Molecular phylogeny and evolution of internal fertilization in South American seasonal cynopoeciline killifishes.

Internal fertilization is a widespread mode of reproduction in chondrichthyans and tetrapods, but uncommon in actinopterygian fishes. In killifishes of the suborder Aplocheiloidei, internal fertilization is restricted to two genera, Campellolebias and Cynopoecilus, both containing species adapted to life in seasonal pools of subtropical South America and exhibiting elaborated inseminating structures. Phylogenetic studies involving these genera are scarce and limited to morphological characters and fragments of mitochondrial DNA sequences available for a few taxa, providing incongruent results and thus impeding hypotheses on the evolution of insemination and related morphological traits. We analyzed three nuclear loci (GLYT1, ENC1, Rho) for 13 aplocheiloid taxa obtaining the first well-supported phylogeny for cynopoecilines, thus providing a significant background to interpret evolutionary changes within the group. Like in killifishes of the suborder Cyprinodontoidei, the evolution of internal fertilization in aplocheiloids is associated with deep changes in the structure of male anal fin. The phylogenetic analyses indicate that internal fertilization corresponds to a single evolutionary event during the evolution of aplocheiloid killifishes. The analyses also indicate that male specialized muscle characters, comprising a muscular ejaculatory pump in the urogenital region and hypertrophied inclinatores and depressores anales, arose in the ancestor of the clade comprising Campellolebias and Cynopoecilus. On the other hand, anal fin specialized structures including the male inseminating tube of Campellolebias and the male inseminating fan of Cynopoecilus evolved independently in each genus.

Estrus traits derived from activity measurements are heritable and closely related to the time from calving to first insemination.

The aim of this study was to estimate genetic parameters for estrus-related traits that could improve selection for increased fertility due to improved ability of the cow to return to cycling and go into heat after calving. We compared the time from calving to first insemination (CFI) to 3 physical activity traits: the interval from calving to first high activity (CFHA), estrus duration (ED), and estrus strength (ES). We calculated CFI based on data from commercial Holstein herds that included the insemination dates for 11,363 cows. The CFHA, ED, and ES traits were derived from electronic activity tags for 3,533 Holstein cows. Estimates of heritability were 0.07 for CFI, 0.16 for CFHA, 0.02 for ED, and 0.05 for ES. We found a strong genetic correlation between CFI and CFHA (0.96). Genetic correlations between ED and CFI and CFHA were -0.37 and -0.68, respectively. Genetic correlations between ES and CFI and CFHA were -0.50 and -0.58, respectively. The heritability of CFHA and its strong genetic correlation with CFI suggest that including CFHA in the genetic evaluation of female cow fertility could improve the effectiveness of selection, because CFHA reflects the ability to return to cyclicity and go into heat after calving.

Evolution of female multiple mating: A quantitative model of the "sexually selected sperm" hypothesis.

Explaining the evolution and maintenance of polyandry remains a key challenge in evolutionary ecology. One appealing explanation is the sexually selected sperm (SSS) hypothesis, which proposes that polyandry evolves due to indirect selection stemming from positive genetic covariance with male fertilization efficiency, and hence with a male's success in postcopulatory competition for paternity. However, the SSS hypothesis relies on verbal analogy with "sexy-son" models explaining coevolution of female preferences for male displays, and explicit models that validate the basic SSS principle are surprisingly lacking. We developed analogous genetically explicit individual-based models describing the SSS and "sexy-son" processes. We show that the analogy between the two is only partly valid, such that the genetic correlation arising between polyandry and fertilization efficiency is generally smaller than that arising between preference and display, resulting in less reliable coevolution. Importantly, indirect selection was too weak to cause polyandry to evolve in the presence of negative direct selection. Negatively biased mutations on fertilization efficiency did not generally rescue runaway evolution of polyandry unless realized fertilization was highly skewed toward a single male, and coevolution was even weaker given random mating order effects on fertilization. Our models suggest that the SSS process is, on its own, unlikely to generally explain the evolution of polyandry.

