Significance of cervico-vaginal microbes in bovine reproduction and pheromone production - A hypothetical review.

The vaginal microbiota has been studied in animal reproduction and fertility, in particular little information of vaginal microbes in reference to bovine reproduction and pheromone production is known. The vaginal mucosa in healthy cow is colonized by an equilibrated and dynamic composition of aerobic, facultative anaerobic and obligate anaerobic microbes. Cervico-vaginal mucus (CVM) composition, viscosity and volume vary with the cyclicity and health status of the reproductive tract. In addition, CVM contains pheromones, volatile compounds, and proteins that attract males for coitus. Commensal microbiota plays a key role in protection of the genital tract from pathogenic microbes by competition effect. In the bovine species, the microbial composition, its abundance and diversity in the female gut, vagina, urine, saliva, and feces, and the associated chemical communication remains poorly documented. The impact of microbes in the reproductive tract of cow, buffalo and certain mammals are discussed in this review. Since the microbial population diversity of CVM is modified during estrus phase it presumes that it may have a role for pheromone production in conspecific. Herein, we would like to critically discuss the current state of knowledge on microbially produced signals in animals and the role of genital and CVM microbiota in estrous cycle and pregnancy.

Reproductive enhancement in buffalo: looking at urinary pheromones and hormones.

The success of conception in buffalo is greatly dependent on precise estrus detection and time of artificial insemination (AI). Various visual, behavioral, biochemical and gyneco-clinical parameters have been tracked closely and a cost-effective combinatorial model has been developed to detect estrus in buffaloes. Pheromones play pivotal roles in reproduction and behavior of mammals. Urine, an easily available biological material which reflects the internal status of an animal, was recruited for profiling the pheromone compounds during the various phases of estrous cycle using gas chromatography-mass spectrometry (GC-MS) analysis. Among the identified compounds, 4-methyl phenol (4-mp, p-Cresol) and 9-octadecenoic acid (Oleic acid) were found to be estrus-specific and would be promising estrus-indicators. Similarly, detection of luteinizing hormone (LH) in urine was also focused to predict the time of ovulation in buffaloes. Partial success has been obtained in the attempt to develop a cost-effective bioassay kit for estrus detection. The ongoing venture of the relevant research team is to develop a biosensor to identify estrus-specific pheromone compounds in urine. Development of a nanoparticle-based bioassay kit for detection of urinary LH for effective prediction of estrus or ovulation is also in progress.

Detection of estrous biomarkers in the body exudates of Kangayam cattle (Bos indicus) from interplay of hormones and behavioral expressions.

Behavioral expressions and biochemical composition of body exudates are significantly altered in concert with the endocrine status, which are all clear indicators of physiological conditions of animals. In this study, we sought to infer about the reproductive physiological status of Kangayam cattle (Bos indicus) by analyzing behaviors, endocrine pattern, and body exudates and further to discover estrous biomarkers so as to facilitate timely artificial insemination/mating and to aid in aspects of conservation of the species. Therefore, in this study, we followed Kangayam cows through pre-estrous to post-estrous phases to correlate the endocrine dependence of biochemical constituents in urine and cervical mucus and sought to identify estrous biomarkers. Behavioral estrus was confirmed in 10 cows, from which urine samples were collected and subjected to determination of LH, FSH, estrogens, progesterone, proteins, and lipids. Furthermore, urinary fatty acids and proteins were profiled using gas chromatography and SDS-PAGE, respectively. The volatile compounds in the urine and cervical mucus were identified by gas chromatography-mass spectrometry analysis. The data revealed that LH, FSH, and estrogen levels increased significantly in estrous urine compared with nonestrous urine, whereas progesterone status was vice versa (P < 0.05). The lipid content was also significantly higher in estrous urine than in pre- and post-estrous urines (P < 0.05). There were also cyclical variations of volatiles and fatty acid profiles across phases of the estrous cycle. More acidic compounds were present in estrous urine, rendering it more acidic, than in pre- and post-estrous urines. Interestingly, oleic acid, which was present as a fatty acid in estrous and post-estrous urines, appeared to be a volatile in post-estrous urine and estrous cervical mucus. In addition, octanoic and butanoic acids were specific to both estrous urine and cervical mucus, indicating their possible candidature as estrous biomarkers. SDS-PAGE analysis showed pronounced expression of a 98 kDa protein in post-estrous urine, which in matrix-assisted laser desorption ionization-time of flight mass spectrometry was identified as albumin. Our results demonstrate multiple biomarkers in estrous urine and specific volatiles in cervical mucus that offer scope to develop viable estrus detection kits for Kangayam cows.

