AN ASSESSMENT OF THE CARRIER STATE AND A NOVEL MARKER OF \u3ci\u3eLEPTOSPIRA\u3c/i\u3e AND ABORTION IN CENTRAL KENTUCKY HORSES

Leptospirosis is a reemerging zoonotic infection of worldwide importance and affects all mammals. The bacterium is transmitted to animals and humans by urine, fetal membranes and body fluids. Leptospira shedding in the urine contaminates both soil and water, exposing both humans and animals to the bacterium. Leptospirosis in horses can cause abortion and is one of the etiologies of equine recurrent uveitis which can lead to blindness. Equine leptospiral abortion in Central Kentucky is primarily caused by serovar Pomona, with occasional cases attributed to serovar Grippotyphosa. There are a few reports in the literature attributing abortion to serovar Bratislava in the United States. Interestingly, Bratislava has the highest seropositivity in the horse in the United States. Two studies were conducted that are included in this dissertation. The first was to determine the prevalence of leptospirosis in horses located in Central Kentucky submitted for necropsy to the University of Kentucky Veterinary Diagnostic Laboratory. Heart, vitreous humor, kidney and urine samples were collected for microagglutination testing (MAT) and real-time PCR (qPCR). Heart blood and vitreous were tested using MAT for serovars Grippotyphosa, Pomona and Bratislava. Kidney, vitreous and urine samples were tested for pathogenic Leptospira by qPCR. MAT test results for heart blood indicated an increased seroprevalence for Bratislava as compared to Grippotyphosa and Pomona. Three horses had positive titers for serovar Bratislava in vitreous and heart blood samples. All urine samples tested negative by qPCR, and only one kidney sample had a weak positive result. Four vitreous samples tested positive for leptospirosis by qPCR, but all samples were negative upon MAT testing. No samples with positive MAT titers were positive by qPCR for any of the samples tested. Females were more likely to have positive MAT titers and were considerably older than males. MAT titers in females were also significantly higher as compared males. Finally, there was widespread seroprevalence in horses, regardless of the reason for necropsy submission. This suggests that exposure to Leptospira on Central Kentucky horse farms is common and the risk of exposure to humans and other animals is possible. The second study evaluated the enzyme heme oxygenase-1 (HO-1) and its potential as a marker for abortion in pregnant mares with elevated MAT titers. HO-1 is the rate-limiting step in the breakdown and degradation of heme to carbon monoxide, free iron and biliverdin, which is converted to bilirubin. Increased levels of HO-1 and its’ by-products are upregulated during inflammation and sepsis. HO-1 together with its by-products are essential in protecting the body from both increased inflammation and in reducing the risk of sepsis in humans, mice and rats. HO-1 together with its by-products are also essential in helping maintain pregnancy in humans, mice and rats. HO-1 in the serum of horses has not been previously investigated. The presence of HO-1 in the serum of healthy non-pregnant mares (NPM), pregnant mares throughout pregnancy (PMOT), pregnant mares 7 months pregnant with and without placentitis (PM) and pregnant mares with high MAT titers (MATS) was investigated. HO-1 levels in both NPM and PMOT were lower than PM, and significantly lower than MATS. Pregnancy alone increased HO-1, the same has been shown in other mammals and humans. Additionally, mares pregnant with elevated leptospiral titers had significantly higher HO-1 levels compared to other mares. This indicates that both pregnancy and high MAT titers increase the animals’ HO-1 response. Further investigation of HO-1 in horses is needed to ascertain its’ importance during infection and/or pregnancy and potential therapeutics

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

AbstractTo understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2−/−mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2−/− mice.The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders

Cytotoxic, anti-inflammatory and adipogenic effects of selected flavonoids on cell lines

