Semen Transfer Research Articles

Evaluation of indirect transfer mechanisms of semen under varying test conditions

There is limited data available on the subject of indirect transfer of non-visible body fluids, particularly semen, and often forensic science practitioner experience alone must be used to guide expectations. It can be difficult to assess the likelihood of proposed transfer scenarios without knowledge of how different variables can affect a transfer. The following work carried out by the Association of Forensic Service Providers UK and Ireland Body Fluid Forum explores how the features of transferred semen change with differences in the primary and secondary surface (porous and non– porous), different contact types (passive, pressure and pressure+) and with wet and dry primary stains. It was concluded that the primary surface type and whether the stain was wet or dry when contact occurred had the most significant effect on the transfer of semen, with wet transfers and transfers from the tested non-porous surface producing significantly more, and larger, visible stains under white light, stains with stronger fluorescence as viewed using Crime-lite® ML2, stains with stronger and faster acid phosphatase reactions and greater numbers of spermatozoa viewed using high power microscopy, compared to dry transfers and transfers from the tested porous surface. Pressure with movement transfers resulted in significantly more visible stains under white light and greater numbers of spermatozoa viewed using high power microscopy compared to passive transfers, however this only occurred when transfers were from a porous primary surface. The secondary surface type was not found to have a significant effect on the numbers of spermatozoa viewed using high power microscopy.

Evaluative approach to semen transfer in a case of alleged sexual assault: A case study

This paper demonstrates a logical framework for evaluating forensic evidence, first described by Cook et al. [1,2], using a casework example of an alleged sexual assault involving semen transfer. Here we show in real time how the case strategy can change with additional information and how to use available experience and published data to interpret the findings obtained, given the background information provided. The findings of the case are interpreted using the Bayesian approach and are reported by giving the strength of support of scientific findings for one proposition rather than a competing proposition, as per the European Network of Forensic Science Institutes (ENSFI) guideline for evaluative reporting. We believe that using this paper as a template will aid other Forensic Science Practitioners (FSP) to add value and weight to their work by assisting them in evaluating and interpreting their own findings.

Differentiation of Multiple Fluorescent Powders, Powder Transfer, and Effect on Mating in Aedes aegypti (Diptera: Culicidae).

Simple SummaryFluorescent powders are one of the most used materials for externally marking mosquitoes, such as Aedes aegypti, a vector of numerous human pathogens. They can be used to explore multiple biological questions related to population size, dispersal, and other interactions related to the spread of mosquito-borne diseases. To ensure marking practical aspects and that these powders do not interfere with mosquito natural behaviors, differentiation of multiple colors applied externally in the same mosquito, their impact on mating, and their transference between individuals after copulation and same-sex interactions were studied. Multiple color differentiation was possible, except for green–yellow combination. No important effect of powder marking was found on any of the mosquito mating phases: coupling (recognition and genital contact), copulation (genitalia engagement and semen transfer), and insemination (deposition of the sperm in the female reservoirs). After copulation with a powder-treated mate, >80% of females and all males had powder in genitalia, legs, and wingtips. The transfer of powder, between same-sex individuals, occurred only in males. In general, fluorescent powders had a little observable effect on Ae. aegypti mating, suggesting that these markers do not alter this important vector life-history trait and are a useful and viable tool for mosquito studies.Five different fluorescent powders (orange, yellow, green, blue, and violet) were tested on Aedes aegypti adults to evaluate the differentiation of multiple fluorescent powder colors applied externally in the same female mosquito, their effect on coupling time, copulation time, insemination success, mate choice, and the extent of transference of powders between marked and unmarked individuals, either during copulation or same-sex interactions. Marking with multiple powders was evaluated after applying different powders in the same female at different times and combinations. The comparative effect of powders on mating was explored using different cross-combinations of marked/unmarked couples. Transference of powders between marked/unmarked individuals after copulation was checked in mated individuals, and between same-sex interactions by allowing them to interact under crowded and uncrowded conditions. Identification of the colors included in multiple markings in the same individual was possible when exploring almost all combinations (exception: green–yellow). No important effect of powder marking between cross-combinations was found on coupling time (overall 95% CI (Confidence Interval) 37.6–49.6 min), copulation time (overall 95% CI 17–20 s), insemination success, nor their mate choice. Transferred powder after copulation activity, concentrated in genitalia, legs, and the tip of wings, occurred in >80% of females and 100% of males. Powder transference in legs and genitalia, between same-sex individuals, occurred only in males (ranged between 23–35%) under both density conditions. The lack of important effects of these powders on the studied aspects of Ae. aegypti provides information about their usefulness and limitations, which should be recognized for future applications and to avoid bias.