Assessment of urinary volatile compounds and proteins in the female goat Capra hircus: A pilot study to reveal potential indicators of oestrus.

Urine samples of female goats in pro-oestrus, oestrus and post-oestrus phases were analysed for finding oestrus-specific volatile compounds using gas chromatograph-mass spectrometry (GC-MS), and proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fourteen urinary volatile were identified covering all three phases among which four compounds, 1-Tetradecanol, n-Pentadecanol, 3-Methylene tridecane and 2-Ethyl-1-dodecene, were unique to oestrus. Also, oestrus urine contained a 25 kDa protein, which was totally absent in pro-oestrus urine, and less-expressed in post-oestrus urine. This protein revealed to be complement C3 fragment. This pilot study, for the first time, reveals the difference in urinary volatile compounds and proteins in the female goat during the different phases of oestrous cycle. The four unique volatile compounds and a 25 kDa protein that appeared as oestrus-specific in this study warrant further investigation to consider them as urinary biomarkers of oestrus in goats.

Heat shock protein(s) may serve as estrus indicators in animals: A conceptual hypothesis.

The estrous cycle consists of episodic phases that eventually regulate reproduction in non-primate mammals. Females are sexually receptive to males only during estrus phase, which lasts from few hours to 2 days. Estrus detection is crucial for reproductive management of animal herd; however, precise estrus detection method/tools are not available. The analysis of body fluids, e.g., facets of proteomics during recent years is promising in the progression of estrus markers. Specifically, heat shock proteins (HSPs) present in body fluids and reproductive organs could be possibly used as markers of estrus. We note the possible vital roles of hyperthermia, stress and steroid hormones during estrus that could positively regulate the expression of HSPs. To validate our hypothesis, we explained the possible mechanism of production of HSPs, specifically during estrus phase. We recommend intensive research on HSPs to develop credible estrus detection tools in animals.

Safety assessment of methanolic extract of Terminalia chebula fruit, Terminalia arjuna bark and its bioactive constituent 7-methyl gallic acid: In vitro and in vivo studies.

Terminalia chebula and Terminalia arjuna were widely used in traditional medicine for the treatment of memory impairment, inflammatory disorders and as an anti-aging agent. However, reports regarding their safety aspects are lacking. Hence, the present study was carried out to investigate the toxicity of methanolic extracts of Terminalia chebula fruit (TCF), Terminalia arjuna bark (TAB) and its bioactive constituent 7- Methyl gallic acid (7MG) under in vitro and in vivo conditions. In vitro toxicity profile of TCF, TAB and 7MG (250-2000 µg/ml) were assessed through cytotoxicity, hemolytic activity, mutagenicity and genotoxicity assays. Results of Ames test, comet assay, MTT and hemolytic assays illustrated that TCF, TAB and 7MG exhibited neither cytotoxic and genotoxic effect in PBMC nor hemolytic activity in RBC and no mutagenic effect in TA 98 and TA 100 up to a limited dose of 2000 µg/ml. Acute and subacute toxicity studies showed no significant change in body weight, behavior, hematology, biochemical parameters, organ weight and histopathology. Over all the results of acute and subacute toxicity studies conclude that oral administration of TCF, TAB and 7MG were observed to be relatively non-toxic and affords practical guidance for selecting safe dose for further clinical trials.

p-cresol and oleic acid as reliable biomarkers of estrus: evidence from synchronized Murrah buffaloes.

Successful reproductive management in buffaloes depends on effective estrus detection. Urinary pheromones identified from natural estrous cycle have been reported to decipher estrus phase. However, its presence has not been analyzed in the urine after synchronization. Thus, our present investigation was to investigate the influence of synchronized estrus urine in bulls and to examine the presence of estrus-specific compounds ascertained in natural estrus in synchronized buffaloes. Mid-stream urine was collected from six synchronized buffaloes during various phases of estrous cycle and volatiles were examined using GC-MS. Sexual provocation in bulls was established by displaying persistent flehmen and frequent mounting towards estrus urine from synchronized animals. Totally forty-two volatile compounds were identified from three phases of estrous cycle, more specifically 4-methyl phenol (p-cresol) and 9-octadecenoic acid (oleic acid) in estrus urine of synchronized animal as similar to natural estrus. Hence, these chemical cues in buffalo urine might be employed as potential marker candidates for the development of an estrus detection aid.

