Effect of Sperm Morphology on Pregnancy Success via Intrauterine Insemination: A Systematic Review and Meta-Analysis.

PURPOSE: Men with abnormal sperm morphology are often counseled that natural conception and intrauterine insemination are ineffective, and in vitro fertilization is the only option. Our objective was to determine the effect of sperm morphology on the pregnancy success of intrauterine insemination. MATERIALS AND METHODS: We systematically searched for studies published prior to January 2017 that 1) reported ultrasound verified clinical pregnancies per intrauterine insemination cycle, 2) assessed sperm morphology using the Kruger strict criteria and 3) described morphology at the greater than 4% and 4% or less and/or the 1% or greater and less than 1% thresholds. In all studies mean female age was between 25 and 40 years and mean total motile sperm count was greater than 10 million. Estimates were pooled using random effects meta-analysis. RESULTS: Data were extracted from 20 observational studies involving a total of 41,018 cycles. When comparing men at the greater than 4% and 4% or less thresholds, the rate of ultrasound verified pregnancy per intrauterine insemination cycle was not statistically or clinically different (14.2% vs 12.1%, p = 0.06) and the risk difference was 3.0% (95% CI 1.4-4.6), indicating 3.0 additional pregnancies per 100 intrauterine insemination cycles. When comparing men at the 1% or greater and the less than 1% thresholds, there were no statistical or clinical differences in the rate of ultrasound verified pregnancy per cycle of intrauterine insemination (14.0% vs 13.9%, p = 0.97) or in the risk difference (1.6%, 95% CI -4.5-7.6). CONCLUSIONS: There appears to be no clinical difference in intrauterine insemination pregnancy success among men with normal and abnormal sperm morphology when accounting for total motile sperm count and female age. Abnormal sperm morphology alone should not exclude couples from attempting intrauterine insemination.

Molecular and physiological analysis of Anopheles funestus swarms in Nchelenge, Zambia.

BACKGROUND: Anopheles funestus has been recognized as a major malaria vector in Africa for over 100 years, but knowledge on many aspects of the biology of this species is still lacking. Anopheles funestus, as with most other anophelines, mate through swarming. A key event that is crucial for the An. funestus male to mate is genitalia rotation. This involves the 135° to 180° rotation of claspers, which are tipped with claws. This physical change then enables the male to grasp the female during copulation. The aim of this investigation was to molecularly characterize wild An. funestus swarms from Zambia and examine the degree of genitalia rotation within the swarm. METHODS: Anopheles funestus swarms were collected from Nchelenge, northern Zambia, during dusk periods in May 2016. All the adults from the swarm were analysed morphologically and identified to species level using a multiplex PCR assay. Anopheles funestus s.s. specimens were molecularly characterized by restriction fragment length polymorphism type and Clade type assays. The different stages of genitalia rotation were examined in the adult males. RESULTS: A total of six swarms were observed during the study period and between 6 and 26 mosquitoes were caught from each swarm. Species analysis revealed that 90% of the males from the swarms were An. funestus s.s. MW-type, with 84% belonging to clade I compared to 14% clade II and 2% failed to amplify. Very few specimens (3.4%) were identified as Anopheles gambiae s.s. Eighty percent of the males from the swarm had complete genitalia rotation. CONCLUSIONS: This is the first time that An. funestus swarms have been molecularly identified to species level. Anopheles funestus swarms appear to be species-specific with no evidence of clade-type differentiation within these swarms. The An. funestus swarms consist mainly of males with fully rotated genitalia, which strongly suggests that swarming behaviour is triggered primarily when males have matured.

Comparative studies of stenogamous behaviour in the mosquito Culex pipiens complex.

