Multidimensional analysis of scent sources with behavioural observation in male and female goat (Capra hircus) pertaining to livestock improvement

Thesis submitted to Bharathidasan University for the award of the degree of

DOCTOR OF PHILOSOPHY IN ENVIRONMENTAL BIOTECHNOLOGY

Submitted by

Mr. D. SANKAR GANESH M.Sc.,

(30660/Ph.D.1/Environmental Biotech/Full Time/January 2010)

Under the guidance of

Dr. S. ACHIRAMAN

Department of Environmental Biotechnology School of Environmental Sciences Bharathidasan University Tiruchirappalli – 620024 Tamilnadu, India

June 2014

Certificate

This is to certify that this thesis entitled “Multidimensional analysis of scent sources with behavioural observation in male and female goat (Capra hircus) pertaining to livestock improvement” submitted to Bharathidasan University, in partial fulfillment of the requirements for the award of Doctor of Philosophy in

Environmental Biotechnology is a record of original work done by Mr. D. Sankar

Ganesh in the Department of Environmental Biotechnology, Bharathidasan

University, under my supervision and the thesis has not been formed the basis for the award of any Degree/ Diploma/ Associateship or other similar title to any candidate of any University/ Institute.

(S. ACHIRAMAN)

Mr. D. SANKAR GANESH Doctoral Research Scholar

DECLARATION

I, D. Sankar Ganesh hereby declare that the thesis entitled with

“Multidimensional analysis of scent sources with behavioural observation in male and female goat (Capra hircus) pertaining to livestock improvement” submitted to

Bharathidasan University, in partial fulfillment of the requirements for the award of

Doctor of Philosophy in Environmental Biotechnology is a record of original and independent work done by me under the supervision of Dr. S. Achiraman, Assistant

Professor, Department of Environmental Biotechnology, Bharathidasan University,

Tiruchirappalli and this thesis has not been formed the basis for the award of any

Degree/ Diploma/ Associateship or other similar title to any candidate of any

University/ Institute.

Place: (D. SANKAR GANESH) Date:

Dedicated this work to my Family & Beloved Guide…

ACKNOWLEDGEMENT

The writing of this thesis has been an incredible journey and a monumental milestone in my academic life. I could not have embarked on this expedition and traveled this far without the passionate and continued support of advisors, colleagues, friends and my family.

Words are not enough express my gratitude to my family members, Mr. G. Devaraj, Mrs. Ponnuthai Devaraj, Mr. D. Muniraj, Mrs. Sumathi Muniraj, Ms. D. Ambika and kutty M. Sharika without their patience and persistence this thesis would not have been possible.

This is my respectful commitment to thank Dr. S. Achiraman, Assistant Professor, Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli - 24 for his responsibility of guidance and supervision on my research carrier. His independency on my research work, friendly in approaches and his kind support are the secret of completion of my work soon. He is great ambassador of real meaning of guide and supervisor and also thanks his family for their love and affection.

I wish to express my sincerest thanks to Dr. M. Krishnan, Professor and Head, for his support and endowing instrument facilities throughout my research.

Dr. G. Archunan, Professor and Head, Department of Animal Science, Bharathidasan University has helped in so many ways to enable and shapes this works while continue to provide firm support and constructive feedback along the way. For that, I will always be grateful. He is a wonderful guide and ally whom one has the good fortune to know.

Dr. S. Kannan, Professor and Head, Department of Zoology, Periyar University, Salem, he is one who initiated and kindled my research interest and makes me as now.

I also thank other faculty members Dr. R. Babu Ranjendran, Professor, Dr. M. Govindaraju, Dr. K. Thamaraiselvi, Dr. T. Sivasudha, Dr. M. Vasanthy and Dr. S. Uma Maheswari, Assistant Professors, Department of Environmental Biotechnology for their support and encouragement.

I thank Dr. M. A. Akbarsha, Director and Chair, Mahatma Gandhi-Dorenkamp Centre (MGDC), for his constant and ever-smiling face in my manuscript correction and critical reading. I thank Dr. V. Ramesh Saravanakumar, Professor and Head, Department of Livestock Production and Management, and The Dean, Veterinary College and Research Institute, Namakkal, for their timely help in sample collection and for the animal facility.

Dr. A. Rajendiran, Assistant Professor, Department of Zoology, Arignar Anna Govt. College, Musiri and Dr. Premkumar, Assistant Professor, Department of Biomedical Sciences, Bharathidasan University, though not formally as my doctoral committee members, has enlightened me in so many aspects about innovations, ideas and other academic matters.

This is my duty to thank Dr. R. Thirumurugan, Associate Professor of Animal Science, Dr. A. Lakshmi Prabha, Assistant Professor of Plant Science, Dr. K. Anbarasu and Dr. R. Rajakumar, Assistant Professor of Marine Biotechnology Dr. Prashanthi Devi, Assistant Professor of Environmental Management and their family members for the constant support and encouragement.

I wish to record my heartful thanks to my brothers Dr. S. Kamalakkannan, Dr. Thirupathi Pichiah, Dr. A. Sankarganesh, present and past fellows of our Laboratory and their family members for their encouragement and timely help whenever I need. I also express my gratitude to Dr. K. Ramesh Kumar, Dr. A. Balasundaram, Dr. V. Masilamani, Dr. S. Alagumanian and Mrs. Shobana Alagumanian for their moral and friendly support in the due course of my research.

My sincerest gratitude also goes to Dr. C. Bharathiraja, Dr. V.R. Muthukumaran, Dr. P. Ponmanickam, Dr. T. Rajagopal, Dr. K. Karthikeyan, Dr. Isai Mathivanan, and past and present scholars of Dr. Archunan’s lab without whose encouragement and support I could not have come this far.

I thank Dr. Rajkumar, A-Star Univesity, Singapore for his timely help in 2- Dimensional Electrophoresis analysis and constant support.

I owe gratefulness to Dr. K. Muthuchelian, Professor and Head, Department of Bioenergy, Madurai Kamaraj University, Madurai and Dr. P. Gunasekaran, Vice- Chancellor, Thiruvalluvar University, Vellore for offering me the summer training programme during my M.Sc., and winter school training during my Doctoral research.

I thank Dr. L. Uma, Professor and Director, NFMC, Dr. D. Prabakaran, Professor, NFMC, for providing me the GC facility for fatty acid analysis. I thank Dr. N. Thajuddin, Professor of Microbiology, BDU for his timely help in lyophilization facility.

I also express my thanks to Dr. Srinivasan, Mrs. Malini Srinivasan, AVRDC, Taiwan, Dr. Padmanabhan, Nanyang Technological University, Singapore and Mrs. Sabarina, Australia for their homely support at Taiwan, Singapore and Australia.

I would also like to thank the non-teaching technical community, especially Mr. Tharasingh, Mr. Murali, Mr. Logesh, Mr. Thirupathi, Mr. Rajangam, Mr. Muruganantham, from Department of Environmental Biotechnology, Mr. Chelladhurai, Mrs. Krishnaveni, Mr. Kumarasamy, Mr. Kumar Administrative building, BDU, among many others, for their professional and administrative backup and for securing a place where we can rest our fatigued minds.

A book with thousand pages starts from single dot. This is my intense to thank my school teacher, Mr. Arockiyaraj, K. H. S. School, Krishnaperi (2000-2004) and Dr. S. Arjunan, Assistant Professor of Biotechnology, K.S.R College of Arts and Science, Thiruchengode. I also thank Mrs. J. Padmapriya, Mrs. Deepa Rani Anna Arivan and Ms. S. Gayathri for their everlasting care, moral support and being with me all the time.

I express my heartfelt thanks to Mrs. Prema Shanmugam, Mrs. Sumitha Achiraman and their whole family members for their immense support, and making me to feel like be in home and part of their family. I also express my gratitude to Mr. Soundararajan, Mrs. Soundari, Mrs. Devi Gayathri Kamalakkannan and Mrs. Kanimozhi Anbarasu Tiruchirappalli for their moral support.

I place my heartfelt thanks to my brother Mr. R. Ramachandran for his cooperation in research and his love and affection towards me and my work.

To my lab mates, I cannot overlook the warmth that your humour and smiles of friendship brought me; Ms. P. Kokilavani, Ms. U. Suriyakalaa, Ms. S. Kalaiselvi, Dr. K. Shanker, Mrs. Sukirtha Bharathiraja, Mr. J. Joe Antony, Mrs. B. Abirami, Mr. D. Siva, Mr. Praveen, Mr. Vinoth, Mr. Anto Thomas, Ms. S. Abinaya, Mr. R.Ashok and the list is endless…

I wish to place on record the immense support and great friends Mr. R. Ravikumar, Mr. S. Muneeswaran, Mr. M. Sakthi Ganesh, Mr. S. Sivaramakrishnan, Mr. S. Balakrishnan, Mr. K. Karhick Kumar, Mr. B. Jeganath, Mr. A. Ganesh, Mr. Dhinesh, Mr. M. Sabari Prakasan, for their love cheered me on. I also express my gratitude to Mrs. Jenis Mery, Mrs. Ranjani, Mrs. Kiruthika, Ms. Jayachitra, Mrs. Geethanjali, Mrs. Kalaiselvi and Mrs. Sujitha for their continuous support during my research.

This is my immense pleasure to thank my well wishers Mr. Sankarasubbu, Mr. Karthi, Mr. N. Kanipandian, Mr. V. Eswaran, Mr. S. Ramkumar, Mr. M. Kanagavel, Dr. B. Sabarathinam, Dr. S. Gopinath, Mr. D. Arulanandh, Mr. V. Raja, Mr. Ganesh, Mr. Ramarajan and Mr. Karthi for their encouragement and time being help.

I thank Mr. Safiullah for his timely help and support during presentations and thesis preparation.

As the whole, my heartfelt thanks to all my seniors, juniors, colleagues, Ph. D., and M. Phil Scholars of Dept. of Environmental Biotechnology, Dept. of Animal Science, Department of Biotechnology and Genetic Engineering, Department of Plant Science, Department of Microbiology, Dept. of Biomedical Science, Dept. of Marine Science, Dept. of Marine Biotech, Dept. of Environmental Management, for their prayers and wishes made me to complete my thesis well and good.

I gratefully acknowledge the financial support offered by Department of Science and Technology (Project Assistant), and also thank Department of Science and Technology (ITS), INSA –CICS (TG), and Department of Biotechnology (CTEP Programme) for their travel grant to participate in the conferences at Australia, and made my stay in the Bharathidasan University possible and to the Staff at Porunai and Cauvery Hostel, who though not so willing all the time, had to attend to my needs any way.

I gratefully acknowledge DST-FIST and UGC Non-SAP Programme for instrumental facilities provided in the Department of Environmental Biotechnology.

As personal note, I thank everyone in my family members and friends for their endless support, encouragement, care, love and affection. Because of them only, my work sailed in smooth way.

To all, I say, thank you and God bless!!!

Sankar Ganesh D

ABBREVIATIONS

GC-MS - Gas Chromatography Mass Spectrometry

SDS-PAGE - Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis mL - milliliter min - Minutes

FSH - Follicle Stimulating Hormone

LH - Luteinizing Hormone

CL - Corpus Luteum

SD - Standard Deviation

MALDI-TOF - Matrix Assisted Laser Desorption Ionization- Time of Flight

µg - Microgram ng - Nanogram m - Meter

L - Litre mg - Milligram mM - Millimolar kg - Kilogram cm - Centimeter hrs - Hours rpm - Revolutions per minute

Contents

Description Page No

General Introduction 1

Review of Literature 12

Chapter-I 21

Chapter-II 44

Chapter-III 64

Chapter-IV 98

Chapter-V 127

Consolidated Discussion 167

Significant findings 173

Conclusion 174

Bibliography 175

LIST OF TABLES

Chapter I

Table 1.1 Behaviours observed in male and female goats during estrus phase

Table 1.2 Behaviours expressed by female goats during different phases of estrous cycle

Table 1.3 Behaviours expressed by male goats towards female in different phases of estrous cycle

Table1.4 Categorization of behaviours exhibited by female goats based on intensity

Chapter II

Table 2.1 Concentration of protein and lipid in urine of female goat during estrous cycle

Table 2.2 List of fatty acids identified in the female goat urine during estrous cycle

Chapter III

Table 3. 1 List of compounds identified in the urine of female goats during Pro-estrus

Table 3. 2 List of compounds identified in the urine of female goats during estrus

Table 3. 3 List of compounds identified in the urine of female goats during post- estrus

Table 3.4 List of compounds identified in the urine of female goats during estrous cycle

Table 3.5 List of compounds identified in the pro-estrus faces of female goat

Table 3.6 List of compounds identified in the estrus faeces of female goat

Table 3.7 List of compounds identified in the post-estrus faeces of female goat

Table 3.8 List of compounds identified in the faeces of pro-estrus, estrus and post- estrus female goat

Chapter IV

Table 4.1 Features of estrus-specific proteins identified using MASCOT tool Chapter V

Table 5.1 List of compounds identified in the urine of intact male goat

Table 5.2 List of compounds identified in the urine of castrated male goat

Table 5.3 List of compounds identified in the urine of prepubertal male goat

Table 5.4 Comparison of volatile compounds identified in the urine of intact, castrated and prepubertal male goat urine

Table 5.5 List of compounds identified in the cornual gland extract of intact male goat

LIST OF FIGURES

Chapter II

Fig. 2.1 Protein concentration among different phases of estrous cycle

Fig. 2.2 Lipid concentration among different phases of estrous cycle

Fig. 2.1 Faecal estradiol and progesterone concentration during estrous cycle

Chapter III

Fig. 3.1 GC-MS chromatogram of urine of goat in pro-estrus

Fig 3.2 GC-MS chromatogram of urine of goat in estrus

Fig 3.3 GC-MS chromatogram of urine of goat in post-estrus

Fig. 3.4 GC-MS chromatogram of pro-estrus faeces of goat

Fig. 3.5 GC-MS chromatogram of estrus faeces of goat

Fig. 3.6 GC-MS chromatogram of post-estrus faeces of goat

Chapter IV

Fig. 4.1 SDS-PAGE analysis of female goat urine collected during estrous cycle

Fig. 4.2 MALDI-TOF analysis of 25 kDa protein using MASCOT tool

Fig. 4.3 MALDI-TOF analysis of 28 kDa protein using MASCOT tool

Fig. 4.4 MALDI-TOF analysis of 32 kDa protein using MASCOT tool

Fig. 4.5 MALDI-TOF analysis of 42 kDa protein using MASCOT tool

Fig. 4.6 MALDI-TOF analysis of 55 kDa protein using MASCOT tool

Fig. 4.7 MALDI-TOF analysis of 65 kDa protein using MASCOT tool

Fig. 4.8 MALDI-TOF analysis of 74 kDa protein using MASCOT tool

Chapter V

Fig. 5.1 GC-MS chromatogram of intact male goat urine

Fig. 5.2 GC-MS chromatogram of castrated male goat urine

Fig. 5.3 GC-MS chromatogram of prepubertal male goat urine

Fig. 5.4 Protein profile of prepubertal and intact male goat urine

Fig. 5.5 Histo-architecture of cornual gland of male goat

Fig. 5.6 GC-MS chromatogram of male goat cornual gland extract

Fig. 5.7 SDS-PAGE profile of male goat cornual gland extract

Fig. 5.8 MALDI-TOF analysis of 28 kDa protein using MASCOT tool

Fig. 5.9 MALDI-TOF analysis of 33 kDa protein using MASCOT tool

LIST OF PLATES

Chapter I

Plate 1. List of materials used for estrus synchronization

Plate 2. Pictorial representation of insertion of vaginal sponges for estrus synchronization

Plate 3. Schematic representation of estrous cycle calculation among synchronized goats

Plate 4. Behavioural expressions by male and female goat during estrus phase

Plate 5. Behavioural expressions by male goat towards estrus female goat

General Introduction

“Most biologists today are familiar with only a handful of model organisms, and more as fancy reagents than as organisms that need to solve real-world problems”.

- Thomas Eisner (1929- 2011) (Father of Chemical Ecology)

Individual consumption of livestock products is closely related to per capita income. That is, with growing incomes people typically increase their consumption of meat, milk and eggs until these products become fully integrated into the daily diet. In addition to higher incomes, increase in human population adds to the demand for animal-source food products

(Steinfeld et al., 2006). Goat is an important livestock animal worldwide. It is popularly called as “poor man’s cow” and accounts for the economy of farmers. Goat has been reared for the purpose of obtaining good source for meat, milk, fiber content, and manure. Developing countries account for the production of goat meat and milk about 93% and 73% respectively, thereby a total of 80% has been given as output of goat products (Aziz, 2010). India is the largest home in goat production which accounts for 115 species of goat out of total of 574 worldwide. The facts that influence the rearing of goats are as follows: high fertility rate, which contribute the generation of young-ones during every six months. It is widely adaptable to variety of environmental conditions and the capital investment for goat rearing in lower than other livestock animals. In addition, the high incidence of disease resistance coupled with the well established local market makes goat as an ideal livestock among the farmers of India. The benefits acquired by the farmers are to be higher productivity, poverty alleviation, improved livelihoods, sustained agricultural and environmental protection (Devendra, 1999). By considering

1 General Introduction the importance of goats, Government of India has started the “Central

Institute for Research in Goat” (CIRG) in July, 1979. Rapidly increasing demand for goat meat and milk products exerts pressure on the livestock sector, which needs to adapt fast in order to cope with high demand.

Studying of reproductive physiology is essential to sustain a good livestock practice. Reproduction determines several aspects of goat production and an understanding the process of reproduction is crucial in goat management. A high rate of reproductive efficiency is important for perpetuation of the species, production of meat, milk, and fiber, and replacement of breeding stock. Males and females play different reproductive roles, and in most animal species, the role of females is not completed until a viable offspring is produced. Female reproductive physiology of non-primate mammals consists of four phases, viz, proestrus, estrus, metestrus, and diestrus. The total length of occurrence of estrous cycle varies in every animal.

For instance, in rat the estrous cycle will be of 4-6 days, while in elephant, it will be around 16 weeks (http://en.wikipedia.org/wiki/Estrous_cycle). Based on the length of the cycle, each phase contributes its own significance effect in the physiology of individuals. Once puberty is reached, the goats display a polyestrous (repeated reproductive cycles) pattern of reproductive activity. The estrous cycle is defined as the number of days between two consecutive periods of estrus (heat), is on average 21 days in does.

The regulation of estrous cycle is entirely depends on the serum hormone profiles of the animal. Among the various hormones, estrogen, progesterone, luteinizing hormone, follicle stimulation hormone has a pivotal roles (Perera, 2011). Gonadotrophin will regulate the sequential steps, such as production of LH surge, leading to surge in estrogen (estrus phase), and reduced level of progesterone, which will favor the synthesis of follicle stimulating hormone. The developed follicle then synthesize more

2 General Introduction progesterone thereby control the surge of estrogen and maintain the diestrus phase (Miller and Takahashi, 2014). Among the four different phases concerned, estrus phase is the crucial one since ovulation occurs during this phase only. The post-ovulatory phase (metestrus and diestrus) involved in the maintenance of ovum in the ovary and process taking place thereof (Forde et al., 2011). The majority of female mammals become sexually-receptive

(express estrus) and ovulates spontaneously at defined intervals. The female will only allow the male to mate during a restricted time coinciding with ovulation. If, mating did not occur at this critical period, the cycle will again repeat. Hence, the estrus phase has paid much attention in the animals in order to manage the success rate of fertilization.

Fig 1. Schematic representation of the different physiological events occurring during estrous cycle in goat: pattern of follicle development, ovarian cycle and endocrine regulations. *Ovulatory follicle(s). Adapted from Baril et al. (1993a); Evans (2003).

3 General Introduction

Communicating the time of ovulation is important to enhance the probability of fertilization. In addition, the co-ordination of sexual behaviour during with the time of ovulation appears to ensure successful fertilization

(Dehnhard et al., 1991; Ziegler et al., 1993). Since ovulation occurs during estrus, the female individuals advertise their readiness to mate through estrus signals. Mammals have evolved many different types of cues to indicate reproductive status. The signals of ovulation (estrus) includes proceptive and receptive sexual behaviours, visual cues such as sexual skin swelling, changes in coloration of genitals, and chemical cues (Zeigler et al., 1993). The chemical cues play an important role in mammalian sexual behaviour and have led to use the term ‘pheromone’ and ‘pheromone responses’ in defining such behaviours.

Karlson and Luscher, (1959) defined the term pheromone as air-borne chemical substances, that are secreted externally by an animal and cause a specific reaction in a receiving individual of same species; the reaction involve either the release of a specific behaviour or physiological change in the recipients’ endocrine or reproductive system (Doty, 1976; Dominic, 1987;

Rekwot et al., 2001). Although originally the term “pheromone” introduced by

Karlson and Luscher (1959) in the context of insect communication, the meaning of the word “pheromone” has since extended to include in the context of chemical communication in broader sense and also in relation to mammalian behaviours (Keverne, 1998; Dominic, 1991; Vandenbergh, 1999).

Based on the type of response by the recipients the pheromones are classified as “primer”, “releaser” and “imprinting pheromone”. Releaser pheromones induce a rapid behavioral response in the recipients generally mediated through the central nervous system. The pheromones involved in sexual attraction, evocation of aggression, recognition, alarming behavior and mother-young interactions are the citations of releaser pheromones. Primer

4 General Introduction pheromones induce a delayed response to prolonged stimulation mediated through central nervous system and endocrine system. Imprinting pheromones organize the central nervous system of the pre-weaning offspring at a critical period that cause permanent alterations of adult behaviour.

Many mammals can discriminate odours produced by conspecifics of different sexes and different physiological status. Estrus detection by the male partner is a classical example for pheromonal communication. The male smells the urine, vaginal mucus of female in estrus and makes the claim as ready for mating. Many studies indicate that males were attracted to estrus females than to di-estrus or non-estrus females. It is believed that all male mammals can detect estrus on the basis of female odours; and only a few species have been investigated so far.

Pheromonal communication is aided by the variety of sources such as urine, faeces, vaginal mucus, exocrine glandular secretions, saliva, wool, etc

(Tirindelli et al., 2009). Among the different sources concerned urine has been consistently reported in mammalian communication starting from mice, rat and also in aquatic animals like fish (Meyerhof and Korsching, 2009). The role of its efficacy in chemical communication particularly opposite sex attraction, mother-young interaction, territorial marking has been extensively studied in mice, rabbit, and tiger respectively (Achiraman and Archunan, 2006; Schaal et al., 2003; Brahmachary et al., 1990). In mice, urine has been reported as a potential carrier of volatile and proteins, which has been functionally proved as an effective communicative source (Beynon and Hurst, 2004). Volatile compounds identified in female mice, in particular, the bound form of volatiles have been well proved as a male attractant (Humphries et al., 1999). In larger animals, particularly in blackbuck, bovine, buffalo, tiger, lion and in elephant urine has been well proved as a source of communication (Archunan, 2009).

The same concept has been established with bovine with well differentiated

5 General Introduction volatile profile during the estrous cycle, in which the estrus phase alone contributes some compounds in the urine (Kumar et al., 2000). In tiger, urine has been well characterized in regard to pheromone analysis, and the role conferred by lipid has been documented in chemical communication (Burger et al., 2008). Moreover, the urinary lipid has been identified as a fixative for keeping the volatile signals for a long time in order to advertise the individuals physiological status. As urine is one of the sample of non-invasive nature, its use has several notable roles in endangered species also (Dehnhard et al.,

2006). Specifically, the urine of endangered species, such as bears, has been critically analysed and the volatile profiles with some other parameters could have been proved as an effective monitor of endocrine status. Indian blackbuck, a critically endangered species also have been studied in view of chemical communication, and the estrus-specific urinary volatiles have been documented (Archunan and Rajagopal, 2013). Thus, excretion of urine is not as a certainly waste product; rather it has multifunctional role in animal communication.

Faeces, another most important source have also been studied in regard to chemo-communication. The ability of using faeces as a source of communication has been characterized initially with the insect, and later it has been established in large mammals. As utilizing the fact of faeces as a non-invasive indicator of physiology, research has been done in neonates for the analysis of difference in the volatile profile in order to differentiate the healthy and non-healthy individuals (De Lacy Costello et al., 2008). The dung of elephant has been analysed in terms of volatile and behaviour aspect and suggested that dung as a potent source of volatiles to be used in chemo- communication (Ghosal et al., 2012). In bovine the faeces has been reported as good source of communication, where it shows consistent variation of its constituents along the phases of estrous cycle (Sankar and Archunan, 2008).

6 General Introduction

Overall, this fact shows that faeces could be an additional source of communication, where the urine has been documented as primary source of communication. Similar concept has been studied with buffalo with the pronounced variation among the different phases of estrous cycle

(Karthikeyan et al., 2013).

Glandular sources, also an important notion in chemical communication. In rat and mice, the major scent gland (preputial gland) has been reported as an effective source in chemical communication. It has been substantiated that the role of scent gland is to trail the volatiles during movement from one place to another place, so as to indicate its presence in the circumstances (Achiraman et al., 2010a). The pronounced effect has been well studied in the glandular pheromones of ants and honeybee. In ants, the concentration of the pheromones released has been checked with the longevity and found that the glandular source have been demonstrated as an effective source (Mashaly et al., 2011). In the case of honey bees, mandibular gland has been well studied and its constituents have been proved to have pleiotrophic effect in colony regulation (Maisonnasse et al., 2010). In elephants, frotalin, a compound isolated from the temporal gland has been reported as pheromone and to have an increasing concentration as the elephant matures, thereby reaching a high concentration in mature males than that of young ones (Rasmussen and Greenwood, 2003). In bovidae family, reports exist for the presence of pre-orbital gland which could be major factor involved in the secretion of compounds mainly involved in territorial markings (Rajagopal, 2010). In goats, the anatomical characterization and histological analysis have been done with the exocrine glands present in the skin (Van Lancker et al., 2005).

In farm animal management, to attain good number of off-springs, estrus synchronization method is practiced. It aids an accurate monitoring of

7 General Introduction phases of the estrous cycle, thereby pave the obstacles in sustaining a good livestock colony. Estrus synchronization is a popular tool in farm animal management and production. There are a variety of techniques used for estrus synchronization. This technique could be useful in regulating the estrous cycle in seasonal animals during non breeding season and also to bring out the cyclicity during same time, so as to benefit via the sequential steps involved in fertilization through artificial insemination (Rodgers et al.,

2012). Various estrus synchronization techniques include altering the lighting regimen, use of exogenous agent, such as vaginal sponges, use of Controlled

Internal Drug Release (CIDR) device, etc (Islam, 2011). At some instance, especially in goats, the introduction of intact males could result in synchronization of estrus in a colony, which is termed as buck effect (Gelez and Fabre-Nys, 2004). The mechanism by which estrus synchronization occurs, is entirely depends on the modulation of synthesis of hormones or the exogenous products contain the hormones which will be released into the blood stream thereby bring the change in the hormonal profile, finally resulted in similarity in estrous cycle expression (Van Werven et al., 2013). In contrast to the exogenous products in synchronizing the estrus, the introduction of male in addition to estrus synchronization tools directly modulate the hormonal profile of female, thereby have a pronounced effect in estrus synchronization (Alvarez et al., 2013). Thus, the technique of estrous synchronization is a useful tool in higher animals.

Even though the female reproductive physiology is the foremost factor in any species, the study of male physiology is equally important in order to coordinate the reproductive success, and thus every colony requires reproductively healthy males. The main purpose of male is in the involvement of sexual partnership and thereby leading to maintenance of a good colony.

However, there are no much difference in the physiological condition of

8 General Introduction normal male as that of females; but based on the reproductive condition, males could be divided into three different categories such as intact, castrated, prepubertal (Longpre et al., 2011a). Similar to females, in males also the involvement of hormones, but instead of estrogen in female, testosterone is the major regulator of male sexual communication (Moncada,

2006). For instance, in intact males the concentration of testosterone is up to the level to perform good sexual activity, while the prepubertal, the level of testosterone will be lower; hence, the mating will be limited as that of intact.

On the other hand, castration, is a condition, where the function of testes will be arrested by gonadectomy or by intervening the flow of sperm to go into ejaculation (Stafford and Meller, 2005).

The male and female produced for the purpose of producing good off- spring to maintain a colony thereby each of them contribute equal importance in the environment. Though, there are factors responsible for the modulation of internal physiology in each animal, the factors favoring the communication and the way in induces the changes in internally is important. Thus, the releasing and receiving potential of the signal by the sender and receiver respectively is another important aspect. In animals, especially in mammals, there exists two systems for receiving and processing of signal (Mucignat-

Caretta et al., 2012). There systems are main olfactory system (MOS) and the vomeronasal organ (VNO). The specificity of action of these sensory systems varied between variety of chemicals, and controversy is still there for proving the particular effect via a specific sensory system (Ihra et al., 2013). At some instance, it has been reported that the sexual attraction is mediated by MOS and sometimes, VNO. Recent reports evidenced that both the systems synergistically act to receive and process the chemo-signals (Achiraman et al.,

2010b).

9 General Introduction

However, none of the study has been focused on goats in view of analyzing the excretory sources and connecting its context with communication purpose or to make a better marker for denoting the physiological status. Since, goat is an essential livestock animal which is in need of the hour, we, here planned to study the behavioural, physiological and biochemical aspect of goat in order to resolve the problems in sexual communication, thereby to pave a milestone in the aspect of livestock improvement.

Rationale of the study Most of the animals have been reported to contain biochemical moieties in their excretory sources which regulate the behaviours in animals, particularly sexual communication. Mating in animals at the right time of ovulation would result in successful reproduction. At present the ovulation period is identified by the farmers by experience which is unreliable all the time and leads to unsuccessful fertilization and breeding. In addition, decrease in fertility of goats makes it difficult to determine the optimal time for artificial insemination. Improving the detection of estrus and identification of heat period at right time of ovulation will lead to increase the fertility rates.

Since, goat is an essential animal the present study is overcome the problems in sexual communication and livestock improvement.

Hypothesis

• Goats have been suspected to contain biochemical constituents and

chemo-signals in their excretory products (otherwise termed as scent

sources) mainly, urine and faeces which may be involved in inter-

individual communication (social or sexual).

• These sources could have been varied during different physiological

conditions, thereby, possess some of the key components to make it as

10 General Introduction

markers of physiological status or used in the enhancement of sexual

performance among conspecifics.

Aim of the study is

• to analyse the behavioural expression and excretory products in terms

of biochemical and signaling aspect (volatile profiles) and,

• to elucidate the possible role of the biochemical parameters and volatile

compounds in order to make it as a better marker to denote the

physiological status of goats and sexual enhancers among conspecifics

respectively.

Objectives of the study is to  synchronize estrous cycle and observe behavioural patterns exhibited

by female and male goats during different phases of estrous cycle,

 analyse the urinary biochemical and faecal steroid of female goat

during estrous cycle,

 analyse the volatile compounds in female urine and faeces across

estrous cycle,

 analyse expression profile and characterization of specific protein in the

urine of female goats and to,

 analyse the volatile compounds and protein in male scent sources

(urine and cornual gland).

Expected outcome of the study

• Identification of ovulatory markers would help to develop a kit which

could enable the accurate estrus detection by the farmers for artificial

insemination and successful reproduction thereby improves goat

productivity.

• Pheromones could also be used for provoking sexual behaviour in

libido males.

• To create awareness to farmers about livestock production.

11 Review of Literature

Livestock production

Domesticated animals, especially ruminants are often paid considerable economical importance. Among the ruminants, goat is an important livestock animal. Goat farming is an important task in the rural parts of India which signifies its role in economy growth. Goat is often referred as poor man’s cow, since most of the farmer rear goats besides their agricultural activities. Goat served as a good source in the production of milk, meat and fiber. The goat meat is popular because of its high nutritive value, particularly less fat and more protein content. It is also a part of the religious practice in India. It was earlier reported that goat milk constitutes higher nutritive value next to cow’s milk (Bosworth and Van Slyke, 1916). Comparing with international level, India is the second largest supplier of goat products among Asian countries thus contribute to great economical status (Dubeuf et al., 2004).

Pheromones

Animals should be reproduced effectively in order to maintain a good healthy colony. The production of young ones is purely based on the sexually reproducing capacity of female goat. However, the role of male animal’s physiology is also pivotal in sexual communication. Between and within animal’s expression of physiological status depends on the semiochemicals.

The fact involved in communication started with silkworm, Bombyx mori, where the discovery of bombykol by the scent gland has been substantially proved as an effective attractant of male moth from few kilometers (Butenendt et al., 1961). Thereafter, intense research has been done in the laboratory animals. Specifically, the field of chemical communication has emerged by the

12 Review of Literature discovery and definition of Peter Karlson and Martin Luscher in 1959. They defined the term “pheromone” which refers to the molecules capable of eliciting/modulating the behavioural/ endocrine system of animals in same colony. Initially it was discovered that the chemical signals has only been implicated in sexual communication, later it was substantially proved to have multiple potentials. Pheromones are classified into many different categories depending on the effect it produces. There are two types; one is involved in sexual activity and another one is involved in social activity. Based on individual’s role, the pheromones are classified into many different categories, which includes primer pheromone, releaser pheromone, aggregation pheromone, alarm pheromone, etc (Tirindelli et al., 2009).

Pheromones classified under the class of semiochemicals (semeon- signal), defined as the class of chemical that contain message or information for the purpose of communication. Based on the response and nature of effect produced, pheromones have been assigned into many classes. As like of that various effect discovered in small animals, pheromones in ruminants, especially male goats have chemical cues that have the capacity to synchronize estrus. The effect termed as buck effect, where the introduction of a male goat in a colony have the capacity to synchronize estrus in female goats (Claus et al., 1990). This may due to the presence/excretion of some specific compounds by the male goats that have the capacity to regulate the hormone pulsatile, thereby regulate the estrous cycle (Iwate et al., 2000).

Sexual communication is an important criterion for any living system to make fingerprint of their young-ones in this world. In order to reproduce effectively, the organisms should possess a good quality in terms of physiology, reproductive system and sexual partner. The physiology of animals is a complicated system which is regulated by the action various parameters inside the body. While comparing the physiology of male and

13 Review of Literature female, the latter is too complicated process to understand the happenings, due to the variation in the physiology brings out the phases of the reproductive cycle.

Estrous cycle

Reproductive cycle of mammals is termed as menstrual cycle and in non-primate mammals it is termed as estrous cycle (Archunan, 2009). In both the conditions, physiological events will occur in order to produce eggs and make ready the uterus to bear the pregnancy. In menstrual cycle, the ruptured endometrium (mensus) will be released with blood, whereas in estrous cycle the endometrium was reabsorbed. Another important difference, in primate mammals’ sexual desire will be there at any time, whereas in non- primate mammals, females allow the males for mating only during estrus.

However, the hormone system in both primate and non-primate mammals are similar. The significance of estrous cycle is, it can last up to death; whereas menstrual cycle last up to age of 45 to 55, then turn into menopause stage

(Vande Weile et al., 1970; Hansel and Convey, 1983).

Estrous cycle is the reproductive cycle exists in non-primate mammals.

The cycle consist of four different phases; namely proestrus, estrus, metestrus and diestrus. Pro-estrus is defined as the phase of pre-ovulatory stage of the animals. At this stage, the animals start to visualize the signs to indicate the male in regard to sexual communication. In contrast, estrus phase is the ovulatory phase involves the production of ovum in the ovary after which the successful fertilization occurs. During metestrus phase the animals starts to implant the ovum in the ovary and after successful implantation the pregnancy will starts during diestrus phase. If mating doesn’t occur during estrus phase, the cycle again repeat, otherwise it will lead to the production of young-ones after gestation period (Larson and Ball, 1992).

14 Review of Literature

The action of gonadotrophin releasing hormone and reproductive hormones needs to be co-ordinate in order to produce substantial effect and thereby regulate the reproductive cycle. First, the follicle stimulating hormone produced in the anterior pituitary will enhance the growth of follicles, and under the influence of luteinizing hormone the follicle attains maximum growth and brings out pre-ovulatory phase. At this instance, the matured follicle will start producing estradiol which is the possible mediator of expression of estrus behaviours. On the continuous secretion of GnRH and the pre-ovulatory surge in LH resulting in estrus phase, in particular the ovulatory phase (Chasombat et al., 2013).

Physiology of goat

Goat reproduces twice a year. Goat attains sexual maturity at age about 3 to 8 months. The total length of the estrous cycle is about 19-21 days which has been divided into four phases namely proestrus (3 days), estrus (2 days), metestrus (6 days) and diestrus (8 days) (Fatet et al., 2011). Once the ovum gets released into the fallopian tube at the end of estrus, and when mated with a male during estrus, the sperm will fertilize the egg and continue the phase with diestrus or pregnancy. If mating doesn’t occur the endometrium will be reabsorbed and the cycle will begin again with proestrus

(de Castro et al., 1999). The gestation period is about 150 days. On average goat will give birth to 2 to 4 off-springs at a time based on the nutritional and adapting condition. The average lifespan of goat is about between 15 to 18 years.

