A quantitative method for detecting ammonia‐oxidizing bacteria in coastal aquaculture systems

There is a need to quantify autotrophic nitrifiers in coastal aquaculture systems for evolving a bioremediation strategy. Autotrophic nitrifiers are extremely slow‐growing organisms, which cannot be detected by traditional methods as they are notoriously difficult to culture. Molecular techniques based on functional genes could be deployed for the detection of nitrifiers. Ammonia monooxygenase (amoA), that catalyses the oxidation of ammonia to hydroxylamine in the rate‐determining step of nitrification is largely unique to ammonia‐oxidizing bacteria (AOB). In the present study, a quantitative real‐time polymerase chain reaction assay targeting amoA was developed to estimate AOB population size in coastal soil, ammonia‐removing bioaugmentors and the solid matrix. To achieve this objective, different set of primers and a dual labelled probe have been designed for SYBR Green and TaqMan real‐time assays. The abundance of AOB ranged from 10⁴ to 10⁶ order of magnitude in the samples. In the present study, biofilm formation of the consortium of nitrifying bacteria onto bagasse has also been quantified. The results demonstrate that the developed method is a rapid and sensitive tool for the quantitative detection of nitrifying bacteria in aquatic and related environment. This helps in making the bioremediation approach for ammonia removal by immobilization of nitrifying bacteria onto the natural substrate.     

Evaluation of genetic architecture and mutation drift equilibrium of Marathwada buffalo population in Central India

In the present study, microsatellite data on 24 loci were generated and utilized to evaluate the genetic architecture and mutation drift equilibrium of Marathwada buffaloes, a Central Indian population maintained under low input system. Sufficient allelic diversity was observed with a total of 109 alleles across different loci. The genetic diversity analysis of Marathwada buffaloes displayed moderate level of within breed variability in terms of mean number of alleles per locus (4.48) and heterozygosity values (Ho = 0.532, He = 0.624). The studied Indian buffalo population showed considerable heterozygote deficiency (F_IS = 0.138) and deviation from HWE at many investigated loci. Three quantitative tests viz. sign test, standardized difference test and Wilcoxon sign rank test and a qualitative test for mode shift distortion of allelic frequencies were employed to evaluate mutation drift equilibrium under three different models of microsatellite evolution. The population was found to deviate significantly under IAM and TPM, while it was reverse under SMM. The qualitative test for mode shift supported the results under SMM indicating the absence of genetic bottleneck in the recent past in Marathwada buffaloes.     

Cytokine profiles, apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P < 0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.     

Objective Sperm Motion Analysis to Assess Dairy Bull Fertility Using Computer‐Aided System – A Review

Motility is one of the most important characteristics associated with the fertilizing ability of spermatozoa and is an expression of their viability and structural integrity. Computer‐assisted semen analyser (CASA) provides precise and accurate information on different sperm motion characteristics. This article reviews various aspects of computer‐aided motility analysis of bull sperm like sample preparation, standardization of instrument settings, importance of various motility parameters evaluated by the system and its impact on basic functional studies of spermatozoa. It gives special emphasis to various aspects of bull sperm motion analysis especially sub‐populations of spermatozoa, hyper‐activation, motion characteristic in different genetic and age groups, etc. and their utility in predicting the fertility of dairy bulls. The need to fill the gap in research and the necessity of universal standardization of the equipment has been discussed.     

Diversity of denitrifying bacteria in the greenwater system of coastal aquaculture

Greenwater technology is based on integrating finfishes inside pens for zero water exchange system of shrimp aquaculture. Nitrogen transformation could be improved by rearing euryhaline finfishes like grey mullet, milkfish which have a broad diet and tolerate poor water quality. The abundance of four denitrifying functional genes coding for nitrate reductase (narG and napA), nitrite reductase (nirS), nitric oxide reductase (qnorB) and nitrous oxide reductase (nosZ) has been examined in the greenwater system through a metagenomic approach. Phylogeny revealed homology of narG clones with uncultured environmental clones, whereas napA clone sequences were found to have homology with cultured (Stappia aggregata) and uncultured microorganisms. The nirS clones show uniqueness with Marinobacter hydrocarbonoclasticus, Aromatoleum aromaticum, and Ruegeria pomeroyi. The qnorB gene has been reported for the first time from culture systems along the Indian coast and clone exhibited 84–87 % identity with different uncultured bacteria. The nosZ clones are closely affiliated with S. aggregate and alpha bacterium. This study revealed denitrifying diversity from a greenwater system which could eventually be used in planning the future strategy for comprehending nitrogen fluxes, greenhouse gases and their mitigation in coastal aquaculture systems.     

Effect of cryopreservation on apoptotic-like events and its relationship with cryocapacitation of buffalo (Bubalus bubalis) sperm

This study investigated the apoptosis-like events associated with cryopreservation process and their relationship with cryocapacitation in buffalo (Bubalus bubalis) sperm. A total of 49 semen ejaculates from seven bulls were studied for structural changes in sperm following cryopreservation. Apoptotic changes were detected by assays specific for translocation of phosphatidylserine (PS) to the cell surface, alterations in membrane permeability and mitochondrial membrane potential (MMP), and DNA integrity. A significant (p < 0.01) percentage of cryopreserved sperm showed externalization of PS and early apoptotic changes and lowered MMP when compared with the fresh sperm. Freezing and thawing of sperm increased permeability to YOPRO-1, an impermeant fluorescent dye. However, on TUNEL staining, cryopreserved sperm showed no breach in DNA integrity. The sperm capacitation status was evaluated by chlortetracycline (CTC) fluorescence pattern, in which a significant (p < 0.01) percentage of cryopreserved sperm were found to be capacitated. The capacitated sperm (Pattern B) was positively correlated with the aforementioned apoptotic events. In conclusion, cryopreservation process induced early apoptosis-like changes in buffalo sperm, and a close link exists between cryocapacitation and apoptosis during cryopreservation of sperm.     

