Theses (M.Sc.)

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Open Access
ASSESSMENT OF MITOCHONDRIAL DNA COPY NUMBER AND FUNCTIONAL CHARACTERISTICS FOR ASSOCIATION WITH SEMEN QUALITY IN BUFFALO BULLS ACROSS DIFFERENT SEASONS
(ICAR-NDRI, KARNAL, 2022) ELDHO MATHAI; R.S. KATARIA
Semen quality and quantity are affected by seasonal changes, particularly heat stress having significant influence. Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, humans, cattle and buffalo. The present study was carried out to correlate the mitochondrial DNA copy number, cellular, biochemical parameters and gene expression data. Based on the semen quality data (HOST, acrosomal integrity, viability) available across the seasons (winter, hot humid, hot summer and comfort seasons), buffalo bulls were classified into seasonally affected (SA) and seasonally non-affected (SNA) by heat stress. The semen samples collected from 8 target animals during winter (Dec 21 – Jan 22) and hot summer (May 22 – June 22) seasons, were used for the study. Fresh semen quality parameters (volume, mass motility and sperm concentration) and postthaw motility of frozen semen of the SA and SNA bulls across summer and winter seasons, were not significantly different (p<0.05). However, fresh semen collected in the winter season had better seminal attributes in both SA and SNA bulls compared to the summer season. Mitochondrial DNA copy number estimation was done by real-time PCR using a mitochondrial coded gene (CYTC) and nuclear-coded gene (ACTB). The mean (±SD) number of mitochondrial DNA copy number for all the animals was 13±2 and had a range from 8 to 18, without any significant variation between the groups. Mitochondrial membrane potential (MMP) values were significantly different for SA animals across the winter and summer seasons. The variation in superoxide levels however was not significant in the animals across the seasons. The dead acrosome reactive (DR) and the live acrosome intact (LI) values showed significant variation across the summer and winter seasons in the SA group of animals. Semen collected in the summer from SA bulls had the least live sperms with intact acrosomes. GSH levels in the seminal plasma were not significantly different across the seasons and animal groups. Catalase concentration in the seminal plasma however was significantly different across the winter and summer seasons in SA animals. There was also no significant variation in lipid peroxidation levels as determined by the malondialdehyde (MDA) marker, irrespective of the seasons and the group of animals. The oxidative stress genes -ATF4, ATF5 and CYTC have shown a 5-fold, 11-fold and 6-fold increase in the expression in the seasonally nonaffected groups in the winter season. Fold changes of apoptosis-related genes BAK1 and MCL2, were 4-fold and 6- fold respectively higher in Sthe NA group in winter compared to summer. In conclusion, the mitochondrial membrane potential (MMP), intact live acrosome (LI), and Catalase levels were positively correlated with semen quality of selected parameters in the fresh as well as frozen semen of Murrah buffalo bulls. There favourably high expression of oxidative stress and anti-apoptotic genes in seathe sonally non-affected group in hot summer compared to affected bull
Open Access
POLYMORPHISM AND EXPRESSION ANALYSIS OF CHROMATIN REMODELLING ASSOCIATED GENES IN BUFFALO BULLS WITH VARYING SEMEN QUALITY UNDER HEAT STRESS
(ICAR-NDRI, KARNAL, 2022) HARSIMRAN KAUR; R.S. KATARIA
During spermatogenesis, the spermatid's nucleus undergoes alterations, including chromatin structural rearrangement and closely packed DNA changes. Tight packaging of DNA is essential for avoiding the DNA fragmentation caused by reactive oxygen species. The replacement of histones by protamines provides a more compact packing of genomic material in sperm nuclei. The goal of the current study was to understand the expressional profile of the candidate genes (PRM1, PRM2, TNP1, and TNP2) involved in chromatin remodelling in spermatozoa of the Murrah buffalo bulls, which were classified as seasonally affected and seasonally non affected by heat stress based on semen quality parameters and characterization of the coding and promoter region of PRM1, PRM2, TNP1, and TNP2 genes. The bubaline PRM1 gene structure has a transcript length of 1.12 Kb, coding for 51 amino acids. Seven variants were found in the protein sequence (p.Q5R, p.R12G, p.G24R, p.C31G, p.L46I, p.S49T, and p.K51R). The TF binding sites varied between the species in the promoter region. In buffalo, the TFs such as CRE-BP1 (64G
