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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Open Access Transcriptome analysis of buffalo bull spermatozoa for identification of fertility associated gene(s)(LUVAS, 2017) Vijayalakshmy.K; Virmani, MeenakshiKeeping in view the importance of sperm RNA and its correlation with fertility, the proposed study was carried out to determine the gene expression between high fertile and sub-fertile buffalo bull spermatozoa and identify the genes potentially associated with bull fertility through transcriptome analysis. These fertilityassociated genes were further validated with qRT-PCR and their possible role in fertilization through in-vitro fertilization. Ten bulls were classified on the basis of conception rates (CR), where bulls having CR <40% and 50% were considered as sub- and high-fertile bulls, respectively. Total motility, rapid motility, average path velocity, curvilinear velocity, amplitude of lateral head, beat cross frequency, straightness, linearity and intactness of plasma membrane of sperm for high-fertile bulls were significantly higher than sub-fertile bulls. Semen samples from these bulls were utilized for sperm enrichment using three different density gradient methods viz., Percoll, BoviPure and Iodixanol. Purified sperm pellets were subjected to RNA isolation using conventional and kit methods with and without TRIZOL. Maximum yield of RNA was achieved when the semen sample was purified using BoviPure as density gradient followed by isolation of RNA by RNAqueous + heated TRIZOL method. Further, AKAP4, CRISP2, Fertilin- â and PLCZ1 transcripts showed significantly higher expression in high- fertile than sub-fertile groups. Transcriptome analysis was done by Next generation sequencing and a total of 33715 and 21788 transcripts were expressed in sub- and high-fertile groups, respectively. When these transcripts were annotated with the available sequence of buffalo, 1248 and 802 transcripts were uniquely expressed in the sub- and high-fertile groups, respectively and 2527 transcripts were identified as common in both the groups. When in silico translated these transcripts, 258 proteins were observed to be commonly present in both the groups. Apart from these, 6 and 18 proteins were specifically present in the high and sub-fertile group, respectively. The expression of AKAP4, CRISP2, Fertilin- â and PLCZ1 was evaluated in IVF derived embryos. All the four transcripts differentially expressed only in sperm and embryo up to cleaved and blastocyst stage, but these genes didn‟t express in immature and mature oocytes. Thus, the present study provides the first evidence of differential expression of AKAP4, CRISP2, Fertilin â and PLCZ1 genes in high and sub-fertile bulls and their transmission to embryo indicating the role of sperm RNA in fertility and embryo development.ThesisItem Open Access Relationship of residual feed intake with rumen fermentation and blood biochemical profile in growing buffalo calves(LUVAS, 2017) Vikramjeet Singh; Madan, JyotsanaThe present study attempts to assess the relationship of residual feed intake (RFI) with rumen fermentation and blood biochemical profile in growing buffalo calves. Twelve healthy buffalo calves of seven to nine month of age were selected and fed with green fodder and concentrate mixture as per ICAR 2013 feeding standards. Weighed amount of feed and fodder was offered and residue was also weighed next day, early in morning. DM of feed and fodder offered and residue left was estimated on daily basis to assess DM intake. After completion of feeding trial, RFI value for individual animals was calculated. Experimental animals were weighed before starting the actual experiment and thereafter at fortnightly intervals. Using the data of fortnightly body weights ADG was calculated. Body growth parameters viz. body length, height, heart girth and abdominal girth of experimental animals were recorded in centimeters at the beginning, in the middle and at the end of experiment. A digestion trial of 6 days was conducted at the end of the experiment to know the nutritive value of feed and fodder. Representative samples of faeces, concentrate mixture, green and left over feed were taken daily and dried in hot air oven for determining dry matter content. The six days dried samples of feaces, feed and fodder