International Journal of Current Biotechnology

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1 Raman Suthanthirakannan and Kamatchi Rameshkumar, Identification of Fatty Acid Profiles in Bovine (Bos Indicus) Saliva using Gas Chromatography in Context of Estrus Detection, Int.J.Curr.Biotechnol., 2014, 2(2):6-10. International Journal of Current Biotechnology ISSN: Journal Homepage : Identification of Fatty Acid Profiles in Bovine (Bos Indicus) Saliva using Gas Chromatography in Context of Estrus Detection Raman Suthanthirakannan and Kamatchi Rameshkumar* Pheromone Research Lab, Post Graduate and Research Dept.of Zoology, Rajah Serfoji Govt. College (Autonomous), Thanjavur , Tamil Nadu. A R T I C L E I N F O A B S T R A C T Article History: Received 22 January 2014 Received in revised form 24 February 2014 Accepted 26 February 2014 Available online 28 February 2014 Key words: Gas chromatography, fatty acids, estrus, reproduction. Introduction India is a country having a variety of indigenous breed of cattle with wide genetic diversity. Many of the indigenous breeds are undergoing genetic degradation due to indiscriminate cross breeding. As a result many indigenous breed with many desirable characters are threatened with extinction (Ramesha, 2001). Likewise in Tamilnadu there are few well known draught breeds of indigenous cattle such as Kangayam, Umblachery, Bargur etc. Umblachery is medium size draught type cattle and it is strong and active with compact body and short legs (Kumarasamy et al., 2008). The reproductive performance of this breed is poor when compare to other breeds. Hence artificial insemination is the outset view to improve the reproduction of this breed. The low rate of success in artificial insemination is due to the lack of effective estrus detection. Animals communicate information concerning reproduction to conspecifics in order to co-ordinate reproductive activities (Doty, 1976; Izard, 1983). In many vertebrates urine, faeces, vaginal secretion as well as endocrine granular secretion can act as sex attractant and serve to facilitate the location and recognition of mates (Aron, 1979; Dominic, 1991). Saliva is one among *Corresponding author. address: rameshnila1@rediffmail.com Mobile no: Improving estrus detection in female bovine is required to ensure successful mating, here saliva which is a complex body fluid containing several different electrolytes which is acting as pheromone cues for olfactory communications. Earlier reports revealed that free fatty acids (FFA) seems to function as pheromones in mammals and also it can be present in urine, saliva, faeces and sebaceous glands may serve as the basis for individual distinction. Based on the above information, the present work is planned to identify the fatty acid profiles in the different reproductive phases by gas chromatography (GC) with flame ionization detector. In the present study 55 fatty acids were identified among them 15 belongs to saturated fatty acids (SFAs), 24 are monounsaturated fatty acids (MUFAs) and 16 are polyunsaturated fatty acids (PUFAs). Among the fatty acid identified, palmitic acid, myristic acid, stearic acid, nonadecyclic acid, oleic acid, trans-7-palmitoleic acid, eicosapentaenoic acid and arachidonic acid were showed maximum levels during estrus phase. Further, many reports informed that the concentration of certain salivary fatty acids are seemed to be high in ovulatory phase of human female saliva, which indicating that the saliva may be considered as a parameter for the detection of ovulation. Hence, the present study concluded that these estrus fatty acids produced from the female saliva may act as an indicator for estrus detection. them which is more popular in recent decades as a medium for the measurement of numerous