Significance of additives to enhance the acceptance of poison bait in poultry rodents of Haripur, Khyber Pakhtunkhwa, Pakistan.

Rodent infestation on poultry farms incurs heavy economic losses to this industry by causing feed loss and disease introduction. Development and continuous improvement of rodents control techniques are vital to minimize and control the damages caused by rodents. Here, we test the feed preference of rodents for locally available and palatable food grains viz. millet (whole), wheat (cracked) and rice (broken) and taste additives namely whole egg (5%), eggshell (5%), peanut cracked (5%) and yeast (2%) that were offered mixed in millet-wheat (50:50 by wt.) bait. We tested the preferences of different food additives through a process of feed choice mechanism. We applied two different techniques to compare the preference of mixed feed baits, these techniques included no-choice with multiple choice feeding tests and paired choice with multiple choices feeding tests. The results indicated that consumption of bait with added whole egg was significantly higher (p > 0.05). Further test for its effectiveness as a carrier for rodenticides revealed 56%, 82% and 92%, reduction in rodent activities with zinc phosphide (2%), coumatetralyl (0.0375%) and Brodifacoum (0.005%) respectively. Our results point to a need on continuous improvement of feed baits by using different combinations to effectively control the rodent infestation.

Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure.

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.

Explorations of the optimal method for isolating oocytes from zebrafish (Danio rerio) ovary.

Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow-up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen-activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time-saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.

Vitamin C modulates Microcystis aeruginosa death and toxin release by induced Fenton reaction.

Cyanobacterial blooms and their associated toxins pose a great threat to human beings. The situation is even worse for those whose drinking water source is a cyanotoxin-polluted water body. Therefore, efficient and safe treatments urgently need to be developed. The present study verified the application of vitamin C on the inhibition of toxic Microcystis aeruginosa. Our results showed that vitamin C drove the Fenton reaction and significantly sterilized cultures of M. aeruginosa. The algicidal activity of vitamin C was dependent on its involvement in iron (Fe) metabolism. Vitamin C enhanced iron absorption leading to high ferrous ion levels. The ferrous ion increased production of reactive oxygen species (ROS) by Fenton reaction, which play a crucial role in the killing process. Interestingly, vitamin C also dramatically decreased the release of microcystins. This study highlights the possible benefits of using a vitamin C-induced Fenton reaction to remove M. aeruginosa and microcystins from drinking water sources.

Pathological damage and immunomodulatory effects of zebrafish exposed to microcystin-LR.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs), especially microcystin-LR (MC-LR), released during cyanobacterial blooms exert great toxicity on fish and even lead to massive death. The present study mainly investigated the pathological damage and immune response of spleen, gut and gill in zebrafish exposed to MC-LR. Fish were exposed to 0, 1, 5 and 20 µg/L of MC-LR for 30 d. In zebrafish exposed to 5 and 20 µg/L MC-LR, edematous mitochondria, deformation of the nucleus and compaction of chromatin were observed in lymphocyte of spleen; frayed gut villi, exfoliation of epithelial cells and widespread cell lyses were observed in intestines; hyperemia in gill lamellae, epithelial tissue edema and uplift and lamellar fusion were observed in gill. Varied changed gene expression was observed in spleen, intestine and gill of zebrafish. The transcriptional levels of IFN-1 and IL-8 in spleen significantly up-regulated in 20 µg/L group, and the transcription of IL-1ß and TNFα in spleen increased in 1 µg/L MC-LR treated fish. In addition, the mRNA levels of IFN-1, IL-1ß, IL-8, TGF-ß and TNF-α dramatically increased in intestine and gill in all MC-LR treated groups. The present studies indicated that MC-LR exposure caused marked pathological damage, however, fish could adjust actively the expression of innate immune-related genes to resist the tissue damage. Our findings provided strong evidence of the recovery potential of fish exposed to microcystins.

Sex-dependent effects of microcystin-LR on hypothalamic-pituitary-gonad axis and gametogenesis of adult zebrafish.

While microcystins (MCs) have been reported to exert reproductive toxicity on fish with a sex-dependent effect, the underlying mechanism has been rarely investigated. In the present study, zebrafish were exposed to 1, 5 and 20 µg/L MC-LR for 30 d. The gonad-somatic index declined in all treated males. 17ß-estradiol (E2), testosterone (T), 11-keto testosterone (11-KT) and follicle-stimulating hormone (FSH) levels increased in serum from all treated females, while T, FSH and luteinizing hormone (LH) levels changed in all treated males. Histomorphological observation showed that MC-LR exposure evidently retarded oogenesis and spermatogenesis. Transcriptional changes of 22 genes of the hypothalamic-pituitary-gonad (HPG) axis exhibited sex-specific responses, and the relationship between gene transcriptions and gametogenesis was evaluated by principle component analysis (PCA). Major contributors to PC1 (gnrh2, gnrhr3, ar, lhr, hmgra, hmgrb and cyp19a) were positively correlated with the number of post-vitellogenic oocytes, while PC1 (gnrh2, lhß, erß, fshr, cyp11a and 17ßhsd) were positively correlated with the number of spermatozoa. The protein levels of 17ßHSD and CYP19a were affected in both females and males. In conclusion, this study first investigated the sex-dependent effects of microcystins on fish reproduction and revealed some important molecular biomarkers related to gametogenesis in zebrafish suffered from MC-LR.