The important notion relies on the time of estrus, since mating occurs out of estrus will not be aid in fertilization, and hence, in most of the animal system alternative tools are being used. Those systems include synchronization of estrous cycle coupled with artificial insemination (Larson and Ball, 1992). Estrus synchronization is widely used in farm animals to

15 Review of Literature enlarge the population size, mainly in livestock animals. Artificial insemination is performed in remote areas, where access to male animals is less and to produce healthy and genetically fit animals.

Words outspoken conveys the message in auditory manner. However, in animals those lack auditory capacity, have an optional way to express its’ feelings by the expression of behaviours. Thus, the behaviours paid much attention in animal communication. Behaviours are of two types in animal system, one is social another one is sexual. The signs of estrus are still poorly studied in most of the mammals which hamper the fertilization rate in animals. The signs of estrus include in most of the animals are, vaginal swelling, redness, vaginal mucus discharge, restlessness, tail raising, bellowing, frequent urination, receptive towards male, chasing of males, mounting on other females thereby express homosexual behaviours, reduced food intake, decrease in milk secretion etc (Layek et al., 2011). Most of the above mentioned signs are not feasible at large-scale application, and at some instance, behaviours could be misinterpreted, thereby leads to false identification of estrus.

Of note among the behaviours of sexual communication, flehmen and mounting behaviours are most important in males. Expression of dominant status by the way of fighting is also observed in some species (Karthikeyan et al., 2013). Especially in hoofed mammals, the status of dominant has been inter-connected with the behaviours they express. It was observed that the maintenance of a whole colony will be taken care by a single male, who is said to be dominant, whereas remaining males are said to be subordinate. The fact relies in the dominance or subordinate is with the external fitness and the internal physiology reflected by the individuals. In general, the intact animals having good body physiology with high concentration of testosterone considered as dominant, whereas others who does not have the characters up

16 Review of Literature to the mark of dominance remains subordinate. It is also observed that, if any subordinate male attain the status of dominance, it will go and compete with the existing dominant male, where the winner of the competition will be the dominant, but the volatile compounds present in the urine of dominant male suppress the sexual activity of other subordinate males (Rajagoapal et al.,

2010).

Even though the estrus phase is of considerable importance, the day length of the phase further decides the success rate of fertilization. In each animals, the length and duration of each phase in estrous cycle varies. The length of estrus phase varies between 24 hours to 7 days in animals. In addition, the living environmental factors and type of species further interfere with the days of estrous cycle and estrus phase.

The list of animals and the length of estrous cycle is tabulated below.

Total no. of Length or Name of the days of duration of Source animal estrous cycle estrus phase Mice Achiraman and 4-6 days 12-24 hours (Mus musculus) Archunan, 2006 Rat Achiraman et al., 4-6 days 12-24 hours (Rattus norvegicus) (2010a) Hamster (Mesocricetus 4 days 6-10 hours Lewis et al., 2002 auratus) Goat 19-21 days 1-2 days Fatet et al., 2011 (Capra hircus) Sheep Bartlewski et al., 16-17 days 1 day (Ovis aries) 2011 Cervids 17-27 days (E.g Capreolus 24 hours Asher, 2011 (average) capreolus) Cow Sartori and Barros, 20 days 6- 30 hours (Bos indicus) 2011 Buffalo Rajanarayanan and 21 days 5-27 hours (Bubalo bubalis) Archunan, 2004

17 Review of Literature

Tiger 18-24 days 1 week Brown, 2011 (Panthera tigris) Bos taurus 18-24 days 2-3 days Forde et al., 2011 Horse 22 days 5-7 days Aurich, 2011 (Equus caballus) Sartori and Barros, Bos indicus cattle 22 days 1-20 hours 2011

The advertisement of sexual desire by the female is the crucial factor in making a good colony of animals. In the presence of male, it is easy to detect estrus and thus in wild condition animals mate naturally and maintain the colony. In small animals also, this fact is working well. However, some of the animals did not express the behaviours outwardly during estrus, thus making hurdle in estrus detection. Particularly in farm animals, if estrus is not detect on time, it leads to great loss in economy. For instance, buffalo is termed as silent heat animals, where the estrus detection is achieved outwardly only by

20%, which pose to have great economic loss. In the series of livestock animals, goat deserves a strong position. The expression of estrus behaviours and estrus detection by male is not that much difficult as compared to other livestock animals. However, detection of estrus in the absence of male animals is a great problem yet to be resolved. This can be solved by the development of reliable tools for detection of estrus, and it can be coupled with artificial insemination to develop and maintain good livestock (Karthikeyan, 2011).

Though, there are some ground level tools are there in estrus detection but their reliability to detect estrus is still poor. For example the basic parameters, such as vaginal temperature, urinary pH, turbidity, viscosity have been studied in terms of estrus detection. The introduction of teaser animals, usually intact males also can help in detecting estrus females. The use of trans-rectal palpation and measuring the length of the follicles using ultrasonography are under practice in higher animals which aid in estrus detection (Gumen et al., 2003). On the other hand, analysis of hormones in

18 Review of Literature serum samples is a better way to detect estrus in animals, however the collection of blood in repeated manner produces stress to the animals and seems to be an invasive one (Henricks, 1972).

Lacunae

Even though goat is considered as an important livestock animals, studies pertain to improvement of goat production has been done very little.

In addition, none of the study has been reported in the aspect solving the hurdles in estrus detection in goat. Moreover, studies relating estrus synchronization and analysis of scent sources which could aid in identification of physiological status in goat have not been documented.

Based on the above literatures, we have important aspects to bring out a key to resolve the difficulty in estrus detection and thereby to improve the goat production. Thus, we have set some sort of key questions to begin the research on goats.

The questions are as follows;

How the internal physiology expressed in behavioural context in both the sex of goats?

Is there any variation in the internal physiology of female goats that reflect in excretory products?

If so, how the alteration of chemical constituents takes place in the scent sources?

If any specific change in the constituents of scent source, could it be utilized for the development of a marker for estrus detection/sexual enhancers in female goats and male goats respectively?

19 Review of Literature

Is this the same case in male goats under different physiological condition (i.e. intact, castrated and pre-pubertal)?

Is there any other sources other than urine and faeces used in goat communication?

How the overall output could be exploited for the purpose of livestock improvement?

20 Chapter I

Estrous cycle synchronization and behavioural analysis

1.1. Introduction

Estrous cycle is the differential form of reproductive cycle expressed in non-primate mammals as that of menstrual cycle in mammals (Ward, 1946).

Estrous cycle of female mammals consists of four phases, such as pro-estrus, estrus, met-estrus and di-estrus (Muthukumar et al., 2013). Pro-estrus phase, the so-called pre-ovulatory phase, in which the dominant follicles in the ovary start to develop under the influence of circulating follicle stimulating hormone (FSH) and the regression of corpus luteum (CL) occurs which profoundly decreases the concentration of progesterone. On later stage of this phase is called as estrus phase. This phase is otherwise termed as “ovulatory phase” in which the follicle development occurs with high concentration of estrogen secretion by the ovary due to the surge in the level of luteinizing hormone. The estrus phase could be visually expressed with the behavioural signs by the animals. During this stage, the animal will be sexually more receptive. Consequently, after estrus phase, the corpus luteum (CL) will develop and produce progesterone, thus the concentration of progesterone will be higher and the estrogen level will be reduced. The estrus signs are no longer expressed and the animal will not allow the male for mating during this phase. Thus, the metestrus and diestrus phases are collectively called as luteal phase (Fatet et al., 2011).

For making a successful fertilization, estrus phase of remarkably important. Hence, much focus has been paid towards the tools for the development of estrus detection strategies. Each method of detection that have developed previously has its own merits and demerits. Since the estrus

21 Chapter I is expressed by the visible estrus signs in some animals, it will be helpful in advertising the estrus phase to conspecifics. However, this is not the same case in all the animals. For example, in buffalo, the estrus signs are not well pronounced outwardly (otherwise termed as silent heat), so that behavioural aspect could not be used for estrus detection (Karthikeyan et al., 2013). Thus, for identification of estrus, techniques are being addressed either field based or laboratory based. But most of the existing techniques are seems to be invasive to the animal, and hence, give stress to animals. Thus, there is an urge to develop a feasible technique for the identification of estrus, especially in livestock animals.

Fortunately, the technique for estrus synchronization was developed and employed in a number of animals, thereby bringing estrus in a scheduled manner (Carrick and Shelton, 1967). The success rate of estrus detection is based on the technique adopted for synchronization. Among the various techniques employed for estrus synchronization, use of vaginal sponges, use of Controlled Internal Drug Release (CIDR) device, and altering the lighting regimen coupled with male effect are used frequently (De Rensis and Lopez-

Gatius, 2007). In vaginal sponge method, the progesterone was used as an effective modulator of estrous cycle in cattle (Freitas et al., 1997). In contrast, in CIDR technique, the T-shaped device will release the progesterone in a slow manner, and upon after removal, the concentration will be reduced and brings the follicular phase (Giles et al., 2013). The third technique is widely used in ruminants, especially in goats (Chemineau et al., 1999). The alteration of lighting regimen directly regulates the expression of gonadotrophic hormones by the production of melatonin in the pineal gland. Thus, extending the lighting time, rather than darker time, coupled with exposure of buck into the colony of anoestrus female colony, the estrus will get synchronized. However, the main focus of all the techniques relies on the identification of estrus. Thus

22 Chapter I for identification of phases accurately, we here used the strategy of vaginal sponges to synchronize estrous cycle and to collect the urine and faeces samples which have been used for the analysis in the following chapters of this thesis.

Female mammals exhibit different types of proceptive and receptive behaviours during its reproductive cycle, among which each phases of the cycle contributes to peculiar behaviours. For instance, in mice, the male exhibit self-grooming at high incidence when it encounter the estrus scent source or estrus female. In behavioural analysis, male mice exhibit high frequency of visit and spent more time near to the estrus urine than to non- estrus urine (Achiraman et al., 2014). Urine samples collected during estrus phase of bovine also exhibit similar pattern in frequency and time of visit when tested with mice (Kumar et al., 2000). However, testing of putative estrus volatiles of faecal origin in dummy buffaloes at various concentrations, bulls exhibit flehmen, mounting and copulation behaviour. In females, the behaviours such as frequent urination, restlessness, vaginal mucus discharge, vaginal swelling are seems to be expressed prominently during estrus than non-estrus phases (Karthikeyan, 2011). Thus, the internal physiology expressed in terms of behaviours by the female which could aid in communicating the male and to have successful fertilization during estrus.

However, estrus synchronization coupled with analysis of behaviours were not studied in detail in goat, we made an attempt to capture the behavioural signs expressed during estrous cycle by female goats and coordinative behaviours by male goats in order validate the standard behavioural signs for easy estrus detection.

23 Chapter I

1.2. Materials and method

1.2.1. Estrous cycle synchronization

Sexually experienced and regularly cycling female goats (n=6) were used in the present study. The experimental animals were maintained separately in the Livestock Farm at Veterinary College and Research Institute,

Tamilnadu Veterinary and Animal Science University, Namakkal, Tamilnadu,

India, during the entire study. The goats were allowed for natural grazing for about 8 hours each day and supplemented with cultivated forages and water ad libitum. The vaginal sponge procured from Central Sheep and Wool

Research Institute (CSWRI), Avikanagar, Rajasthan, India, was used in the present study (Plate 1).

1.2.2. Insertion of vaginal sponges

A veterinarian inserted a single sponge into the cervix region of the vagina of each goat using a plunger and speculum (Plate 2). After the insertion, the animals were kept isolated during housing. The assessment of estrous cycle was done by calculating the days of insertion of sponge. The sponge was retained for about 15 days, and the day it was removed, the goat was considered as in pro-estrus phase. One day after removal of the sponge the goat was considered as in estrus, and two days after removal the goat was considered as in met-estrus and 8 days after removal considered as di-estrus

(Plate 3).

24 Chapter I

Plate 1.

List of materials used for estrus synchronization

a - Estrus synchronization kit consist of a rod, speculum and intra-vaginal sponges, b - Pack of vaginal sponges c - Enlarged view of single vaginal sponge

25 Chapter I

Plate 2. Pictorial representation of insertion of vaginal sponges for estrus synchronization

26 Chapter I

Plate 3. Schematic representation of estrous cycle calculation among synchronized goats

27 Chapter I

1.2.3. Observation of behaviour After removal of the sponges from the goats, the phases of the cycle were calculated as mentioned above. During each phase, the behavioural signs such as vaginal swelling, reddening of vulva, urination frequency, food intake, standing heat, movement inside the fence, vaginal mucus discharge, sexual behaviours were observed in female goats and behaviours exhibited with male such as, flehmen, mounting, expression of dominance, chasing behaviour and follow-up behaviours such as penile protrusion, coitus were observed. All the behaviours were observed in female goats under three different phases of estrous cycle.

1.3. Results

1.3.1. Synchronization of estrus The vaginal sponges have been involved effectively in synchronization of estrous cycle in all the female goats. Upon insertion of the sponges, the synchronized female goats express typical behaviours during each phase of the estrous cycle. Among those behaviours some of the behaviours were seems to be expressed only during estrus phase.

Behaviours observed The list of behaviours observed in female goats was given in Table 1.1,

Table 1.2 and Plate 4. Female exhibit different types of sexually receptive behaviours during estrus phase than during non-estrus phases. Based on the intensity of expression the behaviours were classified and given in Table. 1.4.

Female specific behaviours

Vaginal swelling Vaginal swelling is an important criterion to differentiate the phases of estrous cycle in female mammals. The vaginal region swells with reddening of vulva appeared as a good indicator of estrus. In the present study, we have observed the swollen vagina and the number wrinkles appeared in the vagina

28 Chapter I also reduced during estrus phase of female goats. However, swelling vagina came to normal position and the number of wrinkles also reduced in the case of non-estrus females.

Tail wagging Tail wagging is an important behaviour exhibited by estrus females towards the males (Plate 4 a). This facilitates the advertisement of estrus and helps in sniffing the vulval region by the male. Tail wagging is commonly observed in female goats during all the phases, however during estrus tail wagging was predominantly expressed till than the non-estrus phases.

Restlessness Restlessness is generally appeared as a good indicator of estrus in female mammals. During estrus phase, the female goats of the present study were seems to be in restlessness position which was observed and confirmed by the reduced activities such as moving and roaming inside the fence.

However, during non-estrus, either in pro-estrus or post-estrus the female goats were seems to be active than those of estrus phase.

Vaginal mucus discharge Vaginal mucus discharge is a clear sign of estrus in farm animals. The vaginal mucus discharge usually starts at the end or pro-estrus and clear mucus observed up to the end of estrus phases. In the present study, we have observed clear mucus discharge in the female goats during estrus phase (not up to collectable quantity). In contrast, arresting of vaginal mucus discharge was observed during post-estrus phase.

Frequent urination Since the goats had free access to water and food, an increased consumption of water was increased during estrus phase than that of other phases, and as a result the estrus female urinate frequently than that of

29 Chapter I female in other phases. However, the frequency was reduced when the female was in pro-estrus or post-estrus phases.

Reduced food intake and milk secretion The female goats were allowed for ad libitum feed and water intake.

When compared to the non-estrus phases, the food consumed was seems to be very less during estrus. However there was no variation in water consumption between estrus and non-estrus phases. Reduced milk secretion was observed during estrus and this was confirmed by the reduced size of udder region which was positively correlated with the level of milk secreted.

Also, the female in estrus did not allow the young ones to feed the milk. When the animal reaches post-estrus phases, the milk secretion was seems to be normalized as that of normal goats.

Bellowing and standing heat Deep loud roar sound making in female mammals is called bellowing behaviours. These behaviours could aid in communicating the male by the use of auditory behaviours. Thus, bellowing behaviours were appeared as a standard sign of estrus in female goats and we have observed high incidence of bellowing during estrus phases. However, bellowing was not seems to be appeared in either pro-estrus or post-estrus. In addition to bellowing, standing heat was observed only during estrus phase in female goats. Thus, standing heat is considered as a gold standard tool in estrus detection.

Homosexual behaviours by mounting on other females Homosexual behaviours were well pronounced in female mammals during estrus phase. The increased sexually receptive behaviours make the female goats to exhibit homosexual behaviours in the absence of male goats.

Hence, the female in estrus exhibit homosexual behaviours such as chasing and mounting on other female goats. However, this homosexual behaviours were absent during non-estrus phases.

30 Chapter I

Teasing of male goats Teasing of male goats is a type of receptive behaviour appeared in estrus female mammals. In this case, the estrus females try to insist and tease the male goats which could aid in intimation of the time of estrus to the male goats. While, the estrus female tease the male goats, the male encounter the female and showed sexually receptive behaviours such as flehmen and mounting followed by coitus.

Mock fighting Mock fighting is an important character of estrus in female goats. This behaviour was observed by encountering the male by the female in sidewise and sometimes makes fight by hitting the head region with one another.

Based on the intensity, the behaviours used to characterize estrus may be considered in the following order as tabulated in Table 1. 2.

Male specific behaviours The behaviours expressed by male goats towards estrus female goats were given in Table 1.1, Table 1.3 and Plates 4 & 5.

Chasing of female Estrus females are always chased by the male goats in favor for sexual communication. This behaviour was observed only towards the estrus female in the condition where the female also desire to have mating. When the male had free access to all the females, it did not express chasing behaviours towards the non-estrus females, and thus chasing appeared to as a characteristic male behaviour in identifying estrus females (Video 1).

Penile protrusion Penile protrusion is an important courtship behaviour exhibited by the male mammals in favor of mating. In the present study, penile protrusion was observed towards the estrus female goats. This behaviour was observed next

31 Chapter I to flehmen and mounting. Even though, penile protrusion was also observed towards non-estrus female goats, the female did not allow the male in favor of mating (Plate 4 c; Video 2).

Coitus/copulation Coitus is the end behaviour in the process of mating with estrus female. Coitus will aid in the insertion of penis into the vagina which favors the transport of semen. However, this is an exclusive behaviour appeared only towards the estrus female, however female goats in other phases did not permit the male to have coitus. In the present study, coitus was observed frequently, however the duration coitus was less which is approximately 30 seconds each time (Plate 4 d; Video 2).

Dominance over other male Interestingly, when two males were presented along with estrus female

(separated by a fence), we observed fighting behaviours between the two males to compete with single estrus female. Thus, when estrus female was presented the male hitting their head region in the fence or wall thereby expresses its dominant status to another male. The male separated by the fence which was not allowed to contact the estrus female sexually, often smell the vaginal region of the does and elicit flehmen behaviour (Plate 5 d; Video 3).

Enurination Enurination is a behaviour exhibited by male goats. This behaviour involves the urination of male in their own body, specifically in the stomach region. After urination, the male smells the urine as well as the region where it urinated. This behaviour was observed in male goats when the estrus female was kept nearby and when prevented the male for mating. The urine excreted during enurination process was differed from the normal urine by having the viscous nature. The enurination behaviour was followed by the exhibition of making some characteristic sound by the male goats. This also

32 Chapter I appeared only towards the estrus female goats and not towards females in non-estrus phases (Plate 5 c).

Flehmen and mounting Flehmen behaviour is exhibited by curling of upper and lower lips to perceive the chemical cues present in the scent sources or with when the male goats encounter female goat directly. The expression of flehmen in our study was differed between each phase of the estrous cycle either towards the urine collected or to the female goats. Even though expression of flehmen was observed with the urine of all the female goats, however the intensity and time of expression were comparatively higher towards estrus urine than that of urine from other phases of the estrous cycle. In addition, flehmen was observed repeatedly when the male goat was presented with estrus urine.

Mounting is another characteristic behaviour noted in male goats towards the estrus female goats. Even though male mount female in other phases the intensity of expression was lower and the female did not allow to be mounted with male. However, the female allows the male to be mounted during estrus phase, and hence, mounting behaviour was prominently expressed in male goats when it encounters female goats (Plate 5 a & b; Video 4 & 5).

The male goats smell the vaginal region and excreta (urine and faeces) laid by the female and accelerate the receptive response. This is presumably due to the scent present in the vaginal region, specifically mucus during estrus phase and the excreta may contain some specific signal that may provoke the male receptive behaviours.

Mock fighting Mock fighting behaviour was observed in male goats when the female make attempt of mock fighting. When the estrus female approaches the male by turning its face towards the anal region of the male goat, the male also in a position to turns its face towards the vulval region of the female goat. At some

33 Chapter I instance, male and female goat raised their head and make fight as a fact of meta-communication. However, mock fighting behaviour was not observed in female during non-estrus phases.

Table 1.1. Behaviours observed in male and female goats during estrus phase

S. No Behaviours Male Female goat goat 1 Repeated flehmen ✔ X

2 Mounting ✔ X

3 Penile protrusion ✔ X

4 Enurination ✔ X

5 Dominance over other male ✔ X

6 Chasing of female ✔ X

7 Copulation ✔ X

8 Mock fighting ✔ X

9 Vaginal swelling X ✔

10 Vaginal mucus discharge X ✔

11 Restlessness X ✔

12 Reduced food intake X ✔

13 Reduced milk secretion X ✔

14 Bellowing (Deep loud roar) X ✔

15 Bleating (Wavering cry) X ✔

16 Frequent urination X ✔

17 Homosexual behaviour (chasing other X ✔ females in the absence of males) 18 Standing heat X ✔

19 Mounting other females X ✔

20 Teasing of male goat X ✔

34 Chapter I

Table.1.2. Behaviours expressed by female goats during different phases of estrous cycle

Type of Proestrus Estrus Metestrus Diestrus behaviour

Vaginal -- +++ -- -- swelling

Tail wagging -- +++ -- --

Restlessness -- +++ -- --

Vaginal mucus + +++ -- -- discharge

Reduced food + +++ -- -- intake

Reduced milk + +++ -- -- secretion

Bellowing -- +++ -- --

Standing heat -- +++ -- --

Homosexual -- +++ -- -- behaviours

Mounting on -- +++ -- -- other females

Teasing of male -- +++ -- -- goats

Mock fighting -- +++ -- --

-- Absent; + less intensity; +++ high intensity

35 Chapter I

Table.1.3. Behaviours expressed by male goats towards female in different phases of estrous cycle

Type of Towards Towards Towards Towards behaviour female in female in female in female in pro-estrus estrus met-estrus di-estrus Flehmen + +++ + + Mounting + +++ -- -- Chasing of + +++ -- -- female Enurination -- +++ -- -- Penile + +++ -- -- protrusion Coitus -- +++ -- -- Dominance -- +++ -- -- over other male

-- Absent; + less intensity; +++ high intensity

Table 1.4. Categorization of behaviours exhibited by female goats based on intensity

S. No Behaviours based on the order of intensity 1 Bellowing (Deep loud roar) 2 Bleating (Wavering cry) 3 Standing heat 4 Vaginal mucus discharge 5 Vaginal swelling 6 Homosexual behaviours 7 Mounting other females 8 Frequent urination 9 Restlessness 10 Reduced food intake 11 Reduced milk secretion 12 Teasing of male goat

36 Chapter I

Plate 4. Behavioural expressions by male and female goat during estrus phase

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Plate 5. Behavioural expressions by male goat towards estrus female goat

38 Chapter I

1.4. Discussion

As discussed earlier (introduction section), there are number of techniques used for estrus synchronization in animals. Particularly, the use of vaginal sponge is one of the popular techniques. The vaginal sponges used for estrus synchronization embedded with hormones, usually progesterone or its analogous compounds. Upon insertion, the progesterone embedded in the vaginal sponges slowly released into the bloodstream where it maintains the luteal phase up to removal of the sponge. Consequently after removal of the sponges, the level of progesterone falls, which give raise to estrogen. Thus, together with the action of high progesterone, the vaginal sponges finally resulted in the expression of estrus phase after sponge removal in a scheduled manner. However, the success of synchronization depends on number of days the sponges kept aside and the physiology of individual animals.

Vaginal sponges induced estrus synchronization is ever used technique in ruminants. A study by Hashemi et al. (2006) reported that the use of vaginal sponges is effective one than that of intramuscular injection of hormone for estrus synchronization in ewes. Martınez-Alvarez et al. (2007) also reported that the use of vaginal sponges in estrus synchronization seems to be effective. In support to their findings, they have analysed the serum LH profiles and calculated the time of estrus onset in goats, and found that the vaginal sponges as an efficient method in synchronizing estrus. Likewise, our findings also revealed that the vaginal sponges as one of the suitable methods for estrus synchronization in goats and the hormones embedded in the sponges act in a dose-dependent manner for estrus induction. At some instance, overt signs of estrus are not expressed by the goats; however it was resulted in successful fertilization when artificial insemination was done by calculating the day of sponge removal (Greyling and van der Nest, 2000).

39 Chapter I

Contrary to this, in our study, the female goats express visible and quantifiable estrus signs outwardly after estrus synchronization.

Behavioural expression is a prime fact in animal communication and could easily reflect the internal status to the external world. Animals from small to large mammals have considerable variation in exhibiting various types of behaviours under different physiological conditions (Rekwot et al.,

2001). In particular, non-primate females express behavioural patterns that vary under different phases of estrous cycle, in which estrus phase specifically exhibit some peculiar behaviour prominently (Porto-Filho et al., 2014). In line with this, we observed some of the behaviours seem to be expressed more intensively in estrus female goats, and in accordance with other mammals, those behaviours could be categorized into estrus-specific behaviours. Among the estrus-specific behaviours, bellowing, bleating, standing heat, vaginal mucus discharge, reduced milk secretion, reduced food intake and restlessness were seems to be more intense during estrus than non-estrus.

Similar pattern of behaviours was also observed in British white goat during the onset of estrus among which bleating, restlessness and vaginal mucus discharge seem to be notable (Lelwelyn et al., 1993). Most of the time, we observed homosexual behaviour in female goats during estrus by the expression of mounting behaviour towards other female. This is in accordance with the report of Shearer and Katz, (2006) who revealed that the homosexual behaviours is one of the most important behaviour exhibited by estrus goats.

Standing heat was observed in the female goats while in estrus phase, and in accordance with our report, Roelofs et al. (2005) reported that standing heat could be taken as a good indicator of estrus in cattle. They also suggested restlessness as another indicator of estrus, however, it could not be observed frequently as that of other behaviours in our study. Even though we observed restlessness in estrus female goats, it was not well pronounced often.

40 Chapter I

Bellowing is sometimes observed in estrus animals and taken into account for estrus detection (Layek et al., 2011). In the present study, we have observed bellowing behaviour by female goats only during estrus with high intense.

According to Nair et al. (2009), vocalization is one of the way of communication in animals. Thus, animals from certain distance in the wild utilize this vocal communication in order to make contact with other animal in the colony. In the present context, bellowing observed in female goats could be categorized into vocalization behaviours, and also, it could substantiate that the bellowing behaviour could be a better indicator of estrus, while the male was kept at certain distance. However, in our study, the male was kept inside the pen nearer to the female, and so, the bellowing was not observed at significant level. In contrast to goats, recent report suggests that chasing behaviours was used as a reliable and best indicator of estrus in cows

(Sveberg et al., 2011).

In response to females, males also exhibit different behaviours of which the flehmen and mounting seems to be prominent in ungulates (Doty, 2012).

Ghosal et al. (2012) also denotes that bulls exhibit significant flehmen while it was exposed to the faeces collected from female elephants during ovulatory phase. However, in the present study the faeces was not presented to the animal directly, however, the direct introduction of buck to the doe represented differential attraction during the different phases. The follow-up behaviours of mounting, such as, penile protrusion, self-enurination, chasing of females and coitus, were observed only towards the estrus goat and not to goats of other phases. This is in agreement with Price et al. (1986) that the male expressed self-enurination when prelude with estrus female goat. In an earlier study, we reported that flehmen and mounting behaviours have been expressed with significant difference towards the estrus-specific compounds and non-estrus compounds of faecal origin (Karthikeyan et al., 2013). By

41 Chapter I taking the above fact, we could conclude that the urine and faeces laid by the female (apart from the collected samples) may advertise the male that the female is in estrus, and hence, we could observe significant variation in the male characteristic behaviours towards the estrus female than that of non- estrus, i.e. either pro-estrus or post-estrus (metestrus and diestrus).

Variety of techniques were employed for the identification of estrus in farm animals. Due to the economical importance of goats, studies conveyed the importance of estrus detection and thereby to promote the successive steps in successful fertilization. The estrus detection techniques such as, analysis of serum hormone profile and ultrasonography, often give stress the animal and thereby seems to be invasive. However, in the present study, we have identified the behavioural patterns as one of the standard criteria for the identification of estrus. The added advantage of the present study is the behaviours observed in male goats towards the female goats of various reproductive phases. Thus, the male behaviours confer the fact that, if male goats are there, it will be very easy for the identification of estrus, thus insisted the importance of male goats in estrus detection. Thus, this chapter signifies the importance of behaviours of male and female goats to be used in the visual detection of estrus in female goats. However, in the absence of teaser or male goats and in a condition of small herd of goats without male, alternative tools are being required to confirm the estrus phase. This hurdle could be resolved by developing markers for estrus detection which would lay a milestone in the livestock sector. Thus, we next planned to study about the excretory sources in terms of biochemical analysis to make them as better indicator estrus to detect the estrus phase accurately and effectively.

42 Chapter I

1.5. Summary

 Estrus synchronization was done with vaginal sponges to synchronize

estrous cycle among six goats. Upon synchronization, the phases of the

estrous cycle were determined by the behavioural expression by female

and male goats.

 Among the behaviours exhibited by female tail wagging, restlessness,

reduced food intake and milk secretion was seems to be prominent.

Though, vaginal mucus discharge was observed in pro-estrus also,

however clear and visible discharge was observed only during estrus

which was arrested during post-estrus phases.

 We have observed sexually receptive behaviours such as, standing

heat, bellowing and bleating, vaginal swelling and homosexual

behaviours only during estrus. The increased sexual receptivity

resulted in the exhibition of homosexual behaviours and teasing of

male goats by estrus female goats.

 Mock fighting behaviour was observed both in female and male goats.

 Male goats’ exhibit flehmen and mounting towards the female goats. In

particular, the incidence of flehmen and mounting was in high

frequency towards the estrus samples or estrus female.

 Encountering estrus female elicit a specific behaviour in male goats,

called enurination, which was not observed towards any other phases

of female.

 In particular, the follow-up behaviours such as chasing of female,

penile protrusion and coitus were observed only towards the estrus

female goats.

43 Chapter II

Urinary biochemical and faecal steroid analysis of female goat

2.1. Introduction

Excretory sources often seem to be better indictors of internal physiology in animals. The significant alteration in excretion of metabolites makes urine as a better indicator of physiology (Farr et al., 1984). This has been proved in most of the animals (Achiraman et al., 2011b; Flately et al.,

2013). As a biological waste, urine is the cheapest, easily collectable and largely available source. It contains urea, inorganic salts, creatinine, ammonia, organic acids etc. (Bouatra et al., 2013). In addition, urine has been reported to contain protein. Substantial reports are also available for the occurrence of lipids in the urine (Brahmacharry, 1999). In non-primate mammals, phases of the estrous cycle are profoundly altered by the endocrine status (Fatet et al., 2011). This phase drift contribute to changes in the metabolism inside the body thereby bringing the change in the constituents that are excreted (Saude et al., 2007). A number of studies also suggest the existence of variation in urinary constituents in other animals.

Faeces is the another source of chemo-signal, have been validated for its chemo-signaling properties, from insect to mammals. It is resulted as a waste material after complete digestion of intaken food, but in animals, it could carry some signals (both volatile and non-volatile) to be utilized for the advertisement of physiological status. For favoring chemo-communication, faeces have been reported to contain volatile compounds with consistent variation during different phases of estrous cycle (Ghosal et al., 2012). In addition, it has also been reported to contain metabolites of steroid hormones to be used in non-invasive estrus detection in some of the wild and

44 Chapter II endangered animals (Archunan and Rajagopal, 2013). Thus, it has been ideally believed that the endocrinological status of animal could be identified by the hormonal metabolites present in the faeces.

Female goat reproductive cycle consists of four phases which is profoundly altered by the endocrine status of the animals. This phase drift also contribute to changes in the metabolism inside the body thereby bring the change in the constituents of excretory products (Saude et al., 2007). A number of studies suggest that there exist variation in urinary and faecal biochemical constituents in several animals. In female, estrus phase is of considerable importance, since this phase excrete some specific compounds in their excretory products and proposed to convey the reproductive receptiveness to the conspecifics. This could be resulted in sexual inter-course and aid in successful fertilization. Though identification of estrus phase is crucial for success rate of fertilization, there are variety of techniques updated for estrus detection in animals, but limitations always exists.

Hence, we hypothesize that physiological intervention could change the nature of urine and its bio-constituents. Further, faeces is known contain steroid hormone which could vary between different phases of estrous cycle.

In order to validate the above facts and to utilize urine and faeces for estrus detection strategy, we made an attempt to differentiate the urine of female goat during estrous cycle based on the biochemical pattern and faeces sample based on steroid hormones, with the aim of using the urinary biochemical parameters and faecal steroid levels for non-invasive estrus detection in goats.

2.2. Material and methods

2.2.1. Test animals and sample collection

Six sexually mature female goats were estrus synchronized using vaginal sponges (Central Sheep and Wool Research Institute, Rajasthan,

45 Chapter II

India) by a practiced veterinarian. Estrus synchronization was done as per the protocol as described in Chapter I. After synchronization, the phases of estrous cycle were calculated according the manufacture’s protocol.

Consequently, urine and faeces samples were collected from individual goats and stored at –20 °C until further analysis. The excreta (urine and faeces) were collected directly from the animal during excretion to avoid contamination. During the experimental period, the animals housed separately, given green forage, little fodder with water as ad libitum. The study was carried out at Veterinary College and Research Institute, Namakkal,

India.

2.2.2. Protein Estimation

The total protein in the test samples were determined by adopting the method of Bradford (1976) with Bovine Serum Albumin (BSA) as standard. To the required volume of protein sample, the distilled water was added to make up the volume to 100 µl of Bradford reagent (100 mg of Comassie Brilliant

Blue G-250 to 50 ml of 95% ethanol and 100ml of 85% orthophosphoric acid, made up to 1000 ml) was added.

Composition of Bradford Reagent

Coomassie Brilliant Blue G-250

Ethanol

Orthophosphoric acid

Methodology

Two microlitre of protein sample was added with 2.5 ml of Bradford reagent and incubated in dark for 10 minutes. While the color was changed from green to blue, the solution was read at 595 nm in UV- spectrophotometer. Based on the standard readings, the concentrations of proteins in unknown samples were calculated.

46 Chapter II

2.2.3. Estimation of lipids

Reagents required

Chloroform: methanol mixture (2:1)

0.9% Nacl solution

Concentrated sulphuric acid

Vanillin reagent

Lipids present in the urine samples were estimated according to the protocol of Folch, (1956). To 0.5 ml of urine sample, chloroform: methanol mixture was added. To that, 0.2 ml of 0.9% Nacl was added. The tubes were centrifuged at 3,000 rpm for 1 min. The lower layer was separated and the tubes were kept in water bath at 90-110° C to evaporate the solvent layer. To this, 100 µl of concentrated sulphuric acid was added and again kept in water bath at 90-110° C for 10 min. To that, 5 ml of Vanillin reagent was added and the tubes were read at 625 nm UV- spectrophotometer.

Note: Blank was prepared by adding 100 µl of concentrated sulphuric acid to

5 ml of vanillin reagent.

While evaporating the solvent layer, you could see white colored precipitate in the bottom of the test tubes.

2.2.4. Extraction of fatty acids in urine samples

Reagents required

Saponification reagent

1.5 g Sodium hydroxide in 10 ml methanol: water mixture (1:1 v/v)

Methylation reagent

6 N Hydrochloric acid (6.5 ml) with 5.4 ml methanol

Extraction solvent

1:1 hexane with anhydrous diethyl ether

47 Chapter II

Procedure

Five milliliter of urine sample was mixed with saponification reagent.

The tubes were kept in water bath at 100° C for 30 min. To that, 2 ml of methylation reagent was added and kept again in water bath at 80° C for 20 min. To this 1.25 ml of extraction solvent was added and the tubes were shaken thoroughly for 10 min. The solvent layer (upper layer) was taken and analysed using Gas Chromatography with standards under following procedure.

Gas Chromatography

Two microlitre of the sample was injected in Gas Chromatography

(Perkin Elmer Clarus 500). The temperature regimen is as follows: initial oven temperature 140-240 °C; injection port temperature, 260 °C and detector temperature is 260 °C. The type of column used is SP2650 (0.25 mm diameter, 100 m length). Nitrogen was used as carrier gas with Flame

Ionization Detector. The fatty acid present in the samples were compared with the standards and quantitatively measured with peak height.

2.2.5. Faecal hormone assay

The faecal samples were lyophilized until full water content was get released using a Lyophilizer (CHRiST ALPHA 1-2 LD plus). The dried faecal samples were pulverized in a blender to remove the solid inert materials

(seeds and dietary fiber). About 0.5 g of dried faeces was taken and 5 ml of

80% methanol was added. The tubes were kept in vortex for 30 sec at high speed. Then the tubes were kept in cooling shaker for overnight. After that, the samples were centrifuged at 8,000 rpm for 15 min at 4° C. The resulted supernatant was collected in a fresh tube. From this 100 µl of sample was mixed with assay buffer for hormone analysis.

