Interplay of hormones and metabolite excretion with fern pattern prove saliva as a potent indicator of male reproductive status in Kangayam breed cattle.

Kangayam cattle are one of the drought breeds in India with distinct attributes. Agricultural transformation has led to a decline in many pure-breed indigenous cattle, including the Kangayam breed. Hence, a study on the reproductive physiology of male Kangayam breed cattle is necessary to disentangle problems in the area of livestock improvement. In this study, we investigated the relationship between serum hormones and bio-constituents and ascertained the potential of saliva as an indicator of the reproductive status of Kangayam cattle (Bos indicus). The present study confirms that cholesterol was higher in intact males and lower in prepubertal and castrated males. Testosterone levels were also higher in intact males than in castrated or prepubertal males. Hence, it can be inferred that high cholesterol levels contribute to active derivatization of testosterone in intact males. In contrast, reduced cholesterol availability leads to decreased testosterone synthesis in castrated and prepubertal males. Furthermore, it is reasonable to speculate that testosterone could have influenced salivary fern patterns in intact males, and thus, fern-like crystallization in the saliva was apparent. The unique salivary compounds identified through GC-MS across various reproductive statuses of Kangayam males may advertise their physiological status to conspecifics. In addition, the presence of odorant-binding protein (OBP) in saliva further supports its role in olfactory communication. This study attested to a posssible interlink between gonadal status and serum biochemical profiles. The salivary fern pattern revealed in this study can be used as a predictive tool, and the presence of putative volatiles and OBP adds evidence to the role of saliva in chemical communication.

Mechanistic Insights into the Binding of Boar Salivary Pheromones and Putative Molecule with Receptor Proteins: A Comparative Computational Approach.

Precise estrus detection in sows is pivotal in increasing the productivity within the pork industry. Sows in estrus exhibit exclusive behaviors when exposed to either a live boar or the steroid pheromones androstenone and androstenol. Recently, a study employing solid-phase microextraction-gas chromatography-mass spectrometry has identified a novel salivary molecule in boars, known as quinoline. This finding has intriguing implications as a synthetic mixture of androstenone, androstenol, and quinoline induces estrus behaviors in sows. Nevertheless, the precise pheromonal characteristics of quinoline remain elusive. In this study, we validate and compare the binding efficiency of androstenone, androstenol, and quinoline with porcine olfactory receptor proteins (odorant-binding protein [OBP], pheromaxein, salivary lipocalin [SAL], and Von Ebner's gland protein [VEGP]) using molecular docking and molecular dynamics simulations. All protein-ligand complexes demonstrated stability, as evidenced by the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen-bond (H-bond) plots. Furthermore, quinoline displayed higher binding efficiency with OBP, measured at -85.456 ± 8.268 kJ/mol, compared to androstenone and androstenol, as determined by molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) calculations. Conversely, quinoline exhibited a lower binding efficacy when interacting with SAL, pheromaxein, and VEGP compared to androstenone and androstenol. These findings, in part, suggest the binding possibility of quinoline with carrier proteins and warrant further investigation to support the role of quinoline in porcine chemical communication.

Pheromones, binding proteins, and olfactory systems in the pig (Sus scrofa): An updated review.

Pigs utilize multimodal communication for reproductive and other behaviors, and chemical communication is one of the key components. The success of reproduction relies on chemical communication favored by the steroid pheromones from boar saliva. These steroids were proven to be involved in advancing puberty in gilts (the boar effect) and in promoting estrus behaviors in gilts/sows, thereby helping to detect estrus and facilitating the timing of artificial insemination. The steroid pheromones bound with carrier proteins are evidenced in the mandibular (submandibular) salivary secretions of the boar. These salivary steroids bind with carrier proteins in the nasal mucus and vomeronasal organ (VNO) of the sows, eventually triggering a cascade of activities at the olfactory and endocrine levels. Besides steroid pheromones, pig appeasing pheromones (from mammary skin secretions of sows) have also been demonstrated to bind with carrier proteins in the nasal mucus and VNO of the piglets. Thus far, four different proteins have been identified and confirmed in the nasal mucus and VNO of pigs, including odorant binding proteins (OBPs), salivary lipocalin (SAL), pheromaxein, and Von Ebner's Gland Protein (VEGP). The critical roles of the chemosensory systems, main olfactory systems and VNO, have been comprehensively reported for pigs. This review summarizes the current knowledge on pheromones, their receptor proteins, and the olfactory systems of porcine species.

