Manganese uptake and Mn efficiency of wheat cultivars are related to Mn‐uptake kinetics and root growth

Wheat cultivars differ widely in manganese (Mn) efficiency. To investigate the reasons for different Mn efficiencies, a pot experiment with soil, a solution‐culture experiment, and model calculations were carried out. The pot experiment was conducted with wheat (Triticum aestivum L. cvs. PBW 373, PBW 154, PBW 343, PBW 138, and Triticum durum L. cvs. PBW 34 and PDW 233) grown in a screen house in India. The soil was a loamy sand with pH 8.1, DTPA‐extractable Mn 1.62 mg (kg soil)^–1, and initial soil solution Mn concentration (C_Li) of 0.19 μM. When fertilized with 50 mg Mn (kg soil)^–1, C_Li increased to 0.32 μM. At C_Li 0.19 μM, wheat cv. PBW 373 produced 74% of its maximum shoot dry weight (SDW) with 64% of its maximum root length (RL), while cv. PDW 233 produced only 25% of its maximum SDW with 11% of its maximum RL. The other wheat cultivars were between these extremes. Manganese deficiency caused a reduction in shoot growth, but more strongly reduced root growth. The low Mn efficiency of T. durum cv. PDW 233 was related to a strong depression of its root growth. Manganese influx was similar for all cultivars. In solution culture below 1 μM Mn, under controlled climate‐chamber conditions, Mn influx was linearly related to Mn concentration. Both the efficient cv. PBW 343 and the inefficient cv. PDW 233 had a similar influx. Uptake kinetic parameters from the solution experiment together with soil and plant parameters from the pot experiment were used in a mechanistic nutrient‐uptake model. Calculated values of Mn influx for wheat grown in soil were 55% to 74% of measured values. A sensitivity analysis showed that increasing C_Li or the slope of the uptake isotherm by about 30% would be enough to reach the observed influx. The results of this research indicate that an increase of Mn solubility by microbial or chemical mobilization would increase Mn uptake. But on the other hand, no chemical mobilization would be required to increase Mn uptake if the plant improved its uptake kinetics. Low Mn efficiency of some wheat cultivars was related to their reduced root growth at low soil Mn supply.     

Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time‐dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)‐induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H‐89, PD9809 and GF‐109) and enhancer (dbcAMP, H₂O₂ and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F‐actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.     

Role of crop diversification and integrated nutrient management in resilience of soil fertility under rice-wheat cropping system

A field study conducted for two crop cycles of five cropping systems supplied with six nutrient combinations at the Indian Agricultural Research Institute, New Delhi indicated that the cropping systems having a legume increased organic C content over initial level by 0.02?–?0.05%, available N by 3.5?–?14.1?kg ha^???1, whereas the rice-wheat cropping system resulted in a reduction in organic C and available N over initial level by 0.05% and 1.5?kg ha^???1, respectively after 2 years of study. Rice-potato-mungbean cropping system resulted in a negative balance of available P and rice-clover cropping system had a negative balance of both available P and available K content in soil and thus call for adequate P and K fertilization. Application of P and K helped in building up their content in soil; NPK?+?FYM showed the highest increase in organic C, available N, available P and available K content in soil. These results suggest the inclusion of a legume in a cropping system for maintaining organic C and available N in soil and adequate P and K fertilization for arresting the depletion of available P and K content in soil. Integrated nutrient management is one of the best methods for resilience of soil fertility under rice-wheat cropping system.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

A comparative study on innate immunity parameters in the epidermal mucus of Indian major carps

