6. DISCUSSION

The findings presented in the thesis have yielded some new information to the detection of putative in bovine urine with reference to estrus. Further, the findings of biochemical analysis, behavioural studies have helped to strengthen some of the hypothesis but forward by their workers.

Accurate estrus detection is a major problem in bovine reproduction and therefore, artificial insemination in that successful at satisfactory level. Till now effective, accurate and economical methods are not available for large scale field test of estrus detection. The present study discussed about the method of estrus detection, outlines the problem associated with the activity and describes how they might be solved through estrus detection practice and the proper use of estrus detecting aids. The discovery of estrus related substances often a promising approach to this problem. The benefit of bovine reproductive health programme mainly depends on estrus detection. Therefore it is a need to evaluate a method of estrus detection, which would be economic, reliable and easy, so that the farmer can identify the estrus phase.

The good reproductive performance is essential to efficient management and production as a whole, although specific reproductive targets may depend to extend on local conditions and on individual farm systems and target. Several disruptions are arising with the reproductive process of bovine. In this problems are clarify with the help of to stimulate the animal reproduction. The chemical nature of pheromone is not precisely known, but several body fluids have been reported as potential sources of estrus signaling compounds. The volatile compounds termed or suggestive of pheromonal properties in different body fluids of animal excretion and

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DISCUSSION

other secretion. In the present study, the biochemicals, fatty acids, hormones and active compounds how to use bovine reproduction is discussed below.

The vaginal smears collected from the bovine revealed that the nucleated epithelial cells are highly present during proestrus, where as in the next stage estrus the number of nucleated epithelial cells decreased and the appearance of cornified cells is high percentage. Thereafter, in the metestrus period, there is a decrease in cornified cells and the number of leukocyte increased. During diestrus period, the leukocytes are present in high percentage (Long and Evans, 1922; Freeman, 1988; Mandl, 1951, Rodriguez et al., 1995; Marcondes, 1995; Marcondes et al., 1996; Vanderelei et al., 1996; Chateau et al., 1996; Marcondes, 1998; Spadari-Bratfisch et al., 1999).

The relative increase in percentage of cornified cells and values of total leukocytic infiltration accompanied with the similarity in percentage of both small and large nucleated cells were observed during estrous cycles. This may be attributed to the decrease in plasma progesterone concentration as presented herein and indicated the defense mechanism of the mare during this stage. Many small and large nucleated cells and cornified cells with few leukocytes infiltration were found during 0 day in estrus. The present findings indicated that the vaginal cytological examination is a rapid reliable method for assessment the reproductive pattern in bovine (Bos indicus).

The salivary fern patterns clearly appeared during 0 and 1st day as it may be estrus. Similarly, Alagendran, et al., (2007), reported that the fern pattern of saliva also used to detect the ovulation period. The formation of ferns influenced by the presence of Nacl, which cyclically increases under the influences of estrogen. There may be a chance of estrogen influences in the formation of fern. In the present study,

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DISCUSSION

the fern pattern is clearly visible during the stages of estrus, and this may be influenced by the presence of estrogen.

In the present study the protein content was found to be varied in the urine of all the bovine reproductive stages, which may be due to the changes of hormonal level. However another important finding is that there is an increase in estrogen level during various reproductive stages, there is also a comparatively higher level of protein. Mayer, et al., (1998) demonstrated the highest secretions of estrogen induced protein at estrus in bovine, Further, Rivard and Klemm (1989) postulated the existence of a non- volatile compound that would stimulated the male (Bull) to generate the appropriate mating behaviours at close range. Even though there are variations in the protein content during the various reproductive stages, the estrus urine showed outstandingly higher level of protein due to the higher level of estrogen.

