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Open Access
ANALYSIS OF DIFFERENTIAL EXPRESSION OF microRNA IN LIPOPOLYSACCHARIDE CHALLENGED LYMPHOCYTES OF VECHUR AND CROSSBRED CATTLE
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) DIVYA P. D; Dr. Shynu M.
The present study was carried out to analyse the differences in the expression of miRNAs in response to the in vitro LPS stimulation of PBMC cultures of crossbred and Vechur cattle. The study was performed in adult, apparently healthy, female crossbred and Vechur cattle maintained at University Livestock Farm and Fodder Research and Development Scheme, Mannuthy, and Vechur Conservation Unit, Kerala Veterinary and Animal Sciences University, respectively. The known/ novel miRNAs and the differential expression of miRNAs in response to bacterial endotoxin, LPS in PBMCs of the two genetic groups were identified by Short-read Illumina Next Generation Sequencing. Validation of NGS data of selected differentially expressed miRNAs was carried out by qRT-PCR assay. Prediction of target genes of differentially expressed miRNAs, functional gene enrichment analysis, analysis of cellular pathways involved and protein-protein interaction (PPI) network evaluation of the predicted target genes were also studied using various online bioinformatics tools. Cytokines associated with the immune related pathways of targets of differentially expressed miRNAs were analysed by ELISA. The differences in cytokine expression were also measured after overexpression of selected miRNA with miRNA mimics in the PBMC cultures of Vechur cattle. A total of 0.47 and 0.64 million clean reads, with an average Phred score of 34.92 and 34.75 corresponding 55.3 and 62.1 per cent of the adapter trimmed reads, respectively, for crossbred and Vechur samples were retrieved by NGS. Analysis of miRNAome identified 979 and 853 known miRNAs, and 393 and 139 novel miRNAs in samples from Vechur and crossbred cattle, respectively. Differential expression studies of NGS data revealed significant variation in the expression of miRNAs in LPS challenged PBMCs cultures of Vechur cows with respect to crossbred cattle. The results of real time validation of the expression of selected miRNAs by qRT-PCR assay were also consistent with the results of NGS. Functional gene enrichment analysis, analysis of pathways associated to the targets of differentially expressed miRNAs also revealed significant enrichment of targets of differentially expressed miRNAs in many immune related GO terms, immune associated cellular pathways as well as major cell signalling pathways. The PPI network analysis also showed active involvement of proteins encoded by these target genes in many of the important immune mechanisms. The study could identify differences in the immune related pathways associated to target genes of both up regulated and down regulated miRNAs though some pathways were found to be identical among both. Assessment of cytokines associated to the pathways regulated by the target genes of differentially expressed miRNAs in the supernatants from LPS treated PBMC cultures of also showed significant variations in the level of cytokines viz; TNF α, IL-4 and IFN γ among the crossbred and Vechur samples when compared to the control groups within the breed as well as between the breed. A significantly higher level of TNF α was noticed in LPS treated PBMCs of crossbred cattle whereas, IL-4 level was found to be significantly increased in LPS stimulated PBMCs of Vechur cattle compared to the LPS untreated cells from both groups. However, the present study could not detect any significant difference in IFN γ level among the LPS treated and untreated cells of both crossbred and Vechur cattle. The overexpression studies of miRNAs in Vechur PBMCs by transfecting with selected miRNA mimic could identify significant differences in IL-4 level while the changes were negligible with respect to other cytokines. The findings of the current research work suggest that both Vechur and crossbred cattle are having differences in their potential to tackle the immunological changes in response to an acute inflammation caused by the bacterial endotoxin; LPS. These differences might be contributing to the alleged immunological sturdiness to Vechur cattle compared to crossbred animals although, the specifics needs to be further validated.