were pooled, milled and analysed for proximate analysis according to AOAC (2005). A representative and homogenous sample of rumen liquor was collected through a stomach tube from the rumen of animals of high and low RFI groups and used for total nitrogen, ammonia nitrogen, total volatile fatty acids and individual volatile fatty acids estimation. Blood samples were collected thrice, once at the beginning, in the middle and at the end of trial, from all the animals for estimation of blood biochemical parameters in blood plasma. The results obtained regarding RFI values for high and low RFI groups were 0.235±0.04and -0.235±0.06, respectively. Mean body weight, mean metabolic body weight, average daily body weight gain and mean value of body growth parameters of the high RFI and low RFI groups were non-significant. The DM consumption was lower in low RFI group compared to high RFI group. Dry matter intake per 100 kg body weight (kg/100kg BW) and DMI per kg metabolic body weight was significantly lower in low RFI group (P≤0.01). Total nitrogen and ammonia nitrogen were non-significant in high and low RFI groups, while the TVFAs, acetic acid and butyric acid were negatively correlated with RFI (P≤0.01). Blood glucose, total protein and IGF-1 were negatively but non-significant correlated with RFI. Plasma albumin concentration was significantly positively correlated (r = 0.56) with RFI (P<0.01). BUN concentration was non-significantly positively correlated (r = 0.35) with RFI. RFI was positively correlated with AST (r =0.44) while negatively correlated (r = 0.35) with ALT.ThesisItem Open Access Effect of critical micronutrient supplementation during peri-parturient period on production and reproduction status of buffaloes(LUVAS, 2017) Jyani, Vikas; Gupta, MeenakshiPeri-parturient period is the critical phase in the life of dairy animals affecting future productive and reproductive performance. Present study was undertaken to evaluate the effect of critical micronutrient supplementation during peri-parturient period on production and reproduction status of buffaloes. Study was performed on twenty-two healthy Murrah buffaloes after dividing into two equal groups i.e. control and treatment respectively. Buffaloes in advanced stage of gestation were selected i. e. from 56 days prior to expected date of parturition. Buffaloes of both the groups were maintained on farm’s standard feeding practices as per their requirement. Additional micronutrient supplementation was carried out once in a week in buffaloes of treatment group having Zn, Cu, Co, Cr, Se, vitamin E, vitamin A, and niacin. Blood samples were collected pre-partum at day -56th, -28th, at day of parturition and thereafter at day 28th and 56th after parturition and subjected to plasma separation and estimation of blood biochemical profile and micronutrient status. Milk samples were collected weekly during initial 10 weeks of lactation. Ultrasonography was performed thrice during the study, i.e. on day 25-35, 55-65 and 85-95. Supplementation of critical micronutrient improved production performance of Murrah buffaloes in terms of total milk (18%) and fat corrected milk (P<0.05) production in addition to improvement in fat and protein percentage (P<0.05) of milk. Critical micronutrient supplementation did not affect blood biochemical and micronutrient profile significantly, due to uncompromised level of nutrients and health in buffaloes of control group, except for an improvement (P<0.05) in plasma inorganic phosphorus level in treatment group at day of parturition. Reproductive performance remained unaffected due to supplementation of weekly critical micronutrients in the ration of peri-parturient Murrah buffaloes.ThesisItem Open Access Effects of feed additives rich in essential oils on rumen fermentation, Methanogenesis and nutrient utilization in buffalo(LUVAS, 2017) Krishan Kumar; Rose, Manoj KumarPresent studies were conducted at the Indian Council of Agricultural Research- Central Institute for Research on Buffaloes (ICAR-CIRB), Hisar. Four feed additives (Eucalyptus leaves, Poplar leaves, Clove buds and Dalchini bark) rich in essential oils were selected to examine their effects on rumen fermentation, methanogenesis and nutrient utilization in buffalo. The extracts were prepared (15 g/ 100 ml) either in petroleum ether or n-hexane by Soxhlet’s