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ThesisItem Open Access ROLE OF EUGENOL IN REVERSAL OF VASCULAR DYSFUNCTION INDUCED BY EXPERIMENTAL DIABETES AND HYPERTENSION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-10) Vamsikrishna, Bobba; Srinivasa Rao, G(MAJOR); Ravi Kumar, P; Rama Devi, V; Vinoo, RABSTRACT : Phenylpropanoids are a diverse group of phytochemicals with immense health benefits and found throughout the plant kingdom. Eugenol is a member of the phenylpropanoids and is remarkably versatile molecule which is present abundantly in clove, nutmeg, cinnamon, basil and bay leaf. Epidemiological evidence and clinical trial data indicates that due to presence of biologically active phytochemicals, the plant originated diets can reduce the risk of chronic disease conditions such as cardiovascular disease, hypertension, diabetes and cancer. Hypertension and diabetes are the lifestyle diseases which are considered to be main causes of mortality for decades in humans. Vascular dysfunction is the major change that is associated with diabetes and hypertension. It is well documented that both diabetes and hypertension occur together in most of the human beings that is an additive cause for increase in risk of vascular complications. Though there are standard treatments available at present for these complications, a look for an alternative approach that can better address the vascular problems is most wanted. Hence the present study was designed to know the effect of eugenol against vascular dysfunction associated with either diabetes or hypertension alone and diabetes with hypertension together. The study was carried out in rats that are divided into eight groups with ten rats in each. Group-I (normal control) received vehicle alone for eight weeks whereas group II (eugenol control) received eugenol orally and daily at the rate of 80 mg/kg for eight weeks. Group- III, IV and V constitute experimentally induced hypertension control, diabetic control and diabetic rats with hypertension. Hypertension was induced in rats with administration of L-NAME in drinking water (40 mg/kg/day). Whereas single dose of streptozotocin was injected intraperitoneally at 40 mg/kg for inducing diabetes in rats. Group V rats received both streptozotocin and L-NAME. Group- VI (eugenol treated hypertensive rats), VII (eugenol treated diabetic rats) and VIII (eugenol treated diabetic rats with hypertension) received eugenol 80 mg/kg orally from the day after the onset of diabetes, hypertension or both conditions experimentally. Development of hypertension and diabetes in rats was confirmed by decrease in total nitrate/nitrite levels in serum and high blood glucose levels (>300 mg/dl) respectively. At the end of the study, rats were sacrificed and thoracic aorta was collected for studying vascular reactivity and histopathology. In addition, effect of eugenol in ameliorating the oxidative stress induced by experimental diabetes, hypertension and diabetes associated hypertension was also studied. Moreover, liver and kidney function markers in plasma were estimated in different study groups to know the effect of eugenol on liver and kidney function. Total nitrate (NO3-) and nitrite (NO2-) levels in serum were significantly (P < 0.05) decreased in hypertension control, diabetic control and diabetes associated hypertensive rats. Eugenol treatment had no impact on reversing the nitrate and nitrite levels in diabetes and hypertension back to the normal values noticed in control rats. Hyperglycemia was observed both in diabetic and diabetic hypertensive rats. Eugenol treatment did not have any effect in restoring the blood glucose levels to normal. Eugenol treatment could not show any favorable effect on body weight that had reduced in diabetic and diabetic hypertensive rats. Eugenol treatment had no effect on increased oxidative stress noticed in diabetic, hypertensive and diabetic hypertensive rats. Levels of liver function markers were raised in diabetic and diabetic hypertensive rats indicating liver damage and eugenol had no protective effect on liver damage. But elevated plasma creatinine, blood urea nitrogen levels and reduced plasma total protein in diabetic and diabetic hypertensive rats were restored to normal by eugenol treatment indicating protective effect of eugenol on kidney. Vascular reactivity was studied in-vitro by taking myographic recordings of aorta as described here; 1. Contractile response to phenylephrine and 5-HT. 2. Ach relaxation on phenylephrine and 5-HT induced contraction. 