Open Access
EFFICACY OF CAMPYLOPHAGES AND SELECTED PHYTOCHEMICALS IN THE CONTROL OF CAMPYLOBACTER BIOFILMS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) VIVEKANANDHAN R; Dr. B. Sunil
Campylobacteriosis is one of the most common causes of bacterial foodborne disease. Campylobacter spp. are found as commensals in the intestines of a wide variety of animals, particularly pigs utilised for food production. The present study was undertaken to analyse the efficacy of campylophages and selected phytochemicals in the control of campylobacter biofilms. A total of 130 samples including 40 samples each of faecal and rectal swabs and sewage (n=50) were collected from the two unorganised pig farms. An overall occurrence of 26.15 per cent with higher isolation from rectal swabs (57.5 per cent) than faecal and sewage samples (25 per cent and two per cent) were observed. In both the farms, no effective biosecurity measures were followed. In pork processing line, a total of 80 samples including 20 each of carcass swabs, cutting board swabs, knife swabs and sewage samples were collected. An overall occurrence of 12.5 per cent was observed. All the isolates were subjected to biofilm forming ability at three different temperatures 42℃ (10 per cent CO2 condition), 37℃ & 25℃ (aerobic condition) and compared with two media (Dulbecco’s Modified Eagle’s Medium (DMEM) & Muller Hinton Broth (MHB)). Strong biofilm formers were predominant in aerobic condition at 37℃& 25℃ incubated in MHB. Weak biofilm formers were more on stainless steel and nylon fibre. Carvacrol, trans-cinnamaldehyde, eugenol, ethanolic extract of citrus by-product and bleaching powder had minimum biofilm inhibition concentration of 0.078 mg/mL, 0.156 mg/mL, 0.156 mg/mL, 0.321 mg/mL and 0.625 mg/mL, respectively. Trans-cinnamaldehyde significantly (p<0.001) inhibited 99.39 per cent biofilm formation by the campylobacter isolates on polystyrene surface at MBIC of 0.156 mg/mL. Citrus by-product ethanolic extract significantly (p<0.001) inhibited 99.43 per cent biofilm formation by the campylobacter isolates on stainless steel surface at MBIC of 0.312 mg/mL.Carvacrol significantly (p<0.001) inhibited 97.80 per cent biofilm formation by the campylobacter isolates on nylon fibre surfaces at MBIC of 0.078 mg/mL. Carvacrol, trans-cinnamaldehyde, eugenol, ethanolic extract of citrus by-product and bleaching powder had minimum biofilm inactivation concentration of 0.312mg/mL, 2.5 mg/mL, 1.25 mg/mL, 2.5 mg/mL and 5 mg/mL, respectively. Trans cinnamaldehyde inactivated 21.49, 57.89 and 95.92 per cent pre-formed biofilms by campylobacter isolates after 2, 5, 10 min of exposure, respectively at MBIAC of 2.5 mg/mL on polystyrene surface. Eugenol significantly (p<0.001) inactivated 53.79, 70.87 and 98.90 per cent pre-formed biofilms by campylobacter isolates after 2, 5, 10 min of exposure, respectively on stainless steel surface at MBIAC of 1.25 mg/mL. Trans-cinnamaldehyde significantly (p<0.001) inactivated 87.71, 93.9 and 99.15 per cent pre-formed biofilms by campylobacter isolates after 2, 5, 10 min of exposure, respectively on nylon fibre surface at MBIAC of 2.5 mg/mL. Three campylophages were isolated from sewage samples of farm B. Phage 2 and 3 were characterised as Siphoviridae and phage 5 was as Myoviridae by transmission electron microscope. Phage 5 inactivated 30.3 per cent, 30.55 per cent and 29.60 per cent pre-formed biofilms of campylobacter isolates on polystyrene, stainless steel and nylon fibre surface after 24 h of exposure, respectively. Campylophages were effective in inactivating pre-formed biofilms by the isolates which were lysed by the phages. Campylophages were not effective in inactivating biofilms formed by the campylobacter isolates which did not show lytic activity. Cost analysis showed citrus by-product extract as cost effective (Rs 0.41/L) than other phytochemicals.
Open Access
COMPARATIVE HISTOMORPHOLOGY AND ULTRASTRUCTURE OF THE SKELETAL MUSCLE OF BROILER AND KUTTANAD DUCKS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY ADN ANIMAL SCIENCES UNIVERSITY, 2023-03-23) ALPHINE JOSEPH; Dr. S. Maya
The present study was conducted to investigate the comparative histomorphology and ultrastructure of the skeletal muscles from selected areas in meat type and spent Kuttanad ducks, to determine the comparative proportion of fibre types in skeletal muscles using histochemical methods and to compare the physical, chemical and mechanical properties of the skeletal muscle between the groups by assessing the correlation of meat quality like tenderness and juiciness with muscle fibre properties. A total of 72 birds comprising of 12 birds each of males and females from broiler Vigova SuperM ducks (six to eight weeks of age), Kuttanad ducklings (six to eight weeks) and spent Kuttanad ducks (over 72 weeks) selected at random were used for the study. Muscle samples from six selected regions, viz. neck (Longus coli posticus- LCP), back (Scapulohumeralis caudalis- SHC), breast (Pectoralis- PEC), wing (Biceps brachii- BB), thigh (Iliofibularis- IF) and drumstick (Gastrocnemius lateralis- GL) were collected from the Meat Technology Unit, College of Veterinary and Animal Sciences, Mannuthy, after humane slaughter, processed appropriately and the data collected were analysed statistically using SPSS version 24.0. Morphologically, the breast muscles were the longest among all muscles. The mean length of the muscles was significantly different between groups, in all the regions. Breast muscle was the heaviest muscle in all the groups and among groups, the heaviest was the pectoralis muscle in Kuttanad duckling males. Mean thickness of muscle was maximum for broiler ducks for the breast muscle. Histologically, the muscle was composed of muscle fibres, connective tissue, blood vessels and nerves in all the groups. The connective tissue covering identified were epimysium, perimysium and endomysium with varying degree of thickness in different muscles. Three types of muscle fibres were identified using enzyme histochemical staining techniques, viz. type I, IIA and IIB. Glycogen content was found copious in type IIB fibres under PAS reaction, while fat droplets in the type I fibres were noticed on Oil red O staining. Alkaline phosphatase and acid phosphatase activity showed negative for muscle fibres indicating the absence of any regenerative or degenerative stages of muscles fibres respectively. The endothelial lining of capillaries amidst the muscle fibres showed a positive reaction for alkaline phosphatase in almost all muscles.On micrometry, maximum average fibre density was observed in breast muscle in spent Kuttanad duck males. In all other regions, the maximum average fibre density was seen in broiler duck males with decreasing order of PEC>SHC>BB>GL>LCP>IF. The maximum average fibre diameter was noticed in breast muscle. In breast, wing and drumstick muscles, broiler ducks showed the maximum average fibre diameter whereas in