Understanding the processes of reproductive behaviour in mosquitoes is crucial for improving mating competitiveness and mating specificity for sterile insect release programmes. The Culex pipiens (Linneaus) (Diptera: Culicidae) forms pipiens and molestus (Forskål), two biotypes of the Cx. pipiens complex, are vectors for West Nile virus, St Louis encephalitis virus and lymphatic filariases. Hybridization of these biotypes is known to occur in nature, although form pipiens mates above ground in large spaces (eurygamy) and form molestus preferentially mates in small spaces (stenogamy) such as sewage tunnels. Hybridization may allow gene flow of biotype-specific characteristics that are crucial in the disease transmission cycle. The present study examined and compared mating behaviours, insemination rates, fecundity and fertility in parental and F1 hybrids between Cx. pipiens f. pipiens and Cx. pipiens f. molestus in conditions of stenogamy. Unique mating behaviour sequences were identified in Cx. pipiens f. molestus, including tapping, mounting, co-flying and copulation. Despite the considerably high insemination rates in hybrid crosses, fertility and fecundity rates were varied. This observation could suggest reproductive isolation in the hybrid zone. The study also documents a failure of heterospecific males to produce fertile eggs in Cx. pipiens f. pipiens females, which may be attributable to gametic incompatibilities and may represent an additional barrier to gene exchange.

Fertility traits of Holstein, Brown Swiss, Simmental, and Alpine Grey cows are differently affected by herd productivity and milk yield of individual cows.

Milk yield has a strong effect on fertility, but it may vary across different herds and individual cows. Therefore, the aim of this study was to assess the effects of breed and its interaction with level of milk production at the herd level (Herd-L) and at a cow-within-herd level (Cow-L) on fertility traits in dairy cattle. Data were gathered from Holstein (n = 17,688), Brown Swiss (n = 32,697), Simmental (n = 27,791), and Alpine Grey (n = 13,689) cows in northeastern Italy. The analysis was based on records from the first 3 lactations in the years 2011 to 2014. A mixed model was fitted to establish milk production levels of the various herds (Herd-L) and individual cows (Cow-L) using milk as a response variable. The interval fertility traits were interval from calving to first service, interval from first service to conception, and number of days open. The success traits were nonreturn rate at 56 d after first service, pregnancy rate at first service, and the number of inseminations. The interval from calving to first service, interval from first service to conception, and number of days open were analyzed using a Cox's proportional hazards model. The nonreturn rate at 56 d after first service, pregnancy rate at first service, and the number of inseminations were analyzed using logistic regression. There was a strong interaction between breed and productivity class at both Herd-L and Cow-L on all traits. The effects of herd and cow productivity differed from each other and differed among breeds. The dual-purpose Simmental and Alpine Grey breeds had better fertility than the specialized Holstein and Brown Swiss dairy cows; this difference is only partly attributable to different milk yields. Greater herd productivity can result in higher fertility in cows, whereas higher milk yield of individual cows within a herd results in lower fertility. These effects at both Herd-L and Cow-L are curvilinear and are stronger in dual-purpose breeds, which was more evident from low to intermediate milk yield levels than from central to high productivity classes. Disentangling the effects of milk productivity on fertility at Herd-L and Cow-L and taking the nonlinearity of response into account could lead to better modeling of populations within breed. It could also help with management-for example, in precision dairy farming of dairy and dual-purpose cattle. Moreover, assessing the fertility of various breeds and their different responses to herd and individual productivity levels could be useful in devising more profitable crossbreeding programs in different dairy systems.

Complexity, adaptations and variations in the secondary insemination system of female Dermanyssina mites (Acari: Anactinothrichida: Gamasida): the case of Afrocypholaelaps africana.

Gamasine mites, mainly of the taxon Dermanyssina, possess a secondarily evolved insemination system (sperm access system), of which there are two, generally recognized, structurally different types, the laelapid- and the phytoseiid-type. The ultrastructure of the female sperm access system in Afrocypholaelaps africana is described. It consists of paired insemination pores, opening between the bases of legs three and four, and paired cuticle-lined tubules that converge into a large, sack-like spermatheca, remarkably cuticle-lined as well. The entire spermatheca and part of the tubules are embedded in a peculiar syncytial tissue where numerous sperm cells are present. The general organization of this insemination system is of the laelapid-type. However, it presents striking structural differences, compared with the systems described in Varroa destructor and Hattena cometis, the other gamasine mites having a laelapid-type system studied ultrastructurally until now. The functional morphology, complexity and variations of the sperm access system in Dermanyssina are discussed and correlated with the evolutionary biology of the group.

Machine learning algorithms for the prediction of conception success to a given insemination in lactating dairy cows.