The concentration of these signals may vary according to the phase as well as status of the animal. Among the various sources in communication, urine is one of the chief sources involved in the signal-receptor system (Bimova et al., 2009). The major urinary protein is of minor importance for females. However, at the estrus phase the protein is found at maximum concentration during estrous cycle (Stopka et al., 2007). It clearly shows that the sex attractants produced in estrus females may be necessarily transported by this protein. Estrus specific pheromones are released through the urine, the high level of protein may be required to function as carriers for the ligands and convey the chemical signal. It has been reported that excretion of Major Urinary Protein in urine acts as pheromone carrier (Beynon and Hurst, 2004). The male shows specific behavior and spent more time when exposed to female urine, but stopped by a partition (Amstislavskaya and Popova, 2004) and it suggested that the proteins specific to estrus phase exclusively involved in attraction of male. The nature of the

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DISCUSSION

protein is not identified in the present study, but the appearance of a higher level of protein during estrus seems to be noteworthy.

Most of the workers (Dominic, 1987, 1991; Adams, 1980; Singer 1991; Balakrishnan and Alexander, 1985; Johnston, 1990), believed that the and proteins are actively involved in olfactory communications in most the mammalian species. Patterson (1968) reported that swine used the steroids for enhancing courtship behaviours. This hypothesis is strongly supported by Westphal (1967) that sex attracting lipids are expelled from the cells by the assistance of certain proteins. In the present study, the level of was found to be high during late lactation stage when compared to other stages. In fact the present results clearly indicated that there is positive relationship between lipids and progesterone i.e. when there is an increase in progesterone level the lipid concentration is higher in urine.

The changes in physical properties of the urinary excretions are primarily attributed to the changes in chemical composition. To the best of the authors’ knowledge there is no information on the biochemical composition of cattle urine, although they form the basis for most of the reproductive behaviours. The biochemical components of the urine import the unique odour (Brown, et al., 1996).

The current study reveals that the bovine urine has a considerable amount of carbohydrate, which is comparatively higher than proteins and lipids, since the cow feeds mainly on carbohydrates it is natural to expect a high content of carbohydrates in the urine. The present findings are in agreement with the result of Boyland (1946) who reported that the cervical mucus in estrous cows appeared to be mainly carbohydrate and might be mucopolysaccharide. By contract, the mucus of pregnant cows and those in diestrus contained both polysaccharide and protein. The wide variation in

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DISCUSSION

carbohydrate observed in the present might have been due to several causes as diet, endocrine status and genetic factors etc. There is a close correlation between the activity of endocrine glands and the reproductive behaviour of animals. Rameshkumar et al., (2012) reported that the carbohydrate level was found to be higher in prepubertal stages than other stages because the animal feed on mainly carbohydrates, it is natural to expect a higher amount of carbohydrates in the urine of young animals which need high amount of carbohydrates for their growth.

In the present study, the biochemical estimation revealed that the higher lipid excretion in cow in late lactating period suggests the role of lipids in mother young interaction. However, further research is needed in this field. The presence of considerable amount of lipids suggests their role in olfactory communication and clearly indicates that the proportion of lipid content was varied in various reproductive stages of bovine urine. The present results are further supported by the findings of Brahmachary et al., (1992) that the lipid rich secretion in the tiger urine plays a significant role in sexual attraction and many social involvements.

Rameshkumar and Archunan, (2006) revealed that there is a significant variation in the excretion of lipid content across various reproductive phases that lipids have got specific role in olfactory communication depending upon the physiological state of the individuals and responders. Lipid excreted in urine is certainly not wastage; rather it serves a useful purpose, probably to protect the volatile semiochemicals from quick dispersal by evaporation and washing.

High endogenous gonadal hormone titres are also often necessary to achieve interest and maintain selective responses to sex specific signals. The serum concentration of progesterone and estrogen during the estrous cycle of the cow has

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DISCUSSION

been well documented (Poltka et al., 1969, Stabenfeldt, et al., 1969, Henrickes et al., 1971, Lemon et al., 1974).