neck and thigh by Kuttanad duckling males and in back by Kuttanad duckling females. The average fibre diameter from maximum to minimum from PEC>GL>SHC>IF>BB>LCP. Kuttanad duckling males showed the highest mean cross-sectional area of muscle fibres in the neck muscle. The order of average cross-sectional area of different muscles were LCP>IF>SHC>BB>GL>PEC. The average cross-sectional area of muscle fibres was minimum for broiler duck males in all the muscles except breast muscle. However, the mean cross-sectional area did not differ between groups and between genders in the breast muscle. The highest mean fascicle cross-sectional area was obtained for spent Kuttanad duck females in the iliofibularis muscle. Myosin ATPase, SDH and NADH-TR enzyme histochemistry staining of muscle fibres revealed type I, IIA and IIB fibres in neck, back, thigh and drumstick. Type I fibres were noticed concentrated in particular portions of stained tissue sections of back and drumstick muscles. Breast and wing muscles were identified with only type IIA and type IIB fibres. The presence of type I fibres in neck, back, thigh and drumstick musclesand clear absence of type I fibres in wing and breast muscles were confirmed by immunohistochemistry (IHC). Both immunohistochemistry and enzyme histochemistry staining showed relatively similar qualitative results of different fibre types, with IHC method being the more accurate method. Overall, the breast muscle and wing muscle showed type IIA> IIB pattern except in broiler ducks, where the IIA and IIB are found almost equal. The back and drumstick muscles showed IIB>IIA>I pattern. Considering the ratio of IIA and IIB fibres, more IIB fibre component was observed as SHC>GL>BB> PEC. Both in neck and thigh muscles, spent Kuttanad muscle showed more type I fibres followed by broiler ducks and then by Kuttanad ducklings. Neck muscle showed higher proportion of cross-sectional area for type I fibres than thigh muscle. Scanning electron microscopy of muscle fibres revealed the thick and thin filament arrangement of connective tissue amongst muscle fibres. Under transmission electron microscopy, the, myofibrils, sarcomere, triad, myonuclei and interfibrillar, perinuclear and subsarcolemmal mitochondrial populations were identified in the duck muscle. Interfibrillar mitochondrial concentration was comparatively higher in drumstick muscle than breast muscle that too in spent Kuttanad ducks than Kuttanad ducklings.Serum biochemical analysis showed all the parameters examined within the normal level among all the groups indicating the absence of any injury to the muscles. The pre-slaughter live weight, defeathered weight, mean dressed weight were significantly higher in broiler ducks. The mean carcass yields showed significant interaction with a significantly higher values for spent Kuttanad ducks males (77.44 ± 1.50 per cent) than others in male groups. Physical properties of muscle such as colour and pH were analysed in the study. The highest colour L* value was given by back muscle of broiler duck males and the lowest colour L* value was noticed in the breast muscle of male spent Kuttanad ducks. The highest mean colour a* value was obtained for spent Kuttanad duck (14.6 ± 0.13) males in the neck region and the value was significantly higher than other male groups. Mean b* value was maximum for the neck region of broiler duck and in the wing region of spent Kuttanad ducks. The pH of breast muscle had a drastic fall within the first three hours. Initially, the pH was higher in breast muscle in spent Kuttanad duck males and females. The pH of back, thigh and drumstick muscles showed a higher ultimate pH at 24 h. At 15 minutes, the comparison of pH in hot-boned and intact muscle showed significant differences in the wing, back, thigh and drumstick regions. The pH value of intact muscle showed a significantly higher value than hot-boned muscle. Mechanical property of muscle was analysed by measuring the sarcomere length at 15 min, 1 h, 3 h, 6 h and 24 h. At all the intervals, wing muscle showed the highest sarcomere length in broiler ducks. The R value reached to the value of one in almost all muscles within one hour which indicated the fast onset of rigor mortis. R value was higher in neck muscle in spent Kuttanad ducks. The highest mean moisture content was obtained for broiler ducks males in the neck region. The intramuscular fat was obtained highestin the neck muscle by the spent Kuttanad duck females. The collagen content was evidently and copiously detected in the drumstick muscle of Kuttanad duckling males. The highest mean myoglobin content was obtained for drumstick region followed by thigh region in spent Kuttanad duck males. On SDS PAGE analysis, bands were observed in between 180 to 245 kDa molecular weight and the thigh muscle showed thick and differentiated band in all the groups. The migration of myosin heavy chain isoform bands was greatly affected by the quality of the frozen sample and the temperature of the apparatus.The mean flavour scores did not differ significantly between groups except in the thigh region. There was no significant difference observed in the juiciness and mouth coating score between groups and genders. In the back region, the highest mean ease of fragmentation score was obtained for broiler duck males. The overall tenderness score showed significant difference (p<0.05) in breast muscle and back muscle between groups. The overall tenderness score was significantly higher in broiler ducks in the back region. Overall acceptability score was significantly highest in back muscle followed by breast muscle in broiler ducks compared to spent Kuttanad ducks and similar to Kuttanad ducklings. The correlation study showed that the collagen content had significantly positive correlation on muscle fibre length and negative correlation on cross sectional area. The present study indicated that the muscles can be ranked for better meat quality according to the score of individual muscle fibre properties like the proportion of different fibre types, density of fibres and the cross-sectional area of fibres. Apart from that, meat quality was not only affected by a single factor, but by a number of factors and its combinations, that play a major role in the conversion of muscle to meat as well as its tenderness. Differences in muscle fibre properties may be attributed to the anatomical function of particular muscles. In the present study, the different combination of muscle fibre properties like the increased proportion of fibre type IIB, decreased density of fibres and decreased cross sectional area of fibres of the muscle, together resulted in the overall acceptability of the meat from that muscle. Overall, broiler duck showed better meat quality traits than Kuttanad ducks. Since, each muscle was different in muscle fibre properties, selection of breeds with increased quantity of back and breast muscle will increase the quality of duck meat.
