other thematically related research

In this part of our site another results of studies performed together with  our partners and  connected with the issue of chemical communication and animal behavior are included. 

1. Kokocińska-Kusiak, A., Woszczyło, M., Zybala, M., Maciocha, J., Barłowska, K., & Dzięcioł, M. (2021). Canine olfaction: physiology, behavior, and possibilities for practical applications. Animals, 11(8), 2463.

https://www.mdpi.com/2076-2615/11/8/2463

Simple Summary

Dogs have an extraordinary olfactory capability, which far exceeds that of humans. Dogs’ sense of smell seems to be the main sense, allowing them to not only gather both current and historical information about their surrounding environment, but also to find the source of the smell, which is crucial for locating food, danger, or partners for reproduction. Dogs can be trained by humans to use their olfactory abilities in a variety of fields, with a detection limit often much lower than that of sophisticated laboratory instruments. The specific anatomical and physiological features of dog olfaction allow humans to achieve outstanding results in the detection of drugs, explosives, and different illnesses, such as cancer, diabetes, or infectious disease. This article provides an overview of the anatomical features and physiological mechanisms involved in the process of odor detection and identification, as well as behavioral aspects of canine olfaction and its use in the service of humans in many fields.

Abstract

Olfaction in dogs is crucial for gathering important information about the environment, recognizing individuals, making decisions, and learning. It is far more specialized and sensitive than humans’ sense of smell. Using the strength of dogs’ sense of smell, humans work with dogs for the recognition of different odors, with a precision far exceeding the analytical capabilities of most modern instruments. Due to their extremely sensitive sense of smell, dogs could be used as modern, super-sensitive mobile area scanners, detecting specific chemical signals in real time in various environments outside the laboratory, and then tracking the odor of dynamic targets to their source, also in crowded places. Recent studies show that dogs can detect not only specific scents of drugs or explosives, but also changes in emotions as well as in human cell metabolism during various illnesses, including COVID-19 infection. Here, we provide an overview of canine olfaction, discussing aspects connected with anatomy, physiology, behavioral aspects of sniffing, and factors influencing the olfactory abilities of the domestic dog (Canis familiaris). View Full-Text

Keywords: dogs; olfaction; behavior; chemical communication; disease detection; COVID-19

1. Kokocińska, A., Woszczyło, M., Sampino, S., Dzięcioł, M., Zybała, M., Szczuka, A., ... & Rozempolska-Rucińska, I. (2022). Canine Smell Preferences—Do Dogs Have Their Favorite Scents?

https://www.mdpi.com/2076-2615/12/12/1488

Simple Summary

There are many products that are targeted to pet owners. One category of these products is dog repellents—strongly aromatized solutions designed to stop dogs from approaching and investigating particular areas; the second are cosmetics which should be pleasant for dogs. Dogs have a particularly sensitive sense of smell; therefore, strong scents may be very intense, and not always pleasant, stimuli. It is truly interesting, then, that canine cosmetic products often have very strong fragrances designed mostly to appeal to the dog owners, rather than to the dogs themselves. Indeed, the scents that dogs choose to put on their fur differ strongly from those of common cosmetics. Dogs choose mostly intense, animal-derived smells, such as feces or carcasses, so there is a need to differentiate between canine and human smell preferences. As there is limited scientific data related to canine smell preferences, the purpose of this study was to verify dogs’ reactions to selected scents, which can also be appealing to humans. Our study shows that dogs were more likely to interact with the scents of blueberry, blackberry, mint, rose, lavender, and linalol.

Abstract

The available evidence on dogs’ scent preferences is quite limited. The purpose of this study was to verify the canine response to selected odors that may also be preferred by humans. The experiment was performed using 14 adult dogs (10 female and 4 male) of different breeds, body size, and age (1–14 years). During the experiment, dogs were exposed to 33 odor samples: a neutral sample containing pure dipropylene glycol (control) and 32 samples containing dipropylene glycol and fragrance oils. The dog was brought to the experimental area by its handler, who then stopped at the entrance, unleashed the dog, and remained in the starting position. The dog freely explored the area for 30 s. All dog movements and behavior were recorded and analyzed. The methodology of observing the dogs freely exploring the experimental area allowed us to determine the smells that were the most attractive to them (food, beaver clothing). Our study shows that dogs interacted more frequently with the scents of blueberries, blackberries, mint, rose, lavender, and linalol.  Keywords: dog; olfaction; smell preferences; cosmetics repellents

3. Woszczyło, M., Dzięcioł, M., & Kokocińska, A. (2021). Ocena stresu u zwierząt na podstawie pomiarów stężenia kortyzolu. Weterynaria w Praktyce, 18(10).

