A. K. Dang

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ~60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus)

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): Lactating vis-a-vis heifer

UNLABELLED Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland. BIOLOGICAL SIGNIFICANCE This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

Formation of NET, phagocytic activity, surface architecture, apoptosis and expression of toll like receptors 2 and 4 (TLR2 and TLR4) in neutrophils of mastitic cows

Neutrophils employ both oxidative and non oxidative mechanisms to destroy pathogens. Function of neutrophils coming in milk during mammary invasion is not clearly understood in dairy animals. Therefore, the present study was designed in 36 Sahiwal cows to see the changes occurring in the neutrophil activity of cows suffering from subclinical (SCM) and clinical mastitis (CM). Cows were divided into three groups as healthy (n = 12), SCM (n = 12) and CM (n = 12) groups on the basis of CMT scoring, gross morphological changes in milk, bacteriological examination of milk and by counting milk SCC. Significantly (P < 0.05) higher milk SCC, neutrophil percent and significantly (P < 0.05) lower viability of both blood and milk neutrophils were observed in CM group of cows as compared to SCM and control group of cows. Phagocytic activity (PA) was significantly (P < 0.05) higher in milk neutrophils of SCM and CM cows as compared to control cows. Toll like receptors 2 and 4 in blood and milk neutrophils were found to be significantly (P < 0.05) higher, whereas, apoptosis in terms of altered mitochondrial transmembrane potential, Caspase 3 and 7 activities were found to be significantly (P < 0.05) lower in CM cows. Alterations in surface architecture of neutrophils in terms of formation of pseudopods was observed by scanning electron microscope (SEM) and found to be higher in CM cows. Blood neutrophils were found to be spherical as compared to milk neutrophils. Formation of neutrophil extracellular trap (NETs) were found milk neutrophils of CM cows, whereas, SCM and healthy cows did not exhibit NET formation. The study indicated a positive correlation between lower neutrophil apoptosis and higher expression of TLR2 and TLR4 with the formation of NETs and change in surface architecture. Formation of NET like structures seemed to be an effective mode of defense employed by neutrophils of cows suffering from clinical mastitis.

A comparative study on the blood and milk cell counts of healthy, subclinical, and clinical mastitis Karan Fries cows

Aim: The present study was aimed to study the use of cell counts as an early indicator of mammary health. Materials and Methods: Milk and blood cell counts were estimated from 8 healthy, 8 subclinical (SCM), and 8 clinically mastitis (CM) groups of Karan Fries (KF) cows. Results: Total leucocyte counts and neutrophil percent in blood and milk somatic cells and milk neutrophil percent of healthy cows increased significantly (p<0.05) in SCM cows and CM cows. Viability of blood and milk neutrophils was more in healthy cows, but decreased significantly (p<0.05) in SCM and CM cows. Significant (p<0.05) decrease were also observed in both the blood and milk lymphocytes and monocytes of SCM and CM cows. Phagocytic activity (PA) of blood neutrophils also decreased significantly (p<0.05) in SCM cows. There was no difference between the PA of SCM and CM cows. Milk neutrophil percent was more in the SCM and clinically infected milk than in the blood of these cows. About 96-97% of the neutrophils had segmented nucleus in both healthy and subclinical milk, whereas, 2-3% were having band shaped or immature nuclei. There was a significant decrease in the segmented neutrophils, whereas, band neutrophils increase significantly to about 5% in the infected milk of mastitic cows. Viability of the milk neutrophils decreased more in case of subclinical and clinical milk as compared to that of blood. PA was found to be highest in the milk of healthy group of cows, but decreased significantly (p<0.05) in subclinically infected cows. However, there was no difference between the PA of milk neutrophils of SCM and CM cows. PA of milk was also found to be significantly lower in the milk of healthy cows when compared to that of blood neutrophils. Conclusion: This study indicated that percent neutrophils and their type in conjunction with milk somatic cell counts can be used as a more reliable indicator of mammary health in cows.