Pig pheromones and behaviors: A review.

Chemical signals play indispensable roles in the communication and social behavior of many organisms. Pheromones are a class of chemical signals identified initially in insects. Later, it became evident that diverse animals secrete pheromones in their body exudates and from exocrine glands and use them for social communication. The pig is a vital food animal in which steroid pheromones have been identified and their behavioral effects known since the 1960s. More recently, non-steroidal pheromones have been identified. To date, studies have reported various pheromone sources in boars (saliva, urine, and glandular secretions) and sows (urine, mammary gland secretions, and feces) and pheromone-mediated behavioral consequences are evidenced. These include the boar effect on puberty onset and subsequent estrous behaviors as well as agonistic and avoidance behaviors. Recent research has facilitated the development and evaluation of pheromone-based applications and products to improve the welfare and reproductive performance of pigs. This review aims to summarize the current knowledge of pig pheromones, and their implications in behaviors.

Screening of potential drug for Alzheimer's disease: a computational study with GSK-3 ß inhibition through virtual screening, docking, and molecular dynamics simulation.

The global impact of Alzheimer's disease (AD) necessitates intensive research to find appropriate and effective drugs. Many studies in AD suggested beta-amyloid plaques and neurofibrillary tangles-associated tau protein as the key targets for drug development. On the other hand, it is proved that triggering of Glycogen Synthase Kinase-3ß (GSK-3ß) also cause AD, therefore, GSK-3ß is a potential drug target to combat AD. We, in this study, investigated the ability of small molecules to inhibit GSK-3ß through virtual screening, Absorption, Distribution, Metabolism, and Excretion (ADME), induced-fit docking (IFD), molecular dynamics simulation, and binding free energy calculation. Besides, molecular docking was performed to reveal the binding and interaction of the ligand at the active site of GSK-3ß. We found two compounds such as 6961 and 6966, which exhibited steady-state interaction with GSK-3ß for 30 ns in molecular dynamics simulation. The compounds (6961 and 6966) also achieved a docking score of -9.05 kcal/mol and -8.11 kcal/mol, respectively, which is relatively higher than the GSK-3ß II inhibitor (-6.73 kcal/mol). The molecular dynamics simulation revealed that the compounds have a stable state during overall simulation time, and lesser root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) values compared with co-crystal. In conclusion, we suggest the two compounds (6966 and 6961) as potential leads that could be utilized as effective inhibitors of GSK-3ß to combat AD. Communicated by Ramaswamy H. Sarma.