Naturally-occurring flavonoids have tremendous potential for producing of new therapeutic agents that provide many benefits to mankind.This study focused on the evaluation of the in vitro cytotoxic, anti-inflammatory and adipocyte differentiation effects of the selected flavonoids which were inophyllum D, calanone, and isocordataoblongic acid from Calophyllumsymingtonianum as well as morelloflavone from Garciniaprainiana on MCF 7 human breast cancer cells, RAW 264.7 macrophages and 3T3-L1 pre-adipocytes respectively. The cytotoxicity study on MCF 7 human breast cancer cells was conducted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Meanwhile, the study of anti-inflammatory effects in RAW 264.7 macrophages and adipogenic effects on 3T3-L1 pre-adipocytes were conducted through nitrite determination assay and induction of adipocyte differentiation respectively. In the cytotoxicity study, inophyllum D was the only compounds that exhibited significant cytotoxic effect against MCF 7 human breast cancer with IC50 of 84 μg/mL. Further, all the compounds have shown anti-inflammatory effects in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages with inhibition of nitrite concentration as compared to the positive control. Besides, all the compounds in the range of the tested concentrations also produced adipogenic effects on 3T3-L1 pre-adipocytes and this may suggest that they exhibited potential anti-hyperglycemic property which mimicking the insulin action. Thus, this study may provide significant implication in the discovery of the potential of these selected flavonoids as alternative anti-cancer, anti-inflammatory and anti-hyperglycemic drugs

Effects of Nitric Oxide Synthase Inhibitors on Equine and Bovine Follicular Dynamics and Steroidogenesis.

Current scientific literature has shown that: (1) NO is a pro-ovulatory agent and inhibition of its production inhibits ovulation, and (2) NO has strong antisteroidogenic activity, especially on estradiol (E2) production by granulosa cells. The experiments described in this dissertation were designed to answer the following questions involving effects of NO on ovarian physiology: (1) is the inhibitory effect of nitric oxide synthase (NOS) inhibitors on ovulation irreversible (follicular atresia) or temporary (delayed ovulation)? and (2) what is the relationship between NO and hCG-induced preovulatory luteinization? Accordingly, cycling mares were subjected to treatment with NOS inhibitors (L-NAME or aminoguanidine) during estrus. Results showed that administration of NOS inhibitors temporarily inhibited hCG-induced ovulation for up to 5 d. Using a similar experimental approach, Holstein cows treated with L-NAME had delayed ovulation and an attenuated post-ovulation rise in plasma progesterone (P4) concentration. In a subsequent experiment, analyses of follicular fluid from mares in estrus showed that NO concentrations were increased after hCG administration. Furthermore, equine and bovine granulosa cell cultures treated with NOS inhibitors showed that inhibition of NOS activity in vitro luteinization as evidenced by decreased P4:E2 ratios. Pharmacokinetic and hemodynamic parameters associated with L-NAME administration in horses demonstrated that L-NAME has a short half life (3.6 min) as opposed to its major metabolite L-NNA (19.5 h). The results of these experiments demonstrated that: (1) administration of NOS inhibitors delayed ovulation in mares and cows; (2) intrafollicular production of NO was up-regulated following hCG administration; (3) inhibition of NO prevented preovulatory follicular luteinization and in vitro luteinization of granulosa cells; (4) both nonspecific and iNOS-specific inhibitors caused significant effects both in vivo and in vitro and (5) L-NAME has a short half life in horses and L-NNA is a major metabolite with a relatively long half life. Finally, these results will contribute to designing pharmacological therapies for manipulating ovarian function for experimental or therapeutic purposes. Future studies investigating the involvement of NO with preovulatory luteinization will likely contribute to the understanding of ovarian dysfunction, such as cystic ovarian syndrome or unruptured luteinized follicles. These conditions affect both animals and humans

Aluminum Reproductive Toxicity: A Summary and Interpretation of Scientific Reports

Publications addressing aluminum (Al)-induced reproductive toxicity were reviewed. Key details were compiled in summary tables. Approximate systemic Al exposure, a measure of bioavailability, was calculated for each exposure, based on the Al percentage in the dosed Al species, Al bioavailability, and absorption time course reports for the exposure route. This was limited to laboratory animal studies because no controlled-exposure human studies were found. Intended Al exposure was compared to unintended dietary Al exposure. The considerable and variable Al content of laboratory animal diets creates uncertainty about reproductive function in the absence of Al. Aluminum-induced reproductive toxicity in female mice and rats was evident after exposure to ≥ 25-fold the amount of Al consumed in the diet. Generally, the additional daily Al systemic exposure of studies that reported statistically significant results was greater than 100-fold above the typical human daily Al dietary consumption equivalent. Male reproductive endpoints were significantly affected after exposure to lower levels of Al than females. Increased Al intake increased fetus, placenta, and testes Al concentrations, to a greater extent in the placenta than fetus, and, in some cases, more in the testes than placenta. An adverse outcome pathway (AOP) was constructed for males based on the results of the reviewed studies. The proposed AOP includes oxidative stress as the molecular initiating event and increased malondialdehyde, DNA and spermatozoal damage, and decreased blood testosterone and sperm count as subsequent key events. Recommendations for the design of future studies of reproductive outcomes following exposure to Al are provided