Open Access
Effect of different culture media on developmental competence of In-vitro embryo production in cattle
(ICAR-NDRI, KARNAL, 2021) PANKAJ KARMALI; S.K. DAS
The objective of the present study was to evaluate the effect of the different embryo culture media on early embryonic developmental competence in cattle. In vitro techniques for embryo development involve many steps viz. collection of ovaries from slaughter house, collection of immature oocytes from ovaries, grading of oocytes, in vitro maturation of oocytes, in vitro sperm preparation, in vitro fertilization and culture of presumptive embryos for early embryo development. To conduct the experiments cattle ovaries were collected from Kolkata slaughterhouse and immature oocytes were collected by follicle aspiration technique. After collection, these COCs were thoroughly washed in washing media and then in maturation media before transferring them into IVM media droplets where they were cultured for 24 hours in CO2 incubator at 5% level of CO2 and at 38.5 °C with the maximum humidity. The matured oocytes in vitro were co-incubated with in vitro processed and capacitated sperm for 14-18 hr. After fertilization, presumptive zygotes were washed and cultured in in vitro culture (IVC) media. Three different culture media i.e. (TCM-199, BO-IVF and mCR2aa) were used for early embryo development. The cleavage rate was higher in TCM-199 (74.47±9.63a) and mCR2aa (71.64±10.03a) medium as compared to BO-IVF medium (53.56±3.64b). Morula development rate was significantly higher in TCM-199 (35.39±1.467c) and BO-IVF (26.83±2.18d) medium as compared to mCR2aa (22.83±2.9d) medium. Blastocyst formation rate was observed significantly higher (P<0.05) in BO-IVF medium (14.17±2.85f) compared to TCM-199 (4.63±0.6301e) but not with mCR2aa (10.72±5.42f ) culture medium. From the present study it could be concluded that all three culture medias i.e. TCM-199, BO-IVF and mCR2aa are able to produce blastocyst, but BO-IVF and mCR2aa medias have high potential to produce blastocyst.
Open Access
DEVELOPMENT OF OOCYTE MATURATION MEDIUM FOR BOVINE ASSISTED REPRODUCTIVE TECHNOLOGY
(ICAR-NDRI, KARNAL, 2022) RAMESH P.N.; M.K. SINGH
Oocyte maturation is an inevitable part of assisted reproductive technology (ART) and indigenously developed medium for in vitro maturation is a need of the hour. For the development of oocyte maturation medium for bovine ART, this study was carried out with the following objectives: i) To optimize the culture medium for in vitro maturation of buffalo oocytes, 2) To examine the development competence of in vitro produced embryos using an optimized maturation medium and 3) To study the effect of optimized maturation medium on pluripotency, development and apoptosis-related genes in produced embryos. For this oocytes were cultured in four different maturation media, of which two were formulated in our lab (L-OMM-1 and L-OMM-2) and two commercially available media (C-OMM-1 and C-OMM-2). The buffalo oocytes were cultured for 24 h in CO2 incubator at 5% CO2 in air at 38.5°C for in vitro maturation. The polar body extrusion showed that L-OMM-1 had 79.52±1.48% maturation rate which was significantly higher (P<0.05) than L-OMM-2 (31.51±0.6%); and at par with the maturation rate of C-OMM-1 (82.57±1.96%), but lower (P<0.05) than C-OMM-2 (87.22±1.91%). The oocytes in L-OMM-1 (n=306) and C-OMM-1 (n=288) media were matured in vitro and subjected to IVF and IVC in similar media for both the groups. The cleavage rate observed between above two groups had no difference (64.96 ± 1.33 vs 64.40 ± 2.12) whereas blastocyst rate was found to be at par in both the media (P>0.05) (16.31 ± 1.41 vs 17.71 ± 0.24). TUNEL assay showed no significant (P>0.05) difference in apoptotic index of both the blastocyst groups produced in L-OMM-1 (4.95±0.17%) and C-OMM-1 (4.41±0.19%). The total cell number in blastocyst was higher in L-OMM-1 media (191.88±17.05) compared to C-OMM-1 media (171.63±12.48) but differ non-significantly (P>0.05). The gene expression study revealed that SOD2, BCL-XL, BMP15 and HPRT genes were significantly up-regulated (P<0.05), P53, BAX, GLUT1 and OCT4 were significantly down-regulated (P<0.05), whereas no change in expression of BAD, IGF2, C-MYC in blastocyst obtained from L-OMM-1 compared to C-OMM-1. This all suggests that the lab-formulated medium has competence for in vitro oocyte maturation and subsequent to embryo development as compared to commercial media. In conclusion, the lab-formulated oocyte maturation media (L-OMM-1) can be used for in vitro maturation of oocytes.