apparatus. In in-vitro studies, the production of total gas, methane production, dry matter digestibility, ammonia nitrogen and production of volatile fatty acids were studied. The study revealed a significant (p<0.05) reduction in total gas, methane production, in-vitro dry matter digestibility, ammonia nitrogen and production of volatile fatty acids at high dose (2.0 ml/30ml buffered rumen fluid) as compared to control group. But at low (0.5 ml) and intermediate (1.0 ml) dose level the effects of extracts were comparable to control in all these parameters, with reduction (p<0.05) in methane production. The blends (extracts of poplar leaves, eucalyptus leaves and clove buds) of feed additives examined in this phase also showed a modulation in rumen fermentation with reduction in methane production. An in-vivo trial was conducted to study the effects of selected blend of feed additives on growth and nutrient utilization. Animals were distributed in three groups i.e. CON (basal ration), EOL-1(basal ration + 25 gm each of dry grounded poplar and eucalyptus leaves mixed with concentrate mixture), EOL-2 (Diet same as in control + 75 gm each of dry grounded poplar and eucalyptus leaves mixed with concentrate mixture). There was no significant (p>0.05) difference observed in growth rate and nutrient utilization among the in all treatments. In vivo methane productions were found to be inhibited in both treatment groups, as compared to control group after 3 months of dosing. The heamato-biochemical parameters remained similar (p>0.05) except significant (p<0.05) reduction in blood urea concentration as compared to control group. In conclusion, the essential oil rich feed additives could be used to reduce enteric methane emission and EOL-2 was found to significantly reduced methane production without any adverse effects on digestibility, growth and physiological status of buffaloes.ThesisItem Restricted Analysis of Hariana Cow Urine for Presence of Various Proteins/Peptides in Comparison to Murrah Buffalo(LUVAS, 2016) Sheoran, Maninder Singh; Sandeep KumarModern proteomics technique offers an exciting opportunity in solving some of the most challenging problems of livestock sector, including development of diagnosis based on urinary protein profiling. The present study intended to identify proteins, which may be novel to Hariana cow and Murrah buffalo urine and can subsequently be used for the purpose of disease detection. The urine samples were subjected to diafilteration and TCA precipitation followed by 2-dimensional gel electrophoresis and densitometric analysis in PD Quest software, which revealed the presence of at least 20, 2-D gel spots exhibiting specific appearance in a specific animal. Some of the Identified spots, which are documented to have role in many body functions including body defence, activation of cytoskeleton system, maintenance of muscular activity, suppression of tumour, biomarker of certain diseases, treatment of hypertension, apoptosis regulation includes the adenomatous polyposis coli (APC), vinculin proteins, cystatin-M precursor, melanoma inhibitory activity protein 3, epoxide hydrolase 2, bcl-2-like protein 11. The current work shows light on presence of some of the biologically active substances in the urine which could explain about its medicinal value. Considering that proteomics in animal research is still in its infancy, it is a long way before practical applications of such experiments could actually be realized.ThesisItem Restricted Studies on haematological, Biochemical constituents and hormones in beetal goats(LUVAS, 2010) Shah, Fozia; Gupta, MeenakshiThe present study was conducted with the objective of establishing normal haematological, biochemical constituents and hormones in Beetal goats at various physiological stages of life. Seventy two animals at various physiological stages maintained at goat farm CCS Haryana Agricultural University were included in the investigation. The goats were divided in eight groups with male and female goats at various physiological stages viz. Pre weaning ( 6 male and 6 female), Post weaning ( 6 male and 6 female), Pre pubertal (6 male and 6 female), Post pubertal ( 6 male and 6 female), Pregnant (6 female), Non pregnant (6 female), Lactating (6 female) and Non lactating (6 female), Blood samples were collected from animals prior