48 Chapter II

Constituents of assay buffer

20 mM Tris hydroxyaminomethane

0.3 M Sodium chloride

0.1% Bovine serum albumin

0.1% Tween 80

The pH of the solution is 7.5.

Chemiluminescent Microparticle Immunoassay (CMIA)

The ARCHITECT Estradiol and Progesterone assay in a

Chemiluminescent Microparticle Immunoassay for the quantitative determination of estradiol and progesterone in goat fecal samples.

Summary and explanation of the kit

Estradiol is the most potent natural estrogen in humans. It regulates reproductive function in females, and, with progesterone maintains pregnancy. Most estradiol is secreted by the ovaries (non-pregnant women), although in testes (in men) and adrenal cortex (in men and women) secrete small amounts. During pregnancy, the placenta produces most of the circulating estradiol. Estradiol and estrone interconvert in vivo. In normal non-pregnant women estradiol synthesized by the ovary is the predominant source of both estrone and estriol.

Virtually all circulating estradiol is protein-bound. Reported association constants for estradiol with sex hormone binding globulin and serum albumin are, respectively 6.8 X 108 and 6 X 104. One consequence of this binding is that the conditions of any assay for serum estradiol must release this steroid quantitatively from its binding partners. The amount wand proportion of protein-bound and free estradiol varies by gender, and with pregnancy and menstrual phase in women.

49 Chapter II

Normal estradiol levels are lower at menses and into the early follicular phase and then rise in the late follicular phase to a peak just before the LH surge, which is normally followed immediately after ovulation. As LH peaks, estradiol beings to decrease before rising again during the luteal phase. If conception does not take place, estradiol falls further to its lowest levels, and menses begins shortly thereafter. It conception occurs, estradiol levels continue to rise and doubling during every trimester. At menopause, estradiol levels remain low.

Because the ovaries produce most estradiol in normal women, estimation of this hormone is sometimes a gauge of ovarian function. In addition, monitoring estradiol levels is important in evaluating amenorrhea, precocious puberty, the onset of menopause, and infertility in men and women. Monitoring estradiol levels is essential during in vitro fertilization, because the timing of recovery of oocytes depends on follicular development, which in turn depends on the estradiol level.

Principles of the procedure

The ARTCHITECT estradiol is a delayed one step immunoassay to determine the presence of estradiol using chemiluminescent immunoassay with flexible assay protocols, referred to as chemiflex.

In the first step, sample, specimen diluent, assay diluent, and anti- estradiol (rabbit, monoclonal) called paramagnetic mirocrparticles are combined. Estradiol present tint he samples binds to the anti-estradiol coated microparticles. After incubation, estradiol acridium labeled conjugate is added to the reaction mixture. After a second incubation, and washing, pre-trigger and trigger solutions are then added and the resulting chemiluminescent reaction is measured as relative light units (RLUs). An inverse relationship

50 Chapter II exists between the amount of estradiol in the sample and the RLUs detected by the ARCHITECT I optical system.

ARCHITECT Estradiol Reagent Kit (7K72)

Microparticles: 1 bottle (9.9 mL) anti-estradiol (rabbit, monoclonal) coated microparticle in TRIS/BIS-TRIS buffer with protein (rabbit) stabilizers.

Minimum concentration: 0.0657% solids.

Preservative: ProClin

Conjugate: 1 bottle (5.9 mL) Estradiol acridium-labeled Conjugate in citrate buffer with surfactant stabilizers. Minimum concentration 63.36 ng/mL.

Preservative: ProClin.

Assay Diluent: 1 bottle (5.9 mL) Estradiol assay diluent containing surfactant in citrate buffer. Perservative: ProClin

Specimen diluent: 1 bottle (10 mL) Estradiol specimen diluent containing TRIS buffer with protein (bovine) stabilizers.

Preservative: Sodium Azide

Pre-trigger solution : Pre-trigger solution containing 1.32% (w/v) hydrogen peroxide.

Trigger solution : Trigger solution containing 0.35N sodium hydroxide.

Wash buffer

Wash buffer containing phosphate buffered saline solution.

Preservatives: antimicrobial agents.

51 Chapter II

Progesterone assay

The architect progesterone assay is a Chemiluminescent Microparticle

Immunoassay for the quantitative determination of progesterone in human serum and plasma.

Summary and explanation of the test

Progesterone is produced primarily by the corpus luteum of the ovary in normally menstruating women and to a lesser extent by the adrenal cortex.

At approximately the 6th week of pregnancy, the placenta becomes the major producer of progesterone. The major functions of progesterone are in the preparation of the uterus for implantation and maintenance of pregnancy.

During the follicular phase of the cycle, progesterone levels remain low.

Following the LH surge and ovulation, luteal cells in the ruptured follicle produce progesterone in response to LH. During this luteal phase, progesterone rises rapidly to a maximum. In conception does not occur, progesterone levels decrease during the last four days of the cycle due to the regression of the corpus luteum. If conception occurs, the level of progesterone is maintained at mid-luteal levels by the corpus luteum until about week six. At that time, the placenta becomes the main source of progesterone and levels rise and doubled during first and third trimester.

Serum progesterone is a reliable indicator of either neutral or induced ovulation because of its rapid rise following ovulation. Disorders of ovulation, including anovulation, are relatively frequent and are responsible for infertility in approximately 15-20% of patients. Progesterone levels are abnormally low in these patients during the mid-luteal phase.

Luteal phase deficiency is a reproductive disorder associated with infertility and spontaneous abortion and is thought to occur in 10% of infertile

52 Chapter II women. The infertility and pregnancy loss associated with this disorder are thought to be attributable to inadequate development of the endometrium.

The failure of the endometrium mature is thought to be caused by insufficient production of progesterone by the corpus luteum. Progesterone levels in the luteal phase are lower than normal in women with luteal phase deficiency.

Measurement of progesterone in the first 10 weeks of gestation has been shown to be reliable and effective for the diagnosis and treatment of patients with threatened abortion and ectopic pregnancy. Suppressed progesterone levels in the presence of detectable amounts of hCG is highly suggestive of patients with threatened abortion or ectopic pregnancy, regardless of gestational age.

Biological importance of the procedure

The ARCHITECT progesterone assay is a one-step immunoassay to determine the presence of progesterone in human serum and plasma using

Chemiluminescent Microparticle Immunoassay (CMIA) technology with flexible assay protocols, referred to as Chemiflex.

2.3. Results

2.3.1. Protein level

The protein level varied significantly across all the four phases

(P<0.05). Particularly, the protein concentration was significantly high in estrus followed by diestrus and proestrus. A low level of protein was found in metestrus urine (Table. 2.1; Figure. 2.1).

2.3.2. Lipid level

Urinary lipid concentration also varied significantly among all the phases (P<0.05). Significantly increased level of lipid was found in metestrus followed by diestrus, whereas proestrus and estrus urine revealed significantly low level (Table. 2.1; Figure. 2.2).

53 Chapter II

Fig. 2. Protein concentration among different phases of estrous cycle. Values are expressed in Mean. Superscripts which are not marked with same superscript are significantly different at α=0.05 using Duncan’s Multiple Range Test (post hoc analysis).

Fig. 2.2. Lipid concentration among different phases of estrous cycle. Values are expressed in Mean. Superscripts which are not marked with same superscript are significantly different at α=0.05 using Duncan’s Multiple Range Test (post hoc analysis).

54 Chapter II

Table 2.1. Concentration of protein and lipid in urine of female goat

during estrous cycle

Phases of the cycle Protein Lipid (µg/dl) (µg/ml)

Proestrus 93.8±0.10c 2.2±0.08d

Estrus 96.8±0.07a 2.7±0.11c

Metestrus 89.4±0.12d 5.3±0.12a

Diestrus 95.8±0.12b 4.8±0.18b

Values are expressed in Mean ± SE of 6 animals. Superscripts which are not marked with same superscript are significantly different at α=0.05 using Duncan’s Multiple Range Test (post hoc analysis).

2.3.3 Fatty acid profile

GC analysis revealed three fatty acids (lignoceric, cis 5,8,11,14,17- eicosapentanoic and nervonic acid) during all the four phases with consistent variation in concentration (Table. 1). The level of lignoceric acid was higher in estrus followed by diestrus and proestrus; and the lowest level was observed in metestrus urine. In contrast, cis 5,8,11,14,17-eicosapentanoic acid was higher in metestrus and diestrus, whereas it was found reduced in proestrus and estrus urine. Interestingly, nervonic acid was found elevated in metestrus and reduced during proestrus. However the level was lower in diestrus and it was further reduced in estrus urine (Table. 2.2) (Chromatogram 1 to 4).

55 Chapter II

Chromatogram 1 : GC analysis of pro-estrus urine

Chromatogram 2 : GC analysis of estrus Urine

56 Chapter II

Chromatogram 3 : GC analysis of met-estrus urine

Chromatogram 4 : GC analysis of di-estrus urine

57 Chapter II

2.3.4. Hormone assay

The faecal steroid hormones varied significantly during the phases of estrous cycle (Figure. 2.3). Estradiol concentration was significantly higher during estrus than pro-estrus and post-estrus. In contrast, significantly higher concentration of progesterone was noted during post-estrus than estrus and pro-estrus phase.

Table. 2.2. List of fatty acids identified in the female goat urine during estrous cycle. The concentration of fatty acids is expressed in percentage (%).

Name of the fatty acid Concentration in %

Proestrus Estrus Metestrus Diestrus

Lignoceric acid 93.6 96.8 89.5 95.7

Cis 5,8,11,14,17- 1.1 1.2 2.5 2.3 Eicosapentanoic acid

Nervonic acid 5.2 1.9 7.8 2.5

58 Chapter II

10 150 Progesterone Estrogen 8 140

130

6 120

110 4 Estrogenpg/mL 100 Progesteroneng/mL 2 90

80 0 Pro-estrus estrus Post-estrus Stages of estrous cycle

Fig. 2.3. Faecal estradiol and progesterone concentration during estrous cycle

2.4. Discussion

Excretion is a process of eliminating the metabolic waste through variety of excretory products. Among those, urine is one of the most important due to its potential application in clinical diagnostics. Indeed the constituents of urine make it as a good source in diagnostics, in most of the animal it has also been reported as one of the prime sources in animal communication

(Archunan, 2009). The chemical compounds excreted via urine promote communication between inter-individuals and intra-individuals.

59 Chapter II

The constituents of urine depend on the physiology of individual animal. In male goats, urinary volatiles changed under different physiological status i.e. prepubertal, adolescent and castration. Similarly, it has been reported that the volatiles in the urine changes across the estrous cycle in females (Dehnhard et al., 2006). Further it is evidenced that the female mice excretes different type of biochemical molecules that differ qualitative and quantitative manner among the phases of estrous cycle (Achiraman et al.

2011b).

Among the biochemical molecules, protein, lipid and fatty acids have occupied a major part in animal communication. Hence, we measured the level of protein and lipid and analysed fatty acid in the urine samples of female goat during estrous cycle. Presence of protein in the urine is termed as proteinurea, a condition reflecting disease/illness in animals. However, it has been proven that urinary proteins itself or the volatile bound in it indicates the physiological status and aid in communication in mice (Stopka et al.,

2007). Also, the difference in quantity of urinary protein excretion was determined which further claims that estrus urine contains more amount of protein than the urine of other phases (Achiraman et al., 2011b). In accordance with this, we have observed a higher amount of protein in estrus urine. This led us to speculate that the high level of protein excreted during estrus may be of estrogen dependent and useful in indicating the estrus to the male goats.

The second most important biochemical constituent, the lipid, has been extensively investigated in communication of insects (Rottler et al., 2013).

Even though, presence of lipid in urine related to diseased status of animals, it has also been reported that lipid have pivotal role in animal communication

(Menzies et al., 1992). For instance, the urine of tiger contains more quantity of lipids which could be used for fixing the volatile molecules (Brahmacharry,

60 Chapter II

1999). In cats, the urinary lipids involved in keeping the volatile signals for longer time (Margaret et al., 2008). In the present study, high concentration of lipid was noted during metestrus followed by diestrus. These results confer that the lipids may have a significant function during the follicular phase (pre- ovulatory phase) than in luteal phase (post-ovulatory phase). Zhou et al.

(2009) reported major urinary protein regulates the expression of lipogenic genes in the liver and could contribute to changes in the expression of lipid level. In the present context, we suggest the proteins present in the metestrus phase could regulate the excretion of lipid in urine, however, the specific protein involved in the regulation of lipogenic enzyme has to be studied in detail.

Fatty acids are the most utilized functional mediator in animal communication. Earlier it was reported that the vaginal secretions of human contained the volatile fatty acids which could be the possible mediator of communication (Waltman et al., 1973). Indeed, various studies reported the presence of fatty acids in biological fluids, the role of fatty acid in animal communication has been poorly explored. Alagendran et al. (2011) reported the presence of fatty acids in the female saliva without alteration during menstrual cycle. Foster (2004) reported that fatty acid may act as precursor for pheromone biosynthesis and the level of lipid may directly correlate with the amount of pheromone synthesized. In our study we found three fatty acids namely, lignoceric acid, cis 5,8,11,14,17- eicosapentanoic acid and nervonic acid in the urine of female goats during the estrous cycle. Matsumoto-Oda et al. (2003) reported the presence of fatty acids in vaginal mucus of

Chimpanzees, which differed both qualitatively and quantitatively. In contrast, we have not observed qualitative difference of fatty acids in the goat urine. The important notion in the present study is that the urinary fatty

61 Chapter II acids of the present study were not reported in any other animals, hence, we propose the three fatty acids may be of species-specific.

The estimation of hormones in the faeces is one of the feasible ways to detect the reproductive status of animals and also utilized as a non-invasive method (Kumar et al., 2013). Weltring et al. (2012) analysed the faeces of new world primates in view of steroid hormone profiles and found that estrogen and progesterone were higher in pregnant females than lactating females.

However, our study did not focused on the lactating animals, but we found profound variation of estradiol and progesterone during three different phases of estrous cycle. Among those, we found significantly higher level of estradiol during estrus than any other phases. This has been well documented that the concentration of estrogen in serum is always in higher concentration during estrus (Achiraman and Archunan, 2010), and we infer that the circulating estrogen in serum may contribute to increased concentration of estradiol in the estrus faeces. Earlier study on felids proved that progesterone concentration in the faeces is higher during post-estrus phases (pregnant and pseudo-pregnant condition) than that of ovulatory phase (Brown et al., 1994).

A similar pattern was also observed in our study, that the progesterone concentration was higher in post-estrus faeces, whereas it was significantly less in estrus and pro-estrus faeces. Thus, the overall steroid hormone profile added evidence that the phases of the estrous cycle is accurately determined for the sample collection and the hormone profile data could be used as an alternative tool for non-invasive reproductive assessment in female goats.

Though the level of urinary biochemical and faecal steroid hormones varied significantly, it could be proposed that the volatile compounds may also vary during different phases of the estrous cycle. If so, the volatile pattern would also be used as a non-invasive indicator of physiological status. Hence, the

62 Chapter II next chapter pertains to the analysis of volatile compounds present in the urine and faeces of female goats during different phases of estrous cycle.

2.5. Summary

 The protein concentration in the urine samples of female goats was

higher in estrus phase than that of other phases. The least

concentration of protein was found in metestrus urine.

 The lipid concentration in the urine samples was found elevated in

metestrus urine followed by diestrus phases. However, lipid

concentration was further reduced in estrus phase and least

concentration of lipid was found in proestrus urine.

 In GC analysis, we found three fatty acids, such as, lignoceric acid, cis

5,8,11,14,17-eicosapentanoic acid and nervonic acid in all the urine

samples, with subtle difference in the quantity during estrous cycle.

Among the three fatty acids, lignoceric acid was found in maximum

quantity in estrus urine whereas, cis 5,8,11,14,17-eicosapentanoic acid

and nervonic acid was found to at higher concentration during

metestrus phase.

 In fecal steroid hormone analysis, estradiol was at significantly higher

concentration in estrus, whereas its level was reduced during post-

estrus, and progesterone concentration was significantly higher

concentration during post-estrus phase.

63 Chapter III

Volatile analysis in female urine and faeces

3.1. Introduction

In non-primate female mammals the estrous cycle consists of three phases viz., pro-estrus, estrus and post-estrus (met-estrus and di-estrus combined). In particular, the estrus phase is important, since successful fertilization will occur only if the animal is in sexual contact with males during this phase (Wildeus, 2000). But the identification of individuals in estrus phase in a large herd is difficult. False identification of animals in estrus and consequent out of phase insemination remain a barrier in the improvement of livestock production (Baril et al., 1993). On the other hand, estrus synchronization is an effective tool in the reproductive management of farm animals (Whitley and Jackson, 2004). It involves accurate change in the reproductive phases of animals, so as to be helpful in improving the success rate of fertilization in large herds.

It is an established fact that the male goats detect the females in estrus through chemical cues. Intra-specific communication between animals is most often favored by these chemical cues present in the sources that are excreted. These chemical compounds include both volatiles and non-volatiles, which faithfully convey information about sex, species, and physiological status to the other animals of the same species (Dulac and Torello, 2003).

Particularly, in the female mammals these chemical compounds exhibit considerable variation through different phases in the estrous cycle. This accounts for the changes in the pattern of compounds present in the materials that are excreted (urine, faeces, vaginal mucus, etc.) (Tirindelli et al., 2009). Of note among the excretory sources, urine and faeces are well-

64 Chapter III known non-invasive indicators of internal physiology in higher mammals

(Rajanarayanan and Archunan, 2011; Karthikeyan et al., 2013).

Urine is the one of most primordial source in animal communication.

The use of urine as a source of communication has been documented well earlier with mice (Colby and Vandenberg, 1974). Consequently, urine has been proved as an effective source of communication in other rodents (Lydell and Doty, 1972). Reports revealed that the cat can produce pheromone under the control of a putative pheromone precursor (felinine) (Miyazaki et al.,

2006). Later on, the concept of using urine as a prominent source of communication has been started in higher animals also. For instance, in cow, urine has been observed as a good source of communication which has been substantiated by the variation of volatile compounds and the possibility of those compounds to act as pheromones (Archunan and Rameshkumar, 2012).

Likewise, in buffalo urine has been proved as an effective source of communication which confers the physiological status of females to the males

(Rajanarayanan and Archunan, 2011). In wild condition, urine has been reported as an efficient source of communication by having the long-lasting effect thereby, have potential role in scent marking (Anderson and Vulpius,

1999). In tiger, the urine has been considered as a fluid for territorial marking which imply in the controlled release of volatiles in wild condition (Burger et al., 2008). The fact of using urine as a source of territorial marking has also been established in the endangered Indian blackbuck. The territorial marking could also have been involved in the expression of dominant status in a colony of blackbuck (Archunan and Rajagopal, 2013; Rajagopal et al., 2010).

Faeces is another source of chemo-signal, which has been validated from insect to mammals. Faeces is the digested waste material released as excreta, but in animals, it could carry some chemical signals (both volatile and non-volatile) to be used in the advertisement of physiological status

65 Chapter III between animals. For favoring chemo-communication, faeces have been reported to contain volatile compounds with consistent variation during different phases of estrous cycle in elephant (Ghosal et al., 2012).

In this sense, the present study was aimed at linking estrus synchronization with chemo-signal analysis in the goat. Since, none of the study has been focused on the identification of volatile compounds in the scent sources of Indian goat, Capra hircus, we took urine and faeces as the sources of chemo-signals to monitor the cyclic changes and made an attempt to provide possible evidence for using urine and faeces as reliable non- invasive indicators of estrus in the goat.

3.2. Material and methods

3.2.1. Estrous cycle synchronization

Sexually experienced and regularly cycling female goats (n=6) were used in the present study. The experimental animals were maintained separately in the Livestock Farm at Veterinary College and Research Institute,

Tamilnadu Veterinary and Animal Science University, Namakkal, Tamilnadu,

India, during the entire study. The goats were allowed for natural grazing for about 8 hours each day and supplemented with cultivated forages and water ad libitum. The vaginal sponge procured from Central Sheep and Wool

Research Institute (CSWRI), Avikanagar, Rajasthan, India, was used in the present study.

3.2.2. Insertion of vaginal sponges

A Veterinarian inserted a single sponge into the cervix region of the vagina of each goat using a plunger and speculum. After the insertion, the animals were kept isolated during housing. The assessment of estrous cycle was done by calculating the days of insertion of sponge. The sponge was retained for about 15 days, and the day it was removed the goat was

66 Chapter III considered as in pro-estrus phase. One day after removal of the sponge the goat was considered as in estrus, and two days after removal the goat was considered as in metestrus and 8 days after removal considered as diestrus.

The procedure of estrus synchronization as mentioned in first chapter.

3.2.3. Sample collection

After assessment of the estrous cycle as mentioned above, urine samples were collected individually in screw capped vials directly while animal urinate to avoid contamination. Faeces samples were also collected in zip lock covers. Both the samples were collected during all the above phases, labeled and stored at -20 °C immediately after collection until further use.

3.2.4. Extraction of volatile compounds and Gas Chromatography-Mass

Spectrometry

The GC-MS analyses were made in QP-5000, (Schimadzu, Japan). The

2 µl of extract was injected into the GC-MS on a 30 m glass capillary column with a film thickness of 0.25 µm (30 m X 0.2 mm i.d. coated with UCON HB

2000) using the following temperature programme, initial oven temperature of

40 °C for 4 minutes increasing to 250 °C at a rate of 15 °C for 10 minutes.

The area under each peak was used for quantitative calculations. The detection accuracy was about 1ng/peak. The relative amount of each component was reported as the percent of the ion current. The GC-MS was under the computer control at 70 - ev. Using ammonia as reagent gas at 95 - ev performed chemical ionization. Identification of unknown compounds was made by probability based matching using the computer library built within the NICT 12 system. The GC-MS operating programme is given below.

GC-MS programme

Name of GC-MS: GC-MS QP 5000 (SCHIMADZU, JAPAN)

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GC-parameters

Amount of sample : 2 µl

Oven temperature : 70°C

Oven equal time : 3 min

Injector temperature : 200°C

Interface temperature : 240°C

Sampling time : 1 min

Column length : 30 m

Column diameter : 0.25 mm

Name of the column : DBI - nonpolar column

Column packed material : Dimethyl polysiloxane

Column pressure :14 Kpa

Column flow :1.4 ml/min.

Linear velocity : 38.1

Split ratio : 14

Total flow : 25 ml/min.

MS-Parameters

Start M/Z : 50

End M/Z : 310

Scan Interval : 1 second

Threshold : 1000

Scan speed : 500 amu/sec.

Solvent cut : 4 min.

GC-programming time : 9 min.

Start time : 6 min.

End time : 9 min.

Detector volts : 1.10 KV

Electron impact ionization : 70 ev

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Detector : Electron multiplier

Acquisition time : 4.10 - 64 min.

Mass range : 30 - 600 amu = atomic mass unit

M/Z = Mass to charge ratio.

3.3. Results

3.3.1. GC-MS analysis of proestrus urine samples

The urinary compounds identified in the pro-estrus urine have the molecular weight from 224 to 410 Da. The compounds were identified as 2,4-

Bis 1,1, dimethylethyl phenol,1-Nonadecene, 1-Heptacosanol, 3-Hexadecene,

1-Hexadecene, 1-n-Tetracosanol, Tetra biphenol and Nonadecene. Most of the compounds belong to the chemical group of alcohol and alkenes. The molecular formula consists of minimum of 14 carbon and maximum of 28 carbon atoms. The hydrogen atoms were seems to be between 22 and 50 (Fig.

3.1; Table 3.1).

3.3.2. GC-MS analysis of estrus urine samples

Estrus urine contained a total of 8 volatile compounds in GC-MS analysis. Based on the library search the compounds were identified as 2,4-

Bis 1,1, dimethylethyl phenol, 1-Nonadecene, 1-Heptacosanol, Tetradecanol,

N-Pentadecanol, 3- Methylene tridecane, 2-Ethyl 1- dodecene and Behenic alcohol. The molecular weight of the identified compounds was between 196 and 396. The chemical groups of the compounds were mostly belongs to alcohol and alkenes. The compounds have the carbon atoms from 14 to 22 and hydrogen atoms between 22 and 46 (Fig.3.2; Table 3.2).

3.3.3. GC-MS analysis of post-estrus urine

GC-MS analysis of post-estrus urine revealed a total of 6 compounds which were identified as 2,4-Bis 1,1, dimethylethyl phenol, 1-Nonadecene, 1-

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Heptacosanol, 1-Pentadecene, Nonadecene and Behenic alcohol. The compounds were belonged to the chemical group of alcohol and alkenes. The molecular weight of the compounds was from 210 to 396. The carbon atoms of the identified compounds were from 14 to 27 and hydrogen atoms from 22 to 46 (Fig. 3.3; Table 3.3).

3.3.4. GC-MS analysis of urine samples from estrous cycle

GC-MS analysis revealed a total of 14 compounds, all the phases put together (Table 3.4; Figs. 3.1 to 3.3). Of them, three were common to all the phases; four were specific to pro-estrus phase; four compounds were specific to estrus phase; only one compound was specific to post-estrus phase. The estrus-specific compounds were identified as Tetradecanol, n-Pentadecanol,

3-Methylene tridecane, 2-Ethyl-1-dodecene. Interestingly, we found a compound, namely behenic alcohol which is absent during pro-estrus but present in estrus and post-estrus phases. The molecular weight of the identified compounds was between 196 and 410. The chemical nature of the compounds was found to be alcohol and alkenes but acids and alkanes were also present.

70 Chapter III

Fig. 3.1. GC-MS chromatogram of urine of goat in pro-estrus

Table 3.1. List of compounds identified in the urine of female goats during Pro-estrus

S. No Peak Name of the Molecular Molecular No compound weight formula

1 1 2,4-Bis 1,1, 206 C14H22O dimethylethyl phenol

2 2 1-Nonadecene 266 C19H28

3 3 1-Heptacosanol 396 C27H56O

4 4 3-Hexadecene 224 C16H32

5 5 1-Hexadecene 224 C16H32

6 6 1-n-Tetracosanol 354 C24H50O

7 7 Tetra biphenol 410 C28H42O2

8 13 Nonadecene 266 C19H38

71 Chapter III

Fig. 3.2. GC-MS chromatogram of urine of goat in estrus

Table 3.2. List of compounds identified in the urine of female goats during estrus

S. No Peak Name of the Molecular Molecular No compound weight formula

1 1 2,4-Bis 1,1, 206 C14H22O dimethylethyl phenol

2 2 1-Nonadecene 266 C19H28

3 3 1-Heptacosanol 396 C27H56O

4 8 Tetradecanol 214 C14H30O

5 9 N-Pentadecanol 228 C15H32O

6 10 3- methylene tridecane 196 C14H28

7 11 2-Ehtyl 1- dodecene 196 C14H28

8 14 Behenic alcohol 326 C22H46O

72 Chapter III

Fig. 3.3. GC-MS chromatogram of urine of goat in post-estrus

Table 3.3. List of compounds identified in the urine of female goats during post-estrus

S. Peak No Name of the compound Molecular Molecular No weight formula

1 1 2,4-Bis 1,1, dimethylethyl 206 C14H22O phenol

2 2 1-Nonadecene 266 C19H28

3 3 1-Heptacosanol 396 C27H56O

4 12 1-Pentadecene 210 C15H30

5 13 Nonadecene 266 C19H38

6 14 Behenic alcohol 326 C22H46O

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Table 3.4. Comparison of compounds identified in the urine of female goats during estrous cycle

S. Name of the Molecular Molecular Pro- Estrus Post- No compound weight formula estrus estrus

1 2,4-Bis 1,1, 206 C14H22O √ √ √ dimethylethyl phenol

2 1-Nonadecene 266 C19H28 √ √ √

3 1-Heptacosanol 396 C27H56O √ √ √

4 3-Hexadecene 224 C16H32 √ X X

5 1-Hexadecene 224 C16H32 √ X X

6 1-n-Tetracosanol 354 C24H50O √ X X

7 Tetra biphenol 410 C28H42O2 √ X X

8 Tetradecanol 214 C14H30O X √ X

9 N-Pentadecanol 228 C15H32O X √ X

10 3- methylene 196 C14H28 X √ X tridecane

11 2-Ehtyl 1- 196 C14H28 X √ X dodecene

12 1-Pentadecene 210 C15H30 X X √

13 Nonadecene 266 C19H38 √ X √

14 Behenic alcohol 326 C22H46O X √ √

√- present; X- absent

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(a) Tetradecanol

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(b) n-Pentadecanol

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(c) 3-methylene tricdecane

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(d) 2-Ethyl 1-Dodecene

Mass Spectrum of Estrus specific urinary compounds

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3.3.5. GC-MS analysis of pro-estrus faeces

GC-MS analysis of faeces samples from proestrus goat revealed a total of 11 compounds among which some were belong to the group of alcohol, acid and alkenes. We found three alcoholic compounds such as, 3-methyl-3- buten-1-ol, 1-Dotricontanol and 1-Pentadecanol and one acid, i.e. 1,2-Benzene dicarboxylic acid. The compounds have the molecular weight ranges from 166 to 759 Da. The molecular formula of the identified compounds contains minimum of 6 carbon atoms and maximum of 54 carbon atoms. The number of hydrogen atoms in the identified compounds was between 8 and 40 (Fig. 3.4; Table 3.5).

3.3.6. GC-MS analysis of estrus faeces

Volatile analysis of estrus faeces revealed a total of 9 compounds among which, 5 compounds were identified as estrus-specific. The estrus- specific compounds were Pentadecyl alanine, Ascorbic acid, 1-Octadecanol,

Octadecanoic acid, 1-tetradecanol and Vitamin E. The identified compounds were under the chemical group of alcohol, alkenes and acids. Interestingly, we found two anti-oxidants such as ascorbic acid and Vitamin E only in the estrus faeces. The compounds have the molecular weight ranges from 166 to

430. The number of carbon atoms in the identified compounds was between 8 and 29, whereas the hydrogen atoms were between 6 and 50. In some of the compounds oxygen also present with the number ranging from 1 to 4 (Fig.3.5;

Table 3.6).

3.3.7. GC-MS analysis of post-estrus faeces

The volatile compounds identified in the post-estrus faeces were about

11 compounds. Among which, most of the compounds were alcohol in nature.

In addition, alkene, acid and alkenes were also present. The alcoholic compounds were 3-methyl-3-buten-1-ol, 1-Dotricontanol and 1-Pentadecanol.

The molecular weight of the identified compounds was between 86 and 759.

79 Chapter III

However, the high molecular weight compound tetrapentcontane was a common to pro-estrus and post-estrus faeces. The molecular formula of the volatile compounds comprises of 6 to 20 carbon atoms, and 8 to 110 hydrogen atoms (Fig.3.6; Table 3.7).

3.3.8. GC-MS analysis of faces samples from estrous cycle of female goat

The estrous cycle of female goat revealed 24 compounds, when all the phases put together. Among those, two compounds were present in all the phases and six were specific to pro-estrus (Table. 3.8; Figs. 3.4 to 3.6). In contrast, rest of the phases (estrus and post-estrus) showed 7 specific compounds each. Among the estrus-specific compounds, we observed two anti-oxidants such as ascorbic acid and vitamin E in addition to 1- octadecanol, 1-tetradecanol and octadecanoic acid. Interestingly, the compound, 2-hexadecene was present during pro-estrus and post-estrus whereas absent in estrus faeces. Most of the identified compounds were belonging to alkanes, alkenes, acids and alcohol.

80 Chapter III

Fig. 3.4. GC-MS chromatogram of faeces of goat in pro-estrus

Table 3.5. List of compounds identified in the pro-estrus faces of female goat

S. No Peak Name of the compound Molecular Molecular No weight formula

1 1 Phytol 296 C20H40O

2 2 1,2-Benzene dicarboxylic acid 166 C8H6O4

3 3 2-Hexadecene 224 C16H32

4 4 Tetrapentacontane 759 C54H110

5 5 n-Hexadecanoic acid 256 C16H32O2

6 6 Nonadecene 266 C19H38

7 7 9-Tricosene 322 C23H46

8 8 2,3 oxobutyl Cyclohexane 198 C10H14O4

9 9 4-Chlorobutyric acid 122 C4H7ClO2

10 10 Tetradecence 196 C14H28

81 Chapter III

Fig. 3.5. GC-MS chromatogram of faeces of goat in estrus

Table 3.6. List of compounds identified in the estrus faeces of female goat

S. No. Peak Name of the compound Molecular Molecular No. weight formula

1 1 Phytol 296 C20H40O

2 2 1,2-Benzene dicarboxylic acid 166 C8H6O4

3 11 Pentadecyl alanine 383 C23H45NO3

4 12 Ascorbic acid 176 C6H8O6

5 13 1-Octadecanol 270 C18H38O

6 14 Octadecanoic acid 282 C18H34O2

7 15 1-tetradecanol 214 C14H30O

8 16 Vitamin E 430 C29H50O2

9 17 2-Nonadecene 266 C19H38

82 Chapter III

Fig. 3.6. GC-MS chromatogram of faeces of goat in post-estrus

Table. 3.7. List of compounds identified in the post-estrus faeces of

female goat

S. No. Peak Name of the compounds Molecular Molecular No. weight formula

1 1 Phytol 296 C20H40O

2 2 1,2-Benzene dicarboxylic acid 166 C8H6O4

3 3 2-Hexadecene 224 C16H32

4 4 Tetrapentacontane 759 C54H110

5 18 3-methyl-3-buten-1-ol 86 C5H10O

6 19 1,2-oxathiene 249 C6H8INO2

7 20 1,3,2-dioxaborolane 186 C9H19BO3

8 21 Araldite 320 C18H21ClO3

9 22 1-Dotricontanol 136 C9H12O

10 23 1-Pentadecanol 228 C15H32O

11 24 2,6-octadiyene 289 C16H20ClN3

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Table. 3.8. List of compounds identified in the faeces of pro-estrus, estrus and post-estrus female goat

Peak Name of the Molecular Molecular Pro- Estrus Post- No. compounds weight formula estrus estrus

1 Phytol 296 C20H40O ✔ ✔ ✔

2 1,2-Benzene dicarboxylic 166 C8H6O4 ✔ ✔ ✔ acid

3 2-Hexadecene 224 C16H32 ✔ X ✔

4 Tetrapentacontane 759 C54H110 ✔ X ✔

5 n-Hexadecanoic acid 256 C16H32O2 ✔ X X

6 Nonadecene 266 C19H38 ✔ X X

7 9-Tricosene 322 C23H46 ✔ X X

8 2,3 oxobutyl 198 C10H14O4 ✔ X X Cyclohexane

9 4-Chlorobutyric acid 122 C4H7ClO2 ✔ X X

10 Tetradecence 196 C14H28 ✔ X X

11 Pentadecyl alanine 383 C23H45NO3 X ✔ X

12 Ascorbic acid 176 C6H8O6 X ✔ X

13 1-Octadecanol 270 C18H38O X ✔ X

14 Octadecanoic acid 282 C18H34O2 X ✔ X

15 1-tetradecanol 214 C14H30O X ✔ X

16 Vitamin E 430 C29H50O2 X ✔ X

17 2-Nonadecene 266 C19H38 X ✔ X

18 3-methyl-3-buten-1-ol 86 C5H10O X X ✔

19 1,2-oxathiene 249 C6H8INO2 X X ✔

20 1,3,2-dioxaborolane 186 C9H19BO3 X X ✔

21 Araldite 320 C18H21ClO3 X X ✔

22 1-Dotricontanol 136 C9H12O X X ✔

23 1-Pentadecanol 228 C15H32O X X ✔

24 2,6-octadiyene 289 C16H20ClN3 X X ✔

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(a) Pentadecyl alanine

85 Chapter III

(b) Ascorbic acid

86 Chapter III

(c) 2-Nonodecene

87 Chapter III

(d) 1-Octadecanol

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(e) Octadecanoic acid

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(f) 1-Tetradecanol

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(g) Vitamin E

Mass Spectrum of Estrus specific faecal volatile compounds

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3.4. Discussion The phase shift during the estrous cycle may be exposition of difference in metabolism and contribute to endocrine changes in the physiology and constituents of the excretory products. To validate this, we collected the urine samples and analyzed for volatile compounds during all the phases of the estrous cycle. As expected, we observed difference in the profiles of the urinary volatile among phases. This is in accordance with the earlier report that smaller to higher female mammals exhibit significant difference in the compounds excreted through scent sources on the basis of their reproductive phases (Archunan, 2009). In the present study, we report fourteen urinary volatiles across the estrous cycle, among which only three compounds are common to all the phases of the estrus-synchronized goat. These may be considered as common metabolic products from female goat. We, therefore, speculate that the pre-estrogen surge during initial stage of ovulation may be involved in the release of these estrus-specific volatiles in the urine.

The significant finding of the present study relates to the shift and transition of compounds during the phases. For instance, some of the estrus- specific compounds were reported earlier as pheromones. The compounds

1-nonadecene and 1-heptacosanol appeared as common volatiles in all the phases. However, when the animal comes into pro-estrus phase, it is transited as 3-hexadecene and then as 1-hexadecene and pentadecene, respectively.