3. Eugenol relaxation of phenylephrine and 5-HT induced contraction. The lower mean log EC50 values of phenylephrine (-7.856 M) and 5-HT (-6.967 M) in hypertensive control and diabetic hypertensive rats (Phe: -7.960 M and 5-HT: -7.035 M) demonstrates hyper responsiveness of aortic smooth muscle to phenylephrine and 5-HT in comparison with normal control (Phe: -6.588 M and 5-HT: -5.700 M), and hyper-responsiveness of aorta to phenylephrine was partially reversed by eugenol treatment. But aorta from diabetic control rats showed hyper-responsiveness to phenylephrine (-7.137 M) and hypo reactivity to 5-HT (-5.247 M) compared to normal control rats. Eugenol treatment showed no impact on hyper-reactivity to phenylephrine or hypo-reactivity to 5-HT in diabetic rats. Maximum relaxation (% Emax) by acetylcholine in aorta on phenylephrine and 5-HT induced contractions was significantly reduced in diabetic, hypertensive and diabetic hypertensive rats. The effect was complete in hypertension and diabetic hypertension. Eugenol treatment had no significant change on acetylcholine induced relaxation in diabetic rats but significantly (P<0.001) improved relaxation in hypertensive and diabetic hypertensive rats. Eugenol produced dose dependent relaxation on phenylephrine and 5-HT induced contraction in all experimental groups but its effect was less potent than acetylcholine. Emax of eugenol on phenylephrine induced contraction was reduced in hypertensive, diabetic and diabetic hypertensive rats. Eugenol treatment to diabetic and diabetic hypertensive rats significantly improved Emax of eugenol. Eugenol relaxation on 5-HT induced contraction in diabetic control, hypertensive control and diabetic hypertensive rats were similar to control rats. The pathological changes observed in aorta, heart and kidney due to hypertension, diabetes and diabetic hypertension were not reestablished to normal with eugenol treatment. In conclusion, eugenol partially reversed phenylephrine and 5-HT induced vascular hyper-responsiveness in aorta and augmented the relaxation to acetylcholine in hypertensive and diabetic hypertensive rats but failed to produce a similar response in diabetic rats. However, eugenol had no role in maintaining blood glucose and serum nitrate levels indicating its inability to alleviate diabetes and hypertension. Further, eugenol treatment could not modulate oxidative stress and histopathological changes induced by diabetes and hypertension in plasma, heart and kidney. Further studies are needed to know the molecular mechanism involved in partial reversal of vascular dysfunction by eugenol.ThesisItem Open Access OTOGENESIS IN THE FETUS OF SHEEP (Ovis aries)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-10) KARUNA SRI, VADDI; NAGAMALLESWARI, YAMANI(MAJOR); RAJU, N.K.B.; SREENU, MAKKENA; RAMANI PUSHPA, R.N.The present study was undertaken to elucidate the developmental changes in ear. The study was conducted on 60 embryos and fetuses of Nellore sheep between 22 to 145 gestational days. Morphogenesis revealed that five aural hillocks appeared at 23 days fused to form the pinna later. External acoustic meatus (EAM) appeared first at 24 days. Hairs were apparent on EAM by 126 days. Pinna was pendulous and elongated at 145 days. Tympanic cavity presented three tiny ossicles malleus, incus and stapes at the epitympanic region by 63 days. Rostral process of malleus and Lenticular bone were absent in sheep. The ossified part of tympanic ring appeared in semi lunar shape by 55 days. Ossification initiated in vestibule and cochlea by 70 days. Histogenesis revealed small cone shaped pinna at 24 days. Pharyngeal cleft modified into EAM by 39 days and canalized by 49 days. Epithelium of pinna was keratinized at 99 days. The meatal plug appeared first in 24 day embryos and formed primary external auditory canal (EAC) between 31 to 39 days. The ceruminous and sebaceous glands were first identified by 80 days. Middle fibrous layer of ear drum was formed at 126 days. Tympanic membrane appeared trilaminar and EAC was completely canalized by 140 days. Meckel’s and Reichert’s cartilages developed as mesenchymal condensation by 23 days. The distal part of Meckel’s cartilage modified into malleus and incus. Blastemal cells of Reichert’s cartilage modified as stapes. Tubo tympanic recess (TTR) differentiated between 24 to 27 days. Ossification initiated in malleus, incus and stapes at 