Overexpression of KAT8 induces a failure in early embryonic development in mice.

Embryo quality is strongly associated with subsequent embryonic developmental efficiency. However, the detailed function of lysine acetyltransferase 8 (KAT8) during early embryonic development in mice remains elusive. In this study, we reported that KAT8 played a pivotal role in the first cleavage of mouse embryos. Immunostaining results revealed that KAT8 predominantly accumulated in the nucleus throughout the entire embryonic developmental process. Kat8 overexpression (Kat8-OE) was correlated with early developmental potential of embryos to the blastocyst stage. We also found that Kat8-OE embryos showed spindle-assembly defects and chromosomal misalignment, and that Kat8-OE in embryos led to increased levels of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX by affecting the expression of critical genes related to mitochondrial respiratory chain and antioxidation pathways. Subsequently, cellular apoptosis was activated as confirmed by TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) assay. Furthermore, we revealed that KAT8 was related to regulating the acetylation status of H4K16 in mouse embryos, and Kat8-OE induced the hyperacetylation of H4K16, which might be a key factor for the defective spindle/chromosome apparatus. Collectively, our data suggest that KAT8 constitutes an important regulator of spindle assembly and redox homeostasis during early embryonic development in mice.

The TUNEL assay underestimates the incidence of DNA damage in pig sperm due to chromatin condensation.

Inconsistencies in the relationship between sperm DNA fragmentation and reproductive outcomes as well as the low incidence in farm animals raise concerns on its actual value as a sperm quality parameter. Previous studies suggested that the different sensitivity of techniques evaluating DNA fragmentation could explain variations in the correlation with reproductive outcomes. While the TUNEL assay is one of the most standardized methods to detect DNA damage and cell death, the steric impediment for the terminal nucleotidyl transferase enzyme to access the highly condensed sperm nucleus may decrease the ability of this test to detect internal DNA breaks. In the present study, we sought to determine whether increasing chromatin decondensation makes the TUNEL assay more sensitive to detect DNA damage in pig sperm. We compared three chromatin decondensation treatments (2 mM DTT for 45 min; 5 mM DTT for 8 min and further 45 min; and 5 mM DTT+ 1 M NaCl for 8 min) through the Chromomycin A3 test (CMA3). While incubation with DTT increased the percentages of sperm with decondensed chromatin, regardless of concentration and time of incubation (P < 0.05), the extent of that decondensation was higher when 5 mM DTT was combined with 1 M NaCl. In addition, the TUNEL assay detected a higher number of DNA breaks in sperm with decondensed chromatin (1.89% ± 1.63% vs 8.74% ± 6.05%; P = 0.003). This study shows, for the first time, that previous chromatin decondensation increases the sensitivity of the TUNEL assay to detect DNA damage in pig sperm. These findings also support that larger chromatin decondensation is needed in order for DNA damage to be evaluated properly in species containing protamine P1 only.

Effect of methionine deficiency on the apoptosis and cell cycle of kidney in broilers.

Abundant evidence now supports the idea that methionine deficiency has negative effects on chicken healthy especially in the aspect of cell cycle regulation and apoptosis. But, lacking of knowledge is the evaluation on metabolic organs. To test the effect of methionine deficiency on the kidney, we assessed the apoptosis and the cell cycle of kidney induced by methionine deficiency by the methods of TdT-mediated dUTP Nick-End Labeling (TUNEL), flow cytometry (FCM), quantitative real-time PCR (qRT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) of chickens for 42 days of age. Our results showed that the number of the apoptotic cells was increased (p < .05 or p < .01), while bcl-2 mRNA expression levels were decreased (p < .05 or p < .01) and bax and caspase-3 mRNA expression levels were higher (p < .01) in methionine deficiency group. Furthermore, the cell cycle results showed a time-dependent increase in G2M phase cells and a corresponding decrease in cells at G2M and S stages, the mRNA expression of p53 and p21 was increased (P < .05 or P < .01) and cyclin B and PCNA was significantly lower (P < .05 or P < .01) in the methionine deficiency group than that of the control group. These findings suggested that methionine deficiency could induce renal apoptosis and cell cycle arrest.

The shell gland in laying and natural moulting commercial egg-type chickens: A histomorphological and ultrastructural study.