The estrus-specific compounds such as tetradecanol and n-Pentadecanol have been literally proved the presence of decanol, which is otherwise called as capric alcohol. The term “capric” adds further evidence to the compounds as of goat origin (Capra is the goat genus). The repeated and concurrent appearance of 3-methylene tridecane in all the synchronized goats makes a significant observation. Absence of behenic alcohol during pro-estrus but present during estrus and post-estrus phases imply that it may be useful in

92 Chapter III indication of ovulation (estrus) and thereafter, the consequent implantation

(post-estrus).

Some of the compounds alone have already been reported to have role in chemo-communication system in various animals which adds support to these inferences. For example, tetradecanol (reduced form of myristic acid) and petadecanol have been suggested to have role in chemical communication in mice (Achiraman et al., 2011b). The modified form of 3-methylene tridecane has been proved as an alarm pheromone in sting bugs (Favaro et al., 2012).

The transited form of 2-ethyl-1-dodecene, dodecene, has been studied for electroantennogram response in male obliquebanded moths (Trimble and

Ashraf, 2006). Sun et al. (2012) also showed that dodecene has good affinity towards odorant binding protein in Helicoverpa sp. Another significance of the present study concerns the transition of some compounds from one phase to another during the cycle. Here, we implicate the physiological intervention which would reflect the shift/transition of the compounds. Also, the study suggests a role to alcohols, alkanes and alkenes in goat communication. The report of Gabirot et al. (2012) denotes that alcoholic compounds have interaction with the communication system of lizards which adds support to our suggestion. Further, the number of carbon atoms in estrus-specific compounds provides additional evidence for their biodegradable and volatile nature.

In faeces samples, as many as 24 compounds were totally identified during all the phases; among which 2 compounds were common to all the phases; 7 compounds each represent estrus and post-estrus whereas the proestrus contain 6 compounds. As like of that of urinary volatiles during different phases of estrous cycle (unpublished data), faecal volatiles also varied consistently during estrous cycle with clear shift and transition of chemical groups. For instance, 2-hexadecence appeared in the pro-estrus and

93 Chapter III post-estrus faeces have been shifted as n-hexadecanoic acid and represents pro-estrus phase alone. The compound 1-octadecanol present as estrus- specific has been shifted as octadecanoic acid in the same phase, and again shifted as 1-pentadecanol to represent the post-estrus phase. An important notion, the compound 1-octadecanol appeared in the present study has also been observed in estrus urine of goat, and hence, it may be suggested that the compounds has potent role in goat communication, and so excreted via more than one source in favoring the communication in goats. Regarding the compound profile, there was consistent shift exist in all the three phases.

Most of the alkanes and alkenes were found in proestrus phase, whereas acids and alcohols was predominantly noted in estrus phase. In contrast, there was 3 alcohols were present in post-estrus phase. Moreover, the reported compounds of the present study have already been reported to have a significant role in communication of other animals. For example, 1- octadecanol and hexadecanoic acid have been reported as bound form of volatiles in hamster vaginal protein (aphrodisin) (Briand et al., 2004). In addition, recently we reported that 9-octadecanoic acid as an estrus-specific volatile and have been implicated in eliciting flehmen and mounting behaviour in the bull buffalo (Karthikeyan et al., 2013).

Choi et al. (2005) reported that hexadecanoic acid and octadecanoic acid have been utilized as a precursor for pheromone production in insects.

The interesting notion of the present study is the presence of octadecanoic acid which has been literally termed as capric acid (the term capric pertains to goat) which is having the odor similarities with goat

(http://www.chemicalland21.com/industrialchem/organic/CAPRIC%20ACID. htm). Hence, it could be inferred that the presence of capric acid

(octadecanoic acid) could be a possible factor involving in species-specific communication. Another fact is that the hexadecanoic acid would be a possible precursor for making octadecanoic acid in the same phase. Moreover,

94 Chapter III the faecal extracts of lion has been reported to contain octadecanoic aid and hexadecanoic acid and suggested that the compounds may responsible for repellent activity in deer (Baines et al., 1989). Hexadecanoic acid was also reported as a pheromone compound in organeworm, Amyelosis transitella

(Wang et al., 2010). Earlier evidence of Jurenka, (1997) reported that hexadecanoic acid present in insect undergoes desaturation to form the pheromonal compound in Cadra cautella and Spodoptera exigua. Next to this, the modified form of tetradecanol has been reported as aggregation pheromone in stink bug which adds further evidence that tetradecanol having the pheromonal property (Mori and Wu, 2006). Tetradecanol has also been reported to be present as putative pheromone in the urine of male mouse

(Zhang et al., 2010). Most of the compounds of the faecal origin seem to be alcohols and acids in nature. In support of the present study, Moto et al.

(2003) reported that the alcohol acts as major class of pheromone compounds in silkworm, Bombyx mori.

The interesting finding in the volatile profiles of the present study has some prelude in view of chemical communication of goats. Alanine, the amino acid was found exclusively during estrus faeces. According to the report of

Ponnuswamy et al. (2011) the processing of the faecal metabolite is of microbial dependence, and alanine is excreted in the faeces of humans, under irritable bowel syndrome. However, in the present study, it could be correlated that the microbial biota got altered during estrus phase, and alanine may be excreted in the faeces, however the role of alanine in faeces needs to be analysed further. Ascorbic acid, an important anti-oxidant observed in the faeces of estrus goats, whereas absent during other two phases. Since, the ovulation process is crucial in goats, the anti-oxidant may have some roles in regulation of estrous cycle or vice versa. It is reported that ascorbic acid could be produced in goats by the liver, and its level is notably increased during stress condition (Chatterjee, 1973). Since, the estrus phase, may have a stress

95 Chapter III in animal’s health, the overproduction of ascorbic acid may taken place in liver, which may be excreted through the faeces. We further speculate the role of ascorbic acid in relation to estrus phase, since the food habit of the goats could not affect the level of ascorbic acid synthesis inside the body

(Richmond, 1940). Further, ascorbic acid has a pivotal role in combination with 17-β estradiol in lowering the low density lipoprotein, and thus having anti-atherogenic capacity in addition to anti-oxidant activity (Hwang et al.,

2000). It has been reported in rabbits that exogenous administration of ascorbic acid has modulating effect in the level of tissue estrogen and leads to increased level of estrogen supporting notion of the present study, in which the estrogen level in the faeces samples of estrus phase (Bostanci et al.,

2012).

This is the first study in reference to the volatile profiles analysis of urine and faeces during estrous cycle of estrus synchronized female Indian goat. Overall, the observation is that two alcohols (tetradecanol and N- pentadecanol) with alkane (3-methylene tridecane) and alkene (2-ethyl-1- dodecene) appeared in the goat urine only during the estrus phase of estrus synchronized goats offers a clue that these may be acting as estrus indicators in the goat. However, the role of individual volatile compounds of estrus- specific has to be further validated with behaviour assay to consider them as an effective indicator of estrus. Based on the results obtained we propose that the four compounds of urinary origin and seven compounds of faecal origin are specific to estrus phase and could be considered as estrus-specific. From this inference and together with the results of the previous chapters, it should be possible to develop a marker specific to estrus phase to facilitate the non- invasive detection of estrus in the goat. However, investigation with regard to the efficacy of each compound in relation to behavioral expressions is still unexplored.

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The volatile portion of the scent sources, thus identified give prelude to the existence of proteins in the urine. As that of volatiles, there may be presence of specific protein either qualitative or quantitative manner which could add further evidence to study about the physiology in regard to excretory products. Hence, the next chapter was framed to identify and study about the urinary proteins in the urine samples collected during estrous cycle.

Summary

 Urine and faeces samples collected from three different phases of

estrous cycle were analysed for volatile compounds.

 Urine contain a total of 14 compounds all the phases put together,

among which, estrus phase revealed 4 specific compounds namely,

Tetradecanol, n-Pentadecanol, 3-Methylene tridecane and 2-Ethyl-1-

dodecene.

 Pro-estrus urine contains 4 specific compounds, whereas post-estrus

faeces contain only one specific compound. Pro-estrus and post-estrus

contain a compound which was absent in estrus faeces. Similarly,

estrus and post-estrus faeces contain a compound, namely, behenic

alcohol which was absent in pro-estrus faeces.

 Faeces contain a sum of 24 compounds during all the phases. There

were two compounds common to all the three phases, whereas 6

compounds were specific to pro-estrus faeces.

 In particular, estrus phase contain seven specific compounds which

includes, two anti-oxidants namely ascorbic acid and vitamin E. There

were another two compounds and present in pro-estrus which was

absent in estrus faeces. Post-estrus faeces contain specifically about 6

compounds which was absent in pro-estrus and estrus faeces.

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Proteomic analysis of female goat urine during estrous cycle

4.1. Introduction

Urine is considered as a predominant source of communication in animals. Urine has been reported to contain various biochemical moieties, which encodes the message and favors the communication. The biochemical molecules consist of volatiles, proteins, lipids, fatty acids and other regulatory elements (Bouatra et al., 2013). Urine has been considered as a non-invasive biomarker in disease identification for centuries, since collection of urine never brings any stress to the animals. In biochemistry, the term urinalysis refers to the analysis of urine in terms of biochemical in turn compared with the standard values and makes them use to validate urine as a biomarker

(Delanghe and Speeckaert, 2014). Thus, the process of excretion certainly not be an elimination of digested waste materials, but also provide insightful meaning in terms of advertisement of physiological status (Achiraman et al.,

2011b). Each constituents present in the urine denote specific changes inside the body. In animals, the constituents possess additional roles; most important is the delivery of message to the conspecifics. Thus, the modulatory effect of these biochemical constituents paid greater interest in animal communication. For instance, volatile compounds present in the animal’s urine having the capacity to maintain social regulation, advertisement of dominance, mother-young interaction, and sexual communication and so on

(Tirindelli et al., 2009).

Next to volatiles, proteins are the premier molecules studied extensively in small mammals. Proteins perform variety of functions inside the body, and a trace amount has been excreted in the urine (Berger and Szoka, 1981).

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However, the presence of protein in higher concentration in urine signifies the critical condition called proteinurea. The fact was resolved and the breakthrough was discovered in animals that animals having permanent proteinurea not as an indicator of disease, but as a factor regulating the communication among conspecifics (Cavaggioni and Mucignat-Caretta, 2000).

Initially, it has been reported that urine of mice and rat contains more amount of proteins and later it was discovered that the protein act as carrier for the volatile compounds. Later on the concept was well proved in both the rodents and follow-up structural and functional elucidation clearly determined the role of carrier protein in animal communication. It has been thoroughly reviewed by Flower, (1996) and (2000). Proteins excreted in the urine identified as a promising approach, its endocrine dependency further kindle its importance to study of urinary proteome and it was found that the male and female at estrus have higher concentration of proteins than that of other phases of female. It was proved that testosterone plays a major role in the regulation of excretion of particular protein, the so-called pheromone carrier protein (Ponmanickam et al., 2010). Similarly, estrogen also regulates the protein excretion in urine, since profound changes in the expression pattern of pheromone carrier protein was observed during different phases of estrous cycle. Thus, the phase change in the female’s reproductive cycle often associated with the regulation of excretion (Stopka et al., 2007). However, there are only limited reports available on the concentration of all the proteins rather than a specific protein.

The study of other proteins will provide additional information regarding the molecular mechanism and how the protein gets into the urine and for the purpose what it stands for. Also, the protein profile of urine may change during the different reproductive phases. Also, as like that of smaller mammals, the existence of particular proteins is not always possible in higher

99 Chapter IV mammals. Moreover, reports are available in the study of urinary proteome which is scarce, especially in goats. Hence, we here aimed to study the total proteome of female goat urine during four different phases of estrous cycle to bring out a better marker for differentiation of phases in addition to provide estrus-specific marker protein.

4.2. Material and Methods

4.2.1. Estrus synchronization and urine collection

The method of estrus synchronization and urine collection was followed as mentioned in chapter I. The urine samples were stored in ice condition and transported to lab and kept at –20 ° C until further analysis.

4.2.2. Protein analysis

The urine samples were thawed to room temperature and were processed using molecular weight cut-off membrane (Vivaspin 20, 10 kDa

MWCO, GE Healthcare). Twenty milliliter of each urine samples was poured into upper part of the individual tubes containing the cut-off membrane. The tubes were then centrifuged at 4000 rpm for 10 minutes (Eppendorf 450R).

The centrifuge was repeated with similar time and rpm until minimum volume of sample in the upper layer. After centrifugation, the flow through was discarded and the samples retained in the upper part were taken for further analysis.

Protein Estimation

The total protein in the urine samples was determined by adopting the method of Bradford (1976) with Bovine Serum Albumin (BSA) as standard. To the required volume of protein sample, distilled water was added to make up the volume to 100 µl of Bradford reagent (100 mg of Comassie Brilliants Blue

G 250 to 50 ml of 95% ethanol and 100 ml of 85% orthophosphoric acid, made up to 1000 ml) was added-extract and mixing thoroughly and allowed to

100 Chapter IV stand for 5 minutes. The absorbance was read at 595 nm in a

Spectrophotometer.

Composition of Bradford Reagent

Coomassie Brilliant Blue G-250

Ethanol

Acetic Acid

Methodology

Two microlitre of protein sample was added with 2.5 ml of Bradford reagent and incubated in dark for 10 minutes. After the appearance of clear blue colour, the readings were taken at 595nm in UV-Spectrophotometer.

4.2.3. SDS-PAGE

The protein samples processed using the cut-off membrane were used for SDS-PAGE analysis for protein separation. SDS-PAGE was prepared (12%) and each sample (50 µg) was loaded with the gel loading dye with the molecular weight marker. The molecular weight of the protein was compared with the molecular weight marker, and the protein of interest was cut out from the gel using sterile blade and was used for further analysis.

Principle

A special form of poly acrylamide electrophoresis is sodium dodecyl sulphate –poly acrylamide gel electrophoresis. In this technique, protein mixture is first denatured with SDS and β- mercapto ethanol, which results in reduction of the s-s-bridge in the protein and dissociation of the polypeptide chains. The SDS forms a complex with the polypeptides. These complexes have a strong negative charge that completely overshadows the charge of the polypeptide itself. The SDS polypeptide complexes carry an almost uniform charge density and also the form of the complexes is rather regular. Therefore,

101 Chapter IV the migration velocity through the gel is determined by the molecular mass of the polypeptide.

Procedure

The 12% SDS-PAGE was modified. The Polyacrylamide gels were cast between 11.5x10.3 cm glass plates. Liver, kidney and serum proteins were identified by running molecular mass reference standard (Bangalore Genei cat no: PMWM) containing phosphorylase- 97.7 kDa; serum albumin-66.0 kDa; ovalbumin-43.0 kDa; carbonic anhydrase-29.0 kDa; trypsin inhibitor-

21.1 kDa; lysosome- 14.4 kDa respectively. Electrophoresis was carried out for 4 hours at room temperature.

Sample preparation

Prior to loading the SDS-PAGE, tissue extract was mixed with the 1x sample buffer with equal volume (1:1) and mixed with vortex mixer (CM 101,

REMI equipment) for few seconds and kept for water bath at 100ºC for 4 min, for the purpose to breakdown the polypeptides.

Reagents required

Acrylamide solution (30%)

Acrylamide -14.6 g

Bisacrylamide -0.4g

Water make up to 50 ml (store at 4ºC in dark room)

Running gel buffer (lower Tris) :( pH-8.8)

1.5 M Tris-Cl (MW-121.14) -9.075g

Adjust pH with HCl

Made up to 50 ml with distilled water

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Stacking gel buffer (upper Tris): (pH-6.8) 1M Tris- Cl -3.029 g Adjust pH with HCl Made up to 50 ml with Distilled water

10% SDS SDS -2.5 g Made up to 25 ml with Distilled water

10% Ammonium per sulphate (APS) APS -0.5 g Made up to 5 ml distilled water

Tank buffer (1x): pH –8.2 Tris -750 mg Glycine - 3.6 g SDS - 250 mg Made up to 250 ml with Distilled water

Sample buffer (1x) Tris -1.25 ml of stacking gel buffer (pH-6.8) SDS -2.0 ml solution of 10% SDS Glycerol -1 ml β-mercapto ethanol -0.5 ml A pinch of bromophenol blue added as indicator dye. Water make up to 5 ml.

Staining solution: (Coomassie blue R-250) (0.5% stain) 45% Methanol -50 ml 10% Acetic acid -10 ml Coomassie BlueR-250 -500 mg Made up to 100 ml with Distilled water

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De-staining solution

50% Methanol -50 ml

10% Acetic acid -10 ml

Made up to 100ml with Distilled water

The gel was prepared by mixing the stock solution mentioned above.

Separating gel (12%)

Dis. Water -3.3 ml

30% Acrylamide -4.0 ml

Tris (pH- 8.8) -2.5 ml

10% SDS -0.1 ml

10% APS -0.1 ml

TEMED -0.002 ml

Stacking gel (5%)

Dis. Water -2.1 ml

30% Acrylamide -0.5 ml

Tris (pH-6.8) -0.38 ml

10% APS -0.03 ml

10% SDS -0.03 ml

TEMED -0.003 ml

Preparation of gel

Two glass plates were sandwitched using 1.5 mm space strip and the bottom was sealed with 1% agar. The separating gel (12%) mixture was poured between the two glass plates and was allowed for polymerization. A few drops of separating gel- overlying solution were layered over the separating gel mixture. The solution was then decanted. Then the stacking gel was poured on the separating gel and the comb was inserted. After polymerization of stacking gel, the comb was removed and the wells were

104 Chapter IV rinsed with water. After removing the comb, the glass plates were fixed to the gel chambers.

Solubilization of sample

Samples were mixed with equal volume of sample buffer treated at

100º C for 4 minutes. Then, the samples were loaded into the wells of stacking gel. The chambers were filled with tank buffer and electrophoresis started. Initially a current of 60v was applied till the dye enters the separating gel subsequently current was increased to 100v. Electrophoresis was continued till the marker reached 1cm above the edge of separating gel.

Protein detection by Coomassie Brilliant Blue stain

Once the gel was completed it was rinsed with distilled water for

2 minutes and stained with 0.5% CBB-R-250 stain for 2 hours at room temperature. The stained gel was destained until the appropriate background was obtained. The gel was washed with distilled water and stored in the refrigerator for image analysis.

4.2.4. In-gel Trypsin digestion protocol and MALDI spotting

Solutions needed

100% Acetonitrile

100 mM ammonium bicarbonate

Wash Solution

50% acetonitrile and 50 mM ammonium bicarbonate

Reduction solution

10 mM DTT in 100 mM ammonium bicarbonate

Alkylation solution

50 mM iodoacetamide in 100 mM ammonium bicarbonate

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Trypsin solution

20 μg/ml

Extraction solution

0.1% TFA and 50% Acetonitrile

The desired protein bands (stained gel pieces) were excised mince into pieces (1 -3 mm) and transferred into a sterile microcentrifuge tube. It was washed with 500 µL of wash solution (50% acetonitrile, 50 mM ammonium bicarbonate) and incubated at room temperature for 15 minutes with gentle agitation (vortex mixer on lowest setting). The solution is then removed with a pipette. The gel pieces were washed two more times with 500 µL of wash solution (15 minutes each) or until the Coomassie dye has been completely removed. The gel pieces were dehydrated in 100% acetonitrile for 5 minutes.

When dehydrated, the gel pieces will have an opaque white color and will be significantly smaller in size.

Acetonitrile was removed with a pipette and then the gel was completely dried at room temperature for 10-20 minutes in a centrifugal evaporator. The gel pieces were rehydrated in 150 µL reduction solution (10 mM DTT, 100 mM ammonium bicarbonate) for 30 minutes at 56° C. The reduction solution was discarded with a pipette and 100 µL alkylation solution (50 mM iodoacetamide, 100 mM ammonium bicarbonate) was added and incubated for 30 minutes in the dark at room temperature. The alkylation solution was discarded with a pipette and 500 µL of wash solution was added and incubated at room temperature for 15 minutes with gentle agitation. The wash solution was discarded and dehydrated in 100 µL 100% acetonitrile for

5 minutes. Acetonitrile was discarded and the gel was completely dried at room temperature in a centrifugal evaporator. Typically, this is modified sequencing grade trypsin (Product number V5111, Promega, Madison, WI).

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The lyophilized trypsin (20 µg/vial) was re-suspended in 1 mL of 50 mM ammonium bicarbonate, aliquot (50 µL/tube) and stored at -70° C.

The gel was rehydrated with a minimal volume of protease digestion solution. Approximately 20 µL solution was added for small gel plugs. The gel pieces were digested overnight at 37° C. The sample was centrifuged (12 kg for

30 sec). The supernatant was transferred (containing tryptic peptides) to sterile centrifuge tube. To that 25-50 µL of extraction solution (60% acetonitrile, 0.1% TFA) was added to gel pieces and sonicated in ultrasonic waterbath for 10 min. Alternatively, it was gently agitated by vortexing at lowest setting. The tubes were again centrifuged for 30 sec at 12 k g for 30 sec). The gels were extracted with an additional 25-50 µL of extraction solution. Then the gel pieces were agitated by sonicating in a waterbath for 10 minutes or with gentle vortexing.

Then the tubes were spin down and supernatant was transferred. The pooled extracted peptides were dried by centrifugal evaporation to near dryness. To this, 5 µL of re-suspension solution (50% acetonitrile, 0.1% TFA) was added and sonicated in water bath or gently agitated on a vortex at lowest setting. The samples were spin down and 0.5 µL was spotted on MALDI plate followed by 0.5 µL of alpha-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 50% acetonitrile, 0.1% TFA). The spots were allowed to dry completely and then the plates were loaded into Voyager. The plate was calibrated using internal tryptic peaks of 842.5 and 2211.1 Da.

Note: This protocol contains a reduction and alkylation step. Alternatively, this can be performed prior to gel electrophoresis or after first dimension isoelectric focusing. After peptide extraction mass spec analysis should be performed as soon as possible. Preparation of peptides must be performed with labware that has never been in contact with nonfat milk, BSA, or any

107 Chapter IV other protein blocking agent to prevent carryover contamination. Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination. Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.

4.2.5. MALDI-TOF Mass Spectrometry

The tryptic digests were prepared by mixing equal amounts (2:2) of peptide mixture with the matrix solution (α-cyano-4-hydroxycinnamic acid) saturated with 0.1% TFA and acetonitrile (1:1). Then the samples were analyzed in reflectron mode with delay time of 90 ns and 25Kv accelerating voltage in the positive ion mode. To improve the signal to noise ratio summation of 300 laser shots were taken for each spectrum. External calibration was done using peptide I calibration standard with masses ranging from 1046-3147 Da. Mass spectra were acquired using ULTRAFLEX-TOF/TOF mass spectrometer (Bruker Daltonics, Germany), equipped with a 337 nm pulsed nitrogen laser. MS-MS spectra were acquired by selecting the precursor mass with 8 Da window.

Spectra were processed using flex Analysis software. Monoisotopic peptide masses were assigned and used in the database search. The protein identification was accomplished utilizing the MASCOT data base search engine (Matrix Science, London, UK) (http//www.matrixscience.com, search engine). Probability-based MW search scores were estimated by a comparison of search results against an estimated random match population and were reported as 10 log10 (P), where P is the absolute probability. Scores >63 were shown to be significant (P<0.05) in the mascot search. Proteins identified with scores less than the significant level were reported as unidentified.

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4.3. Results

4.3.1. SDS-PAGE

SDS-PAGE analysis of urine from estrous cycle revealed protein at different molecular weight with consistent variation in concentration.

Proestrus urine revealed seven different proteins with the complete absence of

25 kDa protein and specific presence of 98 kDa, which is absent in other phases. In addition, the concentration of 68 kDa protein was higher in proestrus phase than that of other phases, which was markedly reduced in estrus phase. Estrus phase revealed the presence of seven different proteins with the specific presence of 25 kDa protein and absence of 98 kDa protein.

The concentration of 25 kDa protein was further reduced to metestrus whereas it was completely absent during proestrus and diestrus. Metestrus phase also revealed protein profile as same as estrus. Diestrus phase revealed higher concentration of 68 kDa protein next to proestrus (Figure.4.1).

L1- Diestrus, L2- Metestrus, L3-Estrus, L4- Proestrus, L5& L6- Marker.

Fig. 4.1. SDS-PAGE (12%) analysis of urine samples from female goat during estrous cycle

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4.3.2. MALDI-TOF analysis

The MALDI-TOF analysis of proteins and resulted peptide mass were analysed in MASCOT tool and the score value with sequence coverage were given in Table 4.1.

MALDI TOF analysis of 25 kDa protein

Estrus-specific protein of 25 kDa protein showed matching with

Complement C3 (Fragment) with score value of 30 and sequence coverage about 6%. The sequences given below are the deduced peptide sequences obtained in MASCOT search. The deduced sequences were taken for BLAST search and the sequences matched with the existed protein are given in

Figure 4.2 a and b.

LFPVTRQLNOP ILSSLVVDIM NPDGVVVDRI EKV MELRPFHVPA ITSLGDWK AFTIHIKAM HIYGKPVMGR LLL GQSLYVEASV ISSDAGEIED SILDDIPIVA SPYSIKSK

MALDI TOF analysis of 28 kDa protein

The 28 kDa protein showed matching with Acetyl Co-A carboxylase with 7% sequence coverage and the score value of 52 (Fig.4.3 a & b). When the mass values were given as input in MASCOT tool the deduced sequences were obtained as given below. After that the sequences were taken BLAST search which showed the matching with Acetyl Co-A carboxylase protein.

MVMT LTAAESGCIH YVKRPGAALD PGCVIAKM DIMTSVSGRI PPNVEKSIKK EMAQYASNIT SVLCQFPSQQ IANILDSHAA TLNRKSEREV FFMNTQSIVQ LVQRYR GHMKAVVMDL LRQYLRVETQ FQNGHYDKCV FALREENKSD MNTVLNYIFS HAQVTKK

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MALDI TOF analysis of 32 kDa protein

The 32 kDa protein has showed score value of 54 with Transportin and the sequence coverage about 17% (Fig.4.4 a & b). The mass values resulted in the deduced peptide sequences as given below. When these sequence were taken for BLAST search, it showed matching with Transportin protein.

MEGAKPTLQL VYQAVQALYH DPDPSGK VHAWEISDQL LQIRQDVESC YFAAQTMKMK IQTSFYELPT DSHASLRDL SPVIVTQLAL AIADLALQMP SWKGCVQTLV EK RTEII EDLAFYSSTV VSLLMTCVEK AGTDEK VFRCLGSWFN LGVLDSNFMA NNK

MALDI TOF analysis of 42 kDa protein

The 42 kDa protein showed top score value of 63 with Unconventional

Myosin VII-a and the sequence coverage of 33% (Fig.4.5 a & b). The deduced peptide sequence was obtained as mentioned below for the peptide mass of 42 kDa protein. The deduced sequences were taken for BLAST search where it shows 33% of sequence coverage with Unconventional Myosin VII-a.

YRDHLIYTYT GSILVAVNPY QLLSIYSPEH IRKIG EMPPHIFALA DNCYFNMKRN SRDGCCIISG ESGAGKT KLTLQFLAAI SGQHSWIEQQ VLEATPILEA FGNAKYID IHFNK KLGLGQATDY NYLAMGNCIT CEGREDSQEY ANIRSAMKDRTF ENLDACEVLF STALATAASL LEVNPPDLMN CLTSR

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Table 4.1. Features of estrus-specific proteins identified using MASCOT tool

S. Molecular Proteins matched by Score value Sequence No weight of the MASCOT in MASCOT coverage Protein search 1 25 kDa Complement C3 30 6% (Fragment) 2 28 kDa Acetyl Co-A carboxylase 52 7% 3 32 kDa Transportin 54 17% 4 42 kDa Unconventional Myosin- 63 33% VIIa 5 55 kDa Protein Wnt 47 56% 6 65 kDa Interleukin 40 65% 7 74 kDa Hemoglobin subunit 50 71% epsilon

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a)

b)

VLVIAPAATS SYDDLAVAIL MVDQKKITEV HVLLVNPHTG ATLDEKKVKL QWDNKFIAFT KLQVTPKEVE KWKEDFVRLM VKWDGGQHME IDIPLTSRRG LVFAQTDQPI YTPNNDVNIR LFPVTRQLNOP ILSSLVVDIM NPDGVVVDRI EKNAFEVEKV MELRPFHVPA ITSLGDWKIV SWMKDKPQFN YTSGFKVEEY VLPTFDVSIT SEQPYLHVYD KAFTIHIKAM HIYGKPVMGR AYVRYGVKHQ SKRTLLSTSS ALARFEQGEA MHTLRQKHIL EQYPDPKLLL GQSLYVEASV ISSDAGEIED SILDDIPIVA SPYSIKSKWT VPFFKPGYPY IYKVLVLNPD

Fig.4.2. a) Histogram of the Mascot score for 25 kDa protein showing the score value of 30 with Complement C3 (Fragment)

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 20% sequence coverage is highlighted by being given in red color.

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a)

b)

PSVLRSPSAG KLIQYIVEDG GHVFAGQCYA EIEVMKMVMT LTAAESGCIH YVKRPGAALD PGCVIAKMQL DNPSKVQQAE LHTGSLPRIQ STALRGEKLH RVFHYVLDNL VNVMNGYCLP DPFFSSRVKD WVEGLMKTLR DPSLPLLELQ DIMTSVSGRI PPNVEKSIKK EMAQYASNIT SVLCQFPSQQ IANILDSHAA TLNRKSEREV FFMNTQSIVQ LVQRYRSGIR GHMKAVVMDL LRQYLRVETQ FQNGHYDKCV FALREENKSD MNTVLNYIFS HAQVTKKNLL VIMLIDQLCG

Fig. 4.3. a) Histogram of the Mascot score for 28 kDa protein showing the score value of 52 with Acetyl Co-A carboxylase 1.

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 7% sequence coverage is highlighted by being given in red color.

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a)

b)

MEGAKPTLQL VYQAVQALYH DPDPSGKERA SFWLGELQRS VHAWEISDQL

LQIRQDVESC YFAAQTMKMK IQTSFYELPT DSHASLRDSL LTHIQNLKDL

SPVIVTQLAL AIADLALQMP SWKGCVQTLV EKYSNDVTSL PFLLEILTVL

PEEVHSRSLR IGANRRTEII EDLAFYSSTV VSLLMTCVEK AGTDEKMLMK

VFRCLGSWFN LGVLDSNFMA NNKLLALLFE VLQQDKTSSN LHEAASDCVC

Fig. 4.4. a) Histogram of the Mascot score for 32 kDa protein showing the score value of 54 with Transportin.

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 17% sequence coverage is highlighted by being given in red color.

115 Chapter IV a)

b)

MVILQQGDYV WMDLRSGQEF DVPIGAVVKL CDSGQIQVVD DEGNEHWISP

QNATHIKMPH PTSVTGMMED MIQHLGDLNE AGILRNLLIR YRDHLIYTYT

GSILVAVNPY QLLSIYSPEH IRQYTNKKIG EMPPHIFALA DNCYFNMKRN

SRDGCCIISG ESGAGKTEST KLTLQFLAAI SGQHSWIEQQ VLEATPILEA

FGNAKTIRND NSSRFGKYID IHFNKRGAIE GARIEQYLLE KSRVCRQAPD

ERNYHVFYCM LEGMSEEQKK KLGLGQATDY NYLAMGNCIT CEGREDSQEY

ANIRSAMKVL MFTDTENWEI SKLLAAILHL GNLQYKDRTF ENLDACEVLF

STALATAASL LEVNPPDLMN CLTSRTLITR GETVSTPLSR EQALDVRDAF

Fig. 4.5. a) Histogram of the Mascot score for 42 kDa protein showing the score value of 63 with Unconventional myosin VII-a (Fragment).

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 17% sequence coverage is highlighted by being given in red color.

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MALDI TOF analysis of 55 kDa protein

The 55 kDa protein has showed score value of Protein Wnt and the score value of 56% (Fig.4.6 a & b). The peptide mass value of the 55 kDa protein was taken for MASCOT search which resulted in the deduced peptide sequence as mentioned below. The sequences subjected to BLAST search for identifying the matching sequences which showed the 56% sequence matching with Protein Wnt.

MNAPLGGIWP WLPLLLTWLT PEVSSSWWYM RATGGSSRVM CDNVPGLVSR QRQLCHRHPD VMRAIGLGVA EWTAECQHQF RDHSLFGRV LLRESAF VYAVSSAGVV FAITRA DARALMNLHN NR YNG AIQVVMNQDG TGFTVANKR MDSCEVMCCG FHW CCAVRCQDCL EALDVHTCKA PKSVDWAAPT

MALDI TOF analysis of 65 kDa protein

The 65 kDa protein has showed the score value of 40 with Interleukin and the sequence coverage of 65% (Fig.4.7 a & b). MASCOT search of peptide mass values of the 65 kDa protein showed the peptide sequences as mentioned below. The sequences were subjected to BLAST search to exploit the highly matching protein and it shows about 65% of sequence coverage with Interleukin protein.

MAKVPDLFED LKNCYSENED YSSEIDHLSL NQK FMSLDTSETS KTSKLSFKEN VVMVAASGKI LKLSLNQ FITDDDLEAI ANNTEEEIIK PRSAHYSFQS NVKYNFMRVI JQECILNDAL NQSIIR TQ LFVSAQNEDE PVLLKEMPET PKFFWEKHGSM DYFKSVAHPK LFIATK

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b)

MNAPLGGIWP WLPLLLTWLT PEVSSSWWYM RATGGSSRVM CDNVPGLVSR

QRQLCHRHPD VMRAIGLGVA EWTAECQHQF RQHRWNCNTL DRDHSLFGRV

LLRSSRESAF VYAVSSAGVV FAITRACSQG ELKSCSCDPK KKGTAKDSKG

NFDWGGCSDN IDYGIKFARA FVDAKERKGK DARALMNLHN NRAGRKAVKR

FLKQECKCHG VSGSCTLRTC WLAMADFRKT GDYLWRKYNG AIQVVMNQDG

TGFTVANKRF KKPTKNDLVY FENSPDYCIR DRDAGSLGTA GRVCNLTSRG

MDSCEVMCCG RGYDTSHVTR MTKCECKFHW CCAVRCQDCL EALDVHTCKA

PKSVDWAAPT

Fig.4.6. a) Histogram of the Mascot score for 55 kDa protein showing the score value of 47 with Protein Wnt-2.

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 56% sequence coverage is highlighted by being given in red color.

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b)

MAKVPDLFED LKNCYSENED YSSEIDHLSL NQKSFYDASY EPLREDQMNK

FMSLDTSETS KTSKLSFKEN VVMVAASGKI LKKRRLSLNQ FITDDDLEAI

ANNTEEEIIK PRSAHYSFQS NVKYNFMRVI JQECILNDAL NQSIIRDMSG

PYLTATTLNN LEEAVKFDMV AYVSEEDSQL PVTLRISKTQ LFVSAQNEDE

PVLLKEMPET PKIIKDETNL LFFWEKHGSM DYFKSVAHPK LFIATKQEKL

VHMASGPPSI TDFQILEK

Fig.4.7. a) Histogram of the Mascot score for 65 kDa protein showing the score value of 40 with Interleukin-1 alpha.

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 63% sequence coverage is highlighted by being given in red color.

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MALDI TOF analysis of 74 kDa protein

The protein at 74 kDa has showed the score value with Hemoglobin subunit protein-epsilon with the sequence coverage of 71% (Fig.4.8 a & b).

The peptide mass of the 74 kDa protein showed the deduced peptide sequences as mentioned below. When the deduced sequence was subjected to

BLAST search to identify the sequence coverage it shows 71% with the

Hemoglobin subunit protein-epsilon.

TILSVWGKFFDNFGNLS SPSAIMGNPK VKKVLT SFCEAVKAFAKLS

ELHCDKLHVD PENFKLLGNA MVIILATHFG KEETPDVQAA WQKLVSGVAT

ALAHKYH

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71%

b)

MVJFTAEEKS TILSVWGKVN VEEAGGEALG RLLVVYPWTQ RFFDNFGNLS

SPSAIMGNPK VKAHGKKVLT SFCEAVKNMD NLKGAFAKLS ELHCDKLHVD

PENFKLLGNA MVIILATHFG KEETPDVQAA WQKLVSGVAT ALAHKYH

Fig. 4.8. a) Histogram of the Mascot score for 74 kDa protein showing the score value of 50 with Hemoglobin subunit epsilon.

b) Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 71% sequence coverage is highlighted by being given in red color.

4.4. Discussion

Proteins are the second major macromolecules involved in various biological functions. They have crucial role in animals both by presence or absence. Liver is the major factory for the synthesis of many proteins, where it has been transported to various organs to perform its function. At some

121 Chapter IV instance, proteins are excreted purposely in the excretory products mainly, through urine. The condition has been termed as proteinurea, an unusual condition denoting the status of illness or diseased condition. However, in animals, proteinurea has been observed permanently and later it was discovered that some of the proteins present in the urine have notable function, particularly in communication. The proteins perform the role of communication are said to be the carrier proteins, often have low molecular weight. Its structure and biological functions have been reviewed thoroughly by Flower (1996) and (2000). In smaller mammals, such as mice and rats exhibit a protein at the molecular weight ranging from 18-22 kDa have been proved as a carrier for the volatile molecules (Cavaggioni and Mucignat-

Caretta, 2000). Later on it was discovered that hamster contain a vaginal secretory protein, elephant have albumin and human being possess apolipoprotein D as carriers for volatile molecules (Briand et al., 2004; Zeng et al., 1992). Since, the volatile molecules have significant biological functions, study of volatile-associated molecule, especially the carrier protein has opened a new window in the era of reproductive biology. Since, we have identified biochemical moieties and volatiles in the previous chapters of the thesis, it was suspected and that the urine may contain protein and that may have role as carriers for volatile molecules or its specific presence among different phases of the estrous cycle could consider as a better choice in the estrus detection.

