Toll like receptor 4 (TLR4) gene polymorphism and its association with somatic cell score and milk production traits in Indian dromedary camels.

Our study aimed to explore the genetic variation in the Toll-like receptor 4 (TLR4) gene and establish its association with somatic cell score (SCS) and milk production traits in four Indian camel breeds namely Bikaneri, Kachchhi, Jaisalmeri and Mewari. TLR4 gene fragment of 573 bp spanning 5' UTR, exon-1 and partial intron-1 region was amplified and genotyped using the PCR-sequence based typing method. Only one SNP located at position C472T was identified. Genotyping revealed two alleles (C and T) and three genotypes: CC, CT and TT. The genotype frequencies for CC, CT and TT were 0.116, 0.326 and 0.558 and allele frequencies for C and T alleles were 0.279 and 0.721, respectively. Association study inferred that the effect of genotype on SCS, lactation yield (LY) and peak yield (PY) was non-significant however heterozygote (CT) genotypes recorded lower SCS and higher LY and PY. It can be concluded that the TLR4 gene possesses limited genetic variation, depicting polymorphism at a single locus in Indian camel breeds with a predominance of the TT genotype. The association study indicated that heterozygote animals possess better udder health and production performance, the statistical significance of which needs to be established using a large data set.

The first molecular characterization of Sarcocystis cameli in the Indian dromedary camels (Camelus dromedarius).

In the present study, tissue samples (tongue, esophagus and heart) were investigated from dromedary camels of India for identification and characterization of Sarcocystis spp. using histopathology, PCR and gene sequencing. Genomic DNA extracted from these tissue samples was used for PCR amplification of the cytochrome c oxidase subunit I gene (cox1) of Sarcocystis spp. and the partial sequence of small subunit ribosomal RNA (18S rRNA) gene of the S. cameli. The PCR products were purified, sequenced and analyzed using bioinformatics tools. Based on phylogenetic analysis of the cox1 gene, the sequences of the present study clustered with those of S. cameli, hosted by dromedary camels of Iraq and a close association was observed with S. masoni hosted by dogs and alpacas of China. Until now, there are no 18S rRNA sequences of S. cameli available in GenBank and this is the first study recording 18S rRNA sequences of S. cameli which were grouped with S. masoni from alpaca of China and guanaco and llama of Argentina in phylogenetic analysis. These findings could be useful for further studies on the characterization through molecular epidemiology, genetic diversity and host specificity of S. cameli.

Udder, teat, and milk vein measurements of Indian dromedary camel and its relationship with milkability traits.

The economic viability of the camel in the current scenario can be ensured by improving its dairy potential. The study on the udder and teat characteristics of dromedary camel and understanding its relationship with milk yield and milkability can be of great value in establishing camel as a dairy animal. The present study was conducted on 45 lactating she-camels of four Indian camel breeds, viz., Bikaneri, Jaisalmeri, Kachchhi, and Mewari, stationed at ICAR-NRCC Bikaner, Rajasthan, India. The udder, teat, and milk vein measurements traits, factors affecting these traits and their relationship with milkability traits, were studied in hand-milked Indian dromedary camel. The means ± S.E. of teat lengths (TL), namely, left fore (LF), left rear (LR), right fore (RF), and right rear (RR), were observed as 52.21 ± 1.66, 58.52 ± 2.11, 50.13 ± 1.74, and 54.37 ± 1.82 mm, respectively. The means ± S.E. of teat diameter (TD), namely, left fore, left rear, right fore, and right rear teat diameters, were observed as 42.44 ± 1.60, 46.01 ± 1.68, 39.29 ± 1.31, and 45.20 ± 1.56 mm, respectively. The means ± S.E for udder depth, udder length, udder height from the ground, milk vein diameter, and milk vein length were observed as 25.44 ± 0.42, 37.29 ± 0.80, 114.80 ± 0.80, 2.02 ± 0.08, and 88.70 ± 0.96 cm, respectively. Udder and milk vein measurements did not differ significantly between breeds. Kachchhi breed has largest teat length and diameter. The breed differences were significant (p ≤ 0.05) for TL-LF, TL-RF, and TD-RR only. The effect of parity was non-significant on udder, teat, and milk vein measurement traits except TD-RR (p ≤ 0.05); however, second parity animals had higher values for all the studied traits except udder height from ground. Positive and highly significant (p ≤ 0.01) correlation of milk yield was observed with the majority of udder, teat, and milk vein measurements, milking time, and milk flow rate, while a negative correlation was found with udder height from ground and milk let-down time. It can be concluded that udder characteristics are influenced by various genetic and non-genetic factors and its relationship with milk yield and milkability can be used for selection and dairy management purposes.