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish from relevant infections. The present study is aimed at understanding and comparing the mucosal immunity of Indian major carp, that is, Labeo rohita, Catla catla and Cirrhinus mrigala, by evaluating different immune parameters such as protein content, lysozyme, myeloperoxidase, proteases and alkaline phosphatase activity in the skin mucus. The protein content of mucus sample of these species was compared, and the highest protein content was found in C. catla among the three Indian major carp species. The levels of proteases (40 ± 0.211 units/ml) and myeloperoxidase OD450 nm (1.525 ± 0.108) were found to be highest in the skin mucus of C. catla. However, lysozyme levels were highest in the skin mucus of L. rohita (10.95 ± 0.330 μg/ml), and C. mrigala had the highest alkaline phosphatase activity (30.74111 ± 0.680 U/l). Besides the enzyme activities, the epidermal mucus samples of three Indian major carp species were also tested and compared for the antibacterial activity against seven bacterial strains. Skin mucus of C. catla showed highest antibacterial activity among the three Indian major carps against all the seven bacterial strain, except that Micrococcus lysodeikticus and Vibrio cholerae, however, showed highest activity against mucus of C. mrigala. Also, the epidermal mucus from all the three species successfully agglutinated three freshwater pathogens, viz. Aeromonas hydrophila, Pseudomonas fluorescens and Edwardsiella tarda, with agglutination titres being the highest for Labeo rohita for all the pathogens. The epidermal mucus samples from the Indian major carp species showed haemagglutination activity and successfully lysed human, chicken and goat RBCs showing highest activity in C. catla. These results provide information for a better understanding of the role of epidermal mucus and its components in the innate immune system of Indian major carps.     

Ovsynch Plus protocol improves ovarian response in anovular Murrah buffaloes in low‐breeding season

The objective of the study was to evaluate the efficacy of ovarian response and pregnancy rate in anovular buffaloes following Ovsynch and Ovsynch Plus protocols. Buffaloes (n = 55) were divided into two groups: Ovsynch group (n = 26): GnRH (10 μg, GnRH1) on Day 0, PGF₂α (25 mg) on Day 7, GnRH (10 μg, GnRH2) on Day 9; Ovsynch Plus group (n = 29): 500 IU equine chorionic gonadotropin (eCG) 72 hr (day ?3) prior to Ovsynch protocol, followed by fixed timed artificial insemination (FTAI) 6 and 24 hr after GnRH2 injection in bot groups. Transrectal ultrasonography was performed daily, that is, from day 0 and ?3 in Ovsynch and Ovsynch Plus group, respectively for ovarian response and pregnancy diagnosis at day 30 post‐insemination. In Ovsynch Plus group, administration of eCG prior to GnRH1 increased (p < .001) the diameter (mm) of dominant follicle (DF) from 10.15 ± 0.26 to 12.23 ± 0.34 within 72 hr of treatment resulting higher ovulatory response to GnRH1. Ovulation after GnRH1 was higher (p < .01) in Ovsynch Plus group (96.6%) than Ovsynch group (61.5%). However, ovulation rate to GnRH2 was similar (p > .05) between groups (Ovsynch group: 76.9% vs. Ovsynch Plus group: 70.0%). Mean DF diameter (mm) that ovulated to both GnRHs was higher (p < .01) than non‐ovulated counterparts in both groups (Ovsynch group: 10.80 ± 0.27 vs. 8.47 ± 0.53; Ovsynch Plus group: 11.99 ± 0.24 vs. 9.5 ± 0.63). Pregnancy was established in buffaloes which responded to both GnRHs, irrespective of groups, being higher (p = .52) in Ovsynch Plus group (34.5%) than Ovsynch group (23.1%), though non‐significant. In summary, this study showed that eCG inclusion prior to Ovsynch regimen improves ovulatory response in anovular buffaloes during low‐breeding season.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Ascorbic Acid,Catalase and Chlorpromazine Reduce Cryopreservation‐induced Damages to Crossbred Bull Spermatozoaa

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus × Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris–citric acid–fructose–egg yolk–glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate–Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen‐thawed semen. The functional status of the sperm was assessed using hypo‐osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen‐thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post‐thaw semen quality but when combined with either ascorbic acid or catalse, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post‐thaw semen quality in crossbred bulls.