Comparative evaluation of neutrophil competence and activity of cows and buffaloes around peripartum

To compare immune activities of neutrophils, blood samples were collected from 12 cows and 12 buffaloes on −15, −7, −5, −3, −2 and −1 days prepartum at calving and on 1, 2, 3, 5, 7 and 15 days postpartum. Plasma cortisol levels, phagocytic activity (PA), enzyme (elastase, collagenase and cathepsin G) levels and expression of TLR-2, TLR-4 and IL-8 were also studied. Blood total leukocyte counts and neutrophil percentage increased at calving and decreased faster in buffaloes. Neutrophil PA was significantly (P < 0.01) lower even up to two days post calving in cows. Cortisol levels were significantly (P < 0.01) higher in cows and negatively correlated with neutrophilic functions. Elastase was significantly (P < 0.01) higher in buffaloes. Collagenase levels were significantly (P < 0.01) higher in buffaloes at three days precalving. At 15 days precalving and 7 and 15 days postcalving, expression of TLR-2 gene was significantly (P < 0.01) higher in buffaloes. Expression of TLR-4 gene was significantly (P < 0.01) higher on days 7 and 15 postcalving in buffaloes. Expression of IL-8 was significantly (P < 0.01) higher on day 15 of postcalving in buffaloes. Increased blood neutrophilic function in buffaloes provides disease resistance to them around peripartum.

Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus

Aim: The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM). Materials and Methods: A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy. Results: Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows. Conclusion: The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection.

Comparative expression profiling of insulin-like growth factor binding protein-5 in milk of Bos indicus and Bubalus bubalis during lactation

Insulin-like growth factor binding protein-5 (IGFBP-5) is a key molecule in mammary gland development, which facilitates the removal of mammary epithelial cells (MECs) by apoptosis that takes place during remodeling of the mammary gland during involution. IGFBP-5 binds with IGFs for their bioavailability. IGFBP-5 has been reported to perform pleiotropic roles such as cellular apoptosis, proliferation and differentiation. To understand the role of IGFBP-5 during lactation and clinical mastitis, expression profiling of IGFBP-5 at the protein level was performed in both indigenous cows (Bos indicus) and buffaloes (Bubalus bubalis) belonging to two different breeds - Sahiwal cows and Murrah buffaloes. Reverse-transcriptase PCR (RT-PCR) of IGFBP-5 mRNA confirmed its expression in milk somatic cells and MECs of Sahiwal cows. ELISA was performed for quantitative measurement of IGFBP-5 concentrations in milk during different days (0, 50, 100, 150, 200, 250 and 300) of lactation, during the involution period and in animals exhibiting short lactation and clinical mastitis. The highest concentration of IGFBP-5 in milk was observed during the involution period followed by colostrum, late and early lactation, respectively, in both cattle and buffaloes. No significant difference in the concentration of IGFBP-5 was observed during the first 150 days of lactation between cows and buffaloes. However, higher concentration of IGFBP-5 was observed in cows during late lactation (200 to 300 days) in comparison with buffaloes. To validate the ELISA data, quantitative real-time PCR was performed in MECs of Sahiwal cows. The relative mRNA abundance of IGFBP-5 was found to be significantly (P<0.05) higher on day 15 than between 50 and 150 days of lactation in case of Sahiwal cows. Highest mRNA expression of IGFBP-5 was observed around 300 days of lactation followed by 200 and 250 days (P<0.05), respectively. Murrah buffaloes showed low levels of IGFBP-5 protein in milk as compared with Sahiwal cows during lactation in ELISA. Animals having history of short lactation length (short lactating animals) showed higher levels of IGFBP-5 expression (at protein level) in comparison with normal lactating animals. We propose that higher level IGFBP-5 expression may have functional significance in lactation persistency. As a pro-apoptotic molecule, higher expression of IGFBP-5 was observed to be inversely related to lactation length and milk production.

Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows

The embryonic mortality in cows is a growing concern for an ever-expanding dairy industry. The current study was an attempt to shorten the open period of dairy cows having suffered embryonic loss by diagnosing them at an earlier stage. The blood samples were collected from the Karan Fries (KF) cows on days 0 (day of AI/estrus), 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 post insemination. The experimental animals were then categorized into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. There were 6 animals in each group. The plasma progesterone was higher in pregnant than EEM and LEM cows. Plasma Interferon-tau concentration was significantly (p < 0.05) lower in LEM than pregnant cows where it could be detected from day 14-21 but was non-detectable in EEM cows. The mRNA expression of ISG15, OAS1, MX1 and MX2 in blood neutrophils was significantly (p < 0.05) higher from day 8-42 as against day 0 in pregnant cows. The highest expression was observed around day 18-21 in pregnant cows. The ISG15, OAS1, MX1 and MX2 mRNA expression was significantly (p < 0.05) higher from day 4-42 as compared to day 0 in LEM cows, whereas in EEM cows the expression stayed close to that of day 0 (1.00 ± 0.00). The mRNA expression of ISG15, OAS1, MX1 and MX2 started to decline from day 24 onwards. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The study reveals that the Interferon tau stimulated gene expression in neutrophils can act as peripheral biomarkers for detecting the embryonic mortality in dairy cows.