Case Report: Congenital tuberculosis in an aborted dromedary camel fetus.

Tuberculosis (TB) is a serious public health problem worldwide, especially in tropical developing countries. Nevertheless, reports on congenital TB in humans and animals are extremely rare. In this study, abortion was reported in an 8-year-old she-camel at the 9th month of gestation. The she-camel appeared healthy in clinical examination, had a good body condition score, normal appetite, and had no signs of respiratory disease and fever. The expelled placenta was dark red-colored, thickened, and edematous with multifocal to coalescing ecchymotic hemorrhages on the allantoic surface. The striking finding was multiple, white-yellow, solid nodular lesions in the fetal lung, the pleura, and the liver. On histopathology, typical granulomatous lesions were detected in the lung and the liver characterized by caseous necrosis surrounded by lymphocyte and macrophage infiltration and concentric layers of fibrosis. The Ziehl-Neelsen staining detected scarce acid-fast bacilli in lung and liver tissues. The DNA extracted from tubercular lesions from the lung and liver showed amplification of the IS6110 region of the Mycobacterium tuberculosis complex by PCR. The sequencing and phylogenetic analysis revealed a close association of these sequences with Mycobacterium tuberculosis. The she-camel was detected positive for a single intradermal tuberculin test performed 24 h after abortion. This is the first report on congenital TB caused by M. tuberculosis in a dromedary camel fetus with a possible vertical transmission.

Isolation, purification and characterization of a novel esterase from camel rumen metagenome.

Bacterial esterases are gaining the importance in pharmaceuticals and agrochemical industries due to their excellent biocatalytic properties and a wide range of applications. In the present study, a novel gene encoding an esterase (designated as Est-CR) was identified from shotgun metagenomic sequencing data of camel rumen (Camelus dromedarius) liquor. The open reading frame consisted of 1,224bp, which showed 84.03% sequence identity to Bacteroidales bacterium, corresponding to a protein of 407 amino acids and has a catalytic domain belonging to an esterase. Est-CR belonged to family V with GLSMG domain. The purified enzyme with a molecular mass of 62.64 kDa was checked on SDS-PAGE, and its expression was confirmed by western blotting. The enzyme was active and stable over a broad range of temperature (35-65 °C), displayed the maximum activity at 50 °C and pH 7.0. Individually all metal ions inhibited the enzyme activity, while in combination, K2+, Ca2+, Mg2+ and Mn2+ metal ions enhanced the enzyme activity. The detergents strongly inhibited the activity, while EDTA (10 mM) increased the activity of the Est-CR enzyme. The enzyme showed specificity to short-chain substrates and displayed an optimum activity against butyrate ester. This novel enzyme might serve as a promising candidate to meet some harsh industrial processes enzymatic needs.

Characterizing rumen microbiota and CAZyme profile of Indian dromedary camel (Camelus dromedarius) in response to different roughages.