The ability to objectively assess the stress level in companion animals is an issue of increasing importance in assessing welfare. The use of rapid stress assessment based on blood biochemical parameters during training or during the evaluation of the work progress of behavioral patients is just some of the examples of use. In addition, it enables easy assessment of the level of pressure in the work of service dogs, objective comparison of training methods or easy assessment of the patient’s well-being without the need for behavioral observation. It is therefore not surprising that the evaluation of biochemical parameters correlated with stress in dogs is widely and readily carried out.Stress in animals is evaluated by measuring clinical, behavioral and biochemical parameters.

Acknowledgements

Special thanks for support and assistance in the implementation of the project to:

dr Agata Kokocińska

dr Paweł Pasikowski

dr Jacek Łyczko

dr Paweł Mazurkiewicz

dr Piotr Knap

dr Silvestre Sampino

Students of the Faculty of Veterinary Medicine at Wroclaw University of Environmental and Life Sciences,

Technical and administrative staff of the Wroclaw University of Environmental and Life Sciences

Dogs breeders ( especially Mr. Sebastian Zajączkowski, Kenola).

Research team

Michał Dzięcioł Assoc. Prof.

Tadeusz Jezierski Prof.

Wojciech Niżański Prof.

Martyna Woszczyło

Antoni Szumny Prof.

Ewa J. Godzińska Prof.

Marcin Wrzosek Assoc prof.

Przemysław Podgórski MD

This part contains the observation obtained as a results of the study carried out as part of the project: " Mechanisms of semiochemical communication in canines in the context of sexual behavior: the reserch with the use of the domestic dog                    (Canis familiaris) as a modelspecies", financially supported by National Science Centre (Poland), grant No. UMO-2015/17/B/NZ8/02411

In this project we focused on the identification of the compounds suspected to be a putative canine sex pheromones. We  evaluated the behavioral aspects of the semiochemical communication between females and males of domestic dogs. Using the modern techniques of visualization (MRI and FMRI) we performed studies dedicated to visualization and evaluation of the responsible for detection of semiochemical compounds  organ - VNO-vomeronasal organ ( what was the first in the literature description of this organ with the use this technique!).

The results of our studies and observations prompted us to focus on the less volatile compounds probably playing a n important role in intraindividual communication between males and females during estrus and confirmed our suspicions about the complex nature of the process of identification of the estrus in female domestic dogs.

Most of our results have been already published and the abstracts and links to the full version of the texts are available for you below. 

Some are still in the process of publication ( manuscript sent to journals, being prepared for publication as a doctor's thesis) but we hope it will be attached here soon also.

1. Dzięcioł, M., Woszczylo, M., Szumny, A., Jezierski, T., Kupczyński, R., Godzińska, E. J., ... & Niżański, W. (2018). Identification of putative volatile sex pheromones in female domestic dogs (Canis familiaris). Animal reproduction science, 197, 87-92. https://www.sciencedirect.com/science/article/abs/pii/S0378432018305499

More than hundred urine samples, vaginal secretions, vulval and anal imprints as well as anal sac secretions, collected during various phases of the ovarian cycle, were evaluated with the HS-SPME/GC–MS technique. The results indicate there were differences among samples of urine collected during particular phases of the ovarian cycle. Unequivocally, the amount of carbonyl aromatic compounds, such as acetophenone (hypnone) and benzaldehyde, increased during the period of proestrus and estrus of the estrous cycle. This was accompanied by increases in methylketones, e.g., 2-octanone, 2-pentanone and 3-hexanone. Simultaneously, amounts of sulfide compounds (1-methylotiopropane; 1-methylotiobutane, 1-methylotipentane and dimethyl trisulfide) decreased during the period of estrus and abruptly increased in diestrus. These observations suggest a possible dual mechanism of interaction between males and females during and subsequent to the mating period, including the existence of both attracting and repelling signals. No significant changes were detected in samples other than urine. Further studies including a proteomic approach as well as behavioral assessments are suggested to identify the complete range of estrual semiochemical signals and to verify the semiochemical activities of identified substances

1. Woszczyło, M., Szumny, A., Łyczko, J., Jezierski, T., Krzemińska, P., Szczerbal, I., ... & Dzięcioł, M. (2021). The Case of Atypical Sexual Attractiveness in a Male Domestic Dog—A Case Study. Animals, 11(11), 3156.

