Purification and partial characterization of a sperm motility-inhibitory protein of ram cauda epididymal plasma.

Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE-sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility-inhibitory factor of sheep I and II (MIFS-I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS-I and II, respectively. Specific activities of the partially purified MIFS-I and II were 563 and 261 U/mg of protein, while the fold-purification of the activity were 5119 and 2373, respectively. Both the proteins were heat-labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS-I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS-I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross-reactivity of MIFS-I with polyclonal anti-human SEMA3D antibody. It was concluded that the MIFS-I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.

A novel action of insulin sensitizing drug as a potential promotor of preovulatory follicles, ovulation rate and prolificacy in sheep.

The effect of the insulin-sensitizing drug metformin on preovulatory follicle (POF) number, ovulation rate, fetal rate and prolificacy was studied in forty-six cyclic Malpura ewes. After estrus synchronization, the ewes were equally divided into two groups (n = 23). The treatment group (MET) received a daily oral dose of metformin at a rate of 500 mg/animal for approximately 12 weeks, spanning five estrous cycles, as against untreated control (CON). All the ewes were bred to proven rams at the end of treatment. Ovarian ultrasound scans were performed at each estrus and day 9 of each cycle to assess the number and diameter of POFs and corpora lutea (CL), respectively. A comprehensive assessment of circulating hormones including, estradiol, progesterone, androstenedione, and insulin as well as metabolic indicators such as glucose, and lipid profile parameters was performed. At the end of treatment on the day of estrus (E5D0), the treatment showed a stimulatory effect on follicular development with a 53.2% (P < 0.001) increase in the number of POFs. It also increased the ovulation rate by 67.4% (P < 0.01), with a higher proportion (χ2df1 = 10.7, P < 0.001) of ewes in the MET group having multiple ovulations compared to the CON group (82.6 vs. 30.4%). With 1.48 ± 0.12 prolificacy rate in MET ewes, the proportion of ewes giving birth to multiple lambs was 2.9-fold higher than in the CON group. Plasma estradiol, insulin, glucose, total cholesterol, and LDL-cholesterol concentrations were lower (P < 0.05) in the MET ewes than in the CON. The results of the present study indicate that metformin can increase the number of POF, ovulation rate, fetal rate and prolificacy in ewes, while reducing the plasma estradiol, insulin, glucose and cholesterol in MET ewes.

Dietary n-3 PUFA augments pre-ovulatory follicle turnover and prolificacy in well-fed ewes.

The present study evaluated the effect of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) on preovulatory follicle (POF) turnover, prolificacy, and endocrine and metabolic milieu in Malpura sheep. Fifty cyclic ewes with 3-3.5 body condition scores on a five-point scale were allocated equally to two groups (n = 25) following estrus synchronization and were supplemented with 0.6 mL/kg body weight of n-3 PUFA-rich fish oil (FO) or palm oil (PO) as control, for 60 d following an acclimatization period of 7 d. All ewes were mated with sexually active rams at the end of the supplementation period. On ultrasonographic ovarian scanning at the last fourth estrus, the mean number of POFs was 77.8% greater (P < 0.01) in FO ewes than in the PO ewes. The proportion of ewes with multiple ovulations two months after the beginning of supplementation was 56% in the FO group as compared to 8% in the PO group. The number of fetuses was 46% higher (P < 0.01) in the FO than in the PO ewes at d 45 of gestation. At lambing, the twinning percent in the FO ewes was three times greater than in the PO ewes (27.3 vs. 9.1%). Plasma cholesterol, estradiol, and insulin concentrations were lower (P < 0.01) in ewes fed with FO than those offered PO group at the end of the feeding period. It was concluded that the dietary supplementation of n-3 PUFA-rich FO in well-fed Malpura ewes improved the number of follicles and ovulation rate which led to an increased prolificacy, accompanied by a reduction of plasma cholesterols, estradiol, and insulin.

De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus.

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

Identification and probiotic potential of lactic acid bacteria from camel milk.

In the present study, a total of 80 presumed lactic acid bacteria (LAB) were isolated from camel milk. Selected LAB were identified as Lactococcus lactis (cam 12), Enterococcus lactis (cam 14) and Lactobacillus plantarum (cam 15) and their potential were tested by tolerance & de-conjugation of bile salts, antimicrobial activity, surface hydrophobicity and adhesion potential) along with this of probiotics were evaluated for curd formation and assessed for sensory properties and syneresis. Selected LABs showed antimicrobial activity against wide range of pathogenic bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus and Escherchiaia. coli). LAB (cam 12, cam 14 and cam15) were highly sceptible to chloramphenicol, vancomycin, and tetracyclin. In vitro adhesion studies with Caco-2 cells demonstrated strong adhesion activity with hydrophobicity (99%) was observed. Acute oral toxicity of E. lactis and L. plantarum showed non-toxic, non-virulent and safe for industrial application. The study provides potential LAB which may act as a substitute of functional food, synthetic feed and industrial curd formulation with in the shortest span (240 min at 28-32 °C).