Open Access
TRANSCRIPTOME ANALYSIS OF PECTORAL MUSCLE AMONG PEKIN AND KUTTANAD DUCKS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) S. SANKARALINGAM; Dr. P. Anitha
The study on “Transcriptome analysis of pectoral muscle among Pekin and Kuttanad ducks” was carried out at Department of Poultry Science, College of Veterinary and Animal Sciences, Mannuthy. The experimental birds of male Pekin, Kuttanad control and Kuttanad meat line ducks were reared up to eight weeks of ageand their production parameters, carcass characteristics, physicochemical attributes and compositional characters of meat were also measured from random samples of each replicate at four and eight weeks of age to find the differences among the three types of ducks. Along with that, all the above parameters of heavy- and low-weighing birds of each replicate were also measured in all three groups of ducks to correlate them with the data derived from transcriptome analysis of pectoral muscle of heavy- and low-weighing birds.The study on production parameters among Pekin duck, Kuttanad control population and Kuttanad meat line concludes that the Kuttanad selected line for meat had significant (p<0.05) improvement in body weight from the day-old to eight weeks of age compared to that of Kuttanad control population as a result of four generations of individual selection for eighth week body weight. The mean body weight gain of Kuttanad meat line was not significant compared to that of Kuttanad control population up to four weeks of age and it was significantly (p<0.05) higher at 5-6 weeks period which made the overall body weight gain up to eight weeks significant. During this period, the enrichment of differentially expressed genes (DEGs) for growth process was higher (3%) in GO analysis of transcriptome data between heavy- and lowweighing birds of Kuttanad meat line while that of Kuttanad control population and Pekin duck were zero. The potential markers for enrichment of growth process in Kuttanad meat line were adrenoceptor alpha 2A (ADRA2A), dendrocyte expressed seven transmembrane protein (DCSTAMP), ventricular zone expressed PH domain containing 1 (VEPH1), dual oxidase 2 (DUOX2), maelstrom spermatogenic transposon silencer (MAEL), R-spondin 2 (RSPO2), acleraxis bHLH transcription factor (SCX), matrilin 1 (MATN1), forkhead box C1 (FOXC1), fibroblast growth factor 8 (FGF8) and growth differentiation factor 5 (GDF5) along with the nonsynonymous missense variant of SCX gene c.82G>C. The feed intake of Kuttanad meat line was significantly (p<0.5) more than that of Kuttanad control population after four weeks of age, this was due to the significant (p<0.05) increase in body weight gain of Kuttanad meat line after four weeks of age compared to Kuttanad control population. Even though there was significant (p<0.05) increase in body weight and weight gain in Kuttanad meat line compared to Kuttanad control population, the FCR was similar in both groups and less than that of Pekin duck. The FCR may improve in Kuttanad meat line, if the selection process is continued for few more generations.The study on carcass characteristics reveals that the eviscerated yield, breast yield, deboned breast yield, wings yield, neck yield, abdominal fat yield, heart yield, liver yield and gizzard yield percentage did not have any significant difference among the three types of ducks at four and eight weeks of age. The significant (p<0.05) increase in thigh yield percentage in the Kuttanad meat line compared to that of Kuttanad control group and Pekin duck indicates that the selection for eighth week body weight in Kuttanad duck resulted in significant (p<0.05) increase of thigh muscle compared to proportionate increase of other muscles. The study on physicochemical attributes of meat indicates that the selection for eighth week body weight significantly (p<0.05) improved the myofibril fragmentation index in breast muscle of Kuttanad meat line compared to Pekin duck. This study also concludes that the significantly (p<0.05) lower muscle fibre diameter and shear force in breast muscle compared to that of thigh muscle without affecting the sarcomere length could have led the breast muscle more tender than the thigh muscle. The study on compositional characteristics of meatconcludes that the selection for eighth week body weight significantly (p<0.05) improved the collagen solubility of thigh muscle in Kuttanad meat line compared to that of Pekin duck at four weeks of age. The transcriptome analysis of pectoral muscle of heavy- and low-weighing Kuttanad ducks at four weeks of age revealed that the downregulation of KIF24 gene in low-weighing Kuttanad meat line and its’ 20 SNPs and upregulation of CA3A and cystine/glutamate transporter-like (LOC116494038) genes in heavy-weighing Kuttanad meat line along with more enrichment in catalytic activity and cellular, metabolic, cellular component organization and growth processes could have contributed to the significant (p<0.05) growth of heavy-weighing Kuttanad meat line compared to that of low-weighing duck and also they may indirectly influence theevisceration percentage, deboned breast yield percentage, wings yield percentage and liver percentage and also improve the myofibril fragmentation index in breast muscle. This concludes that the fourth week body weight may also be considered along with selection for eighth week body weight to get more genetic gain per