Urine levels of luteinizing hormone as predictor of the period of ovulation for advantage of timed-artificial insemination in murrah buffalo (Bubalus bubalis).

Assessment of urine levels of luteinizing hormone (LH) for predicting the reproductive status of animals is in practice. The aim of this study was to predict the period of ovulation based on the urine levels of LH for timed-artificial insemination to increase the conception rate in buffaloes, which are naturally silent-oestrous animals. Level of LH in urine was assessed using ELISA, and a cut-off LH concentration for prediction of ovulation period was obtained using receiver operating characteristic analysis. Artificial insemination was performed before- and after -positive prediction of ovulation period adopting this method, and the rates of conception were assessed. Urine LH level of 105 mIU/ml (n = 14) was derived as a cut-off concentration which predicts the ovulation period. The buffaloes in the positively predicted group (day 1 or 2) inseminated via intracervical route had an increase in the conception rate (83.33%); however, the insemination in the before-positive-prediction group resulted in poor conception rates (day 0; 16.66%) compared to that of the naturally inseminated group (day 0; 75.0%). In conclusion, the urinary LH would possibly be a fairly reliable predictor of the ovulation period. The day when cut-off LH concentration is obtained may be taken as the most favourable time for artificial insemination, so as to attain a much better rate of conception in the buffalo.

Identification of pheromone-carrying protein in the preorbital gland post in the endangered Indian male Blackbuck Antelope cervicapra L.

In mammals, a low molecular mass protein (17-20 KDa) reported from the pheromone sources such as urine, saliva, glandular secretion, etc., as ligand-carrier (pheromone carrier) has been associated with chemo-communication. Since the preorbital gland post is one of the major pheromone sources in Indian Blackbuck, an endangered species, we assumed that it possibly contains low molecular mass protein for chemical communication. Hence, we investigated the preorbital gland post in territorial and non-territorial male blackbucks for such low molecular mass proteins adopting SDS-PAGE and LC-MS/MS analysis. The total content of protein was higher in the post of territorial males than non-territorial males of adult and sub-adult. In fact, the protein profiles such as 17, 21, 25, 42 and 61 kDa were noted in the gland secretion of territorial and non-territorial males. The intensity of the 17 kDa protein band was higher in territorial males than non-territorial males. In-gel trypsin digestion of the 17 kDa band was processed and subjected to LC-MS/MS and SEQUEST analyses. The results of LC-MS/MS and SEQUEST search showed the presence of α(2u)-globulin in the 17 kDa band. In addition, the identified α(2u)-globulin sequence possessed GDW residues, which are the characteristic signature for lipocalin family. Since the α(2u)-globulin has been reported from the pheromone-carrying proteins in some mammals, this protein may carry the volatiles (pheromone compounds) in male Blackbucks preorbital gland to evoke the scent marking for maintaining territoriality (home range) and attraction towards female, through the secretion of glandular protein.

Silymarin prevents benzo(a)pyrene-induced toxicity in Wistar rats by modulating xenobiotic-metabolizing enzymes.

Benzo(a)pyrene (B(a)P), which is commonly used as an indicator species for polycyclic aromatic hydrocarbon (PAH) contamination, has a large number of hazardous consequences on human health. In the presence of the enzyme cytochrome-P-450 1A1 (CYP1A1), it undergoes metabolic activation to form reactive intermediates that are capable of inducing mutagenic, cytotoxic, teratogenic and carcinogenic effects in various species and tissues. Research within the last few years has shown that flavonoids exhibit chemopreventive effect against these toxins. In the present study, the protective effect of silymarin (a flavonoid) against B(a)P-induced toxicity was monitored in Wistar rats by evaluating the levels of hepatic phase I (CYP1A1), phase II enzymes (glutathione-S-transferase, epoxide hydroxylases, uridinediphosphate glucuronosyltransferases, NAD(P)H: quinone oxidoreductase 1, sulfotransferases), cellular antioxidant enzyme heme oxygenase and total glutathione. The results reveal that silymarin possesses substantial protective effect against B(a)P-induced damages by inhibiting phase I detoxification enzyme CYP1A1 and modulating phase II conjugating enzymes, which were confirmed by histopathological analysis. Overall, the inhibition of CYP1A1 and the modulation of phase II enzymes may provide, in part, the molecular basis for the effect of silymarin against B(a)P.