Disentangling female postmating responses induced by semen transfer components in a simultaneous hermaphrodite

Optimizing reproductive success is an essential part of evolution for both sexes. Females can optimize mating by avoiding superfluous mating advances and insemination, since both take time away from other activities and may incur costs related to sperm receipt. While many separate-sexed organisms are known to exhibit mate avoidance, much less is known about this for simultaneous hermaphrodites. We addressed this here, using a simultaneously hermaphroditic species that can choose to mate in either of the two sex roles during each mating interaction. Recently, avoidance behaviours in response to natural matings were observed in the hermaphroditic pond snail Lymnaea stagnalis, potentially deterring insemination. To disentangle whether such behaviours are mediated by the mechanical act of mating or the receipt of accessory gland proteins and/or sperm, we intravaginally injected individuals with control or test fluids. Our results show that the avoidance behaviours, crawl-out and biting, were more frequently expressed when individuals were inseminated with accessory gland proteins and/or sperm. These behavioural components of the recipient increased time in courtship prior to insemination, which is concordant with the hypothesis that the partner tries to discourage the potential sperm donor from inseminating. Understanding the mechanism underlying the effects that molluscan accessory gland proteins induce contributes to our understanding of the molecular basis of the recipient's (behavioural) responses as well as how such biochemical postcopulatory strategies evolve.

Correlation between increased postpubertal phallic growth and the initiation of cranial sexual dimorphisms in male Morelet's crocodile.

While puberty is an animal commonality, little is known of its timing or process in crocodylians. Males copulate with an intromittent phallus that has a distinct glans morphology which directly interacts with the female cloaca, putatively effecting effective semen transfer and ultimately increased fecundity. Here we present, during the Morelet's crocodile lifecycle, a well-defined body length (65 cm snout-vent length) inflection point that marks a subsequent increase of phallic glans growth rates. Putatively, this postpubescent growth produces a copulatory-effective phallus. While not as robust of a trend as snout-vent length, this growth inflection concomitantly begins with a body condition index (CI = BM/SVL3 ) between 2.0 and 2.5 and is most distinct above a CI of 2.5. Also, in males, this 65 cm size threshold also aligns with the initiation of more robust growth in caniniform alveoli associated with prominent maxillary and mandibular teeth. This inflection was not observed in females, thus marking a sexual dimorphism that begins to present with the onset of puberty. This bodily manifestation of puberty other than those changes observed in the reproductive tracts is a novel observation for crocodylians and lays a foundation for further study among species of how changing endocrine signaling within sexually maturing males may also influence a broader range of secondary sex characteristics.

Secondary Transfer of Semen

A case is described of a three year old girl who had been treated for urinary tract infections during a longer period. At one incident erythrocytes and spermatozoa were found in the girl’s urine. The mother explained that she had intercourse with her husband early in the morning, and that she used a menstrual cup. As she was emptying the cup during toilet visit with the girl, she had blood on her fingers. When the girl urinated, the urine also touched the mother’s fingers thus washing some of the blood including spermatozoa from the intercourse down into the urine sample. The forensic examination, including DNA-profiling of blood and semen, did not contradict this explanation. No other possible cause was found for this contamination with erythrocytes and semen in the girl’s urine. Child abuse was highly unlikely.