The ability to accurately predict the conception outcome for a future mating would be of considerable benefit for producers in deciding what mating plan (i.e., expensive semen or less expensive semen) to implement for a given cow. The objective of the present study was to use herd- and cow-level factors to predict the likelihood of conception success to a given insemination (i.e., conception outcome not including embryo loss); of particular interest in the present study was the usefulness of milk mid-infrared (MIR) spectral data in augmenting the accuracy of the prediction model. A total of 4,341 insemination records with conception outcome information from 2,874 lactations on 1,789 cows from 7 research herds for the years 2009 to 2014 were available. The data set was separated into a calibration data set and a validation data set using either of 2 approaches: (1) the calibration data set contained records from all 7 farms for the years 2009 to 2011, inclusive, and the validation data set included data from the 7 farms for the years 2012 to 2014, inclusive, or (2) the calibration data set contained records from 5 farms for all 6 yr and the validation data set contained information from the other 2 farms for all 6 yr. The prediction models were developed with 8 different machine learning algorithms in the calibration data set using standard 10-times 10-fold cross-validation and also by evaluating in the validation data set. The area under curve (AUC) for the receiver operating curve varied from 0.487 to 0.675 across the different algorithms and scenarios investigated. Logistic regression was generally the best-performing algorithm. The AUC was generally inferior for the external validation data sets compared with the calibration data sets. The inclusion of milk MIR in the prediction model generally did not improve the accuracy of prediction. Despite the fair AUC for predicting conception outcome under the different scenarios investigated, the model provided a reasonable prediction of the likelihood of conception success when the high predicted probability instances were considered; a conception rate of 85% was evident in the top 10% of inseminations ranked on predicted probability of conception success in the validation data set.

Free fatty acid levels in fluid of dominant follicles at the preferred insemination time in dairy cows are not affected by early postpartum fatty acid stress.

The fertility of high-yielding dairy cows has declined during the last 3 decades, in association with a more profound negative energy balance (NEB) during the early weeks postpartum. One feature of this NEB is a marked elevation in circulating free fatty acid (FFA) concentrations. During the early postpartum period (≤ d 42), circulatory FFA levels were measured weekly, and progesterone concentrations and the diameter of the dominant follicles were determined thrice weekly. Retrospectively, cows that ovulated within 35 d postpartum were grouped as "normal ovulating" cows (n = 5), and the others were grouped as "delayed ovulating" cows (n = 5). In both groups, high total FFA levels (>500 µM) were evident immediately postpartum. Interestingly, cows with delayed ovulation had higher plasma FFA concentrations in the first weeks postpartum compared with normal ovulating cows. In both cow groups, FFA decreased to control levels of non-NEB cows within 3 wk postpartum. The FFA compositions and concentrations in fluids from the dominant follicles of postpartum cows were not different between the normal and delayed ovulating cows when measured at potential insemination points: d 55, 80, and 105 postpartum. Interestingly, the concentration of monounsaturated oleic acid was higher and that of saturated stearic acid lower in follicular fluids of both groups compared with that in blood. The level of FFA in follicular fluid was correlated with the ratio of 17ß-estradiol (E2) to progesterone (P4) in follicular fluid, with a relatively high level of unsaturated FFA in follicles with a low E2:P4 ratio. Taken together, these results indicate that a more severe NEB early postpartum is related to a delay in the first postpartum ovulation and does not affect FFA composition in follicular fluid at the preferred insemination time. The high FFA level in dominant follicles with a low E2:P4 ratio may be due to a different FFA metabolism in these follicles. The diagnostic value of this observation for selective screening of dominant follicles needs further investigation.

Male accessory gland substances from Aedes albopictus affect the locomotor activity of Aedes aegypti females.

Dengue is one of the world's most important mosquito-borne diseases and is usually transmitted by one of two vector species: Aedes aegypti or Aedes albopictus . These two diurnal mosquitoes are frequently found coexisting in similar habitats, enabling interactions between adults, such as cross-mating. The objective of this study was to assess cross-mating between Ae. aegypti females and Ae. albopictus males under artificial conditions and evaluate the locomotor activity of Ae. aegypti virgin females injected with male accessory gland (MAG) homogenates to infer the physiological and behavioural responses to interspecific mating. After seven days of exposure, 3.3-16% of Ae. aegypti females mated with Ae. albopictus males. Virgin Ae. aegypti females injected with conspecific and heterospecific MAGs showed a general decrease in locomotor activity compared to controls and were refractory to mating with conspecific males. The reduction in diurnal locomotor activity induced by injections of conspecific or heterospecific MAGs is consistent with regulation of female reproductive activities by male substances, which are capable of sterilising female Ae. aegypti through satyrisation by Ae. albopictus.

