Mating Behavior Research Articles

Wing spot in a tropical and a temperate drosophilid: C = C enrichment and conserved thermal response

Wings are primarily used in flight but also play a role in mating behaviour in many insects. Drosophila species exhibit a variety of pigmentation patterns on their wings. In some sexually dimorphic Drosophilids, a pigmented spot pattern is found at the top-right edge of the male wings. Our understanding of wing spot thermal plasticity in sexually dimorphic species is limited with wing spots being primarily associated with sexual selection. Here, we investigated the wing pigmentation response of two species with wing spots: D. biarmipes and D. suzukii species to thermal variation. We exposed freshly hatched larvae of both the species to three different growth temperatures and checked for wing pigmentation in adult males. Our results indicate wing pigmentation is a plastic trait in the species studied and that wing pigmentation is negatively correlated with higher temperature. In both species, wings were darker at lower temperature compared to higher temperature. Further, D. suzukii exhibits darker wing pigmentation compared to D. biarmipes. Variation in wing pigmentation in both D. suzukii and D. biarmipes could reflect habitat level differences; indicating a strong G*E interaction. Raman spectral analysis indicated a shift in chemical profiles of pigmented vs. non-pigmented areas of the wing. The wing spot was found enriched with carbon-carbon double-bond compared to the non-pigmented wing area. We report that C = C formation in spotted area is thermally controlled and conserved in two members of the suzukii subgroup i.e. D. biarmipes and D. suzukii. Our study indicated a conserved mechanism of the spot formation in two Drosophila species coming from contrasting distribution ranges.Graphical

Is Drosophila Larval Competition Involved in Incipient Speciation?

Geographical, ethological, temporal and ecological barriers can affect interbreeding between populations deriving from an ancestral population, this progressively leading to speciation. A rare case of incipient speciation currently occurs between Drosophila melanogaster populations sampled in Zimbabwe (Z) and all other populations (M). This phenomenon was initially characterized by Z females refusing to mate with M males. Despite the fact that Z and M flies produce different amounts of cuticular pheromones, their manipulation and that of other sensory signals exchanged during courtship behavior only marginally rescued the behavioral isolation. To further explore the putative mechanisms involved in this phenomenon, we first assessed the fecundity in matings between Z and M flies. Then, we measured the reproduction and survival in adults resulting of co-cultured Z and M larvae. In these two experiments, Z flies rarely emerged. Z and M larvae produced different amounts of food-derived metabolites which were altered in co-culture condition. This maybe related to the different bacteria composition in the gut and body of Z and M flies. However, the mating behavior of co-cultured flies did not change and their cuticular pheromone profile was slightly altered. Thus, the Z/M larval competition could reinforce the barriers induced by gametic and behavioral isolation processes on this incipient speciation phenomenon.

Cellular evolution of the hypothalamic preoptic area of behaviorally divergent deer mice.

Genetic variation is known to contribute to the variation of animal social behavior, but the molecular mechanisms that lead to behavioral differences are still not fully understood. Here, we investigate the cellular evolution of the hypothalamic preoptic area (POA), a brain region that plays a critical role in social behavior, across two sister species of deer mice (Peromyscus maniculatus and P. polionotus) with divergent social systems. These two species exhibit large differences in mating and parental care behavior across species and sex. Using single-nucleus RNA-sequencing, we build a cellular atlas of the POA for males and females of both Peromyscus species. We identify four cell types that are differentially abundant across species, two of which may account for species differences in parental care behavior based on known functions of these cell types. Our data further implicate two sex-biased cell types to be important for the evolution of sex-specific behavior. Finally, we show a remarkable reduction of sex-biased gene expression in P. polionotus, a monogamous species that also exhibits reduced sexual dimorphism in parental care behavior. Our POA atlas is a powerful resource to investigate how molecular neuronal traits may be evolving to give rise to innate differences in social behavior across animal species.

Targeting the Hh and Hippo pathways by miR-7 suppresses the development of insect wings.