generation of selection. The same analysis at eight weeks of age revealed downregulation of TRIM7gene in heavy-weighing birds and its’ 25 SNPs; upregulation of GATM gene in heavyweighing birds and its’ 21 SNPs; upregulation of BCAR3 gene in heavy weighing Kuttanad meat line and its’ 15 SNPs; upregulation of AKR1D1 gene in heavy-weighing birds and its’ six SNPs; downregulation of NCEH1 gene in heavy-weighing Kuttanad meat line and its’ 18 SNPs and upregulation of CA3A, FRAS1, SLC25A48, LOC101802407, ACTC1 and ENAH genes in heavy-weighing birds and also downregulation of PPARGC1A, TAGAP, FAR2, MKNK1, NT5DC1, FABP3, DGAT2, ASB18 and GK genes in heavy-weighing birds along with more DEG enrichment in catalytic activity and cellular component organization, metabolic, biological phase, growth, locomotion, multi-organism process, biological adhesion and cell population proliferation processes could have contributed to significantly (p<0.05) higher eighth week body weight of heavy-weighing Kuttanad meat line compared to that of lowweighing duck. The above potential markers may indirectly contribute to significant increase in breast, deboned breast, drumstick, abdominal fat and gizzard yield percentage and also higher myofibril fragmentation index of thigh muscle and muscle fibre diameter of breast muscle and reduction in shear force of breast and thigh muscles,which ultimately may increase the tenderness of the meat of Kuttanad meat line without affecting the compositional characters.The transcriptome analysis of pectoral muscle of heavy- and low-weighing Kuttanad control population at four weeks of age revealed significant downregulation of MYOM3 gene in low-weighing birds due to the presence of 29 SNPs and upregulation of METTL21C gene in low weighing Kuttanad duck control population due to the presence of four SNPs could have contributed to the significant (p<0.05) growth of heavy-weighing Kuttanad control population compared to that of lowweighing birds at four weeks of age with more enrichment of catalytic activity and metabolic, multicellular organismal and biological regulation processes. The above potential markers may indirectly increase in evisceration percentage, breast, deboned breast, wings, neck and heart yield percentage and also numerical increase in muscle fibre diameter of breast muscle in Kuttanad control population without affecting the compositional characteristics. At eight weeks of age no significant DEG was identified from GO analysis of transcriptome data between heavy- and low-weighing duck of Kuttanad control population.The transcriptome analysis of pectoral muscle of heavy- and low-weighing commercial Pekin ducks at four weeks of age revealed significant downregulation of CCN3 gene in low-weighing Pekin duck due to the presence of seven SNPs and also upregulation of MATN1 gene in heavy-weighing duck along with more enrichment of transporter, structural molecule and molecular transducer activities and multicellular organismal, localization, locomotion, response to stimulus, signaling and biological adhesion processes could have contributed to significantly (p<0.05) higher growth of heavy-weighing Pekin duck compared to that of low-weighing duck at four weeks of age. The above potential markers may indirectly influence the significant (p<0.05) increase in evisceration, deboned breast, wings and gizzard percentage and also myofibril fragmentation index in Pekin duck without affecting the compositional characteristics. The same analysis at eight weeks of age revealed upregulation of ATP2A2 gene in heavy-weighing Pekin duck and presence of seven SNPs; upregulation of TNNI1 gene in heavy-weighing birds and itsfour SNPs; downregulation of MYBPC1gene in low-weighing Pekin duck and its 17 SNPs and also upregulation of CA3A/CA3, NR4A3, LMOD2, TFF2, IRX2, ACTC1 and ALB genes in heavy-weighing Pekin duck along with more enrichment of binding and transcription regulator activities and multicellular organismal, growth, locomotion, multi-organism, biological adhesion, cell population proliferation and immune system processes could have contributed to the significantly (p<0.05) higher body weight in heavy-weighing Pekin duck compared to that of low-weighing Pekin duck at eight weeks of age. The above potential markers may indirectly influence the significant (p<0.05) increase in muscle fibre diameter of thigh muscle of Kuttanad duck without affecting the compositional characteristics.The present study concludes that the Kuttanad meat line evolved from Kuttanad duck breed through selection for eighth week body weight over four generations is moving in the right direction of selection with moderate response. The response to selection is only around 250g body weight gain after four generations of individual selection which is not enough to cope up with the demand for duck meat. So, it is advisable to go for genomic selection using the candidate genes identified from this study along with the individual selection to improve growth, carcass characteristics, physicochemical attributes and compositional characteristics of Kuttanad meat line. It is advisable to go for combined selection for fourth and eight week body weight to get more genetic gain. This study also concludes that the above potential markers can be used for genomic selection in Kuttanad meat line, Kuttanad control population and commercial Pekin duck, respectively after validation of SNPs.