Ovarian Follicle Growth and Yolk Formation in the New World Marsupial Monodelphis domestica

The adult Monodelphis domestica ovary was found to be similar to that of the eutherian mammal possessing follicles in various stages of development, corpora lutea and interstitial tissue. Polyovular follicles, containing between two and eleven oocytes, were found in approximately 25% of the individuals observed. Folliculogenesis proceeded in an identical manner to that in the eutherian but oocyte growth continued throughout follicular development. It therefore did not conform to the strict biphasic pattern of follicle growth previously described in eutherian and marsupial species. Oestrous cycles were monitored and, contrary to previous studies, female M domestica isolated from males continued to exhibit oestrous cycles. All females appeared to exhibit repetitive cycles of ovarian follicle growth accompanied by changes in the reproductive tract. If exposed to the stimulus of a male, a group of large follicles completed their development and ovulation occurred. In the absence of such stimulus, these follicles became atretic and were replaced by a successive wave of follicles. At the ultrastructural level, the growth and development of the oocyte was accompanied by an increase in the number of its organelles and a change in their distribution which reflected the metabolic activity involved during such growth. Apart from the conventional organelles found within oocytes, lamellar complexes and two unusual forms of mitochondria were observed at different stages of development. Smooth endoplasmic reticulum and Golgi bodies appeared to be responsible for the formation of multivesicular bodies which were first observed in the oocytes of primary follicles. As the multivesicular bodies increased in size they became more flocculent in nature until they formed transparent vesicles which exhibited no polarity. These then coalesced to form a large vesicular mass, occupying most of the central region of the oocyte, which was identical to the "yolk" mass described in other marsupial species. Histochemical analyses revealed that in smaller oocytes the cytoplasm consisted mostly of carbohydrate and protein but during development this was replaced by the yolk vesicles. The nature of these vesicles remained undetected by histochemical staining and it was only by means of molecular probe labelling that lipid was detected. Autoradiographical analysis revealed that metabolites were transported from the circulation into the oocytes and that this uptake was fairly uniform in all developmental stages of oocyte. For the first time in a marsupial, individual preantral follicles were cultured in vitro. Although follicles grew for up to 8 days, and sometimes exceeded the size where antrum formation was observed in vivo, definite antrum formation was not achieved

Absence of accessory sex gland secretions in the ejaculate alters the profile of proteins in uterine fluid after mating and early pregnancy in the golden hamster (Mesocricetus auratus).