Wings are important organs of insects involved in flight, mating, and other behaviors, and are therefore prime targets for pest control. The formation of insect wings is a complex process that is regulated by multiple pathways. The Hedgehog (Hh) pathway regulates the distribution of wing veins, while the Hippo pathway modulates wing size. Any interventions that can manipulate these pathways have the potential to disrupt wing development and could be used for pest control. In this study, we find that overexpression of miR-7 in Drosophila results in smaller wings with disordered veins. Mechanistically, miR-7 directly targets both ci and yki via different mature miRNAs (miR-7-5p and miR-7-3p), thereby disrupting the Hh and Hippo pathways. Importantly, this regulatory mechanism is also observed in another insect species, Helicoverpa armigera. Finally, by utilizing a nanocarrier delivery system, we show that introducing miR-7 via star polycation (SPc) leads to wing defects in H.armigera. In conclusion, these findings uncover that miR-7 inhibits wing formation by targeting both the Hippo and Hh pathways, indicating its potential for use in pest control strategies.

Eumelanic Coloration Impacts Mating Behavior, Stress Response, and Predator Preference in the Polymorphic Eastern Mosquitofish

ABSTRACTThe expression of melanic or black coloration is ubiquitous and has significant behavioral and ecological relevance. Although black coloration is common, melanic morphs within populations are often considered rare. The mechanism for the maintenance of rare melanic morphs is often associated with differences in morph behavior, predator preference, or the interaction of both. Mechanistically, the genetic loci associated with black coloration in rare morphs can also influence behaviors, and these pleiotropic effects may provide some benefit to black morphs. We predicted that different mating behaviors, antipredator responses, and stress response, as well as predator preference influenced the maintenance of a rare black morph in freshwater Eastern mosquitofish. We created predator and control treatments to measure differences in melanic and silver mosquitofish behavior and cortisol levels. We also measured predator approach and attack of melanic and silver morphs. Overall, melanic morphs exhibited a higher number of mating behaviors compared to silver morphs and returned to mating attempts quicker than silver morphs after predator exposure. Melanic males also exhibited higher baseline cortisol levels, but silver males produced more cortisol after predator exposures compared to melanic males. These specific responses may provide the mechanism by which melanic males continue to persist in populations where they are rare.

The effect of size on mate selection, fecundity and survival in Culex pipiens mosquitoes

AbstractCulex pipiens L. is a medically important mosquito due to its abundance and ability to transmit West Nile virus. Despite being the focus of many mosquito control strategies, very little is known about its mating behaviour. Several control strategies rely on knowing female mate preferences to ensure their efficacy. Therefore, the purpose of this study was to characterize mate selection based on mate quality. Because larger individuals should have more resources available to be used towards mating behaviour, it was hypothesized that larger mates (males or females) would be of higher value and preferentially mated with compared to smaller ones. By manipulating food availability, males and females were reared to be either large or small adults and were mated with either a large or small mate. Through a series of experiments, the effect of size on mate success and post‐mating responses was assessed via insemination rates, blood feeding, egg laying, fertility and survival. Despite larger females living longer and being more fecund, males did not preferentially mate with larger females, nor did male size influence female survival or fecundity. Because larval rearing environment had an influence on adult morphology and fitness, it should be taken into consideration in mosquito control programmes.

Novo plant-based mosquito repellent shows promise for exclusion of Aedes mosquitoes from "window" entry.

Mosquitoes threaten over half of the world's population through vectored diseases such as malaria, zika, yellow fever, dengue, and chikungunya. Mosquitoes have a highly developed olfactory system attuned to chemotaxis relating to host-seeking, mating, and oviposition behavior. In this study, we aimed to determine the spatial efficacy of 2 plant-based repellent blends (Blend3 and Blend4 that had previously been found to successfully repel Aedes, Anopheles and Culex mosquitoes in wind tunnel assays) in excluding Aedes aegypti from the window entry. A new cage system was developed for parallel "no-choice" and "choice" olfactometric assays. In the no-choice trial, Blends 3 and 4, as well as commercial products (N, N-diethyl-3-methylbenzamide, p-menthane-3,8-diol [PMD], 3-(N-n-butyl-N-acetyl)-amino-propionic acid ethyl ester, and 2-(2-hydroxyethyl)-1-methylpropylstyrene 1-piperidine carboxylate), were adsorbed into filter papers of different sizes and placed in a window created between 2 attached bug dorms. Then, the number of mosquitoes entering the window was counted through a 6-min period. In choice olfactometric assays, Blends 3, 4, and PMD were adsorbed into filter paper and the number of mosquitoes moving away from Blend 3 and PMD were compared. No-choice assays showed that Blend3 (P < 0.001) and Blend4 (P = 0.0012) were more repellent than the best commercial product PMD. Additionally, while Blend 4 was significantly more repellent than Blend 3 (P = 0.012) in the choice assay, overall, these 2 blends show promise as new repellents for the spatial exclusion of Aedes aegypti from window entry alone or as part of a "push-pull'' strategy.

