Thumbnail Image

Anand Agricultural University, Anand

Anand Agricultural University (AAU) was established in 2004 at Anand with the support of the Government of Gujarat, Act No.(Guj 5 of 2004) dated April 29, 2004. Caved out of the erstwhile Gujarat Agricultural University (GAU), the dream institution of Sardar Vallabhbhai Patel and Dr. K. M. Munshi, the AAU was set up to provide support to the farming community in three facets namely education, research and extension activities in Agriculture, Horticulture Engineering, product Processing and Home Science. At present there seven Colleges, seventeen Research Centers and six Extension Education Institute working in nine districts of Gujarat namely Ahmedabad, Anand, Dahod, Kheda, Panchmahal, Vadodara, Mahisagar, Botad and Chhotaudepur AAU's activities have expanded to span newer commodity sectors such as soil health card, bio-diesel, medicinal plants apart from the mandatory ones like rice, maize, tobacco, vegetable crops, fruit crops, forage crops, animal breeding, nutrition and dairy products etc. the core of AAU's operating philosophy however, continues to create the partnership between the rural people and committed academic as the basic for sustainable rural development. In pursuing its various programmes AAU's overall mission is to promote sustainable growth and economic independence in rural society. AAU aims to do this through education, research and extension education. Thus, AAU works towards the empowerment of the farmers.

Browse

201421
Reset filters
End date: 2014 × Start date: 2014 × Type: Thesis × Thesis Type: Ph.D ×
Reset filters

Search Results

Now showing 1 - 9 of 21
  • Thumbnail Image
    ThesisItemOpen Access
    EXPRESSION PROFILING, SNP DETECTION AND VALIDATION IN SQUAMOUS CELL CARCINOMA OF HORN IN KANKREJ CATTLE (Bos indicus) USING NEXT GENERATION SEQUENCING
    (AAU, Anand, 2014) KORINGA, PRAKASHKUMAR G.; Joshi, Chaitanya G.
    Horn cancer is a widely prevalent cancer amongst Kankrej cattle (Bos indicus) seen sporadically, especially in case of working class of castrated male animals i.e. bullocks. A transcriptome envisaged characterization as well as correlation to known genomic changes such as structural and copy number alterations, focused ins/dels and single nucleotide mutations. Here, we employed high throughput RNA-seq using GS-FLX Titanium for characterization and comparison of normal and cancerous horn transcriptome in Bos indicus. A total of 909,362 reads with average read length of 405bp for horn cancer (HC) and 583,491 reads with average read length of 411bp for horn normal (HN) were obtained by sequencing gene transcripts derived from HC and HN tissues. Assembled data were analyzed for identifying novel as well as differentially expressed transcripts using CLC Genome Workbench. RNA-seq analysis using different bioinformatics pipelines and software identified differentially expressed genes i.e. upregulation of KRT6A, KRT6B, KRT6C, KRT14, SFN, KRT84, PI3, CAl, C0L17A1, ANLN, SERPINB5 etc., as well as down-regulation of NR4A1, FOSB, LRIGl, BOLA, SCGBIAI, CXCL17, KRT19, BPIFBl, NR4A1 and TFF3 etc., in HC tissues. The signaling pathway investigation in this study revealed many of the cancer related pathways which mainly include cell cycle regulation pathways, p53 tumor suppressor pathways, NFKB and MAPKs pathways, LPS signaling pathway and PI3K-Akt pathways. The resuh of transcriptome expression profiling was validated using RT-qPCR in nine randomly selected genes. It revealed concordance of gene expression profile with RNA-seq analysis. We also used transcriptome data to elucidate complexity of the alternative splicing in HC transcriptome. We identified potential candidate splice variants that might be helpful in development of relevant biomarkers for early diagnosis of HC. The fiiture studies targeted at in depth characterization of these potential candidate splice variants might change the currently used clinical approaches. Herein we characterized global landscape of alternative splicing events exhibited by pair of HC and HN tissue and confirmed selected alternative splicing events with significant association to HC by RT-qPCR. Ine analysis of the same RNA-seq data using SeqMan Pro Version 10.0.0 resulted in to a 9532 and 7065 SNPs as well as 1171 and 1172 Indels in HC and HN, respectively. Out of total, 7889 SNPs and 1736 Indels uniquely present in HC, 5886 SNPs and 1146 Indels uniquely present in HN are novel and reported first time in Bos indicus, whereas rest are already reported in Bos taurus dbSNP database at NCBI. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN tissues. SNPs identified in RNA-seq analysis were validated in fiirther studies in two groups consisting of 50 animals each of HC and HN bullocks. DNA from HC tissue and blood of HN individual was extracted and 96 pairs of primers were used to generate amplicons of an average 300bp to get sequenced using Ion Torrent PGM. The resulting reads were assembled using SeqMan N Gen of DNASTAR and data were analyzed using Arraystarll. Case control analysis was carried out to find SNP significantly associated with HC. SNP at position 63251805 (dBSNP ID rsl36870681) identified in BPIFAl can serve as a potential candidate genetic marker in HC. The SNPs and Indels identified in this study will be useful resource for future studies to understand genetic basis for phenotypic variation between Bos taurus and Bos indicus as well as cancers in animals. A very large number of SNPs are essential for the designing and construction of arrays. SNPs identified in this study will enrich the dbSNP database of NCBI (http://www.ncbi.nlm. nih.gov/projects/SNP/) and will be useful resource for array designing. This study is the first attempt to reveal novel transcripts, differentially expressed genes as well as identification and validation of SNPs using digital expression analysis in Bos indicus and provides novel insights into bovine transcriptome. Our study will serve as a step further in detailed characterization of HC transcriptome and provide firm base to explore and mitigate HC at finer resolution. The present findings would provide basis for further screening of genes and identification of markers for early diagnosis and therapeutic intervention of HC.
  • Thumbnail Image
    ThesisItemOpen Access
    REGULATION OF ACTIVIN TYPE II RECEPTOR B (ACVR2B) EXPRESSION THROUGH RNA INTERFERENCE IN GOAT MYOBLAST CELLS
    (AAU, Anand, 2014) PATEL, AMRUTLAL KALUBHAI; Joshi, Chaitanya G.