Mimicking the cauda epididymal plasma-like osmolality in extender improves liquid preservation of ram semen at 3-5°C.

Cauda epididymis in mammals is known to store mature sperm largely in quiescent state for several weeks without significantly affecting fertility. Hence, the aim of this study was to evaluate the effects of mimicking cauda epididymal plasma (CEP)-like conditions in extender on liquid preservation of ram semen at 3-5°C. Four experiments were conducted in this study: (1) evaluation of physicochemical properties of ram CEP, (2) effect of hyperosmotic solution on sperm motility and functional membrane integrity (FMI), and the effects of (3) CEP-like hyperosmolality (390 vs. 360 mOsmol/kg) and (4) pH in extender (pH 6.5 vs. 6.8) on liquid preservation of ram semen. Sperm treatment with hyperosmotic solution (450 mOsmol/kg) resulted in a decline (P < 0.05) in mass motility (3.5 ± 0.2 vs. 4.3 ± 0.2) and FMI (30.4 ± 3.2 vs. 52.1 ± 2.1%) compared to that with isoosmotic solution (360 mOsmol/kg). Overall, sperm viability, acrosomal integrity, and progressive motility were similar (P > 0.05) while straight-line velocity (77.8 ± 3.1 vs. 71.3 ± 2.7µm/s), linearity (47.4 ± 0.4 vs. 39.5 ± 0.9%), straightness (79.7 ± 0.5 vs. 74.0 ± 0.5%) and beat cross frequency (28.6 ± 0.8 vs. 26.0 ± 0.5 Hz) were higher (P < 0.05) and FMI (65.7 ± 1.5 vs. 75.4 ± 1.1%) was lower (P < 0.05) following liquid-preservation in hyperosmotic extender compared to that in isoosmotic extender. Both total motility (83.3 ± 1.8 vs. 75.4 ± 1.5%) and progressive motility (51.7 ± 2.3 vs. 39.5 ± 1.9%) were higher (P < 0.05) at 48 h of storage in hyperosmotic extender compared to the control. Overall, the seminal attributes were similar (P > 0.05) between the two pH's of the extender. In conclusion, semen extender having CEP-like osmolality but not the pH was superior to extenders having conventional osmolality and pH for liquid preservation of ram semen.Abbreviations: AI: artificial insemination; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; CASA: computer-assisted semen analyzer; CEP: cauda epididymal plasma; ELON: elongation; EYTF: egg yolk-Tris-citrate-fructose; FMI: functional membrane integrity; GLM: general linear model; GPC: glycerophosphatidylcholine; HOS: hypoosmotic swelling; LIN: linearity; pHe: external pH; PROG: progressive motility; S.E.M.: standard error of the mean; SLTF: soya lecithin-Tris-fructose extender; SP: seminal plasma; STR: straightness; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight-line velocity; TM: total motility.

Production and preclinical assessment of camelid immunoglobulins against Echis sochureki venom from desert of Rajasthan, India.

Snakebite is a significant cause of death and disability in subsistent farming populations of rural India. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses. Because of complex fiscal reasons, the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region has been questioned time to time. In this study, we report successful immunisation of dromedaries (Camelus dromedarius) against the venom of Indian saw-scaled Viper- Echis carinatus sochureki. This study assessed the specificity and potential of camels immunised with venom of medically most important snake of Western India, the saw-scaled viper (Echis c. sochureki). Using WHO standard pre-clinical in vivo tests the neutralisation of the venom responsible for the lethal, haemorrhagic, coagulant and local necrotizing activities were measured, since these are the most significant effects that characterize envenoming by this species. The anti-venom was found significantly effective in the neutralisation of all these effects tested and thus, revealed further an immunological perspective, that camel IgG anti-venom (monospecific) would be as efficacious as specific equine anti-venoms or even of better choice in treating snake specific envenoming.

Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity.