A review of the Use of Acoustic Organs in Insects in Terms of Predatory Behaviors

Acoustic organs, generally involved in sound generation and hearing, play a crucial role in insects, particularly in communication and mating behaviors. However, although rare, they also contribute to predatory behaviors. Some well-known examples include parasitism in various parasitic flies like Emblemasoma sp., aggressive mimicry in bush crickets (Chlorobalius leucoviridis), which imitate the female cicadas response song, and the use of stridulation by myrmecophilous social parasites like butterflies (Maculinea sp.) and beetles (Paussus sp.) to infiltrate ant nests. Despite the significance of these behaviors, research in this area is limited due to barriers such as lack of facilities and difficulties in collecting samples. Consequently, few studies have been conducted on acoustic behaviors in predatory contexts compared to other major uses like mating. This article will review several general studies on these three aspects, summarizing the methods and findings. The studies indicate that playback experiments and observations play a crucial role, that these behaviors are highly specialized for survival, and that challenges such as limited sample collections remain

Behavioral corroboration that Saitis barbipes jumping spiders cannot discriminate between males’ red and black ornaments

Physiological or genetic assays and computational modeling are valuable tools for understanding animals’ visual discrimination capabilities. Yet sometimes, the results generated by these methods appear not to jive with other aspects of an animal’s appearance or natural history, and behavioral confirmatory tests are warranted. Here we examine the peculiar case of a male jumping spider that displays red, black, white, and UV color patches during courtship despite the fact that, according to microspectrophotometry and color vision modeling, they are unlikely able to discriminate red from black. To test whether some optical or neurological component could have been missed using these methods, we conduct mate choice experiments. Some females are presented with a choice between males with their red leg coloration painted over with either red or black paint, while other females are presented with a choice between males with the same coloration painted over by either red or white paint. This latter pairing of red and white males should have been easily distinguishable to the spiders and served as a control to ensure our experimental setup was conducive to natural mating behavior. Red males were more likely to mate than white males (P = 0.035), whereas red and black males had identical mating success (P = 1.0). This suggests that previous physiological and computational work on these spiders was correct in concluding that they are unable to discriminate between red and black. Any functional significance of displaying both colors, rather than only black, remains unresolved.

Phytochemical Profiling by UHPLC-ESI-MS/MS, In Vitro Antioxidant, In Vivo Antidiabetic, and Pro-Sexual Effects of Salvia balansae Noë Ex Coss. Flower Extract in Diabetic Male Rats.

Our study assessed the phytochemical composition and the effects of Salvia balansae flower aqueous extract (FAE) on sexual dysfunction in diabetic rats. Total phenolic and flavonoid content, quantification of phytochemicals by ultrahigh performance liquid chromatography (UHPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) method, and in vitro antioxidant capacity of FAE extract were elucidated. For the in vivo study, diabetes was induced by one intraperitoneal injection of streptozotocin (STZ) (40mg/kg), and treated diabetic rats were given FAE at 200mg/kg. Rats were subjected to a sexual incentive motivation test (SIM) and a mating behavior test, and then serum testosterone levels and sperm quality were analyzed. Our study indicates that FAE contained a large amount of phenolic compounds and flavonoids, and 13 constituents were quantified; cynaroside (3.8mg analyte/g extract) was the main compound, followed by rosmarinic acid (3.145mg analyte/g extract), thus confirming its high antioxidant capacity. FAE treatment significantly reduced blood glucose levels, improved sexual motivation and copulatory behavior, increased testosterone levels, and enhanced sperm quality in diabetic-treated rats compared to diabetic untreated rats. The present work demonstrates the therapeutic potential of S. balansae flowers in enhancing reproductive function in diabetic rats, potentially through its antioxidant, antihyperglycemic, and aphrodisiac properties.

Eurasian beaver ( Castor fiber ) mating behavior.

