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Open Access
Studies on pre-pubertal indices in cattle through nutritional interventions under field conditions
(G.B. Pant University of Agriculture and Technology, Pantnagar, District Udham Singh Nagar, Uttarakhand. PIN - 263145, 2022-08) Mahajan, Mohit; Shiv Prasad
The present study was undertaken to evaluate the effect of bypass protein, bypass fats, rumen protected niacin, rumen protected choline and probiotics on the prepubertal heifers, repeat breeders, anestrus and advance pregnant cattle. The study was conducted under field conditions covering animals from the Tarai region of Udham Singh Nagar, Uttarakhand, India and under controlled conditions at Instructional Dairy Farm (IDF), G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India. Prepubertal heifers were divided into T1 (3 months feeding, field), T2 (5 months feeding, field), T3 (3 months feeding, farm) and T0 control groups. Repeat breeders, anestrus and advance pregnant cattle groups were fed experimental diet for 1 month. Heifers were subjected to monthly analysis of physical parameters i.e., body condition score, body weight, age at first heat, ovarian status, age at first conception, number of services per conception, hematological parameters, biochemical parameters and hormonal profile, estrogen, progesterone and leptin. The incidence of metabolic and reproductive diseases and milk yield were done in advance pregnant animals. It was recorded that with supplementation of experimental diet in heifers under field and controlled condition, a significant increase in the body condition score and body weight was recorded. Estrus induction rate was recorded to be 85%, 90% and 70% in T1, T2 and T3 groups respectively significantly more than control group i.e., 35%. Similarly, a higher conception rate of 76.47 %, 83.33 % and 71.42 % for T1, T2 & T3 groups, respectively, significantly higher than T0 with 14.42 %. Hematological examination recorded no significant change. Biochemical examination recorded a nonsignificant decrease in BUN and non-significant increase in blood glucose during the experiment. Simultaneously a significant increase in the blood total protein was recorded. Experimentally fed pre pubertal heifers reported a significant higher estrogen level from the 1st (93.09 ± 2.02 pg/ml), 2nd (89.91 ± 1.71 pg/ml) and 3rd (87.11 ± 2.25 pg/ml) months compared to the control group. Estrogen concentration was higher in all experimentally fed heifers. A non-significantly higher progesterone concentration was recorded for all heifers fed experimental diet compared to the control group. This is relative to the high estrus induction rate and high conception in heifers fed experimental diet. Leptin concentration was recorded to be non-significantly higher in the heifers fed experimental diet at all stages in experiment for both those in estrus as well as those conceived. Anestrus animals recorded 70% estrus expression with a 40% conception rate in contrast to 40% estrus expression and 10% conception in the control group. Repeat breeder cattle recorded conception rate of 55% in contrast to the control group with 30 %. Experimentally fed pregnant cattle recorded a remarkable reduction in the incidence of metabolic and reproductive diseases from 17 to 4 cases as compared to 10 to 5 cases in the control group. The milk production post-treatment (14.85 ± 0.44 liters/day/cow) was significantly high compared to the control (11.20 ± 0.88 liters/day/cow) group.
Open Access
Study on physico-morphology, biochemical constituents and effect of different levels of egg yolk on cryopreservation of Pantja buck semen
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-09) Rashmi; Gupta, H.P.
