Urinary Proteins of Female Domestic Dog (Canis familiaris) during Ovarian Cycle.

The presence and identity of non-volatile chemical signals remain elusive in canines. In this study, we aim to evaluate the urinary proteins of female domestic dogs in the estrus and anestrus phases to evidence the presence of non-volatile chemical signals and to elucidate their identities. We collected urine samples from eight female dogs in the estrus and anestrus phases. A total of 240 proteins were identified in the urine samples using liquid chromatography-mass spectrometry (LC-MS analysis). The comparison of the proteins revealed a significant difference between the estrus and anestrus urine. We identified proteins belonging to the lipocalin family of canines (beta-lactoglobulin-1 and beta-lactoglobulin-2, P33685 and P33686, respectively), one of whose function was the transport of pheromones and which was present only in the estrus urine samples. Moreover, proteins such as Clusterin (CLU), Liver-expressed antimicrobial peptide 2 (LEAP2), and Proenkephalin (PENK) were more abundant in the estrus urine when compared to the anestrus urine. LEAP2 was recently described as a ghrelin receptor antagonist and implicated in regulating food intake and body weight in humans and mice. Proenkephalin, a polypeptide hormone cleaved into opioid peptides, was also recognized as a candidate to determine kidney function. As of yet, none of these have played a role in chemical communication. Clusterin, an extracellular chaperone protecting from protein aggregation implicated in stress-induced cell apoptosis, is a plausible candidate in chemical communication, which is a claim that needs to be ascertained further. Data are available via ProteomeXchange with the identifier PXD040418.

The influence of manual semen collection in male trained dogs (Canis familiaris), in the presence or absence of a female in estrus, on the concentrations of cortisol, oxytocin, prolactin and testosterone.

Sex pheromones are chemical substances secreted into the environment that affect the physiology and behavior of recipients. Females use these compounds during oestrus to attract males, which leads to attempts of mating. This study evaluates the influence of manual semen collection in male dogs, in the presence or absence of a female in estrus, on the blood concentrations of cortisol (CRT), oxytocin (OXT), prolactin (PRL) and testosterone (T), as hormones involved both in the physiology of reproduction and stress. Ten male dogs were used in Experiment 1 to measure the serum and plasma concentrations of the aforementioned hormones in the absence of semen collection. Subsequently in the same animals, the concentrations of these hormones were evaluated before and after semen collection in the presence (Exp. 2) or in absence of a female in estrus (Exp. 3). No significant changes in hormone concentration caused by the semen collection were found, either with, or without the presence of female in estrus. Obtained results suggest that the procedure of manual semen collection in dogs, probably due to its passive character, does not stimulate endocrine glands to secrete hormones, and the process of ejaculation is probably controlled by neural pathway. The lack of effect of semiochemical stimulation to the CRT, PRL, OXT and T level, could be caused by a short contact with female during semen collection. Further studies on involvement of the hormones during the process of natural mating, especially preceded by long courtships, similar to that observed under natural conditions, should shed a light on the physiology of mating and the connection between the endocrine system and semiochemical stimulation in dogs.

Canine Smell Preferences-Do Dogs Have Their Favorite Scents?

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.

The Case of Atypical Sexual Attractiveness in a Male Domestic Dog-A Case Study.

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.

Canine Olfaction: Physiology, Behavior, and Possibilities for Practical Applications.

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).

The Role of Urine in Semiochemical Communication between Females and Males of Domestic Dog (Canis familiaris) during Estrus.

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.

MRI Features of the Vomeronasal Organ in Dogs (Canis Familiaris).

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.

Identification of putative volatile sex pheromones in female domestic dogs (Canis familiaris).

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.