Open Access
ANTIMICROBIAL USE AND PRESCRIBING BEHAVIOUR OF VETERINARIANS IN LARGE ANIMAL PRACTICE IN KERALA
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-23) G.VIJAYAKUMAR; Dr. P. Reeja George
A mixed method research design was used to investigate the perception of veterinarians engaged in large animal practice about antimicrobial use and resistance as well as their knowledge, attitudes and practices regarding antimicrobial resistance and stewardship. Four focus group discussions were conducted in four districts of Kerala, each with five veterinarians engaged in large animal practice, as part of the qualitative part of the study. Themes emerging from these discussions were used for questionnaire development for the quantitative part of the study. The structured, pre tested questionnaire was sent by Google form linksto 1580 veterinarians working in Animal Husbandry Department, Kerala. A total of 336 responses were obtained and these formed the sample for the study. Majority of the respondents were middle aged (55.1 per cent), male (55.1 per cent), married(94.6 per cent), undergraduate (45.2 per cent), veterinary surgeons (78.6 per cent), working in veterinary dispensaries (63.4 per cent) situated in panchayat localities (74.7 per cent). Most of the respondents had between 11 and 22 years of total work experience (39.28 per cent), had not attended any training on antimicrobial use and resistance (66.7 per cent) and had not participated in any antimicrobial stewardship programmes (93.2 per cent). It was also evident that 47.02 per cent of the veterinarians had medium level of confidence with regard to appropriate antimicrobial prescribing and 71.4 per cent had a low level of general awareness about antimicrobial resistance and stewardship. Clinical signs and prior experience were the major clinical and non-clinical factors that influenced the prescribing decision of veterinarians on whether to prescribe an antimicrobial or not. Spectrum of activity of the drug and clinical signs were the major pharmacological and nonpharmacological factors that influenced the prescribing decision of veterinarians to choose a particular antimicrobial. Entrofloxacin, oxytetracycline and ceftriaxone were the antibiotics frequently used by the veterinarians in large animal practice. The results of the study also indicated that 47.61 per cent of the veterinarians had a medium level of perception about antimicrobial use and resistance; 47.32 per cent had a medium level of knowledge and 34.82 per cent had a moderately favourableattitude towards antimicrobial resistance and stewardship. It was also observed that 40.18 per cent of the veterinarians had a high level of adherence to optimum antimicrobial stewardship practices. Pressure or demand from clients, lack of access to antibiotic sensitivity testing laboratory facilities and insufficient fund allotment for drug purchase were the major barriers to optimum prescribing perceived by veterinarians. Establishing more antibiotic sensitivity testing laboratory facilities and providing sufficient veterinary manpower in the field were the major interventions suggested by the respondents to optimise prescribing practices.
Open Access
EVALUATION OF ANTICANCER PROPERTY OF RECOMBINANT GOAT LACTOFERRIN
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-08) VASUDHAR BHAT S. V.; Dr. Uma R.
The present study was conducted with the objective to express and purifyrecombinant Malabari goat lactoferrin in Escherichia coli (E. coli). The study was carried out in three phases. In the phase I, a portion of lactoferrin (Lf) gene encoding the N lobe of Malabari goat lactoferrin (cLf-N) was cloned into pJET1.2/blunt cloning vector and transformed into E. coli strain JM109 and was sequenced which revealed a 793 bp fragment with 100 per cent similarity to goat Lf in the database. This ampliconwas subcloned into pET28a(+) expression vector and transformed into BL-21 (DE3) pLysS bacterial host. The transformed E. coli BL21 (DE3) pLysS containing the recombinant plasmid expressed maximum recombinant protein (rcLf-N) upon induction with 1mM IPTG at 37°C for five hours. The recombinant protein, rcLf-N, containing polyhistidine tag was purified using Ni-NTA affinity column chromatography and confirmed as a derivative of Lf by Western blotting. In phase II, rcLf-N was analysed for its in vitro anticancer activity in Daltons Lymphoma Ascites (DLA) cell lines. In MTT assay there was a significant (p<0.05) reduction in the per cent inhibition of cell proliferation in a concentration-dependent manner from 800μg/mL to 8.75μg/mL, indicating the cytotoxic effect of rcLf-N in a dose-dependent manner upon DLA cell lines. The half-maximal inhibitory concentration (IC50) value was found to be 263.5 μg/mL using Graph pad prism. Further, upon Acridine orange/ Ethidium bromide (AO/EB) and Hoechst staining of the treated cells, the apoptotic changes produced by rcLf-N and standard drug Cisplatin in DLA cells were highlighted. During phase III trial, the in vivo anticancer activity of rcLf-N was analysed on Swiss albino mice bearing DLA induced solid tumour. Based on preliminary studies the concentrations 50μg and 75μg of rcLf-N per animal were selected for comparison with the standard drug Cisplatin. A significant (p<0.05) reduction in tumour weight, tumour volume and tumour weight to body weight ratio was observed in the rcLf-N and cisplatin treated groups compared to the control group. Maximum reduction was observed in group