biomolecules (Streckfus and Bigler, 2002). The greatest advantage, of saliva is readily accessible and easy for biochemical investigations. The animal may produce several volatile compounds from different sources but the influence of pheromone activity may due to single or combination of compounds. For instance, among the number of volatile substances identified in the urine of male mice (Achiraman and Archunan, 2005) a few volatiles such as 2-sec- buthylthiazole, 3, 4- dehydro-exobrevicomin, hydroxyl-6 methyl- 3-heptanone (Novotny, et al., 1999) have exhibited primer pheromonal activity. A broad array of biochemicals is now monitored in saliva (Lawrence, 2002 and Tabak, 2001). Lipids and fatty acids were extensively studied in saliva for biomarker research. It is well known that the fatty acids and their derivatives are involved in the olfactory communication. For instance, FFA seems to function as pheromones in a number of mammals including tiger (Bramhachary et al., 1991, 1992) and African Cheetah (Poddar-Sarkar and Bramhachary, 1997). Recently hormone analysis in female saliva was used for fertility monitoring (Read, 1993). However, findings indicate that these assays may be useful beyond the study of reproductive concerns. Hence, saliva is considered as the best non- invasive source for biochemical study including fatty acid analysis (Freundl et al., 1996). The saliva is a very good source of hormones Volume 2; Issue 2; Feb, 2014 Int.J.Curr.Biotechnol. 6

2 and fatty acids and their levels changed in accordance with the menstrual cycle (Flynn and Lynch, 1974). Further, it is reported that, the volatile fatty acids present in mice estrus urine can act as a biomarker (Achiraman, et al., 2011). It is of interest that similar changes may occur in salivary fatty acids constituents of women during various reproductive stages, which may also act as sex arousal for males of the same species (Martin et al., 2005). Thus the present work is aimed to detect the fatty acid profiles in bovine saliva during different reproductive phases like prepubertal, estrus, pregnant and lactation. Material and Methods Animals and Sample collection With the help of veterinarian the saliva samples were collected from six cows of Bos indicus (Umblachery) maintained in the District Livestock farm, Korukkai, Thiruthuraipoondi, Tamilnadu. The samples were pooled together in order to avoid individual variations and used for GC analysis. The stages of the estrous cycles were carefully examined for two to three consecutive cycles. The animals were fed with standard diet and water ad libitum. Fatty acid profile The lipid was extracted from the collected saliva by using chloroform and methanol (1:1) and the total lipid was estimated by Folch et al., (1957). Five ml of sample was taken and mixed with saponification reagent. The tubes were tightly closed and kept for 30 minutes at 60 C in a water bath. 2 ml of methylation reagent was added to each tube and kept again in the water bath at 80 C for 20 minutes. Finally, a sufficient amount of extraction solvent (200 ml hexane ml of diethyl ether) was added to each tube, and then closed tightly, and shaken thoroughly for 10 minutes. About 2/3 of the organic phase (upper layer) containing the fatty acid methyl esters (FAME) were transferred into screw cap glass vials. From each vial 1 ìl of the FAME was injected into the GC column (Miller and Berger, 1985). Statistical Analysis The obtained data were computed by ANOVA test followed by the post hoc Duncan s test. All the data analyses were significant at P < 0.05 (Zar, 1982). Results The extracts prepared from the saliva were analyzed and the results were shown in Table fatty acids were present in different reproductive phases. In that, 15 of them belong to saturated fatty