In dromedary camels, which are pseudo-ruminants, rumen or C1 section of stomach is the main compartment involved in fiber degradation, as in true ruminants. However, as camels are adapted to the harsh and scarce grazing conditions of desert, their ruminal microbiota makes an interesting target of study. The present study was undertaken to generate the rumen microbial profile of Indian camel using 16S rRNA amplicon and shotgun metagenomics. The camels were fed three diets differing in the source of roughage. The comparative metagenomic analysis revealed greater proportions of significant differences between two fractions of rumen content followed by diet associated differences. Significant differences were also observed in the rumen microbiota collected at different time-points of the feeding trial. However, fraction related differences were more highlighted as compared to diet dependent changes in microbial profile from shotgun metagenomics data. Further, 16 genera were identified as part of the core rumen microbiome of Indian camels. Moreover, glycoside hydrolases were observed to be the most abundant among all Carbohydrate-Active enzymes and were dominated by GH2, GH3, GH13 and GH43. In all, this study describes the camel rumen microbiota under different dietary conditions with focus on taxonomic, functional, and Carbohydrate-Active enzymes profiles.

Differential expression of BMP/SMAD signaling and ovarian-associated genes in the granulosa cells of FecB introgressed GMM sheep.

The present study was conducted to analyze the mRNA expression of the BMP/SMAD signaling and steroidogenesis associated genes in the granulosa cells (GCs) of newly developed Booroola homozygous carrier GMM (FecBBB ) and non-carrier GMM (FecB++ ) ewes through qRT-PCR. Results showed that the expression of BMP2 (P < 0.05) and BMP6 (P < 0.01) was significantly higher in the GCs of the homozygous carrier GMM (FecBBB ) than the non-carrier GMM (FecB++ ) ewes, while the expression of BMP4 was significantly higher (P < 0.001) in the GCs of non-carrier GMM (FecB++ ) than the homozygous carrier GMM (FecBBB ). In comparison, the expression of TGFßR1, BMPR1A, BMPR1B, and BMPRII was not significantly different between GCs of the homozygous carrier GMM (FecBBB ) and non-carrier GMM (FecB++ ) ewes. Similarly, the expression of SMAD1, SMAD2, SMAD3, SMAD4, and SMAD5 was not significantly different between GCs of homozygous carrier GMM (FecBBB ) and non-carrier GMM (FecB++ ). Further, expression of the INHIBIN, P4R, CYP11A1, and 3ßHSD1 genes were not significantly different among the GCs of homozygous carrier GMM (FecBBB ) and non-carrier GMM (FecB++ ), while the expression of StAR was significantly higher (P < 0.01) in the GCs of homozygous carrier GMM (FecBBB ) than that of GCs of non-carrier GMM (FecB++ ) ewes. It is concluded that the FecB mutation significantly up-regulates the expression of BMP2, BMP6, and StAR genes and down-regulate the expression of BMP2 in granulosa cells of newly developed GMM ewes. ABBREVIATIONS: BMP: Bone morphogenetic protein; TGFß: Transforming growth factor-beta; SMAD: Fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic; GCs: Granulosa cells; GMM: Garole x Malpura x Malpura; FecB: Booroola fecundity; BMPR: Bone morphogenetic protein receptor; CYP11A1: Cytochrome P450 family 11 subfamily A member 1; StAR (Steroidogenic acute regulatory protein); 3ßHSD1: 3 Beta-hydroxysteroid dehydrogenase; IGF: Insulin-like growth factor; ActR2: Activin receptor 2; ACVR1: Activin A receptor, type I; ACVR2: Activin A receptor, type II; ACVRL1: Activin A receptor like type 1; ACVR1B: Activin A receptor type 1B; ACVR1C: Activin A receptor type 1C; RFLP-PCR: Restriction fragment length polymorphism-polymerase chain reaction; qRT-PCR: Quantitative real-time PCR; ANOVA: Analysis of variance; P4R: Progesterone receptor: AMH: Anti mullerian hormone; RT-PCR: Reverse transcriptase-polymerase chain reaction; ER: Estrogen receptor; FSHR: Follicle stimulating hormone receptor.

Use of peripheral blood for production of buffalo (Bubalus bubalis) embryos by handmade cloning.

