Assessment of carbon footprint of milk production and identification of its major determinants in smallholder dairy farms in Karnataka, India.

Indian dairy enterprise is dominated by smallholder dairy farms that contribute 72% of the country's total milk production. These smallholder dairy farms are often considered to emit substantial greenhouse gases (GHG) but are poor in productive performances. Therefore, it is crucial to estimate the carbon footprint (CF) of milk production of the smallholder Indian dairy farms. The primary objectives of the study were (1) Assessing the CF of milk production of smallholder dairy farms through life cycle analysis in south-interior Karnataka, India; (2) Identifying the hotspots of GHG emissions and significant factors influencing the CF of milk production in smallholder dairy production system. The study accounted GHG emissions from different sources and considered multiple functions of the smallholder production system. Estimations were made based on primary data collected from 47 farms and associated secondary data. For estimating the CF of milk production, the emissions of carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) on a CO2-equivalent (CO2-eq) basis from feed production, enteric fermentation, manure management, transport and energy usage were allocated to fat- and protein-corrected milk (FPCM) based on mass balance, price (crop byproducts and residues) and feed digestibility. Principal component analysis and stepwise linear regression analysis were performed to identify the major factors influencing the CF. The average total GHG emissions (kg CO2-eq yr-1 farm-1) attributable to milk production based on mass, economic, and digestibility allocations were 8,936, 8,641, and 8,759, respectively. The contributions of CH4, N2O, and CO2 to the total farm GHG emission were 70.6%, 20.5%, and 7.69%, respectively. The major emission hotspots were CH4 emission from enteric fermentation (66.8%) and GHG emission from feed production (23.0%). The average CF of cradle-to-dairy cooperative milk production varied from 1.45 to 1.81 kg CO2-eq kg FPCM-1. The CF of milk production was more than 2-fold greater, when milk yield was below 3,500 kg lactating cow-1 yr-1. The FPCM yield 100 kg body weight-1, dry matter intake, and CH4 emission from manure management were the strongest determinants of the CF and explained 83.4% of the observed variation. The study emphasized the importance of considering multiple functions of a mixed crop-livestock-based dairy production system for estimating CF per unit of product. The results suggest that maintaining high-yielding dairy animals and adopting appropriate feeding strategies for better feed utilization are the possible effective interventions for reducing the CF of milk production.

Effect of silkworm (Bombyx mori) pupae oil supplementation on enteric methane emission and methanogens diversity in sheep.

In vitro and in vivo studies were conducted to examine the effect of silkworm pupae oil on methane (CH4) emission and methanogens diversity. Five graded levels (2, 4, 6, 8 and 10%) of silkworm pupae oil were tested in vitro. Eighteen Mandya adult sheep were divided into three groups. All the animals were fed on similar basal diet except the oil supplementation in test groups. Oil level for supplementation was decided on the basis of in vitro study. In vitro study indicated a reduction of 22% in CH4 production with 2% oil supplementation. Animals in test groups were supplemented with oil (2%) either daily (CON) or intermittently (INT) on every alternate week for all the seven days. A significant reduction of 17-20% in enteric CH4 emission (g/d) was achieved due to oil supplementation in sheep. However, No variation was established between test groups CON and INT. In present study, Methanobrevibacter was major genus contributed ∼90% of the total rumen methanogens; whilst Methanobrevibacter gottschalkii was the most abundant methanogens species. Abundance of Methanobrevibacter ruminantium was affected with the oil supplementation. It can be concluded that the silkworm pupae oil at 2% can decrease CH4 emission by 15-20%.

Synthesis, biological evaluation, and molecular docking study of some new rohitukine analogs as protein tyrosine phosphatase 1B inhibitors.