Stud male-originating chemosignals: a luteotrophic agent.

The chemosignals from mating male are found to be responsible for protecting his coital partner against pregnancy failure induced by strange male or food-deprivation. The stud male pheromone not only provides luteotrophic support in female of vulnerable condition but also exerts luteotrophic effect in pregnancy-blocked females by inducing pseudopregnancy. The luteotrophic stimulus rendered by stud male to prevent pregnancy failure is mediated through the main olfactory system, and not through the accessory olfactory system, since the accessory olfactory system is primarily involved in perceiving the luteolytic stimulus produced from strange male for causing pregnancy failure. It has been shown that pericopulatory period seems to be crucial in females in the formation of stud male chemosignals, and the olfactory luteotrophic memory of stud male is further proved to be a short-term one. The precise mechanism involved in accessing and retaining the stud male chemical cues is unclear. In this brief review an attempt has been made to bring out the luteotrophic process of stud male chemosignals, the olfactory pathway and critical period to access the signals. The possible neural mechanism and neural chemistry underlying the formation and recognition of mating male chemical cues are also highlighted.

Safety and toxicological evaluation of Rhizopora mucronata (a mangrove from Vellar estuary, India): assessment of mutagenicity, genotoxicity and in vivo acute toxicity.

The present was carried out to evaluate the toxicity of methanolic leaf extract of Rhizophora mucronata (MERM) under in vivo and in vitro conditions. Mutagenicity of MERM (up to 4,000 µg/plate) evaluated by Salmonella/microsome assay (TA98, TA100, TA1535 and TA1538 strains), with or without metabolic activation showed no mutagenic effect in any of the tester strain. Evaluation of genotoxicity (comet assay) and cytotoxicity in PBMC revealed that MERM showed no significant difference in comet tail moment (TM) and tail scores and cytotoxicity up to 24 h respectively. In acute toxicity studies, oral administration of single doses of MERM (250-2,000 mg/kg) in Wistar rats produced neither mortality nor any noticeable changes in behavior. Hematological and biochemical parameters showed no difference, except for a significant increase in ALT and AST at the highest dose. Histopathological findings revealed hepatotoxicity and neurotoxicity at highest dose of extract. In subacute toxicity studies administration of MERM (1,000 mg/kg) for 28 consecutive days neither altered the body weight gain nor behavioral parameters. No significant change was observed in the hematological and biochemical parameters analyzed. Histopathological examination showed normal architecture suggesting no morphological disturbances. Collectively, these data demonstrate that consumption of MERM for various medicinal purpose is safe.

Investigations of Ramachandran disallowed conformations in protein domain families.

In peptide and protein structures, occurrence of (φ,ψ) angles in the disallowed region of the Ramachandran map almost always suggests local regions of error or poor accuracy. However, very rarely genuine disallowed conformations occur as noted in the current study in proteins of known structure available at ultra-high resolution (≤1.2 Å). In the current work, extent of conservation of genuine disallowed conformations in homologous proteins of known structures has been analyzed. From a dataset of 124 protein domain families, with structure of at least one constituent member in each family available at a resolution of 1.2 Å or better, we have analyzed the conservation of 221 disallowed conformations. It is observed that the disallowed conformation is only moderately conserved in protein domain families. In the gross dataset no particular residue type adopting disallowed conformation elicit high conservation of residue type though there are alignment positions in the dataset with complete conservation of both the residue type and the disallowed conformation. Conserved disallowed conformation in protein domain families play biologically significant role in roughly 50% of the cases. The residues with the disallowed conformation or its flanking residues are often located within or around the functional site of the protein.

Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS.