Endometrial response of beef heifers on day 7 following insemination to supraphysiological concentrations of progesterone associated with superovulation.

Ovarian stimulation is a routine procedure in assisted reproduction to stimulate the growth of multiple follicles in naturally single-ovulating species including cattle and humans. The aim of this study was to analyze the changes induced in the endometrial transcriptome associated with superovulation in cattle and place these observations in the context of our previous data on changes in the endometrial transcriptome associated with elevated progesterone (P4) concentrations within the physiological range and those changes induced in the embryo due to superovulation. Mean serum P4 concentrations were significantly higher from day 4 to day 7 in superovulated compared with unstimulated control heifers (P < 0.05). Between-group analysis revealed a clear separation in the overall transcriptional profile of endometria from unstimulated control heifers (n = 5) compared with superovulated heifers (n = 5). This was reflected in the number of differentially expressed genes (DEGs) identified between the two groups with 795 up- and 440 downregulated in superovulated endometria. Ten times more genes were altered by superovulation (n = 1,234) compared with the number altered due to elevated P4 within physiological ranges by insertion of a P4-releasing intravaginal device (n = 124) with only 22 DEGs common to both models of P4 manipulation. Fewer genes were affected by superovulation in the embryo compared with the endometrium, (443 vs. 1,234 DEGs, respectively), and the manner in which genes were altered was different with 64.5% of genes up- and 35.5% of genes downregulated in the endometrium, compared with the 98.9% of DEGs upregulated in the embryo. In conclusion, superovulation induces significant changes in the transcriptome of the endometrium which are distinct from those in the embryo.

Morphological and morphometrical assessment of spermathecae of Aedes aegypti females.

The vectorial capacity of Aedes aegypti is directly influenced by its high reproductive output. Nevertheless, females are restricted to a single mating event, sufficient to acquire enough sperm to fertilize a lifetime supply of eggs. How Ae. aegypti is able to maintain viable spermatozoa remains a mystery. Male spermatozoa are stored within either of two spermathecae that in Ae. aegypti consist of one large and two smaller organs each. In addition, each organ is divided into reservoir, duct and glandular portions. Many aspects of the morphology of the spermatheca in virgin and inseminated Ae. aegypti were investigated here using a combination of light, confocal, electron and scanning microscopes, as well as histochemistry. The abundance of mitochondria and microvilli in spermathecal gland cells is suggestive of a secretory role and results obtained from periodic acid Schiff assays of cell apexes and lumens indicate that gland cells produce and secrete neutral polysaccharides probably related to maintenance of spermatozoa. These new data contribute to our understanding of gamete maintenance in the spermathecae of Ae. aegypti and to an improved general understanding of mosquito reproductive biology.

Multiple deleterious effects of experimentally aged sperm in a monogamous bird.

Sperm aging is known to be detrimental to reproductive performance. However, this apparently general phenomenon has seldom been studied in an evolutionary context. The negative impact of sperm aging on parental fitness should constitute a strong selective pressure for adaptations to avoid its effects. We studied the impact of sperm aging on black-legged kittiwakes (Rissa tridactyla), a monogamous seabird. Kittiwakes comprise a model system because (i) of evidence that females eject their mates' sperm to prevent fertilization by sperm that would be old and degraded by the time of fertilization and result in reduced reproductive performance and (ii) the lack of extra-pair fertilization in this species makes cryptic female choice an unlikely explanation of postcopulatory sperm ejection by females. We experimentally manipulated the age of the sperm fertilizing kittiwake eggs by fitting males with anti-insemination rings for variable periods of time preceding egg-laying. We found evidence that sperm aging negatively affected four sequential stages of reproduction: fertilization potential, rate of embryonic development, embryonic mortality, and chick condition at hatching. These results may be produced by a continuum of a single process of sperm aging that differentially affects various aspects of development, depending on the degree of damage incurred to the spermatozoa. The marked impact of sperm age on female fitness may thus drive postcopulatory sperm ejection by females. These results provide experimental evidence of deleterious effects of sperm aging on a nondomestic vertebrate, underlining its taxonomic generality and its potential to select for a wide array of adaptations.

In vivo measurement of pH and CO2 levels in the uterus of sows through the estrous cycle and after insemination.