Simple Summary

The period of heat is a time of naturally increased interest of the male in the female. Males recognizing specific chemical signals are able to find the female in estrus and try to mate with her. According to current knowledge, there is a specific hormonal play accompanying this process, with the rise of estrogens and progesterone in the female thought to be responsible for stimulating sexual arousal in males. In this paper we describe the cases of atypically increased attractiveness in a male, that clearly influenced the behavior of adult, intact males, which made mating attempts during contact with him, even though he had no detectable signs of clinical estrogenization. The “case” animal had a basal level of the hormones considered to be involved in the stimulation of sexual arousal. The case animal was a castrated male with confirmed genetic sex and confirmed lack of gonads, and a urinary chemical profile similar to that of a female in estrus. Even though similar cases are noted in breeding and veterinary practice, to our knowledge this is the first report of cases that include clinical and laboratory examination. As a hypothesis, we propose the involvement of other hormones in the creation of incidental attractiveness, or an increased production of compounds responsible for attractiveness (sex pheromones) resulting from metabolic events unrelated to reproductive processes. Further studies are needed to determine the cause of this phenomenon, which would expand our knowledge about the mechanisms involved in the creation of semiochemical communication and the production of the compounds responsible for the modification of behavior in the signal recipients.

Abstract

During the ovarian cycle in domestic dogs, females do not accept males during the first days of estrus but become attractive to males from the beginning of proestrus, with this attractiveness persisting until the end of the estrus phase. It is believed that increased estradiol is responsible for the female attractiveness to the males. In this paper we describe the case of strong, but atypical attractiveness of a castrated male to various, adult, intact males, influenced by the emitted semiochemical signals. Any significant changes in the level of hormones typically involved in the process connected with estrus and responsible for sexual arousal in the males were assessed. The case animal was a 4 year old castrated male Border Collie that was extremely attractive to various males, which presented high levels of sexual arousal, with intensive sniffing and licking of the preputial area, specific vocalization, increased salivation and, finally, mating attempts. Clinical examination of the castrated male revealed a lack of testes in the scrotum and abdominal cavity confirmed by USG. Laboratory tests indicated basal levels of estradiol, testosterone, and progesterone (15.23 pg/mL, <0.05 ng/mL, 0.25 ng/mL), and sex was confirmed via cytogenetic and molecular analysis. Chemical analysis (HS-SPME) of the urine indicated a huge similarity to the profile obtained from a bitch in estrus, with an elevated level of acetophenone, which has been previously postulated in the literature as being a characteristic of the estrus phase in female domestic dogs. This case presented very atypical sexual attractiveness, particularly when taking into account the basal levels of hormones which, according to current knowledge, are responsible for the creation of attractiveness. As a hypothesis requiring verification, we propose the idea of involvement of other hormones in the creation of incidental attractiveness or increased production of compounds responsible for attractiveness (sex pheromones) resulting from metabolic events unrelated to reproductive processes. To our knowledge it is the first described case presenting this phenomenon, which, with more detailed study, could shed new light on the process of creation of sexual attraction in the domestic dog.