CONTEXT: Predicting ovulation is the basis on which the fertile period is determined. Nowadays there are many methods available to detect the ovulatory period. Unfortunately, these methods are not always effective for accurate detection of ovulation. Hence, an attempt was made to detect ovulation through single dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein with the help of saliva ferning. AIMS: The aim of this study was to determine the association of protein level with endogenous reproductive hormone level across the menstrual cycle. SETTINGS AND DESIGN: Salivary protein and its confirmation were evaluated during menstrual cycle followed by SDS-PAGE and Mass spectrometry. STATISTICAL METHOD USED: The protein content present in saliva throughout menstrual cycle is trail by SPSS statistical software version. MATERIALS AND METHODS: Salivary proteins were investigated serially during pre-ovulatory, ovulatory and post-ovulatory periods of normal menstrual cycle in eighteen healthy volunteers. The samples were collected in three consecutive menstrual cycles. Salivary protein was estimated and analyzed by single dimension SDS-PAGE. RESULTS: The results revealed significant variations in protein concentrations during the menstrual cycle. Protein levels were maximum during ovulation and minimum during postovulatory phase. Further, single dimension SDS-PAGE analysis showed seven different fractions of proteins is from 14-90 kilo Dalton (kDa) in the three phases of the menstrual cycle. CONCLUSIONS: Among the proteins, 48 kDa protein was more predominantly exhibited during ovulatory phase than pre and post-ovulatory phase. The present study indicates that the protein level and the specific protein band (48 kDa) through MALDI-TOF MS analysis might serve as an indicator for ovulation.

Gas chromatographic/mass spectrometric analysis of volatile metabolites in bovine vaginal fluid and assessment of their bioactivity.

The chemical profiles of vaginal fluid collected from cows in oestrus and nonoestrus were analysed by gas chromatography-mass spectrometry (GC-MS) to establish any qualitative differences that might have potential value in bovine biocommunication. Eight different organic compounds were detected using the two chromatograms. The chemical profiles of oestrus vaginal fluid were distinguished significantly by the presence of three specific substances, namely, trimethylamine, acetic acid, and propionic acid that were not present in nonoestrus phase. The oestrus specific synthetic compounds were rubbed onto the genital region of nonoestrus animals (dummy cows), and the bulls were allowed to sniff the genital region and observed sexual behaviours. The statistical significance was higher (P < 0.001) in bulls exhibiting repeated flehmen and mounting behaviours towards the mixture of acetic acid, propionic acid, and trimethylamine as compared to test these compounds separately. It was concluded that the volatile substances present in the bovine vaginal fluid during oestrus may act as chemical communicators.

Diversity and community structure of butterfly of Arignar Anna Zoological Park, Chennai, Tamil Nadu.

Investigation was carried out on the diversity of butterfly fauna in selected localities of conservation and breeding center of Arignar Anna Zoological Park (AAZP), Chennai, Tamil Nadu. Atotal of 56 species were recorded, 15 of them belonged to Pieridae, 12 Nymphalidae, 9 Satyridae, 8 Papilionidae, 7 Danaidae, 3 Lycaenidae and 1 species each belonged to the families Acraeidae and Hesperidae. Qualitatively and quantitatively Pieridae family were comparatively dominant than that of other families. The notable addition to the 25 more species listed during this observation were compared to previous field survey. Comparison of butterfly species distribution between the different localities revealed that butterfly species richness was higher at mountain region with 52 species and lowest of 25 species at public visiting areas. Visitor's activities may be that reason for effects on butterfly distribution and lack of vegetation. Each five endemic and protected species (i.e. endangered) listed under the Wildlife (Protection)Act were highlighted greater conservation importances of the AAZP. It is suggest that butterfly species diversity generally increase with increase in vegetation and declines with the increase in disturbance.

Spermidine may decrease ER stress in pancreatic beta cells and may reduce apoptosis via activating AMPK dependent autophagy pathway.

The risk for diabetes increases with increasing BMI<25. Insulin resistance is the key factor for type 2 diabetes; studies revealed that endoplasmic reticulum stress is the main factor behind this disease. With increase in ER stress, pancreatic beta cells start to undergo apoptosis, leading to a decline in the pancreatic beta cell population. The ER stress arises due to unfolded protein response. Recently, spermidine get importance for increasing the longevity in most of the eukaryotes including yeast, Caenorhabditis elegans, Drosophila and human peripheral blood mononuclear cells via induction of autophagy pathway. Autophagy is also involved in regulation of scavenging of proteins. One of the major cellular pathways for scavenging the aggregated intracellular protein is autophagy. Hence spermidine can be a candidate for the treatment type 2 diabetes. Autophagy genes are regulated by mTOR (mammalian Target Of Rapamycin) dependent or independent pathway via AMPK. Hence either inhibition of mTOR or activation of AMPK by spermidine will play two crucial roles, first being the activation of autophagy and secondly the reduction of endoplasmic reticulum stress which will reduce beta cell death by apoptosis and thus can be a novel therapeutic candidate in the treatment of insulin resistance in type 2 diabetes and preserving pancreatic beta cell mass.