Open Access
DEVELOPMENT OF BuPAG ANTIBODIES BASED IMMUNODIAGNOSTIC ASSAYS FOR EARLY DETECTION OF PREGNANCY IN BOVINES
(ICAR-NDRI, KARNAL, 2022) SHWETA YADAV; SUDARSHAN KUMAR
A good reproductive management in livestock depends on an early and precise diagnosis of reproductive dysfunctions or aberrations. Good reproductive efficiency is necessary for dairy animals to produce more milk throughout their lives. The most significant dairy animal on the Indian subcontinent, the buffalo (Bubalus bubalis), is known for issues with a long calving gap, delayed adolescence, and a high prevalence of anoestrus. The problem is further made complicated by the lack of accurate early pregnancy diagnosis techniques at field level. In bovine species, a number of pregnancy diagnosis techniques are used to detect early pregnancy, but none of them is optimal due to the test's inherent limitations in terms of sensitivity, accuracy, specificity, speed, and convenience of use. Pregnancy associated glycoproteins (PAGs) are potential pregnancy biomarkers in farm animals that are secreted during days 22 to 28 after fertilization from conceptus to maternal circulation. A total of 12 PAG isoforms and their variants were identified in buffalo during the early stages of pregnancy, i.e., BuPAG 1, 2, 4, 6, 7, 8, 9, 13, 15, 16, 18, and one novel isoform. In our study we have used two isoforms, i.e. BuPAG-1 and BuPAG-7 to develop two major immunodiagnostic assays that are enzyme linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). The goal of the present study was to develop and validate a Sandwich-ELISA which can detect pregnancy in maternal serum at 35 days of post insemination. Therefore, serum samples of both pregnant and non-pregnant animals were collected from cows and buffaloes from 0-60 days after insemination. The developed BuPAG-1 & 7 based ELISA assays were able to identify pregnancy at day 35 p.i. in serum with accuracy of 86-93%. This is time saving for farmers and more efficient for laboratories. Since ELISA requires laboratories and technicians, which can’t be done at field level, so we have also tried to optimize and develop BuPAG-1 and 7 based lateral flow immunoassay (LFIA), which can give results in 2-5 minutes after applying serum samples with accuracy of 75-80% at day 35 p.i. Being affordable, sensitive, specific, user-friendly, rapid, robust, and equipment-free, this point of care testing can be considered as one of the most promising pregnancy diagnostic tool for farmers.