We subjected the urine of female goats to analyse the protein profile.

We observed difference in the expression profile of proteins among four different phases of estrous cycle. As we found protein concentration varied significantly among different phase of estrous cycle, the present data has additional clue that the physiological intervention play a major role and control the excretion of protein through urine. In support of this,

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Muthukumar et al. (2013) identified proteins at four different phases of estrous cycle in mice, but they found concentration dependent of excretion of similar type of proteins. They further inculcate that a specific protein at 14.5 kDa have profound variation among different phases, and in particular estrus and metestrus phase contained higher concentration of that particular protein. In contrast, we found more proteins at higher concentration in proestrus. In addition, we found a protein at 25 kDa which was specific to estrus and metestrus phase and it was completely absent during proestrus and diestrus. However, diestrus phase revealed a good expression of 65 kDa protein next to proestrus. This is in accordance with the report of

Ponmanickam et al. (2013) that the vaginal mucus protein concentration was higher in diestrus next to estrus. Excluding the 25 kDa protein, all the other proteins as same as in all the phases however the expression pattern was varied in every phases. For instance, 65 kDa protein highly expressed in proestrus which was remarkably reduced in estrus phase.

To further elucidate the specific role govern by each protein, we next sought to characterize the proteins estrus urine. We observed the estrus specific protein of 25 kDa with high score value with complement C3 protein.

Uterine luminal fluid is responsible for transport and support medium for spermatozoa. It was studied that the secretion of luminal fluid is hormone- dependent, especially estradiol than progesterone, and hence it play a major role in implantation. It has also been proved that under the control of estrogen, the uterus has good quantity of luminal fluid. Florescin lebelled dextran was used to study the movement of the fluid, where under the surge of estradiol the concentration of fluid was higher, but dropped significantly when estradiol was reduced in presence of high level of progesterone (Selleh et al., 2005). It is interesting to note that the presence of complement C3 was observed in the uterus luminal fluid collected during estrous cycle of mouse.

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The expression of complement C3 protein was proved to be under the control of estrogen, and hence, detectable amount of mRNA was observed during estrus and undetectable during metestrus and diestrus (Li et al., 2002). It is also substantiated with the ovariectomized mice with exogenous administration of estradiol. However, the level of complement C3 was again reached a detectable quantity during late pregnancy and lasts up to delivery

(Li et al., 2002). In line with this, the 25 kDa protein of the present study showed high score value with Complement C3. It was interesting to note that the protein was appeared during estrus and metestrus, and, hence, it could be postulated that the 25 kDa protein has some crucial role during estrus and metestrus phase.

The second protein at estrus phase 28 kDa protein has showed top score with Acetyl Co-A carboxylase. In literature, it provides melonyl- Co A for the synthesis of fatty acids. Acetyl Co- A carboxylase was found in the mammary gland of goat and its expression was correlated with the production of milk (Travers and Barber, 1993). However, none of the reports were available in regard to the presence of this protein in urine. Since, this protein is presented during all the phases this may considered as female-specific protein which may confer important role in the synthesis of fatty acids in goats, however its presence and role in urine has to be studied in detail. The protein at 32 kDa has showed top score with Transportin. Eventhough there are only scanty reports are available regarding this protein, it has been reported that it play crucial role in neurodegenerative diseases namely,

Amyotrophic lateral sclerosis and frontotemporal dementia (Brelstaff et al.,

2011). The fourth protein at 42 kDa has shown top score value with

Unconventional Myosin VII-a. This protein has several important roles in mammalian species and mutation of this gene revealed several impairments in mice (Gerdin, 2010). However, its presence in goats or urine has not been

124 Chapter IV reported. The fifth protein at 55 kDa showed top score with Protein Wnt, a protein having signaling property. They pass the signals from outside of the cell to inside through cell surface receptors. Wnt proteins have several notable functions in physiology, including development of embryos and interplay in several important diseases including cancer (Moon et al., 2004). This receptor protein, otherwise termed as Frizzleds have many characteristics similar to G- protein coupled receptor (Wang and Malbon, 2004). Thus, in our study we speculate that Wnt protein may have notable function in intracellular communication.

The protein at 65 kDa of the present study has top score value with

Interleukin, a group of proteins known to be involved signaling mechanism.

Its function has been implicated well in immune system (Staal et al., 2008).

However, no other specific functions have been studied in terms reproductive biology for this protein, and hence, the presence of this protein in the urine of goats has to be validated further and studied in detail to elucidate the functional aspect of this protein in goats. The 74 kDa protein at estrus phase showed top score with Hemoglobin subunit epsilon. Hemoglobin is expressed in the embryo for the function of transport of oxygen. In addition, the embryo is known to contain hemoglobin subunit epsilon. Taken together, the function of the protein could be correlated with the oxygen transport and also it is surprising to note that the protein was expressed in the female goats.

Even though MALDI-TOF and MASCOT analysis of estrus-specific protein provide a fundamental data for identification of proteins, further study at peptide level is warranted.

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4.5. Summary

 The urine sample of female goats collected during estrous cycle were

analysed for protein profile.

 The estrus urine sample revealed the presence of 25 kDa protein which

was absent during pro-estrus and diestrus. However, the intensity of

65 kDa protein was higher in proestrus and reduced in estrus.

 Overall all the phases expressed similar pattern of proteins except

fewer proteins.

 The proteins at estrus phase was analysed for peptide mass fingerprint

by MALDI-TOF analysis revealed the 25 kDa protein as Complement

component C3. The 32 kDa protein has showed score value with

Transportin.

 The 42 kDa protein showed top score value with Unconventional

Myosin VII-a and 55 kDa protein has showed score value with Protein

Wnt

 The 65 kDa protein has showed the score value with Interleukin and

the sequence coverage of 65%. The protein at 74 kDa has showed the

score value with Hemoglobin subunit protein-epsilon with the sequence

coverage of 71%.

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Volatile and protein analysis in male scent sources

Volatile and protein analysis in male goat urine in prepubertal, intact and castrated condition

5.1. Introduction

Animal rely on chemical cues for inter-individual communication. The signals emitted from the male and female animals convey the physiological status to one another. Chemical signals dedicate the prime channel of communication among animals. These chemical signals are mainly excreted through scent sources. The chemical signals are present in the excreta and involved in the context of social or sexual communication. The excretion of chemical signals in male and female animals based on its reproductive condition may be under the influence of circulating steroid hormones. The excretory products studied in detail, pertaining to animal communication includes urine, faeces, saliva, vaginal mucus, glandular secretions, milk, tear etc. (Tirindelli et al., 2009). Specifically, urine seems to be a potent source.

The mode of communication facilitated by these excretory sources comprises of individual recognition, puberty acceleration, social organization, mother-young interaction, sexual communication and so on. (Archunan et al.,

2014). Among the sources investigated urine seems to be a prime and potent source of communication among many animals which includes mice

(Achiraman and Archunan, 2006), rat (Selvaraj and Archunan, 2002), cat

(Miyazaki et al., 2005), blackbuck (Archunan and Rajagopal, 2013), cow

(Archunan and Rameshkumar, 2012), buffalo (Rajanarayanan and Archunan,

2011), and elephant (Goodwin et al., 2006). The well pronounced effect of urine in higher animal has been implicated in sexual communication. Number

127 Chapter V of studies implies the importance of urine in the aspect of sexual communication.

The fact of urine to act as a better source of communication relies in the constituents present in it. The constituents analysed in the urine mostly contains volatiles which acts to deliver the message between individual animals in a colony. The variation in urinary volatiles has been reported in normal and castrated male mice. Further, the level of attraction to individual’s urine varied significantly at different physiological condition of male

(Achiraman et al., 2014). In rat urine, variation was observed under normal and prepubertal condition and the presence of specific volatiles has been reported to driven the sexual attraction of female (Osada et al., 2009).

Moreover, it has been proved that age as one of the major factors which determine the sexual communication and thus, only aged males has been preferred by females for mating (Osada et al., 2003). This is same as in higher mammals, where the urine shows significant variation in attracting the conspecifics. The variation in urinary volatile profiles have also been implicated in lion which further corroborate that the volatile changes in the same sex animals (Anderson and Vulpius, 1999). In wild/endangered species, the analysis of urine aid in the identification of physiological status of the animals in non-invasive manner (Dehnhard et al., 2006). In addition to volatiles, urine has been reported to contain other biochemical molecules which could also vary under different physiological condition. As like that of volatiles, at some instances, proteins have also been reported to be involved in sexual communication among animals. Particularly, major urinary protein in rats has been reported to act as a molecule of attraction and significant changes have been observed when the animal attains sexual maturity

(Vettorazzi et al., 2013). Thus, proteins have significant roles during the development process of animals. However, most of the studies have been

128 Chapter V focused only on female mammals, and hence, lack of knowledge exists in regard to volatile analysis in male’s urine. Further, to our knowledge there has been no study conducted on goats in this aspect.

In goats, buck effect is an important character in which the odor of intact male plays a significant role in synchronizing estrus in anoestrous females. Further it is believed the odor of male plays a major role in buck effect. However, the chemistry of this odor is not explored, and hence, we aimed to analyse the urinary volatile and proteins in male goat under different physiological condition.

5.2. Materials and methods

5.2.1. Test animals

Male goats with three different physiological status have been utilized for urine sample collection in the present study. The groups (each group contains 4 animals) were, intact (24 months old), prepubertal (30 days old) and castrated (post-pubertally castrated (wether)) i.e. castrated after 18 months. The urine samples from the above groups were collected in screw cap vials to avoid contamination and stored at –20 ºC until analysis. During the experimental period, the prepubertal animals were fed with milk from does and rest of the groups were given with cultivated forage, green fodder and provided with water ad libitum. The study was carried out at Veterinary

College and Research Institute, Namakkal, India.

5.2.2. GC-MS analysis

The urine samples collected from individual goats were extracted with dichloromethane (1:1) and the extract was filtered with Whatman filter paper and used for analysis of volatile compounds using GC-MS (QP-2010 plus,

Shimadzu, Japan). Two micro-liters of extract was injected into the GC-MS on a 30 m capillary column with a film thickness of 0.25 µm (30 m x 0.2 mm i.d.,

129 Chapter V coated with UCON HB 2000) under the following temperature regimens: initial oven temperature, 40 °C for 1 minute and then 170 °C for 1 minute, afterwards increasing up to 240 °C for 5 minutes. The detection accuracy was

1 ng/peak. Mass spectrometer was operated in CI mode at 70 eV, with ammonia as reagent gas. Identification of unknown compounds was made by probability-based matching, using WILEY RegistryTM, NIST05 and NIST05s based library.

5.2.3. Protein precipitation and SDS-PAGE analysis

The urine samples collected from intact, castrated and prepubertal male goats were precipitated using TCA method. The individual samples were precipitated with 100% TCA followed by 0.1% TCA and the resulted pellet was washed with acetone. The final pellet was dissolved in PBS (pH –7.2) analysed for presence of proteins using spectrophotometer and protein estimation was done by adopting the method of Bradford (1976). An equal amount of protein

(50µg of protein) of all the samples were loaded on 12% SDS-PAGE gel along with molecular weight marker (Genei, Cat. No: 105979). The molecular weight identification of unknown proteins was done using the marker proteins.

5.3. Results

5.3.1. Volatiles present in the urine of intact goats

GC-MS analysis of intact goat urine revealed a total 14 volatile compounds. Among those, 5 compounds were common with all the phases and two compounds were specific to intact and castrated goat but not present in prepubertal urine. Interestingly, a compound, cyclohexane was found in intact and prepubertal urine but absent in castrated goat urine. There were six compounds specifically present in intact goat urine, which was exclusively absent in castrated and prepubertal goats. Among the intact urine specific compounds, three were alkenes and one was two were alcohol in nature. The molecular weight of the compounds identified in intact goat urine was about

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72 to 330 Da. The molecular formula of the compounds had 3 to 36 hydrogen atoms and 1 to 18 carbon atoms.

5.3.2. Volatiles present in the urine of castrated goats

Volatile analysis of castrated urine contained a total of 18 compounds in GC-MS analysis. There were 5 compounds which was also present in other urine samples and considered as common volatiles. However, there were two compounds namely, 5- Eicosene and 2,6, bis (1,1-dimethyethyl) phenol which was present in both castrated and prepubertal goat urine and found absent in intact urine. Interestingly, castrated goats contain 9 specific compounds which belong to chemical groups mostly of alkane, alkene and alcohol. The molecular weight of the compounds from castrated urine ranged from 68 to

354. The molecular formula of the compounds contains 1 to 20 carbon atoms and 2 to 36 hydrogen atoms.

5.3.3. Volatiles present in the urine of prepubertal goats

The urine of prepubertal goat contains 11 compounds among which 5 compounds were common to other two groups, i.e. intact and castrated.

Interestingly, we found a compound present in prepubertal which was also found in intact and another two compounds present in prepubertal and in castrated goat urine. In contrast to other two groups, there were only three compounds specific to prepubertal goats, such as 8-mehtyl-1- decene,

Cyclopentane and Butane. The compounds identified in the prepubertal goat urine have the molecular weight ranges from 83 to 280 Da. The molecular formula of the identified compounds were seems to contain 4 to 20 carbon atoms and 3 to 40 hydrogen atoms.

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5.3.4. Volatiles present in the urine of intact, castrated and

prepubertal goats

GC-MS analysis revealed the presence of 28 compounds altogether in all the phases. Among which, 5 compounds were specific to all the phases; 6 were specific to intact male, 9 were specific to castrated goat, and only 3 were exclusive to prepubertal urine (Table 5.1; Figures. 5.1, to 5.3). Two compounds were present during castrated and prepubertal urine.

Interestingly, the compounds chloromethane and 1-chlorohexadecene were present during intact and castrated male, whereas found absent in prepubertal urine. Notably, cyclopropane was present in intact and prepubertal urine but was absent in castrated male goat. In contrast, two compounds namely, 2,6, bis (1,1-dimethylethyl) phenol and 5-Eicosene were present during castration and prepubertal urine whereas absent in intact urine. The molecular weight of the identified compounds was between 50 and

354. The chemical groups of the identified compounds were mostly belonged to alkenes, alkanes and some were alcohol, however, other compounds were also present. More specifically, alkenes were noted in high numbers during all the animals. In intact urine, alkenes, alkanes and alcohol were present. In contrast, most of the compounds in castrated male urine have been identified as alkenes and alcohols.

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Fig. 5.1. GC-MS chromatogram of intact male goat urine

Table 5.1. List of compounds identified in the urine of intact male goat

S. No Peak Compound Name Molecular Molecular No. weight formula

1 1 1-cyclopropyl-3-methyl-allene C7H10 94

2 2 1- Buten-3-yne C4H3Cl 86

3 3 5- Tetradecene C14H28 196

4 4 5- Octadecene C18H36 252

5 5 1- Octadecene C18H36 252

6 6 Chloromethane CH3Cl 50

7 7 1-Chloro hexadecene C16H33Cl 260

8 8 Cyclopropane C8H16 112

9 11 Propane C15H12 72

10 12 2-Methyl-2-Butanal C5H8O 84

11 13 7- Hexadecene C16H32 224

12 14 3,5- bis (1,1-dimethylethyl) phenol C14H22O 206

13 15 1,2- Benzenedicarboxylic acid C20H26O4 330

14 16 1- Pentadecene C15H30 210

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Fig. 5.2. GC-MS chromatogram of castrated male goat urine

Table 5.2. List of compounds identified in the urine of castrated male goat S. Peak Compound Name Molecular Molecular No. No. weight formula 1 1 1-cyclopropyl-3-methyl-allene C7H10 94

2 2 1- Buten-3-yne C4H3Cl 86

3 3 5- Tetradecene C14H28 196

4 4 5- Octadecene C18H36 252

5 5 1- Octadecene C18H36 252

6 6 Chloromethane CH3Cl 50

7 7 1-Chloro hexadecene C16H33Cl 260

8 9 2,6 bis (1,1- dimethylethyl) phenol C14H22O 206

9 10 5- Eicosene C20H40 280

10 17 2- Dodecene C12H24 168

11 18 1-Phenyl- 2- Butanone C10H12O 148

12 19 Diflouromethane CH2F2 52

13 20 1,3 Pentadiene C5H8 68

14 21 3- Penten-1-ol C5H10O 86

15 22 Pyrazine C4H4N2 80

16 23 Dichloromethyl sulfinyl chloride C2H3Cl3 244

17 24 2- methyl-1-pentanol C6H14O 102

18 25 1- Tetracosanol C20H50O 354

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Fig. 5.3. GC-MS chromatogram of prepubertal male goat urine

Table 5.3. List of compounds identified in the urine of prepubertal male goat

S. No. Peak Compound Name Molecular Molecular No. weight formula

1 1 1-cyclopropyl-3-methyl-allene C7H10 94

2 2 1- Buten-3-yne C4H3Cl 86

3 3 5- Tetradecene C14H28 196

4 4 5- Octadecene C18H36 252

5 5 1- Octadecene C18H36 252

6 8 Cyclopropane C8H16 112

7 9 2,6 bis (1,1- dimethylethyl) C14H22O 206 phenol

8 10 5- Eicosene C20H40 280

9 26 8-mehtyl-1- decene C11H22 154

10 27 Cyclopentane C8H16 112

11 28 Butane C5H9N 83

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Table. 5.4. Comparison of volatile compounds identified in the urine of intact, castrated and prepubertal male goat urine

Peak Molecular Molecular Intact Castrated Prepubertal Compound Name No. weight formula male male male 1-cyclopropyl-3- 1 C7H10 94 ✔ ✔ ✔ methyl-allene

2 1- Buten-3-yne C4H3Cl 86 ✔ ✔ ✔

3 5- Tetradecene C14H28 196 ✔ ✔ ✔

4 5- Octadecene C18H36 252 ✔ ✔ ✔

5 1- Octadecene C18H36 252 ✔ ✔ ✔

6 Chloromethane CH3Cl 50 ✔ ✔ X

7 1-Chloro hexadecene C16H33Cl 260 ✔ ✔ X

8 Cyclopropane C8H16 112 ✔ X ✔ 2,6 bis (1,1- 9 C14H22O 206 X ✔ ✔ dimethylethyl) phenol

10 5- Eicosene C20H40 280 X ✔ ✔

11 Propane C15H12 72 ✔ X X

12 2-Methyl-2-Butanal C5H8O 84 ✔ X X

13 7- Hexadecene C16H32 224 ✔ X X 3,5- bis (1,1- 14 C14H22O 206 ✔ X X dimethylethyl) phenol 1,2- 15 Benzenedicarboxylic C20H26O4 330 ✔ X X acid

16 1- Pentadecene C15H30 210 ✔ X X

17 2- Dodecene C12H24 168 X ✔ X 1-Phenyl- 2- 18 C10H12O 148 X ✔ X Butanone

19 Diflouromethane CH2F2 52 X ✔ X

20 1,3 Pentadiene C5H8 68 X ✔ X

21 3- Penten-1-ol C5H10O 86 X ✔ X

22 Pyrazine C4H4N2 80 X ✔ X Dichloromethyl 23 C2H3Cl3 244 X ✔ X sulfinyl chloride

24 2- methyl-1-pentanol C6H14O 102 X ✔ X

25 1- Tetracosanol C20H50O 354 X ✔ X

26 8-mehtyl-1- decene C11H22 154 X X ✔

27 Cyclopentane C8H16 112 X X ✔

28 Butane C5H9N 83 X X ✔ X – Absent; ✔ - Present

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(a) 2-Methyl-2-Butanal

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(b) 7- Hexadecene

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(c) 1,2- Benzenedicarboxylic acid

Mass spectrum of intact male specific volatile compounds

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5.3.2. Proteins present in the intact and prepubertal goats

The protein analysis of prepubertal and intact goat urine represents distinct protein profile. The prepubertal goat urine excretes more protein, whereas intact male goats excrete fewer proteins in urine. Specifically, the proteins expressed at the molecular weight of 14, 20, 25, 55, 65, 68 and 85

KDa. In contrast, 17 kDa protein expressed in the intact male goat urine was absent in prepubertal urine, whereas the protein at 65 and 68 kDa present in both intact and prepubertal urine. Thus, there are three specific proteins were observed in prepubertal urine and one protein was specific to intact male goat urine (Figure. 5.4).

kDa

Lane 1- Marker; Lane 2- Prepubertal male goat urine; Lane 3- Intact male goat urine

Fig. 5.4. Protein profile of prepubertal and intact male goat urine

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5.4. Discussion There has been considerable report in the level of attracting power

(urine) of the above category of male with the females. Study by Longpre and

Katz, (2011b) corroborate that the varying attracting capacity may depend on the internal physiological status that reflect on external morphology. However, there are three major physiological status have been noticed in male animals, i.e. prepubertal, intact, castrated (wether & stag). The exact role of the internal physiology which underscore the attracting capacity has not been elucidated. Prepubertal is a condition of which the role of testosterone has not been fully established, and thereby the animal lack sexual characteristics. In contrast, castration is a condition to prevent or inhibit the action of testosterone. It is usually performed in animals to reduce the aggressive/dominant characters expression in a colony of animals. Further, castration in turn reduces the unpleasant smell in the meat, since the intact male (i.e. non-castrated) gives the buck smell to the meat in the case of goats

(Kebede et al., 2008). All these reports underscore the importance of the testosterone in internal physiology of goats. On the other hand, the appearance of secondary sexual characteristics is fully regulated by the androgen, which may potentiate differential sexual performance among goats in different physiological condition. However, none of the study has been reported in the variation of physiology in the context of analyzing excretory products. Also, in order to differentiate the sexually intact animals, females should have some clues, and these may be presented by the excretory products. So, we analysed the urine of intact, prepubertal and castrated male goat’s urine in the context of volatile identification. We noticed a significant variation in the number of volatiles during each stage of male goats, and found 5 volatiles as common to all the status. Study in mice model by Harvey et al. (1989) reported that the volatile compounds present in the urine vary with dominant and subordinate status, notably, the compounds presented at higher concentration in dominant males is not observed in castrated males.

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Similarly, in our study we found 7 volatiles specifically in intact male urine; however castrated males showed another 10 specific compounds which were not present in intact urine. In mice, the intact male specific compounds were noted in higher concentration in aged males, and so they are mostly preferred by the female for sexual communication for the purpose of maintaining a genetically healthy colony (Osada et al., 2008). This could be the reason for the selection of intact animals for mating by the female.

Consistently, some of the urinary volatiles compounds which were absent in castration and prepubertal conditions seemed to be present in the status of intact and when the castrated animals were treated with exogenous hormone which signifies the role of testosterone in alteration of metabolism

(Achiraman and Archunan, 2005). In line with this, we suggest the compounds present in the intact males, which were absent in the castrated and prepubertal conditions may be of testosterone dependent. Castration enhances the development of fat tissue rather than muscle tissue especially in higher animals (Moran d-Fehr et al., 2000). Usually, the chemical cues emitted by the animals are resulted from the fat metabolism and hence seem to be the end products of it. For instance, the fatty alcohols such as 2-methyl-

1-pentanol and 1-tetracosanol have been noted only in the urine of castrated animals, and the fact may be due to the higher fat content, since fatty alcohols are derived from the fatty acids presented by the fat tissue. Thus, it may suggest that high fat content contribute higher number of volatiles than other male animals as observed in the present study. However, in prepubertal goats, we found only three specific compounds. The report of Ahmad et al.

(1996) clearly demonstrated gradual increase in the testosterone level and surge in luteinizing hormone level while puberty develops in prepubertal goats. Thus, in prepubertal condition, the signal from GnRH to testosterone may not be developed and hence, the action of testosterone may be prohibited in the metabolism. It is believed that the compounds excreted in the intact

142 Chapter V goats may not be appeared in prepubertal while there are only three specific compounds were excreted. This clearly indicates that the testosterone level in the serum may play a major role in production of metabolites which act as chemical signals in goat, however the level of testosterone has to checked in all the groups.

The compounds exhibit consistent shift in every stage of male goats.

For instance, the compounds tetradecene and octadecene has been shifted as dodecene, pentadecene and hexadecane in intact urine. However, the alkenes represented as common compounds have been shifted as alcoholic compounds in castrated goats and thus presented 3-Penten-1-ol, pentanol and tetracosanol. In particular, cyclopropane has been appeared in intact and prepubertal males, however it was absent in castrated goats. By contrast, another two compounds 2,6 bis (1,1- dimethylethyl) phenol and 5-Eicosene was found to be absent in intact males but present in prepubertal and castrated. These compounds were of testosterone independent and present when there is the reduced action level of testosterone inside the body. Since, the physiology of prepubertal goats resemble the stage of castration, both the phases presented two specific compounds in the present study, however it was absent in intact goats.

The chemical nature of the compounds has significantly altered in each animal’s urine. It was noted that the alkenes were generally represented as common compounds. Among those, the modified form of 1-octadecene was reported to have pheromonal property in the peach leafminer moth (Zhang et al., 2013). The well known compound of 2-methy-2-butanal in mother-young interaction which stimulates the feeding behaviour in rabbit pups have been identified in our present study (Schaal et al., 2003). Even though, their report supports our study in the context of the pheromonal property of the compounds, they have reported the compound in the female milk, in contrast

143 Chapter V we have identified in the urine of intact male goat. Though, the compounds possess pheromonal property, it may combine with other compounds at different ration, to confer the cumulative effect in communication. The study by Arnaud et al. (2002) reported that 1-pentadecene as a common pheromonal compound in flour beetles. It has been experimentally proved that the compound, 1-dodecne has pheromonal property which have good affinity with the pheromone binding protein in Helicoverpa species (Sun et al., 2012).

The compound, 1-dodecne, has been implicated in various insect chemical communication system (http:www.pherobase.com/databases/compound/ compounds-detail-delta1-10Hy.php). Altogether, the volatile analysis of present study positively supported by the previous reports by conferring the pheromonal property of the compounds. The compounds may have proportionately blends at specific concentration resulting in the production of peculiar smell in each animal’s urine.

The prepubertal goats excreted more number of proteins than that of intact male goats. It has been reported that testosterone plays a major role even in the maturation of kidney, and thereby in controlling the weight of the kidney and also the androgen receptors present in the orchitectomized and intact condition were varied which further substantiate the role of testosterone which maintains the integrity of the kidney and its metabolism

(Dairiki Shortliffe et al., 2014). The study by Ozgo et al. (2009) revealed that the glomerular filtration as an important fact in excretion of protein, and thus the underdeveloped status resulted in excretion of higher amount of protein in prepubertal male’s urine. Further, the report of Cebo et al. (2010) confirmed the presence of mere higher level of proteins in the milk of the goat as like as bovine, and thus, these proteins may have passed to the prepubertal while feeding. Notably, Ziegler, (2007) reported that the animals in prepubertal condition takes much more protein than the required amount from the milk of its mother, and thus, the excretion level of protein seems to be higher in the

144 Chapter V urine of prepubertal animals. In consistent with the above findings, we also report higher number of protein in the prepubertal’s urine. During prepubertal condition, the undeveloped condition both physically and morphologically, glomerular region generally filter the proteins and the resulted protein fractions usually again catabolized by the epithelial cells in renal tubule. Perhaps, the alteration in the catabolism of renal epithelial cells causes significant alteration in the protein level during prepuberty and intact condition. It is well studied that the epithelial cells present in the kidney undergoes transformation from young to adult stage, thereby differentially regulate the excretion of protein in the urine (Baud, 2003). Thus, it may confer that the growth and development of goats may have participated in the excretion of protein through urine.

Testosterone is the key hormone in regulating the typical male physiology. Its level has been drastically reduced in the prepubertal condition; however, the level seems to be at normalcy in intact condition to perform the function related to physiological change. In rats, it has been reported that the level of testosterone could be attributed to the concentration of a specific carrier protein which is involved in chemo-communication (Ponmanickam et al., 2010). Investigation of Knopf et al. (1983) clearly elucidated the level of expression of mRNA responsible for Major Urinary Protein production in mice, and consistent variation has been observed in castrated mice and the level was significantly increased to 1000 fold when testosterone was administered exogenously. Thus, the proteins present in the urine of intact male goats may be under the control of testosterone level present in the serum. Further, since we have observed more number of proteins in the urine of prepubertal goats and less number of proteins in the intact male goats, it may be suggested that the increased testosterone level may limit the number of proteins to be excreted in the urine of goats.

145 Chapter V

The findings of the present study summarize the presence of specific volatile compounds during different physiological status of male goats. It may be due to the difference in the metabolic process that brings out expression of unique volatiles in each physiological condition of male goats. Further, the protein excretion in the urine seems to be a good marker to denote male goat at what stage they are, since consistent variation is exhibited in the protein profile visually.

5.5. Summary

 The urine of intact, castrated and prepubertal male goats were

analysed for volatile compounds. GC-MS analysis revealed a total of 18

compounds, among which 5 compounds were common to all the

phases.

 Specifically, intact male contain only 6 compounds. These compounds

may have a role in attracting the conspecifics and may be involved in

buck effect. Interestingly, a compound, cyclohexane was found in intact

and prepubertal urine but absent in castrated goat urine.

 Castrated goat contains totally 9 compounds. However, there were two

compounds namely, 5- Eicosene and 2,6, bis (1,1-dimethyethyl) phenol

present in both castrated and prepubertal goat urine which was absent

in intact urine.

 The urine of prepubertal goat contains 11 compounds, among which 3

compounds were specific to prepubertal which might be involved in

mother-young interaction.

 In protein analysis, prepubertal urine contains more number of

proteins, whereas, there were fewer proteins in intact goats.

Specifically, 17 kDa protein present in intact goats was absent in

prepubertal urine.

146 Chapter V

Histological, volatile and protein analysis in the cornual gland of male goat, Capra hircus

5.6. Introduction

Physiological status can be assessed by conspecifics through a variety of scent sources, including scent glands. Mammalian pheromones are characterized by multiple compounds used in intra-specific communication

(Tirindelli et al., 2009). In goats, the ‘buck effect’ is produced in the skin/hair of intact males and has the capacity to synchronize/regulate the reproductive behaviours of female. Mori and colleagues (Iwate et al., 2000; Iwate et al.,

2001; Okamura and Mori, 2005) have demonstrated skin and cutaneous gland products that reveal the mechanism underlying the buck effect. Recent work by this group has identified compounds produced in the head skin of intact males that stimulate the reproductive system of female goats (Murata et al., 2014).

Scent gland is present in animals. For instance, Atta laevigata, an ant, has mandibular gland (modified scent gland) and the concentration of the compound it synthesizes varies in relation to the castes (Hernandez et al.,

1999). In male lizards the femoral gland contains granular cells which are a source of pheromones (Chamut et al., 2009). Among the mammals, the mammary gland of the rabbit secretes milk, which contains 2-methylbut-enal

(2MB2E), which is believed to be a mediator of mother-young interaction

(Schaal et al., 2003). The volatiles secreted by the different glands have roles in communication among conspecifics. Especially, the scent gland, preputial gland of rodents, has been reported to play a key role in communication between sexes. For instance, in an earlier investigation we reported that the clitoral gland of female laboratory rats contains an estrus-specific compound which could be a mediator of sexual communication (Achiraman et al., 2011).

In addition, synthesis of the key constituents of preputial gland of male rat is

147 Chapter V testosterone dependent, which indicates that there is a relationship between glandular secretions and sexual status of animals (Ponmanickam et al.,

2011).

In hoofed mammals, (e.g., deer) the preorbital gland is a good source of chemical compounds that facilitate chemo-communication, and some of these compounds have been implicated in the expression of dominant status by a particular male animal in the colony (Bartos, 1983). Considerable difference in the histoarchitecture of this gland has been shown between territorial and non-territorial Indian Blackbuck (Rajagopal and Archunan, 2011). In elephants, the temporal gland secretes a fairly good quantity musth, which is of androgen-dependent (Rasmussen et al., 1990). The axillary gland secretion is one of the important sources of human pheromones. The secretory material has peculiar smell due to microbial action and confers the message of maleness (Zeng et al., 1992). Considering the large population size and great diversity in mammals, only scanty reports are available in support of the glandular sources performing roles in chemical communication. On this sense, the glands that play roles in chemical communication in goats (Capra hircus) have been poorly studied, and the concept of buck odor production has not been clearly explained. Therefore, we embarked on a study of buck odor production by the cornual gland by subjecting the gland to histological, volatile and proteomic analysis.

5.7. Materials and methods

5.7.1. Test animal

Adult male goats, Capra hircus were fed with cultivated forage, green fodder and water ad libitum. When the animals were about 24 months old, they were sacrificed at the slaughter house. As soon as the animal was sacrificed, the head was taken and the cornual gland was dissected out and

148 Chapter V kept in ice until further use. The gland was located following the method of

Van Lancker (2003).

5.7.2. Histological analysis

Slices of the gland were fixed in 10% neutral buffered formalin and processed for routine paraffin embedding. Five micrometer thick serial sections were stained with heamatoxylin and eosin and mounted in DPX mountant. The sections were examined in a research microscope (Wetzlar

Hund, Germany) and photographed.

5.7.3. Sample preparation

A part of the gland was homogenized quantitatively in PBS (pH-7.2) in sterilized homogenizer. The supernatant was extracted with dichloromethane and a part was used for GC-MS analysis of volatiles. Remaining part of the supernatant was solubilized in gel loading buffer and used for protein analysis adopting SDS-PAGE.

5.7.4. Gas Chromatography-Mass Spectrometry

The homogenate of cornual gland sample in PBS (pH-7.2) was extracted with dichloromethane (1:1). The extract was filtered with Whatman filter paper and used for analysis of volatile compounds using GC-MS (QP-2010 plus,

Shimadzu, Japan). Two micro-liters of the extract was injected into the GC-

MS on a 30 m capillary column with a film thickness of 0.25 µm (30 m x 0.2 mm i.d., coated with UCON HB 2000) under the following temperature regimens: initial oven temperature, 40 °C for 1 min and then 170 °C for 1 min, afterwards increasing up to 240 °C for 5 min. The detection accuracy was 1 ng/peak. Mass spectrometer was operated in CI mode at 70 eV, with ammonia as reagent gas. Identification of unknown compounds was made by probability-based matching, using WILEY RegistryTM, NIST05 and NIST05s based library.

149 Chapter V

5.7.5. SDS-PAGE analysis

The supernatant of the extract was subjected to estimation of protein adopting the spectrophotometeric method of Bradford (1976). The proteins were separated adopting SDS-PAGE (Laemmli, 1970) by loading equal amount of proteins in all wells when appropriate molecular weight marker (Genei,

Bangalore, India, Cat. No: 105979) was also run alongside the samples for detection of molecular weight of the proteins of interest. The protein bands were stained with Coomassie brilliant blue.

5.7.6. Destaining and in-gel digestion

The protein bands of interest were excised and the gel plugs were de- stained separately using 100 μl of 25 mM NH4HCO3 in 50% (v/v) acetonitrile

(1:1) by incubation at 37 °C for 30 min. Each gel plug was sliced into small cubes, and placed in 1.5 ml eppendorf tube. After drying in a Speed-Vac

(Savant), the gel was incubated in 100 μl of 2% β-mercaptoethanol/25 mM

NH4HCO3 for 20 min at room temperature. The same volume of 10% 4- vinylpyridine in 25 mM NH4HCO3/50% acetonitrile was added for cysteine alkylation. After 20 min the gel was soaked in 1 ml of 25 mM NH4HCO3 for 10 min, dried and incubated overnight with 25 mM NH4HCO3 containing 100 ng of modified trypsin (Promega). The tryptic digest was removed from the gel and the proteins were extracted first with 300 μl of 25 mM NH4HCO3 and then in

50% acetonitrile. The two fractions were pooled and dried in a Speed-Vac and then stored at -20 °C for further analysis. When resumed, each fraction was resuspended in 0.1% formic acid immediately before use (Ponmanickam and

Archunan, 2006).

5.7.7. MALDI -TOF MS analysis

The MALDI-TOF MS data were acquired using an Ultraflex TOF/TOF spectrometer (Bruker Daltonics, Billericia, MA, USA, and Bremen, Germany), equipped with 50 Hz pulsed nitrogen laser (337 nm), operated in positive ion

150 Chapter V reflectron mode using a 90-ns time delay, and a 25 kV accelerating voltage.

External calibration was done using peptide I calibration standard

(Angiotensin II, Angiotensin I, substance P, bombesin, ACTH clip 1-17, ACTH clip 18-39, somatostatin 28) with molecular masses ranging from 1000 to

3200 Da. The samples were prepared by mixing an equal amount of peptide

(0.5 μl) with dihydroxybenzoic acid/α-Cyano-4-hydroxycinnamic acid (CHCA) saturated with 0.1% trifluroacetic acid (TFA) and acetonitrile (1:1) as matrices.