Detection of occurrence of a recent genetic bottleneck event in Indian hill cattle breed Bargur using microsatellite markers

The effective number of breedable individuals is a crucial determinant for maintaining genetic variability within a population. The population of Bargur, the hill cattle of South India, has gone down drastically by more than 93 % in the past three decades, and only a few thousand animals are available at present. The present study was undertaken to evaluate Bargur cattle for mutation drift equilibrium and to detect the occurrence of recent genetic bottleneck event, if any, in this population. About 50 unrelated animals, true to the type, were sampled and genotyped at 25 microsatellite loci. The mean observed heterozygosity (0.808?±?0.023) was higher than the mean expected heterozygosity (0.762?±?0.008) with 15 out of 25 microsatellite loci exhibiting heterozygosity excess when assumed under Hardy–Weinberg equilibrium. To evaluate Bargur cattle for mutation drift equilibrium, three tests were performed under three different mutation models, viz., infinite allele model (IAM), stepwise mutation model (SMM) and two-phase model (TPM). The observed gene diversity (H _e) and expected equilibrium gene diversity (H _eq) were estimated under different models of microsatellite evolution. All the 25 loci were found to exhibit gene diversity excess under IAM and TPM, while 22 loci were having gene diversity excess under SMM. All the three statistical tests, viz., sign test, standardized differences test, and Wilcoxon sign rank test, revealed significant (P?<?0.01) deviation of Bargur cattle population from mutation-drift equilibrium under all the three models of mutation. Furthermore, the qualitative test of allele frequency distribution in Bargur cattle population revealed a strong mode shift from the normal L-shaped form suggesting that the population had experienced genetic bottleneck in the recent past. The occurrence of genetic bottleneck might have led to the loss of several rare alleles in the population, which point towards the need for efforts to conserve this important cattle germplasm. The present study is the first report in demonstrating the genetic basis of demographic bottleneck in an Indian cattle population.     

Microsatellite-based genetic monitoring to detect cryptic demographic bottleneck in Indian riverine buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The present study was undertaken to evaluate different Indian riverine buffalo breeds (Bubalus bubalis) for mutation drift equilibrium and occurrence of any recent genetic bottleneck. A total of 330 animals from seven different breeds were analyzed with a set of 24 heterologous microsatellite markers. Three different tests revealed significant heterozygosity excess in all the seven buffalo breeds studied when assumed under infinite alleles model of microsatellite evolution, while it was the reverse with no significant heterozygosity excess when assumed under conservative stepwise mutation model. Under the two-phase model, all the buffalo breeds except Mehsana were found to be in mutation drift equilibrium when evaluated by all the three statistical methods. Standardized differences test and Wilcoxon signed-rank test revealed significant heterozygosity excess suggesting possible cryptic demographic bottleneck in Mehsana buffaloes of Western India.     

Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker-based analysis

The present study aimed at assessing the status of the Chilika buffalo population of eastern India employing cytogenetic and molecular markers. The Chilika buffaloes investigated cytogenetically possess a somatic chromosome count of 50, identical to that of typical riverine buffaloes. Various diversity estimates, viz. observed number of alleles (4.68), effective number of alleles (2.79), and observed (0.487) and expected (0.602) heterozygosity across 25 heterologous microsatellite markers indicated the presence of a moderate level of genetic diversity in Chilika buffaloes, comparable with three other prominent Indian riverine buffalo breeds (Murrah, Nagpuri and Toda) included in this study. Across the four buffalo populations, mean estimates of F -statistics from Jackknifing over loci were significantly different from zero (p < 0.05), with F _IT (total inbreeding estimate) = 0.315 ± 0.038, F _IS (within-population inbreeding estimate) = 0.178 ± 0.038, and F _ST (population differentiation) = 0.166 ± 0.025. Inter-breed analysis reflected Chilika buffaloes to be genetically close to Nagpuri followed by Murrah and Toda buffaloes. Factorial correspondence analysis (FCA) revealed low breed-specific clustering of Chilika and Nagpuri buffaloes. Additionally, the neighbour-joining tree structure of mitochondrial DNA D-loop haplotypes indicated clear grouping of the Chilika haplotypes with the riverine buffalo. Thus the cytogenetic, microsatellite and mitochondrial data analysed in the present study classify Chilika buffalo of eastern India to be of the riverine type and not swamp-type buffalo.     

Seven novel single nucleotide polymorphisms identified within river buffalo (Bubalus bubalis) lactoferrin gene

The present study aimed at identifying single-nucleotide polymorphic (SNP) sites in different coding and non-coding regions of lactoferrin gene in Indian riverine buffaloes. A total of 102 animals from six different river buffalo breeds were screened at six bubaline lactoferrin gene loci. Single-strand conformation polymorphism (SSCP) analysis revealed monomorphic patterns at three loci LtfE2, LtfE11, and LtfE14 while a total of eight distinct patterns were observed in the other three loci viz. LtfE5, LtfE10, and LtfE16 which correspond to respective exons and their flanking regions. Sequence analysis of different SSCP variants revealed the presence of two SNP sites within the coding (exon 16) region and five SNP sites in flanking non-coding regions (intron 4 and intron 9). Both SNPs within exon 16 were found to be synonymous. The SNPs and haplotypes identified in the present study could serve as potential markers for association with susceptibility/resistance to mastitis in buffaloes.