    Enhancement of skeletal muscle mass through genetic manipulation has drawn attention to increase the meat production in the farm animals. Among the various techniques of regulating gene expression, RNA interference (RNAi) has been proposed as a promising tool to suppress the target gene expression. Attempts have been made to increase the skeletal muscle mass in transgenic animals through knockdown of Myostatin, a gene with potential negative effect on muscle growth. It has been well established that myostatin mediates its action through binding to its cell surface receptor mainly to activin type II receptor B (ACVR2B). Besides regulating myostatin activity, ACVR2B has also been known to regulate the activity of other Transfonning Growth Factor beta (TGF-(3) superfamily ligands which negatively regulates muscle growth. The inhibition of ACVR2B signaling has shown dramatic increase in the muscle mass to a greater extent than myostatin inhibition. Hence, in the present study we aimed to investigate the possibility of ACVR2B knockdown to enhance the myogenesis in goat through various RNAi methods such as expression of short hairpin RNAs (shRNAs) under U6 and CMV promoters and expression of artificial microRNAs (amiRNAs) under CMV and muscle specific muscle creatine kinase (MCK) promoters. Further we studied effect of ACVR2B knockdown on the expression of myogenic regulators and assessed induction of undesired interferon response against RNAi vectors. Among the seven shRNAs tested, the U6 promoter driven shRNAs showed 63 (p= 0.0004), 76 (p= 0.0001), 75 (p=0.0000), 74 (p= 0.0005), 80 (p= 0.0001), 74 (p= 0.0000) and 57% (p= 0.0013) silencing with sh1 to 7, respectively in HEK293T cells whereas 24 (p= 0.1497), 24 (p= 0.2243), 15 (p= 0.3988), 31 (p= 0.1263), 14 (p= 0.4425), 46 (p= 0.0318), and 26 % (p= 0.1288) silencing of endogenous ACVR2B with shl to sh7, respectively and 53 (p- 0.0005), 32 (p= 0.0171), 38 (p= 0.0025), 66 (p= 0.0002) and 51% (p= 0.0008) with sh3, sh4, sh5, sh6 and sh7, respectively against ectopically expressed goat ACVR2B in goat myoblasts ceils. The knockdown of endogenous ACVR2B resulted in the 116, 105, 84, 64, 119, 102, 121. and 157% expression of MyoD; 131, 128, 128, 123, 104, 103, 69, and 157% of MyoG in sh1 to sh7 transfected cells, respectively. The transfection of U6 driven shRNA resulted in the induction of OAS1, a marker for innate interferon response by 3 to 1861 fold in 293T cells and up to 94 fold in the goat myoblasts cells. In an attempt to overcome the undesired cellular toxicity associated with U6 driven shRNAs as reported in the number of studies, we expressed these shRNAs under CMV promoter. The CMV driven shRNAs showed weak silencing of 37 (p= 0.1622) and 18% (p= 0.4877) by sh1 and sh3, respectively in HEK293T cells whereas 7% (p= 0.5749) by shl and 4% (p= 0.7493) by sh5 in goat myoblasts cells. Unlike suggested earlier, we observed significant induction of interferon response to CMV driven shRNAs up to 46 fold in 293T cells and 105.3 fold in goat myoblasts cells. Alternatively, we assessed another RNAi approach using amiRNAs which mimics the endogenous miRNA biogenesis pathway. Among the four amiRNAs tested by placing them in 5'-UTR region of GFP reporter, we observed 64 (p=0.0004), 77 (p=0.0002), 1 (p=0.8712), and 41% (p=0.0115) silencing in 293T cells by ami204, ami318, ami735 and ami878, respectively against exogenously expressed goat ACVR2B; 19 (p=0.3593) and 9% (p=0.4977) by ami204 and ami318, respectively against endogenous ACVR2B and 23% (p=0.0444) by ami318 against exogenously expressed ACVR2B in goat myoblasts cells. Since, amiRNAs placed in 5'-UTR were shown to affect the translation of reporter GFP, we further placed them in 3'-UTR of GFP which resulted in enhanced expression of GFP thereby enabling the monitoring of expression of amiRNAs. The 3'-UTR derived amiRNAs showed 50% (p=0.0002) silencing only by ami3I8 in 293T cells whereas 47 (p=0.0193), 16 (p=0.2959), 19 (p=0.1547), and 28% (p=0.0770) by ami204, ami318, ami735 and ami878, respectively against endogenous and 67%) (p=0.0004) by ami318 against overexpressed ACVR2B in goat myoblasts cells. The expression of myogenic regulators MyoD remained unchanged by amiRNAs cloned in the 5'-UTR whereas expression of MyoG was significantly up regulated by ami878 (p=0.0089). However, amiRNAs cloned in 3'-UTR showed significant down regulation of MyoD by 51 (p=0.0007), 27 (p=0.0232), 29 (p=0.0074), and 31% (p=0.0104) and MyoG by 36 (p=0.0034), 12 (p=0.2532), 22 (p=0.0303), and 37% (p=0.0026) by ami204, ami318, ami735 and ami878, respectively. As observed for U6 and CMV driven shRNAs, CMV driven amiRNAs showed significant induction of interferon response in 293T (up to 121.7 fold) and myoblasts (212.5 fold) cells. As ACVR2B has been shown to be essential for embryonic development, we tested the possibility of its knockdown in skeletal muscle using muscle specific MCK promoter. Among the MCK and MSTN promoters with and without two repeats of MCK enhancer, we observed maximum transcriptional activity by MCK promoter in goat myoblasts cells. We thus tested best amiRNAs (ami204 and ami318) by expressing under MCK promoter which showed 22% silencing efficacy by ami318 in 293T cells and 32% silencing efficacy in goat myoblasts cells by transient transfection assay. Further to test the possibility of ACVR2B knockdown after stable integration of amiRNAs into goat myoblasts genome, we generated lentivirus particles carrying amiRNAs expression cassettes and transduced the goat myoblasts. The myoblasts cells stably integrated with amiRNAs showed ~8% silencing by ami318 which was increased to 34% upon induction of differentiation under muscle specific promoter. Western blot analysis revealed 41% and 57% silencing by 5'-ami204 and 5'-ami318, respectively whereas 14% and 35% silencing by 3'-ami204 and 3'-ami318, respectively in stable myoblasts upon induction of differentiation. Unlike transient transfection assay vv'hich showed positive correlation of expression of ACVR2B with myogenic regulators, its stable knockdown resulted in up regulation of MyoD in 5'-UTR derived ami204 and ami318 and overall down regulation of MRFs in 3'-UTR derived ami204 and ami318 integrated goat myoblasts cells. The 5'- UTR derived ami204 and ami318 showed increased rate of cell proliferation as well as myoblasts fusion in stable goat myoblasts compared to scramble control indicating growth promoting effect of ACVR2B knockdown. The skeletal muscle specific partial knockdown of ACVR2B is unlikely to affect the embryonic survival and it will be interesting to further assess its possible growth promoting effect in adult animal by generating transgenic goat through somatic cell nuclear transfer of goat myoblast cells stably integrated with amiRNAs.
  • Thumbnail Image
    ThesisItemOpen Access
    COMPARATIVE APPRAISAL OF PHYSICAL, CHEMICAL, INSTRUMENTAL AND SENSORY EVALUATION METHODS FOR MONITORING OXIDATIVE DETERIORATION OF GHEE
    (AAU, Anand, 2014) MEHTA, BHAVBHUTI MANOJBHAI; Aparnathi, K. D.
    Ghee is the most famous traditional dairy product in India, now also gaining popularity in Asian countries as well as other continents like Australia, Europe and America. This fat rich dairy product is highly regarded for typical pleasing flavor. Ghee, being a lipid, prone to oxidation. Lipid oxidation is a degradation process considered to be a major cause of deterioration in the quality of fat products. It imparts rancid and unpleasant flavors to the products and thus decreases their organoleptic value. The reaction of fat oxidation is autocatalytic and occurs in two stages. In primary stage of the fat oxidation reaction between the unsaturated fatty acid and oxygen leads to formation of hydroperoxides and during secondary stage of the reaction the hydroperoxides decomposed into off smelling volatile compounds. This is very complex process that resuh in the formation of several different compounds. Therefore, there is no single method to measure all these compounds, consequently, the use of more than one testing method is recommended to monitor oxidative changes taking place in lipid. Numerous physical, chemical and instrumental methods have been reported to measure oxidative deterioration of oils and fats. The selection of appropriate analytical method is very essential to obtain correct information about oxidative deterioration of product like ghee. Analytical methods most widely reported are weight gain, conjugated dienes, Kreis number, iodine value, free fatty acids content, peroxide value, TBA value, p- Anisidine value, TOTOX value, Carbonyl value and Rancimat. However, no systematic work has been carried out so far for selection of the most appropriate methods to monitor oxidative deterioration of ghee. Therefore, this study was undertaken with a goal to compare performance of selected physical, chemical and instrumental methods to evaluate oxidative deterioration of ghee and to establish correlation between results obtained by these methods with results obtained by sensory evaluation of the ghee for flavor score. The entire work of study was divided as (1) selection of methods used for determination of peroxide value of ghee, (2) evaluation of methods used for monitoring oxidation of ghee on the bases of changes taking place during primary stage of lipid oxidation, (3) examination of methods used for monitoring oxidation of ghee on the bases of changes taking place during secondary stage of lipid oxidation, (4) testing of Rancimat for suitability to study oxidation of ghee, (5) comparison between performance of the method selected from stage of primary oxidation, method selected from secondary stage of oxidation and Rancimat to study oxidative deterioration of ghee and (6) work out the correlation between the results obtained by accelerated storage study and the results obtained from sensory evaluation of ghee for flavor score on storage at normal temperature. In selection of methods for determination of chemical changes taking place during primary stage as well as secondary stage of oxidation in ghee, accelerated storage study was employed. The samples of ghee were prepared by creamy butter method stored at 80°±2°C and monitor for chemical changes using selected methods at regular interval of every 48 hours. The samples of ghee were simultaneously analyzed for changes in flavor score by sensory evaluation using 9 point hedonic scale. For testing the suitability of Rancimat to study the oxidative deterioration of ghee, oxidation curves for the ghee were obtained at 90°C, 100°C, 110°C and 120°C and Arrhenius plots were obtained to predict the shelf life of ghee. Simultaneously ghee samples were also stored at 35°±2°C and monitor for changes in flavor score by sensory evaluation using 9 point hedonic scale at an interval of 10 days. Amongst the BIS, AOAC, AOCS/IUPAC, FOX and IDF methods used to monitor the peroxide formation in ghee, the FOX method was found most consistent. Moreover, the best correlation between changes in flavor score of ghee and peroxide formation was given by FOX method. Amongst the weight gain. Conjugated dienes, Kreis number. Iodine value, free fatty acids content and Peroxide value by FOX method based on chemical changes in the primary stage of the oxidation of ghee, except the FOX method no other method gave significant correlation with changes in flavor score of ghee during storage. Amongst the TBA value, p-Anisidine value, TOTOX value and Carbonyl value based on chemical changes in the secondary stage of the oxidation of ghee, the carbonyl value was found consistent. In addition it gave the highest correlation with changes in flavor score of ghee during storage. The changes in temperatures of Rancimat were highly correlated with induction period of ghee samples and changes in its flavor scores on storage at 80°±2°C. The predicted shelf life of ghee at 80°±2°C obtained from the prediction models prepared using the results of peroxide value by FOX method and carbonyl value of the ghee stored at 80°±2°C and that obtained from Rancimat was almost in corroboration with the actual shelf life of ghee stored at 80°±2°C. The predicted shelf life of ghee at 35°±2°C obtained from the prediction models prepared using the results of peroxide value by FOX method, carbonyl value and flavor score of the ghee stored at 80°±2°C was almost in corroboration with the actual shelf life of ghee stored at 35°±2°C. However, the predicted shelf life of ghee at 35°±2°C obtained from Rancimat was 2.47 times higher than the actual shelf life of ghee stored at 35°±2°C. Therefore, it appeared from the results that use of peroxide value by FOX method, carbonyl value and flavor score of ghee on storage at 80°±2°C are promising for predicting shelf life of ghee at normal temperature, but use of Rancimat does not appear promising to predict the shelf life of ghee on storage at normal temperature.