63, 78 and 80 days respectively. Incudo-malleal and incudo-stapedial joints were established as diarthrodial joints by 80 and 85 days respectively. Eustachian tube lined by psedostratified ciliated columnar epithelium and Meckel’s cartilage disappeared by 85 days. Ossicular ligaments were differentiated at 104 days. The tympanic cavity comprised of fully grown ossified ossicles as adult by 140 days. Otic placode of inner ear differentiated into the otocyst and acoustico-facial ganglion was formed from otocyst by 22 days. Otocyst underwent extensive modification to form semicircular canals, vestibule and cochlea. Endolymphatic duct developed at 23 days. Posterior semicircular duct appeared first than anterior and lateral. The cochlear duct located ventrally within the developing otic capsule during 24 to 27 days. Utriculosaccular chamber (vestibule) differentiated into utricle and saccule with macula utriculi and macula sacculi respectively; Crista ampullaris developed in the ampulla of canal at 31 days. Thickening of epithelium in the cochlear duct formed organ of Corti by 46 days. Cochlea differentiated into scala vestibuli, scala tymapani and scala media at 69 days. The hair cells of organ of Corti matured first in basal turn at 104 days; cochlea was well developed with 21/2 turns indicated the complete inner ear formation as adult in 140 days sheep fetuses.ThesisItem Open Access ANTICARCINOGENIC ACTIVITY OF CINNAMALDEHYDE AND NANO CINNAMALDEHYDE AGAINST MAMMARY CANCER IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-05) Ravi, Killi; Ravi Kumar, P(MAJOR); Bharavi, K; Rama Devi, V; VINOO, RABSTRACT: Cancer is a growing health problem in both developing and developed countries. Cancer chemoprevention is defined as the use of natural or synthetic agents that reverse, suppress or arrest carcinogenic and/or malignant phenotypic progression towards invasive cancer. The effectiveness of many anticancer drugs is limited due to their inability to reach the target site in sufficient concentrations and efficiently exert the pharmacological effect on affected cells without harming the healthy cells. Therefore, the search for new anticancer agents with better efficacy and fewer side effects is an ongoing phenomenon. Plant phenolic compounds are a group of secondary metabolites with wide pharmacological activities (Al-Rimawi et al., 2016). Plant polyphenols have drawn increasing attention due to their potent antioxidant properties and their marked effects in the prevention of different oxidative associated diseases such as cancer (Dai and Mumper, 2010). trans- Cinnamaldehyde is a phenolic compound found naturally in various species of the genus Cinnamomum and is used in preparing beverages, medicinal products, perfumes and cosmetics. Nanoparticles (NPs) are versatile agents with a variety of biomedical applications including drug delivery. Keeping this in view, the present study was undertaken to evaluate the anticancer potential of cinnamaldehyde (CNMA) and its nano preparation, the nano zinc cinnamaldehyde (CZN) in chemical induced rat breast cancer model. Further, cinnamaldehyde was also studied for its disposition kinetics in rats. For induction of mammary tumors, fifty days old virgin female Sprague-Dawley rats were administered with single oral dose of 20 mg dimethyl benz(a)anthracene (DMBA). Those rats positive for mammary tumors on 90th day were only selected for further studies. The study was carried out in seven experimental groups viz. normal control (C), DMBA control (DMBA-C), cinnamaldehyde prophylactic (CNMA-P), cinnamaldehyde treatment (CNMA-T), nano cinnamaldehyde control (CZN-C), nano cinnamaldehyde treatment (CZN-T) and tamoxifen (TAM). CNMA-P group rats received cinnamaldehyde (50 mg/kg b.wt) orally starting from the 45th day i.e. five days prior to the DMBA administration and continued to received the same until the end of the study on 120th day. CNMA-T, CZN-C and TAM treatment groups received the respective treatments for a period of 30 days beginning from the 90th day. All the animals were sacrificed on 120th day. Plasma concentration of cinnamaldehyde and cinnamic acid were estimated in rats administered with cinnamaldehyde at a dose rate of 500 mg/kg b.wt. From the pharmacokinetic study it was evident that cinnamaldehyde is rapidly converted into cinnamic acid (CA). Apparent volume of distribution (Vd) and plasma clearance (Cl) were high for CNMA but its AUC0-t, MRT and t1/2 values were low when compared