Open Access
GENERATION OF OOCYTE-LIKE CELLS FROM BUFFALO UMBILICAL CORD BLOOD- MESENCHYMAL STEM CELLS
(ICAR-NDRI, KARNAL, 2022) PRIYA SEHRWAT; D. MALAKAR
The aim of present study was to perform in vitro differentiation of Umbilical Cord Blood- Mesenchymal Stem Cells to Oocytes like cells. UCB-MSC were isolated from the umbilical cord of calf just after a delivery with the help of 18-gauge syringe and brought to lab for further process. Blood was equally diluted with DPBS and loaded on HiSepTM LSM 1077 gradient and centrifuged at 400 g for 30 min and a buffy coat was formed at interface was taken and seeded in flask containing DMEM/F12, 20% FBS,1% P/S solution at 5% CO2 at 37°C in incubator. Media was changed twice a week for 3 weeks. After 3-week RNA of cultured cells were isolated and characterized for MSCs confirmation through RT-PCR, Alkaline Phosphatase Assay and immunostaining for MSC specific gene. After confirmation of MSCs which appear to be spindle in shape and adherent in nature and having CD73, CD90 and CD105 surface markers present on them, were seeded in well for differentiation into Primordial Germ Cell Like Cells (PGCLCs) then to Oocyte Like Cells (OLCs) using differentiation media containing DMEM/F12, FBS, BMP4 and P/S antibiotic solution. Bone Morphogenic Protein 4 (BMP4) is a member of the transforming growth factor-b (TGFb) group of proteins, to initiate signal transduction in target cells. BMP4 due to its signal transduction pathway, it activates different proteins used in germ cell development. Confirmation of PGCLCs on 7th day was done by immunostaining using STELLA and VASA as primary antibody, also by doing RT-PCR for PGCLCs specific genes (Oct4, VASA, STELLA and SCY3). OCLs formed on 14th day were also confirmed by immunostaining taking ZP2 as primary antibody, also confirming by RT-PCR for OLCs specific genes (ZP2 and ZP3). The results were found positive for PGCLCs and OLCs formation. It can be concluded that UCB-MSC can be directed differentiation into Oocyte Like Cell.
Open Access
EFFECT OF CURCUMIN ON THE DEVELOPMENT AND GENE EXPRESSION OF HEAT-STRESSED IN VITRO PRODUCED BUFFALO (Bubalus bubalis) EMBRYOS
(ICAR-NDRI, KARNAL, 2022) RITIKA; M.K. SINGH
The present study was carried out with the following objectives: (i) To examine the effect of curcumin on the developmental competence of heat-stressed in vitro-produced embryos. 2) To analyze the level of apoptosis and expression of some important genes in heat-stressed buffalo embryos treated with curcumin. For this immature oocytes (n=1330) were isolated from slaughterhouse-based buffalo ovaries and were subjected to in vitro maturation (IVM), fertilization (IVF), and culture (IVC) in presence of a different concentration of curcumin (0 μM, 5 μM, and 10 μM) in culture media at 38.5°C and in combination with curcumin doses and heat stress (39.5°C) to presumed zygote for 2 h once after 48 h of IVF. The cleavage rate was found to be significantly higher (P<0.05) at 5 μM curcumin (64.11±1.15%) compared to control (58.75±2.25%) or 10 μM of curcumin (44.66±4.28%) at 38.5°C. When these cleaved embryos were further cultured till the blastocyst stage, found a significant increase (P<0.05) in 5 μM curcumin (21.46±0.67%) compared to 10 μM (6.50±1.17%) and control (16.63±1.49%), respectively. When presumed zygotes were exposed to 39.5°C during IVC cleavage rate was found significantly higher (P<0.05) in 5 μM curcumin supplementation (57.03±2.50%) compared to control (47.79±4.30%) and 10 μM (37.54±0.83%). The blastocyst rate was significantly higher (P<0.05) at 5 μM curcumin (15.28±1.31%) as compared to control (8.59±1.20%) which in turn significantly higher (P<0.05) than 10 μM curcumin (3.03±1.20%). When the embryos produced under the above conditions were subjected to a gene expression study, found that at 38.5°C, the expression of HSP genes significantly (P<0.05) reduced when adding the curcumin, but at the higher temperature, both the HSP10 and HSP60 genes highly up-regulated (P<0.05). At both temperatures, all anti-oxidant-related genes SOD2, SOD3, GPX1 and GPX2 had higher expression in presence of 5 μM curcumin compared to 10 μM or control. The expression of anti-apoptotic (BCL-XL, P53) genes was higher compared to pro-apoptotic genes (BAD and BAX) at 38.5°C, whereas at higher temperature all apoptosis-related genes had significant high (P<0.05) expression, but the ratio of anti-apoptotic vs pro-apoptotic was higher. The pluripotency genes (OCT4 and C-MYC) at both temperatures had higher compared to the control. In conclusion, this study suggests that curcumin supplementation in embryo culture medium effectively combated the adverse effects of heat-stress and supports the embryo production in vitro.