  • Thumbnail Image
    ThesisItemOpen Access
    BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF COLD-INDUCED SWEETENING IN POTATO (SOLANUM TUBEROSUM L.) VARIETIES DURING STORAGE
    (AAU, Anand, 2014) GALANI, YAMDEU JOSEPH HUBERT; TALATI, J. G.
    Potato (Solatium tuberosum L.) is the third most important food crop, the most important non-grain food crop and one of the most essential basic vegetable worldwide as well as in Indian subcontinent. After harvest, potatoes are stored in cold storage to provide round the year supply to markets and consumers. But during storage at cold temperatures, many cultivars accumulate free reducing sugars derived from breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose in a metabolic process known as cold-induced sweetening (CIS). Understanding the basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars adversely affect the quality of processed food products. Investigations were carried out to characterize at biochemical and molecular levels the CIS in 11 potato varieties namely DSP 287, DSP 186, Kufri Surya, K. Chipsona-3, K. Sutlej, K. Sadabahar, K. Jyoti, K. Lauvkar, K. Himsona, K. Bahar and K. Badshah stored at 3 different temperatures viz., room temperature (25-32°C), incubator (15°C) and cold storage (4°C). Samples, collected every 15 days intervals for 105 days storage were analyzed for different parameters associated with carbohydrate metabolism on one side, and antioxidant capacity on the other side. Analysis of carbohydrate dynamics showed that low temperature storage negligibly influenced dry matter, starch and maltose contents of tubers, but a significant increase in reducing sugars, total soluble sugars, fructose, glucose, hexoses:sucrose ratio and a decrease of sucrose content were observed at 4°C as compared to room temperature. A strong positive correlation was found between reducing sugars and total soluble sugars, and between fructose and glucose. Additionally, important shrinkage and sprouting of tubers was observed at 15°C, they were less intense at room temperature, and no any shrinkage and sprouting occurred on tubers stored at 4°C. The potato varieties also appeared to be suitable for processing immediately after harvest or short storage at room temperature. The activity of β-amylase was considerably increased by storage at low temperature, and a weak correlation with starch content indicated an important role of other enzymes in starch degradation while absence of maltose accumulation with increased β-amylase activity implied a possible significant activity of maltase in potato tubers. Acid invertase activity drastically rose at low temperature and strongly paralleled reducing sugars, glucose, fructose and hexoses:sucrose ratio. Moreover, as acid invertase activity increased, sucrose content decreased, indicating the essential role of acid invertase in development of CIS. The above findings allowed to group the 11 potato varieties into low to high sugar-forming groups and thereby select K. Jyoti as CIS-tolerant and K. Badshah as CIS-susceptible. Banding pattern of both native and denaturated proteins of potato tubers could not clearly discriminate the varieties. Zymograms of p-amylase revealed differentially induced bands at low storage temperatures, which might justify the observed increase of enzyme activity. Screening of the 11 potato varieties with 10 SSR primers detected a total of 42 alleles arranged in 44 different configurations, among which 37 alleles (88%) were polymorphic. The polymorphic information content value of the SSR locus ranged from 0.473 to 0.787 thus indicating a high utility of these markers for study of genetic diversity in potato. The dendrogram derived from Dice's similarity coefficients among the 11 varieties could partially but efficiently differentiate close parents and sugar-forming groups. Differential gene expression analysis showed that during storage expression of vacuolar acid invertase gene StvacINV1 and p-amylase gene BAMl increased at low temperature and their transcripts were more expressed in the CIS-tolerant variety than the CIS-sensitive. Expression of invertase inhibitor gene INH2a however was higher in the CIS-tolerant variety than the CIS-sensitive. Correlating StvacINVl and INH2a expressions with reducing sugar content and acid invertase activity established that post-translational regulation of acid invertase by the invertase inhibitor protein could be an important component of resistance to CIS. Besides, correlation between BAM1 expression and (β-amylase activity affirmed the hypothesis of several enzymes and pathways involved in starch degradation during cold storage of potato. Analysis of antioxidant capacity parameters revealed that low temperature storage greatly influenced vitamin C content as well as the phenolic content. During storage, both parameters initially increased, then a fluctuated decline was observed but until the last day of observation, they remained above the initial level. Phenolic acids profiling by UPLC identified 12 compounds among which the most abundant was chlorogenic acid followed by gallic acid, sinapic acid and ellagic acid which is reported for the first time, while trans-cinnamic acid was the lowest. Except paracoumaric acid which decreased at 4°C, all the phenolic acids increased with storage among which sinapic acid and feruhc acid appeared to be most enhanced. Correlation analysis showed that gallic acid, caffeic acid. chlorogenic acid and protocatechuic acid significantly contributed to total phenolic content. Evaluation of antioxidant activity showed a close relationship between DPPH and ABTS methods. Antioxidant activity estimated by both the methods increased up to 60 days storage then at 90 days, they dropped to a level comparable or lower than the original value, irrespective of the storage temperature. Correlation study revealed that chlorogenic acid, gallic acid and ferulic acid mostly contributed to antioxidant activity. Activity of the antioxidant enzymes superoxide dismutase and ascorbate peroxidase both increased initially but then decreased to values lower than the initial level and were not influenced by storage temperature. Correlation with antioxidant activity indicated that the enhancement of reactive oxygen scavenging species in tubers could result mainly from ascorbate peroxidase activity. Isoforms of the two enzymes showed interesting polymorphism and changes in bands intensity as well as differential induction or suppression of bands during storage. However, isozymes of ascorbate peroxidase showed higher similarity and better discrimination of the varieties. Although a clear relationship between CIS and antioxidant capacity was not established, nevertheless it appeared that low sugar-forming varieties K. Jyoti, K. Himsona and K. Surya were also having high antioxidant capacity whereas K. Chipsona-3 and K. Bahar both high sugarforming had low antioxidant capacity. Hence, it is not unreasonable to suggest that antioxidant capacity of potato tubers should be taken into account in development of CIS-resistant varieties. Nonetheless, additional evidences are needed to confirm this suggestion as well as there is an urgent need to develop new varieties capable to cope with this cold stress.
  • Thumbnail Image
    ThesisItemOpen Access
    ULTRASONOGRAPHY IN FOLLICULAR DYNAMICS, FERTILITY MANAGEMENT AND EARLY PREGNANCY DIAGNOSIS IN CATTLE
    (AAU, Anand, 2014) HADIYA, KAMLESHBHAI KHODABHAI; Dhami, A. J.