Reproductive Strategies of Aedes albopictus (Diptera: Culicidae) and Implications for the Sterile Insect Technique

Male insects are expected to optimize their reproductive strategy according to the availability of sperm or other ejaculatory materials, and to the availability and reproductive status of females. Here, we investigated the reproductive strategy and sperm management of male and virgin female Aedes albopictus, a mosquito vector of chikungunya and dengue viruses. The dynamics of semen transfer to the female bursa inseminalis and spermathecae were observed. Double-mating experiments were conducted to study the effect of time lapsed or an oviposition event between two copulations on the likelihood of a female double-insemination and the use of sperm for egg fertilization; untreated fertile males and radio-sterilised males were used for this purpose. Multiple inseminations and therefore the possibility of sperm competition were limited to matings closely spaced in time. When two males consecutively mated the same female within a 40 min interval, in ca. 15% of the cases did both males sire progeny. When the intervals between the copulations were longer, all progeny over several gonotrophic cycles were offspring of the first male. The mating behavior of males was examined during a rapid sequence of copulations. Male Ae. albopictus were parceling sperm allocation over several matings; however they would also attempt to copulate with females irrespective of the available sperm supply or accessory gland secretion material. During each mating, they transferred large quantities of sperm that was not stored for egg fertilization, and they attempted to copulate with mated females with a low probability of transferring their genes to the next generation. The outcomes of this study provided in addition some essential insights with respect to the sterile insect technique (SIT) as a vector control method.

A technique to artificially inseminate leafcutter ants

Artificial insemination (AI), the instrumental transfer of semen from the male to female reproductive organs, offers excellent opportunities to study mating system adaptations as it allows paternity to be experimentally manipulated. AI techniques have been developed for many animals, but rarely for ants, where they would be particularly useful as most species do not mate under controlled lab conditions. Here, we describe an AI technique for Atta leafcutter ants involving (1) the collection of ejaculates via induction of natural ejaculation, (2) storage in glass capillaries, and (3) transfer to queens using a modified AI equipment as used for honeybees. Queens were fixed and anesthetized in a queen holder, after which the sting chamber was opened with two steel hooks, the tip of the semen-containing glass capillary was inserted into the bursa copulatrix and the semen slowly expelled. Sperm was successfully stored in the spermatheca of queens, and some queens produced a small colony as a result. We could furthermore confirm the earlier observations that Atta semen is directly transferred to the spermatheca rather than to the bursa copulatrix as in most other eusocial insects. The technique that we present here can offer novel opportunities to study mating events such as sperm transfer, sperm competition, and cryptic female choice in ants. At present, the number of queens that produce colonies after AI remains low. However, this number will likely increase, as our results indicate that rearing conditions after AI influence colony founding success of artificially inseminated Atta queens.

Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species

Approximately 503 of the known species of birds are classified as ‘endangered’ or ‘critical’. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific.

Physiological determinants of male mating performance in aphids (Hemiptera: Aphididae)

When investigating sexual strategies of aphids, it is necessary to set up multiple replicates to compensate for the small number of eggs laid per female. Genetic variation among replicates can be minimised if members of the same clone are used, but problems can arise unless the participants are also physiologically equivalent. A series of experiments on Myzus persicae (Sulzer) was performed to investigate aspects of male maturation and semen transfer that should be considered in planning or interpreting experiments on aphid sexual strategies.

Potential relationships between physical traits and male broiler breeder fertility

Genetic selection in primary broiler breeders may modify skeletal structure, possibly impeding semen transfer, and could alter the size and degree of fluctuating asymmetry (FA) of bilateral traits associated with fertility. Hence, we hypothesized specific morphometric traits could predict differential fertility. Sixty primary broiler breeder males from Strains A and B (n = 30/strain) were individually housed with an average of 10 females per male. Male fertility and sperm penetration (SP) through the perivitelline layer were estimated on fresh eggs. At 50 wk, BW, keel length (KL), posterior pelvic width and length (PPW, PPL), dorsal pelvic width and length (DPW, DPL), tarsometatarsal length and width (TL, TW), comb length and width (CL, CW), and wattle length, width, and area (WL, WW, WA) were measured. Results indicated that Strain A had smaller BW, KL, WL, WW, WA, CL, CW, PPL, DPL, and DPW. A higher degree of FA was found in Strain A TL and WL (P < 0.05), yet DPW FA was greater for Strain B (P < 0.001). In addition, DPW FA negatively correlated with Strain B fertility (r = -0.369; P < 0.01); however, other FA measurements did not correlate with estimated fertility or SP. Strain A WL correlated with SP (r = 0.383; P < 0.01) and fertility (r = 0.346; P < 0.01). Results indicate DPW alteration may impact semen transfer upon copulation, as Strain A fertility negatively correlated with DPW (r = -0.298; P < 0.05). This research provides evidence that morphometric traits might be useful to predict fertility in broiler breeders.