to watering and feeding in morning from the jugular vein under aseptic conditions in the heparinized vials. Haematological examination for haemoglobin (Hb), packed cell volume (PCV), erythrocyte sedementation rate (ESR), total leucocyte count (TLC) and total erythrocyte count (TEC) were done immediately in fresh samples by standard techniques. Plasma was collected by centrifuging blood at 2500 rpm for 30 minutes and was stored in clean and dry glass vial with cap and was kept at -20°C till analyzed for the estimation of tri-iodothyronine (T3), thyroxin (T4), sodium, potassium, chloride, aspartate amino transferase (AST), alanine amino transferase (ALT), acid phosphatase and alkaline phosphatase. The results revealed that the Haematological values, electrolyte and thyroid hormones were higher in males than females, whereas, enzymes were higher in females than male in Beetal goat. The Hb, TEC, TLC, PCV, T3, T4, sodium, potassium and chloride showed maximum value in pre weaning stage similarly, activity of alkaline phosphatase and acid phosphatase was also highest in pre weaning stage in both sexes compared to rest of groups in study. The value of Hb, TEC, PCV, sodium, potassium , chloride , T3, T4, and acid phosphatase did not exhibit any significant change in between post weaning, pre pubertal and post pubertal stages of study in both sexes. Lactation and pregnancy showed decreased value of Hb, TEC, PCV, sodium, potassium and chloride whereas value of T3, T4, AST, ALT, AKP and ACP increased in lactating and pregnant goat as compared to non lactating and non pregnant goat.ThesisItem Restricted Effects of cryoprotectants on freezing, Culture behaviour and apoptosis of buffalo umbilical cord matrix cells(LUVAS, 2011) Preeti Singh; Rose, M.K.Umbilical cord represents the link between mother and fetus during pregnancy. Postnatally umbilical cord is a discarded organ and the collections of umbilical cord cells do not require an invasive procedure with ethical concerns. It is composed of a special embryonic mucous connective tissue called Whartson’s jelly and its cells have the properties of stem cells. Umbilical cord matrix cells are cryopreserved to study their future applications. Cryopreservation is a method to protect cells from freezing injury. Cells are vulnerable to cryopreservation induced apoptosis due to artificial cleavage of apoptotic protein during freezing and thawing. A family of cysteine proteases present in most cells is responsible for apoptosis. In the present study confluent cultures from buffalo umbilical cord matix cells were frozen at three different passages (P3, P6, P9) in three different combinations of cryoprotectants viz. DMEM culture medium containing 20% FBS and 10% DMSO, 1.5 M ethylene glycol and 0.2 M sucrose in PBS and 4% DMSO, 6% trehalose and 90% FBS. Pre and post freezing cell viability (%) was assessed using trypan blue dye exclusion method. The cells in (FBS-DMSO) and (EG-Sucrose) had more post thaw viability than (DMSO- trehalose –FBS) cryoprotectant combination. In post thaw culture behavior studies cells cryopreserved in (FBS-DMSO) started adhering to the surface of flask within 4 hrs and could proliferate well with similar morphology as that of continuous culture. Most of the cells cryopreserved in (EG-Sucrose) could not adhere to the surface of flask even after 24 hrs. The cells cryopreserved in (DMSO- trehalose –FBS) could adhere to surface of flask after 10-12 hrs and could grow well without any marked change in morphology. The RT–PCR analysis of antiapoptotic gene, Bcl-2, and proapoptotic gene, Bax with house keeping gene (β actin) of pre-freezing and post thawed cells showed that both the genes were expressed from cells of each group at P3, P6 and P9 before freezing and after thawing. In conclusion, the umbilical cord matrix cells have similar post thaw viability in (FBS-DMSO) and (EG-Sucrose) cryoprotectanat combinations but post thaw culture behavior is better in (FBS-DMSO). The expression of Bcl-2 and Bax genes in pre and post thawed cells indicate that quantitative expression studies are required to ascertain their level of expression.ThesisItem Restricted Effect of turmeric supplementation in feed on hematobiochemical, mineral and antioxidant profile of post kidding goats and their kids(LUVAS, 2014) Krishna Kumar; Sindhu, SoniaAn experiment was conducted