acids, 24 are MUFAs and 16 are PUFAs. Majority of the fatty acids were significantly higher in estrus phase when compared to prepubertal, pregnant and lactation. The SFAs palmitic acid, stearic acid and mystric acid were present in high concentration during estrus phase when compared to other fatty acid. Among the 24 MUFAs identified oleic acid was present in high concentration and PUFAs like eicosapentaenoic acid and erachidonic acid were also present in high concentration in estrus phase when compared to other stages. In the comparison, SFAs present significantly higher to MUFAs and PUFAs (Table 2). Discussion The fatty acid analysis in bovine saliva showed that the palmitic acid is present in maximum level during estrus phase followed by stearic acid and mystric acid. Similar kind of report had been published by Mattina, et al., (1991), which revealed that palmitic acid was excreted in reproductive phase of bobcat urine and is certainly involved in sexual attraction. It is also well documented that male preferred the saliva of estrus female to that of non-estrus females in Mongolian gerbels (Block, et al., 1981). Further, the oleic acid is present in high amount during estrus when compared to other fatty acids. Similar kind of report had been published by Michael, (1961) that oleic acid was found in higher concentration during estrous cycle followed by proestrus and they suggested that oleic acid may be involved in sexual attraction. Stearic acid and oleic acid are primarily responsible for differences in fat softness (Smith et al., 1998) and the concentrations of these fatty acids in subcutaneous adipose tissue are affected by breed, sex, age, and nutrition (Clemens, et al., 1973, Eichhorn et al., 1986; Huerta-Leidenz et al., 1993; Mandell, et al., 1998). Further the results showed that next to oleic acid, trans-9- palmitoleic acid, trans-5 myristoleic acid and cis-7- octadecenoic acid were present in high level in estrus phase which is similar to the result of Prabu and Rameshkumar, (2013). They revealed that 26 different types of MUFAs like Cis-7-myristoleic acid, Cis-6- pendecenoic acid, Cis-6-palmitoleic acid, trans-9- palmitoleic acid, trans-7-palmitoleic acid, oleic acid, nonadecenoic acid and Cis-7-docosenoic acid etc., were detected during estrous cycle. Among these, Cis-5- heptadecenoicacid, Cis-7-heptadecenoic acid, oleic acid and nonadecenoic acid are mostly present in higher concentration in estrus phase when compared to proestrus and post estrus phases. PUFAs like arachidonic acid, eicosapentaenoic acid and docosa hexaenoic acid were present in high level in estrus when compared to other fatty acids in prepubertal, pregnant and lactation. Over all the estrus phase has significant level of fatty acids when compared to other phases, in that SFAs were found to be significantly present when compared to MUFA and PUFA. Rameshkumar and Archunan, (2006), reported that different type of fatty acids in bovine urine like palmitic acid, myristic acid and pentadecanoic acid were in higher amount than other fatty acids. Alagendran, et al., in 2011 reported that notable amount of palmitate was present during ovulatory phase when compared to that of other reproductive phases and they suggest that it may be involved in chemoattraction. The ovulatory phase of women saliva has the capacity to attract the opposite partner through pheromonal signals, by the presence of these fatty acids in ovualtory phase which appears to play a role in the day 13. The concentration of volatile fatty acids in the saliva of estrus and non estrus bovine differ. Hence, the present work concluded that volatile fatty acids may act as a source for pheromone in olfactory communication. Even though the present study did not reveal any estrus precise fatty acids seem to be higher during estrus phases, this finding indicates that the quantification of salivary fatty acid may be considered as one of the parameters in detection of estrus in bovine by developing biochemical kit. Further study is needed to gather more information about the functional role of the fatty acids through behavioural analysis. Acknowlegement The authors sincerely thank UGC, New Delhi, Government of India, (F.No /2012 (SR) date ) for providing financial assistance to carry out this work very successfully. 