Rohitukine (RH) was extracted from the stem bark of Dysoxylum binectariferum Hook. It was derivatized to different arylsulphanmides by treating with the corresponding aryl sulphonyl chlorides. These derivatives were tested in-vitro on protein tyrosine phosphatase 1B (PTP1B) inhibition. Among these the active compounds K2, K3, K5, and K8 significantly inhibited the PTP1B by 51.3%, 65.6%, 71.9%, and 55.9% respectively at 10 µg/ml, the results were also supported by in-silico docking experiments. The most potent compound K5 was analyzed for antidiabetic and antidyslipidemic activity in vivo. It showed a marked reduction in blood glucose level (random and fasting) and serum insulin level in db/db mice. It improved glucose intolerance as ascertained by the oral glucose tolerance test (OGTT). These NCEs (New Chemical Entities) also lowered cholesterol and triglyceride profiles while improved high-density lipoprotein cholesterol in db/db mice. The K5 was further evaluated for antiadipogenic activity on MDI (Methylisobutylxanthine, dexamethasone, and insulin)-induced adipogenesis. where it significantly inhibited MDI-induced adipogenesis in 3 T3-L1 preadipocytes, at 10 µM and 20 µM concentration. These results were compared with the parent compound RH which inhibited 35% and 45% lipid accumulation while the RH analog K5 inhibited the lipid accumulation by 41% and 51% at 10 and 20 µM concentration, respectively. These results well corroborated with in-silico studies.

Goat as the ideal climate-resilient animal model in tropical environment: revisiting advantages over other livestock species.

In the agriculture sector, livestock are considered extremely resilient to climate change and are tipped to play a significant role in ensuring food security to meet the increased demands of growing human population by 2050. Compared to other domestic species, goats are considered the ideal animal model for climate change due to its high thermal and drought resilience, ability to survive on limited pastures, and high disease resistance. This review is therefore a revisit to the advantages of rearing goats over other livestock species under current and future trends of changes in climate, particularly to cope with recurrent multiple stressors such as heat load, and lack of water and feed. In summary, goats, also called as poor man's cow, are preferred by the small-scale landless farmers due to their low input and assured higher output system, as they require low initial investment, with minimum specialized facilities and labors. Furthermore, they perceive goats as better resilient animal to cope with multiple stressors such as heat load, and water and feed scarcity, and possess better skills to cope with bush, when compared with sheep and cattle. The unique capacity for employing behavioral plasticity and morphological features of goats gives them clear advantage over sheep and cattle, when coping with seasonal biotopes, and experiences of water and feed shortage. When facing with low-quality feed, they also are superior to cattle and sheep to digest dry matter and to recycle nitrogen. Additionally, goats have superior ability to desiccate feces and concentrate urine, when compared with sheep and cattle. These advantages make goat the go-to species for efficiently countering the adversities associated with climate change and to optimize appropriate economic return through sustained production. Therefore, goats are tipped to be the future animals with extreme potential to counter the projected alarming climate change impacts and expected to play a significant role in ensuring food security to meet the demands of the growing human population by the end of this century.

EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro.

PURPOSE: The objective of the present study was to purify sheep spermatogonial stem cells (SSCs) from testicular isolate using combined enrichment methods and to study the effect of growth factors on SSC stemness during culture. METHODS: The testicular cells from prepubertal male sheep were isolated, and SSCs were purified using Ficoll gradients (10 and 12%) followed by differential plating (laminin with BSA). SSCs were cultured with StemPro®-34 SFM, additives, and FBS for 7 days. The various doses (ng/ml) of growth factors, EGF at 10, 15, and 20, GDNF at 40, 70, and 100 and IGF-1 at 50, 100, and 150 were tested for the proliferation and stemness of SSCs in vitro. The stemness in cultured cells was assessed using SSC markers PLZF, ITGA6, and GFRα1. RESULTS: Ficoll density gradient separation significantly (p < 0.05) increased the percentage of SSCs in 12% fraction (35.1 ± 3.8 vs 11.2 ± 3.7). Subsequently, purification using laminin with BSA plating further enriched SSCs to 61.7 ± 4.7%. GDNF at 40 ng/ml, EGF at 15 and 20 ng/ml and IGF1 at 100 and 150 ng/ml significantly (p < 0.05) improved proliferation and stemness of SSCs up to 7 days in culture. GDNF at 40 ng/ml outperformed other growth factors tested and could maintain the ovine SSCs proliferation and stemness for 36 days. CONCLUSIONS: The combined enrichment method employing density gradient centrifugation and laminin with BSA plating improves the purification efficiency of ovine SSCs. GDNF at 40 ng/ml is essential for optimal proliferation and sustenance of stemness of ovine SSCs in vitro.

Summer season induced changes in quantitative expression patterns of different heat shock response genes in Salem black goats.