The purpose of the present study was to determine the histological and ultrastructural changes in the luminal epithelium of the shell gland associated with natural moulting. Samples of the shell gland from laying (32 weeks old) and moulting (75 weeks old) hens were studied using histological, histochemical and electron microscopic techniques. In addition, TUNEL was used to demonstrate the distribution of apoptotic cells in the luminal epithelium of the shell gland. Autophagy, characterized by the presence of autophagosomes and autolysosomes, was evident in the early stages of degeneration in non-ciliated, ciliated and mitochondrial cells. The intermediate and advanced stages of regression in non-ciliated as well as mitochondrial cells occurred via apoptosis, while both apoptotic and necrotic ciliated cells were observed during the later stages of degeneration. The results of the present study suggest that a synergy of autophagy, apoptosis and necrosis is involved in the involution of the shell gland during natural moulting.

Resveratrol promotes in vitro activation of ovine primordial follicles by reducing DNA damage and enhancing granulosa cell proliferation via phosphatidylinositol 3-kinase pathway.

We aimed to study the effects of resveratrol on the morphology, DNA fragmentation, follicular activation and cell proliferation after in vitro culture of ovine ovarian tissue, and to verify if PI3K pathway is involved in resveratrol action in the sheep ovary. Ovaries were collected and divided into fragments. One fragment was fixed for histology (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+ ) alone or with resveratrol (2, 10 or 30 µM). After culture, ovarian tissue was destined to morphological analysis. TUNEL and proliferating cell nuclear antigen (PCNA) analyses were performed in the fresh control, α-MEM+ and 2 µM resveratrol. Inhibition of PI3K activity was performed through pre-treatment with LY294002. The percentage of normal follicles was similar between α-MEM+ and 2 µM resveratrol, and higher than those in other resveratrol treatments. An increase in follicular activation was observed in all treatments compared to fresh control. DNA fragmentation decreased in tissues cultured in 2 µM resveratrol compared to α-MEM+ . Moreover, PCNA-positive cells were higher in 2 µM resveratrol than in α-MEM+ . LY294002 inhibited follicular activation stimulated by α-MEM+ and 2 µM resveratrol. In conclusion, 2 µM resveratrol promotes primordial follicle activation compared to the fresh control by reducing DNA fragmentation and stimulating granulosa cell proliferation through activation of the PI3K pathway.

The toxic effect of aflatoxin B1 on early porcine embryonic development.

Aflatoxin B1 (AFB1) is a type of mycotoxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus. AFB1 is considered as the most toxic mycotoxin owing to its toxic effect on health. In the present study, the toxic effect of AFB1 on early porcine embryonic development and its possible mechanism were investigated. Blastocyst formation was impaired with treatment of 1 nM AFB1 compared with control, 0.01, 0.1 group (40.13 ±â€¯2.10%, 28.21 ±â€¯1.62%, 32.34 ±â€¯2.07% vs 19.01 ±â€¯1.06%). Further study showed that the presence of AFB1 induced the generation of reactive oxygen species (ROS). And excessive ROS caused DNA damage which confirmed by the comet assay. Additionally, AFB1 also disrupted the DNA damage repair through the regulation of 53BP1. The AFB1 treatment significantly increased the γH2A foci and decreased the 53BP1 foci. TUNEL assay confirmed the generation of apoptosis, further resulting in the occurrence of autophagy. Moreover, AFB1 significantly increased the expression of pro-apoptosis genes Bax and Casp3 and reduced the expression of anti-apoptotic genes Bcl2 and Bcl-xl. In addition, the AFB1 also significantly increased the expression of autophagy related genes Lc3 and Beclin1. The presence of AFB1 significantly impaired the cell proliferation, a parameter of blastocyst quality for outgrowth. These results showed that the presence of AFB1 impaired porcine early embryonic development through oxidative stress, as well as DNA damage and repair, apoptosis, autophagy.

Effect of methionine deficiency on oxidative stress and apoptosis in the small intestine of broilers.

The aim of this study was to investigate the effects of methionine (Met) deficiency on antioxidant functions (in the duodenal, jejunal and ileal mucosa) and apoptosis in the duodenum, jejunum and ileum of broiler chickens. A total of 120 one-day-old Cobb broilers were divided into two groups and fed a Metdeficient diet and a control diet, respectively, for six weeks. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), the ability to inhibit hydroxyl radicals, and glutathione (GSH) content were significantly decreased in the Met-deficient group compared to the control. In contrast, malondialdehyde (MDA) content was significantly higher in the Met-deficient group. As measured by terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'- triphosphate dUTP nick end-labelling (TUNEL) and flow cytometry (FCM), the percentages of apoptotic cells were significantly increased. In conclusion, dietary Met deficiency can cause oxidative stress and then induce increased apoptosis in the intestine. Oxidative stress contributes to intestinal apoptosis. This results in the impairment of local intestinal mucosal immunity due to oxidative stress and apoptosis in the small intestine. The results of this study provide new experimental evidence for understanding the negative effects of Met deficiency on mucosal immunity or the functions of other immune tissues.