Present study was conducted to assess the cryopreservation of extended Pantja buck semen in Tris-citric acid-fructose-egg yolk-glycerol (TCF-EY) extender containing 4 different levels (5, 10, 15 and 20%) of egg-yolk and to compare the neat, post diluted, post equilibrated and post-thawed quality of semen. Artificial Vagina (AV) method was used to collect semen from four reproductively mature, 2.5 to 4 years old, healthy breeding Pantja bucks. A total of thirty-two ejaculates were collected twice a week. After semen collection from all bucks, neat semen was examined for pH, volume, concentration, mass motility, live spermatozoa count, HOST, acrosome and abnormal spermatozoa percent. Thereafter semen samples were pooled and evaluated for some biochemical parameters viz; Malondialdehyde (MDA), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvate Transaminase (GPT) and Glutathione Peroxidase (GSH-Px). Pooled semen sample was divided and diluted into four groups as D1[Neat semen + tris-fructosecitric acid-egg yolk (@5%)-glycerol], D2[Neat semen + tris-fructose-citric acid-egg yolk (@10%)-glycerol], D3[Neat semen + tris-fructose-citric acid-egg yolk (@15%)-glycerol] and D4[Neat semen + tris-fructose-citric acid-egg yolk (@20%)-glycerol]. Pooled semen samples were processed and evaluated without washing after dilution, post equilibration and after thawing for progressive motility, live and dead sperm percentage, sperm abnormalities, plasma membrane integrity (HOST) and acrosome integrity. Evaluation of MDA, GOT, GPT and GSH-Px was also done at post thawing stage. Biochemical parameters were evaluated in pooled semen before dilution and at post-thawing stage only. Dilutions were made according to the SOP and equilibrated at 5°C for 3 h and then the French mini (0.25 ml) straws were filled with semen and prefrozen in liquid nitrogen (LN2) vapours followed by stocking in LN2 container for seven days. After 7 days frozen semen straws were thawed in a water bath at 37˚C for 30 seconds and post thaw evaluation was done for above mention parameters. The mean value for neat seminal parameters of Pantja buck semen were recorded as volume 0.51±0.090 ml, pH 6.83±0.004, concentration 3.94±0.228, mass motility 4.49±0.078, live spermatozoa 84.75±1.75%, head abnormality (%) 2.13±0.057 %, mid piece abnormality 0.46±0.021 %, tail abnormality 4.85±0.29%, total abnormality (%) 7.48±0.34 %, plasma membrane integrity (%) 83.53±1.696 %, Intact acrosome (%) 82.63±1.760 %, MDA 2.22±0.16 (nmol/ml), GOT 127.36±0.556 Units/l, GPT 16.99±0.348 Units/l and GSH-Px 7.94±0.56 Units/ml. Significant difference (P< 0.05) was observed for all the parameters in different stages of freezing viz; post dilution, post equilibration and post thawing and in various diluters viz; D1, D2, D3 and D4. All seminal parameters except total abnormality showed higher value in D2 (10% EY) group at post dilution, post equilibration and post thawing stages compared to other groups. Likewise, significantly lower value for MDA, GOT, GPT and GSH-Px was observed in 10% egg yolk at post thawing stage. It was therefore concluded that the Pantja buck semen can be cryopreserved successfully at 10% egg yolk level (V/V) in Tris-citric acid-fructose-egg yolk -glycerol extender.
Open Access
Insilico characterisation and epitope profiling of differentially expressed plasma membrane proteins of ‘x’ and ‘y’ chromosome bearing bovine sperm
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-09) Sharma, Prachi; Sharma, Mridula
The specific and differentially expressed plasma membrane proteins of bovine sex sorted semen were selected after reviewing numerous research publications on proteomic profile of sexed semen of bovines. The insilico characters such as physicochemical properties, primary, secondary, tertiary structures, membrane topology, sub-cellular localisation, domain analysis, protein interactions and epitope profiling, both linear and discontinuous peptides was done for all the proteins by using various bioinformatics tools and software. For X specific protein CLRN3, 6 and 4 and for Y chromosome specific protein, SCAMP1, 6 and 8 linear and discontinuous epitopes were predicted, respectively. The proteins were localized on Plasma membrane like SPACA1, Leucine-rich repeat and fibronectin type III domain containing 2, CLRN3, SCAMP1 and Uncharacterised Protein may act as antigenic protein to be used for further production of sex- specific antibodies and separation of X and Y sperm. The results of insilico characterisation and epitope prediction of various selected X and Y bovine sperm plasma membrane specific and differentially expressed proteins would be helpful in selection of some efficient antigenic proteins to be used for further X and Y sperm specific antibody production or synthesis of antibodies artificially.