The pH-CO2-HCO3- system is a ubiquitous biological regulator with important functional implications for reproduction. Knowledge of the physiological values of its components is relevant for reproductive biology and the optimization of Assisted Reproductive Technologies (ARTs). However, in situ measurements of these parameters in the uterus are scarce or null. This study describes a non-invasive method for in situ time-lapse recording of pH and CO2 within the uterus of non-anesthetized sows. Animals were at three different reproductive conditions, estrous with no insemination and two hours after insemination, and diestrous. From pH and CO2 data, HCO3- concentration was estimated. The non-invasive approach to the porcine uterus with novel optical probes allowed the obtaining of in situ physiological values of pH, CO2, and HCO3-. Variable oscillatory patterns of pH, CO2 and HCO3- were found independently of the estrous condition. Insemination did not immediately change the levels of uterine pH, CO2 (%) and HCO3- concentration, but all the values were affected by the estrous cycle decreasing significantly at diestrous condition. This study contributes to a better understanding of the in vivo regulation of the pH-CO2-HCO3- system in the uterus and may help to optimize the protocols of sperm treatment for in vitro fertilization.

Microscale laser surgery reveals adaptive function of male intromittent genitalia.

The leading hypothesis for the evolution of male genital complexity proposes that genital traits evolve in response to post-insemination sexual selection; that is, via cryptic female choice or sperm competition. Here, we describe a laser ablation technique for high-precision manipulation of microscale body parts of insects, and employ it to discern the adaptive function of a rapidly evolving and taxonomically important genital trait: the intromittent claw-like genital spines of male Drosophila bipectinata Duda. We demonstrate experimentally and unambiguously that the genital spines of this species function to mechanically couple the genitalia together. The excision of the spines by laser ablation sharply reduced the ability of males both to copulate and to compete against rival males for mates. When spineless males did succeed to copulate, their insemination success and fertilization rate were not statistically different from controls, at odds with the post-insemination sexual selection hypothesis of genital function and evolution. The results provide direct experimental support for the hypothesis that genital traits evolve in response to sexual selection occurring prior to insemination.

Sophisticated sperm allocation in male fowl.

When a female is sexually promiscuous, the ejaculates of different males compete for the fertilization of her eggs; the more sperm a male inseminates into a female, the more likely he is to fertilize her eggs. Because sperm production is limited and costly, theory predicts that males will strategically allocate sperm (1) according to female promiscuity, (2) saving some for copulations with new females, and (3) to females producing more and/or better offspring. Whether males allocate sperm in all of these ways is not known, particularly in birds where the collection of natural ejaculates only recently became possible. Here we demonstrate male sperm allocation of unprecedented sophistication in the fowl Gallus gallus. Males show status-dependent sperm investment in females according to the level of female promiscuity; they progressively reduce sperm investment in a particular female but, on encountering a new female, instantaneously increase their sperm investment; and they preferentially allocate sperm to females with large sexual ornaments signalling superior maternal investment. Our results indicate that female promiscuity leads to the evolution of sophisticated male sexual behaviour.

Male but not female olfaction is crucial for intermolt mating in European lobsters (Homarus gammarus L.).

Chemical signals are common in most crustacean social interactions and are often perceived via chemosensory (olfactory) organs on the first antenna. Intermolt courtship behaviors and mating were investigated in size-matched pairs of intermolt European lobsters (Homarus gammarus) where the olfactory receptors of either the male or the female were lesioned with distilled water (olfactory ablation) or seawater (control). Matings or advanced male courtship behaviors (mounting and turning) were common in seawater controls and olfactory-ablated females. In contrast, when male olfaction was ablated with distilled water, there was not a single mating, and the only male courtship behaviors seen were a few very brief and unsuccessful mounting attempts. Individual females mated up to 5 times with different males, showing that previously inseminated females were still attractive to males. Thus, male but not female olfaction is crucial for intermolt mating in H. gammarus, indicating the presence of a female sex pheromone during the entire female molt cycle, not only at the time of molting. Female sex discrimination may be based on other cues from the male in combination with typical male behaviors.

Sugar deprivation reduces insemination of Anopheles gambiae (Diptera: Culicidae), despite daily recruitment of adults, and predicts decline in model populations.