Eurasian beavers ( Castor fiber ) typically mate in the water at night, and observations of mating beavers are extremely scarce. As a result, little is known about their mating behavior. Here, I provide video documentation of beavers mating in the River Elbe during daylight, offering a unique opportunity to observe their mating behavior. The mating cycle consisted of: 1) a phase of self-grooming on land, 2) followed by the male pushing the female into the water and following her, and 3) copulation taking place in the water. This cycle was repeated several times throughout the same day. These observations contribute to our limited understanding of Eurasian beaver mating behavior.

The Influence of Hypoxic Conditions on Mating Behavior in a Bimodal Breather, the Great Pond Snail (Lymnaea stagnalis).

AbstractReduced oxygen availability can become an environmental stressor that imposes constraints on the energy and/or time budget of aquatic animals. This also seems to be the case for bimodal breathers, since it was previously reported that the amount of oxygen in the water interacts with feeding and egg-laying activities. The great pond snail (Lymnaea stagnalis) is a typical bimodal breather and a simultaneous hermaphrodite for which both male and female mating behaviors have been studied in detail. The aim of this study was to test whether hypoxic conditions affect mating behavior in this bimodal breather and to investigate whether mating motivation and the choice of sexual role is influenced by this environmental condition. Our results show that when oxygen cannot be acquired via cutaneous respiration or aerial respiration, mating and locomotion are inhibited. When oxygen is available only via air, the occurrence of unilateral insemination, reciprocal insemination, and biting behavior is reduced and courtship duration is decreased compared to when oxygen is also available in the water. Our results indicate that depending on the oxygen availability in the environment, water, and air, this bimodal breather has to weigh its motivation to mate against its need for cutaneous and/or aerial respiration. These findings contribute to our understanding of how animals optimize their behavioral investment decisions under changing environmental circumstances.

Non-random mating behaviour between diverging littoral and pelagic three-spined sticklebacks in an invasive population from Upper Lake Constance.

Adaptive divergence and increased genetic differentiation among populations can lead to reproductive isolation. In Lake Constance, Germany, a population of invasive three-spined stickleback (Gasterosteus aculeatus) is currently diverging into littoral and pelagic ecotypes, which both nest in the littoral zone. We hypothesized that assortative mating behaviour contributes to reproductive isolation between these ecotypes and performed a behavioural experiment in which females could choose between two nest-guarding males. Behaviour was recorded, and data on traits relevant to mate choice were collected. Both females of the same and different ecotypes were courted with equal vigour. However, there was a significant interaction effect of male and female ecotypes on the level of aggression in females. Littoral females were more aggressive towards pelagic males, and pelagic females were more aggressive towards littoral males. This indicates rejection of males of different ecotypes in spite of the fact that littoral males were larger, more intensely red-coloured and more aggressive than the pelagic males-all mating traits female sticklebacks generally select for. This study documents the emergence of behavioural barriers during early divergence in an invasive and rapidly diversifying stickleback population and discusses their putative role in facilitating reproductive isolation and adaptive radiation within this species.

Male medaka continue to mate with females despite sperm depletion.

In animals where males engage in multiple matings, sperm depletion can substantially reduce the reproductive success of both sexes. However, little is known about how successive matings affect sperm depletion, fertilization rates and mating behaviour. Here, we investigated this phenomenon under laboratory conditions. Medaka (Oryzias latipes), an externally fertilizing fish, is an ideal model to test predictions of sperm depletion because there are established methods to observe its mating and count sperm. Medaka males mated with multiple females (19 per day, on average; range, 4-27), experiencing significant sperm depletion, with sperm release declining markedly after the first few matings, reaching only 0.5-6.3% by the last mating of the day. Fertilization rates decreased, particularly after approximately 10 consecutive matings, although there was some recovery in the next-day's matings. The decline in courtship effort and mating duration probably resulted from the males becoming increasingly fatigued. Despite the reduced sperm availability, females did not adjust their clutch size as a counterstrategy. These results suggest substantial reproductive costs for males and the potential for sexual conflict owing to limited sperm availability. For species with frequent successive mating, these findings highlight the need to reconsider reproductive strategies and their impact on sexual selection.

Sexual dimorphism alters seasonal chelae muscle mechanisms in spiny-cheek crayfish (Faxonius limosus).