    These investigations were undertaken on 60 Gir and HF x K Crossbred cattle of University farm in Anand from January to June 2014 with the objectives to study the follicular dynamics of spontaneous as well as hormonally (Mid-cycle PG, Ovsynch and CIDR) induced estrous cycles and even during early pregnancy in postpartum cows and postpubertal heifers using real time B-mode transrectal ultrasound scanning; to detect early pregnancy and embryonic mortality using clinical diagnosis and ultrasonography; to evaluate the efficacy of estrus synchronization protocols toward fertility improvement in subfertile cows; to monitor blood biochemical and plasma endocrine profile of spontaneous and induced cycles during treatment and thereafter at weekly interval until next estrus or 42 days of pregnancy; and to correlate plasma progesterone and estradiol profile with ovarian changes during estrous cycle and early pregnancy. The normal cyclic (24) and infertile (36) animals were thoroughly screened for their genital health through gynaeco-clinical examinations. They were subjected to transrectal ultrasonographic scanning to study the follicular dynamics during various reproductive phases and during estrus synchronization treatments. The animals found in spontaneous or induced estrus were inseminated with good quality frozen-thawed semen. Pregnancy was confirmed in non-return cases on day 23, 28, 35 and 42 using transrectal ultrasonography and per rectal palpation 60 days post-AI. Infertile animals (subestrus and repeat breeders) were included in estrus synchronization protocols, viz.. Mid-cycle PG, Ovsynch and CIDR, and subsequent ovarian follicular/luteal dynamics, blood profile and fertility evaluation. Six subestrus/ repeat breeding postpartum cows each of Gir & Crossbreds (6x2 = 12) having mid cycle mature CL were treated with single i/m injection of 500 ng cloprostenol, and fix timed AI was performed at 72 and 96 hrs post-treatment. Ultrasound scanning of the animals was performed daily from start of treatment till expression of induced estrus/FTAI and/or ovulation with blood sampling. Similarly, six infertile cyclic cows each of Gir and Crossbred category (6x2=12) were treated with standard CIDR protocol and equal numbers (6x2=12) with Ovsynch protocol using FTAI on day 9 and 10. The ovarian changes were monitored daily with blood sampling on day 0 (initiation of treatment), 2, 4, 6, 7 (PG injection), 8,9 (GnRH injection), and day 10 (induced estrus/FTAI). All 36 induced cycling and fix timed inseminated cows together with 24 spontaneously cycling and inseminated cows {cows (n=12) and heifers (n=12), 6 each of Gir and Crossbred type} were further monitored for follicular dynamics daily from the day of AI till day 21 post-AI and then for early pregnancy in non-return cases through uterine scanning with 6.5 MHz transducer, together with jugular blood sampling at weekly interval, i.e. on day 0 (AI), 7, 14, 21 post-AI and then on day 23, 28, 35 and 42 post-AI in non-return cases for assay of blood glucose and plasma total cholesterol, estradiol and progesterone profile. All these inseminated animals (n=60) not returned to estrus again were monitored through transrectal USG (6.5 MHz) for detection of early pregnancy and/or embryonic mortality on day 23, 28, 35 and 42 post-AI, and the findings were correlated with plasma P4 on day 21 and per rectal palpation on day 60 post-AI. The characteristics of dominant and ovulatory follicles observed between two consecutive estruses in 24 unbred animals, comprising of 12 heifers and 12 cows, 6 each of Gir and HF x K crossbred genotype, using 7.5 MHz tranrectal linear array transducer indicated the presence of two- and three-wave cycles in 66.66 (n=12) and 33.33 (n=6) per cent, respectively, of all heifers and crossbred cows, while in Gir cows the frequency of 2- and 3-wave cycles was equal. None of the animals showed single-wave cycle. The second wave appeared earlier in the estrous cycle with 3-wave than with 2-wave (9.00±1.00 vs 10.00±0.58 days; P<0.01). The persistence of the first dominant follicle and duration of regression phase were significantly (P<0.05) longer in 2-wave than in 3-wave cycles (20.25±0.25 vs 19.00±0.00 days and 7.25±0.62 vs 5.00±0.00 days). The maximum diameter of ovulatory follicle was significantly higher (P<0.05) in 3-wave as compared to 2-wave pattern (13.70±0.10 vs 12.55±0.27 mm). The linear growth rate of first dominant follicle was significantly (P<0.01) higher in 2-wave as compared to 3-wave cycle (1.09±0.19 vs 0.46±0.11 mm/day). Similarly, the linear growth of ovulatory follicles was significantly (P<0.05) higher in 3-wave than the 2-wave cycles in all animals. The duration and end day of second dominant follicle were significantly (P<0.05) longer in 2-wave as compared to 3-wave cycle (12.00±0.70 vs 6.00±0.00, 21.50±0.28 vs 14.50±0.50). The emergence of ovulatory follicle was significantly (P<0.01) earlier in 2- wave (9.50±0.50 days) as compared to 3-wave cycle (14.50±0.50 days). Similarly, the duration of ovulatory follicles differed significantly (P<0.01) between 2- and 3-wave cycles (12.00±0.70 vs 7.00±0.00 days). The diameter of ovulatory follicle of 2-wave cycle was significantly (P<0.05) larger than 3~wave cycle (17.22±0.79 vs 14,65±0.45 mm). The ovulatory follicle of 2-wave cycle in crossbred cows appeared significantly (P<0.01) earlier than 3-wave cycle (days 9.00±0.71 vs 14.50±0.50). The persistence of ovulatory follicle was significantly longer (P<0.01) in 2-wave as compared to 3-wave cycle (12.00 ±0.70 vs 7.50±0.50 days). Two- and three-wave cycles differed significantly (P<0.05) with respect to the mean interovulatory interval (22.33±0.33 vs 24.00±0.00 days). In Crossbred cows with 2-wave cycle, there was no relationship between the development of first dominant follicle and presence of CL in either of the ovaries, while in 3-wave cycle the first dominant follicle and the ovulatory follicle developed on opposite side to the ovary bearing the CL, while the second dominant follicle developed on the same ovary bearing the CL in both Gir and Crossbred cows. Similarly, no pattern was observed for the development of ovulatory follicle with respect to first dominant follicle on the ovaries in 2-wave cycle. However, the proportion of ovulatory follicles was higher on opposite side as compared to same side to the first dominant follicle in 2-wave cycle in both the breeds. In 3-wave cycle the second dominant follicle developed opposite to first dominant follicle and the ovulatory follicle developed opposite to second dominant follicle in all the animals. Thus, there was no difference in the pattern of this phenomenon between two breeds studied. In Gir and Crossbred cows, there were positive correlations between CL diameter and plasma P4 values (r = 0.82 & 0.72), and follicle size and E2 values (r = 0.69 «& 0.36). The average plasma P4 values were higher (P<0.01) and E2 values lower on day 7 and 14 than on day 0 and 21 post-estrus, with corresponding larger CL and smaller follicular size in both Gir and Crossbreds. The profiles of follicular growth and regression compared for first 21 days post-AI in conceived and in non-bred cyclic Gir & Crossbred cows revealed the fi-equency of occurrence of two follicular wave up to 75.00 (6/8) per cent in pregnant and 58.33 (7/12) per cent in non-bred cyclic cows, while the remaining seven cows (2 pregnant and 5 cyclic) showed three follicular waves. Almost similar patterns in the follicular growth characteristics, viz., beginning day, end day and duration (days) of first dominant follicle were observed between pregnant and non-bred cyclic cows (0.75±0.29 vs 0.50±0.29, 9.25±0.48 vs 8.25±0.25 and 8.50±0.48 vs 7.75±0.48). However, the linear growth rate (mm/day) of first dominant follicle was significantly lower (P<0.05) in pregnant as compared to non-bred cyclic cows (0.93±0.07 vs 1.21±0.12). The difference was also significant (P<0.01) in the beginning day (15.75±0.25 vs 14.00±0.41) and the maximum diameter of first dominant follicle (11.72±0.22 vs 14.40±0.24 mm) between above two groups. Similarly, the duration (days) of regression phase and linear regression rate (mm/day) of first dominant follicle (4.75±0.65 vs 7.00±0.40 and -1.07±0.07 vs -1.39 vs 0.09) and the maximum diameter of second dominant follicle (11.70±0.22 vs 16.40±1.04 mm) were significantly (P<0.01) lower in pregnant as compared to normal cyclic cows. In all, 60 spontaneous (n=24) or induced cyclic (n=36) cows were inseminated and those did not return to estrus by day 21 after AI were further scanned four times on day 23, 28, 35 and 42 using trans-rectal linear array transducer of 6.5 MHz frequency to detect early