Buffalo embryos were produced by handmade cloning using peripheral blood-derived lymphocytes as donor cells. Although the blastocyst rate was lower (P < 0.01) for lymphocyte- than control skin fibroblast-derived embryos (6.6 ± 0.84% vs. 31.15 ± 2.97%), the total cell number (152.6 ± 23.06 vs. 160.1 ± 13.25) and apoptotic index (6.54 ± 0.95 vs. 8.45 ± 1.32) were similar. The global level of H3K9ac was higher (P < 0.05) in lymphocyte- than that in skin-derived blastocysts; whereas in IVF blastocysts, the level was not significantly different from the two cloned groups. The level of H3K27me3 was similar among the three groups. The expression level of DNMT1, DNMT3a, HDAC1, and IGF-1R was higher (P < 0.01) in lymphocytes than that in skin fibroblasts. The expression level of CDX2 was higher (P < 0.05) than that of DNMT3a, IGF-1R, OCT4, and NANOG was lower (P < 0.05) in lymphocyte-derived than in IVF blastocysts; that of DNMT1 and HDAC1 was similar in the two groups. The expression level of all these genes, except that of NANOG, was lower (P < 0.05) in lymphocyte- than in skin fibroblast-derived blastocysts. It is concluded that, peripheral blood-derived lymphocytes can be used for producing handmade cloning embryos in bubaline buffaloes.

Handmade Cloned Buffalo (Bubalus bubalis) Embryos Produced from Somatic Cells Isolated from Milk and Ear Skin Differ in Their Developmental Competence, Epigenetic Status, and Gene Expression.

We compared the cloning efficiency of buffalo embryos produced by handmade cloning (HMC) using ear skin- and milk-derived donor cells. The blastocyst rate was lower (p < 0.05) for milk-derived than that for skin-derived embryos, whereas the total cell number and apoptotic index were similar. The global level of H3K9ac was higher (p < 0.05) in skin- than in milk-derived cells, whereas the level of H3K27me3 was similar in the two groups. The global level of H3K9ac was similar between milk-derived and in vitro-fertilized (IVF) blastocysts, which was higher (p < 0.05) than that in skin-derived blastocysts. The level of H3K27me3 was similar among the three groups. The expression level of IGF-1R and G6PD was higher (p < 0.05) in skin- than in milk-derived cells, whereas DNMT1, DNMT3a, and HDAC1 expression level was similar. In the blastocysts, the expression level of DNMT1, HDAC1, OCT4, and CDX2 was higher (p < 0.05) in skin-derived than that in IVF blastocysts. The expression level of DNMT3a and IGF-1R, was in the order (p < 0.05) skin-derived and IVF > milk-derived blastocysts and that of NANOG was (p < 0.05) IVF-> milk-derived > skin-derived blastocysts. The expression level of all these genes, except NANOG, was lower (p < 0.05) in milk- than in skin-derived or IVF blastocysts. In conclusion, milk-derived cells can be used for producing HMC embryos of quality similar to that of skin-derived embryos, although with a lower blastocyst rate.

Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.

This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine- and tail skin-derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic index was lower (p<0.05) than that of control blastocysts. The global level of histone H3 acetylated at lysine 9 (H3K9ac) was similar in the three types of donor cells and in urine- and tail skin-derived HMC blastocysts and in vitro-fertilized (IVF) blastocysts (controls). The global level of histone H3 trimethylated at lysine 27 (H3K27me3) in the cells was in the order (p<0.05) urine≥tail skin>ear skin-derived cells, whereas in blastocysts, it was higher (p<0.05) in urine- and tail skin-derived HMC blastocysts than that in IVF blastocysts. The expression level of CASPASE3, CASPASE9, P53, DNMT1, DNMT3a, OCT4, and NANOG, which was similar in HMC blastocysts of three the groups, was lower (p<0.05) than that in IVF blastocysts, whereas that of HDAC1 was similar among the four groups. Following transfer of urine-derived embryos (n=10) to five recipients (two embryos/recipient), one of the recipients delivered a normal calf that is now 5 weeks old.