Estradiol secreted by the ovary during follicular stage of the estrous cycle produces behavioural changes necessary to allow mating, the appearance of confiscation in vagina and characteristic changes in the oviduct and the uterus (Finn and Martin, 1970). Mating within a narrow (12 to 22 h) period on the day of estrus is a priquiste for optimal fertilization in cow (Schams et al., 1977). The time requirement is mainly regulated by female lordosis behaviour, which allows copulation. In the present study estrogen level is significantly high during estrus period when compared to that of pro and post estrus period. The results are in agreement with the earlier findings of Carrick and Shelton (1969) and Lavasseur and Thibault (1980). Progesterone levels must be low at the time of peal estrogen levels in order to express behavioural estrus (Lavasseur and Thibault, 1980). Dehnhard et al., (1991) stated that the presence of the ovary seems to be essential for the synthesis of pheromones. At estrus, estradiol stimulates vascularity, muscular activity and increases the number of epithelial cells in bovine tubular genetalia (Asdell, 1964; Roberts, 1956).

The progesterone level was significantly less is estrus period when compared to all other reproductive phases. Milvae et al., (1984) also reported the lower concentration of progesterone during estrous cycle in heifers. Progesterone release occurs in an episodic fashion in correlation with LH release during mid and late luteal phase (Ellinwood et al., 1984). Similarly in the present study the progesterone level was higher in late pregnant period while the significantly lower level of progesterone in all other reproductive phases. Although previous studies have shown that estrogen alone was sufficient to induce estrus behaviour in the ovaractomized cow progesterone may act to govern the intensity of estrus behaviour. However, there have been conflicting reports on the relationship between estrogen and progesterone in regulating

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DISCUSSION

estrus behaviour using ovaractomized cows. Cows received high doses of estradiol repeatedly failed to display estrus behaviour (Carrick and Shelton, 1969). However, when progesterone was given prior or along with estradiol, the animals displayed estrus. The role of progesterone in regulating prior to the rise of estrogen may regulate the occurrence and intensity of estrus behaviour (Carrick and Shelton, 1969).

Mandal and Prakash (2003), Mehrotra et al., (2005), Venkatesan et al., (2005), and Selvaraju et al., (2002) who recorded lower concentrations of progesterone on different days of estrous cycle as compared to the concentration of progesterone in Punganur cattle. A recent study investigated seasonal changes in circulating serum progesterone and estrogen in free ranging and captive female sea lions. Progesterone concentrations increased in the fall irrespective of pregnancy status and progesterone concentrations were higher in pregnant animals when compared with non pregnant animals in the spring. In the majority of animals, there was no clear pattern in the estrogen concentration (Greig et al., 2007).

The level of hormone in each individual depends on the genotypes of the animal, environmental and other physiological condition like age, growth, pregnant, lactation etc. The present study concluded that estrogen, LH and FSH high level found to be high in estrus period when compared to all other stages. Further, the present investigation clearly shows that the progesterone level was high in pregnant than all other phases.

The fatty acids like hepatanoic, isohexanoic acids identified in tiger urine are absent in cow urine, (Bramachary, et al., 1992). Among the fatty acids identified in cow urine, and are predominantly present in estrus proestrus

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DISCUSSION

and post estrus urine whereas the concentration of other fatty acids are comparatively less. Since the estrous urine has the capacity to attract the opposite partner through pheromonal signals, the present in estrus phase appears to play a role in this context.

This statement was supported by Bramachary et al., (1992) that the free fatty acids seem to function as pheromones in number of mammals. The difference in the fatty acids composition may be due to the alterations of lipid metabolism especially of metabolic events affecting ketogenesis and would reflect in the chemical alteration of urine. Furthermore, the variation in fatty acid concentration of urinary samples may be serving as the basis for individual recognition. The present results may recall with earlier findings of Mykytowycz (1970) Welsch et al., 1988, Flood et al., 1989, Decker et al., (1992) in which the sebaceous gland of rabbit rat and fishing but have many kinds of fatty acid and glycerides and appear to be involved in individual recognition and scent marking. In the present study, saturated fatty acids are predominantly found in almost all the urine samples. By contrast, the bobcat urine predominantly contains unsaturated fatty acids (Mattina et al., 1991).