pregnancy and embryonic mortality, if any. The sensitivity of USG was cent per cent on all the 4 days, but the specificity was lower on day 23 and 28 (68.00 % each) as compared to day 35 (92.00 %) and 42 (100%). On day 23 and 28, 8 animals were incorrectly diagnosed pregnant, while on day 35, two and zero animals were wrongly diagnosed pregnant. However, the progesterone assay on day 21 post-AI revealed 9 animals being diagnosed incorrectly pregnant. The sensitivity and negative predictive values were cent per cent on all days by both the methods, but specificity and positive predictive values were lower for pregnancy diagnosis using progesterone assay (64.00 % and 79.55 %) as compared to ultrasound scanning results on all 4 days (day 23 & 28, 68.00 to 81.40 %; day 35, 92.00 - 94.59 % and day 42, 100 %). Similarly, the accuracy was relatively higher with ultrasound scanning than the progesterone assay (100 vs 86.44 %). Based on plasma progesterone profile, 35 cows were correctly diagnosed as pregnant (P4, 6.48±0.38 ng/ml) and 16 cows as non-pregnant (P4, 0.45±0.13 ng/ml). However, 9 animals were incorrectly diagnosed as pregnant (P4, 4.14±0.12 ng/ml) and no animal was incorrectly diagnosed as non-pregnant as compared to the results of pregnancy diagnosis on day 60 by rectal palpation. Out of 9 cows, which were incorrectly diagnosed pregnant on the basis of plasma P4 profile on day 21 post-AI, 6 were found pregnant on day 23 and 28, but only three were found pregnant on day 35 by ultrasonography indicating early embryonic mortality in three animals (3/35; 8.57%) between day 28 and 35, The technique of ultrasound scanning facilitated the diagnosis of all non-pregnant animals as early as on day 23 post-service. The results indicated that day 35-42 is the earliest possible time when pregnancy diagnosis should be attempted using ultrasound for maximum accuracy and specificity. The conception rates of 12 subfertile cows, each subjected to CIDR, Ovsynch and Mid-cycle PGF2a treatment protocols were 41.66, 41.66 and 33.33 per cent, respectively, at induced estrus. The corresponding conception rates at second cycle post-treatment were 28,57, 42,85 and 25,00 per cent; and at third cycle 40,00, 50.00 and 33.33 per cent, with the overall conception rates of 3 cycles as 75.00, 83.33 and 66.66 per cent. In untreated cyclic control group, out of 24 animals (12 Gir and 12 Crossbreds) inseminated, the conception rates at first, second and third service and overall of 3 services were 20.83, 21.05, 26.66 and 54.16 per cent, respectively. The results obtained using all 3 treatment protocols were much better than that of untreated cyclic control animals. Gir cattle in fact responded better with CIDR, while Crossbreds showed better results with Ovsynch protocol, and the response with Mid-cycle PGF2a treatment was relatively poor and almost same in both the classes of animals. The follicular dynamics, CL size, plasma progesterone, estradiol-17p, total cholesterol and blood glucose profiles were studied in Mid-cycle PGF2a treated as well as CIDR and Ovsynch treated Gir and Crossbred cows from the day of initiation of treatment until day of induced estrus/FTAI. The pooled mean CL size in cows as monitored by transrectal ultrasonography at 0, 24, 48 and 72 (estrus/AI) hrs of PG treatment was found to be 14.02±0.46, 12.68±0.49, 10.26±0.62 and 7.70±1.16 mm, respectively, and that of follicle 4.42±0.22, 5,37±0.35, 8.52±0.59, 12.94±0.74 mm. The corresponding plasma progesterone concentrations were 5.22±0.43, 3.10±0.36, 1.14±0.32 and 0.48±0.31 ng/ml, respectively, and those of estradiol-17p 19.42±2.07, 24.33±2.33, 41.25±4.40, 51.41±2.02 pg/ml. The values of CL size and plasma P4 were significantly (P<0.01) higher and those of follicle size and E2 lower on the day of PG treatment indicating the presence of mature functional CL on the ovary. The CL size reduced and plasma progesterone concentration dropped significantly with parallel increase in follicle size and plasma E2 within 48-hrs of PG treatment. The values of CL size and plasma P4 were the lowest or at basal level and those of follicle and E2 at peak on the day of induced estrus/AI, around 72-hrs posttreatment. No significant variation was noted in any of the traits between breeds or at any of the intervals studied within the breed, except at 72-hrs of PG treatment wherein the values of CL size and plasma P4 were significantly (P<0.05) higher in crossbreds as compared to Gir, suggesting breed difference in the action of PG and duration of induced luteolysis. The conceiving and non-conceiving cows revealed identical pattern and values of CL and follicle size as well as plasma P4 and E2 concentrations till 48-hrs of mid-cycle PG injection, but by 72-hrs of treatment the drop in CL size and plasma P4 were drastic and significant over 48-hrs values in conceiving cows than the non-conceiving cows. The pooled mean concentration of blood glucose recorded at 0, 24, 48, 72 hrs after PG treatment was 56.78±2.25, 57.34±2.00, 58,76±2.06, 60.78±1,61 mg/dl, respectively. The corresponding plasma total cholesterol concentrations were 166.84±3.73, 166.36± 2.33, 173.42±2.65, 169.19±3.89 mg/dl, respectively. The values of none of these traits varied significant between different intervals post-PG-treatment, and the values were more or less similar in both the breeds, and no significant variation was noted in blood glucose or plasma cholesterol profile at any of the intervals studied between breeds. The follicular dynamics, CL size, plasma progesterone, estradiol-17p, cholesterol and blood glucose profiles were also studied in Gir and Crossbred cows on day 0 (Wg CIDR insertion), 2, 4, 6, 7 (CIDR removal & PG injection), 8 and 9 (GnRH injection) of CIDR (1.38 g hydroxyl-progesterone in silastic coil) treatment, and on day 10 (induced estrus/FTAI). The pooled mean CL size in cows on these was found to be 11.09±0.71, 10.97±0.69, 10.80±0.50, 10.60±0.43, 10.63±0.49, 8.25±0.18, 5.65±0.38 and 5.21±0.67 mm, respectively and that of follicle size 7.70±0.59, 7.23±0.36, 8.68±0.71, 8.63±0.63, 9.02±0.67, 10.50±0.61 12.48±0.72, 7.50±1.00 mm, respectively. The corresponding plasma P4 concentrations were 4,55±0.34, 5.54±0.49, 5.12±0.31, 4.73±0.24, 4.66±0.36, 1.78±0.21, 0.67±0.12, 0.13±0.02 ng/ml, respectively, and those of estradiol-17p 14.16± 1.94, 25.50±2.40, 29.16±2.94, 37.16±3.39, 30.75±1.92, 40.75±2.75, 49.66±4.03, 33.50 ±7.02 pg/ml. The values of CL size and plasma P4 were significantly (P<0.01) higher and follicle size and E2 lower on the day of CIDR insertion indicating that the animals were cyclic with presence of mature functional CL on the ovary when the treatment was initiated. The CL/ follicle size and plasma progesterone/estradiol concentrations were maintained for 7 days until CIDR was removed and PG was injected i/m, which resulted in luteolysis with sudden and significant drop in CL size and plasma progesterone within 24- hrs with further drop to basal level with concurrent rise in follicle size and plasma E2 levels by next 48-hrs, i.e. the day of induced estrus/FTAI due to withdrawal of negative feedback effect of plasma P4 on the pituitary and hypothalamus, thus inducing ovulatory estrus within 72-hrs. No significant variation was noted in any of these traits at any of the intervals studied, between or within Gir and Crossbred cows. The values of blood glucose and cholesterol were more or less similar in both the breeds, as were found in Mid-cycle PG treated group, and no significant variation was noted at any of the intervals post-CIDR treatment between breeds or within breed. Although the blood glucose levels were apparently higher and cholesterol lower in Gir cows as compared to Crossbreds during the CIDR treatment phase until induced estrus. The effect of Ovsynch treatment on the follicular dynamics, CL size, plasma progesterone, estradiol-17p, total cholesterol and blood glucose profiles studied in infertile cyclic Gir and Crossbred breed cows on the same days as with CIDR protocol was almost similar with identical pattern and values to those of CIDR treatment group. All these parameters were also studied in normal cyclic control post-pubertal heifers (6) and postpartum cows (6) of Gir and Crossbred breeds each at weekly interval, together with those cows induced to estrus and inseminated at fixed time under three synchronization protocols, from day 0 (estrus/AI) to day 21 post-AI and then on day 23, 28, 35 and 42 in non-return cases. One cows in each synchronization treatment underwent embryonic mortality between day 28 and 35 post-AI as confirmed by transrectal USG. The pooled mean CL size in 12 heifers as monitored by ultrasonography on day 0 (estrus/AI), 7, 14, 21, 