Research efforts of elucidating the molecular mechanisms governing heat shock response which imparts thermo-tolerance ability to indigenous breeds are very scanty. Therefore, a study was conducted with the primary objective to determine the impact of heat stress on the expression pattern of different heat shock response genes in the hepatic tissues of indigenous Salem Black goat. The study was conducted for a period of 45 days in twelve 1-year-old female Salem Black breed goats. The animals were randomly allocated into two groups of six animals each, C (n = 6; Salem Black control) and HS (n = 6; Salem Black heat stress). The C animals were maintained in the shed in comfort condition while HS animals were exposed outside to summer heat stress between 10:00 h to 16:00 h during experimental period. The animals were slaughtered at the end of study and their liver samples were collected for assessing the different heat shock response genes. Based on the results obtained from the study it was established that the heat shock protein 70 (HSP70), HSP90, super oxide dismutase (SOD), nitrous oxide synthase 1 (NOS1) genes were significantly (P < 0.05) down regulated. However, heat stress did not influence the expression pattern of heat shock factor-1 (HSF1) gene. The lower level of expression of all heat shock response genes may be due to less magnitude of heat stress in the study to induce cellular stress response in Salem Black goats.

Microalbuminuria as a Predictor of Pre-eclampsia in Pregnant Women Presenting in the Antenatal Clinic at Dhulikhel Hospital.

Background Pre-eclampsia is described as a pregnancy-specific syndrome that can affect virtually every organ system. The appearance of proteinuria remains an important diagnostic criterion. Proteinuria is an objective marker and reflects system-wide endothelial leak, which characterizes the pre-eclampsia syndrome. Objective To determine protein values using the spot urinary albumin/creatinine ratio amongst the pregnant women and to identify whether this test can be used as the investigation of choice for predicting pre-eclampsia in near future. Method A hospital based observational cross-sectional study conducted from February 2016 to August 2017. The study included pregnant women from 20-28 weeks of gestation. They were tested for spot urinary albumin and creatinine. They were all followed up till term, delivery and post partum. Result The mean age of the study patients was 28 years (N+335), the most common age group being 25 to 29 years (43.6%). Pregnant women with higher values of urinary albumin/creatinine ratio were found to develop hypertension compared to women with normal values of urinary albumin/creatinine ratio in the third trimester with insignificant difference (p < 0.283) statistically. Conclusion Microalbuminuria is one of the predictor of pre-eclampsia however it cannot be used as routine investigation for predicting pre-eclampsia in future; further studies in large scales are needed to prove whether urinary albumin/creatinine ratio can be utilized as predictor for pre-eclampsia or not.

Effect of heat stress on the quantitative expression patterns of different cytokine genes in Malabari goats.

A study was conducted to determine the influence of exposure to hot environment on different cytokine gene expression patterns in Malabari goat. The study was conducted in 12 (10 months to 1 year old) goats of Malabari breed for 45-day duration. The control (n = 6; C) group goats were kept under comfortable condition in the shaded pens while heat stress (n = 6; HS) group goats were kept out in the open hot environment during summer season for the study duration between 10.00 h to 16.00 h. The representative mesenteric lymph node (MLN) samples were collected from the slaughtered goats at the end of study for assessing the different cytokine gene expression and histopathological changes. Compared with C group, the expression patterns of interleukin 18 (IL-18), tumor necrosis factor-α (TNF-α), interferon-ß (IFN-ß), and IFN-γ downregulated (P < 0.05) in the HS group. The histopathological changes of MLNs showed paucity of lymphocyte distribution in follicular areas as wells as decreased density of lymphocytes in the germinal centers of the HS group (P < 0.05). The findings from this study reflected the compromised immune functions during heat stress in Malabari goats. Further, the study established that IL-18, TNF-α, IFN-ß, and IFN-γ genes could serve as reliable immunological markers for quantifying heat stress-mediated immune response alterations in Malabari goats.

Study of Prescription Pattern and Factors Associated with Clinical Outcomes in Type 2 Diabetes Mellitus with Reference to Short Term Glycemic Control.