Open Access
COMPARATIVE EXPRESSION PROFILING OF IMMUNE RESPONSE GENES IN ANAPLASMA MARGINALE INFECTED AND HEALTHY CATTLE
(ICAR-NDRI, KARNAL, 2022) RASHMEET KAUR; SONIKA AHLAWAT
In this study, changes in expression profiles of genes encoding 14 cytokines (IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL16, IFNA, IFNB, TGFB1, and TNFA) were investigated amongst six Anaplasma marginale infected and six healthy crossbred cattle. Health status of the animals was determined based on clinical signs, blood smear examination and molecular detection using A. marginale-specific primers. Total RNA was isolated from the peripheral blood mononuclear cells (PBMCs) of the infected animals as well as healthy controls, which was further reverse transcribed to cDNA. Primers for real time PCR were designed using Primer3 software and the results were analyzed by the 2–ΔΔCT method with RPS15 and GAPDH as the reference genes. Selection of RPS15 and GAPDH as normalizers for gene expression data for this study was based on the comprehensive ranking achieved through the RefFinder tool by analyzing the CT values of 14 housekeeping genes (RPLP0, ACTB, RPS28, YWHAZ, SDHA, PPIA, RPS9, RPS15, UXT, GAPDH, B2M, BACH1, HMBS, and PPIB) in blood PBMCs of cattle and buffaloes infected with different tick-borne diseases (anaplasmosis, theileriosis, babesiosis, and trypanosomiasis). The expression levels of IL1A, IL1B, IL6, IL10, IL12A, IL12B, and TNFA varied significantly between the two groups, with higher expression in the infected cattle. The transcript abundance of IL4, IL16, and TGFB1 did not vary between the infected and healthy animals. The expression of IL2 and IL8 was higher in the healthy animals, but the results were non-significant. Taken together, this study provides evidence for differences in expression of cytokine genes in response to anaplasmosis in crossbred cattle.
Open Access
Characterization of Biofilm Forming Genes in Staphylococcus aureus Isolates Causing Bovine Mastitis
(ICAR-NDRI, KARNAL, 2022) ILA SINGH; SACHINANDAN, DE.
India is regarded as the highest milk producer in the world; however, the productivity of animals is threatened because of infectious diseases like mastitis. Among the various mastitis pathogens, Staphylococcus aureus is identified as chief etiological agent responsible for subclinical and chronic mastitis. S. aureus expresses various virulence factors, including toxins and immune-evasive surface molecules and enzymes that promote tissue invasion and biofilm formation. Chronic biofilm infections are challenging to eradicate with antibiotics, and biofilm formation could be a possible explanation for mastitis cases that are not resolved by standard treatment. The intercellular gene cluster adhesion operon (ica) is one of the gene clusters that has been investigated for its role in biofilm formation for isolates from bovine mastitis. The present study was designed to screen the S. aureus isolates recovered from the cases of bovine mastitis for in vitro biofilm production with the following objectives: (i) To isolate S.aureus from Mastitis Milk sample (ii) Phenotypic Characterization of Biofilm-producing S.aureus and (iii) To study the Adhesion genes of S.aureus isolates A total of 83 S.aureus strains were isolated from 300 mastitis milk sample. These 83 isolates were further subjected to phenotypic and genotypic biofilm characterization. Phenotypic detection of biofilm was done by CRA and MTP methods, which revealed that out of 83 isolates, 55 (66.2%) were able to produce slime, and the rest, 28 (33.73%), were non-slime producers. However, results of the microtiter plate test (MTP) revealed that among 83 S. aureus isolates, 5 (6.02%) were strong biofilm producers, 9 (10.8%) were moderate biofilm producers, and 30 (36.1%) were weak and 39 (46.9%) were non-biofilm producers. Genotypic characterization of intercellular adhesion genes IcaA and IcaD revealed that out of 83 isolates, 71 (85.5%) were found positive for icaA, while 75 (90.3%) were positive for icaD. Among surface adhesion genes, the prevalence of genes bap, cna, eno, FnbpA, ebps, sasG were 2 (2.4%), 29 (34.9%), 65(78.3%), 71(85.5%), 47(56.6%) respectively. These findings suggest that regulation of biofilm formation is primarily mediated by PIA-dependent genes. Thus, more research is needed to fully comprehend the processes and genetic foundations that lead to biofilm development, which are crucial in designing successful biofilm circumvention measures.

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