23, 28, 35 and 42 post-AI was found to be 4.72±0.33, 12.30±0.55, 12.72±0.62, 8.74±1.39, 9.47±1.17, 11.83±0.87, 14.44±0.61 and 14.50±0.67 mm, respectively, and that . of follicle size 12.70±0.51, 5.86±0.52, 5.23±0.45, 9.21±0.96, 7.4U0.91, 6.14±0.48, 4.80±0.51 and 4.94±0.30 mm, respectively. The corresponding plasma progesterone concentrations were 0.49±0.10, 3.16±0.21, 5.18±0.41, 3.01±0.99, 3.50±0.96, 5.66±0.73, 8.54±0.21 and 8.96±0.15 ng/ml, respectively, and those of estradiol-17p were 56.08±2.98, 29.58±2.52, 20.25±2.43, 38.08±5.70, 22.66±4.97, 21.58±2.84, 15.80±2.53 and 22.40±3.29 pg/ml, respectively. Very similar values and trend of observations were recorded for CL and follicular size as well as plasma progesterone and estradiol-17p profile in postpartum cows also from the day of estrus/breeding till day 42 post-AI. The values of CL size and plasma P4 were the lowest or at basal level on the day of estrus/AI, increased highly significantly (P < 0.01) by day 7 and 14, dropped down around day 21-23 and again rose significantly at day 35 and 42 post-AI, with concurrent inverse trend in follicle size and plasma E2 profile. These trends were associated with establishment of pregnancy and return of rest of the animals to next cycle around day 21- 23. No significant variation was noted in CL or follicular size and plasma progesterone or estradiol-17P profile at any of the intervals studied and the trend was the same in both the Gir and Crossbred heifers as well as cows. Further, the conceiving and non-conceiving animals of Gir and Crossbred breed (irrespective of parity) revealed identical pattern and values of CL/follicle size and plasma P4, E2 concentrations till day 14 post-AI, and there was significant drop in CL size as well as progesterone profile in non-pregnant animals with increase in follicle size and plasma E2 profile due to luteolysis, folliculogenesis and res-establishment of next cycle around day 21-23; and persistence of CL and continuance of progesterone production in conceived ones. The pooled mean concentration of blood glucose in heifers on day 0 (estrus/AI), 7, 14, 21, 23, 28, 35 and 42 post-AI was found to be 64.35±3.21, 68.71±2.54, 66.41±2.63, 68.67±2.10, 66.91±2.08, 64.43±2.30, 60.96±1.69 and 61.18±1.58 mg/dl, respectively. The corresponding cholesterol levels were 172.73±5.27, 175.91±3.69, 181.01±1.85, 176.90± 3,33, 173.90±2.65, 174.81±2.19, 170.50±2.60 and 169.79±1.43 mg/dl, respectively. Very similar values and trend of observations were recorded for blood glucose and plasma total cholesterol profile in postpartum cows from the day of estrus/breeding till day 42 post-AI. The values of none of these traits varied significantly between intervals post-estrus/AI, No significant variation was noted between Gir and Crossbred heifers as well as cows in blood glucose or plasma cholesterol profile at any of the intervals studied and the trend was the same. Further, the conceiving and non-conceiving animals of Gir and Crossbred breed (irrespective of parity) revealed identical pattern and values of blood glucose and plasma cholesterol concentrations till day 42 post-AI. Very similar trend and values of follicular dynamics, CL size, plasma P4, E2, total cholesterol and blood glucose were also found in all three groups of estrus synchronized cows of Mid-cycle PG, CIDR and Ovsynch protocols (12 each) at weekly interval from day 0 (induces estrus/FTAI) to day 21 post-AI and then on day 23, 28, 35 and 42 in nonreturn cases, hence the data are not repeated again for these groups. It is concluded that the ultrasound scanning is a very usefiil technique to study follicular dynamics as well as to detect early pregnancy and embryonic mortality in bovines. The Gir and Crossbred cows evince either 2- or 3-follicular waves per cycle, but not the single wave cycle. Ultrasonographic technique is helpful in detection of nonpregnancy as early as day 23 post-service with cent percent negative predictive value. There is regular growth and development of dominant follicles during early stages of pregnancy as a safe guard in the event of early embryonic mortality to evince next cycle in time. Plasma progesterone profile on day 21 post-breeding is also a very good index for detection of pregnancy with 79.55 per cent positive predictive value and cent per cent negative predictive value. The CL diameter and plasma P4 values as well as follicle size and E2 values were positively correlated. The results in general did not reveal any specific role of glucose or cholesterol in reproductive processes. Of the different synchronized treatment protocols, the highest overall conception rate was achieved with Ovsynch (83.33%) followed by CIDR (75.00%) and Mid-cycle PGF2a (66.66%) treatment and the least in control group (54.16%), hence the Ovsynch and CIDR treatments can be recommended to the field veterinarians for improving the fertility of infertile cattle.
  • Thumbnail Image
    ThesisItemOpen Access
    COMPARATIVE EFFICACY OF COCCIDIOSTATS ON EXPERIMENTALLY INDUCED EIMERIA TENELLA INFECTION ALONG WITH EFFECTS ON GROWTH HAEMATO-BIOCHEMISTRY AND PATHOLOGY IN BROILERS
    (AAU, Anand, 2014) HIRANI, NITINKUMAR DEVRAJBHAI; Hasnani, J. J.
    The efficacy of three commonly used feed coccidiostats named Diclazuril (T1) Salinomycin (T2), Diclazuril + Salinomycin (T3) in shuttle programme and Maduramicin (T4) on experimentally induced Eimeria tenella coccidial infection and their effects on growth, haematology, biochemical and histopathological changes were undertaken in three hundred Cobb400 strain of broiler at University Poultry Complex, Anand Agricultural University, Anand during year 2012. Birds were given feed containing Diclazuril (T1), Salinomycin (T2), and Maduramicin (T4) coccidiostats at dose rate of 1 ppm, 60 ppm and 5 ppm upto 42 days. Weekly body weight and feed consumption were recorded. Various parameters considered for comparative efficacy were studied. Experimental infection of 50,000 oocysts of E.tenella was given on 22nd day of age. Blood was collected before experimental infection at 3 weeks and after experimental infection at 4 weeks of age for haemato-biochemical study. The results of faecal score, oocyst per gram (OPG), lesion score, oocyst index value and mortality indicated better efficacy of coccidiostats as compared to non medicated birds in experimental infection with better efficacy of Maduramicin and Salinomycin as compare to Diclazuril and Diclazuril + Salinomycin shuttle treatment. Coccidiostats proved to have growth promoting action in broiler chickens during the experimental infection. Birds fed with Maduramicin medicated (5 ppm) performed well in terms of live weight gain and feed conversion ratio and it was followed by salinomycin (60 ppm) for weight gain and Diclazuril (Ippm) for feed efficiency in broiler birds. Result of sensitivity against E. tenella indicated good efficacy of Maduramicin (82%), whereas limited efficacy of Salinomycin (76%), Diclazuril (74%) and Diclazuril + Salinomycin Shuttle group (71%)) on the basis of Global index value (GINNC % ) Haematological studies revealed that haemoglobin concentration, packed cell' volume and total erythrocytes counts were significantly (P < 0.05) reduced, while total leukocytes counts were significantly increased on account of coccidial infection in all coccidiostat treatment and infected non treated groups. Different Leukocytes Count (DLC) value revealed significant increase in heterophills, lymphocytes and eosinophills and significant decrease in monocytes and basophills on account of coccidial infection. Results on haematological studies indicated comparatively less pathological damage by Salinomycin. Studies on biochemical profile revealed significantly (P < 0.05) lower serum glucose and serum total protein, while significant increase in serum total cholesterol. Serum Glutamic Oxalo-acetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT) and Alkaline Phosphatase (AKP) activities was observed due to coccidial infection as compared to pre infection levels in birds. Results of biochemical studies indicated comparative less pathological damage by coccidiostats treatment as compared to infected non treated group, but there was no consistent trend for drug choice. From histopathological study it was clear that the Maduramicin and Salinomycin treated group showed very less mechanical damage to tissue hence it could be used as a curative remedy against the caecal coccidiosis. The presence of clusters of large schizonts in the caecum was pathognonomic for E. tenella. The magnitude of infection type and dose of coccidiostat and stage of development of the disease could be established by histopathological observation.