Background Diabetes is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. Though oral hypoglycemic agents (OHAs) or/and insulin are prescribed to control the hyperglycemia, still glycemic control is not seen in most of the cases. Evaluating the treatment pattern including the glycemic control and associated factors are of utmost importance for better patient care. Objective To investigate the treatment pattern and short term glycemic control among Type 2 Diabetes Mellitus (DM2) patients and study the factors associated with it. Method Patients meeting the inclusion criteria with DM2 attending laboratory or Out-Patient Department or Emergency Department in Dhulikhel Hospital and patients who were admitted to the ward taking various OHAs and/or insulin at the time of data collection were considered. Informed consent was taken prior to the interview. The collected data was entered as well as analyzed using the SPSS (Statistical Package for Social Sciences) version 21.0 and the p-value <0.05 was considered statistically significant. Result Among the 118 patients, 50.8% of them were males. Majority (28%) of the patients belonged to the age group of 51-60 years. Out of the 118 patients, 22% had their fasting blood glucose (FBG) level under control whereas 78% had uncontrolled FBG level. Metformin was the most (28%) commonly prescribed drug. Significant association was found between gender; knowledge about disease and medication with short term glycemic control (p=0.034, p=0.004 ans p=0.039 respectively). Conclusion All the patients enrolled in this study were under various anti-diabetic medications, however, majority of them didn't have their glycemia under control. Significant association was found between FBG level and gender, knowledge about disease and knowledge about medication.

Expression patterns of candidate genes reflecting the growth performance of goats subjected to heat stress.

The study is an attempt to elucidate the molecular mechanisms governing growth performance during heat stress in goats. The primary objective of the study was to establish the influence of heat stress on the expression patterns of different growth related genes in Malabari goats. The study was conducted for a period of 45 days in 12 Malabari goats randomly allocated into two groups: MC (n = 6; Malabari control) and MHS (n = 6; Malabari heat stress). At the end of study period, all 12 animals were slaughtered and their liver tissues were collected for gene expression and histopathological studies. The temperature-humidity-index (THI) inside the shed (74.9) proved that the animals were not stressed while in the outside environment (86.5) the animals were extremely distressed. The hepatic growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor-1 (IGF-1), leptin (LEP) and leptin receptor (LEPR) gene expression patterns were significantly (P < 0.05) lower in heat stress group as compared to the control group animals. In addition, negative correlation (P < 0.05) was also established between THI and all the growth related gene expression in the study. The hepatic histopathological section showed more fatty and degenerative changes (P < 0.05) in hepatocytes in MHS group as compared to MC group. The study offers the first thorough insight into the expression patterns of different growth related genes during heat stress exposure in goats. Further, the study established GH, GHR, IGF-1, LEP, LEPR genes to be the ideal markers to reflect growth potential in Malabari goats.

Resilience of three indigenous goat breeds to heat stress based on phenotypic traits and PBMC HSP70 expression.

The objective of this experiment was to evaluate the influence of summer heat stress on physiological and behavioral responses of Osmanabadi, Salem Black, and Malabari goats. The study also evaluated the differences in heat shock protein 70 (HSP70) expression pattern between these breeds. The study was conducted over 45 days during summer (April-May) using 36 1-year-old female goats by randomly allocating them into six groups with six animals in each group: Osmanabadi control (Osmanabadi CON), Osmanabadi heat stress (Osmanabadi HS), Malabari control (Malabari CON), Malabari heat stress (Malabari HS), Salem Black control (Salem Black CON), and Salem Black heat stress (Salem Black HS). The Osmanabadi CON, Malabari CON, and Salem Black CON animals were housed in a shed while the Osmanabadi HS, Malabari HS, and Salem Black HS groups were subjected to heat stress by exposing them to outside environment between 1000 and 1600 h during the experimental period. All 36 animals were provided with ad libitum feed and water. The data generated were analyzed by general linear model (GLM) repeated measurement analysis of variance. Results indicated that the drinking frequency (DF) was higher (p < 0.01) in heat stress groups (12.58, 12.25, and 10.75 times for the Osmanabadi HS, Malabari HS, and Salem Black HS, respectively) as compared to their respective control groups (5.67, 6.25, 5.58 times for the Osmanabadi CON, Malabari CON, and Salem Black CON, respectively). Water intake (WI) also showed similar trend to DF. The urinating frequency also (UF) differed between breeds with lower value (p < 0.05) recorded in the Salem Black HS (1.5 times) compared to the Malabari HS (2.92 times). The highest (p < 0.05) rumination time (RuT) was recorded in the Malabari HS (48.00 min) than both the Osmanabadi HS (20.91 min) and Salem Black HS (23.67 min). The heat stress increased (p < 0.05) all physiological variables at 1400 h. The findings of this study suggest RR, RT, and PBMC HSP70 are reliable biological markers for evaluating thermo-tolerance capacity of indigenous goat breeds.