Upregulation of Cyp19a1 and PPAR-γ in ovarian steroidogenic pathway by Ficus religiosa: A potential cure for polycystic ovary syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Quite a few plants are in use to treat female infertility and associated problems. Availing the cues from traditional knowledge, phytochemical studies and ethnopharmacological evidences, the aphrodisiac plant Ficus religiosa (F. religiosa) is widely in use to cure infertility in women. For instance, the juice of leaf and aerial root of F. religiosa is reported to normalize the dysregulated menstrual cycle in women. Besides, it is believed that regular circumambulation of F. religiosa during the early hours of the morning helps women in alleviating infertility which could be attributed to the potential phytovolatiles released from F. religiosa. However, the evidences for therapeutic potential of F. religiosa in treating female infertility are arbitrary and mostly anecdotal. AIM OF THE STUDY: The present study was aimed at examining if extracts of fresh and/or dry leaf of F. religiosa would cure polycystic ovary syndrome (PCOS) in the rat model. METHODS: Rats were divided into seven groups; control (Group I), PCOS-induced (P.O, Letrozole -1 mg/kg BW for 21 days) and untreated (Group II), PCOS-induced and treated with the leaf extracts of F. religiosa (Groups III-VI), and, PCOS-induced and treated with pioglitazone (Group VII). The estrous intervals, body and organ weights (ovary and uterus), and serum hormones (testosterone, luteinizing hormone [LH], estrogen, and progesterone) were measured, and the expression of Cyp19a1 (aromatase), and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) were assessed in the experimental rats. The levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and antioxidants (MDA, GSH, GPx, SOD, and CAT) were also quantified. Besides, the putative volatile compounds in the esterified leaf extracts were identified using Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: Letrozole treatment induced irregular estrous and altered weight of organs and hormonal milieu, which were reverted to normal in leaf extracts-treated PCOS-induced rats. Remarkably, fresh leaf treatment up-regulated Cyp19a1and PPAR-γ and increased the levels of 3ß-HSD and 17ß-HSD. We found 3-acetoxy-3-hydroxy-propionic acid in fresh and dry leaf extracts, which we attribute to efficacy of the extracts in alleviating PCOS. CONCLUSION: Put together, our findings suggest the leaves of F. religiosa as potential in alleviating PCOS, mainly due to the presence of putative volatile molecules. Further screening of the leaves of F. religiosa is recommended to identify other key molecules and to develop a systematic therapeutic intervention for PCOS.

Adipose-Derived Molecules-Untouched Horizons in Alzheimer's Disease Biology.

The global incidence of Alzheimer's disease (AD) is on the rise with the increase in obesity and metabolic disease epidemic. Obesity is co-morbid with the increase in mass of adipose tissue, which secretes numerous molecules that are biologically important. Obesity and its associated conditions are perhaps involved in the causative pathway of AD. Immunologically important cytokines such as IL-1ß, IL-10, and IL-18, which are released by adipose tissue, are also found to be associated with AD. Besides, the expression of IL-6, IFNγ, and TNF alpha are also associated with AD. Ang-I and Ang-II are found to mediate the progression of AD. Complement factors B, C4b, and H are differentially expressed in AD. Overall, several adipocyte-derived cytokines are found to be dysregulated in AD, and their role in AD remains to be studied. The induction of autophagy is a very promising strategy in the treatment of AD. A variety of adipose-derived molecules have been shown to modulate autophagy. However, very little literature is available on the role of adipose-derived molecules in inducing autophagy in microglial cells of AD. Understanding the role of adipose-derived molecules in the development of AD, especially in the induction of autophagy, would open up new avenues in devising strategies for the treatment of AD.

Identification of potential pheromone source in sows.

Pheromones play a pivotal role in intra-species communication for reproduction and social behavior in a variety of mammals, such as boars. For boars, saliva is a rich source of pheromones, however, the identification of additional sources and relative abundance of pheromones in various body fluids of sows is also essential to understand the reproductive behaviors of pigs. The present study was designed to identify the source(s) of pheromones in sows. We collected urine, feces, saliva and cervical mucus/vaginal wash samples from sows at pre-estrus, estrus and post-estrus phases, and from gilts and exposed boars to each of these potential sources of pheromones. All the boars tested spent more time sniffing and hyper-salivating in response to urine from sows in estrus than that from sows not in estrus. The sniffing behavior of boars towards estrus samples differed from that towards the samples from non-estrus sows (P < 0.005) and gilts (P < 0.001). Further, hypersalivation behavior of boars differed between estrus samples and gilt samples (P < 0.05) and estrus samples compared to pre-estrus samples (P < 0.05). This is an indication that pheromones are abundant in the estrus samples. We conclude that urine of estrus sows can be a rich source of pheromones and the same can be used to identify, purify and characterize novel pheromone molecules.

Urinary volatile metabolomics as a viable alternative diagnostic tool for polycystic ovary syndrome: An exploratory hypothesis.