Keywords: semiochemical communication; behavior; sex pheromones; dog; hormones; attractiveness

1. Jezierski, T., Dzięcioł, M., Szumny, A., Niżański, W., Woszczyło, M., Pieczewska, B., & Godzińska, E. J. (2019). Discrimination of estrus odor in urine by male dogs in different experimental settings. Journal of Veterinary Behavior, 29, 25-30.

https://www.sciencedirect.com/science/article/abs/pii/S1558787818301606

This study assessed the most suitable behavioral test for discrimination of the odor of female dog urine during estrus and anestrus, to identify chemical compounds acting as female pheromones. Twelve male dogs were used for testing. Urine samples were collected from 16 females in estrus, 5 females in anestrus, 5 spayed females, and 8 males. In test 1, five samples were arranged randomly in a lineup outdoors, and the dogs showed no spontaneous interest or preference toward the estrus samples. In test 2, urine samples from a female in either estrus or anestrus were spilled on the grass in two diverging tracks. The dogs seemed to be unable to localize the estrus urine sample, and when found accidently, they showed only a weak interest. In test 3, two urine samples from a female either in estrus or in anestrus were placed into artificial vaginas of two dog models. The dogs sniffed the samples longer (P = 0.004) than in test 2 and were able to distinguish estrus versus anestrus samples by longer sniffing of estrus samples (P < 0.02); however, their interest decreased in consecutive trials. Test 4 involved responses of dogs that had been previously trained in the scent lineup to discriminate cancer odor. The trained dogs indicated the estrus urine sample correctly amidst four anestrus and one male samples in 98.6% and 74.6% of trials, respectively, with a probability of correct indications by chance of 50% and 20%, respectively. Longer sniffing of estrus samples was observed only during the first trial. It was concluded that the lineup using trained dogs was the most useful method to discriminate estrus odor if multiple testing is required. Although not all typical sexual behaviors could be observed with this method, quantification of the dogs' responses was helpful for future studies on the chemical structure of pheromones.

1. Dzięcioł, M., Podgórski, P., Stańczyk, E., Szumny, A., Woszczyło, M., Pieczewska, B., ... & Wrzosek, M. A. (2020). MRI features of the vomeronasal organ in dogs (Canis familiaris). Frontiers in Veterinary Science, 7, 159. https://www.frontiersin.org/articles/10.3389/fvets.2020.00159/full

            According to current knowledge, the vomeronasal organ (VNO, Jacobson's organ) is the structure responsible for semiochemical signal detection. In dogs and other mammals, it is located close to the vomer and palatine processes of the incisive and maxillary bones. Although there are reports describing the anatomy and histology of this structure, there are limited available reports assessing this organ in live individuals and no direct visualization reports in dogs. The aim of this study was 2-fold: (1) preparation and optimization of a protocol for magnetic resonance imaging (MRI) examination of the VNO in a cadaver study with precise visualization and localization, and (2) characterization of the physiological VNO image features in MRI of live dogs. The first part of the study was performed on 10 beagle cadavers, the second on 8 live beagle dogs. For the VNO visualization, a 1.5T MRI (Philips^® Ingenia) scanner and 20-channel digital head-neck spine coil were used (Philips^®, Holland). The cadaver study allowed confirmation of the organ's location by the topical application of an MRI contrast agent (gadolinium) via the external entrance of the VNO canal. Accurate delineation of the VNO was obtained using a high resolution submillimeter three-dimensional T1-fast field echo (FFE) 3D sequence. Imaging of the VNO in 8 living dogs allowed the description of the morphological MRI features and direct evaluation of its shape and size. The results obtained demonstrate the ability to visualize the VNO in vivo and to evaluate its structure in dogs.

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1. Dzięcioł, M., Niżański, W., Jezierski, T., Szumny, A., Godzińska, E. J., Ochota, M., ... & Pieczewska, B. (2017). The efficiency of synthetic sex pheromones in sexual arousal stimulation in domestic dogs. Polish Journal of Veterinary Sciences, 429-437. https://journals.pan.pl/Content/116799/PDF/v20_n3_429.pdf

The aim of this study was to evaluate the suitability of synthetic bitch sex pheromones (Eau’ De Estrus®, Synbiotics USA) for the stimulation of the reproductive reflexes in adult male dogs. In experiment I: anoestral bitches were applied synthetic (n=6) or natural (n=6) pheromones and their attractiveness was compared to the attractiveness of bitches in natural oestrus, In experiment II: swabs socked in natural estrual discharge or Eau D’Estrus were presented to male dogs and behavioral arousal (Exp. II a) and changes in blood flow in the penile artery reflecting erection (Exp. II b) were recorded. In experiment III the time dedicated for sniffing in experimental dogs was analyzed in relation to the type of attractant presented (natural estrual discharge or Eau D’Estrus). In all three experiments we observed the signs of sexual arousal, increased blood flow in the penile artery and increased time of sniffing only with natural pheromones. No signs of excitation or changes in penis perfusion were noted after the use of artificial pheromones. Our results did not support the suitability of Eau D’Estrus for male dog sexual stimulation.