7 Int.J.Curr.Biotechnol. Volume 2; Issue 2; Feb, 2014

3 Table 1: Fatty acids analysis of bovine (Bos indicus) saliva using GC Car.chain Fatty acids Prepubertal Estrus Pregnant Lactation C10:0 Capric acid C11:0 Undecyclic acid C12:0 Lauric acid C13:0 Tri decyclic acid C14:0 Myristic acid C15:0 Penta decyclic acid C16:0 Palmitic acid C17:0 Margaric acic C18:0 Stearic acid C19:0 Nonadecyclic acid C20:0 Arachidic acid C21:0 Heneicosanoic acid C22:0 Pehenic acid C23:0 Tricosanic acid C24:0 Lignoceric acid Σ Of SFAs C14:1ω-3 Cis-3 Myristoleic acid C14:1ω-5 Trans-5 Myristoleic acid C14:1ω-7 Cis-7 Myristoleic acid C15:1ω-6 Cis-6-Pentadecenoic C16:1ω-5 Cis-5-Palmitoleic acid C16:1ω-6 Cis-6-Palmitoleic acid C16:1ω-7 Trans-7-Palmitoleic acid C16:1ω-9 Trans-9-Palmitoleic acid C17:1 ω-5 Cis-5- Heptadecenoic acid C17:1ω-7 Cis-7- Heptadecenoic acid C17:1ω-8 Trans-8-Heptadecenoic acid C18:1ω-5 Cis-5-Octadecenoic acid C18:1ω-7 Cis-7-Octadecenoicacid C18:1ω-9 Oleic acid C19:1ω-8 Nonadecenoic acid C20:1ω-5 Cis-5- Eicosenoic acid C20:1ω-7 Cis-7- Eicosenoic acid C20:1ω-9 Cis-9- Eicosenoic acid C20:1ω-11 Trans 11- Eicosenoic C22:1ω-7 Cis-7- Docosenoic acid C23:1ω-9 Trans-9- Docosenoic acid C24:1ω-3 Cis-3- Tetrasenoic acid C24:1ω-6 Cis-6- Tetrasenoicacid C24:1ω-9 Trans-9-Tetrasenoic acid Σ Of MUFAs C16:2ω-6 Hexe decenoic acid C18:2ω-3 Trans-3-linoleic acid C18:2ω-6 Linoleic acid C18:3ω-3 Alfa linolenic acid C18:3ω-6 Gamma linolenic acid C18:4ω-3 Stearidonic acid C19:2ω-6 Octadecenoic acid C20:2ω-6 Eicosadienoic acid C20:3ω-6 Dihomogamma linolenic C20:4ω-6 Arachidonic acid C20:5ω-3 Eicosapentaenoic acid C20:5ω-6 Cis-6 Eicosapentaenoic C22:3ω-3 Docosa trienoic acid C22:4ω-6 Docosa tetraenoic acid C22:5ω-3 Docosa Pentaenoic acid C22:6ω-3 Docosa Hexaenoic acid Σ Of PUFAs Volume 2; Issue 2; Feb, 2014 Int.J.Curr.Biotechnol. 8

4 Table 2: Analysis of fatty acids in bovine (Bos indicus) saliva during various stages S t a g e s S A F s M U F A s P U F A s P r e p u b e r t a l b b a E s t r u s c d b P r e g n a n t a a c L a c t a t i o n a c c Dissimilar alphabets in vertical column are significantly different at P < 0.05% level References Achiraman S. and Archunan G., I-iodo 2 methylundecane, a putative estrus specific urinary chemosignal of female mouse (Mus musculus). Theriogenol. 66: Achiraman S. and Archunan G., Ethyl-2,7- dimethyl octane, a testosterone dependent unique urinary sex pheromone in male mouse (Mus musculus). Anim. Reprod. Sci. 87: Alagendran S, Rameshkumar K., Rengarajan G., Fernandez R., Guzman G. and Archunan G. (2011). Detection of fatty acids profile in human saliva with special reference to ovulation. Inter. J. Biol. 3 : Aron C., Mechanisms of control of the reproductive function by olfactory stimuli in female mammals. Physiological Reviews. 59: Block M.L., Volp L.C. and Hayes M.J Saliva as a chemical cues in the development of social behaviour. Science. 211: Brahmachary R.L., Dutta J., and Poddar-Sarkar M The marking fluid of tiger. Mammalia. 