Polycystic Ovary Syndrome (PCOS) is a metabolic disorder prevalent globally. Female infertility cases are also on the increase during the recent times which almost matches with the increasing incidence of PCOS. The NIH-USA-defined symptoms for clinical confirmation of PCOS include oligo-ovulation, elevated androgen level and presence of cysts in the ovary. Therapeutic approaches to PCOS require confirmatory diagnostics such as measurement of hormones and ultrasound scan of the ovary, which are in part, invasive. Conversely, the volatile organic compounds (VOCs) that are present in body fluids (urine, feces, saliva, etc.) and exhaled breath are reported to be endogenously altered in diseased state, which may be indicative of diseases including cancer. We hypothesize that the hindered metabolic state in PCOS condition would conditionally alter the VOCs that eventually are excreted in urine, which may offer a template to develop a viable and non-invasive diagnostic tool.

Squalene is a potential endocrine modulator in rat: A proof-of-principle study with 3-methylcholanthrene-induced toxicity.

Oestrus urine was proved as a potential endocrine modulator in alleviating the toxicity induced by 3-methylcholanthrene (3-MC) in male rats. We, in this study, aimed to prove the attributing potential of toxicity alleviation to squalene, an oestrus-specific pheromone in rats. A single dose of 3-methylcholanthrene (25 mg/kg BW, i.p.) was administered to male Wistar rats with concurrent exposure to squalene sprayed in bedding material (Group III). Group II rats did receive 3-MC treatment but did not expose to squalene. Group I rats were intact control neither administered 3-MC nor sprayed with squalene. In consequence of 3-MC toxicity, liver and testes weights were increased and the components of blood cells (RBC and WBC count, Hb level) and testosterone concentration were significantly reduced in Group II rats. Moreover, sperm count was reduced and antioxidants (testes and epididymis) were significantly altered. Exposure to squalene in Group III rats comparatively normalised all the variable components towards baseline and reorganised the histological architecture of reproductive tissues that were exacerbated with 3-MC toxicity. This study ultimately proved squalene as a potent molecule in alleviating the toxicity induced by 3-methylcholanthrene.

A correlation of fecal volatiles and steroid hormone profiles with behavioral expression during estrous cycle of goat, Capra hircus.

Chemical signals (both volatile and non-volatile) form the major communication channels in animals. These signals are transferred mainly through excretory sources to facilitate inter-individual communication. In particular, the reproductive cycle of female mammals, including goats, exhibits significant changes in the constituents of their excretory products, and female mammals also express different behavioral patterns. We propose that feces is one of the important sources of chemo-signals in goats. However, the behavioral patterns and analysis of excretory sources based on chemical communication have not yet been studied in the Indian goat, Capra hircus. To validate our hypothesis, we analyzed the behavioral patterns and the volatiles and steroid hormone profiles in the feces samples of female goats during the estrous cycle. Here, we synchronized the estrous cycle in six female goats and obtained feces samples. The samples were extracted with dichloromethane and analyzed using gas chromatography-mass spectrometry. A portion of the sample was used for hormone assay to confirm the phases in the estrous cycle. Induction of she-goats into estrus was detected from the vaginal swelling, mucus discharge, restlessness, reduced milk secretion, bellowing, bleating, frequent urination, standing heat, allowing the male to mount, mounting on other females and teasing of males. The repeated male behaviors viz., flehmen, mounting, penile protrusion, body rubbing, dominance over other males and finally coitus with estrus female by male goats were observed. Analysis of volatiles revealed a total of twenty-four compounds combining all the phases in the estrous cycle. Among those, some of the volatile compounds and two antioxidants (ascorbic acid and vitamin E) were estrus-specific. Based on the fecal steroid analysis, higher level of estradiol during estrus and higher level of progesterone during post-estrus were observed. The behavioral patterns of female and male goats combined with qualitative differences in the volatile compounds and the two antioxidants rendered the estrus identifiable. Furthermore, the fecal steroid analysis also supported the detection of hormonal status during the estrous cycle. To the best of our knowledge, this is the first report correlating the behavior with volatiles and hormones in the feces samples from female Indian goats. It is concluded that the volatile pattern and hormone profile in feces, supported by specific behavioral patterns, should be considered a better modality of non-invasive estrus detection in goats.