In the present study urine samples were collected during various phases of estrous cycle and detected 62 different fatty acids using gas chromatography. Margaric acid and stearic acid was noted in higher concentration during in estrus phase when compared to that of all other fatty acids. This finding is consistent with the report of Mattina et al., (1991) that was excreted in bobcat urine which is involved in sexual attraction of conspecifics. The concentrations of fatty acids were maximum in estrus phase when compared to proestrus and postestrus phase. The saturated fatty acids like margaric acid and stearic acid were predominantly present during estrus phase. It is also reported that increased amount of these fatty acids in

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DISCUSSION

bovine urine involved for odour production and they may act as pheromones (Ramesh kumar and Archunan, 2006). However, in the proestrus urine, palmitic acid was present in higher quantity than all other stages. Brahmachary, et al., (1992) also stated that the free fatty acids seem to function as pheromones in tigers. It is also to be remembered that the heptanoic and isohexanoic fatty acids, which are present in tiger (Bramhachary, et al., 1991) and Cheetah (Bramhachary, et al.,1992; Poddar Sarkar, 1996) as marking fluid but these fatty acids are absent in bovine urine (Ramesh kumar and Archunan, 2006).

In the present study, 26 different types of monounsaturated fatty acids were detected from of bovine urine. Alagendran, et al., (2011), reported that there are 17different types of fatty acids were identified in human saliva. The fatty acids like as oleic acid, palmitic acid and were comparatively higher in concentration than the other fatty acids. These three fatty acids were mostly present in ovulatory phase when compared to follicular and luteal phases of menstrual cycle.

Kumar et al. (2000) reported that the tridecanoic, pentadecanoic and are present predominantly in ovulatory phase and these fatty acids may act as estrus specific in bovine urine. It is known that the ovulatory phase contains higher levels of estrogen and the notion of present report increases the possibility of role of estrogen with the fatty acid excretion. It is additionally hypothesized that fatty acids along with other biochemical components of urine play a prominent role in opposite sex attraction.

These cyclic fluctuations in lipid levels do occur under the influence of both endogenous and exogenous sex hormones. Likewise the bobcat urine predominantly contains unsaturated fatty acids (Mattina, et al., 1991). Oleic acid is found to be in

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DISCUSSION

higher concentration as compared to that of all other fatty acids, by contrast, Cis-5- heptadecenoic acid and Cis-7- heptadecenoic acid showed maximum level of concentration during in estrus period. Likewise, available report indicates that the mice glandular tissues like preputial and cheek contains long chain double bonded fatty alcohols (Albone 1984). These reports are consistent with our present findings and suggest that the fatty acids in higher concentration emitted during ovulation probably act as sexual attractants in bovine. It indicates that these fatty acids in estrus stage appeared due to the high circulation of steroidal hormones. The difference in the fatty acid composition may be due to the alterations of lipid metabolism especially of metabolic events affecting ketogenesis and would have reflected in the chemical alteration of urine. Reliable and accurate measures of fatty acids and its volatiles are easily detected through gas chromatography. The present study may provide evidence that the presence of one are more fatty acids in estrus urine along with other compound might involve in sexual attraction and behaviour.

The present interpretations clearly indicate that biochemical constituents such as proteins, carbohydrates and lipids are varied in the urine during different reproductive phases of bovine. In addition to this, the quantitative and qualitative estimation of fatty acids also varied. The presence of one or two fatty acids in estrus phase along with other components may involve in the estrus indicating compounds. The presence of hormones such as estrogen, progesterone, LH and FSH may provide evidence their relationship with the excretion of biomolecules.

The biochemical constituents such as proteins, carbohydrates and lipids either individually or combindly appear to play a vital role in the bovine reproductive behaviour since the amount of biomaterials are higher in estrus phase. It is, therefore,

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DISCUSSION

possible that proteins, carbohydrates and lipids may indicate as biochemical marker to identify the estrus phase.

