Results

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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 Dzięcioł1* "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