Open Access
In silico studies on efficacy of various antibacterial drugs against bovine endometritis
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-09) Arya, Damini; Sharma, Mridula
The present study was conducted with the objective to find effective drug against bacterial bovine endometritis using Molecular docking. The antibacterial property of selected plants (already studied in vivo and new) was studied its comparison with conventional antibiotics was done. The plants selected for study were Tulsi, Garlic, Aloe vera and Kalmegh, among them Tulsi and Garlic were previously studied in vivo and in vitro for treating bovine endometritis and Aloe vera and Kalmegh were selected on the basis of its potent antibacterial activity. The phytochemicals of Tulsi were thymol, eugenol, linalool, carvacrol, apigenin and caryophyllene. Phytochemicals of Garlic were allicin, ajoene, diallyldisulphide and diallyltrisulphide. Phytochemicals of Aloe vera were aloe-emodin, anthracene, campesterol, lupeol and sitosterol β. Phytochemicals of Kalmegh were andrographolide, 14-deoxyandrographolide, neoandrographolide and 3-O-β-D-Glucopyranosyl-14,19-Dideoxyandrographolide. Selection of bacteria (S. aureus and E. coli) for the present study was done on the basis of most predominantly found bacteria causing bovine endometritis as observed in previous studies. From S. aureus Gyrase B, DNA ligase, dehydrosqualene synthase and FtsA protein and from E. coli DNA gyrase, FtsZ and ZapD protein were selected. The selected bacterial proteins were docked against phytochemicals of plants. To conduct molecular docking the 3-D structure of receptors and ligands is needed. The structures of receptor or bacterial proteins were obtained from Protein Data Bank in PDB format. The 3-D structures of ligand were retrieved from PubChem and ChemSpider databases in SDF and Jsmol format respectively. Based on molecular docking it was concluded that, Aloe vera showed highest binding affinity followed by Kalmegh, Tulsi and Garlic i.e., -8.05, -7.55, -6.40 and -3.89 Kcal/mol respectively. When the antibacterial property of plants and antibiotics were compared it was concluded that the Aloe vera and Kalmegh showed higher binding affinity as compared to gentamicin (-6.95 Kcal/mol) and enrofloxacin (-6.97 Kcal/mol) against pathogenic proteins of Staphylococcus aureus and E. coli. On comparing binding energy obtained by each phytochemical of each plant it was observed that Apigenin of Tulsi, Ajoene of Garlic, Lupeol of Aloe vera and 3-O-β-D-Glucopyranosyl-14,19- Dideoxyandrographolide of Kalmegh showed the better efficacy as they had minimum binding energy against all / maximum bacterial proteins as compared to other phytochemicals of the respective plant. Thus, the combination therapy of Aloe vera, Tulsi and Kalmegh or the specific most efficient phytochemicals (Apigenin, Lupeol, sitosterol β, Anthracene, Neoandrographolide and 3-O-β-DGlucopyranosyl-14, 19-Dideoxyandrographolide) combination may be used for further in vivo or in vitro trials in a direction of effective drug discovery against bacterial bovine endometritis.
Open Access
Differential proteomic profiling of X and Y sperm enriched Sahiwal bull semen
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-10) Sharma, Vishaka; Sharma, Mridula
Pre-determining the foetal sex is of prime importance for economic and scientific growth of both dairy farmers and livestock owner. Hence, the present study was aimed to compare the protein profile of X- and Y-sorted semen of Sahiwal bull. Semen sample (n=6) were categorized into three groups i.e. group I (X-sorted), group II (Y-sorted) and control group (both X- and Y- sperms). Total protein estimation SDS PAGE and nano LC-MS of all three samples was performed. In this study, significant total protein concentration in control group, X-sorted and Y sorted Sahiwal semen was 2.189 ± 0.035 mg/ml, 2.098 ± 0.033 mg/ml, and 1.79 ± 0.042 mg/ml respectively. Significant difference (P < 0.05) was observed in total protein concentration between control group and Y-sorted semen. Also, between X-sorted and Y sorted semen the difference was found to be significant (P < 0.05) at 5% level of significance. In SDS PAGE, specific proteins of molecular weight between 18-24KDa and between 30-37KDa were present in X-sorted sperms. Also, band corresponding to 25KDa was specific to Y-sorted sperms. In Nano LC/MS, total 241 proteins were identified, out of which 113 were differentially expressed between X- and Y-sorted sperms, in which 54 proteins are having at least two unique peptides. Out of 54 proteins, 27 were upregulated in X-sorted sample, 3 were upregulated in Y-sorted sample and 24 were differentially downregulated. Highly upregulated protein viz. Armadillo repeat containing 12 protein, NDC1 transmembrane nucleoporin, Beta-nerve growth factor, C-type natriuretic peptide, Nucleobindin-2, Phosphoglycerate mutase 2, Calmodulin along with one uncharacterised protein having accession number F1MN9 may have potential to be used as biomarker for separating X and Y sperm.