to CA. Tumor volume, tumor multiplicity, tumor burden, body weight, tissue antioxidant and peroxidation status and various plasma biochemical parameters including total sialic acid (TSA) levels were assessed in all experimental groups. In addition tumor latency period was recorded in CNMA-P group. Mammary tumor tissues were subjected to light and electron microscopic examination for pathological changes and immunohistological studies for the presence of estrogen and progesterone receptors. Prophylactic treatment with cinnamaldehyde increased the tumor latency period and decreased the tumor volume, tumor burden and tumor multiplicity. Compared to DMBA control group, cinnamaldehyde and its nano preparation showed favourable results in terms of tissue antioxidant profiles and plasma biochemical parameters. Mild to moderate regressive changes were observed in various treatment groups. However tamoxifen treated rats showed greater extent of favourable changes in terms of tumor histology. The study revealed that, cinnamaldehyde (CNMA) and nano zinc cinnamaldehyde (CZN) have mild degree of anticancer effects, with CZN being little more effective than CNMA. However CNMA was more effective when used prophylactically than when used as a therapeutic agent. Hence, it is likely that CNMA can reduce the possibility of breast cancer occurrence and severity when used prophylactically.ThesisItem Open Access EVALUATION OF RATIONS CONTAINING SLOW RELEASE UREA ON PRODUCTION PERFORMANCE OF RUMINANTS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-03) RAVI KANTH REDDY, P; Srinivas Kumar, D(MAJOR); Raghava Rao, E; Venkata Seshiah, Ch.; Satheesh, KABSTRACT: In the present study, two in vitro and four in vivo trials were conducted to evaluate the usage efficiency of slow release urea (Optigen II) product in buffalo bulls, adult rams, and ram lambs fed crop residue based total mixed rations and in lactating buffaloes fed green fodder and conventional concentrate mixture. An in vitro nitrogen release test was conducted to analyze the damage caused to SRU coating during handling and processing. The urea release from the coated urea (SRU) as supplied by manufacturer and SRU collected from the total mixed rations fed to adult rams, ram lambs, buffalo bulls, and lactating animals were 69.01, 85.68, 84.51, 82.16, and 76.29 per cent as much urea as uncoated urea in 1 h, revealing a considerable damage caused to the coating at varying levels in the four in vivo trials conducted. Another in vitro trial was conducted to assess the rate of NH3-N release from various protein sources used in the in vivo experiments. The rate of increase in NH3-N release was much more pronounced in urea group followed by SRU (Optigen II), CSM, SBM, and urea, with a significant (P<0.01) treatment x time interactions. In the 1st trial, four graded Murrah buffalo bulls (avg. b. wt. 365.47 ± 16.45 kg), arranged in a 4 x 4 LSD, were randomly allotted to four dietary treatments viz. total mixed ration (TMR) with maize stover and concentrate in 70: 30 proportion, incorporated with SRU at 0 (T1), 1.0 (T2), 2.0 (T3), and 3.0 (T4) per cent level, and evaluated for their effect on rumen fermentation pattern, mineral balances, and nutrient utilization in buffalo bulls. The rumen fermentation studies in fistulated Murrah buffalo bulls revealed that the pH, TVFA and nitrogen fractions i.e. NH3-N, TCA insoluble N and residual N were higher (P<0.01) in SRU fed groups (T2, T3, and T4) compared to control (T1). The concentration of TVFA, TCA ppt N, and Residual N peaked at 4 hours post feeding, while the NH3-N, total N, and food and protozoal N showed a dose-dependent relationship with the peak values at 4, 4, 6 and 8 hrs post feeding for T1, T2, T3 and T4, respectively. The digestibility coefficients of DM, OM, CP, NDF and hemi-cellulose were higher (P<0.05) in SRU incorporated rations as compared to the control. However, incorporation of SRU in rations had no effect (P>0.05) on the digestibility of EE, CF, NFE, ADF and cellulose. All the buffalo bulls were in positive balance for N, Ca and P. The intake (g/d) of N, Ca and P were similar among all treatments. Incorporation of SRU in the diets of buffalo bulls had no effect (P>0.05) on N, Ca and P retentions expressed either as g/d, per cent of intake or as per cent absorbed. The average DMI of buffalo bulls expressed as kg/d or as % BW was comparable among the treatments. The present study indicated that incorporation of SRU in the ration