Comparative assessment of growth performance of three different indigenous goat breeds exposed to summer heat stress.

A study was conducted to assess comparatively the growth performance of three different indigenous goat breeds during exposure to summer heat stress. The primary objective of the study was to observe the heat stress impact on the growth performance based on the body weight changes, allometric measurements, growth hormone (GH) concentration and peripheral blood mononuclear cell (PBMC) Insulin-like growth factor-1 (IGF-1) mRNA expression pattern during the summer season in comparison with the local breed (Osmanabadi). Thirty-six ten-month- to one-year-old female goats of Osmanabadi, Malabari and Salem Black breeds were randomly divided into six groups, OC (n = 6; Osmanabadi control), OHS (n = 6; Osmanabadi heat stress), MC (n = 6; Malabari control), MHS (n = 6; Malabari heat stress), SBC (n = 6; Salem Black control) and SBHS (n = 6; Salem Black heat stress). Body weight was recorded at weekly intervals, whereas other growth and allometric measurements and blood collection were carried out at fortnightly intervals. Breed factor significantly (p < .05) influenced only few growth variables such as body weight, body mass index (BMI) and body condition score (BCS). However, heat stress treatment significantly (p < .05) reduced all growth parameters expect BMI. Further, the heat stress significantly (p < .01) increased plasma GH concentration in goats with significantly higher (p < .05) concentration recorded in OHS. Among the stress groups, the lower (p < .05) PBMC IGF-1 mRNA expression was recorded in OHS, while the higher (p < .05) expression was observed in SBHS indicating the extreme adaptive capability of Salem Black breed. Thus, the results indicated that the Salem Black breed performed much better compared to both Osmanabadi and Malabari breeds indicating the superior ability of this breed to adapt to heat stress challenges. The results also indicated that plasma GH and IGF-1 gene may act as ideal biomarkers for assessing the heat stress impact on growth performance in indigenous goats.

Medication Adherence to Levothyroxine Therapy among Hypothyroid Patients and their Clinical Outcomes with Special Reference to Thyroid Function Parameters.

Background Non-adherence to medication in hypothyroid patients leads to poor clinical outcome and increased health care costs. Study related to adherence to levothyroxine in hypothyroid patients is limited worldwide. Objective To determine the adherence pattern and factors affecting adherence in hypothyroid patients and compare the clinical outcomes among adherent and non-adherent patients. Method A cross-sectional study was conducted in Dhulikhel hospital, Kathmandu University hospital. Patients with hypothyroidism on levothyroxine fulfilling the inclusion criteria were enrolled in this study. After obtaining the informed consent from the patients, structured questionnaire was used to interview the patients. Statistical analysis was performed by using SPSS 23.0. P-value < 0.05 was considered as statistically significant. Result Among 113 patients enrolled in this study, 85.8% of them were females. Majority (76.10%) of them belonged to the age group of 31-40 years. More than half (51.3%) of the patients were adherent to medication. Non-adherence was significantly associated with literate people following medication (p=0.003, OR=0.305), missed appointment to follow-up (p=0.007, OR=0.346) and longer duration of medication (p=0.019, OR=4.385). However, there was no statistically significant association of clinical outcomes with reference to TSH, fT4, fT3 and patients' age, adherence to medication and concomitant use of other drugs. Conclusion More than half of the patients were adhered to the Levothyroxine therapy. Forgetfulness was the main reason for missing the dose and non-specific reasons attributed to discontinuation of medication for these patients. Good adherence was seen among illiterate people. Treatment related factor, patient-provider relationship and patient related factor were determined as the major barrier to medication adherence. Most of the non-adherent patients had uncontrolled TSH.

Assessment of Clinical Pharmacokinetic Drug-Drug Interaction of Antimalarial Drugs α/ß-Arteether and Sulfadoxine-Pyrimethamine.

