Preliminary study

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)