Camelpox is an important viral disease of camels, which may produce mild skin lesions or severe systemic infections. Camelpox virus (CMLV) isolates retrieved from an incidence of camelpox in camels at Bikaner, India were characterized on the basis of genotype and pathotype. Histopathological examination of the CMLV scab revealed intracytoplasmic-eosinophilic inclusion bodies. The phylogenetic analysis of all eight CMLV isolates for C18L gene nucleotide sequence revealed its clustering with its strains M-96 from Kazakhstan and CMS from Iran. The study will help to understand the transmission chain, pathobiology, and epidemiology of circulating CMLV strains. The full genome sequencing of some of the exemplary samples of CMLV is recommended in order to plan and implement a suitable control strategy.

Camel milk ameliorates hyperglycaemia and oxidative damage in type-1 diabetic experimental rats.

This study was designed to assess anti-diabetic potential of goat, camel, cow and buffalo milk in streptozotocin (STZ) induced type 1 diabetic albino wistar rats. A total of 48 rats were taken for the study where one group was kept as non-diabetic control group (8 rats) while others (40 rats) were made diabetic by STZ (50 mg/kg of body weight) injection. Among diabetic rats, a control group (8 rats) was kept and referred as diabetic control whereas other four groups (8 rats each) of diabetic rats were fed on 50 ml of goat or camel or cow or buffalo milk for 4 weeks. All the rats (non-diabetic and diabetic) were maintained on standard diet for four weeks. STZ administration resulted in enhancement of glucose, total cholesterol, triglyceride, low density lipoprotein, HbA1c and reduction in high density lipoprotein in plasma and lowering of antioxidative enzymes (catalase, glutathione peroxidase and superoxide dismutase) activities in pancreas, kidney, liver and RBCs, coupled with enhanced levels of TBARS and protein carbonyls in pancreas, kidney, liver and plasma. OGTT carried out at the end of 4 week milk feeding indicated that all milks helped in early maintenance of glucose level. All milks reduced atherogenic index. In camel milk fed diabetic group, insulin concentration enhanced to level noted for non-diabetic control while goat, cow and buffalo milk failed to restore insulin level. HbA1c level was also restored only in camel milk fed diabetic group. The level of antioxidative enzymes (catalase, GPx and SOD) in pancreas enhanced in all milk fed groups. Camel milk and to a reasonable extent goat milk reduced formation of TBARS and PCs in tissues and blood. It can be concluded that camel milk ameliorates hyperglycaemia and oxidative damage in type-1 diabetic experimental rats. Further, only camel milk completely ameliorated oxidative damage in pancreas and normalised insulin level.

Camelpox: A brief review on its epidemiology, current status and challenges.

Camelpox caused by a Camelpox virus (CMLV) is a very important host specific viral disease of camel. It is highly contagious in nature and causes serious impact on health even mortality of camels and economic losses to the camel owners. It manifests itself either in the local/mild or generalized/severe form. Various outbreaks of different pathogenicity have been reported from camel dwelling areas of the world. CMLV has been characterized in embryonated chicken eggs with the production of characteristic pock lesions and in various cell lines with the capacity to induce giant cells. Being of Poxviridae family, CMLV employs various strategies to impede host immune system and facilitates its own pathogenesis. Both live and attenuated vaccine has been found effective against CMLV infection. The present review gives a comprehensive overview of camelpox disease with respect to its transmission, epidemiology, virion characteristics, viral life cycle, host interaction and its immune modulation.

Characterization of GM-CSF-inhibitory factor and Uracil DNA glycosylase encoding genes from camel pseudocowpoxvirus.

The present study describes the PCR amplification of GM-CSF-inhibitory factor (GIF) and Uracil DNA glycosylase (UDG) encoding genes of pseudocowpoxvirus (PCPV) from the Indian Dromedaries (Camelus dromedarius) infected with contagious ecthyma using the primers based on the corresponding gene sequences of human PCPV and reindeer PCPV, respectively. The length of GIF gene of PCPV obtained from camel is 795 bp and due to the addition of one cytosine residue at position 374 and one adenine residue at position 516, the open reading frame (ORF) got altered, resulting in the production of truncated polypeptide. The ORF of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. Comparison of amino acid sequence homologies of GIF and UDG of camel PCPV revealed that the camel PCPV is closer to ORFV and PCPV (reference stains of both human and reindeer), respectively.

Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels.

The dsRNA binding protein (RBP) encoding gene of parapoxviruses (PPVs) from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV) from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV) and PCPV (reindeer PCPV and human PCPV) shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV) from different geographical areas of the world shared 69.5-71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV) in comparison to caprine and ovine PPV (ORFV).