Antimalarial drug combination therapy is now being widely used for the treatment of uncomplicated malaria. The objective of the present study was to investigate the effects of coadministration of intramuscular α/ß-arteether (α/ß-AE) and oral sulfadoxine-pyrimethamine (SP) on the pharmacokinetic properties of each drug as a drug-drug interaction study to support the development of a fixed-dose combination therapy. A single-dose, open-label, crossover clinical trial was conducted in healthy adult Indian male volunteers (18 to 45 years, n = 13) who received a single dose of AE or SP or a combination dose of AE and SP. Blood samples were collected up to 21 days postadministration, and concentrations of α-AE, ß-AE, sulfadoxine, and pyrimethamine were determined by using a validated liquid chromatography-tandem mass spectrometry method. Pharmacokinetic parameters were calculated and statistically analyzed to calculate the geometric mean ratio and confidence interval. Following single-dose coadministration of intramuscular AE and oral SP, the pharmacokinetic properties of α/ß-AE were not significantly affected, and α/ß-AE had no significant effect on the pharmacokinetic properties of SP in these selected groups of healthy volunteers. However, more investigations are needed to explore this further. (This study has been registered in the clinical trial registry of India under approval no. CTRI/2011/11/002155.).

Molecular Characterization and Computational Modelling of New Delhi Metallo-ß-Lactamase-5 from an Escherichia coli Isolate (KOEC3) of Bovine Origin.

Emergence of antimicrobial resistance mediated through New Delhi metallo-ß-lactamases (NDMs) is a serious therapeutic challenge. Till date, 16 different NDMs have been described. In this study, we report the molecular and structural characteristics of NDM-5 isolated from an Escherichia coli isolate (KOEC3) of bovine origin. Using PCR amplification, cloning and sequencing of full blaNDM gene, we identified the NDM type as NDM-5. Cloning of full gene in E. coli DH5α and subsequent assessment of antibiotic susceptibility of the transformed cells indicated possible role of native promoter in expression blaNDM-5. Translated amino acid sequence had two substitutions (Val88Leu and Met154Leu) compared to NDM-1. Theoretically deduced isoelectric pH of NDM-5 was 5.88 and instability index was 36.99, indicating a stable protein. From the amino acids sequence, a 3D model of the protein was computed. Analysis of the protein structure elucidated zinc coordination and also revealed a large binding cleft and flexible nature of the protein, which might be the reason for broad substrate range. Docking experiments revealed plausible binding poses for five carbapenem drugs in the vicinity of metal ions. In conclusion, results provided possible explanation for wide range of antibiotics catalyzed by NDM-5 and likely interaction modes with five carbapenem drugs.

Effects of graded levels of tannin-containing tropical tree leaves on in vitro rumen fermentation, total protozoa and methane production.

AIMS: This study was carried out to determine the effect of graded levels of tannin-containing tropical tree leaves, Autocarpus integrifolis, Azardirachta indica and Ficus bengalensis, on the in vitro rumen fermentation pattern, total protozoa and methane suppression in order to establish the optimum dose of these leaves for inclusion in the ruminant diets. METHODS AND RESULTS: The air-dried and ground samples of Au. integrifolis, Az. indica and Ficus bengalensis were subjected to in vitro incubation using 30 ml buffered rumen fluid at 0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0% (dry matter refers to moisture-free basis) of a total mixed ration (TMR: refers to mixture of roughage and concentrate containing cereals and oil cakes) devoid of tannin. The TMR for the experimental incubation was prepared by mixing 40 parts of ground Elusine coracana straw as roughage source with 60 parts of concentrate mixture. The leaves contained an average 130 g kg(-1) CP with 7·0 MJ of ME kg(-1) DM. The average neutral detergent fibre (NDF) content was <400 g kg(-1) DM. Highest total phenol (TP; g kg(-1) DM) was recorded in Az. indica (108) followed by F. bengalensis (103) and Au. integrifolis (76), and total tannin (TT) content also showed similar trend. However, condensed tannin (CT) was highest in F. bengalensis (260) followed by Au. integrifolis (186) and Az. indica (138). There was significant (P < 0.05) reduction in the NH3 concentration in the presence of all the leaves. Autocarpus integrifolis tannins did not cause inhibition of total volatile fatty acid (TVFA: refers to the concentration of volatile fatty acids, viz. acetic, butyric and propionic) concentration, whereas F. bengalensis and Az. indica tannins at higher level of incubation (>5.0%) reduced TVFA concentration. Protozoa (cells per mL) were similar at all levels of inclusion with Au. integrifolis, but reduced in case of F. bengalensis and Az. indica. As the level of tannin increased in the incubation medium, there was a linear reduction in methane concentration. Highest methane reduction (%) was recorded in incubations supplemented with Az. indica (61.5) followed by F. bengalensis (46.8) and Au. integrifolis (30.3). CONCLUSIONS: It was established from this study that tropical leaves of F. bengalensis, Au. integrifolis and Az. indica suppress methanogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Ficus bengalensis, Au. integrifolis and Az. indica leaves are of interest in the enteric methane ameliorative strategies. Total mixed ration containing 10-15% ground F. bengalensis or Au. integrifolis or Az. indica leaves could be fed to ruminants to reduce enteric methanogenesis. However, in vivo trials need to be carried out to validate these in vitro results.