Análisis de la dinámica de la fragmentación de ADN en espermatozoides para la optimización de la Inseminación Artificial Intrauterina / Dynamic analysis of sperm DNA fragmentation for optimizing intrauterine artificial insemination

Introducción y Objetivos: De forma normal existe un porcentaje de espermatozoides con ADN fragmentado en el eyaculado. Sin embargo, la manipulación de las muestras de semen para R.A. puede aumentar el grado basal de fragmentación. Aunque los mecanismos de procesamiento del semen que pueden afectar a la fragmentación no son todavía conocidos, el objetivo es analizar la influencia del swim-up en la dinámica de fragmentación del ADN espermático a lo largo del tiempo en las mismas condiciones en las que se procesa el semen para IAC, y evaluar si la fragmentación del ADN espermático tras swim-up, así como su evolución en el tiempo puede predecir la posibilidad de gestación en IAC. Material y métodos: Las muestras de semen utilizadas se han obtenido de 25 parejas en tratamiento de IAC el mismo día de la Inseminación. La fragmentación del ADN se midió con el Kit Halosperm (SCD). Se empleó la T de Student para la comparación estática de medias entre ambos grupos y tests de regresión lineal, exponencial y logarítmica para el estudio dinámico. Resultados: En todo momento, dentro del intervalo estudiado, el índice de fragmentación del semen capacitado se mantiene significativamente inferior al del semen fresco. Conclusiones: Los resultados apoyan la idea de que la centrifugación que conlleva el swim-up no induce daño adicional en el ADN espermático y que es recomendable utilizar las muestras de semen post swimup para inseminación en un intervalo de una hora (AU)

Introduction: A certain level of damaged DNA is present in mature sperm cells after a natural ejaculation. Additionally, sperm management for artificial reproduction techniques (ART) may increase the basal rate of DNA damage. Unfortunately, the mechanisms and processes involved in Sperm DNA Fragmentation (SDF) are not fully illuminated. The main aim of this investigation was to analyse the influence of swim-up on the evolution of sperm DNA fragmentation values over time, emulating the experiment within the same set-up of sperm handling as used for insemination and to evaluate whether the degree of sperm DNA fragmentation as well the evolution over time can predict IUI outcome. Material and methods: Semen samples were obtained from couples undergoing IUI (only one cycle per couple was included) for infertility treatment at the Clinica Tambre in Madrid, Spain. A total of 25 males provided 25 samples that were analysed. SDF fragmentation was assessed using the sperm chromatin dispersion (SCD) test using the Halosperm kit (Halotech DNA, Madrid). Statistical analysis was performed using T-Student and regression analysis . Results: Mean values of DNA fragmentation in the spermatozoa prepared by swim-up are lower than before selection in all times studied. Conclusion: The results of this study support the notion that the swim-up technique, as practised in our laboratory, does no induce DNA damage to human spermatozoa, as assessed by SCD assay. We therefore propose that sperm samples be used before an hour after processing in intrauterine insemination (AU)

Thin and fat cows, and the nonlinear genetic relationship between body condition score and fertility.