  • Thumbnail Image
    ThesisItemOpen Access
    METAGENOMIC ANALYSES OF GERIATRIC GUT MICROBIOME DURING PROBIOTIC Lactobacillus helveticus MTCC 5463 INTERVENTION
    (AAU, Anand, 2014) SENAN, SUJA; Prajapati, J. B.
    Age related changes in the gastrointestinal tract, as well as changes in diet and host immune system reactivity, inevitably affect gut microbial population composition. Therapeutic strategies to counteract these changes have been suggested in ageing people. These include dietary supplements contairung prebiotics, probiotics and a combination of both of these, synbiotics. In this study a double blind randomized crossover trial was carried out where 72 elderly subjects were fed with fermented drink containing probiotic Lactobacillus lielveticus MTCC 5463 for 30 days. A real-time quantitative PCR assay based on bile salt hydrolase gene targeting primers and 3' minor groove binder (MGB) probes for accurate detection and quantification of Lactobacillus helveticus MTCC 5463 in human faecal samples was developed. Out of the 57 strains of Lactobacilli tested by in silico PCR, only two strains L. helveticus HIO and L. helveticus R0052 showed the predictive amplification while the rest 55 tested negative. The primers do not amplify any strains of Streptococcus thermophilus that were added to the fermented milks as starter culture. Genomic DNA standards were prepared with six different serial dilutions (2.68 X 106 to 2.68 X 10). The curve was found to be linear, with R2 values > 0.98, over the ranges of 10^ to lOi CPU/ml for MTCC 5463 strain. The slope of the standard curve was -3.372 which predicted the assay efficiency as 97.95%. At the end of 30 days the strain appeared in the faeces of all subjects in the treated group, reaching a level as high as 8.32 to the lowest amount of 6.17 log gene copies/g faecal matter at end of feeding period. After wash out, L. lielveticus MTCC 5463 was detected at gradually reduced levels. It can be observed that the lowest count of probiotic strain post washout was 3.75 log gene copies/g faecal matter at the end of 8 weeks of discontinuation of feeding. This proves the trarisient and consumption-dependent nature of this probiotic bacterium. The strain was not detected in any of the subjects before active test feeding. Traditional plate counts of lactobacilli at genus level on selective medium ranged from a baseline reading of 8.6 log CFU/g of wet fecal matter, which rose to 9.3 log CFU/g at the end of feeding period and a gradual decrease to 8.7 log CFU/gm at the end of the placebo feeding. The qPCR primers targeted the bile salt hydrolase gene of MTCC 5463 which made the gene copy count a fraction of the plate count. From the plate count results it can be said that the recovery of Lactobacilli from stool samples was at IxlO^ colony forming units/g (CFU/g) by week 4 giving a recovery of 82%. A more precise picture of the recovery of the strain calculated using qPCR results came to 18%. Among the 72 elderly subjects who participated in the trial, we could identify 10 respondents who showed positive results in the primary outcome of cholesterol reduction and 10 who showed an increase in cholesterol with a decreasing lactobacilli population indicating non response to probiotic therapy. DNA from the faecal samples of these 20 respondents during baseline and end of feeding was analyzed. Amplicons from the hypervariable region of the 16S rRNA gene were generated and sequenced each on a 316 chip. The data sets for before feeding have a total of reads ranging from 13,061 to 980,628 with read length ranging from 201 to 251 bps and a total amount of 42,52,62,470 bases. The data sets for after feeding showed reduction in reads ranging from 65 to 102,507 with read lengths varying from 268 to 165 with a total of 59,962,912 bps. After a washout of 4 weeks and before placebo feeding the sequencing data can be summarized having reads ranging from 1.386 to 172,304 with read lengths of 158 to 198 bps and a total of 24,10,52,754 bps. Post placebo feeding saw a similar trend of reads from 425 to 171,896 with sequence length from 133 to 155 bps with a total base pairs of 29,14,22,863. Sequencing reads were clustered into operational taxonomic units described by community metrics and taxonomically classified. Reads per sample were clustered and studied for diversity and richness using MG-RAST. All the community members in our samples were from the domain bacteria. The most prevalent phyla in all samples were: Firmicutes, Proeohacteria, Actinohacteria and Baderoidetes with Firmicutes dominating in all samples. All the samples taken prior to treatment showed an abundance in Blautia, Bifidobacterium, Clostridium, Escherichia, Eubacterium, Faecalibacterium, Lactobacillus, Prevotella, Roseburia, Ruminococcus and Shigella. It was strikingly evident that the non respondents harboured more Shigella, Escherichia and less Runinococcus and Clostridium (compared to positive respondents). Lactobacilli and Prevotella showed an increase in abundance values after probiotic treatment with a decrease in Shigella, Ruminococcus, Bacillus and Bifidobacterium. The shifts in gut community structure during probiotic therapy was also studied using QIIME, an open-source software pipeline able to perform, starting from raw sequence data, a wide range of analyses on microbial communities, that is, sequence aligiunent, identification of operational taxonomic units (OTUs), elaboration of phylogenetic trees, and phylogenetic or taxon-based analysis of diversity within and between samples. The responders showed 52.1 % Firmicutes compared to 40.6 % in non responders. The other major phyla Proteobacteria was higher in non responders at 49% than 38% in responders. The class based assignments of responders showed profound shifts in Bacilli, Clostridia and Gammaproteobacteria. Among non responder subjects, the relative proportion of Lactobacillales, Clostridials and Enterobacteriales could be the deciding biomarkers as in the case with responders. We could assume that interplay of Firmicutes and Proteobacteria and specific classes like Clostridiales, Enterobacteriales and Lactobacillales could be indicative of the amenability of the gut microbiota to dietary modification. To examine the difference in responders and non responders for the genera of major phyla Firmicutes and Proteobacteria we used STAMP software for statistical techiuques. We could identify a few microbial biomarkers that differentiate the responders from non responders. The STAMP analysis revealed that among responders and non responders the chief genera of Firmicutes that showed significant difference are Lactobacillus, Clostridium, Euhacterium, and Blautia (q< 0.002) while the genera of Proteobacteria included Shigella, Escherichia, Burkholderia and Camphylobacter (q-value<0.002). This proof-of-principle study introduces for the first time in India, potential microbial biomarkers for probiotic MTCC 5463 responsiveness in geriatric volunteers and reveals the potential of microbiota signatures for personalized nutrition.