Ho Wing Ki Vicky.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 191-211).Abstracts in English and Chinese.Abstracts --- p.iAbstracts in Chinese --- p.iiiAcknowledgements --- p.vTable of Abbreviations --- p.viTable of Contents --- p.ixList of Figures and Tables --- p.xivChapter Chapter 1 --- General Introduction --- p.1Chapter 1.1. --- The Male Accessory Sex Glands --- p.2Chapter 1.1.1. --- The Prostatic Complex --- p.4Chapter 1.1.2. --- The Coagulating Gland --- p.4Chapter 1.1.3. --- The Seminal Vesicles --- p.5Chapter 1.1.4. --- The Ampullary Gland --- p.6Chapter 1.1.5. --- The Cowper's gland --- p.6Chapter 1.1.6. --- The Littre's Gland --- p.7Chapter 1.2. --- The Uterus --- p.7Chapter 1.2.1. --- The Structure of Uterus --- p.7Chapter 1.2.2. --- The Endometrium --- p.8Chapter 1.3. --- Identifications of Proteins by Two Dimensional Gel Electrophoresis and Matrix-assisted Laser Adsorption / ionization Time-of-flight (MALDI-TOF) Mass Spectrometry --- p.9Chapter 1.3.1. --- Principle of Two Dimensional Gel Electrophoresis --- p.9Chapter 1.3.2. --- Principle of MALDI-TOF Mass Spectrometry --- p.11Chapter 1.4. --- Hypothesis and Aim of this Study --- p.12Chapter 1.4.1. --- Hypothesis --- p.13Chapter 1.4.2. --- Aim --- p.13Legends and Figures --- p.14Chapter Chapter 2 --- The Effect of Omission of Male Accessory Sex Gland Secretions on Post Coital Uterine Fluid --- p.17Chapter 2.1. --- Introduction --- p.18Chapter 2.1.1. --- Uterine Fluid at the Time of Mating --- p.18Chapter 2.1.2. --- Ventral Prostate Secretory Proteins --- p.20Chapter 2.1.3. --- Proteins Specifically Bind to Sperm Membrane --- p.22Aim of Study --- p.23Chapter 2.2. --- Materials and Methods --- p.24Chapter 2.2.1. --- Animal Model --- p.24Chapter 2.2.1.1. --- Female golden hamsters --- p.24Chapter 2.2.1.2. --- Male golden hamsters --- p.25Chapter 2.2.2. --- Collections of Biological Samples --- p.26Chapter 2.2.2.1. --- Female Hamster Serum --- p.26Chapter 2.2.2.2. --- Uterine Fluid --- p.26Chapter 2.2.2.3. --- Uterine Tissues --- p.27Chapter 2.2.2.4. --- Ejaculated Sperm in Uterus --- p.27Chapter 2.2.2.5. --- ASG Secretions --- p.27Chapter 2.2.2.6. --- ASG Tissues --- p.28Chapter 2.2.2.7. --- Epididymal Sperm --- p.28Chapter 2.2.3. --- Isolation and Purification of Sperm Binding Ventral Prostate Proteins (SBVPP) --- p.28Chapter 2.2.3.1. --- Plasma Membrane Protein from Epididymal Sperm --- p.28Chapter 2.2.3.2. --- Affinity Chromatography --- p.29Chapter 2.2.3.3. --- FPLC Gel Filtration --- p.30Chapter 2.2.4. --- Histology of Flushed Uterus --- p.30Chapter 2.2.5. --- Quantification of Protein --- p.31Chapter 2.2.6. --- Two dimensional Electrophoresis --- p.31Chapter 2.2.6.1. --- First DimensiońؤIsoelectric Focusing --- p.32Chapter 2.2.6.2. --- Second DimensiońؤSDS PAGE --- p.32Chapter 2.2.6.3. --- Visualization of Protein Spots --- p.33Chapter 2.2.7. --- Identification of Proteins --- p.34Chapter 2.2.7.1. --- Protein Spots Detection --- p.34Chapter 2.2.7.2. --- MALDI-TOF --- p.34Chapter 2.2.7.2.1. --- In-Gel Digestion for MALDI-TOF --- p.34Chapter 2.2.7.2.2. --- MALDI-TOF Analysis --- p.35Chapter 2.2.7.2.3. --- MALDI-TOF Data Analysis --- p.36Chapter 2.2.8. --- Confirmation of Prostate Specific Antigen (PSA) --- p.37Chapter 2.2.9. --- Confirmation of Prostatic Acid Phosphatase (PAP) --- p.37Chapter 