1. Woszczyło, M., Jezierski, T., Szumny, A., Niżański, W., & Dzięcioł, M. (2020). The role of urine in semiochemical communication between females and males of domestic dog (Canis familiaris) during estrus. Animals, 10(11), 2112. https://www.mdpi.com/2076-2615/10/11/2112/htm?ltclid=

Simple Summary

Canine reproductive behavior can be easily observed; however, the mechanism of semiochemical signaling in this species is still not well understood. Despite numerous studies, no efficient, artificial canine sex pheromones are available. In most studies of canine semiochemical communication, female urine was believed to be a source of volatile compounds that attract males. We hypothesized that urine is also a source of compounds that are very important in the process of the mating decision but are not so volatile. These compounds are collected by licking urine or the vulva and are transferred into the vomeronasal organ. Such behavior always precedes the male’s mating decision. In two experiments, we assessed the reactions of male dogs in response to air containing odor molecules from estrous females’ urine, from a live female in estrus, and from food, as well as during direct sniffing of urine samples from females in estrus, in anestrus, from male dogs and from humans. It was concluded that urine odor is not used for long-distance semiochemical communication in dogs but rather for close distance signaling.

Abstract

This study aimed to assess the mechanisms of semiochemical signal detection in dogs. In the first experiment, five males were exposed to volatile semiochemicals emitted by a live female in estrus and the female’s urine sample collected during estrus. The odor of canine food and clean air were used as controls. In the second experiment, 25 males could directly sniff and lick the urine samples from females in estrus, from females in anestrus, from males and from humans, placed in a lineup. Sniffing, licking and salivation, as well as keeping dogs at different distances from the source of odor, were recorded in both experiments. Experiment 1 showed that food odor was sniffed by males longer than estrous urine. Volatile semiochemicals from females in estrus evoked interest in males but without visual cues did not cause overt symptoms of sexual arousal. In Experiment 2, the estrous urine evoked interest in males and provoked significantly longer sniffing. Licking accompanied by salivation was observed in all instances only during direct contact with estrous urine. The results suggest a complex character of detection of female reproductive status, in which both volatile and nonvolatile compounds emitted by females and present in female urine are involved.

Keywords: canine reproduction; chemical communication; pheromones; urine; VNO

1. Martyna Woszczyło¹, Antoni Szumny², Piotr Knap³, Tadeusz Jezierski⁴, Wojciech Niżański¹, Agata Kokocińska⁵, Michał Dzięcioł^1* "Manual collection of semen in dogs does not affect the concentrations of sex hormones  in the presence or absence of a female in estrus". In press

1. Woszczyło M. 1 , Pasikowski Paweł 2 Sankar Ganesh Devaraj 3 , Kokocińska Agata 4 , Szumny A 5 .,Niżański W. 1 , Dzięcioł M. 1 * Proteomic evaluation of urine of the female of domestic dog ( Canis familiaris), during different stages of ovarian cycle. In press

This part contains the results of a study published between 2012 and 2014, which gave the impulse to focus on the issue of mechanism of chemical communication in dogs during estrus. Semiochemical signaling is supposed to be essential for identification of the female in heat and evaluation of her attractiveness. Natural semiochemicals could influence the male behavior and other physiological parameters.  

Described below results were originally published in listed below publications.

1. Dzięcioł M, Stańczyk E, Noszczyk-Nowak A, Niżański W, Ochota M, Kozdrowski R. Influence of bitches sex pheromones on the heart rate and other chosen parameters of blood flow in stud dogs (Canis familiaris). 2012, Res Vet Sci 93: 1241-1247

2. Dzięcioł M, Niżański W., Ochota M., Kozdrowski R., Stańczyk E. Observation on possibility to identify by the stud dogs the signs of the fertile period in bitches. 2012 Journal of Animal and Veterinary Advances: 11, 962-967.