Urine is a reliable indicator of the physiological status of an animal because of its function as the major means by which metabolic waste is eliminated (Albone, 1984). As many urinary compounds are also found in the blood, it is not surprising that urine can convey to the external world much information concerning the internal physiological state of the animal, and so provide a source of chemical signals in many species. The potential as pheromones of the urinary compounds of some laboratory rodents has been thoroughly studied, and their relative objects have been established (Bronson, 1971, 1974).

This study focused on the analysis of the GC-MS profiles of the volatile compounds of cows' urine in three different phases proestrus, estrus and post estrus and has provided evidence that different urinary compounds are present in the different stages. In particular, two peaks present during the estrus phase, relating to 1- iodo- methyl undecane and Tetratetracontane, were not found in the other two phases.

The appearance of specific peaks in the estrus urine may represent behaviorally important chemical signals. Thus, the presence of 1- iodo- 2-methyl undecane in the urine may indicate that this is a putative pheromone. Since a natural estrous cycle is caused by hormonal changes, the observed variations in the bovine urine may also be caused by endocrine changes. The endocrine dependence of some urinary volatile compounds has been reported in mouse (Jemilo et al., 1987; Andreolini et al., 1987; Schwende et al., 1984) vole (Boyer et al., 1988), wolf (Raymer et al., 1984). The presence of volatile compounds 1- Dodecene, 5- Octadecene, 5- Eicosene and 1- Pentadecene, in all three phases of estrus cycle suggests that these compounds appear

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DISCUSSION

to be common metabolic by products in urine throughout estrous cycle. However, functional activities of these compounds are not known at present. The compounds 1- iodo- methyl undecane and tetratetracontane present in estrus phase have an important role in sexual attraction. However, those compounds identified in the estrus phase, need to be confirmed through testing with bull behaviour and this would give more information in relation to olfactory communication of bovine urinary compounds.

The innovation of estrus related substances offer a capable approach to this problem. Such substances have been identified in equine urine and reported to act as markers for estrus detection (Ma and Klemm, 1997). Available reports indicate that the estrus-related odours present in the cow urine are discriminated by rat (Dehnhard and Claus, 1998). The trained dogs are also capable of recognizing the milk and urine odours of estrus stage of the cow (Hawk et al., 1983). Furthermore, the estrus unique markers have found in bovine milk (Ma and Klemm, 1997). The presents findings would give additional support in favour of identifying the estrus phase of the cow by using urinary pheromonal compounds. Estrus – related chemicals might also be useful as sexual stimulates for increasing the libido of unmotivated males.

Comparisons of the urinary profiles between estrus and pregnant sample indicate that there are some specific volatile compounds observed in estrus, which are found in pregnant urine. Since a natural estrous cycle is caused by hormonal changes, the above qualitative variations in the urine may also be due to endocrine changes.

The compound octyl cyclopropane, found in early prepubertal is also present in estrus and post estrus urine but its biological importance is not yet known. It is noteworthy that the early pregnant urine contains high molecular weight compounds as compared mid and late pregnant urine samples. Therefore, it is reasonable to

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DISCUSSION

presume that the compounds present in the late gestation may be used a biochemical markers for identifying the parturition time in cow. Further, investigation is required to confirm the biological functions of each compounds identified in the pregnant urine.

The compounds identified in three different phases differ from one stage to other. While comparing the lactating urine with estrus and pregnant urine, it is interesting to note that the compound, 1- iodo- methyl undecane, which is identified in the estrus phases.

The present investigation clearly indicated 2.0% concentration of 1-iodo-2- methylundecane compound is significant in flehmen and mounting behaviours. It is important to note that increases the concentration of compounds did not enhance the bull behaviour. It indicators that the optimum concentration required in the cow urine to influence the bull behaviour appears to be 2% regardless of the volatiles involved in attractant and mounting activity. Similarly, Rasmussen, et al., (1997) reported 0.5mm concentration of (Z) 7-dodecene – l- yl acetate consistently elicited high levels of flehmen responses equivalent to bio responses elicited by pre – ovulatory urine.