The mass spectrum of the 26 kDa protein was obtained and the mono-isotopic masses of 26 kDa protein spectrum were analyzed. Masses below 50 m/z were not considered due to interference from the matrix.

5.7.8. Database analysis

The spectra were processed using FLEX analysis software. Database search was accomplished with the assigned masses of monoisotopic peptide.

MASCOT search engine (Matrix Science, London, UK)

(http//www.matrixscience.com) was utilized for the protein identification.

MASCOT analysis was carried out using carbamidomethyl as fixed modification and oxidation (M) as variable modification. For 33 kDa protein search was made in the taxonomy of eukaryota and for 28 kDa protein search was made with all taxonomical entries.

5.8. Results

5.8.1. Histological organization of cornual gland

The gland is located close to the apical region of the dermal papilla. It opens at the dermal pore and is surrounded by the roots of the hair follicles.

The surrounding area is filled with smooth muscle fibres, running at different direction, i.e., cross, obligue and longitudinal. Smooth muscle fibres were also noticed between lobules of the holocrine gland. The contraction of the smooth muscle fibre between the lobules and the fibres located around the gland

151 Chapter V could be involved in extrusion of sebum. Acini are serous, i.e., formed of small lobules, and the acinar gland is lobulated (Figure. 5.5).

5.8.2. Volatile compounds in the cornual gland extract and their

features as revealed in GC-MS analysis

Fourteen volatiles were observed in the cornual gland extract, among which most of the compounds were alkanes and alkenes whereas some were alcohols. The molecular weights of the compounds were between 84 and 270

Da. There were 4-18 carbon atoms, and 3-38 hydrogen atoms in the compounds (Table. 5.2; Figure. 5.6).

152 Chapter V

a) Vacuoles; b) Hair follicles; c) Duct

Fig. 5.5. Histo-architecture of cornual gland of male goat.

153 Chapter V

Fig .5.6. GC-MS chromatogram of male goat cornual gland extract

Table.5.5. List of compounds identified in the cornual gland extract of intact male goat, Capra hircus

Peak Compound Name Molecular Molecular No. formula weight

1 Cis-1-Chloro Buten-3-yne C4H3Cl 86

2 1-Cyclopropyl octane C11H22 154

3 Tri cyclohexane C6H12 84

4 Alpha Dodecene C12H24 168

5 5-Octadecene C18H36 252

6 1,2 Hydroxyethoxy tridecane C13H28 184

7 2,4-bis 1,1 dimethyl ethyl Phenol C6H6O 94

8 1-Pentadecene C15H30 210

9 5-Eicosene C20H40 280

10 2,3 Dimethyl Hexane C8H18 114

11 1,2 Benzenedicarboxylic acid C9H8O 164

12 2-Methyl 2-Butenal C5H8O 84

13 1-Hepten-5-one C7H12O 112

14 1-Octadecanol C18H38O 270

154 Chapter V

(a) 2-methyl 2-butanal

155 Chapter V

(b) 5-Octadecene

156 Chapter V

(c) 1-Octadecanol

Mass spectrum of volatile compounds in the cornual gland

157 Chapter V

5.8.3. Proteins in the cornual gland extract as revealed in SDS-PAGE

analysis

The protein extract of the gland was separated in 12% SDS-PAGE which revealed the presence of seven different protein fractions, and their molecular weights ranged from 15 to 58 kDa. Among the different proteins, the intensity of 48 kDa protein fraction was the highest (Fig. 5.7).

5.8.4. Identification of the protein as revealed in MALDI-TOF

Among the different protein fractions two protein fractions, at 28 and

33 kDa, were subjected to MALDI-TOF analysis, which revealed high score matching with DNA mismatch repair protein and Abietadiene synthase, respectively, in MASCOT analysis. The sequence coverage for DNA mismatch repair protein was 22% and that for Abietadiene synthase was 20%. The score value (Figs. 5.8 & 5.9).

158 Chapter V

kDa

Lane 1- Marker; Lane 2- Cornual gland extract

Fig. 5.7. SDS-PAGE profile of male goat cornual gland extract.

159 Chapter V a)

x104 Intens. [a.u.]

1.25

1.00

0.75 1676.748 691.164

0.50 2059.984 1135.650 1771.944 2211.070 1274.740

0.25 1029.764 845.112 1475.761 718.252 1994.001 2283.155 1836.761 1568.724 1365.660 2145.024 2807.359 2584.210 2462.067

0.00 1000 1500 2000 2500 3000 3500 4000 m/z b)

KSFIKVSDNG CGIPADQLTL AISRHCTSKI TDDVHNIYFL GFRGEALPSI

GSVAKLKLTS RTQEAENATE IIVTAGKIIG PKPAAANPGT IVEVRDLFFV

TPARLKFMKT DRAETNAISD MIKRIAIAFP HIRFSLSGLD RTMELPATA

NSTQGQLQRI TQIMGKEFAP NSIALNAKRE SIRLTGFACL PSFNRSNSLH

QFAYVNRRPV RDKFLWGTIR GAYADVMARD RYPVSILFID LPPADVDVNV

Fig. 5.8. a. Mass Spectra of 28 kDa protein obtained from MALDI-MS.

b. Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 22% sequence coverage is highlighted by being underlined in red color.

160 Chapter V a)

x104 Intens. [a.u.]

1.5

1.0 1515.705 1790.856

0.5 2211.047 1434.716 1643.818 1993.914 691.005 1301.701 842.473 1179.592 2283.155 1707.799 1029.719 2399.111 759.328 1365.630 2144.979 1847.883 2807.363 2510.152 2922.404

0.0

1000 1500 2000 2500 3000 3500 4000 m/z b)

IMKLQSKDGS FLSSPASTAA VFMRTGNKKC LDFLNFVLKK FGNHVPCHYP

LDLFERLWAV DTVERLGIDR HFKEEIKEAL DYVYSHWDER GIGWARENPV

PDIDDTAMGL RILRLHGYNV SSDVLKTFRD ENGEFFCFLG QTQRGVTDML

NVNRCSHVSF PGETIMEEAK LCTERYLRNA LENVDAFDKW AFKKNIRGEV

EYALKYPWHK SMPRLEARSY IENYGPDDVW LGKTVYMMPY ISNEKYLELA

KLDFNKVQSI HQTELQDLRR WWKSSGFTDL NFTRERVTEI YFSPASFIFE

Fig.5.9. a. Mass Spectra of 33 kDa protein obtained from MALDI-MS.

b. Number in the mass spectrum gives precise m/z (M+H) values for the detected peptide ion signals. The matched 20% sequence coverage is highlighted by being underlined in red color.

161 Chapter V

5.9. Discussion

Histological technique was adopted to have an idea about the structural organization of the cornual gland and the observation confirmed that the cornual gland is a sebaceous gland that produces sebum. According to Ponmanickam et al. (2010) preputial gland produces volatile compounds and also some proteins involved in animal communication. It is an established fact that sebaceous gland produces sebum, which is an oily and waxy substance as documented in the humans (Zouboulis, 2004). According to Abbasi et al. (2009) the interdigital gland of sheep produces sebum and is discharged by the hair follicles present adjacent to the lobules. We also made a similar finding, and the cornual gland is formed of numerous lobules, located adjacent to the hair follicles indicating that sebum is produced and it is extruded via the hair follicles. In the deer, the fluid secretion release is visible, whereas in the goat the extrusion of the secretory material is not visible, but one can sense the pungent odor of the glandular secretion, which is indicating that the secretion is released as a volatile substance. However, in several occasions the gland is ruptured by hitting against the wall of fence especially when the buck was encountered with a female goat in estrus

(unpublished observation). Thus, it is comprehensive that the gland does secrete a material, and when the buck desires to mate with a female in estrus, the gland is ruptured and the secretion discharged as a volatile substance.

In his pioneering study on goat scent gland, Van Lancker (2005) suggested that cornual gland lacks the capacity to produce any pheromones; however, the study did not speak about volatiles. To validate and support our hypothesis we carried out some preliminary studies so as to conclude that cornual gland is one of the major scent glands and may be involved in buck odor production. Earlier, it was reported that the scent gland of insects has distinctive roles in producing the compounds, mostly volatiles, and these

162 Chapter V compounds have been connected with eliciting behavioural responses. In this context, we analyzed the volatile portion of the gland to conclude that cornual gland is a scent gland. We found 14 volatile compounds in GC-MS analysis, of which most of the compounds were alkanes, but some belonged to amine, ester, ketone, and alcohol groups. The compounds under the class alkanes, isoalkanes, alcohols, ketones, carboxylic acids, oxiranes, furanoid linalool oxides, branched-, unbranched-, saturated- and unsaturated aldehydes have been reported in interdigital secretions and suggested to have a role in the expression of the dominant status (Reiter et al., 2003). Therefore, we have reason to believe that the volatile compounds identified in the cornual gland of goat in the present study must have some crucial role in goat communication. Thus, we propose that the compounds identified in the present study are putatively pheromones. Very recently, Murata et al. (2014) reported that the skin of goats contains a compound called octanol but, in our study, we found octadecanol in the glandular extract. It could be postulated that there may be a possible derivatization of octadecanol to octanol

(reduction of 2 carbon atoms in the octanol) in the gland. In addition, these authors found a number of ketones, but the prominent fractions were alkanes and alkenes. In our finding octadecanol occurs only in the glandular extract of intact goat, whereas it is totally absent in the scent sources (urine and faeces) of female animals and urine of male irrespective of their reproductive status

(unpublished data). Hence, we assign the role of producing the specific smell so as to deliver the buck effect to octadecanol.

Most of the compounds identified in the present study are not new but have already been reported to act as pheromones in other animals and insects. For instance, the compound identified in the present study 1,2- benzene dicarboxylic acid has already been found in the clitoral gland of rat

(Kannan and Archunan, 2001). The importance of this compound in the

163 Chapter V present context lies in its source, i.e., the compound is of glandular origin in both the studies. The compound 2,7-dmiethyl octane was identified as an androgen-dependent urinary pheromone in mouse (Achiraman and Archunan,

2005). In the present study, we found octane (1-chloropropyl octane) group in the cornual gland and it could be inferred that this may also be androgen- dependent; another compound identified in the present study, Cyclohexane, has been earlier reported as anti-aggregation pheromone in spruce beetle,

Dendroctonus rufipennis (Holsten et al., 2003). Another compound, 1- dodecene, identified in the present study, has been documented as a semiochemical which would bind with the odorant binding protein (OBP-10) in sibling moth species (Sun et al., 2012). The compound 2-methyl 2-butanol identified in the cornual gland has been earlier reported in rabbit’s milk where it facilitates typical nipple searching behavior in rabbit pups (Luo, 2004).

Another compound 5-hepten-2-one has been reported in the rodent urine and is believed to be responsible for the delay in puberty in grouped females

(Burger, 2005). The compound 1-dodecanol has been reported as bound form volatile in vaginal fluid of hamster and constitutes the second most important compound in the hamster vaginal lipocalin (Briand et al., 2004).

Characterization of the other compounds also revealed most of them to be pheromones but the exact role has not been evaluated. The database available on the website pherobase gives adequate details about each of the pheromonal compounds. Thus, some of the compounds identified in the present study (e.g., 5-Eicosene) have been reported in the pherobase.

Similarly, the present study revealed the repetitive presence of a compound namely, 1-Buten 3-yne, which has also been detected in the urine of pubertal male goat and not in the prepubertal male (unpublished data).

The compounds produced from scent sources are generally volatile in nature and, hence, they are associated with carrier molecules which provide

164 Chapter V for sustained release of the signal. Flower (2006) in his review, has elucidated the structural and functional aspects of the lipocalin superfamily proteins, to which the carrier proteins generally belong. A number of original studies have explained the possible role of lipocalin proteins which are related to chemical communication. Further, histological and protein analyses of the scent gland of muskrats provide for influence of androgens and estrogens in the secretion of volatiles (Lu et al., 2011). Corroborating with these findings, we also found most of the compounds as of pheromonal property and two compounds were found to be volatiles which bind to proteins. Therefore, we looked for carrier molecules that would bind and deliver volatile compounds. Seven protein bands, in the molecular weight range from 15 to 58 were identified. To elucidate the functional aspect of the proteins we subjected two of the proteins to MALDI-TOF analysis. The two protein fractions were identified as

DNA mismatch repair protein and Abiedatiene synthase respectively. But these two proteins are not carrier proteins, but they have other roles.

DNA mismatch repair protein (MMR protein) has been reported in the sebaceous gland of other animals, where its function is crucial in inhibition of tumor formation, since the absence of this protein have been linked to Lynch syndrome (Plocharzyk et al., 2013). Tome et al. (2013) identified DNA mismatch repair protein in various mouse tissues and found its concentration to be higher in the proliferative tissues than non-proliferative tissues. Since cornual gland is one among the proliferative tissues the presence of MMR protein must associate with some crucial role although its relative concentration in other tissues of goat needs to be verified. Alterations in MMR protein has been implicated in the development of Muir-Torr syndrome

(Shalin et al., 2010). Though the MMR protein has been implicated in repairing the errors that occur during DNA replication by avoiding mismatch pairing of nucleotides, how the defective protein is involved in the

165 Chapter V development of this syndrome is not yet clearly understood. We infer that cornual gland is an important organ that produces the sebum and plays role in communication of goats, and MMR protein may be necessary to keep the gland in a healthy condition.

The cornual gland of goat is diversely lobulated, reflecting sebaceous gland organization. The gland produces volatile compounds and proteins.

Inferring from the structural analysis, and based on the existing literature, several of these compounds could be pheromones, of which octadecanol is suggested to trigger the buck effect, in which the protein also may have role.

5.10. Summary

 The histological analysis of the cornual gland revealed the structure of

sebaceous gland with the presence of vacuoles, ducts and hair follicles.

 Volatile analysis in the cornual gland showed 14 different compounds,

among which the presence of octanol seems to be important.

 In protein analysis, the gland depicts seven different proteins ranging

from 15 to 58 kDa.

 Of them, two proteins (28 & 33 kDa) were analysed with MALDI-TOF,

which shows significant score value and matching of peptide sequences

with DNA mismatch repair protein and Abietadiene synthase,

respectively.

166 Consolidated Discussion

Estrus synchronization was done in mature female goats with vaginal sponges. After synchronization the phases of the estrous cycle were determined by the expression of various behaviours in female and male goats.

Female goat express typical estrus signs such as vaginal swelling, reddening of vulva, tail wagging, vaginal mucus discharge, restlessness, reduced food intake and milk secretion. Specifically the behaviours like, standing heat, bellowing, bleating, vagina mucus discharge and homosexual behaviours by mounting on other female goats were noted in high frequencies only during estrus and not in non-estrus phases. The female express its sexual interest by exhibition of the estrus specific signs to intimate the male about its endocrinological status and readiness for mating. The male goats also express usual sexually receptive behaviours like flehmen and mounting in higher frequencies. However, the follow-up behaviours such as, penile protrusion, mounting and copulation were observed only towards with estrus female goats. A specific behaviour, called enurination (urination in the udder region of own body and smelling it) was noted in male goats while encountering with estrus female. Both the male and female goats express mock fighting behaviour during estrus phases. Even though the behaviours could used for estrus detection, development of alternative tool using the chemical signals would be a promising approach. Hence, the analysis of scent sources in terms of analysis of biochemical parameters is need of the hour.

The urine samples collected during the four different phases of the estrous cycle were subjected to protein, lipid and fatty acid analysis. The faeces samples were analysed for steroid hormones such as estradiol and progesterone. The level of protein was higher during estrus urine and least concentration was found in metestrus urine. This denotes that the high level

167 Consolidated Discussion of urinary proteins may be important in advertisement of estrus to the male while the in metestrus it may have different function such as in implantation.

In contrast, the high lipid content in urine of metestrus goat denotes that the function of lipid in luteal phase, whereas the less lipid content in pro-estrus urine denotes the function in onset of estrus. All the phases exhibit three different fatty acids with varying concentration. Among which, lignoceric present at high concentration in estrus urine and other two fatty acids such as cis 5,8,11,14,17- eicosapentanoic acid and nervonic acid are present at high concentration in metestrus urine. To our knowledge, the fatty acids identified in the present study have not been reported in any other animal’s excreta, hence, it could be suspected that these may be of species-specific and may confer role in advertisement of physiological status when coupling with other biochemical moieties. The faecal steroid analysis revealed high concentration of estradiol in estrus faeces whereas progesterone concentration high in diestrus faeces, which further corroborate the possible interaction of hormones in the regulation of estrous cycle. Moreover, this is the first evidence reporting the faecal steroid analysis in goat, and it may useful in non-invasive estrus detection. Though the biochemical parameters appeared as a good tool analysis of analysis of other parameters such as volatile analysis could be efficient for use them in estrus detection. Hence, the next chapter framed on the analysis of volatile compounds in the urine and faeces of female goats.

The volatile analysis of urine and faeces samples collected during pro- estrus, estrus and post-estrus samples have expressed different compounds with consistent shift and transition during every phases. There were 14 compounds identified in the urine of female goat during estrous cycle. Among them, estrus phase exhibit 4 specific compounds namely, tetradecanol, n- pentadecanol, 3-methylene tridecane and 2-ethyl-1-dodecene. It has already

168 Consolidated Discussion been reported that the alcoholic groups have notable function in acting as pheromones. And based on the earlier literature most of the compounds identified in the present study have been reported as semiochemcals in other animal system. Similarly, the faeces samples of pro-estrus, estrus and post- estrus faeces revealed a total of 24 compounds. Among them, two compounds were present in all the phases and six compounds were specific to pro-estrus phase. Estrus phase consist of seven specific compounds, with the inclusion of two anti-oxidants such as Ascorbic acid and Vitamin E. It is interesting to note that the ascorbic acid could be synthesized by goat themselves to meet out the vitamin necessity also, the specific presence of ascorbic acid in the estrus phase insist its role in estrus phase. Another important notion is the presence of octadecanoic acid which has been literally termed as capric acid.

The volatile compounds identified as estrus specific could be a better choice in estrus detection, however, use of proteomics would be utilized as a non- invasive method of estrus detection.

The urine samples collected during estrous cycle were subjected to

SDS-PAGE analysis which revealed the presence difference in the expression pattern during every phases. In addition, some of the proteins were seems to be vary in concentration. Proestrus urine revealed seven different proteins with the complete absence of 25 kDa protein and presence of 98 kDa, which is absent in other phases. In addition, the concentration of 68 kDa protein was higher in proestrus phase than that of other phases, which was remarkably reduced in estrus phase. Estrus phase revealed the presence of seven different proteins with the specific presence of 25 kDa protein and absence of

98 kDa protein. The concentration of 25 kDa protein was further reduced to metestrus whereas it was completely absent during proestrus and diestrus.

Metestrus phase also revealed protein profile as same as estrus. Diestrus phase revealed higher concentration of 68 kDa protein next to proestrus. We

169 Consolidated Discussion have observed the estrus specific protein of 25 kDa with high score value with complement C3 protein. The complement C3 protein was studied during the estrous cycle of mice and it has been postulated that the expression of the protein is under the control of estrogen. It is also interesting to note that the level of complement C3 was again reached a detectable quantity during estrus, pregnancy and lasts up to delivery. It was interesting to note that the protein was appeared during estrus and metestrus, and, hence, it could be postulate that the 25 kDa protein has some crucial role during estrus and metestrus phase. The second protein at estrus phase 28 kDa protein has showed top score with Acetyl Co-A carboxylase. In literature, it has been suggested that melonyl- Co A for the synthesis of fatty acids. Acetyl Co- A carboxylase was found in the mammary gland of goat and its expression was correlated with the production of milk. Thus, the Acetyl Co-A carboxylase may be involved in the production of synthesis of fatty acids in female goats. The analysis of various biochemical and behavioural characteristics could be served as better indicator of physiological status in female, however analysis of male scent sources seems to be necessary.

The urine samples of male goats collected during prepubertal, castrated and intact conditions were subjected to GC-MS analysis for analysis of volatile compounds. GC-MS analysis revealed a total of 18 compounds, among which

5 compounds were common to all the phases. Specifically, intact male contain only 6 compounds. Interestingly, a compound, cyclohexane was found in intact and prepubertal urine but absent in castrated goat urine. Castrated goat contains totally 9 compounds. However, there were two compounds namely, 5- Eicosene and 2,6, bis (1,1-dimethyethyl) phenol present in both castrated and prepubertal goat urine which was absent in intact urine.

Literally, it has been suggested that castration is being done in livestock animals to increase the body weight. It was evidently proved that the fat mass

170 Consolidated Discussion of the castrated goats is higher than that of intact condition, and thus the increased fat concentration could contribute the specific presence of two fatty alcohols in the urine of castrate goats. The urine of prepubertal goat contains

11 compounds, among which 3 compounds were specific to prepubertal. In protein analysis, prepubertal urine contains more number of proteins, whereas, there were fewer proteins in intact goats. Specifically, 17 kDa protein present in intact goats was absent in prepubertal urine. The prepubertal goats have underdeveloped kidney and in addition it takes more amount of protein from the milk. It could be suggested that the reduced filtration capacity and high consumption of proteins from the milk results in the excretion of more number of proteins in the prepubertal urine. The urinary volatile and proteins could be used in the differentiation of physiological status in males. The promising fact in goat, is the production of male effect, which has not been studied in detail. However, literatures revealed that the scent gland in goats may contribute some odors that may account for buck effect.

The scent gland mediated communication has been well documented in animals. To substantiate this fact in goat, we have analysed a scent gland in goat, called cornual gland. The histological analysis of the cornual gland depicted the structure of sebaceous gland with the presence of vacuoles, ducts and hair follicles. Further, volatile analysis in the cornual gland revealed a total of 14 compounds, among which the presence of octanol seems to be important. The compound, octadecanol has been reported in cornual gland of goat and implicated its role in buck effect. Since we have dissected out the gland and went for volatile analysis, and hence, the compounds octadecanol may be reduced into octanol. To substantiate the presence of carrier proteins, we have subjected the gland to protein analysis and we found

7 different proteins in the cornual gland extract of intact goat. Of them, two proteins (28 & 33 kDa) were analysed with MALDI-TOF, which shows

171 Consolidated Discussion significant score value and matching of peptide sequences with DNA mismatch repair protein and Abietadiene synthase, respectively. Among them

DNA mismatch repair protein has been shown to be present in the sebaceous gland of other animals in healthy condition and its function has been modulated with the diseased condition. Even though, there was no significance was found with DNA mismatch repair protein with chemo- communication its role has to be further studied.

172 Significant Findings

 The male goat exhibited enurination behaviour when encounter estrus female.

 The increased protein and reduced lipid concentration coupled with high concentration of lignoceric acid favors the estrus detection. In addition, faecal concentration of estrogen and progesterone would be useful for non-invasive estrus detection in goats.

 Transition in the urinary and faecal volatiles was recorded along with the endocrinological status during estrous cycle. The estrus-specific urinary compounds were identified as Tetradecanol, n-Pentadecanol, 3- Methylene tridecane, 2-Ethyl-1-dodecene and in faeces the estrus- specific compounds includes two anti-oxidants.

 The estrus urine sample revealed the specific presence of 25 kDa protein which was absent during pro-estrus and diestrus. The protein has shown to be matched with Complement component C3, in which the expression was under the control of estrogen.

 Castrated male goat contains more number of volatile compounds with two fatty alcohols which may be contributed by the increased fat mass during castration. In protein analysis, prepubertal goats excrete more proteins which is presumably due to the intake of high protein from milk and reduced filtration capacity of the kidney.

 The histology, volatile and protein analysis positively support the cornual gland as a major scent gland in goat and the compounds it synthesize may involve in buck odor production.

173 Conclusion

Findings of the present study in biochemistry aspect from female and male goats concluded that the change in internal physiology (endocrine changes) has been reflected in the excretion. A comprehensive transition of urinary and faecal volatiles across the estrous cycle was reported for the first time and these volatiles are speculated to act as estrus signals to attract conspecifics and therefore could be utilized for the development of non- invasive estrus markers in goat. Further, the intact male specific volatiles of urine and cornual gland could be further validated for its estrus synchronizing ability. Overall, this fundamental data would be better choice in concerning the improvement of goat production.

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192 Publications

Papers under revision

1. D. SankarGanesh, R. Ramachandran, V. Ramesh Saravanakumar, U. Suriyakalaa, S. Kannan, G. Archunan, S. Achiraman. A correlation of faecal volatiles and steroid hormone profiles with behavioural expression during estrous cycle of goat, Capra hircus. General and Comparative Endocrinology (I.F. 2.8).

2. D. SankarGanesh, R. Ramachandran, R. L. Rengarajan, P. Ponmanickam, V. Ramesh Saravanakumar, G. Archunan, S. Achiraman. Physiological influence on urinary volatile and proteins in goat, Capra hircus- A comparative study in prepubertal, intact and castrated male goats. The Journal of Physiological Sciences (I.F. 1.0)

Papers communicated

1. D. SankarGanesh, R. Ramachandran, K. Karthikeyan, S. Muniasamy, V. Ramesh Saravanakumar, S. Kannan, G. Archunan, S. Achiraman. Biochemical and volatile analysis in urine of estrus synchronized goat during estrous cycle that facilitate estrus detection. Animal Reproduction Science (I.F. 1.8).

2. D. SankarGanesh, R. Ramachandran, R. Ashok, S. Muthukrishnan, V. Ramesh Saravanakumar, S. Kamalakkannan, R. Sukirtha, G. Archunan, S. Achiraman. Buck odor production in the cornual gland of male goat, Capra hircus- Validation with histological, volatile and proteomic analysis. Zoology (I.F. 1.4).

193

Animal Reproduction Science 138 (2013) 163–167

Contents lists available at SciVerse ScienceDirect

Animal Reproduction Science

jou rnal homepage: www.elsevier.com/locate/anireprosci

Faecal chemical cues in water buffalo that facilitate estrus detection

a a

Kandasamy Karthikeyan , Samuthirapandi Muniasamy ,

b b

Devaraj SankarGanesh , Shanmugam Achiraman ,

c a,∗

Veluchamy Ramesh Saravanakumar , Govindaraju Archunan

a

Center for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli 620024, Tamilnadu, India

b

Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamilnadu, India

c

Department of Livestock Production and Management, Veterinary College and Research Institute, Namakkal-637002, Tamilnadu, India

a r t i c l e i n f o a b s t r a c t

Article history:

Chemo-signals are among the reliable non-invasive methods for estrus detection in mam-

Received 5 September 2012

mals. Water buffalo is a silent heat animal and, hence, there is search for chemo-signals

Received in revised form 21 February 2013

which would be effective non-invasive indicators of estrus state. We analyzed the fae-

Accepted 23 February 2013

cal chemical cues during the estrous cycle in buffalo and to find the estrus-specific faecal

Available online 5 March 2013

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

Keywords:

Buffalo 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

Estrus-specific cues

Faeces only in estrus faeces. The faecal samples of estrus buffaloes and the estrus-specific com-

GC–MS pound(s) (4mp + tv) at three different concentrations were tested for behavioral responses

4-Methyl phenol (flehmen and mounting behavior) in the bull. The bulls exhibited repeated flehmen when

Trans-verbenol

exposed to a combination of the two compounds (i.e., 4mp + tv) as compared to the indi-

Bioassay

vidual 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.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction is no outwardly manifested clear signs of estrus (Suthar

and Dhami, 2010). Realizing this problem, there have been

Estrus detection is a prerequisite for efficient reproduc- several attempts to device methods to overcome the dif-

tive management in farm animals (Drost, 2007). Further, ficulties of estrus detection in buffaloes, but most of the

artificial insemination performed in farm animals is suc- methods developed till date have not yet been successful

cessful only when it is done exactly during the estrus and reliable. There is, thus, pertinent need to develop an

phase. Very specially, in buffalo the detection of estrus is efficient estrus detection method in buffaloes in order to

problematic since it is a silent-heat animal in which there enhance the success rate of conception through artificial

insemination.

In ungulates, volatile cues are one of the primitive com-

∗ munication channels (Booth and Signoret, 1992). Most

Corresponding author. Tel.: +91 431 2407040.

E-mail address: [email protected] (G. Archunan). often, the volatile cues are released in urine, faeces, vaginal

0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.02.017

164 K. Karthikeyan et al. / Animal Reproduction Science 138 (2013) 163–167

fluid etc. (Epple, 1986). Among the various sources of compounds. The extracts were filtered through cheese

volatile cues, faeces is noteworthy in intra-specific chemi- cloth or nylon mesh (60–120 ␮m) (Sankar and Archunan,

cal communication in many vertebrates (Archunan, 2009). 2008). Two microlitres of the extract was injected into

An earlier evidence provided by Kimura (2001) suggested the GC–MS system (QP-5050, Schimadzu, Japan) on a 30 m

that faeces carry cues about the reproductive status of glass capillary column with a film thickness of 0.25 ␮m

animals, especially in females of different species. Devel- (30 mm × 0.2 i.d. coated with UCON HB 2000). The tem-

oping on this suggestion adopting bull behavior assay we perature regimen was as follows: initial oven temperature

◦ ◦ ◦

also showed that faecal constituents would provide for of 40 C for 4 min, increasing up to 250 C at 15 C/min, and

◦

estrus detection in bovine (Sankar and Archunan, 2008). then held at 250 C for 10 min. The detection accuracy of

Further, the analysis of chemo-signals offers a promising the substance was 1 ng/peak. Mass spectrometer was oper-

approach to assess the reproductive status of the ani- ated in CI mode at 70 eV, using ammonia as reagent gas.

mal non-invasively. This has been adequately proved in The identified compounds were matched with the library

bear that analysis of urinary volatiles could be one of the of chemical substance (NIST 6221B).

methods to assess the reproductive status non-invasively

(Dehnhard et al., 2006). 2.4. Behavioral analysis

To date, analysis of volatiles of faeces from buffaloes

(Bubalus bubalis) belonging to different phases of estrous The experimental setup consists of 3 bulls with six

cycle has not been attempted, and the possibility of mak- female dummy buffaloes. Raw faeces sample at three dif-

ing use of the chemical cues in faeces in estrus detection ferent concentrations (0.5%, 1.0%, 2.0%) was choked in

remains unexplored. Hence, keeping in view the diffi- cotton and applied on the perineal region of external gen-

culty in estrus detection and the possibility of making italia of the dummy (non-estrus) buffalo (Rajanarayanan

use of chemical cues in faeces as reliable estrus indicator, and Archunan, 2004). The bulls were allowed to sniff it

the present study was aimed at identification of spe- for about 15 min. The flehmen and mounting behaviors in

cific volatile compounds in faeces and confirmation of the response to faeces sample were recorded.

estrus-specific compounds adopting bull behavior assay so Based on the findings from GC–MS analysis, and litera-

as to develop estrus indicators using faeces. ture survey, two estrus-specific compounds viz., 4-methyl

phenol and trans-verbenol (Sigma–Aldrich, St. Louis, MO,

2. Materials and methods USA), were chosen for bull behavior assay. The methodol-

ogy for preparation of samples and the assay as in previous

2.1. Test animals experiment was adopted. The two compounds were tested

individually and, also, in combination. The non-estrus com-

Six sexually mature female buffaloes (Bubalus bubalis, pound, hexadecanoic acid, was used as the positive control.

Murrah breed) were used for sample collection. For behav-

ior assay three bulls were used against the six female 2.5. Statistical analysis

dummy buffaloes. The animals were maintained at exotic

Data were compiled using SPSS statistical software

cattle-breeding centre, Orathanadu Livestock Farm, Tan-

(Version 17, SPSS Inc., Chicago, IL, USA) and subjected to

jore District, Tamil Nadu, India, and fed with conventional

analysis of variance (ANOVA) with post hoc comparison

diet (cultivated forage crops, supplemented with a little

(one-way) using Duncan’s Multiple Range Test (DMRT).

green fodder) and water ad libitum.

2.2. Determination of estrous cycle and sample collection 3. Results

3.1. Identification of volatile compounds

The phases of the estrous cycle were determined with

the help of the conventional estrus behaviors in female

GC-MS analysis revealed a total of 27 compounds, all

buffaloes such as vaginal swelling and secretions, fern pat-

the three phases put together (Table 1). Among these com-

tern, restlessness, frequent urination and tail wagging, and

pounds 18 were present during proestrus, 21 during estrus,

the response of the male such as exhibition of flehmen,

and 11 during postestrus. Two compounds viz., 4-methyl

mounting etc. (Rajanarayanan and Archunan, 2004). The

phenol (4mp) and trans-verbenol (tv) were present only

trans-rectal palpation of the uterus and fern pattern also

during the estrus phase. Three compounds, 2, 4-bis cyclo-

were checked for the confirmation of estrus phase. The

hexanal, 8-methyl-1-decane and 2-ethyl-1-decanol, were

total length of the estrous cycle was 21 days. The day on

present during all three phases.

which the above behaviors were observed was considered

as estrus. Two to five days prior to this day was consid-

ered as proestrus, while 2 days after estrus was taken as 3.2. Behavioral assay

postestrus, and faecal samples were collected accordingly.

The flehmen and mounting behaviors exhibited by

2.3. Sample analysis bulls towards the dummy buffalo applied with raw fae-

cal samples differed significantly between concentrations

The faecal samples (collected in two consecutive of the faeces (P < 0.05). The higher flehmen was observed at

cycles from six female buffaloes) were extracted with 1.0% concentration whereas 0.5% was sufficient in eliciting

dichloromethane (1:1) for the fractionation of volatile mounting behavior (P < 0.05) (Table 2).

K. Karthikeyan et al. / Animal Reproduction Science 138 (2013) 163–167 165

Table 1

Volatile compounds identified in the faecal sample of buffalo during estrous cycle. Samples were collected in duplicate from six animals from three phases

of estrous cycle (Twelve samples from each phase).

a

Retention time Compound Proestrus Estrus Postestrus

√ √

6.183 1,1,3,3,3-Pentachloropropane X √

6.867 3-Methyl phenol √X X

6.933 2-Nonen-1-ol X√ X

7.000 4-Methyl phenol X√√X

8.333 2-Propyl-1-heptanol X√

8.500 3-Pentanamine X X √ √

9.733 Cyclohexane X

√ √

11.533 4,5-Dimethyl -2-undecene X

√ √

12.783 1-Bromo-5- chloropentane X √ √

12.800 Tetradecane X

√ √ √

14.083 2,4-bis cyclohexanol √√

14.367 1,1-Oxybis –decane√√ X

14.500 1-Hexadecanol √ X√

15.000 Hexadecane √√X

15.083 Pentadecane X

√ √ √

15.783 8-Methyl-1-decane √ √

16.283 2,6,10-Trimethyl-dodecane √ X √

16.950 Tetratetracontane √ X

17.650 Trans-verbenol X√√X

17.867 1-Iodo tetradecane √X

18.867 Hexadecanoic acid X√ √X √

20.050 2-Ethyl-1-decanol √ √

20.933 Oxirane √X

22.083 Hexadecanoic acid√√ X X

22.550 Octacosane √ X √

24.433 Heptadecane √ X

√

26.750 2,6,10,14-Tetramethyl heptadecane X

a

The time at which different molecules are retained by the column and then elute from the column in GC–MS analysis.

With reference to the isolated compounds, the highest The combination of the two estrus-specific compounds

flehmen behavior was recorded at 0.5% concentration of produced significant attraction in bull towards the dummy

the two compounds (4-methyl phenol and trans-verbenol) buffalo with repeated increase in flehmen and mounting

separately (P < 0.05). However, 4-methyl phenol was found behaviors (P < 0.05). Interestingly, significantly high level

to be more effective than trans-verbenol in eliciting the of flehmen behavior was observed towards combination

flehmen behavior (P < 0.05). The highest mounting behav- of the two compounds at 0.5% concentration each than all

ior was recorded with 1.0% 4-methyl phenol, and 0.5% other samples/compounds (P < 0.05). However, a moderate

trans-verbenol combined (Table 2). decrease in flehmen and mounting behaviors was recorded

Table 2

Bioactivity of raw sample, and estrus-specific compounds in single and in combination mode at different concentrations.