  • Thumbnail Image
    ThesisItemOpen Access
    THE EFFECT OF FEEDING BYPASS FAT AND YEAST (SACCHAROMYCES CEREVISIAE) SUPPLEMENTED TOTAL MIXED RATION ON THE GROWTH PERFORMANCE OF WEANER SURTI KIDS
    (AAU, Anand, 2014) SHANKHPAL, S. S.; PARNERKAR, SUBHASH
    The studies were conducted to evaluate the effect of feeding bypass fat and yeast {Saccharomyces cerevisiae) supplemented total mixed ration comprising of 60: 40 concentrates: Jowar hay on the growth performance of weaner Surti kids. Based on the in vitro studies, the improvement in digestibility of DM, OM and maximum gas production; the optimum levels of live yeast {Saccharomyces cerevisiae) and bypass fat was decided as 2% for incorporation in TMR. Twenty-four growing Surti kids of similar body weight were randomly allotted to four groups, six in each and were individually fed for 15 days preliminary feeding and 120 days experimental period to meet their energy and protein requirements as per ICAR (1998) standards. The kids were fed TMR with no bypass fat and yeast (TO; TMR with 2% yeast (T2); TMR with 2% bypass fat (T3) and TMR with a combination of 2% each of yeast and bypass fat (T4) The average daily gain was 63.74 ± 0.54, 80.22 ± 0.38, 67.43 ± 0.68 and 88.50 ± 0.26 g in T1, T2, T3 and T4 groups, respectively. The treatment groups differed significantly (P<0.01) from each other. The highest gain was in T4 followed by T2, T3 and T1 groups. Similarly, the increase in the body measurements viz. increase in body length (10.00 ± 0.82, 10.83 ± 1.08, 10.83 ± 1.08, 9.67 ± 0.67 and 12.00 ± 0.82 cm), height (15.00 ± 0.58, 16.67 ± 0.67, 14.17 ± 1.19 and 16.33 ± 1.20 cm) and heart girth (15.33 ± 0.67, 16.83 ± 0.75, 15.67 ± 0.49 and 17.33 ± 1.09 cm) of kids in T1, T2, T3 and T4 groups, respectively, indicating that the gain in body length, height and heart girth was numerically higher in T4 kids compared to Ti, T2 and T3 groups. However, the treatment groups did not differ (P>0.05) from each other significantly. The average daily DMI of experimental kids in Ti, T2, T3 and T4 groups during digestion trial was 603.13 ± 17.04, 625.51 ± 15.48, 615.02 ± 11.89 and 639.40 ± 10.94 g/day and when expressed as kg/lOOkg body wt. was 4.37 ± 0.10, 4.25 ± 0.10, 4.40 ± 0.13 and 4.21 ± 0.09 and the same in terms of g/kg wt. was recorded as 84.12 ± 1.37, 84.13 ± 1.32, 84.95 ± 1.82 and 83.00 ± 1.24. The DM intake of experimental kids did not differ significantly (P>0.05). The average digestibility coefficients of nutrients in Ti, T2, T3 and T4 groups for DM (62.24 ± 0.37, 63.90 ± 0.31, 62.82 ± 0.23 and 64.52 ± 0.36%); OM (65.70 ± 0.38, 67.28 ± 0.32, 65.82 ± 0.23 and 67.80 ± 0.36 %); CP (67.37 ± 0.56, 68.34 ± 0.22, 68.14 ± 0.30 and 68.83 ± 0.23%)%); EE (71.05 ± 0.42, 70.90 ± 0.30, 75.68 ± 0.21 and 77.67 ± 0.25%); CF (63.27 ± 0.25, 66.79 ± 0.44, 62.69 ±0.16 and 67.89 ± 0.23%); NDF (56.69 ± 0.39, 60.17 ± 0.23, 57.70 ± 0.21 and 60.24 ± 0.22%); ADF (49.65 ± 0.45, 51.57 ± 0.23, 48.87 ± 0.05 and 50.50 ± 0.33%) and hemi-cellulose (68.37 ± 0.98, 75.63 ± 0.65, 72.76 ± 0.65 and 76.70 ± 0.36%) were significantly (P<0.01) higher in groups supplemented with Saccharomyces cerevisiae alone (T2) and combination of Saccharomyces cerevisiae and bypass fat (T4) group. But the groups did not differ with respect to digestibility coefficient of NFE (65.99 ± 0.68, 67.06 ± 0.54, 65.99 ± 0.35 and 66.95 ± 0.53%). The average DCP content of T1, T2, T3 and T4 groups (7.34 ± 0.06, 7.34 ± 0.02, 7.42 ± 0.02 and 7.42 ± 0.02 %) were statistically similar, but the TDN content of the T4 group (63.17 ± 0.32) was significantly (P<0.01) higher than T1 (61.46 ± 0.34), T2 (62.54 ± 0.29) and T3 (62.45 ± 0.21) groups which did not differ from each other. The respective cumulative intakes in Ti, T2, T3 and T4 groups during 120 days of DM (72.13 ± 1.83, 74.22 ± 1.89, 73.24 ±1.61 and 74.92 ± 1.47 kg), CP (11.51 ± 0.29, 11.71 ± 0.30, 11.67 ± 0.26 and 11.78 ± 0.23 kg) and that of TDN (44.35 ± 1.23, 46.42 + 1.25, 45.75 ±1.12 and 47.32 ± 0.90 kg) were statistically (P<0.05) similar.
  • Thumbnail Image
    ThesisItemOpen Access
    DEGRADATION AND DOWNWARD MOVEMENT OF VARIOUS AGROCHEMICALS IN CLAYEY, SANDY AND SANDY LOAM SOILS
    (AAU, Anand, 2014) LEKSHMI., S; SHAH, P. G.
    An experiment comprising of soil column study and microplot study were conducted based on degradation and downward movement of various agrochemicals in clayey, sandy and sandy loam soils was carried out in Pesticide Residue Laboratory at AINP on Pesticide Residues, Anand Agricultural University, Anand. The soil column study was conducted to see the leaching behavior and depth wise movement of agrochemicals in soil columns for duration of 15 days. The microplot study was carried out to know the dissipation of pesticides in clayey, sandy and sandy loam soils for duration of 60 days. Thi-ee types of soils viz., clayey collected from Navsari (T1), sandy from Dantiwada (T2) and sandy loam from Anand (T3) were used in this study. Soil columns were prepared to scmtinize the downward movement of agrochemicals in soil. Soil columns were wetted to their maximum water holding capacity by applying aqueous solution of 0.01 M CaCl2 and fortified with pesticides @ of 10µg g-1 on soil basis from the formulations on the top of the column. Each column was inigated @ 100 m L of aqueous solution of 0.01 M CaCl2 per day until the end of the experiment (15 days). Leachates collected after 5th, 10th and 15th day was analysed for agrochemical residues. The residue concentration in the leachate after leaching the soil column for 6 to 10 days was higher compared to the leachate collected during 0 to 5 and 11 to 15 days in clayey, sandy and sandy loam soils, The highest residue concentration was obtained in the leacliate collected from sandy soil which was followed by sandy loam and clayey soil. Soil columns were cut and soils at different depths Dl (0-6 cm), D2 (6-12 cm), D3 (12-18 cm), D4 (18-24 cm) and D5 (24-30 cm) were dried under shade and analyzed for agrochemical residues. The considerable amount of residue concentration of agrochemicals were obtained in all depths from Dl to D5 in clayey, sandy and sandy loam soil in the case of third and fourth group of agrochemicals. But for the first and second group of agrochemicals the depth wise distribution of residue concentration of agrochemicals in clayey, sandy and sandy loam soil was observed to be below determination level (BDL). The residue concentration of agrochemicals were mainly confirmed in the top soil layer in the case of clayey soil whereas in sandy and sandy loam soil, the depth wise distribution of agrochemicals found to be increasing as deptli increases. The depth wise residue retention found to be higher in clayey soil followed by sandy loam and sandy soil. This promotes the higher agrochemical residue concentration in the leachate obtained from sandy soil followed by sandy loam soil. The above results revealed the fact that the sandy soil possesses the highest leaching potential followed by sandy loam and clayey soil. In order to study the dissipation of applied pesticides from clayey, sandy and sandy loam soils a microplot study was conducted for duration of 60 days. Twelve microplots each of size 60x60 cm with a depth of 60 cm was considered in the experiment. A representative 300 kg bulk soil was taken and fortified at concerned level (10 times the recommended dose) by spraying of pesticide formulation. Samples taken at 0, 1,3,5,10,20, 30 and 60 days were analyzed for pesticide residues. The highest concentration of most of the pesticides was observed till 60 days in clayey soil. The samples of sandy loam soil indicated appreciable pesticide residue concentration up to 20 days whereas in sandy soil pesticide residue concentration was observed only up to 10 days. The lower content of organic matter might contribute to the faster rate of dissipation of pesticides from sandy soil thus resulting in lesser rate of adsorption of chemicals to sandy soil particles.