Effects of Size and Flight Performance on Intermale Mate Competition in the Cicada Killer, Sphecius speciosus Drury (Hymenoptera: Sphecidae)

The characteristics of successfully mating male cicada killers (Sphecius speciosus Drury) were compared to those of unsuccessful males. In the field, successful males were larger and had higher ratios of flight muscle mass to body mass (flight muscle ratios) than unsuccessful males. The elevated flight muscle ratio of successful males was not because of semen transfer to the female nor because of an allometric effect. Better territory-holding ability and flight maneuverability probably contribute to the success of large males with a high flight muscle ratio in nature under most conditions. However, temporal or situational conditions may also exist whereby small males may occasionally have opportunities to mate.

The reproductive capacity of Panstrongylus megistus (Hemiptera: Reduviidae) males treated with precocenes

Reproductive performance including mating, semen transfer, egg production and hatching rate were investigated in pairs of Panstrongylus megistus in wich the male was treated with either precocene II or ethoxyprecocene II, topically applied shortly after imaginal molt. Eighty percent of the males were not able to mate for a period as long as 40 days after treatment. Those males wich did mate (20%) produced small spermatophores; a few (10%) were unable to transfer spermatozoa to female, while the others (10%) exhibited reproductive performance similar to that of non-treated males. The cumulative effects of the precocene on the different stages of reproduction resulted in a drastic reduction of offspring production. The anti-allatal compound precocene seems to inhibit successful reproduction through a corpus allatum inactivation and subsequent lack of secretory activity of the male accessory glands.

Routine AI application in the Hannoverian Sport Horse Breeding Association

The Hannoverian Sport Horse Breeding Association registers about 16 000 brood-mares, 9500-10 000 of which are yearly booked to the stallions of the Landgestüt Celle. This is a state-owned depot where 180 active sires are kept and offered for breeding service for mares kept in farms throughout the Lower Saxon county. The other brood-mares are served by stallions owned privately, competing with the Celle stallions. Each year, 10% of the stallions are recruited from a selected and intensely tested group of young stallions. During the breeding season — late January to late July—the sires are stabled in breeding subcenters where they serve as semen donors for AI (10 locations) or for natural breeding (38 locations). In the subcenters for AI, semen is processed for fresh, cooled and shipped semen transfer. Stallions, selected for breeding, undergo a strict prebreeding and breeding health management program. AI is performed mainly by using fresh cooled semen, and only occasionally by frozen semen. Stallions are not allowed to serve simultaneously in artificial and natural breeding. Semen collection is confined to one single collection per day and per stallion to adjust daily sperm output to a predictable level. The number of sperm cells per insemination dose is set at 5 × 10 8 progressively motile spermatozoa. The insemination volume varies from 10 to 30 cm −3. Semen is extended with skim milk extender ( Kenney et al., 1975) or in a glycine-egg-yolk extender ( Van der Holst, 1984) with or without removal of seminal plasma following centrifugation of the semen. A new container for shipment has been developed which maintains the preferred temperature of 5 °C up to 36 h. Fertility results from AI with fresh semen are 67.1% registered foals, i.e. almost a five points better result than those obtained after natural breeding (62.8 registered foals) during the last three seasons preceding the introduction of AI. The use of AI tends to become progressively the zootechnique of equine breeding practice in almost all of the horse breeds except the thoroughbred. AI systems for frozen semen and some systems using fresh semen have been presented for practical use in the field.