Response of male mice to odours of female mice in different stages of oestrous cycle: self-grooming behaviour and the effect of castration.

The behavioural assays were carried out in a Y-maze wherein intact, castrated and testosterone-treated male mice were exposed to oestrus and non-oestrus urine samples. The intact male mice investigated more frequently and spent more time in the Y-maze arm with oestrus urine than in that with non-oestrus urine. In contrast, the castrated mice were not attracted to oestrus urine, whereas testosterone-treated mice showed preference for oestrus urine. The rate of self-grooming was higher in intact males in case of exposure to oestrus urine while the rate was lower with respect to non-oestrus urine. However, castrated mice exhibited less self-grooming behaviour which was partially restored by testosterone treatment. The results suggest that self-grooming behaviour is an indicator of detection and discrimination of oestrus by males, and supports the androgen role in male chemosensory ability to discriminate between oestrus and non-oestrus female odours.

Faecal chemical cues in water buffalo that facilitate estrus detection.

Chemo-signals are among the reliable non-invasive methods for estrus detection in mammals. Water buffalo is a silent heat animal and, hence, there is search for chemo-signals which would be effective non-invasive indicators of estrus state. We analyzed the faecal chemical cues during the estrous cycle in buffalo and to find the estrus-specific faecal volatile compounds adopting bull behavior assay. The faecal samples were collected at three phases of the estrous cycle (i.e., proestrus, estrus and postestrus) and subjected to gas chromatography-mass spectrometry analyses. We found 27 volatile compounds in the faeces of buffaloes, of which 4-methyl phenol (4mp) and trans-verbenol (tv) were found only in estrus faeces. The faecal samples of estrus buffaloes and the estrus-specific compound(s) (4mp+tv) at three different concentrations were tested for behavioral responses (flehmen and mounting behavior) in the bull. The bulls exhibited repeated flehmen when exposed to a combination of the two compounds (i.e., 4mp+tv) as compared to the individual compounds or raw faecal sample collected from buffalo when in estrus (P<0.05). However, higher number of mounting behavior was recorded when bulls were exposed to 4mp followed by a combination of the two compounds (4mp+tv) and trans-verbenol (P<0.05), in that order. By contrast, less number of mounting behavior was exhibited by bulls when exposed to the control sample (i.e., Hexadecanoic acid) (P<0.05). As inferred from the bull behavior assay, the present study suggests that the two compounds, 4 methyl phenol and trans-verbenol would be reliable indicators of estrus in buffaloes.

Increased squalene concentrations in the clitoral gland during the estrous cycle in rats: an estrus-indicating scent mark?

Squalene in the rat clitoral gland is reported to be semi-volatile and may serve as a chemo-signal. The objective was to determine squalene concentrations in the clitoral gland throughout the reproductive cycle. Clitoral glands were extracted with dichloromethane; 23 compounds were identified with Gas Chromatography linked Mass Spectrometry (GC-MS). Since squalene concentrations were significantly higher during proestrus and estrus, and remarkably reduced during metestrus and diestrus, we inferred that it could be an ovulation-indicating chemosignal in the female rat, acting as a scent mark for the male. This hypothesis was tested by investigating its efficacy to attract males, including studying the role of the olfactory-vomeronasal system of the male in perceiving squalene. For detection of squalene, males used their conventional olfactory system when at a distance from the female, whereas the vomeronasal organ was used when they were in close proximity to the female. We concluded that squalene was a female-specific chemosignal that attracted males, and furthermore, that the olfactory-vomeronasal system had an important role in the perception of squalene.

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection.

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.