Sex-specific behaviours of freshwater crayfish are key elements in sustaining species persistence and successful conquering of new habitats in freshwater ecosystems. However, to date, information on molecular mechanisms that underpin the anatomy and physiology of crayfish sexes in successful mating behaviour was scarcely presented. In this study, Faxonius limosus females and males were sampled in spring and autumn to assess the impact of sexes and seasons on body parameters and activity of arginine kinase (ak), ferritin (fr), crustacean calcium-binding protein 23 (ccbp-23), troponin c (tnnc), and skeletal muscle actin 8 (actinsk8) genes related to the functioning of muscles in chelae. Comparison of body parameters showed significant differences in the weight and size of individuals in two seasons, underlining that large chelae are essential for males in mating behaviours and male-male competitive interactions. The gene expression analysis showed that activities of the five genes in the chelae muscle of F. limosus were influenced by the season- and sex-specific drivers. Multivariate analyses specifically identified the key genes (e.g., tnnc in males from spring) that were directly involved in metabolisms of chelae muscles of males and females collected in spring and autumn. The study, for the first time, described the direct impact of two key seasons and sexes on the anatomical features and molecular mechanisms that shaped the behaviour of F. limosus.

Reading between the lines: Novel insights on wild Pacific harbour porpoise (Phocoena phocoena vomerina) social communication through narrow-band high frequency click trains.

For cetaceans that produce narrow-band high-frequency click trains such as the Pacific harbour porpoise (Phocoena phocoena vomerinae), social acoustic behavior is poorly understood. While harbour porpoises have a reputation of being generally non-social and often solitary, few studies have aimed to quantify acoustic social communication for this species. In the waters surrounding the Port of Prince Rupert in British Columbia, Canada, harbour porpoises are often seen in groups where they have been observed attempting mating and surface-active behaviors. To assess the extent of social communication amongst porpoises in this region, we analyzed a long-term passive acoustic monitoring F-POD dataset collected from two sites, coupled with a detailed social acoustic criterion for detecting patterns of non-foraging click trains. Based on these criteria, porpoises were found to be producing patterns of obvious, discrete, and repetitive click trains that were marked as social. Generalized additive models were used to identify significant temporal trends in the dataset. On average, 5.3% of click trains produced by porpoises were social. Monthly and diurnal fluctuations in social detection positive minutes (DPM) followed a similar trajectory to non-social DPM, with peak activity observed during periods of darkness and from spring to early summer with a smaller increase in the fall. At one site, 11.1% of the DPM in May were classified as social. In general, proportionally more social DPM were found during periods of more overall DPM, suggesting that porpoises were socially communicating while in proximity to one another. Notably, overall DPM significantly decreased by 53.7% over three years. This novel methodology can be replicated in other regions to gain further insight into the social acoustic behavior of harbour porpoises.

Bonobo Optimizer Inspired PI‐(1+DD) Controller for Robust Load Frequency Management in Renewable Wind Energy Systems

With the growing presence of renewable energy sources (RESs), the necessity for adaptive and robust control strategies becomes more pronounced. This article proposes a self‐adaptive bonobo optimizer (SABO)‐based proportional integral one plus double derivative (PI‐(1+DD)) controller that offers a novel solution to the load frequency control (LFC). It draws inspiration from the reproductive strategies of bonobos, employing unique mating behaviors to enhance optimization processes. This innovative approach introduces memory capabilities, repulsion‐based learning, and diverse‐mating strategies. It is developed to tune the PI‐(1+DD) controller for handling the LFC in a two‐area power system involving a thermal plant and RESs of a wind farm. The proposed SABO algorithm is applied in a comparative manner to the standard bonobo optimization algorithm (BOA), Coot algorithm, particle swarm optimizer (PSO), and Pelican optimization approach (POA). Also, the SABO‐based PI‐(1+DD) controller is contrasted to PI and PIDn controllers. The simulation findings distinguish the proposed SABO‐based PI‐(1+DD) controller as a versatile and adaptive controller offering a more resilient and efficient approach to tackle the complexities introduced by the evolving energy landscape. It demonstrates its potential to significantly improve the dynamic response of power systems, particularly in the face of step load changes and random fluctuations. The proposed SABO‐based PI‐(1+DD) controller shows significant enhancement compared to BOA, Coot, POA, and PSO with 38.81%, 46.27%, 16.79%, and 37.40%, respectively. Also, it demonstrates an impressive percentage improvement of 97.1% compared to the PIDn controller and 74.88% over the PI controller considering random consecutive fluctuations in the system.