Name of the sample Duration of Flehmen (in minutes) Number of mounting

0.5% 1.0% 2.0% 0.5% 1.0% 2.0%

d b a c b c

± ± ± ± ± ±

Raw faeces sample 3.81 0.08 4.13 0.05 3.95 0.17 3.88 0.17 3.71 0.14 3.83 0.06

c a a d b b

3.83 ± 0.04 4.1 ± 0.09 3.88 ± 0.6 3.80 ± 0.15 3.75 ± 0.10 3.65 ± 0.11

c c a c a b

3.86 ± 0.20 3.81 ± 0.07 3.78 ± 0.06 3.85 ± 0.16 3.86 ± 0.12 3.55 ± 0.13

b b a a a a

± ± ± ± ± ± 4-Methyl phenol 4.83 0.24 4.33 0.66 4.01 0.09 5.06 0.07 5.23 0.04 4.70 0.07

b a a b b ab

4.83 ± 0.18 4.1 ± 0.08 3.65 ± 0.18 5.03 ± 0.16 4.16 ± 0.07 4.03 ± 0.16

± ab b a ab a ab

5.01 0.18 4.40 ± 0.15 4.26 ± 0.12 5.11 ± 0.18 4.30 ± 0.12 3.58 ± 0.14

c b a b a b

Trans-verbenol 4.38 ± 0.13 4.25 ± 0.08 3.96 ± 0.08 4.53 ± 0.06 4.33 ± 0.07 4.23 ± 0.12

b a a c b ab

4.42 ± 0.12 4.14 ± 0.07 3.68 ± 0.16 4.40 ± 0.15 4.20 ± 0.07 3.92 ± 0.03

b bc a bc a b

4.74 ± 0.19 4.12 ± 0.08 4.22 ± 0.17 4.60 ± 0.17 4.32 ± 0.13 3.42 ± 0.18

a a a a a b

± ± ± ± ± ±

4-Methyl phenol + trans- 6.05 0.10 4.88 0.09 4.06 0.05 4.93 0.08 4.21 0.08 4.33 0.19

a a a a a a

± ±

± ± ± ±

verbenol (4mp + tv) 5.38 0.22 4.44 0.26 4.08 0.10 5.68 0.24 5.00 0.25 4.15 0.08

a a a a a a

5.68 ± 0.12 4.92 ± 0.03 4.22 ± 0.13 5.68 ± 0.31 4.62 ± 0.28 4.10 ± 0.07

e c b d c d

± ± ± ± ± ± Hexadecanoic acid 2.35 0.11 2.46 0.10 2.23 0.06 2.33 0.12 2.46 0.10 2.16 0.07

d b b e c c

(control) 1.36 ± 0.15 1.61 ± 0.20 1.78 ± 0.10 1.45 ± 0.13 1.68 ± 0.12 2.11 ± 0.17

d d b d b c

1.63 ± 0.17 1.26 ± 0.12 1.45 ± 0.05 2.10 ± 0.06 2.13 ± 0.07 1.88 ± 0.06

Values are expressed as Mean ± SEM.

a–e

Within a column, mean without a common superscript differed (P < 0.05).

166 K. Karthikeyan et al. / Animal Reproduction Science 138 (2013) 163–167

with further increase in concentration of the two com- estrus indicator in urine of buffalo (Rajanarayanan and

pounds. The compound used as the control produced lesser Archunan, 2011) and is also an estrus indicator in faeces.

attraction in terms of duration as well as number of both Thus, 4-methyl phenol emerges as a common metabolic

the behaviors (P < 0.05) (Table 2). product excreted for the purpose of chemical communi-

cation between buffaloes. Second, based on the previous

4. Discussion and present evidence it is understood that estrus-specific

signals are excreted via more than one source, so as to

Very many studies have documented the role of diverse effectively aid in attracting the opposite sex. Third, it is

sources of chemo-signals, such as urine, faeces, saliva, vagi- well known that estrus phase is estrogen-dependent, and

nal mucus and specialized glands. In the present study, hence, the compounds excreted during the estrus phase

faecal volatiles are found to be yet another most efficient provide a clue that estrogen should be playing an impor-

source of chemo-signals in buffalo communication. Faeces tant role in secretion of the estrus-specific compounds. This

is one of the major media by which metabolic products has been supported by the observation of Mohamadi et al.

are eliminated from the animal and, therefore, it is not (2011), who evidenced that estrogens play a key role in the

surprising that faeces can potentially convey much infor- regulation of pheromones secretion in goat.

mation about the internal physiology of the animal to Because our sample contains volatiles, particularly two

the external world and thus, provide a source of chemo- compounds which are estrus-specific in nature, we next

signals in many species. It is a point to note that faeces focused on testing these compounds in bull behavior assay

is considered to be a potent source of chemical signals to confirm them as estrus-specific and to use them as reli-

in rats (Lee and Moltz, 1985), pigs (Morrow-Tesch and able estrus indicators. In the context of behavior assay, bulls

McGlone, 1990), lizards (Lopez et al., 1998) and blackbucks exhibited two behaviors i.e., flehmen and mounting, which

(Rajagopal et al., 2011) which all support the present opin- are important in assessing bull’s sexual desire towards

ion. the estrus-specific faecal sample and estrus-specific syn-

Twenty seven compounds have been detected with thetic compounds. This is in accordance with the previous

consistent variation among themselves throughout the reports that flehmen behavior is higher towards estrus-

three phases. This is in accordance with our earlier report specific samples in goat, elephant and buffalo (Pugh, 2002;

that bovine has significant variation in volatile compounds Sukumar, 2003; Archunan and Ramesh Kumar, 2012). But

in its faeces across estrous cycle (Sankar and Archunan, the level of attraction varies according to the nature and

2008). A comparison of the compounds between the concentration of the compounds, individually or in combi-

three phases revealed that two compounds, 4-methyl nation, as in the previous evidence where bulls exhibited

phenol and trans-verbenol, are present only during the differential expression of flehmen and mounting behaviors

estrus phase, which is interesting. Studies on urine of towards the estrus-specific vaginal fluid prepared at 4 dif-

mare support the present finding that the compound p- ferent concentrations. In the present study, the maximum

and m-cresols (derivatives of methyl phenol) identified flehmen and mounting behaviors were recorded towards

during the estrus phase of mare as an ovulation indicating the combined 4mp + tv (0.5%) and individual 4-methyl phe-

marker (Mozuraitis et al., 2012). Incidentally, 4-methyl nol (1.0%), respectively. Further, there was no significant

phenol binds with the olfactory receptors in Drosophila difference between 1.0% and 2.0% concentration in elic-

melanogaster (Shiraiwa, 2008) and this indicates the pos- iting response towards all the samples (except positive

sibility of 4-methyl phenol playing role as a pheromone. control) in flehmen behavior and this implies that these

The second compound, trans-verbenol, has been found to concentrations can be considered as the olfactory threshold

be involved in pheromonal communication in bark beetles level.

(Romon et al., 2007; Zhang et al., 2008). Trans-verbenol,

which is estrus-specific, has already been reported as

5. Conclusion

a sex attractant in insects (Pitman et al., 1968). An

online database (pherobase, http://www.pherobase.com/

Faeces would serve as a chemo-signal in inducing

database/compound/compounds-detail-trans-verbenol.

behavioral response in conspecifics. In this study estrus-

php) also evidences that most of the insects use trans-

specific chemical cues were identified in the faeces of

verbenol in their chemo-communication system. Thus, the

female buffalo; the bull behavior assay substantiates the

two compounds appearing in the faeces only during the

role of these chemical signals, particularly, 4-methyl phe-

estrus phase would be helpful in communicating the status

nol and trans-verbenol, in buffalo communication. We

of estrus to the bull. This is in accordance with earlier

therefore, propose the two compounds as estrus indicators

reports which suggest that small to large mammals release

in buffalo, which would be better as markers of estrus in a

specific compounds in the faeces during estrus, which

non-invasive manner.

may help the bull to perceive the estrus state effectively

(Rekwot et al., 2001). Of note among the compounds,

cyclohexane is present during proestrus and estrus phases, Acknowledgements

and we suggest that this may be an indicator of initiation

of follicular development and ovulation, since it is totally We thank Prof. M. A. Akbarsha, Director & Chair,

absent during postestrus. Mahathma Gandhi- Dorenkamp Center, Bharathidasan

There are three major outcomes of the present study. University, for the encouragement and the critical reading

First, 4-methyl phenol which was earlier shown to be an of the manuscript. We acknowledge the financial support

K. Karthikeyan et al. / Animal Reproduction Science 138 (2013) 163–167 167

from DBT, UGC-SAP & Non-SAP Programmes, and DST- Mozuraitis, R., Buda, V., Kutra, J., Borg-Karlson, A.K., 2012. p- and m-

Cresols emitted from estrous urine are reliable volatile chemical

PURSE, Government of India.

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Pitman, G.B., Vite, J.P., Kinzer, G.W., Fentiman Jun, A.F., 1968. Bark beetle

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Available online at www.sciencedirect.com

Theriogenology 76 (2011) 1676–1683 www.theriojournal.com

Increased squalene concentrations in the clitoral gland during the estrous cycle in rats: An estrus-indicating scent mark? Shanmugam Achiramana,b, Govindaraju Archunanb,*, Bethunaicken Abiramia, Palanivel Kokilavania, Udhayaraj Suriyakalaaa, Devaraj SankarGanesha, Soundararajan Kamalakkannanb, Soundarapandian Kannanc, Yoshiaki Habarad, Ramaiyan Sankare a Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli -620 024, Tamilnadu, India b Centre for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli -620 024, Tamilnadu, India c Department of Zoology, Bharathiar University, Coimbatore 641 046, Tamilnadu, India d Laboratory of Physiology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan e Department of Animal Behavior and Physiology, School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, Tamilnadu, India Received 17 May 2011; received in revised form 24 June 2011; accepted 25 June 2011

Abstract 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. © 2011 Elsevier Inc. All rights reserved.

Keywords: Pheromone; Clitoral gland; Squalene; Estrous cycle; Rat

1. Introduction alert males that she is ready for mating [1]; olfactory signals during estrus are used to communicate recep- The estrous cycle consists of four phases, viz. proes- tivity [2–4]. Due to their secretive nature, rodents de- trus, estrus, metestrus, and diestrus. Ovulation occurs pend solely on chemosignals for sexual attraction. In during late proestrus to early estrus. The female has to that regard, sources of chemosignals include urine, fae- ces, vaginal fluids, and scent gland secretions [5,6]. * Corresponding author. Tel.: 00-91-431-2407040. Regarding the latter, the preputial gland has an impor- E-mail address: [email protected] (G. Archunan). tant role in scent marking behavior in rodents. This

0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.06.033 Author's personal copy

S. Achiraman et al. / Theriogenology 76 (2011) 1676–1683 1677 gland has been studied in many mammals, including To understand chemo-signals in female rats and primates, rodents, carnivores, proboscids, and ungu- their importance, it is necessary that the chemicals lates [7–9]. excreted are collected, analyzed qualitatively and quan- The preputial gland is formed of modified sebaceous titatively, and evaluated for bioactivity. Therefore, the acini and is located on either side of the penis or clitoris objectives of the present study were to: in males and females, respectively [10]. In females, this i) confirm the presence of squalene in the clitoral gland is also called the clitoral gland. It is believed that gland among phases in the reproductive cycle; the secretion of this gland is released in two ways: i) via ii) determine the concentration of squalene in the cli- the skin, so it can be smeared on stones, mud, wood, toral gland during the estrous cycle; etc., during animal movement, to leave scent marks for iii) demonstrate the role of squalene as a chemosignal, counterparts to perceive; and ii) into voided urine. adopting a bioactivity-guided assay; and Scent gland secretion is a rich source of lipids involved iv) evaluate the role of the olfactory-Vomeronasal Or- in communicating information regarding species, sex, gan (VNO) in the perception of estrus and non- dominance status [11–13], as well as for attracting estrus odors. conspecifics [7,14]. It is noteworthy that male rats were more attracted by odors derived from the clitoral gland during estrus than those derived from this gland in other 2. Materials and methods phases of the reproductive cycle [15]. 2.1. Animals Although scent marks are particularly well-suited for providing reliable signals regarding estrus, it is Twenty adult female Wistar rats, Rattus norvegicus, assumed that their main function is in defense [16]. 12–15 wk old, were maintained in polypropylene cages ϫ ϫ Recently, Zhang et al [15] reported a high level of (40 25 15 cm) with 2 cm of rice husk lining the squalene in the preputial gland when compared to other bottom as bedding material. The rats were housed sep- glands. In contrast, Natynczuke et al [9] reported the arately in rooms under laboratory conditions (12 h L:D) without males, and fed pelleted feed (Sai Durga Feeds presence of a homologous series of aliphatic acids and Ltd., Bangalore, India) and water ad libitum, in accor- their ethyl esters (squalene) in the clitoral gland of dance with guidelines for animal care by the Institu- brown rats. It is well known that physiological and tional Animal Ethics Committee (IAEC), Bharathi- biochemical profiles of females, particularly lipids, dasan University, Tiruchirappalli, India. vary according to reproductive status. For example, body odors of female rats contained estrogen-depen- 2.2. Determination of the estrous cycle dent pheromones that attracted males [17,18]. In fact, The estrous cycle was assessed in adult female rats squalene was reported to be involved in biosynthesis of (n ϭ 6), by observing vaginal smears, which were pre- estrogen during cholesterol breakdown. Therefore, pro- pared and examined under light microscopy for propor- duction of squalene in the clitoral gland presumably tions of the three main cell types, viz. leukocytes, epithe- varied according to phases of the estrous cycle, al- lial cells, and cornified cells [27]. At proestrus, the smear though this has apparently not been studied. has a large proportion of nucleated epithelial cells; at There are several chemosensory systems that can me- estrus it primarily consists of cornified cells; at metestrus, diate social recognition in animals through odor detection it consists of an almost equal proportion of leukocytes and [19]. In rodents, the main olfactory and vomeronasal sys- cornified cells; and at diestrus, it consists predominantly tem appeared to be of primary importance in this regard of leukocytes. This assessment was done, by the same [20,21]. It is generally believed that the Main Olfactory technician, between 07:00 and 08:00 each morning. System (MOS) was responsible for odor discrimination, whereas the Accessory Olfactory System (AOS) was pri- 2.3. Method of extract preparation marily involved in pheromonal communication [22–25]. Adult female rats were killed by cervical dislocation The MOS perceives primarily relatively volatile chemical under light diethyl ether anesthesia. The clitoral gland substances (which are air-borne), whereas the AOS deals was dissected out, placed in dichloromethane, thor- with non-volatile molecules [26]. Since squalene is semi- oughly homogenized for ϳ5 min under ice-cold (0 °C) volatile, it would be pertinent to determine which of the conditions in a sterile glass homogenizer, and centri- olfactory systems (i.e., MOS or AOS) perceives squalene fuged at 4472 ϫ g for 2 min. The supernatant was if it acts as chemosignal. immediately filtered through a pre-equilibrated silica Author's personal copy

1678 S. Achiraman et al. / Theriogenology 76 (2011) 1676–1683 gel column (50 cm). The filtrate was collected in clean responder (i.e., test animal) was released. On the right glass vials sealed with an airtight, screw-type cap and arm, various samples were kept, i.e., proestrus, estrus, stored at Ϫ20 °C until further analysis. metestrus, diestrus, squalene, and dichloromethane (control). In the left arm, distilled water was kept as 2.4. Chemical identification by Gas Chromatography control. An odor preference test was conducted; the and Mass Spectrometry (GC-MS) frequency and duration of visits made by the responders The GC-MS analyses were conducted in a QP-5000 to various samples were recorded separately [30]. (Schimadzu, Kyoto, Japan). An aliquot (2 ␮L) of ex- Frequency of visit: Number of visits made by the tract was injected into the GC-MS on a 30 m glass responder towards right or left arm (5 min/test). capillary column, with a film thickness of 0.25 m (30 m ϫ Duration of visit: Time spent by the responder in 0.2 mm id, coated with UCON HB 2000) using the investigation near the Petri dish containing the samples following temperature program: initial oven tempera- (10 min/test). ture 40 °C for 4 min, increasing to 250 °C at a rate of Grooming behavior: Number of grooming acts made 15 °C every 10 min. The gas chromatography facility by the responder near the Petri dish containing the (Schimadzu GC 15A) was equipped with FID detector samples (10 min/test). connected to an integrator. The area under each peak was used for quantification. The detection accuracy was 2.6. Statistical analysis ϳ 1 ng/peak. The relative concentration of each com- Data were compiled using SPSS statistical software ponent was recorded as percentage of the ion current. (Version 10; SPSS Inc., Chicago, IL, USA) and subjected The GC-MS was operated under computer control at 70 to two-way ANOVA, with post-hoc comparison using eV, using ammonia as reagent gas at 95 ev chemical Duncan’s Multiple Range Test. ionization mode. Identification of unknown compounds was made by probability-based matching, using the computer library within the NICT 12 system. 3. Results 2.5. Experimental design 3.1. GC-MS analysis 2.5.1. Test animals The GC-MS profile (Table 1 and Figs. 1–4) were Eighteen adult male Wistar rats were equally allo- representative compounds obtained from the clitoral gland cated into three groups, intact males (Group I), VNO- during the four phases of the estrous cycle. Twenty three ablated (Group II), and ZnSO4-irrigated (Group III). peaks were recorded during one cycle. The chemical con- stituents identified in the samples were alkanes, alde- 2.5.2. VNO-ablation and ZnSO4-irrigation hydes, acids, and amides, with a predominance of alkanes. The VNO-ablation was carried out in the males as Visual examination of all chromatograms revealed a con- previously described [28]. Rats were anesthetized un- sistent qualitative difference in the chemical profiles der light dose of diethyl ether and 5% ZnSO4 solution among the various phases in the estrous cycle; there were was administrated intranasally, to rupture the main ol- only 14 compounds present throughout the estrous cycle factory epithelial cells [29]. (Table 1 and Fig. 1). 2.5.3. Odor Preference Test (OPT) The proestrus sample contained 15 peaks and estrus Odor preferences of intact, VNO-ablated and sample contained 14 peaks in which the relative abun- dance (RA) of squalene was 100. The subsequent me- ZnSO4–irrigated male rats were tested. Six sexually experienced male individuals were used for each test, testrus and diestrus rats showed 14 compounds, among without repetition. The animals were exposed only which RA of squalene was 24 at metestrus and 48 at once to each sample. Fresh samples were used for each diestrus. trial and the samples were placed in an open Petri dish. Comparison of the compounds, thus identified, across The behavioral study was carried out using a “Y” maze the estrous cycle revealed a remarkable variation. For apparatus made of tin sheet, and consisting of three instance, among the 23 compounds, only one compound, arms. Each arm was ϳ80 cm long and 15 cm wide. tricosane, was detected only during proestrus and estrus. The lateral sides were closed with glass plates, whereas However, another compound, 9 ehtyl-9-heptyl octadec- the top portion was covered with wire mesh. In this ane, was detected only during the estrus phase. Com- apparatus, feed and water were available ad libitum.In pounds such as octacosane and octadecanoic acid were the middle of the common space of the apparatus, the present only in the metestrus sample, whereas acetamide Author's personal copy

S. Achiraman et al. / Theriogenology 76 (2011) 1676–1683 1679

Table 1 List of compounds identified in the clitoral glands of female rats during the estrous cycle. Peak No. Retention time Compound Chemical class Pro Est Met Die 1 01.45 Undecane Alkane ϩϪϩ Ϫ 2 03.33 2,6-11, trimethyl dodecane Alkane ϩϩϩ ϩ 3 05.78 2 methyl tridecane Alkane ϩϩϩ ϩ 4 08.22 2,6, 11, 15, trimethyl hexa decane Alkane ϩϩϩ ϩ 5 09.70 Trocosane Alkane ϩϩϪ Ϫ 6 10.47 Heneicosane Alkane ϩϩϩ ϩ 7 12.52 Pentatricontane Alkane ϩϩϩ ϩ 8 13.15 Pentacosane Alkane ϩϪϪ Ϫ 9 14.03 Heptacosane Alkane ϩϪϪ ϩ 10 14.39 Octacosane Alkane ϪϪϩ Ϫ 11 14.87 0-nitrobenzaldehyde Aldehyde ϩϪϩ Ϫ 12 15.69 Tetratetraconatne Alkane ϩϩϪ ϩ 13 16.10 Demecolcine ϪϪϪ ϩ 14 16.48 3-methyl eicosane Alkane ϩϩϩ ϩ 15 17.05 Betulin ϪϪϪ ϩ 16 17.25 3 ethyl 5-(2ethylbuthyl octadecane) Alkane ϩϪϩ ϩ 17 17.90 9 ehtyl-9-heptyl octadecane Alkane ϪϩϪ Ϫ 18 18.12 Octadecanoic acid Acid ϪϪϩ Ϫ 19 18.52,54 Squalene ϩϩϩ ϩ 20 19.30 Octadecatrienoic acid Acid ϩϩϩ ϩ 21 20.75 Aristolochic acid Acid ϩϪϪ ϩ 22 23.02 Cholic acid Acid ϩϩϩ 23 23.61 Acetamide ϪϪϪ ϩ ϩ, Present; Ϫ,Absent; Pro, Proestrus; Est, Estrus; Met, Metestrus; Die, Diestrus. was found only in the diestrus sample (Table 1 and Figs. 3.2. Number of visits 1–4). Comparison of the RA of compounds also revealed Male rats more frequently visited clitoral gland ex- a remarkable difference across the estrous cycle. Inter- tract at estrus, followed by proestrus, than metestrus estingly, squalene had high intensity at proestrus and and diestrus (Table 2). A similar trend was noticed in highest intensity during estrus, although it was greatly the VNO-ablated rats. Conversely, the ZnSO4-irrigated reduced to below half the intensity at metestrus and rats did not show any variation in the preference to- diestrus phases (Fig. 3 and Fig. 4). Thus, there was a wards the various samples. Intact, VNO-ablated and marked fluctuation in intensity of this compound during the estrous cycle (Figs. 1–4).

Fig. 1. Gas chromatographic profile of the clitoral glands of proestrus Fig. 2. Gas chromatographic profile of the clitoral glands of estrus rats. Squalene was expressed at peak 19. rats. Squalene was expressed at peak 19. Author's personal copy

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Table 2 Frequency of visits made by three groups of male responder rats towards preputial gland extracts of female rats collected in various phases of the estrous cycle, as well as squalene and dichloromethane (DCM).

Sample Intact rat VNO ZnSO4 ablated rat irrigated rat Proestrus 5.6 Ϯ 0.2b 4.4 Ϯ 0.6b 1.3 Ϯ 0.5b Estrus 7.4 Ϯ 1.2a 6.9 Ϯ 0.8a 1.9 Ϯ 0.2b Metestrus 2.0 Ϯ 0.3d 3.1 Ϯ 0.4c 2.3 Ϯ 1.2a Diestrus 3.4 Ϯ 0.4c 3.7 Ϯ 0.7c 1.8 Ϯ 0.2b Squalene 3.3 Ϯ 0.9c 2.0 Ϯ 0.3d 2.6 Ϯ 0.3b DCM (Control) 0.2 Ϯ 0.0e 0.3 Ϯ 0.2e 0.3 Ϯ 0.1c Values are expressed as mean Ϯ SEM of six animals, with 5 min/test. a-e Within a column, means without a common superscript differed Fig. 3. Gas chromatographic profile of the clitoral glands of metestrus (P Ͻ 0.05). rats. Squalene was expressed at peak 19.

that of the intact rat, but the response was not equal to that ZnSO4-irrigated rats did not make frequent visits to- for the estrus samples (Tables 2–4). Interestingly, the wards squalene, as compared to that of clitoral gland intact rats which visited the squalene samples spent more extract. However, none of the male responders exhib- time in investigating the estrus sample; however, only a ited any preference towards dichloromethane. The fre- few rats visited the squalene sample. Although the VNO- quency of visits by the three groups of male responders ablated rats did not spend much time in the sample area, varied significantly (Table 1). There was a main effect they spent the most time near squalene. The ZnSO4- for frequency of visits by all three responders (P Ͼ irrigated rats showed a similar trend as intact rats. The 0.001) and the two way interaction was also significant time spent by all the three responders towards various (F ϭ 29.883; d.f. 10.54; P Ͻ 0.001). samples differed with a two way interaction (P Ͻ 0.001). 3.3. Duration of visits 3.4. Self-grooming The duration of visits of rats which visited the sample All responders from all three groups exhibited alone was recorded (Table 3). The time spent by the three grooming behavior when exposed to the various sam- sets of responders varied significantly (Tables 2–4). The ples (Table 4). Intact males spent more time in self- intact rats spent more time in investigating the proestrus grooming activity during exposure to proestrus and and estrus samples than the other samples. Even though estrus samples than to the other samples. It was note- the VNO-ablated rats visited the sample area, the time worthy that intact rats spent more time and exhibited spent by them was less when compared to the intact rat. In more grooming activity when exposed to squalene than contrast, the ZnSO4- irrigated rats had a trend similar to

Table 3 Duration of visits (in seconds/10 min/test) made by the responders by the three groups of male responders towards female preputial glands at various stages of the rat cycle, as well as squalene.

Samples Intact rat VNO ablated ZnSO4 rat irrigated rat Proestrus 428.3 Ϯ 104.3b 128.5 Ϯ 47.2c 289.6 Ϯ 87.4c Estrus 537.4 Ϯ 142.3a 152.5 Ϯ 62.7b 421.5 Ϯ 134.2a Metestrus 189.3 Ϯ 83.9d 148.5 Ϯ 105.3b 243.9 Ϯ 64.3c Diestrus 162.6 Ϯ 68.8de 174.7 Ϯ 63.3ab 126.3 Ϯ 82.4e Squalene 289.7 Ϯ 89.7c 197.5 Ϯ 127.9a 387.3 Ϯ 105.5b DCM 147.5 Ϯ 88.6f 201.3 Ϯ 12.4a 159.3 Ϯ 55.8d (Control) Values are expressed as mean Ϯ SEM of six animals. Fig. 4. Gas chromatographic profile of the clitoral glands of diestrus a-f Within a column, means without a common superscript differed rats. Squalene was expressed at peak 19. (P Ͻ 0.05). Author's personal copy

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Table 4 duction by the clitoral gland during the estrous cycle. Grooming behavior exhibited by the responders by the three Squalene has also been reported to be a major compo- groups of male responders towards female preputial gland of various stages of rat and squalene (in sec/10 min/test). nent of the pheromonal scent marks of saddleback tamarins (Saguinus fuscicollis), a new world primate Samples Intact rat VNO ablated ZnSO4 rat irrigated rat [36]. Furthermore, squalene acted as male pheromone in the giant panda, Aliuropoda melanoleuca [37]. Proestrus 45.32 Ϯ 12.75c 18.53 Ϯ 7.29c 67.86 Ϯ 13.46b Estrus 47.42 Ϯ 7.84b 23.65 Ϯ 10.32ab 59.38 Ϯ 9.53c Interestingly, squalene was present in sweat samples of Ϯ e Ϯ c Ϯ Metestrus 29.43 8.61 18.93 6.67 36.49 14.53d female humans; its concentration was much lower in pre- Diestrus 37.87 Ϯ 10.62d 19.43 Ϯ 12.65c 28.57 Ϯ 8.04e menstrual than menstruating females. Therefore, squalene Squalene 63.66 Ϯ 4.76a 14.5 Ϯ 5.84d 72.23 Ϯ 6.45a might be an indicator of ovulation and act as a potential Ϯ f Ϯ a Ϯ DCM 18.53 8.45 25.37 12.83 29.64 8.94e pheromone to attract men [38]. As proestrus and estrus (control) rats are believed to emit pheromonal signals, any com- Values are expressed as mean Ϯ SEM of six animals. a-f Within a column, means without a common superscript differed pound expressed at a high level specifically during these (P Ͻ 0.05). phases may be a behaviorally important chemical signals that attracts males. Similarly, it was previously reported that urinary volatiles differed only quantitatively, but not other samples. The VNO-ablated rats spent more time qualitatively, during the estrous cycle of mice [39]. How- at the estrus sample than all other samples. However, they spent significantly less time in grooming when ever, a specific volatile compound was found in the mouse compared to intact rats. Similar to intact rats, the (1- Iodo 2-methyl undecane) [4] and elephant (7-dodecen 1-yl acetate) [2]. Since squalene is already known to be ZnSO4-irrigated rats also engaged in grooming activity when exposed to squalene. involved in chemo-signaling, the surge of squalene at ovulation is a candidate for indicating estrus and scent marking. In the present study, squalene concentrations 4. Discussion increased during proestrus and peaked during estrus. It is The GC-MS profiles of clitoral gland during the four well known that glandular chemo-signals are involved in phases of the female reproductive cycle contained up to 23 effective communication [9,10] and, hence, the phero- volatile compounds. Several of the compounds identified monal role of the compounds identified in the glandular in this study were similar to volatiles identified in urine samples in the present study were confirmed with behav- from the Californian mouse [31] and Swiss mouse [4,32]. ioral analysis. The number seemed relatively high compared to the vola- The male rats made frequent visits towards the vial tiles identified in the preputial gland of house rat, Rattus consisting of clitoral glands of estrous females and spent rattus, in which the presence of farnesol was suggested to considerable time near to the sample, suggesting its role in act as a female attractant [12]. However, farnesol was not sex attraction. Similarly, male golden hamsters were at- detected in the clitoral gland in the present study. In the tracted to the urinary volatiles of females [40]. It is well present study, squalene was detected in the clitoral gland, known that odors of estrus females are usually more consistent with a previous report [9]. In contrast, Zhang et attractive to males than those in other phases [40,41]. The al [15] reported the presence of both farnesol and squalene attraction to estrus odor by male individuals is presumably in female and male rats, respectively, and suggested that due to the presence of specific chemical signals present in squalene was a female-attractant produced in excess by estrus female scent sources. the male. It was also suggested that sebaceous and clitoral Although the precise functions of the two olfactory glands had high levels of squalene, cholesterol esters, and systems are not known, there is some evidence indicat- provitamin D (7-dehydrocholestero1) [33]. In the present ing they have distinct roles [42]. Although a single study, squalene was present during all phases of the es- system is frequently used for testing, Baxi et al [43] trous cycle, with significantly greater relative abundance suggested that investigators should use a two-by-two during proestrus and estrus, and lesser abundance during design for testing both the olfactory as well as vomer- metesrus and diestrus. onasal system to draw a definite conclusion. In the Estradiol concentrations increase and peak during present study, both intact and VNO-ablated rats made proestrus and estrus [34]. Fatty acid metabolism is frequent visits to the late proestrus and estrus samples, controlled by sex hormones [35]; therefore, it seemed whereas they were not attracted by squalene. This may plausible to propose that changes in hormone concen- be due to the semi-volatile nature of this compound. trations may be the basis of variation in squalene pro- However, ZnSO4-irrigated males were unable to detect Author's personal copy

1682 S. Achiraman et al. / Theriogenology 76 (2011) 1676–1683 glandular extract as well as squalene, due to the loss of In conclusion, squalene, a semi-volatile chemical ability to detect volatile odors. In spiny mice, ZnSO4- present in the clitoral gland of the female, reached peak irrigation eliminated the preference for conspecific’s concentrations during estrus, and acted as a chemosig- odor, suggesting that the MOS was necessary for indi- nal. In males, the olfactory-vomeronasal system had a vidual discrimination [44]. Conversely, the extent of synergistic role in the perception of this chemosignal. time spent by male rats had different trends. Intact male and ZnSO4-irrigated rats had a preference for the cli- Acknowledgments toral gland of late proestrus and estrus females, whereas the VNO-ablated rats did not show any significant re- We thank Professor M.A. Akbarsha, Director & Chair, Mahathma Gandhi Dorenkamp Center, Bhara- sponse. Interestingly, the intact and ZnSO4-irrigated male rats had similar responses towards squalene, in thidasan University for his encouragement and critical contrast to VNO-ablated rats, which failed to perceive reading of the manuscript. This work was partially squalene. It is noteworthy that interruption of the ol- supported by a grant from CSIR (SA). We gratefully factory system, particularly the VNO, disrupted repro- acknowledge financial support received from UGC, ductive activities in some species of rodents [40,45]. UGC-SAP, Non-SAP, DBT, DST-Fast Track and Rodents use self-grooming behavior to encounter CSIR, New Delhi, Government of India. Both GA and their conspecific’s scent marks [46]. In the present YH acknowledge visiting fellowships awarded by study, intact males spent considerable time in self- Heiwa-Nakajima Foundation, Japan. grooming activity during exposure to clitoral gland extracts of late proestrus and estrus phases, as well as to References squalene. 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Biochemical Analysis of Female Mice Urine with Reference to Endocrine Function: A Key Tool for Estrus Detection

Shanmugam Achiraman1,2*, Govindaraju Archunan2, Devaraj SankarGanesh1, Thangavel Rajagopal2,3, Rengasamy Lakshminarayanan Rengarajan2, Palanivel Kokilavani1, Soundararajan Kamalakkannan2,4 and Soundarapandian Kannan5

1Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, India 2Centre for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, India 3Department of Biotechnology, Ayya Nadar Janaki Ammal College, Sivakasi 626 124, Tamilnadu, India 4University of Lausanne, Institute of Physiology, Rue du Bugnon 7, 1005 Lausanne, Switzerland 5Department of Zoology, Bharathiar University, Coimbatore 641 046, Tamilnadu, India

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 pres- ent study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lip- ids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogen- treated 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 pres- ent during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. How- ever, 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.

Key words: protein, carbohydrate, lipid, fatty acids, ovariectomy, estrus, mouse

nals to the external environment through urine, saliva, feces, INTRODUCTION and specialized scent glands (Vandenberg, 1999). Probably Female reproductive physiology is a complex process the concentration of these signals may vary according to the and the macromolecules produced from females depend phase as well as status of the animal. Among the various upon hormonal regulation and physiological status. Commu- sources in communication, urine is one of the chief sources nicating at the time of ovulation and the co-ordination of involved in the signal-receptor system (Bimova et al., 2009). sexual behavior appears to be an important task for the suc- The male has more attraction to estrus female than non- cessful fertilization. The odors produced from females may estrus due to the presence of some specific compounds vary according to the reproductive phase (Michael, 1975; (Dominic, 1991; Archunan, 2009). O’Connell et al., 1981). All mammals excrete chemical sig- Some animals have permanent proteinurea due to the occurrence of abundant concentrations of protein, which * Corresponding author. Phone: +91-431-2407088; Fax : +91-431-2407045; serve as chemical signals among the animals and not as an E-mail: [email protected] indicator of disease. The Major Urinary Protein (MUP) pres- doi:10.2108/zsj.28.600 ent in mice is a member of the lipocalin protein family, which Biochemical Analysis in Mice Urine 601 has a central cavity lining with tryptophan residues (Robertson sodium (Abbot Lab Ltd., Mumbai, India) diluted in 0.9% saline and et al., 1998). The cavity has the capacity to bind with volatile injected by IP. Ovariectomy was performed as described by Kannan chemical signals delivered into the external environment and Archunan (1998). Six ovariectomized mice were allowed to (Novotny et al., 1999). The expression of this protein is com- recover for approximately two weeks and then treated with estradiol paratively weaker in females than males (Finlayson et al., 0.1% gel (Sandrena Organon, Holland) over the flank region approximately 8–10 mg, once daily for 15 days after ovariectomy. 1968), but this does not mean that MUP is of minor impor- tance for females. However, at the estrus phase the protein Estrous cycle determination is found at maximum concentration during estrous cycle The estrous cycle was observed in all mice through the obser- (Stopka et al., 2007). It clearly shows that the sex attractants vation of vaginal smear (Archunan and Dominic, 1991). The smears produced in estrus females may be necessarily transported were analyzed under light microscopy for the observation of three by this protein. types of cells: leukocytes, eptithelial and cornified cells. A proestrus The pheromones and their carrier proteins are involved smear consists of predominance of nucleated cells while the estrus in the chemical communication apart from the lipid fractions. smear exhibit only cornified cells. The metestrus smear consists of Brahmachary (1996) identified lipids from the marking fluid equal proportion of leukocytes and cornified cells and diestrus of the tiger and considered that it could be responsible for smear consists of predominance of leukocytes. The assessment was done by a single technician at 07:00–08:00 a.m. the characteristic odor of the animal. Fatty acid levels vary from one trimester to trimester in pregnant, as well as in lac- Sample collection tating, bovine urine (Rameshkumar and Archunan, 2001; The urine sample was collected at all the reproductive phases Rameshkumar et al., 2003). Mucopolysaccharide layer from all the groups. While collecting the urine sample a gentle serves as chemical signal in Asian elephant (Rasmussen, abdominal massage was applied and the animal was held over a 1998). Rameshkumar et al. (2000) revealed that the feeding watch glass. The samples were pooled and screened through habit and the breakdown of sugar may lead to production of cheesecloth or nylon mesh (16–120 μm) at the time of collection many metabolites that in turn may possibly act as phero- and stored at –20°C to be analyzed within a week. monal signals. The composition of the excretory products may vary Biochemical constituents The biochemical constituents such as proteins, (Lowry et al., according to the various reproductive phases. Males are 1951) carbohydrates, (Dubois et al., 1956) and lipids, (Folch et al., more attractive to females in estrus than in all other phases 1957) were analyzed in all the urine samples. (Dominic, 1991) due to the release of putative chemical sig- nals. It is therefore strongly believed that the biochemical Fatty acid profile constituents of urine vary during estrous cycle. However, Five ml of urine sample was taken and mixed with saponifica- these profiles have not yet been evaluated. Further, the role tion reagent. The tubes were tightly closed and kept for 30 minutes of estrogen in the regulation of biochemical constituents in at 60°C in a water bath. 2 ml of methylation reagent was added to urine is not known. each tube and kept again in the water bath at 80°C for 20 minutes. The identification of the biochemical constituents of the Finally, a sufficient amount of extraction solvent (200 ml hexane + urine in female mice during various reproductive phases will 200 ml of diethyl ether) was added to each tube, and then closed tightly, and shaken thoroughly for 10 minutes. About 2/3 of the aid us in elucidating the functional aspects of these com- organic phase (upper layer) containing the fatty acid methyl esters pounds in the estrus phase. Hence, the present investigation were transferred into screw cap glass vials. From each vial 1 μl of was carried out to evaluate the biochemical constituents such the fatty acid methyl ester (FAME) was injected into the Gas Chro- as proteins, carbohydrates, lipids, and fatty acids, and hor- matography (GC) column (Miller and Berger, 1985). mones such as estradiol and progesterone during various reproductive phases among the normal, prepubertal, and Blood collection and serum separation ovariectomized and estrogen-treated ovariectomized mice so The blood was collected in clean microcentrifuge tubes from as to use these constituents as an indicator of estrus. mice at various reproductive phases by puncturing the jugular vein. The serum was separated from blood by centrifugation at 3000 rpm MATERIALS AND METHODS for 10 minutes and stored at 20°C for hormone assays.