Identification of urinary sex pheromones in female buffaloes and their influence on bull reproductive behaviour.

The present study was carried out to identify the urinary sex pheromones of buffaloes and their role in relation to bull reproductive behaviour. Urinary samples were collected from 12 buffalo heifers at four different phases of estrous cycles. Fourteen compounds were identified throughout the cycle, which included phenol, ketone, alkane, alcohol, amide, acid and aldehyde. Among the 14 chemical profiles, three compounds were only found on the day of estrus, viz. 1-chlorooctane, 4-methylphenol and 9-octadecenoic acid. Behavioural investigation clearly showed that bulls were attracted and exhibited repeated flehmen behaviour towards the 4-methyl phenol. The bulls displayed penile erection and mounting behaviour while exposed to 9-octadecenoic acid. However, the other compound, 1-chlorooctane, did not influence such sexual behaviours. The present results provide evidence that the estrus-specific urinary volatile compounds appear to be sex pheromones which initiate the bull's reproductive behaviour.

Zooplankton diversity and physico-chemical conditions in three perennial ponds of Virudhunagar district, Tamilnadu.

Plankton diversity and physico-chemical parameters are an important criterion for evaluating the suitability of water for irrigation and drinking purposes. In this study we tried to assess the zooplankton species richness, diversity and evenness and to predict the state of three perennial ponds according to physico-chemical parameters. A total of 47 taxa were recorded: 24 rotifers, 9 copepods, 8 cladocerans, 4 ostracods and 2 protozoans. More number of zooplankton species were recorded in Chinnapperkovil pond (47 species) followed by Nallanchettipatti (39 species) and Kadabamkulam pond (24 species). Among the rotifers, Branchionus sp. is abundant. Diaphanosoma sp. predominant among the cladocerans. Among copepods, numerical superiority was found in the case of Mesocyclopes sp. Cypris sp. repeated abundance among ostracoda. Present study revealed that zooplankton species richness (R1 and R2) was comparatively higher (R1: 4.39; R2: 2.13) in Chinnapperkovil pond. The species diversity was higher in the Chinnapperkovil pond (H': 2.53; N1: 15.05; N2: 15.75) as compared to other ponds. The water samples were analyzed for temperature, pH, electrical conductivity alkalinity salinity, phosphate, hardness, dissolved oxygen and biological oxygen demand. Higher value of physico-chemical parameters and zooplankton diversity were recorded in Chinnapperkovil pond as compared to other ponds. The zooplankton population shows positive significant correlation with physico-chemical parameters like, temperature, alkalinity phosphate, hardness and biological oxygen demand, whereas negatively correlated with rainfall and salinity. The study revealed that the presence of certain species like, Monostyla sp., Keratella sp., Lapadella sp., Leydigia sp., Moinodaphnia sp., Diaptomus sp., Diaphanosoma sp., Mesocyclopes sp., Cypris sp. and Brachionus sp. is considered to be biological indicator for eutrophication.

Using mass spectrometry to detect buffalo salivary odorant-binding protein and its post-translational modifications.

A large number of mammalian odorant-binding proteins, which are lipocalins, have been studied. These proteins participate in peri-receptor events by selecting and carrying odorant molecules. The present study aimed at identifying the buffalo salivary odorant-binding protein (sOBP), and to determine its post-translational modification using mass spectrometry. The buffalo salivary 21 kDa protein was initially separated adopting sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it was identified as sOBP with high statistical reliability using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and SEQUEST, for the first time. Further, the post-translationally modified peptides were screened adopting MS/MS. A total of four post-translational modifications, namely glycation at lysine-(59), hydroxylation at lysine-(134), ubiquitination at lysine-(121), and dihydroxylation in lysine-(108), were recorded. Moreover, these modifications have not been identified in buffalo salivary odorant-binding protein.