increased (P<0.05) the DCP and TDN content expressed as % in the diet consumed or as kg/d. Further, the DM, DCP, TDN and ME intakes per kg W0.75 were similar among the treatments and were higher than the values recommended by ICAR (1998) standards. In the 2nd trial, twelve lactating Murrah buffaloes were randomly allotted to two dietary treatments and fed hybrid Napier (APBN-1) ad libitum and concentrate mixture incorporated with SRU at 0 (T1) and 2.0 (T2) per cent level, and evaluated for their effect on nutrient utilization, milk yield and composition. The per cent digestibility of gross nutrients and cell wall fractions increased in lactating Murrah buffaloes fed rations incorporated with SRU as compared to the control, but the differences were not significant (P>0.05). Incorporation of SRU in the diet had no effect (P>0.05) on the DCP and TDN content expressed as % in the diet consumed or as kg/d. Further, the DM, DCP, TDN and ME intakes per kg W0.75 were similar among the treatments and were higher than the values recommended by ICAR (1998). The average yield of milk, fat, SNF, total solids, lactose, protein, butter fat yield, 6% FCM yield, and energy corrected milk yield were higher in buffaloes fed SRU compared to control but the differences were not significant (P>0.05). The feed efficiency (kg DMI per kg 6% FCM) was similar in both the rations. The cost of feed/kg 6% FCM was higher (P>0.05) in T1 compared T2, with a net increase in benefit over feed cost by 4.67% in SRU fed rations. In the 3rd trial, four adult rams (avg. b. wt. 43.02 ± 0.76 kg), arranged in 4 x 4 LSD were randomly allotted to four dietary treatments viz. TMR with green gram straw and concentrate in 60: 40 proportion, incorporated with SRU at 0 (T1), 1.0 (T2), 2.0 (T3), and 3.0 (T4) per cent level, and evaluated for their effect on serum urea nitrogen and nutrient utilization. The mean SUN concentration (M mol/L) was higher (P<0.01) in T4 and lower in T1 compared to other treatments. Time after feeding significantly (P<0.01) affected the SUN concentration with a peak at 4 h postprandial period. Further, Incorporation of SRU in the rations had no effect (P>0.05) on the digestibility of nutrients except CP which increased significantly (P<0.05) at 3.0% level as compared to the control. The DCP content expressed as per cent in the diet consumed or as intake (kg/d) was higher (P<0.05) in T4 as compared to T1. The present study indicated that incorporation of SRU in the diet had no effect (P>0.05) on the TDN content expressed as % in the diet consumed or as kg/d. Further, the DM, DCP, TDN and ME intakes per kg W0.75 were similar among the treatments and were higher than the values recommended by ICAR (2013). In the 4th trial, twelve ram lambs were randomly distributed to two dietary treatments and fed TMR with green gram straw and concentrate in 50: 50 proportions, incorporated with SRU at 0 (T1) and 2 (T2) per cent level, and evaluated for its effect on growth, nutrient utilization and carcass characteristics. The DMI expressed as Kg/d (P<0.05) or g/kg W0.75 (P<0.01) was lower in SRU fed group as compared to the control. Incorporation of SRU in the rations had no effect (P>0.05) on ADG, FCR (kg feed / kg gain) and cost of feed ( /kg gain). Incorporation of SRU in the TMR had no effect (P>0.05) on the digestibility of nutrients, except CP, which increased significantly (P<0.05) as compared to the control. Incorporation of SRU in the diet had no effect (P>0.05) on the DCP and TDN content expressed as % in the diet consumed or as kg/d. Further, the DM, DCP, TDN and ME intakes per kg W0.75 were similar among the treatments and were higher than the values recommended by ICAR (1998). Carcass studies revealed that the SRU incorporation had no effect (P>0.05) on slaughter data, dressing per cent, proportion of wholesale cuts and carcass composition, when compared to the control. Further, it is observed that both yield of visceral organs and chemical composition of longissimus dorsi muscle were unaltered upon replacing SBM by SRU in the diets. Based on the results of the present study, it is concluded that slow release urea (Optigen II) can be incorporated up to 3% in total mixed rations for buffalo bulls and adult rams, up to 2% in ram lambs and up to 2% in the concentrate mixture for lactating buffaloes thus reducing the cost of feed per kg gain in ram lambs and cost of feed per kg 6% fat corrected milk in lactating buffaloes without any adverse effects.