Test animals Hormone assay The subjects of this study were adult female mice 24 ± 03.0 g The hormones such as estradiol and progesterone were esti- (± S.D.) of Swiss strain. They were 8–10 weeks old and separately mated in the serum sample of various reproductive phases by solid housed in polypropylene cages with 2 cm of rice husk as bedding phase radioimmunoassay using standard kits (Alpco Diagnostics). material. The animals were maintained under controlled tempera- ture, light and dark cycle (12 hr/12 hr), provided with pellet food Statistical analysis (Sai Durga Feeds and Foods, Bangalore, India) and water ad The results obtained in the present investigation were sub- libitum. jected to statistical analysis using SPSS statistical software (version 11.0) and Two-way ANOVA with Duncans test. Experimental procedure Twenty-four adult female mice were divided into three groups: RESULTS normal (12 animals), ovariectomized (six animals) and ovariecto- Protein levels mized and estrogen-treated (six animals). The prepubertal group consisted of six animals. The urinary protein content significantly differed across the different reproductive phases of female. The estrus urine Ovariectomy and estrogen treatment contained the highest level of protein, followed by proestrus Twelve female mice were anesthetized with 60 mg/kg pentothal phase. The metestrus and diestrus urine contained more or 602 S. Achiraman et al. less similar concentrations of protein. The ovariectomized Duncan’s post hoc analysis). mice excreted the lowest level of protein of all the phases. On estrogen treatment the ovariectomized mice excreted a Lipid levels higher level of protein. The prepubertal mice also excreted A considerable amount of lipids was excreted and the a notable concentration of protein (Table 1: Duncan’s post- concentration significantly varied among the phases of hoc analysis). estrous cycle (F = 72.647, d.f = 9, 49; P < 0.001). The high- est level of lipid was excreted during the estrus followed by Carbohydrate levels the proestrus phase of the estrous cycle. The metestrus and The carbohydrate content in urine significantly varied diestrus animals excreted a lower concentration of lipids in across various reproductive phases of female mice (F = urine. Removal of ovaries in mice caused a significant 4.660, d.f = 9, 49; P < 0.001). A significantly higher level of reduction in urinary lipids, in fact estrogen treatment to ova- total carbohydrate was found in metestrus phases than in riectomized mice enhanced the urinary lipids excretion other phases of estrous cycle. The lowest level of urinary (Table 1: Duncan’s post hoc analysis). carbohydrates was noted during the proestrus phase. Further, the ovariectomized mice exhibited a reduced level Fatty acid profile during the estrous cycle of total carbohydrates and the estrogen replacement caused The GC analysis showed that free fatty acids in urine a notable increase in carbohydrate excretion (Table 1: varied qualitatively and quantitatively across the estrous cycle. Twenty different fatty acids were detected in the female urine sample; among them six fatty acids, namely Table 1. Biochemical constituents in the urine of female mice dur- ing various reproductive phases. Values are expressed in Mean ± lauric, tridecanoic, myristic, palmitic, palmitoleic and oleic SE. Means in the same vertical column that are not marked with the acid were present in all four phases of the estrous cycle. But same superscript (alphabets) letters are significantly different at α = the concentration of all the six fatty acids varied consider- 0.5 level (Duncan’s test). ably across the estrous cycle and three fatty acids namely tridecanoic, palmitic and oleic acid were notably higher in Biochemical constituents mg/ml S.No. Urine samples the estrus followed by proestrus phase. Pentadecanoic acid Protein Carbohydrates Lipids was present in higher concentration in estrus phase, absent 1 Proestrus 3.74 ± 0.13a 67.6 ± 4.22b 221.4 ± 16.04b in proestrus and little quantity was detected during diestrus 2 Estrus 4.11 ± 0.05a 75.6 ± 5.40ab 279.8 ± 06.59a and metestrus phase. The nondecanoic acid was present 3 Metestrus 2.71 ± 0.31b 76.4 ± 8.08ab 157.8 ± 09.52d during proestrus and estrus, which was totally absent during 4 Diestrus 2.50 ± 0.22b 63.0 ± 5.02b 156.0 ± 07.72d metestrus and diestrus phase. Henecosanoic acid was pres- 5 Ovx 0.81 ± 0.08d 66.8 ± 5.26c 073.4 ± 08.34e ent during all phases except estrus (Table 2). 6Ovx + 2.52 ± 0.19b 81.0 ± 6.01ab 195.2 ± 06.37c estrogen Fatty acids in ovariectomized mice with estrogen treat- treatment ment 7 Prepubertal 1.73 ± 0.17c 45.2 ± 5.37d 070.8 ± 05.68f The urine of ovariectomized group contained six fatty

Table 2. Fatty acid profile of female mice urine. Values are expressed in Mean ± SE. Means in the same vertical column that are not marked with the same superscript (alphabets) letters are significantly different at α = 0.5 level (Duncan’s test).

Fatty acids mg/g of lipid Name of the Fatty acids Ovx + estrogen Proestrus Estrus Metestrus Diestrus Ovx Prepubertal treatment Lauric acid 0.44 ± 0.08ab 1.38 ± 0.03a 0.69 ± 0.02b 0.65 ± 0.01b – – 0.04 ± 0.02c Tridecanoic acid 14.8 ± 0.22b 16.3 ± 0.05a 7.71 ± 0.07d 0.05 ± 0.07fg 2.34 ± 0.02e 9.48 ± 0.57c 0.53 ± 0.02f Myristic acid 0.03 ± 0.07b 0.04 ± 0.03b 0.02 ± 0.01b 0.02 ± 0.01b – – 2.07 ± 0.65a Pentadeccanoid acid 6.04 ± 0.16a 5.98 ± 0.18ab 0.00 ± 0.02d 2.03 ± 0.14b – 0.72 ± 0.01c – Palmitic acid 6.12 ± 0.18b 12.2 ± 0.25a 2.43 ± 0.11c 0.01 ± 0.02de 1.11 ± 0.35cd 5.62 ± 0.21b 0.02 ± 0.01d Heptadecanoic acid – 0.08 ± 0.01b – 0.01 ± 0.04c 0.04 ± 0.02b 0.24 ± 0.01a – Stearic acid 2.02 ± 0.07a 0.04 ± 0.09b 0.07 ± 0.01b –– – – Nondecanoic acid 0.02 ± 0.01b 0.02 ± 0.02b – – 0.67 ± 0.01ab 0.87 ± 0.05a 0.87 ± 0.05a Henecosanoic acid 0.03 ± 0.02bc – 0.36 ± 0.03a 0.01 ± 0.02b – 0.23 ± 0.01ab – Behenic acid – – – – 0.60 ± 0.02b 3.35 ± 0.52a 0.43 ± 0.02b Tricosanoic acid – – – 1.07 ± 0.05a ––– Lignoceric acid – 0.01 ± 0.05c – 0.04 ± 0.01b – 0.08 ± 0.02b 3.05 ± 0.15a Palmitoleic acid 0.08 ± 0.02a 0.09 ± 0.01a 0.07 ± 0.02a 0.01 ± 0.01ab ––– Oleic acid 6.17 ± 1.05bc 10.1 ± 1.85a 3.11 ± 0.30c 0.05 ± 0.01d 3.06 ± 0.35c 6.03 ± 1.25bc 7.32 ± 0.56b Mysteric acid – 0.39 ± 0.02b 0.04 ± 0.01c 1.05 ± 0.25a – 0.04 ± 0.02c – Linolenic acid – – – 2.36 ± 0.42a 0.05 ± 0.01c 0.74 ± 0.02b 0.34 ± 0.12bc Cislinolenic acid 0.09 ± 0.01a –––– – – Elaidic acid – 2.06 ± 0.05a – – – 0.85 ± 0.01b – Eicosanoic acid 0.22 ± 0.02a –––– – – Biochemical Analysis in Mice Urine 603 acids (tridecanoic, palmitic, nondecanoic, behenic, oleic, Progesterone and linolenic acid). Among these oleic and behenic acids The progesterone concentration was also found to be were present in higher concentrations. Interestingly, behenic vary significantly across the estrous cycle (F = 77.051, d.f = acid was observed in the urine of ovariectomized mice, 5, 29; P < 0.001). A significantly higher level of progesterone which was found to be absent in estrous cycle of normal was noted during the metestrus phase, followed by diestrus females. Further, tridecanoic and palimitic acids, which were phase, than in the proestrus and estrus period. Progester- present in higher concentrations in normal females, but were one was higher during estrus phase when compared to found reduced drastically in the urine with complete absence proestrus phase of the estrous cycle. Similarly, the ovariec- of pentadecanoic, henecosanoic, lignoceric, mysteric and tomized mice with estrogen treatment showed a lower level elaidic acids in ovariectomized mice. The estrogen treat- of progesterone. The prepubertals revealed considerably a ment showed an increase in quality and quantity of fatty higher level of progesterone than that of ovariectomized acids and presence of twelve fatty acids was noted in the mice treated with estrogen (Table 3: Duncan’s posthoc anal- estrogen-treated group. In addition, tridecanoic, oleic and ysis). palmitic acids were excreted in higher concentrations when DISCUSSION compared to ovariectomized females. Further, behenic acid was present only in ovariectomized mice, whereas absent in The present study established the variation of biochem- estrogen-treated ovariectomized mice (Table 2). ical constituents in urine among the estrous cycle of female. In general, mouse urine contains proteins usually at higher Prepubertals concentrations in male than in female (Achiraman and The prepubertal urine contains nine fatty acids in lower Archunan, 2006). In the present study, a notable amount of concentrations. Among them, the lignoceric and myristic protein was present in the female urine of estrus phase. acids were present at notable levels. The elaidic acid shown Since, some estrus specific pheromones are released to be present in estrus female urine was absent in prepuber- through the urine, the high level of protein may be required tal urine. However, the concentration of tridecanoic acid was to function as carriers for the ligands and convey the chem- found high in proestrus and estrus, as compared to that of ical signal. It has been reported that excretion of Major diestrus. The prepubertal urine contained a higher concen- Urinary Protein in urine acts as pheromone carrier (Beynon tration of myristic acid when compared to other phases of and Hurst, 2004). It is therefore possible to suggest that the estrous cycle (Table 2). higher level of protein excreted from female at estrus is use- ful to communicate her conspecifics. Further, the male Hormone analysis shows specific behavior and spent more time when exposed Estradiol to female urine, but stopped by a partition (Amstislavskaya The estradiol concentration varied significantly across and Popova, 2004) and it suggested that the proteins spe- the estrous cycle of female mice (F = 66.663, d.f = 6, 34; P < cific to estrus phase exclusively involved in attraction of 0.001). A higher level of estradiol concentration was male. The nature of the protein is not identified in the pres- reported during the estrus phase than in all other phases in ent study, but the appearance of a higher level of protein the cycle. Next to the estrus phase, the estradiol concentra- during estrus seems to be noteworthy. tion was significantly higher in proestrus phase, when com- It has been reported that the nature of feeding habits pared to other phases. The lowest level of estrogen was may have a major impact on excretion of bio-molecules noted in ovariectomized mice. In fact, estradiol replacement through urine (Galef, 1994; Rameshkumar et al., 2000). This in ovariectomized mice showed an elevation in estradiol. In may be the reason for a considerable release of carbohy- the case of prepubertal animals, a significantly lower level of drates in urine. For instance, alteration in diet changes the estradiol was noticed as compared to that of all other urinary odours of guinea pig and mice (Beauchamp, 1976; phases with the exception of ovariectomized mice (Table 3: Schellinck et al., 1997). It is also observed that a minor com- Duncan’s post-hoc analysis). ponent of Major Urinary Protein complex of the house mouse revealed a glycoprotein containing N-linked oligosac- charide (Ziegler et al., 1993). In this regard, the insignificant Table 3. Hormonal profiles in female mice of various reproductive changes in carbohydrate content during the estrous cycle ± phases. Values are expressed in Mean SE. Means in the same are probably due to the changes in hormonal level. Ma et al. vertical column that are not marked with the same superscript (1995) identified that the high level of circulating estrogen (alphabets) letters are significantly different at = 0.5 level (Duncan’s test). stimulates the breakdown of glycogen and other materials into glucose in mammals, which will be utilized for energy in Hormone titers cells during the time of ovulation. Thus the reduction of car- S.No. Blood Samples Estrogen Progesterone bohydrates in blood in turn released through urine. pg/ml ng/ml Lipids in the urine are not certainly a waste product. 1 Proestrus 2.03 ± 0.30b 01.03 ± 0.19c Reports have come out regarding tiger, in which the urinary 2 Estrus 2.68 ± 1.37a 02.84 ± 0.21b lipids can be used as fixatives (Poddar-Sarkar, 1996). The 3 Metestrus 0.88 ± 0.16c 13.05 ± 1.32a mechanism of fixing volatile molecules by “lipids” has been 4 Diestrus 0.95 ± 0.08c 03.10 ± 0.05b observed in Tulpaia belangeri (shrew) (Stralendroff, 1987). 5 Ovx 0.31 ± 0.04d 0.31 ± 0.05c Our earlier study also indicated that lipids play a crucial role 6 Ovx + estrogen treatment 1.12 ± 0.20c 0.34 ± 0.25c in the sexual attraction in rat, and vary considerably on basis 7 Prepubertal 0.41 ± 0.04d 0.54 ± 0.06c of physiological status (Kannan et al., 1998). Due to their 604 S. Achiraman et al. distinctive properties, even the presence of small amount of level during the ovulatory phase (Day, 2004). In accordance lipids in urine describes the status of the animals (Burger et with this view in the present study, progesterone levels were al., 2008). Despite their key protective effect, the present higher in the metestrus phase declining towards the diestrus results revealed that the endocrinological status would have phase. Progesterone release occurs in an episodic fashion a major impact on the excretion of lipids, and hence, the in correlation with LH release, during mid and late luteal greater expression noted in the estrus phase than that of phase (Eisthen et al., 1987). Similarly, a surge of progester- other phases of estrous cycle. one was observed in the late metestrus and early diestrus Several lines of evidence suggest that the urinary fatty in laboratory mice (DeLeon et al., 1990). However, the role acids act in individual identification as well as sex attractant of progesterone and estradiol was not obviously reported, in certain mammalian species (Mattina et al., 1991; Poddar- and the level of progesterone prior to the raise of estrogen Sarkar and Brahmachary, 1999). Similarly, in the present may help to regulate the occurrence and intensity of estrus study as many as 20 fatty acids were detected. Among behavior (Hansel and Convey, 1983). Furthermore, the them, six (namely, lauric, tridecanoic, myristic, palmitic, dynamic property of the hormones revealed in the present palmitoleic and oleic acid) were present in all four phases of study has added knowledge not only on estrous cycle regu- the estrous cycle, and three fatty acids viz., tridecanoic, lation, but also seems to serve as the control switch for palmitic and oleic acid were found in higher concentrations biomolecular excretion through urine. only during estrus followed by proestrus phase, suggesting CONCLUSION that they may be involved in sexual attraction. Consistent with this, some volatile fatty acids namely, acetic, propionic, The present findings revealed that the protein and lipid and butanoic acids, varied during menstrual cycle in level was significantly higher during estrus than in other humans, and these fatty acids reached a peak near the time phases. Further, the estrogen replacement also showed a of ovulation in humans (Michael, 1975). similar trend. Likewise, specific fatty acids such as palmitic Interestingly, ovariectomized mice show some deletion and oleic acids were excreted in higher amount at estrus as of fatty acids, and the estrogen replacement showed the well as ovariectomized with estrogen treated groups. Thus, reappearance of some fatty acids such as pentadecanoic, these findings suggested that the excretion of biomolecules henecosanoic, lignoceric, mysteric and elaidic which are in urine is linked to the hormonal status of the animals. strongly suggested to be estrogen specific. This interference Hence, the appearance of biochemical constituents can be was further supported by Rameshkumar et al. (2000) considered as estrus indicators. Since, urine is the potent reported that the tridecanoic, pentadecanoic and myristic source of communication, this technology can be used as a acid are present predominantly in ovulatory phase and these non-invasive method in estrus detection. fatty acids may act as estrus specific in bovine urine. It is known that the ovulatory phase contains higher levels of ACKNOWLEDGEMENTS estrogen and the notion of present report increases the pos- We thank Professor Chellam Balasundaram, Bharathidasan sibility of role of estrogen with the fatty acid excretion. It is University for critical reading of the manuscript. We gratefully additionally hypothesized that fatty acids along with other acknowledge the funding agencies such as UGC, DST, UGC-SAP, biochemical components of urine play a prominent role in UGC-Non SAP, and CSIR, Government of India, for their financial opposite sex attraction. support. S. A. acknowledges DST-Fast Track scheme for the Young The palmitic and oleic acid were seen in higher concen- Scientist award. G. A. acknowledges Heiwa Nakajima Foundation, trations during proestrus, estrus phases and in ovariecto- Japan for the award of visiting fellowship. mized plus estrogen-treated females, which clearly indicates REFERENCES that these compounds may also be estrogen-dependent. This finding is consistent with the report of Mattina et al. Achiraman S, Archunan G (2006) 1-Iodo-2methylundecane, a puta- (1991), which showed that palmitic acid was excreted in the tive estrus-specific urinary chemo-signal of female mouse (Mus reproductive phase of bobcat urine and is certainly involved musculus). Theriogenology 66: 1913–1920 in sexual attraction of conspecifics. Later, Kannan and Amstislavskaya TG, Popova NK (2004) Female-induced sexual arousal in male mice and rats: behavioral and testosterone Archunan (1999, 2001) reported the presence of palmitic response. 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Neuroscience Letters

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Detection of estrus by male mice: Synergistic role of olfactory–vomeronasal system

Shanmugam Achiraman a,b, Ponnirul Ponmanickam a, Devaraj Sankar Ganesh b, Govindaraju Archunan a,∗

a Center for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India b Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India article info abstract

Article history: In rodents, olfactory pathway comprises two distinct systems viz, the main olfactory and vomeronasal Received 21 January 2010 systems, both differing in anatomy, physiology and function. The precise role of the main olfac- Received in revised form 21 April 2010 tory/vomeronasal system in estrus detection is yet to be explored. Therefore, the present investigation Accepted 21 April 2010 was planned to elucidate the role of main olfactory and vomeronasal system in the estrus discriminat- ing ability of male mice. Female urine samples of proestrus, estrus, metestrus, diestrus, ovarectomized, Keywords: ovarectomized plus estrogen treated and prepubertal mice were used for the present study. In addition, MOS the urine from intact, castrated and castrated with testosterone treated mice was also tested for odour AOS VNO preference by male mice. The male responders were categorized into three groups namely (a) normal, Estrus detection (b) ZnSO4-irrigated and (c) vomeronasal organ (VNO)-ablated. The behavioural responses such as fre- Urine quency and duration of visits to urine samples were carried out in a Y-maze apparatus to assess odour Mice preference. The normal mice displayed more frequent visits to estrus urine samples than to non-estrus

samples. In contrast, ZnSO4-irrigated mice showed significant reduction in the frequency of visits towards estrus urine, whereas, the vomeronasal (VNO)-ablated mice did not show any noticeable preference. With regard to the duration of visits the VNO-ablated mice showed significant reduction in visiting time when

compared to ZnSO4-irrigated mice. This finding indicated that the main olfactory system (MOS) was involved primarily in the attraction from a distance, while the VNO played a major role in close prox- imity (pre-copulatory behaviour). The males spent less time with the urine of same-sex; however, the response was higher with castrated male urine which was reduced on testosterone treatment indicating that a specific odour in intact male causes aversive behaviour in male. This study provides support to the fact that volatile compounds could also be perceived by VNO, probably when the main olfactory system is in functional state. The study implies that the olfactory–vomeronasal system plays a synergistic role in the detection of estrus. © 2010 Elsevier Ireland Ltd. All rights reserved.

The reproductive cycle of the female mammals excluding primates Several chemosensory systems that can mediate social recog- is called estrous cycle which is characterized as proestrus, estrus, nition in animals through odour detection, but in rodents, the metestrus and diestrus. The ovulation occurs during the end of main olfactory and vomeronasal system seem to be of primary proestrus to the beginning of estrus stage [17]. Female individ- importance [31,44]. The olfactory and vomeronasal organ main- uals advertise their readiness to mate through urinary olfactory tain separate central projections through several synapses [16]. signals during estrus phase [4,38,40]. Experimental evidence sug- The consistent presence of separate projections suggests that the gests that many rodents are capable of following and detecting two systems have different roles in physiology and behaviour [7] subtle differences in odour trail laid by conspecifics. It is believed but the main olfactory system (MOS) is responsible for odour that almost all male mammals can detect estrus on the basis of discrimination, while the accessory olfactory system (AOS) is pri- female odours [4,21]. Urine odours of rodents contain information marily involved in pheromonal communication [45,47]. The MOS about the species, gender, sexual state (estrus), dominance status, primarily handles relatively volatile chemical substances that are maternal state and the individual identity [3,10,12,48]. air-borne, whereas, the AOS deals with non-volatile molecule [16]. Surprisingly, the mouse vomeronasal organ also detects odorants [27,41].

∗ In mice, the role of MOS is evaluated using ZnSO4-irrigation and Corresponding author. Tel.: +91 431 2407040; fax: +91 431 2407045. the AOS by VNO ablation [5]. In spiny mice, zinc sulphate irriga- E-mail address: [email protected] (G. Archunan).

0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.04.051 S. Achiraman et al. / Neuroscience Letters 477 (2010) 144–148 145 tion eliminates the preferences for conspecific’s odour, suggesting that vomeronasal system is not necessary for individual discrimi- nation [22,29]. In golden hamster, the removal of VNOs disrupts the mating behaviours of males [22] and the ablation of VNO interferes with the pheromone reception [13]. Zinc sulphate treatment on the olfactory mucosa of male hamster eliminates attraction to vaginal secretions [37]. Although the above studies indicate the importance of main and accessory olfactory systems in the perception of dif- ferent odours, it is still difficult to know which system is involved in estrus detection. The removal of gonads dramatically reduces the urinary chemo- signals in mice and steroidal administration restores this ability in both the sexes [3]. In this regard the male preference to urine of different reproductive state (intact female), ovarectomized female and ovarectomized female plus estrogen treatment needs to be tested. Further, the response of male towards the same-sex urine and urine of castrated male and castrated male with testosterone treatment is equally important to analyze. In order to under- stand the mode by which signals are used to co-ordinate sexual behaviour, a study on the changes in the signals and their responses throughout the reproductive stages is required. Therefore, the present study was designed to: Fig. 1. Haematoxylin and eosin stained section from mice cranium (OE, olfactory epithelium; VN, vomeronasal organ). (i) evaluate the role of olfactory–vomeronasal system involved in the perception of estrus odour. Thirty adult female mice at various reproductive phases (ii) know the endocrine dependency of chemo-signals released by (proestrus, estrus, metestrus, diestrus, ovarectomized, ovarec- female and male mice. tomized with estrogen treated females, normal males, castrated and castrated-testosterone treated and prepubertal mice) were Thirty regularly cycling female virgin mice, Mus muscu- used as donors. The stages of estrous cycle were confirmed by lus [24 ± 03.6 g] of 8–10 weeks old, 12 prepubertal females microscopic observation of vaginal smear [6]. The urine was col- [13 ± 0.05 g] of 3–4 weeks and 20 intact males [27 ± 06.0 g] of 10–12 lected by gentle abdominal massages while the mouse was held weeks old, were used as donors. Another 15 adult males were over a watch glass. Samples were screened through cheese cloth or used as test animal for odour preference test. They were reared nylon mesh (16–120 ␮m). Fresh urine samples were used for each on pelleted food (Sai Durga Feeds Pvt. Ltd., Bangalore) and water behavioural analysis. ◦ ad libitum, in laboratory condition (12 h D:L; 35 ± 5 C). The bedding Odour preferences by normal, ZnSO4-irrigated and vomeronasal material was changed before every odour preference test. The pro- organ (VNO)-ablated male mice were tested. Five sexually expe- cedural aspects of this study were approved by Institutional Animal rienced male individuals were used for each test. The animals Ethics Committee (IAEC), India. were exposed only once to each urine sample. Fresh urine sam- Castrated and oophorectomized [23] mice were maintained ples were used for each trial. The samples were placed in glass under normal laboratory conditions for about 2 weeks. phials (50 mm × 17 mm), the top lids of which had small holes for Testosterone enanthate (Testoviron; Schering, Germany) was the release of volatile compounds from urine into the surrounding diluted with arachis oil and administered intramuscularly to environment. The behaviour study was carried out with the help gonadectomized mice, at a dose of 0.1 mg/day/male for 2 weeks. of “Y” maze apparatus consisting of three arms [42]. Odour prefer- 1 ml of Testoviron contained 250 mg of testosterone enanthate. ence test was carried out in which the frequency and duration of Oophorectomized mice were applied with estradiol (0.1%) gel (San- visits made by the responders to the urine samples were recorded drena; Organon, Holland) over the flank region for 2 weeks. for each animal. VNO ablation [39] was carried out in five males. Five adult male mice were anesthetized and administered intra-nasally with 5% Frequency of visit: Number of visits made by the responder zinc sulphate solution using [25] gauge hypodermic needle, cut to towards right or left arm (5 min/test). a length of 4 mm and ground smooth at the tip [5,43]. Duration of visit: Time spent by the responder in investigation For histological study, intact mice were sacrificed by cervical glass phials (in s, 5 min/test). dislocation under a mild dose of diethyl ether anesthesia, and decapitated. The mandible and all soft tissues, with the exception Data were analyzed with two-way ANOVA with Duncan’s post of the mucosa on the oral surface of the hard palate, were removed. hoc analysis to compare the behaviour of males in response to vari- The cranium was decalcified in an acid path, which consisted of 10% ous urinary compounds and t-test was also carried out. All analyses formic acid and 6% paraformaldehyde in 0.1 M phosphate buffer were conducted with SPSS package version 11 (Chicago, IL, USA). [11]. Decalcification of mice heads were performed in three acid The histoarchitecture of cranial sections of one mouse showed baths each for about 15 h. When sodium oxalate mixed with a test the appearance of both the olfactory epithelium and the quantity of the formic acid failed to produce a calcium oxalate vomeronasal organ (Fig. 1). precipitate, the decalcified cranium was transferred to 5% sodium The responders (normal, ZnSO4-irrigated mice and VNO-ablated sulphate for about 15 h, then washed in running tap water. mice) showed preference towards urinary samples. The frequency Tissues were dehydrated and embedded in paraffin. For observ- of visits by the three categories of responders towards various ing vomeronasal and nasal mucosae, the decalcified cranium was urine samples differed significantly (P < 0.001) and the two-way sectioned horizontally at a thickness of 10 ␮m. Each section was interactions were also significant (P < 0.001). The visits of normal mounted on a glass slide and stained with haematoxylin and eosin. male to estrus urine were more frequent than to all other sam- The slides are viewed at 40×. ples. The frequency of visits towards the urine samples significantly 146 S. Achiraman et al. / Neuroscience Letters 477 (2010) 144–148

Table 1 Frequency of visit made by the male responders towards various urine samples of mice (5 min/test).

S. No. Urine samples Test Animals

Normal VNO-ablated ZnSO4-irrigated 1 Distilled water (control) 2.00 ± 0.31g 1.40 ± 0.24e 1.60 ± 0.24b 2 Proestrus 6.40 ± 0.67c 5.40 ± 0.51b 2.20 ± 0.37ab 3 Estrus 11.60 ± 0.92a 6.40 ± 0.67b* 2.80 ± 0.40ab**,a 4 Metestrus 4.40 ± 0.67de 4.60 ± 0.51bc 1.40 ± 0.60b 5 Diestrus 5.40 ± 0.51cd 4.40 ± 0.24bc 1.80 ± 0.20b 6 OVX 3.80 ± 0.37de 4.80 ± 0.37bc 1.40 ± 0.40b 7 OVX + estrogen treatment 9.20 ± 0.37b 9.20 ± 0.86a 2.40 ± 0.81ab 8 Male 2.60 ± 0.40fg 5.60 ± 1.36b 1.60 ± 0.51b 9 Castrated male 5.40 ± 0.67cd 6.00 ± 1.22b 2.00 ± 0.70ab 10 Castrated male + testosterone treatment 2.80 ± 0.48ef 4.00 ± 0.70bc 2.60 ± 0.74ab 11 Prepubertal 1.80 ± 0.80g 3.00 ± 0.44cd 3.80 ± 1.01a

Values are expressed in mean ± SE. Those means in the same vertical column that are not marked with the same letters are significantly different at ˛ = 0.05 level (Duncan’s test). * t-Test between normal versus VNO-ablated at P < 0.001. ** t-Test between normal versus ZnSO4-irrigated at P < 0.001. a t-Test between VNO-ablated versus ZnSO4 at P < 0.001.

(P < 0.001) varied among the responders (normal, VNO-ablated and the male mouse is able to discriminate various urinary odours of zinc sulphate-irrigated mice). The VNO-ablated mice were able to its own species indicating the possibility for opposite sex urine discriminate the different urine odours significantly and showed to be involved in the attraction of mates. The results also sug- more preference to the urine of ovarectomized mice treated with gested that a specific change in female odours during the time estrogen, than to the estrus urine of intact mice. The ZnSO4- of ovulation could create greater interest in males towards con- irrigated mice exhibited significantly (P < 0.001) lesser number of specifics. The observed increase in attractiveness of males to female visits to urine samples, than by normal and VNO-ablated mice. Male odours during estrus was consistent with earlier observations in mice showed significantly (P < 0.001) less preference for the con- other species [1,8,22,26,46,49]. In a pilot study, male meadow voles trol sample and the prepubertal urine (Table 1: Duncan’s post hoc were found to spend more time in investigating the arm of a Y- analysis). maze that contained materials of an estrus female [15]. The normal The efficacy of the VNO lesions was assessed by the time spent male exhibited their preference towards estrus urine by frequently by VNO-ablated mice near various urine samples. The duration of visiting and spending more time in investigating the estrus sam- visits by all the three sets of responders was found significantly ple. This suggests that the estrus urine may contain estrus-specific vary (P < 0.001) and the two-way interaction was also significant pheromones. Our recent findings provide further evidence in sup- (P < 0.001). The VNO ablation significantly disrupted the male pref- port of this suggestion that a compound, 1-iodo 2-methyl undecane erence towards estrus: the responder spent equal time near all was found to be a putative estrus-specific chemo-signal in the urine the urine samples. In contrast, the ZnSO4-irrigation did not disrupt of female mouse [4]. estrus detection. The normal male mice showed significantly less Although the precise function of the two olfactory systems is attraction towards the same-sex urine. The urine of castrated plus not known, there is some evidence suggesting the different roles testosterone treated mice showed effects similar to those of normal of both the systems. The main olfactory system seems to be the animal, in fact, there was a slight reduction in attraction which is primary one attracting of males to female odours, from a distance. statistically insignificant (Table 2: Duncan’s post hoc analysis). In this investigation the frequency of visits of the ZnSO4-irrigated The study revealed that the female mouse could change its mice towards urine samples was affected, while the duration of odour dramatically during estrus period of estrous cycle as indi- visit was unaltered. This finding suggests that the main olfactory cated by male preference. Exposure of female urine to the male system is probably involved only in attraction, whereas, other mice increases their investigating ability. The results indicated that behavioural activities may depend on the vomeronasal system.

Table 2 Duration of visit made by the male responders towards various urine samples of mice (in s/5 min/test).

S. No. Urine samples Test animals

Normal VNO-ablated ZnSO4-irrigated 1 Distilled water (control) 34.8 ± 3.24f 30.0 ± 2.00d 33.6 ± 1.72f 2 Proestrus 107.6 ± 3.96b 32.4 ± 3.69cd 71.0 ± 4.21ab 3 Estrus 135.6 ± 3.93a 58.2 ± 4.28a* 75.2 ± 3.50a**,a 4 Metestrus 75.6 ± 7.36cd 60.4 ± 9.44a 76.6 ± 5.85a 5 Diestrus 76.8 ± 20.51c 47.4 ± 5.02ab 56.8 ± 5.92bc 6 OVX 48.2 ± 3.44ef 40.2 ± 3.70bc 58.8 ± 4.35bc 7 OVX + estrogen treatment 77.6 ± 10.06c 38.6 ± 3.95bc 69.0 ± 4.11ab 8 Male 43.0 ± 6.58ef 48.2 ± 5.45ab 42.2 ± 3.27ef 9 Castrated male 56.2 ± 2.45de 59.4 ± 2.42a 48.4 ± 9.30cd 10 Castrated male + testosterone treatment 37.8 ± 5.55f 45.0 ± 5.03ab 43.2 ± 3.05de 11 Prepubertal 50.4 ± 7.77ef 51.0 ± 3.20ab 57.8 ± 2.59bc

Values are expressed in mean ± SE. Those means in the same vertical column that are not marked with the same letters are significantly different at ˛ = 0.05 level (Duncan’s test). * t-Test between Normal versus VNO-ablated at P < 0.001. ** t-Test between Normal versus ZnSO4-irrigated at P < 0.001. a t-Test between VNO-ablated versus ZnSO4 at P < 0.001. S. Achiraman et al. / Neuroscience Letters 477 (2010) 144–148 147

Results of the present study are consistent with the previous volatile constituents: namely 3-cyclohexene-1-methanol (I); 3- observation, wherein the male hamster the MOS rather than AOS aminotriazole (II); 4-ethylphenol (III); 3-ethyl-2,7-dimethyloctane mediates attraction to female odours from a distance [37]. Further, (IV); 1-iodoundecane (V) [2]. Among these compounds, only 3- MOS seems to be important in the discrimination and recognition ethyl-2,7-dimethyl octane (IV) reappeared following testosterone of conspecifics. For example, zinc sulphate incapacitation exhibits treatment into castrated males [3]. Therefore, the presence of this a drastic reduction in nipple searching behaviour in rabbit pups compound may be the reason for the aversion in male mice. [20]. Previous studies have demonstrated that interruption of an Our results conclude that even after the destruction of olfac- olfactory system, particularly the VNO, disrupts the reproductive tory epithelium, the mice, with the help of AOS, are still able to activities in some species of rodents [22,28]. Likewise, in this study detect estrus, even though, the frequency of the visit is reduced also the VNO ablation has disrupted the investigatory ability of male to some extent indicating the MOS may be involved only from responders towards female urine. In fact, the ZnSO4-irrigated and a distance. Whereas the AOS controls the specific behaviour like normal males spent more time in investigating estrus urine sample. estrus detection, it probably controls general purpose behaviours Similarly, in male hamsters, vomeronasal lesion causes a decrease like attraction, sniffing, etc. Taken together, the present findings in copulatory behaviour and the removal of the entire olfactory bulb support and provide circumstantial evidence that the main and eliminates the mating behaviour completely [22,34]. Present study accessory olfactory system show a synergistic effect in detecting thus clearly indicated that the ZnSO4-ablated mice could detect estrus. The male mouse uses (i) MOS to detect estrus through estrus and suggested the involvement of accessory olfactory bulb volatiles released by female even from a distance and (ii) VNO in estrus detection through perception of volatile compounds. to confirm the estrus by follow-up pre-copulatory behaviours like The vomeronasal organ is thought to play a crucial role in the sniffing, licking and mounting indicating that accessory olfactory perception of pheromones, which affect instinctive reproductive vomeronasal system has a synergetic role in detecting estrus and as well as social behaviours in mammals [32]. Removal of VNO in mating process. decreases the investigating ability of male mice towards estrus urine sample, thus suggesting the positive role of VNO in pre- Acknowledgements copulatory behaviour. It has been previously reported that removal of the VNO decreases male sexual behaviour such as ano-genital We thank Dr. Chellam Balasundaram, Emeritus Professor, sniffing, vocalizations, mounts and copulations [14,30,36,47] and in Bharathidasan University, for critical reading of the manuscript. pregnancy block, puberty acceleration and lordosis behaviour [16]. This work was supported by grants from Council of Scientific and Even though several reports, including the present study, have Industrial Research, DST-FAST Track, DST-FIST, UGC, UGC-SAP and indicated that the removal of VNO decreases male behaviour. UGC non-SAP, Government of India. GA acknowledges Heiwa Naka- Other studies show that VNO does not have role in some of jima Foundation, Japan, for the award of visiting fellowship. the pheromonal activities [15]. For instance, the removal of VNO in male guinea pig does not eliminate preferential investigation References of female urine [9]. 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