The results further showed that bulls are attracted and exhibited repeated flehmen behaviour significantly towards the 1-iodo- 2-methylundecane in contrast other compound tetetratetracontane did not elicit significantly. Identification of (2) – 7- Dodecen-1-yl-acetate in female Asian elephant that is released in the pre- ovulatory urine was found to influence the specific behaviour include flehmen in male elephants (Rasmussen et al., 1997).

In addition, Archunan and Rameshkumar, (2012) identified two estrus specific compounds in cow urine and conducted the bull bioassay using synthetic compounds

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DISCUSSION

are found that the compound 1-iodoundecane elicits flehmen behaviour in bull. Likewise, sex specific pheromones have been characterized structurally identified and linked to sexual mammals (Singer et al., 1976, Jemilo et al., 1985, Novotny et al., 1985, Tannoundji et al., 1994, Drickamer, 1995, Selvaraj and Archunan, 2002). In addition, the compound 1- iodo- 2-methylundecane is known to putative estrus specific urinary chemo signals of female mouse (Achiraman and Archunan, 2006).

The urine testing or soliciting sequence of which flehmen is a competent has been described anecdotally in many species and was called harnfordem by Schenkel (1966). The absence of a display function for flehmen judged by the lack of response by adjacent conspecifics is consistent with earlier reports (Estes, 1972, Kramer, 1969). Because urine testing usually precedes courtship, it perhaps surprising those adjacent males do not respond to flehmen. However, it is the subsequent behaviour of the male that should be indicated of the reproductive status of the orienting and approaching if another male inhibits courtship (Coblentz, 1974).

The present study is the occurrence of flehmen behaviour in bulls towards the dummy cow that received the samples, irrespective of the source at estrus. The chemosenory responses and pre-mating behaviour of adult males to the female urinary cues have been well demonstrated in Bos taurus (Klemm et al., 1987., Paleologou, 1977). Furthermore, the flehmen behaviour exhibited peak in early and met estrus and decreased in late estrus in cows (Blazquez, 1988) and buffaloes (Rajanarayan and Archunan, 2004; Brito, 2002). Under field conditions visual stimuli probably plays a role in stimulating male sexual behaviours, but in vitro tests with the cervico- vaginal mucus clearly show that there is an olfactory component produced from the vagianal region, independent of vision, specifically important in the stimulation of male reproductive behaviours, such as sniffing, licking, and repeated flehmen (Jacobs et al.,

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DISCUSSION

1980, Hradecky et al., 1983). The present study in consistent with earlier report that vaginal secretions of heifers at estrus stimulated sexual activity and mounting behaviour (Nishimura et al., 1991). The temporal relationships between estrus related odours in vaginal mucus and milk might possibly be expected because estrus related odours have also been found in urine and blood (Kiddy et al., 1984).

The compounds 1- iodo- 2-methylundecane and tetratetracontane appeared during only estrus period were not found in other phases. It is interesting to note that these compounds are very characterstic of the estrus period. The present study concludes that the following two compounds namely 1- iodo- 2-methylundecane and tetratetracontane constancy were specific to estrus. The behaviour analyses clearly demonstrated repeated that the bulls are attracted by 1- iodo- 2-methylundecane and displayed mounting behaviour. It is also concluded that the compounds 1- iodo- 2- methylundecane identified in the estrus urine appear to be a putative pheromone and it is responsible for the effect the precopulatory behaviours in bull.

An important findings in the present study is that the identification of specific compounds in estrus urine and confirmation of the identified compounds with bull by behavioural studies. Since the response of bull to the synthetic compounds are very much satisfactory, this findings would give a real impact in farm animal reproduction with economic important. As a biochemical approach, it should be possible to use these compounds as a marker to develop a biomarker system for effective estrus detection.

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