at least in part, to a major reduction in the effective insemination of female worms and to a minor impairment of oogenesis.

Inhibitory effect of chlorpromazine on rat reproduction: a two week administration test before mating.

The effect of chlorpromazine (CPZ) administration before mating on reproduction was studied using male and female Sprague Dawley strain rats, and was evaluated over three generations. CPZ in dose of 12.5, 25, 50 and 100 mg/kg was orally administered to both male and female rats (F0 generation) every day for 2 weeks before mating with the following results: 1) The incidence of insemination and pregnancy of the F0 generation rats decreased but did not decrease in the F1 generation. While no influence of CPZ on the number of myometrial glands, on the length of pregnancy or on delivery was noted in either the F0 or F1 females, the number of surviving fetuses and newborns decreased in a dose-dependent fashion. 2) The body weight of rats in the F0 and F1 generations decreased depending on the dosage of CPZ, but the body weight of the F2 generation increased. 3) The wet weight of the major organs of the male and female rats in the F0 generation was affected by CPZ when administered at 100 mg/kg. The weight of the adrenal, pituitary and prostate glands in males, the adrenal gland and ovary in females increased, whereas the weight of the pituitary and uterus in females decreased. These results suggest that administering CPZ to the F0 rats before mating exerts an influence on the reproduction in both the F0 and F1 generations.

Inhibitory effect of chlorpromazine on rat reproduction: a test of administration for nine weeks before breeding.

The effect of chlorpromazine (CPZ) administration prior to insemination on reproduction was studied using male and female Sprague-Dawley strain rats evaluated over three generations. CPZ in doses of 12.5, 25, 50, and 100 mg/kg was administered to both male and female rats orally every day for 9 weeks before mating with the following results: 1) The duration of the estrous cycle was prolonged in the F0 females. 2) The incidence of insemination and pregnancy of the F0 males and females as well as the number of surviving fetuses and newborns showed a tendency to decrease. 3) The body weight of the rats in F0 and F1 generations decreased depending on the dose of CPZ, but the body weight of the F2 generation increased. 4) The wet weight of the major organs of the rats in F0 and F1 generations was affected by administration of CPZ at 100 mg/kg. The weights of the liver, kidney, adrenal, pituitary, testis, and prostate glands in males and the kidney, adrenal gland, and ovary in females were increased, whereas the weights of the pituitary and the uterus were decreased in females. These results suggest that premating administration of CPZ to the parent rats exerts influence on the reproduction of the F0 and F1 generations.