Sexual behaviour and fertility in broiler breeder domestic fowl

An experiment was designed to investigate the reasons for the decrease in fertility which commonly occurs in broiler breeder domestic fowl in the later part of the laying year. Male broiler breeders were penned individually with 15 females and fed separately from them at various levels of energy, in order to attain different target body weights, and at two levels of protein. Sexual behaviour and fertility were recorded at 28, 38 and 58 weeks of age. The frequency of all sexual behaviour showed a large decrease with age which was suggestive of a decline in libido. There was also a significant effect of body weight, with heavier cocks showing more sexual displays and slightly more matings. Level of protein had little effect on sexual behaviour and fertility. Cocks which were more severely restricted showed a reduction in fertility and libido in Weeks 38 and 58. The decline in libido was not enought to account for reduced fertility in heavy cocks at 58 weeks and it was concluded that this was most probably a consequence of the large bulk or the conformation of the males at this age interfering in some way with the transfer of semen during copulations which otherwise looked normal.

The Effects of Isolation, Grouping and Population Density on Fecundity ofBulinus truncatus

Summary Bulinus truncatus is the intermediate host of Schistosoma haematobium, a trematode parasite causing bladder bilharziasis in man. In the struggle against this disease, control of snail populations is an important goal and expansion of our knowledge of the reproductive activity of Bulinus is relevant in this regard. In this study the effects of various factors on the fecundity of Bulinus were investigated in specimens kept under continuous water refreshment conditions. The results are considered to be more indicative of the natural situation than results obtained under the current use of discontinuous water refreshment. Isolated snails produce considerably higher numbers of egg masses and eggs than grouped snails. The effect of grouping on isolated snails, and the reverse situation, is very rapid, being fully effective within one week. The increase in fecundity of isolated snails is most probably caused by the absence of copulatory activity, since transfer of semen is known to curtail egg laying in freshwater pulmonate snails. Egg mass production was shown to decrease with increasing population density. This may be caused either by the difference in space available or by the possible difference in copulatory activity. Isolated snails showed a faster body growth than grouped snails. The possible role of hormones involved in establishing the above effects and the consequences of the results for snail control programs are discussed.

Transfer of semen accelerates the onset of egg-laying in female copulants of the hermaphrodite freshwater snail,Lymnaea stagnalis

Egg-laying in the hermaphrodite pond snail Lymnaea stagnalis starts sooner in group-reared animals than in isolated snails. This holds for animals kept at 12-h L:12-h D as well as at 16-h L:8-h D photoperiods. From observations on the onset of egg-laying in snails in which semen transfer was prevented by surgical removal of parts of the vas deferens, it is concluded that semen transfer is the main factor accelerating the onset of egg-laying. Detailed observations revealed that the onset of egg-laying is accelerated only in female copulants. Growth of the albumen gland is faster in group-reared snails than in isolated animals, but that of the prostate gland is similar in the two groups. The effects of copulation on egg-laying are discussed in relation to the physiology of hermaphroditism in L. stagnalis.

Feeding and oviposition behavior in the mosquito Aedes aegypti (L.). I. Preliminary studies of physiological control mechanisms.

1. When female Aedes aegypti were given a daily opportunity to take a blood meal over a 9-day period, the number of mated females feeding on days 3, 6, and 9 was significantly less than the number of virgins feeding. This low period of feeding coincides with the terminal phases of the first, second, and third gonotrophic cycles, respectively.2. The "mated" feeding pattern could be established in a virgin female by the implantation of a male accessory gland. Copulation without semen transfer or the implantation of a testis was ineffective in inducing the pattern of biting.3. Functional ovaries in the female receiving the accessory gland implant are required in order for the implant to exert its effect. Implantation of a normal ovary plus an accessory gland into females lacking ovaries produced the mated biting pattern.4. The material from the male accessory gland also controls oviposition behavior by female mosquitoes. Mated females lay their eggs readily on completion of oogenesis. Virgins mature an equal number of eggs but retain rather than deposit them. Implantation of male accessory gland into virgin females causes them to oviposit on maturation of their eggs.