3. Dzięcioł M., Niżański W., Stańczyk E., Kozdrowski R., Najder-Kozdrowska L., Twardoń J. The influence of antibiotic treatment of bitches in oestrus on their attractiveness to males during mating. 2013 Pol J Vet Sci 16, 509-516
4. M. Dzięcioł, J. Politowicz, A. Szumny, W. Niżański. Methyl paraben as sex pheromone in the canine urine - is the question still open? 2014 Pol J Vet Sci 17, 601-605,

Research topic of papers presented in this dissertation contains current issues related to the phenomenon of semiochemical communication in dogs in the context of the reproductive behavior of domestic dog (Canis familiaris). Semiochemical communication is the oldest and most widespread in nature way of communication used by all species of living organisms (from unicellular organisms through plants, to vertebrates). Substances responsible for caring the information are used to be called semiochemical substances. Those substances may have a different function and could be connected with defense, luring, can act as a repellents, modulate social behavior as well as modulate physiology, including processes related to reproduction. A special group of these substances are pheromones used in communication between individuals of the same species. Among the large number of pheromones the most affecting appear to be a sex pheromones. The modulatory effect on behavior as well as on selected physiological parameters has been demonstrated in many animals’ species and in humans. Modulation of behavior, as well as reproductive parameters such as puberty, stimulation of the sexual reflexes, synchronization of the cycles and attracting the opposite sex, as shown by previous studies are related to the modification of the activity of endocrine glands. A prime example of the impact of sex pheromones in domestic animals is known as “Ram effect”, responsible for the appearance of synchronized ovarian cycles of sexually mature ewes following the introduction to the group of female a sexually mature ram. Although this phenomenon is most relevant in a controlled breeding of small ruminants (the effectiveness of this method is comparable with hormonal synchronization), similar effect of activation of the hormonal axis, responsible for stimulation of the gonads used to be also observed in other species, including dogs. At the turn of the seventies and eighties of the last century, the rapid progress in the field of analytical chemistry associated with technological progress (development of analytical techniques like GC/MS), gave hope for the rapid identification of pheromones - substances for which existing there was no doubt, however, still in absolute most of cases there were no any responsible for the semiochemical communication specific chemicals identify. In 1979 in prestigious journal Science, Goodwin et al. published the results of theirs research on sex pheromones of dogs. Despite the opposite opinion of the another researchers about the findings of Goodwins` team, this work became a canon of the subject, and still is the most frequently cited publication in this field. Adequate high level of attractiveness of females to the male dogs is an essential element for proper conduct of sequences of reflexes leading to copulation. It means that sexual attractiveness is a key element for ensuring the ability of proper, natural reproduction of the animals. Also, in many cases, during performing the assisted reproductive techniques such as artificial insemination, the presence of a bitch in heat, or at least the use of chemical signals (pheromones) obtained from the female is necessary to collect semen from the male dog. The first two papers of the presented cycle: "Influence of bitches sex pheromones on the heart rate and other chosen parameters of blood flow in stud dogs (Canis familiaris)" and "Observation on Possibility Identify it by the Stud Dogs of the Signs of the Fertile Period in Bitches" are focused on the analysis of the physiological phenomena associated with the perception of sex pheromones in dogs, while the third article in the series describes the modulating effect of external factors on the identification of the pheromone signal . The observations made during our researches led us furthermore to attempt to verify, using modern and sensitive analytic methods, the theses describing semiochemical compounds contained in the urine of bitches during oestrus. The last paper from the cycle titled “Methyl paraben as a sex pheromone in the canine urine – is the question still open?” is dedicated to this issue. Identification of semiochemical substances bases on the simple logic diagram of experiment consists of the following elements: collection of the material containing pheromones, identification of compounds, synthesis of analogues, and verification of the activity of those compounds. This simple scheme unfortunately in practice is extremely difficult to carry out. This is mainly due to two key difficulties. Firstly, the sensitivity of modern detection methods is still far below the sensitivity of the natural senses of animals (especially dogs), what is the reason that we can not be sure that all chemical substances contained in the sample actually were detected. The second critical point is the verification of the initial results, namely the analysis of the activity of putative pheromones. This difficulty is due to the fact that the pheromone signal rarely is based on one active compound, and more usually it is a mixture of several substances. Moreover