2.2.10. --- Confirmation of Tumor Necrosis Factor Stimulated Gene-6 (TSG-6) --- p.37Chapter 2.2.11. --- Scanning Electron Microscopy (SEM) --- p.38Chapter 2.3. --- Results --- p.39Chapter 2.3.1. --- Morphology of Flushed Uterus --- p.39Chapter 2.3.2. --- Protein Concentration --- p.39Chapter 2.3.3. --- "Serum, Pre and Post Coital Uterine Fluid Protein Profiles" --- p.39Chapter 2.3.4. --- ASG Secretory Proteins and Their Fate After mating --- p.40Chapter 2.3.4.1. --- ASG Secretory Proteins and 1 hp.c. Uterine Fluid --- p.40Chapter 2.3.4.2. --- VP Secretory Proteins and SBVPP --- p.40Chapter 2.3.4.3. --- PAP and PSA --- p.41Chapter 2.3.5. --- Protein Profile at 1 h p.c --- p.41Chapter 2.3.6. --- Protein Profile at 4 h p. c --- p.42Chapter 2.3.7. --- Identification of Proteins Differentially Expressed by the Three Groups at 1 h p .c --- p.43Chapter 2.3.7.1. --- Confirmation of TSG-6 --- p.43Chapter 2.3.8. --- SEM --- p.44Chapter 2.4. --- Discussion --- p.45Chapter 2.5. --- Conclusion --- p.55Legends and Figures --- p.56Chapter Chapter 3 --- The Effect of Omission of Male Accessory Sex Glands Secretions on Periimplanation Uterine Fluid Proteins --- p.114Chapter 3.1. --- Introduction --- p.115Chapter 3.1.1. --- Cytokine & Growth Factor --- p.115Chapter 3.1.1.1. --- Vascular Endothelial Growth Factor (VEGF) --- p.115Chapter 3.1.1.2. --- Leukaemia Inhibitory Factor (LIF) --- p.116Chapter 3.1.1.3. --- Insulin-like Growth Factors (IGFs) --- p.116Chapter 3.1.1.4. --- Interleukin 1 (IL-1) --- p.117Chapter 3.1.1.5. --- Adhesion Molecule --- p.118Chapter 3.1.2. --- Uterine Secretory Proteins --- p.118Chapter 3.1.2.1. --- Estrogen Dependent Protein --- p.119Chapter 3.1.2.2. --- Progestin-Dependent Endometrial Protein --- p.120Chapter 3.1.2.3. --- Uteroglobin --- p.121Chapter 3.1.2.4. --- Uteroferrin --- p.122Chapter 3.1.2.5. --- Prolactin --- p.123Chapter 3.1.2.6. --- Glycodelin --- p.123Chapter 3.2. --- Aim of Study --- p.124Chapter 3.3. --- Method and Materials --- p.124Chapter 3.3.1. --- Animal and Surgery --- p.125Chapter 3.3.2. --- Collection of Biological Samples --- p.125Chapter 3.3.2.1. --- Uterine Fluid --- p.125Chapter 3.3.2.2. --- Uterine Tissue --- p.125Chapter 3.3.3. --- Quantification of Protein --- p.125Chapter 3.3.4. --- Two Dimensional Electrophoresis --- p.125Chapter 3.3.5. --- Identification of Proteins --- p.126Chapter 3.3.6. --- Confirmation of Growth Differentiation Factor-8 (DGF-8) by PCR --- p.126Chapter 3.3.7. --- Confirmation of GDF-8 by Western Blotting --- p.126Chapter 3.4. --- Results --- p.128Chapter 3.4.1. --- Protein Concentration --- p.128Chapter 3.4.2. --- 60 Hours Post Coitum --- p.128Chapter 3.4.2.1. --- Protein Profile --- p.128Chapter 3.4.2.2. --- Differentially Expressed Protein Spots and Proteins Identification --- p.129Chapter 3.4.3. --- 72 Hours Post Coitum --- p.129Chapter 3.4.3.1. --- Protein Profile --- p.129Chapter 3.4.3.2. --- Differentially Expressed Protein Spots and Proteins Identification --- p.130Chapter 3.4.3.3. --- Confirmation of Growth differentiation factor 8 (GDF-8) --- p.130Chapter 3.5. --- Discussion --- p.132Chapter 3.6. --- Conclusion --- p.139Legends and Figures --- p.140Chapter Chapter 4 --- General Conclusion --- p.186Chapter 4.1. --- Conclusion --- p.187Chapter 4.2. --- Further Studies --- p.190References --- p.191Appendices --- p.21