Copulatory mechanism and genital coupling of the longhorn beetle Moechotypadiphysis (Coleoptera, Cerambycidae).

The function of insect external genitalia has played a significant role in exploring insect mating mechanisms and male fertilization strategies. However, due to the privacy of genital coupling, insect copulatory mechanisms have only been investigated in a few insect groups. In this study, we observed the mating behavior using freeze-fixated pairs in copula to reveal the copulatory mechanism of the longhorn beetle Moechotypadiphysis (Pascoe, 1871). At the beginning stage of mating, the male M.diphysis usually takes 30 min to control the female and then extends its median lobe and endophallus. Approximately 80% of males (19/24) of M.diphysis exhibit multiple expansions (the membranous endophallus expands and enters into the female genital tract), ranging from two to five times. There are two types of expansions: short ones lasting for 1.4 to 49 s and long ones ranging from 1.03 to 7.23 min. During copulation, male tarsi continuously grasped the female elytra, thorax, and abdomen to help the male to initiate and maintain copulation. Male genital structures are closely connected to female genital structures: the apical phallomere and flagellum on the male endophallus contacting the bursa copulatrix duct and the spermathecal duct of the female, and the abundant microstructures on the surface of the everted male endophallus directly anchoring the female genital tract. Finally, we discuss the possible reasons for the evolution of their complex mating-related structures. Our research will help to explore the evolutionary mechanisms of insect genital structures.

Patterns of paternity: insights into mating competition and gene flow in a recovering population of humpback whales.

Variation in reproductive success is a fundamental prerequisite for sexual selection to act upon a trait. Assessing such variation is crucial in understanding a species' mating system and offers insights into population growth. Parentage analyses in cetaceans are rare, and the underlying forces of sexual selection acting on their mating behaviours remain poorly understood. Here, we combined 25 years of photo-identification and genetic data to assess patterns of male reproductive success and reproductive autonomy of the New Caledonian (Oceania, South Pacific) humpback whale breeding population. Paternity analysis of 177 mother-offspring pairs and 936 males revealed low variation in male reproductive success (average 1.17 offspring per father) relative to other polygynous species. The observed skew in success was higher than expected under random mating and skewed overall towards males (93%) without evidence of paternity over the study period. Finally, an updated male gametic mark-recapture abundance estimate of 2084 (95% confidence interval = 1761-2407, 1995-2019) fell between previous census estimates of the New Caledonian population and the wider Oceanian metapopulation. Our results provide critical insights into the mating competition of male humpback whales and population dynamics across Oceanian populations, two important factors affecting the slow recovery from whaling across the South Pacific region.

Absence of mating behaviors in the female dogs neonatally treated with estrogen and progesterone.

This study aimed to develop a non-surgical method to neutralize reproduction in female dogs. Female Beagle puppies, aged 6 days, were treated with pellets designed to release estradiol benzoate (EB; 1.0 mg) and progesterone (P4; 5.0 mg) over approximately 3 weeks. Their estrous cycles were monitored from 6 to 34 months of age by examining their vulvas daily and measuring their serum P4 levels once a month. Vulvar edema and discharge, followed by a serum P4 level above 5 ng/ml, indicated the potential estrus. Each time a dog showed these signs, breeding was attempted by housing with a proven male Beagle. All the treated dogs displayed cyclic progesterone surges with 5 to 6-month-long anestrous intervals. Surprisingly, none exhibited sexual behaviors, and no mating occurred (i.e., no intromission and copulatory tie), resulting in no pups being born. This phenomenon was further explored in laboratory animals. Neonatal female rats were treated with microspheres containing smaller doses of the same steroids (0.3 mg EB + 3.0 mg P4) at 1 or 2 days old. At 3 months old, the rats were ovariectomized, chemically stimulated to exhibit estrus behaviors using a standard protocol and tested for receptivity to proven male rats. Untreated control rats showed normal receptivity (i.e., lordosis) and allowed males to mate. However, rats treated with EB+P4 did not exhibit lordosis or allow mating. These results indicate that the combined use of estrogen and progesterone could be an effective non-surgical method for inhibiting mating behavior and, consequently, neutralizing female dog reproduction.