Effect of plant secondary compounds on in vitro methane, ammonia production and ruminal protozoa population.

AIMS: The objective of this study was to evaluate the potential of secondary plant metabolites from 38 sources to serve as antimethanogenic additives in ruminant diets. The effect of leaf tannins from these different plant sources on rumen fermentation, protozoal populations and methanogenesis was also studied. METHODS AND RESULTS: Samples (200 mg dry matter, DM) were incubated without and with polyethylene glycol (PEG)-6000 (400 mg DM) as a tannin binder during 24-h incubation in the in vitro Hohenheim gas system. In the leaf samples, total phenol (g kg(-1) DM) was maximum in Pimenta officinalis (312) followed by Oenothera lamarckiana (185) and Lawsonia inermis (105). Of the 38 samples, condensed tannins exceeded 4.0 g kg(-1) in only Alpinia galanga (7.50), Cinnamomum verum (4.58), Pelargonium graveolens (18.7) and Pimenta officinalis (23.2) and were not detected in seven samples. When the bioactivity of the leaf samples was assessed using the tannin bioassay, the percentage increase in the amount of gas produced during incubation of samples with the tannin-binding agent PEG-6000 over the amount produced during incubation without the tannin binder ranged from nil (zero) to 367%, with the highest being recorded with A. galanga leaves. The ratio of methane reduction per ml of total gas reduction was maximum with Rauvolfia serpentina (131.8) leaves, followed by Indigofera tinctoria (16.8) and Withania somnifera (10.2) leaves. Total and differential protozoal counts increased with added PEG in twenty-two samples, maximum being in Pimenta officinalis. Increased accumulation of total volatile fatty acids during incubation with added PEG-6000 was recorded, and the values ranged from zero to 61%. However, the increase was significant in only 11 of the 38 tannin sources tested indicating noninterference of tannin on in vitro fermentation of carbohydrates by the majority of samples tested. Conversely, in 26 of 38 plant sources, the leaf tannins reduced N-digestibility as evidenced by increased accumulation of NH3 -N with added PEG. CONCLUSIONS: Our study unequivocally demonstrated that plants containing secondary metabolites such as Rauvolfia serpentine, Indigofera tinctoria and Withania somnifera have great potential to suppress methanogenesis with minimal adverse effect of feedstuff fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: It was established that methanogenesis was not essentially related to the density of protozoa population in vitro. The tannins contained in these plants could be of interest in the development of new additives in ruminant nutrition.

Effect of medicinal and aromatic plants on rumen fermentation, protozoa population and methanogenesis in vitro.