analytical methods used for verification of results are still far from perfect. Still, the main method of verification in vertebrates are the behavioral tests, which, unfortunately, are often insufficiently sensitive, and often difficult to interpret. The aim of the study being the first paper from the cycle within the dissertation was to analyze the impact of natural sex pheromone of bitches on selected physiological parameters of the sexually mature male dogs. The overall objective of this experiment was to develop a method to objectively assess the impact of the sex pheromones on the subject receiving the signal (males) and thus obtain a tool that allowed for reliable analysis of semiochemicals` activity in dogs. During the experiment, we used to evaluate heart rate (HR), as well as using the Doppler technique we analyzed the blood flow in the vessels supplying blood to the corpora cavernous of the penis. In the first case, analysis of records from holter installed on  sexually mature and experienced males showed significantly elevated HR in response to the pheromone signal coming from females in estrus, while signals coming from females in anoestrus did not cause a similar reaction. Observed in the experiment cardiovascular reactivity to the pheromones is in conformity with the findings of other researchers who have demonstrated a modulating (in this case, lowering heart rate) impact of the appeasing pheromones to hear rate of horses in stressful situations. During our study except the evaluation of the blood flow in vessel leading blood to the corpora cavernosa of the penis, as a control also perfusion of the vessels supplying the kidneys and testes were evaluated. Analysis of the results showed that in response to the pheromone signal increased perfusion was observed only in the vessel leading blood to the corpora cavernosa of the penis. There were no changes in blood flow in vessels of testis and kidney. The obtained results showed the usefulness of both evaluated methods (heart rate, blood flow in penis) for the evaluation of influence of sex pheromones on the organism of recipient. It should be noted, however, that the analyzes heart rate appears to be less useful due to the limited specificity (also non-specific stimuli can cause a similar reaction), while the increased perfusion of the corpora cavernosa seems to be a specific response (specificity of this reaction has been previously confirmed by other researchers (Sue and Sachs et al). In the second paper entitled “Observation on possibility to identify by the stud dogs the signs of the fertile period in bitches “ we took attempt to verified suggested by some authors (eg MVR Kustritz, Theriogenology, Reproductive behavior in small animals. Theriogenology 2005 ), although never verified experimentally thesis, that some dogs are able to identify the optimal time for mating in females. During experiment the sexual behavior (level of interest of dogs towards bitches in heat, mating attempts etc.) and the results of  routine laboratory tests (including progesterone levels) determining the optimum time for mating in females were compared. The ovarian cycle of the bitch consists of anoestrus, proestrus, estrus and metoestrus. The so-called heat lasting about 18 - 23 days consist of two phases: proestrus and estrus. In the first phase of the heat bitch however thanks to intensive secretion of volatile pheromone is attractive to dogs, does not allow for mating (no tolerance reflex). From the biological point of view, this period is used for luring more males that can compete for the female (the best chance of covering the female and the transfer of its genetic material has the strongest, dominant individual). Estrus phase, in which the female usually allows for mating contains a fertile period, which is optimal for effective fertilization. It begins approximately four days after ovulation and lasts about three to four days. Routinely this time could be detected by evaluation of the of progesterone level in serum (10-15 ng / ml) and by evaluation of the cyclic changes in the vaginal epithelium (vaginal cytology). Comparative analysis of the behavior of dogs used in the experiment (interest in bitch, mating attempts) and laboratory parameters used for the determination of optimal for mating time showed that seven of the nineteen tested dogs perform mating attempts only during the period of optimal fertility. The obtained results not only verify the suggested positive phenomenon, but also gave very valuable suggestions on the specifics of the secretion of sex pheromones in bitches. On the basis of obtained results it can be concluded that the semiochemical signal emitted by females during heat has got no uniform character, but changes during the course of the cycle. This may suggest the fractional nature of this signal (composition changing during the cycle), as well as, taking into account the high dynamics of hormonal changes accompanying the subsequent stages of cycle, a close connection between the endocrine activity and semiochemical signaling, which was previously reported in the context of sex pheromones in other canines (Raymer, J., Wiesler, D., Novotny, M., Asa, C., Seal, U.S., Mech, L.D., 1986. Chemical scent constituents in urine of wolf (Canis lupus) and their dependence on reproductive hormones. Journal of Chemical Ecology 12, 297-300). It should be noted that the ability to recognize the optimum time for mating is not widespread and is only observed in some individuals. Similar