55: 150. Brahmachary, R.L., Poddar-Sarkar, M. and Dutta J., Chemical signal in the tiger. In: Chemical signals in vertebrate VI. D. Muller-Schware and R.L. Doty (Eds), Plenum press, New York Clemensn E., Arthaud V., Mandigo R. and Woods W., Fatty acid composition of bulls and steers as influenced by age and dietary energy level. J. Ani. Sci. 37(6): Dominic, C.J., Chemical communication in animals. J. Sci. Res. 41: Doty, R.L Mammalian Olfaction, Reproductive Processes and Behaviour. Academic Press, New York, USA. Eichhorn J.M., Coleman L.J., Wakayama E.J., Blomquist G. J., Bailey C.M. and Jenkins T. G., Effects of breed type and restricted versus ad libitum feeding on fatty acid composition and cholesterol content of muscle and adipose tissue from mature bovine females. J. Ani. Sci. 63(3): Flynn A.M, Lynch S.S., Cervical mucus and identification of the fertile phase of the menstrual cycle. Br. J.Obstet Gynaecol. 83: Folch J., Less M. and Slone Stanley H., A simple method for the isolation and purification of total lipid from animal tissues. J. Bio. Che. 226: Huerta-Leidenz N.O., Cross H.R., Savell J.W., Lunt D.K., Baker J.F. and Pelton L.S., Comparison of the fatty acid composition of subcutaneous adipose tissue from mature Brahman and Hereford cows. J Ani. Sci. 71(3): Izard, M. K., Pheromones and reproduction in domestic animals. In: Vandenberg, J G, (Ed.), Pheromones and Reproduction in Mammals. Academic Press, New York, Kumarasamy P., Sivaselvam S.N., Rajendran R., Thangaraju P. and Mahalinga Nainar., Chromosomal characterization of Umblachery breed of cattle (Bos indicus) a famous South Indian breed of Tamilnadu, India. Ind. J. Sci. Tech. 6: 1-3 Lawrence H.P., Salivary markers of systemic disease: noninvasive diagnosis of disease and monitoring of general health. J. Can. Dent. Assoc. 68(3): Mandell I.B, Buchanan-Smith J.G. and Campbell C.P., Effects of forage grain feeding on carcass characteristics, fatty acid composition, and beef quality in Limousin-cross steers when time on feed is controlled. J. Ani. Sci. 76(10): Martins Y., Preti G., Crabtree C.R., Runyan T., Vainius A.A. and Wysocki C.J., Preference for human body odors is influenced by gender and sexual orientation. Psychol Sci. 16(9): Mattina M.J.I., Pignatello J.J. and Swihart R.K., Identification of volatile components of Bobcat (Lynx rufus) urine. J. Chem. Ecol. 17 (2): Michael, R.P., Observation upon the sexual behavior of the domestic cat (Fellis Cattus). J.Under laboratory conditions. Behaviour. 18: Miller L. and Berger T., Bacteria identification by GC of whole cell fatty acids GC Hewlett Packard Appl. Note Novotny M., Jemiolo B., Wiesler D., Ma W., Harvey S., Xu F., Xie T. M. and Carmack M., A unique urinary constituent, 6-hydroxy-6-methyl-3-heptanone, is a pheromone that accelerates puberty in female mice. Chem. Biol. 6: Int.J.Curr.Biotechnol. Volume 2; Issue 2; Feb, 2014

5 Poddar-Sarkar M. and Brahmachary R.L., J Lipid Med Cell Sign Prabu T. and Rameshkumar K., Detection of fatty acids in Bovine (Bos indicus) urine during different phases of estrous cycle using gas chromatography. Int. J. Advan. Res. 1: Ramesha K.P., Commissioned paper in the thematic working groups on domesticated bio-diversity, National biodiversity strategy and action plan, Ministry of Environment and Forestry, Government of India. Rameshkumar K. and Archunan G., Analysis of urinary fatty acids in bovine (Bos taurus): An effective method for estrus detection. Ind. J. Ani. Sci. 76 (9): Read G.F., Status report on measurement of salivary estrogens and androgens. Ann. Acad. Sci. 694: Smith S.B., Yang A., Larsen T.W. and Tume R.K., Positional analysis of triacylglycerols from bovine adipose tissue lipids in degree of unsaturation. Lipids. 33(2): Streckfus C.F. and Bigler L.R., Saliva as a diagnostic fluid. Oral Dis. 8: Tabak L.A., A revolution in biomedical assessment: the development of salivary diagnostics. J Dent Educ. 65(12): Zar J.H., In Biostatistical Analysis, Englewood Cliffers, N.J; Prentice hall. Inc. 3: Volume 2; Issue 2; Feb, 2014 Int.J.Curr.Biotechnol. 10