Open Access
Comparative study on Ravenna grass (Saccharum ravennae) in relation to retention of foetal membranes in bovines
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-10) Himanshu Kumar; Gupta, H.P.
The present research work was organized for comparative study on ravenna grass(Saccharum ravennae) in relation to retention of foetal membranes in bovines. Thirty six primiparous and pluriparous recently calved crossbred cows without the history of dystocia were selected for the study and divided into five treatment and one control group as T1, T2, T3, T4, T5 and C. Cows in T1 group were given 2kg bamboo leaves, orally once after mixing with jaggery. Whereas cows in group T2, T3 and T4 were given green ravenna grass @ 50, 150 and 300gm, orally, single dose respectively. A single dose of methylergometrine maleate @ 5mg IM was given to cows in T5 group. All treatment was given immediately after parturition. Cows in control group were left untreated. Cows were examined for expulsion of foetal membranes and onset of first postpartum visible estrus. The mean time of expulsion of foetal membranes in groups T2, T3 and control was significantly (p˂0.05) higher than the groups T1, T4 and T5. The mean time (days) for first postpartum visible estrus was significantly (p<0.05) short in T1, T3, T4 and T5 as compared to control group. On day 0, glucose concentration was significantly (p˂0.05) low in control as compared to T1 and T5. In T1, T2, T4, T5 and control group, the significant (p˂0.05) increase in the blood glucose concentration was observed between day 0 to 30 and 45 postpartum. In T1, T2, T3, T4, T5 and control group, there was a significant rise in the total protein level from day 0 to 15, 30 and 45. Significantly (p˂0.05) lower calcium level in blood was observed in T2 and control as compared to the T1, T4 and T5, on the day of calving (day 0). Significantly (p˂0.05) lower phosphorus level in blood was observed in control group as compared to the T1, T4 and T5, on the day of calving (day 0). In group T1, T3 and T5, the concentration of selenium was significantly increased from day 0 to 30 and 45 postpartum. While, in the T4 group, the concentration of selenium was significantly increased from day 0 to 15 postpartum. In the control group, the zinc concentration was significantly increased from day 0 to 15, 30 and 45 postpartum. The finding from the present study suggests that there is no effect on hemoglobin, TLC and DLC concentration by different treatment protocols. Ravenna grass is also play an important role to prevent the case of RFM as similar to methylergometrine maleate and bamboo leaves.
Open Access
Studies on cryopreservation of Pantja buck semen
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-11) Dhara, Souvik; Gupta, H.P.
Present study was conducted with the objectives of evaluation of seminal attributes and development of suitable extender for cryopreservation of Pantja buck semen. The experiment was divided in two parts as Experiment I and experiment II. For experiment I, the ejaculates from four sexually mature bucks were pooled and divided into three sub samples A, B, C and D. Each sub samples were diluted in tris based medium, supplemented with varying concentration of soybean lecithin like, A (0.1% SL), B (0.2% SL), C (0.3% SL) and D (0.4% SL). The sub samples were examined for progressive motility, plasma membrane integrity, liveability and acrosome integrity at post-dilution, post-equilibration and post-thawing stages. For experiment II, ejaculates from four sexually mature bucks were pooled after evaluation of each sample separately. Pooled semen was divided into three equal aliquots as I, II and III and rest portion was centrifuged to separate seminal plasma for bio-chemical evaluation. Group I sample was diluted into two parts as D1 [Neat semen+ tris-fructose-citric acid-egg yolk (7.5%)-glycerol] (n=8) and D2 [Neat semen+ tris-fructose-citric acid-Soya lecithin (0.2%)-glycerol] (n=8). Group II was centrifuged to removal of seminal plasma and diluted into two parts as D3 [Washed semen+ tris-citric acid-egg yolk (7.5%) glycerol] and D4 [Washed semen + tris-citric acid- SL (0.2%)-glycerol]. Again group III sample was centrifuged to remove the seminal