suggestions can be found in the literature. Wierzbowski described similar phenomenon in respect of certain stallions. The question of the quality of semiochemical signals and aspects of its detection are also included in the next paper from the cycle. Both, reports from the literature and our own clinical experience led us to raise the question about the importance of the presence of bacteria in the vagina of the bitches in context of quality of the semiochemical signal emitted during estrus by females. In a paper titled " The influence of antibiotic treatment of bitches in oestrus on their attractiveness to males during mating ", we attempted to assess the importance of reducing the amount of bacteria colonizing the vagina of bitches in heat for the attractiveness of females to experienced male dogs. Bitches vaginal environment under physiological conditions, most of the time is occupied by a small number of microorganisms or persist sterile. The rapid and significant increase in the number of bacteria is observed from the beginning of the heat, while the end of this period is also associated with effective elimination of microorganisms, by large number of neutrophils (beginning of metoestrus). The evaluation of the results of bacteriological examination of the vagina during estrus has been performed by many researchers, but most of them reached the common conclusion that the increased number of bacteria in this period is a physiological phenomenon They used to conclude also that the interpretation of the results of this study in the context of the potential risk to the health of the female is difficult and hence this examination has a limited diagnostic value. The problems with unequivocal a clear diagnosis based on the bacteriological examination and the potential high risk for the males of being infected during the often repeated natural mating was the reason why males` owners often required from the females` owners a health certificate, including the presentation of the results of bacteriological examination of the vagina. This situation has contributed to creation of strong pressure on the antibiotics use prior to mating, which guaranteed negative results of bacteriological examination. Unfortunately, the severity of these practices in a very short period of time has led to numerous reports that despite the detection of optimal mating time and strongly presented tolerance reflex in bitches, males were not willing to mating females. Analysis of the literature in the context of factors that modulate the attractiveness of females in other species, and numerous reports confirming the involvement of the bacterial flora in the creation of the chemical signals, prompted us to undertake studies on the importance of reducing the vaginal bacterial flora in bitches in heat in the context of attractiveness of females to males. During study experienced stud dogs, who earlier confirmed the ability to identify the optimal mating time have been used. The results confirmed that females with sterile vagina were less attractive to males. This observation is consistent with reports of other authors, who conducted similar experiments in other species (sheep, rat) (Ungerfeld and Silva 2005, Merkx et al. 1998 ). The fact that oestrus in treated females was recognized by all males while mating attempt was not taken in most cases, seems to confirm the previously postulated thesis of the complexity of the semiochemical signal emitted by females during the heat. As mentioned earlier, the most recognizable work about sex pheromones in dogs is the study of Goodwin et al. (1979), which postulated methyl paraben as the main sexual attractant isolated from the environment of the bitchs` vagina. Although since that time a few reports questioning those thesis were published, they are not widely known and the most recent publications that address the issue of sex pheromones in dogs refer to the work of Goodwin et al. (Santos et al. 2013, Fatjo 2010). In order to complex analyze the question of sex hormones in females, we attempted to re-verify the earlier conflicting reports. Taking into account both the number of females used in previous experiments (3 to 5 units), the methodology of collection material (collected urine from the kennels` floor) and significantly higher sensitivity of modern methods and GC/MS apparatus, we attempted to re-examine whether methyl paraben actually may be suspected for being the main sex pheromone in bitches. Our previous clinical experience with this substance have not confirmed its activity (Dzięcioł et al. 2011). In our last paper from the presented cycle entitled "Methyl paraben as sex pheromone in canine urine-is the question still open?", despite the development of a new, modern and much more sensitive method of paraben detection, in any samples of urine collected from ten females throughout the all heat we have not found the methyl paraben. The results, together with previously published reports, indicating a complete lack of efficacy commercially available product (E`du estrus) containing methyl paraben just proves that the results published by the team of Goodwin et al. did not identify the sex pheromones of dogs. It should be noted that the methyl paraben is and has been very widely used chemical, both as a food preservative and a component of many cosmetics. Also another substances presented in the work of Goodwin et al., identify as a components of bitches urine are the common identified artifacts (plastelizers)