treated with rcLf-N @75μg intratumourally for three days. Histopathological examination of tumour tissue in all the groups treated with rcLf-N and cisplatin showed the presence of apoptotic changes with decreased spread of neoplastic cells into surrounding tissues and decreased neovasculature.Relative expression of VEGF and Caspase-3 genes was analysed in both in vivo and in vitro studies with GAPDH as the reference gene via quantitative real-time PCR. A dose-dependent downregulation of VEGF and upregulation of Caspase-3 was revealed in the cells/tissues treated with IC50 and double IC50 doses of rcLf-N both in vitro and in vivo. Similar results were observed with IC50 Cisplatin. On comparison between intratumoural and intraperitoneal routes of treatment in vivo, the intratumoural route of treatment was better in downregulating VEGF and activating Caspase-3 on day 7 and 14, although the fold change was non-significant. From the present study, it could beconcluded that the novel recombinant protein produced antineoplastic activity through apoptosis and rcLf-N @75μg exhibited most potent anticancer activity against DLA cells.
Open Access
DEVELOPMENT OF REGION-SPECIFIC PHAGE RESISTANT DAIRY STARTER COMBINATIONS FOR THE STATE OF KERALA
(VERGHESE KURIEN INSTITUTE OF DAIRY AND FOOD TECHNOLOGY MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-12-08) ARCHANA CHANDRAN; Dr. A.K Beena
A total of 200 curd samples were collected from the two agroecological zones of Kerala covering the districts of Idukki, Ernakulam, Thrissur, Palakkad and Wayanad. Consumer preference for medium low acid curd was significantly higher than high acid curd in both zones. Average acidity of curd samples in per cent lactic acid was1.65 ± 0.06 in high range and1.49± 0.04 in central mid land. The average counts of coliforms, S.aureus (coagulase positive) and yeast and mold in the samples were higher than the permissible limits prescribed by FSSAI in both zones. The predominant lactic acid bacteria isolated from curd samples were L.plantarum (26 per cent) followed by L.fermentum (22 per cent). The presence of E.faecaliswas detected at 31 per cent level in central midland. Detection of virulence genes gel E and asa 1 in E.faecalis isolates from curd samples is a safety concern. Study on the yeast microflora of curd suggested the synergistic association of yeast and LAB to be decisive of technotextural properties of curd. Lactose fermenting yeasts like Kluyveromyces marxianus, Kluyveromyces lactis and Pichia kudriavzveii were also isolated from the curd samples. Bacteriophages attacking Lactobacillus sp were detected in effluent samples at a level of 105-107 PFU/ml. The four Lactobacillus phages belonged to Siphoviridae family. Seventy three per cent of LAB strains in the study were found to be phage resistant. L.fermentum (ADMH 12) and Pichia kudriavzveii (Y01) were combined in 1:1 ratio for developing phageresistant starters. Overall acceptability of curd (7.58 ± 0.21) was significantly higher for the product prepared using starter combination than those prepared using single starters. Skim milk lactose combination (1:1) was found to be the ideal cryoprotectant for ADMH12 and skim milk-trehalose combination (1:1) for Y01
Open Access
PROCESS STANDARDIZATION OF CASEIN HYDROLYSATE INCORPORATED FUNCTIONAL PREMIX FOR WOMEN
(DEPARTMENT OF DAIRY TECHNOLOGY VERGHESE KURIEN INSTITUTE OF DAIRY AND FOOD TECHNOLOGY MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-11-28) BEENA R.L; DR. S.N. Rajakumar
Calcium is the most abundant mineral in the human body and is vital for bone health and many physiological functions. Cereals and legumes are staple foods in India. They generally contain phytates and oxalates that tightly bind calcium and other divalent cations. Bioactive peptides produced from protein hydrolysis are found to have many functional properties, one of which is facilitating better calcium absorption in the body even from a high phytate food. This concept was taken to formulate the hypothesis of the present work. An attempt was made to formulate a diverse composite premix for women based on Recommended Daily Allowance by ICMR (2020). The premix base was made with selected cereals, pulses, and an oil seed which are nutrient-dense and locally available. The proportion of their addition was optimized using CCRD of RSM taking sensory scores as responses. The optimized composite flour mix had an ingredient composition of 65.95 parts of cereal blend, 36.01 parts of pulse blend, and 3.98 parts of oilseed. The functional ingredient, casein hydrolysate was prepared and the level of addition to the premix was optimized. Characterization of the casein hydrolysate was done with respect to calcium binding and calcium solubilizing properties and amino acid profile of the hydrolysate was also studied. SDS-PAGE was done to determine the molecular weight of the peptide fractions. The final optimized composite flour was kept for storage study in metalized polyethylene pouches sealed inside a carton box. It was kept at room temperature for 100 days and evaluated its sensory, physico-chemical and microbial properties once in every 20 days. The product was found to be shelf-stable even after 100 days. In-vivo study was conducted in female Wistar rats (two-month-old) for four weeks. Calcium content in the femur was analyzed after the study period and the treatment group with casein hydrolysate was found to have significantly (p< 0.01) higher calcium than the control groups.