to study the effect of turmeric powder (TP) on haematobiochemical, mineral and antioxidant profile in post kidding goats and their kids. A concentrate diet containing 0.0, 0.5% and 1% turmeric was fed in three groups of goats, first group was control group and two treatment groups were 0.5% and 1% respectively. For measuring haematological parameters [haemoglobin, erythrocyte sedimentation rate (ESR), packed cell volume (PCV), differential leucocyte count (DLC)and total leucocytes count (TLC)], biochemical parameters[glucose, total protein, ketone bodies and blood urea nitrogen(BUN)], enzyme assay [alkaline phosphatase(ALP), aspartate aminotransferase (AST),alanine aminotransferase (ALT)] electrolytes[sodium (Na),potassium (K) and chloride(Cl)] and antioxidants level [vitamin E, beta carotene, vitamin C and super oxide dismutase (SOD)] blood samples from each group were taken on 0,30,60 and 90 days of post kidding from goats and on 30,60 and 90 days in their kids respectively. The level of haemoglobin was significantly higher on 90th day of study in goats of 1% turmeric supplemented group than other groups. The level of glucose was significantly lower at 90 days of post kidding in goats. The level of ketone bodies decreased in goats from day 0 to 90 days of post kidding. The level of vitamin E was higher at 60 days and 90 days in 0.5% and 1% turmeric fed group than control group. No significant change in vitamin E level in 0.5% and 1% turmeric fed group goat kids between and within group was observed. The level of SOD increased at 30 days and at 90 days in 0.5% and 1% turmeric fed group compared to control group. No significant change in SOD level in kids of 0.5% and 1% turmeric fed goats between and within groups was found. No significant change in other parameters like PCV, ESR, neutrophil%, lymphocyte%, basophil%, eosinophil%, monocyte%, TLC count, total protein, BUN, ALP, AST, ALT, Na, K and Cl among all groups at different days of studies both in goat and their kids was observed. The study suggested some positive effect of turmeric on the health status of goats after kidding.ThesisItem Restricted Effect of antioxidants on the quality of liquid preservation of Beetal buck semen(LUVAS, 2016) Sarangi, Archana; Pardeep SinghThe mammalian sperm membrane contains a large number of unsaturated fatty acids and is therefore sensitive to lipid peroxidation. Oxidative damage to spermatozoa during preservation of liquid semen at 4°C is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. Vitamin E (DLα-Tocopherol) and glutathione are believed to be the primary component of antioxidant system of the spermatozoa. The objective of this study was to evaluate the role of vitamin E and glutathione in improving the seminal parameters during storage for 72 hours. The thirty-six semen ejaculates were collected with the help of artificial vagina from six bucks (Beetal) during the normal reproduction season. The samples were centrifuged at 1500rpm for 10 minutes and the seminal plasma was removed. The sperm pellet was diluted with Tris based extender and divided into 3 groups. Group T1 was taken as control; group T2 were supplemented with tocopherol @ 3mM and group T3 were supplemented with glutathione @ 1mM respectively. The samples were evaluated for morphological parameters viz., progressive sperm motility, percent livability, percent abnormal spermatozoa , acrosome integrity after liquid preservation for 0, 24, 48 and 72 hours and oxidative stress parameters viz., lipid peroxidation, glutathione peroxidase (GPx), superoxide dismutase (SOD) after liquid preservation for 0 and 72 hours. It was observed that after 0, 24, 48 and 72 h of storage at 4°C, the progressive sperm motility, percent livability and percent intact acrosomes were significantly (P˂0.05) higher in group T2 and T3 samples as compared to control. But, percent abnormal spermatozoa and level of lipid peroxidation in T2 and T3 group was significantly (P˂0.05) lower after 72hours of incubation at 4°C. Similarly, GPx and SOD values were highly significant (P˂0.05) in T2 and T3 group after 72 hours of storage at 4°C. Thus it can be concluded that supplementation of vitamin E and glutathione to the semen samples @ 3mM and 1mM concentration respectively, helps in maintaining the seminal parameters during liquid preservation of semen at 4°C upto72 hours.