Indian Journal of Biochemistry & Biophysics Vol. 55, June 2018, pp. 183-190 Buck odor production in the cornual gland of the male goat, Capra hircus– Validation with histoarchitecture, volatile and proteomic analysis Devaraj Sankarganesh1,#, Rajamanickam Ramachandran1,2, Radhakrishnan Ashok1, Veluchamy Ramesh Saravanakumar3, Raman Sukirtha1, Govindaraju Archunan4* & Shanmugam Achiraman1,4* 1Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India 2Department of Microbial Biotechnology, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India 3Department of Livestock Production and Management, Veterinary College and Research Institute, Namakkal-637 002, Tamil Nadu, India 4Center for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli- 620 024, Tamil Nadu, India Received 28 January 2018; revised 14 May 2018 In many animals, glandular secretions or pheromones that possess biological moieties contain messages encoded by the intrinsic smell. In male goats, the cornual gland (a sebaceous gland), may synthesize and excrete relevant chemical components that are responsible for the ‘buck effect’. To test this, cornual glands from freshly-slaughtered male goats (N=6) were subjected to histoarchitecture analysis, to infer about the structural alignment, to the GC–MS analysis for volatile compounds and to SDS–PAGE for protein profiling followed by MALDI-TOF to characterize specific protein bands. The gland possesses sebum, vacuoles and hair follicles inferring its capability to synthesize and extrude the scent. We found 14 volatiles in GC–MS analysis, in which 1-octadecanol might be a putative pheromone of buck odor. We identified seven different proteins in SDS-PAGE. Two proteins, 28 and 33 kDa, were highly matched with DNA mismatch repair protein and Abietadiene synthase, respectively, as inferred from MALDI-TOF. Conclusively, the volatiles identified in the cornual gland suggest that the structural microelements of the gland may synthesize (sebum and vacuoles) and release the key volatiles through the hair follicles. The volatile(s) thus produced in male goats either solely or synergistically may confer the buck odor. Keywords: GC–MS, Goaty odor, MALDI-TOF, Putative pheromone, Scent gland, Sebum Physiological status of animal can be assessed by synchronization of estrus by increasing the LH pulse conspecifics through a variety of scent sources, rate and leads to ovulation (buck effect). Perhaps all including glandular secretions. Mammalian the stimuli (odor, sight, sound, and touch) are pheromones are characterized by multiple compounds mutually involved; odor seems to be a key element in used in intra-specific communication1. In goats, ‘buck regulating the buck effect2. Hamada et al.3 utilized the odor’ is believed to be produced in the skin/hair/gland hair samples of males as an odor source and exposed of intact males and has the capacity to synchronize/ it to the female goats for a short period and found an regulate the reproductive cycle and ovulation in the increase in GnRH pulses in females. Subsequently, female. The introduction of sexually naïve bucks Ichimura et al.4 exposed the female goats with the to a group of non-ovulating females results in pooled hair samples of males in a muzzle-fitted mask relatively for a longer period (4 h) and observed —————— *Correspondence: greater GnRH pulses. The compound responsible for 5 Phone (M): +91-9894835753 (S. A.); +91-9443922228 (G. A.) the buck odor was identified as 4-ethyloctanoic acid . E-mail: [email protected] (S. A.); [email protected] (G. A.) Lancker et al.6 reported a sebaceous gland #Present address: Department of Biotechnology, School of Bio underneath the horns of the male goats. Earlier, the and Chemical Engineering, Kalasalingam Academy of Research gland was swabbed and volatile compounds were and Education, Krishnankoil-626 126, Tamil Nadu, India identified in an attempt to validate the buck effect, but Abbreviations: GC–MS, Gas Chromatography-Mass Spectrometry; no conclusive information was derived as decades-old SDS-PAGE, Sodium dodecyl sulfate-Polyacrylamide gel chemical separation (chromatography) techniques electrophoresis; MALDI-TOF/MS, Matrix-assisted laser 7 desorption/ionization-time of flight/Mass Spectrometry; PBS, were adopted . Interestingly, the upper dermal layer Phosphate-buffered saline; MMR protein, Mismatch repair protein extracts of sebaceous glands from intact and castrated 184 INDIAN J. BIOCHEM. BIOPHYS., VOL. 55, JUNE 2018 plus testosterone administered goats were shown to clearly indicate that hair/skin samples of head area induce greater multiple unit activities in female contain compounds of goaty odor, which is goats8. Further, it is also demonstrated that the extract presumably