Toxicological effects and recovery of the corneal epithelium in Cyprinus carpio communis Linn. exposed to monocrotophos: an scanning electron microscope study

This study was conducted based on the evidence of fish habitats in North India being affected by organophosphate pesticides draining from agricultural fields into bodies of water, especially during the rainy season. Various tissues of fish such as scales, gills ovaries, kidney, and liver have been studied from the toxicological point of view, but the toxicological effects of aquatic pollutants on fish cornea have not been investigated to date. We conducted comparative toxicological studies on the cornea of Cyprinus carpio communis using two sublethal (0.038 and 0.126 ppm) concentrations of monocrotophos pesticide for 30 days. Corneas from all the groups were evaluated by a scanning electron microscope. The fish exposed to the monocrotophos pesticide developed corneal necrosis due to the formation of crystalloid‐like structures, thinning and shrinkage of microridges on the corneal epithelium. After 30 days, fish from the monocrotophos‐treated tank were transferred to normal environmental conditions. After 60 days under natural condition, epithelial cells did not fully recover. In conclusion, exposure to monocrotophos induces irreversible changes in the cornea of C. carpio communis. As fish and mammalian visual systems share many similarities, the reported finding may offer useful insights for further toxicological and ophthalmological studies in humans.     

An assessment of benzimidazole resistance against caprine nematodes in Central India

Current status of resistance to benzimidazole (BZ) group of anthelmintic drugs against caprine nematodes in Central India at Amanala goat farm, Jabalpur, Madhya Pradesh (M. P.), was systematically investigated using faecal egg count reduction (FECR) test and egg hatch test (EHT). Besides, allele-specific PCR (AS-PCR) was deployed to ascertain the susceptible genotype (alleles) especially of the Haemonchus contortus. Randomly selected 30 goats, irrespective of age and sex, were divided into three groups of 10 each, to serve as treated and untreated controls. It was ensured that the animals were not administered with an anthelmintic drug for the past 3 months prior to undertaking the study, and faecal egg counts were estimated. FECR test evidenced fenbendazole resistance by partial elimination (24.90%) copro-egg counts in the treated group of animals vis-à-vis controls with a lower confidence interval of ?26%. Further, EHT revealed ED-50 value of 0.335 μg of thiabendazole/ml, confirming benzimidazole resistance in the animals of that farm. AS-PCR showed that 62% of H. contortus larvae were homozygous resistant (rr), 24% heterozygous (rS) and 14% homozygous susceptible (SS). The genotypic frequencies of three genotypes (rr, rS and SS) were significantly (P < 0.01) different. The prevalence of benzimidazole resistance allele (r) was also significantly (P < 0.01) higher (74%) as compared to susceptible allele (S) (26%). The resistance to benzimidazole has been discussed while emphasizing improved managemental practices designed to reduce exposure of the goat population to parasites, minimize frequency of anthelmintic use at optimum dose and rotational use of different chemical groups of medicines with different mode of action, so as to overcome and combat the upcoming problem in the field.     

Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.     

Effect of immunization with bovine luteinizing hormone receptor on ovarian function in cats

OBJECTIVE: To determine the effect of immunization with bovine luteinizing hormone receptor (LH-R) on ovarian function of cats. ANIMALS: 9 adult female domestic cats. PROCEDURE: 7 cats were immunized with 0.5 mg of LH-R encapsulated in a silastic subdermal implant (3 x 10 mm); 2 served as control cats. Receptors had 80% specific binding to 125I-human chorionic gonadotropin with a binding capacity of 2,682 pM/mg. Cats received booster injections of LH-R. Cats were induced to ovulate with luteinizing hormone (LH) releasing hormone on day 345. Samples of venous blood and vaginal cells were collected through day 395. Observation of estrus behavior continued until day 516. Serum concentrations of estradiol, progesterone, thyroid gland hormones, LH, and LH-R antibody were determined. RESULTS: LH-R antibody was detected in the sera of immunized cats within 21 days after implantation. Detection of LH-R antibody was associated with suppression of serum progesterone to < or = 0.5 ng/mL during the study period, compared with concentrations of 5 to 10 ng/mL in control cats. Immunized cats did not display signs of estrus. Release of LH after administration of LH-releasing hormone indicated an intact hypothalamic-pituitary axis but poor corpus luteum function. Serum estradiol concentrations remained between 30 to 40 pg/mL in immunized and control cats. With the decrease antibody titers, hormone concentrations returned to a pattern consistent with that during fertility. CONCLUSIONS AND CLINICAL RELEVANCE: Active immunization with LH-R suppressed corpus luteum function in cats. The effect was reversible. An LH-R-based antifertility vaccine may have clinical application in other vertebrates.     

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.     

Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration

1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.     

Fertility following CIDR based synchronization regimens in anoestrous Nili-Ravi buffaloes

The objective of this study was to compare oestrus expression and fertility rate in used and new controlled internal drug releasing (CIDR) device treated anoestrous buffaloes. Furthermore, to determine the timing of ovulation, and fertility rate in estradiol benzoate (EB) and GnRH-administered CIDR-treated anoestrous Nili-Ravi buffaloes. In experiment 1, buffaloes received either a used CIDR (UCIDR, n = 35) or a new CIDR (NCIDR, n = 36) for 7 day and PGF2α on day 6. Oestrous expression was similar (p > 0.05) between UCIDR (88.5%) and NCIDR (96.6%) buffaloes. The pregnancy rate did not differ (p > 0.05) because of treatment (37.1% in UCIDR vs 36.6% in NCIDR). In experiment 2, buffaloes (n = 55) received CIDR device for 7 days and PGF2α, on day 6 and randomly assigned into three treatment groups: (i) CIDR-EB (n = 17) received EB on day 8, (ii) CIDR-GnRH (n = 18) received GnRH on day 9 and (iii) control (n = 20) received no further treatment. Mean interval from CIDR removal to ovulation in CIDR-EB, CIDR-GnRH and CIDR group were 61.3 ± 0.8, 64.9 ± 1.8 and 65.1 ± 16.7 h, respectively. However, the buffaloes in the CIDR-EB and CIDR-GnRH group had lesser variability in the timing of ovulation compared to control. The pregnancy rate of both CIDR-EB group (58%) and CIDR-GnRH group (61%) were tended to be higher (p < 0.1) than control (30%). In conclusion, compared to NCIDR devices, previously UCIDR devices are equally effective to induce oestrus in anoestrous buffaloes resulting optimal pregnancy rate. Administration of EB and GnRH after CIDR removal results in tighter synchrony (less variability) and improved fertility in anoestrous buffaloes. CIDR based synchronization regimens have great potential in fertility improvement in anoestrous buffaloes.