Thin and fat cows are often credited for low fertility, but body condition score (BCS) has been traditionally treated as a linear trait when genetic correlations with reproductive performance have been estimated. The aims of this study were to assess genetic parameters for fertility, production, and body condition traits in the Brown Swiss population reared in the Alps (Bolzano-Bozen Province, Italy), and to investigate the possible nonlinearity among BCS and other traits by analyzing fat and thin cows. Records of BCS measured on a 5-point scale were preadjusted for year-season and days in milk at scoring, and were considered positive (1) for fat cows if they exceeded the value of 1 residual standard deviation or null (0) otherwise, whereas positive values for thin cows were imputed to records below -1 residual standard deviation. Fertility indicators measured on first- and second-parity cows were interval from parturition to first service, interval from first service to conception, interval from parturition to conception, number of inseminations to conception, conception at first service, and nonreturn rate at 56 d after first service. Production traits were peak milk yield, lactation milk yield, and lactation length. Data were from 1,413 herds and included 16,324 records of BCS, fertility, and production for first-parity, and 10,086 fertility records for second-parity cows. Animals calved from 2002 to 2007 and were progeny of 420 artificial insemination bulls. Genetic parameters for the aforementioned traits were obtained under univariate and bivariate threshold and censored linear sire models implemented in a Bayesian framework. Posterior means of heritabilities for BCS, fat cows, and thin cows were 0.141, 0.122, and 0.115, respectively. Genetic correlations of body condition traits with contemporary production were moderate to high and were between -0.556 and 0.623. Body condition score was moderately related to fertility in first (-0.280 to 0.497) and second (-0.392 to 0.248) lactation. The fat cow trait was scarcely related to fertility, particularly in first-parity cows (-0.203 to 0.281). Finally, the genetic relationships between thin cows and fertility were higher than those between BCS and fertility, both in first (-0.456 to 0.431) and second (-0.335 to 0.524) lactation. Body condition score can be considered a predictor of fertility, and it could be included in evaluation either as linear measure or as thin cow. In the second case, the genetic relationship with fertility was stronger, exacerbating the poorest body condition and considering the possible nonlinearity between fertility and energy reserves of the cow.

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.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference.

Mosquitoes are the culprits of some of the most important vector borne diseases. A species' potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female's physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti. We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila, clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti.

Circadian clock of Aedes aegypti: effects of blood-feeding, insemination and RNA interference

Mosquitoes are the culprits of some of the most important vector borne diseases. A species’ potential as a vector is directly dependent on their pattern of behaviour, which is known to change according to the female’s physiological status such as whether the female is virgin/mated and unfed/blood-fed. However, the molecular mechanism triggered by and/or responsible for such modulations in behaviour is poorly understood. Clock genes are known to be responsible for the control of circadian behaviour in several species. Here we investigate the impact mating and blood-feeding have upon the expression of these genes in the mosquito Aedes aegypti . We show that blood intake, but not insemination, is responsible for the down-regulation of clock genes. Using RNA interference, we observe a slight reduction in the evening activity peak in the fourth day after dstim injection. These data suggest that, as in Drosophila , clock gene expression, circadian behaviour and environmental light regimens are interconnected in Ae. aegypti .

Influencia de la calidad embrionaria y seminal en el síndrome del embrión evanescente / Embryo and seminal quality and its influence on the vanishing embryo syndrome

Introducción. La evanescencia embrionaria consiste en la pérdida espontánea de uno o más embriones durante una gestación múltiple sin que el embarazo se interrumpa. Nuestros objetivos fueron: determinar la tasa de evanescencia en mujeres con embarazos múltiples concebidos por microinseminación espermática (ICSI: Intracytoplasmic Sperm Inyection), analizar la evolución de estos embarazos y relacionar la presencia de este fenómeno con posibles causas cuantificables en laboratorio como la calidad embrionaria y la calidad seminal, así como con la edad de la mujer. Material y métodos. Estudio retrospectivo de 145 embarazos múltiples de ciclos de ICSI realizados entre el 1/01/2004 y el 31/12/2008, las pacientes fueron divididas en dos grupos (112 no evanescentes y 33 con saco/s evanescentes). Se realizó seguimiento ecográfico de las embarazadas y los parámetros se recogieron de la base de datos del centro. Posteriormente realizamos un estudio de casos-controles histórico para analizar los resultados neonatales de los embarazos simples procedentes de evanescentes, comparando con un grupo control de nacimientos procedentes de embarazos simples obtenido mediante técnicas de reproducción asistida (TRA) en nuestro centro que fueron registrados en el mismo período de estudio (n=163). Resultados. La tasa de evanescencia se situó en un 22,7%, siendo de un 17,5% en embarazos gemelares y de un 50% en embarazos triples. La evanescencia en los embarazos múltiples fue significativamente menor en el grupo con dos sacos gestacionales que en el grupo con tres o más sacos gestacionales (p<0,001). Los resultados perinatales de los nacidos vivos en embarazos con evanescencia fueron similares a los obtenidos en nacidos procedentes de embarazos inicialmente simples. La tasa de embriones de buena calidad fue significativamente mayor en el grupo sin evanescentes (p<0,001). No se observaron diferencias en términos de edad y de calidad seminal entre los grupos de estudio. Conclusiones. La tasa de evanescencia embrionaria aumenta significativamente en los embarazos de más de dos sacos gestacionales, existiendo una relación entre este fenómeno y la calidad de la transferencia embrionaria (AU)