Selenium

prepared by Syracuse Research Corporation under contract no. 205-1999-00024 ; prepared for U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry."September 2003."Chemical manager(s)/author(s): John Risher ... [et al.]--P. ix."A Toxicological Profile for selenium, Draft for Public Comment was released in September, 2001. This edition supersedes any previously released draft or final profile"--P. iii."This toxicological profile is prepared in accordance with guidelines developed by the Agency for Toxic Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA). The original guidelines were published in the Federal Register on April 17, 1987"--P. v.Also available via the World Wide Web.Includes bibliographical references (p. 315-411)

Evaluation of spermatogenic, aphrodisiac and anti-oxidant activity of classical siddha drug Thathu Viruthi Chooranam in rodents

Herbal preparations in Siddha system of medicine always have a unique range of beneficial effect because of its wonderful therapeutic value without causing adverse effects. Here the drug Thathu Viruthi Chooranam was pharmacologically evaluated and scientifically validated and standardized. The ingredients of the drug was identified and authenticated by Gunapadam experts. The drug was prepared as per classical Siddha literary procedure and subjected to various studies to reveal its potency and efficacy of the drug. The organoleptic character and physicochemical studies were made into standardization of the drug TVC. From the above studies the TVC is standardized as per AYUSH guidelines. The biochemical and instrumental analysis was made to know the presence of active ingredients in the drug which is responsible for its activity. Here, the biochemical analysis showed the presence of Calcium, Magnesium, Zinc, Sulphate, Chloride by its synergistic effects, the drug as activity against the disease. In instrumental analysis, FTIR showed the OH group has higher potential towards inhibitory activity against microorganisms. Sometimes the presence of Phenols in medicinal plants possess highly Anti-Oxidant property which enhances the drug effect against the disease. SEM picture explained the particle size of the drug. In ICP-OES described about the absence of heavy metals and its permissible limits which showed the safety of the drug. TLC plate showed different colour phytoconstituents of chloroform extract of TVC. The bands revealed presence of seven violets, four reds, two greens, one blue, one white and one yellow showing the presence of alkaloids, glycosides, phenols, triterpenes, flavonoids and quinines. The drug Thathu Viruthi Chooranam was proved that it is free from toxicity through the acute and 28 days repeated oral toxicity study as per the OECD guidelines. In acute toxicity study there was no mortality of rats observed. In 28 days repeated oral toxicity study, the obtained results of haematological, biochemical, urinary parameters were normal. The histopathological findings did not show any abnormalities. And this herbal formulation possess more potent Aphrodisiac activity in ethanol treated male rats. This trial drug Thathu Viruthi Chooranam exhibits high range of Spermatogenic activity in ethanol induced male rats. Most of the research findings already reported that the role of antioxidant is essential to treat male infertility. Here the trial drug possess strong antioxidant activity. These results conclude that the drug Thathu Viruthi Chooranam is a best drug of choice for the treatment of male infertility. By analyzing all those findings, it is proved that the drug Thathu Viruthi Chooranam has high range of therapeutic value and the safety of the drug for clinical use is ensured. So, it is confirmed that the herbal formulation Thathu Viruthi Chooranam may never cause any adverse effects in clinical use. The preparation of the drug is cost effective too. So, the clinical trials have to be followed on the drug Thathu Viruthi Chooranam. Thus can treat the infertile males effectively and help them to achieve their dream of producing their own offspring. CONCLUSION: The drug “Thathu Viruthi Chooranam” was prepared as per the classical Siddha literature Sarabenthirar Vaithiya Rathnavali. It fulfills all the standardization parameters of Chooranam as mentioned in AYUSH guidelines. The results of biochemical analysis showed the presence of acid and basic radicals in the sample. The presence of alkaloids, flavonoids, glycosides, tannins, triterpenoids were identified in the choloroform and methanolic extract of Thathu Viruthi Chooranam through Phytochemical analysis. TLC plate showed different colour phytoconstituents of chloroform extract of TVC. FT-IR Study results exhibits the presence of some organic functional groups such as alcohols, phenols, alkenes, amines, aromatics, aliphatic amines, alkyl halides. SEM analysis showed the objects of sizes ranging from 3μm to5 μm. XRD pattern of the trial drug Thathu Viruthi Chooranam shows good crystalinity. Based on OECD 423 the trial drug Thathu Viruthi Chooranam is considered as non toxic up to the dose of 2000mg/kg. The drug Thathu Viruthi Chooranam possess the potent Aphrodisiac activity in ethanol treated male rats. The drug Thathu Viruthi Chooranam could be confirmed as no-observedadverse-effect level (NOAEL) drug as it acts harmlessly under the current normal usage and this phenomenon is considered to be of no toxicological concern. High antioxidant therapeutic nature was evaluated in the drug Thathu Viruthi Chooranam through DPPH Scavenging assay. Because of this anti-oxidant property, it can be used as a drug to treat the male infertility. Hence it is proved that the drug Thathu Viruthi Chooranam was pharamacollogicaly evaluated for the property of Spermatogenic, Aphrodisiac and Antioxidant activity

Heavy Metal Toxicity in Public Health

It is often said that the “dosage” of any substance determines its remedy or poison effect. Heavy metal sources encompass sewage, pesticides, fertilizers, environmental contamination, occupational exposure/contact through inhalation, ingestion, and skin. Before the advent of technology/the industrial revolution, communicable diseases ravaged the human race but this seems to have given way to non-communicable diseases such as cancers, renal failure, hormonal distortion enzymes, inhibition of fetal growth, and DNA damage causing negative health issues due to heavy metals. This book brings to the fore probably the most recent experimental research/review on heavy metal contamination, remediating techniques, cellular tissue damage, and toxicological and antioxidant effects of heavy metals. It is hoped that its contents will make interesting reading for all