due to the presence of sebaceous glands of cutaneous gland possesses active ingredients to (cornual gland). However, the study of the cornual 9-11 11 produce buck odor . Remarkably, Murata et al. gland in the perspectives of histology, and volatile identified a single olfactory molecule (4-ethyloctanal) analysis in the extracts of the gland is not performed, that is responsible for the elicitation of buck effect by which may further shed light on buck effect. confirming the multiple unit activity and induction of Therefore, we embarked a study on the cornual gland LH pulses in female goats. However, hitherto no to 1) Confirm the secretory and excretory nature of study was performed in the extracts of the cornual the gland by subjecting it to histological analysis, 2) gland to identify the complete volatiles. Detect the complete volatiles (putative pheromones) In many animals, the scent gland and the volatile in the extract of the gland by GC–MS analysis, and 3) compounds produced in it have pivotal roles in To elucidate the protein profile to look for the communication among conspecifics. For instance, presence of carrier proteins, if any. Atta laevigata, an ant, has a mandibular gland (modified scent gland) and the concentration of the Materials and Methods compound it synthesizes varies in relation to the Test animals and collection of gland samples castes12. In male lizards, the femoral gland with Adult male goats (N=6), Capra hircus fed with granular cells is the major source of pheromones13. standard ration and water ad libitum were used for the The mammary gland of the rabbit secretes milk that present study. The inclusion criterion was sexually contains 2-methyl but-2-enal, which is believed to be intact male goats and they were not castrated. The a mediator of mother-young interaction14. The goats were sacrificed at the slaughter house during preputial gland of rodents has been reported to play a April-May when their age was <24 months, and key role in communication between sexes. For 12 gland samples (2 from each) were obtained. The instance, in an earlier investigation, we reported that gland was located as described previously6. The hairs the clitoral gland of female laboratory rats contains an in the caudomedial border beneath the horns were estrus-specific compound which could be a mediator shaved and the gland was located as a distinct 15 of sexual communication . In addition, synthesis of oval-shaped structure with 1–3 cm in length and the key constituents of the preputial gland of male rat 0.5–1.5 cm wide. We used one gland from each goat is testosterone-dependent, which indicates that there is for extract preparation by homogenizing with PBS. a relationship between glandular secretions and sexual This extract was used for volatile analysis in GC–MS 16 status of animals . and remnant of the extract was processed for In hoofed mammals, (e.g., deer) the preorbital SDS–PAGE analysis. Another gland from each goat gland is a good source of chemical compounds that was subjected to histological sectioning and facilitate chemo-communication, and some of these representative of single gland structure was shown in compounds have been implicated in the expression of results. The study does not involve live animals, and dominant status by a particular male animal the glands were collected from the monitored animals 17 in the colony . Considerable difference in the after sacrification at the slaughter house. histoarchitecture of preorbital gland has been shown between territorial and non-territorial Indian Histoarchitectural analysis blackbuck18,19. In elephants, the temporal gland The glands were dissected and fixed immediately secretes a fairly good quantity of musth, which is in 10% neutral buffered formalin, and subsequently reported to be androgen-dependent20. The axillary embedded in paraffin. The sections (4 µm) were gland secretion is one of the important sources of made, and stained with haematoxylin and eosin. The human pheromones. The secretory material has a stained sections on glass slides were examined under peculiar smell due to microbial action and confers the a light microscope (Wetzlar Hund, Germany) and message of maleness21. photographs were captured with real-time Considering the large population size and great interpretations by a veterinary anatomist. diversity in mammals, reports are fragmentary in Sample preparation support of the glandular sources performing roles in The glands from six goats were sliced into small chemical communication. In goat, previous reports pieces using sterilized blades, which were SANKARGANESH et al.: BUCK ODOR PRODUCTION IN THE CORNUAL GLAND OF THE MALE GOAT 185 homogenized using a sterilized homogenizer