Background. The vanishing embryo phenomenon is defined as the spontaneous loss of one or more embryos in a multiple pregnancy without its interruption. The aim of this study was to determine the rate of vanishing embryo syndrome in women with multiple pregnancies conceived by Intracytoplasmic Sperm Injection (ICSI), to analyse the evolution of these pregnancies, and to find a relationship with possible causes, such as embryo and seminal quality. Material and methods. A retrospective analysis was performed of 145 multiple pregnancies conceived by ICSI, from January 2004 to December 2008. They were divided in two groups (112 without vanishing syndrome and 33 with any embryo suffering from vanishing syndrome). We then carried out a case-control study in order to compare the neonatal results of single pregnancies with vanishing embryo syndrome. We carried out transvaginal ultrasound monitoring until 12weeks of gestation. The data were obtained from the centre data base. Results. Vanishing embryo syndrome was observed in 22.7% of our patients, with 17.5% of cases being observed in twin pregnancies, and 50% in triple pregnancies. The vanishing embryo rate in multiple pregnancies was significantly lower in the group with two gestational sacs than in the group with three or more gestational sacs (P<.001). The rate of good quality embryos was significantly higher in the group without vanishing embryos (P<.001). No differences were observed in terms of age and seminal quality among study groups. Perinatal outcomes of live births were similar in both groups. Conclusion. Vanishing embryos rate seems to increase in pregnancies with over two gestational sacs. We observed a relationship between this syndrome and embryo transfer quality (AU)

Udder health and female fertility traits are favourably correlated and support each other in multi-trait evaluations.

Genetic parameters were estimated for protein yield (PY), clinical mastitis (CM), somatic cell score, number of inseminations (NI) and days from calving to first insemination (CFI) in first-parity Swedish Red cows by series of tri-variate linear animal models. The heritability of PY was moderate (0.34 ± 0.004), and the heritabilities of the functional traits were all low (0.014 ± 0.001-0.14 ± 0.004). The genetic correlation between CM and CFI (0.38 ± 0.05) was stronger than the correlation between CM and NI (0.05 ± 0.06), perhaps because CM and CFI usually are observed in early lactation when the cow is likely to be in negative energy balance, whereas NI generally is recorded when the cow is not in negative energy balance any more. The genetic correlation between NI and CFI was very close to zero (-0.002 ± 0.05), indicating that these two fertility traits have different genetic backgrounds. All genetic correlations between PY and the functional traits were moderate and unfavourable, ranging from 0.22 ± 0.02 to 0.47 ± 0.03. In addition, the effect of including genetic and phenotypic correlations between the trait groups milk production, udder health and female fertility on the accuracy of the selection index was quantified for a heifer, a cow and a proven bull. The difference between the accuracy obtained by multi-trait and single-trait evaluations was largest for the cow (0.012) and small for the heifer and the bull (0.006 and 0.004) because the phenotype of the cow for one trait could assist in predicting the Mendelian sampling term for a correlated trait.

The effects of mating and instrumental insemination on queen honey bee flight behaviour and gene expression.

Mating is fundamental to most organisms, although the physiological and transcriptional changes associated with this process have been largely characterized only in Drosophila melanogaster. In this study, we use honey bees as a model system because their queens undergo massive and permanent physiological and behavioural changes following mating. Previous studies have identified changes associated with the transition from a virgin queen to a fully mated, egg-laying queen. Here, we further uncouple the mating process to examine the effects of natural mating vs. instrumental insemination and saline vs. semen insemination. We observed effects on flight behaviour, vitellogenin expression and significant overlap in transcriptional profiles between our study and analogous studies in D. melanogaster, suggesting that some post-mating mechanisms are conserved across insect orders.