plasma and diluted into two parts as D5 [Washed semen + tris- citric acid-egg yolk (7.5%) - Bovine seminal plasma- glycerol] and D6 [Washed semen + tris- citric acid-SL (7.5%) - Bovine seminal plasma- glycerol]. Each sample was examined for progressive motility, liveability, plasma membrane integrity, acrosome integrity and morphological abnormality at postdilution, post-equilibration and post-thawing stages. Evaluation for Malondialdehyde (MDA) assay, Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvate Transaminase (GPT) and Glutathione Peroxidase (GSH-Px) were done at post-thawing stage. A total 12 (6 from group D1 and 6 from group D6) does were artificially inseminated by frozen thawed semen to evaluate fertility rate. The mean value of seminal attributes of four Pantja bucks were volume 0.36±0.06 ml, pH 6.86±0.04, mass motility (0-5 Scale) 4.47±0.22, progressive motility 92.28±1.17%, concentration 3.54±0.14 x 109/ ml, liveability 87.28±0.78%, acrosome integrity 94.31±1.05%, plasma membrane integrity 90.27±0.86%, head abnormality 1.64±0.12%, mid-piece abnormality 0.34±0.04%, tail abnormality 2.76±0.10%, total abnormality 4.70±0.17%, GPT 18.10±1.20 U/L, GOT 124.04±8.48 Un/L and GSH-Px 10.90±0.53 U/ml. In experiment I, group B (0.2% SL) showed significantly higher value of post-thawed seminal attributes compared to other groups (p< 0.05). In experiment II, Significant difference was observed in seminal attributes between soybean lecithin based extender and egg yolk based extender, washed and nonwashed semen (p< 0.05). Significantly higher post-thawing seminal parameters were observed in group D6 compared to rest of the groups (p< 0.05). In both of the experiments significant reduction of seminal attributes were observed at each steps of freezing (p< 0.05). Conception rate of group D1 and D6 were 33.33% and 66.67% respectively.
Open Access
Post service urinary and blood protein profiling in Sahiwal cattle
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2020-09) Bahuguna, Chandni; Sharma, Mridula
For economic and scientific dairy farming, dairy cattle and buffaloes should calve every year and to maintain this strategy, early pregnancy diagnosis is of utmost importance. Hence, the present research was aimed to study the protein profiling in blood and urine of pregnant and non-pregnant Sahiwal cattle for early pregnancy diagnosis. Blood and urine samples were collected from 30 Sahiwal cattle (n= 15 pregnant and n=15 non pregnant). After studying total protein, SDS PAGE and nano LC-MS a comparison was made between the data obtained from pregnant and non pregnant animals. In this study, it was found that total protein concentration of serum increased in pregnant group as compared to non pregnant group. A continuously increasing trend of protein was observed in pregnant group after day 12 (6.36 ± 0.76 g/dl) to day 22 (6.87 ± 0.39 g/dl) whereas non specific trend was found in non pregnant group. In urine, total protein concentration increased significantly (p < 0.1) in pregnant group as compared to non pregnant group from day 16 to 22. A trend of increase in concentration from day 0 to day 16 and then decrease till day 22 was observed in both groups. Maximum protein concentration was observed on day 16 (31.6 ± 0.68 mg/dl) and minimum on day 0 (23.32 ± 0.81 mg/dl) in urine of pregnant group. In SDS PAGE of serum, specific proteins of molecular weight between 29 to 43kDa and over expression of 66 kDa protein was present in pregnant group as compared to non pregnant group. In case of urine, proteins of molecular weight 43kDa and 66kDa were over expressed in pregnant cows as compared to non pregnant cows. In nano LC-MS, total 248 proteins were identified. Out of which, 129 were up regulated and 54 were down regulated. There were 17 highly up regulated proteins having more than 30 fold change. 20 up regulated proteins in nano LC-MS corresponded to the specific band present in the SDS PAGE. Highly up regulated proteins like like lactotransferrin, Golgin A4, MYRIP, PKD1 and PWWP domain containing protein MUM1 may have the potential to be used as biomarker for the pregnancy diagnosis and development of pregnancy diagnostic kit.

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