The potential of tannins from 21 medicinal and aromatic plant leaves as antimethanogenic additives in ruminant feeds was investigated. The effect of tannin from these leaves on rumen fermentation parameters, protozoa population and methanogenesis was studied by incubating the samples [200 mg dry matter (DM)] without and with polyethylene glycol (PEG)-6000 (400 mg DM) as a tannin binder during 24-h incubation in the in vitro Hohenheim gas method. Based on the methane percentage estimated in the total gas produced, methane production in millilitre was calculated [methane volume (ml) = methane % × total gas produced (ml) in 24 h]. In the samples, crude protein and neutral detergent fibre (g/kg DM) ranged from 113 to 172 and from 352 to 444 respectively. The total phenol (TP; g/kg DM) content was highest in Terminalia chebula (274) followed by Hemigraphis colorata (71) and Sapindus laurifolia (51) respectively. In the remaining samples, it was <43 g/kg DM. Activity of tannins, as represented by the increase in gas volume on addition of PEG, ranged from 0 to 133%, with the highest being recorded in T. chebula. The per cent increase in methane on PEG addition was 0 for Ammi majus, Aristolochia indica, Cascabela thevetia, Ipomea nil and Lantana camara, illustrating that tannins present in these samples had no effect on methane concentration. The PEG addition increased the total protozoa count by >50% in A. indica and C. thevetica. One of the important findings of our study was that of the 21 samples screened, Entodinia population increased in 12 with PEG as compared to 7 where Holotricha increased, indicating higher susceptibility of Entodinia to tannin. There was no increase in the protozoa population with PEG when incubating Cardiospermum halicacabum, Clerodendrum inerme, Dioscorea floribunda, Nerium oleander and Selastras paniculatus, which strongly suggested that methane suppression recorded in these samples was not because of a defaunating effect of their tannins per se. The fermentation pattern reflected increased total volatile fatty acid (TVFA) concentration from 0 to 28.3% with PEG addition among the leaves. Our results confirmed further observations that methanogenesis in vitro is not essentially related to density of protozoa population. Secondly, medicinal and aromatic plants such as C. inerme, Gymnema sylvestre and Sapindus laurifolia containing tannins appear to have a potential to suppress in vitro methanogenesis.

Chebulinic acid alleviates LPS-induced inflammatory bone loss by targeting the crosstalk between reactive oxygen species/NFκB signaling in osteoblast cells.

Chebulinic acid (CA), a plant ellagitannin derived from Triphala, is reported to exhibit both anti-inflammatory & anti-oxidant activity apart from anti-tumour property. However, its role in inflammatory bone loss conditions was unexplored. We hypothesized that CA may prevent the bone loss under inflammatory conditions induced by lipopolysaccharide (LPS) in 10-week-old male C57BL/6J mice. Micro-CT analysis and histomorphometric evaluations were carried out where it was found that CA significantly improved the bone micro-architectures by enhancing trabecular connectivity and strength of the bone. CA also increased the bone regeneration as examined by calcein labelling and ex-vivo mineralisation along with maintaining the bone serum markers. Further, CA ameliorated the reduction in osteoblast cell differentiation, proliferation and viability after LPS stimulation. DCFDA and Mitosox staining revealed that CA presented remarkable protective effects against LPS treatment by attenuating oxidative stress, both at cellular & mitochondrial levels. In addition, CA significantly decreased the production of pro-inflammatory cytokines, and down-regulated the phosphorylation of NFκB and IκBα, indicating that CA could attenuate the inflammatory impairment to primary osteoblast cells by suppressing the NFkB signalling pathway. Taken together, the protective role of CA against LPS-induced bone loss & inhibitory effect on total ROS levels hold promise as a potential novel therapeutic strategy for the inflammatory diseases in bones.

Simultaneous estimation of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide from Polyalthia longifolia and its metabolite in hamster plasma: application to pharmacokinetic study.

A selective and sensitive LC-MS-MS method was developed and validated for simultaneous estimation and pharmacokinetic studies of 16α-hydroxycleroda-3,13(14) Z-dien-15,16-olide (K-09) obtained from Polyalthia longifolia and its metabolite (K-9T), a novel antidyslipidemic agent. Sample clean-up involved liquid-liquid extraction of both the analytes and internal standard (rosuvastatin) from 200 µL of hamster plasma. The analytes were chromatographically separated on a Symmetry-Shield C18 (5 µm, 4.6 × 150 mm) column, using acetonitrile-0.1% aqueous formic acid (92:08, v/v) as the mobile phase. Detection was performed using negative ion electrospray ionization in multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 1.56-200 ng/mL, with a correlation coefficient (r²) of 0.998 or better. The within- and between-batch precisions (relative standard deviation, %RSD) and the accuracy (percentage bias) were within acceptable limits as per FDA guidelines. The validated method was successfully applied to reveal the pharmacokinetic parameters of K-09 and metabolite after oral administration. This method will therefore be highly useful for future studies of K-09 and metabolite K-9T pharmacokinetics in preclinical and clinical studies.