The genetics of postmating, prezygotic reproductive isolation between Drosophila virilis and D. americana.

Many studies have demonstrated the rapid diversification of reproductive genes that function after mating but before fertilization. This process might lead to the evolution of postmating, prezygotic barriers between species. Here, I investigate the phenotypic and genetic basis of postmating, prezygotic isolation between two closely related species of Drosophila, Drosophila virilis and D. americana. I show that a strong barrier to interspecific fertilization results in a 99% reduction in progeny production. A genetic interaction among maternal and paternal alleles at only a few loci prevents the fertilization of D. virilis females by D. americana males. These loci are autosomal and isolation acts recessively; the fertilization incompatibility is caused by at least two loci in the maternal D. virilis parent in combination with at least three loci in the paternal D. americana parent. These findings, together with results from classical experiments, suggest that male-female coevolution within D. americana may have driven postmating, prezygotic isolation between species.

Genetic analysis of days from calving to first insemination and days open in Danish Holsteins using different models and censoring scenarios.

The objectives of this study were to estimate genetic parameters and evaluate models for genetic evaluation of days from calving to first insemination (ICF) and days open (DO). Data including 509,512 first-parity records of Danish Holstein cows were analyzed using 5 alternative sire models that dealt with censored records in different ways: 1) a conventional linear model (LM) in which a penalty of 21 d was added to censored records; 2) a bivariate threshold-linear model (TLM), which included a threshold model for censoring status (0, 1) of the observations, and a linear model for ICF or DO without any penalty on censored records; 3) a right-censored linear model (CLM); 4) a Weibull proportional hazard model (SMW); and 5) a Cox proportional hazard model (SMC) constructed with piecewise constant baseline hazard function. The variance components for ICF and DO estimated from LM and TLM were similar, whereas CLM gave higher estimates of both additive genetic and residual components. Estimates of heritability from models LM, TLM, and CLM were very similar (0.102 to 0.108 for ICF, and 0.066 to 0.069 for DO). Heritabilities estimated using model SMW were 0.213 for ICF and 0.121 for DO in logarithmic scale. Using SMC, the estimates of heritability, defined as the log-hazard proportional factor for ICF and DO, were 0.013 and 0.009, respectively. Correlations between predicted transmitting ability from different models for sires with records from at least 20 daughters were far from unity, indicating that different models could lead to different rankings. The largest reranking was found between SMW and SMC, whereas negligible reranking was found among LM, TLM, and CLM. The 5 models were evaluated by comparing correlations between predicted transmitting ability from different data sets (the whole data set and 2 subsets, each containing half of the whole data set), for sires with records from at least 20 daughters, and chi(2) statistics based on predicted and observed daughter frequencies using a cross validation. The model comparisons showed that SMC had the best performance in predicting breeding values of the 2 traits. No significant difference was found among models LM, TLM, and CLM. The SMW model had a relatively poor performance, probably because the data are far from a Weibull distribution. The results from the present study suggest that SMC could be a good alternative for predicting breeding values of ICF and DO in the Danish Holstein population.

Success at first insemination in Australian Angus cattle: analysis of uncertain binary responses.

Field data from Australian Angus herds were used to investigate 2 methods of analyzing uncertain binary responses for success or failure at first insemination. A linear mixed model that included herd, year, and month of mating as fixed effects; unrelated service sire, additive animal, and residual as random effects; and linear and quadratic effects of age at mating as covariates was used to analyze binary data. An average gestation length (GL) derived from artificial insemination data was used to assign an insemination date to females mated to natural service sires. Females that deviated from this average GL led to uncertain binary responses. Two analyses were carried out: 1) a threshold model fitted to uncertain binary data, ignoring uncertainty (M1); and 2) a threshold model fitted to uncertain binary data, accounting for uncertainty via fuzzy logic classification (M2). There was practically no difference between point estimates obtained from M1 and M2 for service sire and herd variance; however, when uncertain binary data were analyzed ignoring uncertainty (M1), additive variance and heritability estimates were greater than with M2. Pearson correlations indicated that no major reranking would be expected for service sire effects and animal breeding values using M1 and M2. Given the results of the current study, a threshold model contemplating uncertainty is suggested for noisy binary data to avoid bias when estimating genetic parameters.