Our research tests the hypothesis that the inability to sugar-feed reduces the insemination rate in mosquito populations. To test this, we measured the effects of sugar availability on cumulative insemination performance of male Anopheles gambiae Giles s.s. (Diptera: Culicidae) during 10-d periods of continual emergence of equal numbers of both sexes, and we evaluated the implications at the population level with a matrix population model. On each day of each of four replicates, 20 newly emerged mosquitoes of each sex were recruited into the populations within two mesocosms, large walk-in enclosures with simulated natural conditions. Each mesocosm contained a cage to replicate the experiment on a small scale. Scented sucrose was absent or present (control). A human host was available nightly as a bloodmeal source in both mesocosms. Sugar availability and enclosure size significantly influenced female insemination. In the mesocosms, with sugar 49.7% of the females were inseminated, compared with 10.9% of the females without sugar. In the small cages, the insemination rates were 76.0 and 23.5%, respectively. In the mesocosms, cumulative survival of females after 10 d was 51.6% with sugar and 25.6% without sugar. In the cages, female survival was 95 and 73%, respectively. Sensitivity analysis of the population projection matrix shows that both reduced male survival and reduced mating capability due to a lack of sugar contributed to lower insemination rates in females, and in the absence of sugar the insemination rate was lowered to an extent that led to population decline.

Evidence for costs of mating and self-fertilization in a simultaneous hermaphrodite with hypodermic insemination, the Opisthobranch Alderia willowi.

Simultaneous hermaphrodites offer the chance to study antagonistic coevolution between the sexes when individuals function in both roles. Traumatic mating by hypodermic insemination has repeatedly evolved in hermaphroditic taxa, but evidence for the fitness costs of such male-advantage traits is lacking. When reared in isolation, specimens of the sea slug Alderia willowi (Opisthobranchia: Sacoglossa) initially laid clutches of unfertilized eggs but 4 days later began self-fertilizing; this is only the third report of selfing in an opisthobranch. Hypodermic insemination may allow selfing in Alderia if penetration of the body wall bypasses internal mechanisms that promote outcrossing. Selfing specimens and slugs reared in pairs had reduced fecundity compared to isolated slugs laying unfertilized clutches, suggesting that hypodermic insemination imposes a cost of mating. Egg production increased for field-caught slugs separated after mating compared to slugs held in pairs, a further indication that accessibility to mates imposes a fitness cost to the female function. Such antagonism can confer a competitive advantage to slugs mating in the male role but diminish reproduction in the female role among hermaphrodites capable of long-term sperm storage. Alderia willowi is also a rare case of poecilogony, with adults producing either planktotrophic or lecithotrophic larvae. Our rearing studies revealed that most slugs switched between expressed development modes at some point; such reproductive flexibility within individuals is unprecedented, even among poecilogonous species.

Cross-mating between Malaysian strains of Aedes aegypti and Aedes albopictus in the laboratory.

Reciprocal and homologous mating experiments between Malaysian Aedes aegypti and Aedes albopictus mosquitoes were conducted in the laboratory. Two methods were employed, namely an artificial mating technique and a natural cage mating technique. The study demonstrated there exists a strong reproductive isolation between Ae. aegypti and Ae. albopictus. Insemination occurred in cross-mating experiments between Ae. aegypti females and Ae. albopictus males and also between Ae. albopictus females and Ae. aegypti males. Cross mating between Ae. aegypti females and Ae. albopictus males produced more eggs than that between Ae. albopictus females and Ae. aegypti males with both artificial mating and natural cage mating techniques. The matings did not result in the production of viable eggs by the females. No embryonation of these eggs was observed when the eggs were bleached. With homologous mating Aedes aegypti produced significantly greater numbers of eggs compared to Aedes albopictus mosquitoes, and all the eggs hatched successfully.

Sperm interactions from insemination to fertilization.

The task of spermatozoa is to transport its DNA-load as efficiently and safely as possible from the male organism to the female. Before it reaches its destination, it has to pass almost through the entire female reproductive tract, a potentially hostile environment. During passage, it is confronted by a sophisticated system that provides sperm storage sides but also possibly facilitates selection. The present review attempts to summarize the current knowledge of sperm interactions during that journey. A better understanding of the highly complex processes taking place between insemination and fertilization will be necessary to improve the efficiency of conventional reproductive techniques as well as for enabling the development and establishment of new ones.