Open Access
CLINICO-THERAPEUTIC AND ULTRASTRUCTURAL STUDIES ON CANINE ATOPIC DERMATITIS WITH SPECIAL REFERENCE TO FILAGGRIN GENE POLYMORPHISM
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-11-10) AMBILY V. R.; Dr. Usha Narayana Pillai
In the present study conducted on canine atopic dermatitis, among the total 2032 dermatological cases in dogs presented to the clinics from different parts of Kerala, 245 cases were diagnosed as various allergies over a period of three years. Out of these, 63 cases were recorded as canine atopic dermatitis based on Favrot’s criteria with the exclusion of other differentials. All the 63 cases diagnosed as CAD were utilized for epidemiological as well as clinical lesion distribution pattern studies using CADESI-04 scoring system. A total of hundred animals, 50 each of atopic and healthy controls were subjected to polymorphism studies. Sixteen atopic animals, which satisfied atleast six out of eight Favrot’s criteria with alesional pruritus at onset and pruritus score above two with clinical lesional scores above 20 were selected for detailed study to reduce variations due to extent and severity of clinical signs. In the present study, occurrence rate of allergic dermatitis recorded was 13 per cent, atopic dermatitis being second to flea allergy dermatitis. Male dogs below 3 years of age were found more affected with CAD. Distribution of lesion follows a pattern in atopic dermatitis with both paws and flexural areas were affected followed by axillae and ear pinnae, which can be better utilized for diagnostic purpose. Haemato-biochemical studies of atopic dogs revealed hypoalbuminaemia with reduced AG ratio, anaemia with leukocytosis, neutrophilia and eosinophilia. Absolute eosinophil count with positive correlation with neutrophil to lymphocyte ratio (NLR) in atopic dogs was of diagnostic significance. Serum Ig E and IL-31 were found significantly elevated in atopic dogs. The IL-31which was correlated significantly with clinical scores, indicated the role of IL-31 as key cytokine in atopic dermatitis. Invitro serum allergen testing revealed allergens such as grass mix, weeds, pollen grains of mulberry and acacia tree, house dust, house dust mites and storage mites sensitive to dogs in the region studied. Histopathological examination of atopic skin was characterized by diffuse epidermal hyperplasia with acanthosis, spongiosis and prominent rete ridges, as well as orthokeratotic hyperkeratosis as major findings. Immunohistochemical staining of filaggrin protein in lesional and non lesional skin of atopic animals exhibited faint granular staining, where as normal skin of healthy animals exhibited intense discrete staining in the stratum granulosum. Transmission electron microscopic studies revealed varying changes in the epidermis, dermis and subepidermal areas. The corneal area, epidermal cells belonging to stratum basale, stratum spinosum, stratum granulosum and stratum lucidum could be seen with retrograde changes of varying intensity. Further non keratinocyte cells that inhabit the epidermis such as melanocytes, Langerhan cells also showed changes. Dermal components were seen loosely textured and contained collagen fibrils, fibroblasts, macrophages and blood vessels. Polymorphism studies revealed 12 SNPs in the amplified fragment of exon 3 FLG, out of which 11 were novel. Genotype frequencies revealed that one novel SNP (Exon 3: c.2584G>T) and other reported SNP (c.2337T>G) were associated with CAD. The novel variation was found to have deleterious effects on protein function as analysed by five bioinformatics tools in combination and, it was also identified as a disease causing one, which affects protein stability and function. The treatment response was evaluated by the periodic reduction in clinical lesional score, heamato-biochemical, serum Ig E and IL-31 values. Cyclosporine @ 5mg/kg body weight was an effective and well tolerated drug for treating atopic dermatitis in dogs, with considerable improvement noticed in pruritus and skin lesions during the first month of treatment followed by tapering the dose by reducing the frequency of administration to every other day. Regular bathing with a nonirritant shampoo helped in reducing the pruritus and this might be due to the restoration of skin barrier function by its moisturizing and cleansing action. Omega-3 fatty acids could lower Ig E production in allergic individuals and could lower the overall drug requirement. Based on the above studies, it was concluded that follow-up evaluation of these parameters could be a relevant approach to find out the therapeutic effectiveness. A therapeutic plan should consist of both calcineurin inhibitors like cyclosporine and clinically supportive treatments with moisturising shampoos and essential fatty acids to improve epidermal barrier function in atopic skin. Further studies are warranted to uncover new therapeutic targets like filaggrin monomers or drug candidates that upregulate FLG in skin, which can provide more treatment options rather than the current symptomatic therapy, in the increasing eve of atopic dermatitis in dogs

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