Relationship between profitability and type traits and derivation of economic values for reproduction and survival traits in Chianina beef cows.

The objectives of this study were 1) to propose a profit function for Italian Chianina beef cattle; 2) to derive economic values for some biological variables in beef cows, specifically, production expressed as the number of calves born alive per year (NACY), age at the insemination that resulted in the birth of the first calf (FI), and length of productive life (LPL); and 3) to investigate the relationship between the phenotypic profit function and type traits as early predictors of profitability in the Chianina beef cattle population. The average profit was 196 Euros/(cow.yr) for the length of productive life (LPL) and was obtained as the difference between the average income of 1,375 Euros/(cow.yr) for LPL and costs of 1,178 Euros/(cow.yr) of LPL. The mean LPL was equal to 5.97 yr, so the average total phenotypic profit per cow on a lifetime basis was 1,175 Euros. A normative approach was used to derive the economic weights for the biological variables. The most important trait was the number of calves born alive (+4.03.cow(-1).yr(-1) and +24.06 Euros/cow). An increase of 1 d in LPL was associated with an increase of +0.19 Euros/(cow.yr) and +1.65 Euros/cow on a lifetime basis. Increasing FI by 1 d decreased profit by 0.42 Euros/(cow.yr) and 2.51 Euros/cow. Phenotypic profit per cow had a heritability of 0.29. Heritabilities for eight muscularity traits ranged from 0.16 to 0.23, and for the seven body size traits between 0.21 and 0.30. The conformation trait final score can be used as an early predictor of profitability. The sale price of the animal and differences in the revenue and costs of offspring due to muscularity should be included in a future profit function.

Frequency of multiple inseminations in field-collected Anopheles gambiae females revealed by DNA analysis of transferred sperm.

We investigated the frequencies of single and multiple matings in field-collected female Anopheles gambiae by conducting microsatellite DNA analyses on the sperm contained within their spermatheca. Amplifcation by a polymerase chain reaction (PCR) at four loci allowed the detection of sperm extracts exhibiting more than two alleles per locus, thereby revealing the occurrence of multiple inseminations. Polyandry was found in six of 239 females examined, or 2.5% of the samples. Previous analyses of the molecular form of the sperm and female extracts using a PCR-based diagnostic procedure showed that two of these multiple inseminations involved cross-mating between two chromosomal/molecular forms of An. gambiae s.s. Thus polyandry occurred within-form in 1.7% of examined females while other multiple inseminations may be linked to processes of reproductive isolation between forms of An. gambiae.

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice.

Sertoli cells play a pivotal role in spermatogenesis through their interactions with germ cells. To set up a strategy for treating male infertility caused by Sertoli cell dysfunction, we developed a Sertoli cell gene transfer system by using an adenovirus vector, which maintained long-term transgene expression in the testes of infertile mice. Introduction of an adenovirus carrying the mouse Steel (Sl) gene into Sertoli cells restored partial spermatogenesis in infertile Steel/Steel(dickie) (Sl/Sl(d)) mutant mouse testes. Although these males remained infertile, round spermatids and spermatozoa from the testes produced normal fertile offspring after intracytoplasmic injection into oocytes. None of the offspring showed evidence of germ line transmission of adenoviral DNA. Thus, we demonstrate a successful treatment for infertility by using a gene therapy vector. Therefore, adenovirus-mediated gene delivery into Sertoli cells not only provides an efficient and convenient means for studying germ cell-Sertoli cell interactions through manipulation of the germ cell microenvironment in vivo, but also is a useful method to treat male infertility resulting from a Sertoli cell defect.