Cryopreservation (-20 °C) of canine corneoscleral tissue: histological, microbiological, and ultrastructural study.

OBJECTIVE: To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) canine corneoscleral tissue (<1 year) and to compare it with long-term cryopreserved (LTC) tissue (>6 years). ANIMALS STUDIED: Thirty-six healthy canine globes. PROCEDURE: After a decontamination protocol, globes were enucleated and stored at -20 °C. Corneoscleral tissue was evaluated at different periods: <1 year (20 eyes) and >6 years (12 eyes). Four eyes were used as controls. Microbiologic study included direct (blood, McConkey and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy (TEM). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was also used for keratocyte characterization. RESULTS: Corneal microbial growth was observed in 25% of the direct STC cultures, and in 47.4% and 16.7% of the enriched STC and LTC cultures, respectively. Scleral STC direct cultures were 30% positive, while enrichment cultures were positive in 66.7% and 16.7% of the STC and LTC, respectively (P = 0.011). Cryopreservation artifacts were higher in LTC tissues (P < 0.001). Apoptotic keratocytes were predominant by TEM and TUNEL, in both STC and LTC. Minimal structural differences were detected in collagen organization between STC and LTC. CONCLUSIONS: Cryopreservation of canine corneoscleral tissue seems to reduce bacterial contamination over time. Apoptosis is the main way of death of cryopreserved canine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 8 years without structural or microbiological impediment.

Re-oxygenation after anoxia induces brain cell death and memory loss in the anoxia-tolerant crucian carp.

Crucian carp (Carassius carassius) survive without oxygen for several months, but it is unknown whether they are able to protect themselves from cell death normally caused by the absence, and particularly return, of oxygen. Here, we quantified cell death in brain tissue from crucian carp exposed to anoxia and re-oxygenation using the terminal deoxy-nucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and cell proliferation by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) as well as PCNA mRNA expression. We also measured mRNA and protein expression of the apoptosis executer protease caspase 3, in laboratory fish exposed to anoxia and re-oxygenation and fish exposed to seasonal anoxia and re-oxygenation in their natural habitat over the year. Finally, a behavioural experiment was used to assess the ability to learn and remember how to navigate in a maze to find food, before and after exposure to anoxia and re-oxygenation. The number of TUNEL-positive cells in the telencephalon increased after 1 day of re-oxygenation following 7 days of anoxia, indicating increased cell death. However, there were no consistent changes in whole-brain expression of caspase 3 in either laboratory-exposed or naturally exposed fish, indicating that cell death might occur via caspase-independent pathways or necrosis. Re-oxygenated crucian carp appeared to have lost the memory of how to navigate in a maze (learnt prior to anoxia exposure), while the ability to learn remained intact. PCNA mRNA was elevated after re-oxygenation, indicating increased neurogenesis. We conclude that anoxia tolerance involves not only protection from damage but also repair after re-oxygenation.

Proliferation-apoptosis balance in Staphylococcus aureus chronically infected bovine mammary glands during involution.

The objective of this study was to determine whether Staphylococcus aureus chronic intramammary infection (IMI) influences expression of proteins related to regulation of proliferation and apoptosis processes and proliferation/apoptosis index during active involution in bovine mammary gland. Twenty-one Holstein non-pregnant cows in late lactation either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. Cows were slaughtered at 7, 14 and 21 d after cessation of milking and samples for immunohistochemical analysis were taken. Protein expression of Bcl-2, Bax, Fas and active caspase-3 in mammary tissue was significantly affected by chronic S. aureus IMI, all showing increased immunoexpression in S. aureus-infected quarters at all involution stages. The percentage of apoptotic cells was increased by IMI in both mammary parenchyma and stroma, and the percentage of parenchymal and stromal cell proliferation was also increased. The proliferation/apoptosis ratio was significantly increased by IMI only in stromal cells. This imbalance to favour proliferation in S. aureus-infected mammary quarters could be one of the underlying causes that induce aberrant involution with permanence of nonsecretory tissue and increase of stromal components.

Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines.

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

Relationship between Eimeria tenella development and host cell apoptosis in chickens.

Coccidiosis causes considerable economic losses in the poultry industry. At present, the pathology of coccidiosis is preventable with anticoccidials and vaccination, although at considerable cost to the international poultry industry. The purpose of the present study was to elucidate the relationship between Eimeria tenella development and host cell apoptosis in chickens, which provides a theoretical basis for further study of the injury mechanism of E. tenella and the prevention and treatment of coccidiosis. Cecal epithelial cells from chick embryo were used as host cells in vitro. In addition, flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling, and histopathological assays were used to detect the dynamic changes in E. tenella infection rates, DNA injury rates, and apoptosis rates in groups treated with and without the caspase-9 inhibitor Z-LEHD-FMK. Following E. tenella infection, we demonstrated that untreated cells had less apoptosis at 4 h and, inversely, more apoptosis at 24 to 120 h compared with control cells. Furthermore, after the application of Z-LEHD-FMK, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays, and translation of phosphatidyl serines to the host cell plasma membrane surface, the treated group chick embryo cecal epithelial cells exhibited decreased apoptosis and DNA injuries (P<0.01) at 24 to 120 h. However, light microscopy showed that E. tenella infection rates of treated cells were higher (P<0.01) than untreated cells during the whole experimental period. Together, these observations suggest that E. tenella can protect host cells from apoptosis at early stages of development but can promote apoptosis during the middle to late stages. In addition, the inhibition of host cell apoptosis can be beneficial to the intracellular growth and development of E. tenella.

Interactions of highly and low virulent Flavobacterium columnare isolates with gill tissue in carp and rainbow trout.

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.

Effect of methotrexate on cerebellar development in infant rats.

Six-day-old rats were treated intraperitoneal injections with methotrexate 1 mg/kg, and the cerebellum was examined. Both the length and width of the vermis decreased in the methotrexate-treated group instead of the control from 4 day after treatment (DAT) onward. A significant reduction in the width of the external granular layer was detected on 2 and 3 DAT in the methotrexate group. By 4 DAT, the width of the external granular layer of the methotrexate group was indistinguishable from the control, and by 8 DAT, it was greater than that of the control. The molecular layer of methotrexate group on 8 and 15 DAT was thinner than that of the control. On 1 DAT, in the methotrexate group, there were many TUNEL and cleaved caspase-3-positive granular cells throughout the external granular layer, and they decreased time-dependently. On 1 DAT, in the methotrexate group, phospho-histone H3-positive cells in the external granular layer were fewer than in the control and tended to increase on 2-4 DAT. The p21-positive-rate of the external granule cells in the MTX group was higher than in the control on 1-4 DAT. These results suggested that methotrexate exposure on postnatal day 6 induces a delay, slowing in the migration of external granular cells to the inner granular layer, attributed to decrease or inhibition in the production of external granular cells that had arisen from apoptosis and the decrease in cell proliferative activity, resulting in cerebellar hypoplasia.

Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification.

This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-µL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%).

Fasciola hepatica: a comparative survey of adult fluke resistance to triclabendazole, nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and fluke histology.

In order to investigate the incidence and distribution of adult fluke resistance to the fasciolicide tricalbendazole (TCBZ) amongst populations of Fasciola hepatica in sheep flocks in Northern Ireland (NI), individual rectal faeces samples were collected from 3 groups of 20 sheep, before (pre-dose), and 21 days after (post-dose) treatment of the animals with TCBZ, nitroxynil or closantel, on each of 13 well-managed sheep farms distributed across the province. The efficacy of each flukicide was determined for each farm, using faecal egg count reduction (FECRT) and F. hepatica coproantigen ELISA testing. In certain flocks, 2 sheep with high pre-dose faecal egg counts (FEC) were killed 3 days and 21 days respectively after TCBZ treatment, and the histology of the fluke reproductive organs was compared with that of flukes from untreated sheep, and from sheep treated with nitroxynil or closantel 2 days prior to death, using haematoxylin and eosin (H&E) staining and an in situ hybridisation method (TdT-mediated dUDP nick end labelling [TUNEL]) to demonstrate apoptosis. Results from FECRT revealed that in all flocks with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis, and this finding was generally supported by the results of the coproantigen reduction test (CRT). The histology of reproductive organs of flukes from TCBZ-treated sheep in these flocks was normal, when compared with untreated flukes, and this, together with the FECRT and CRT findings, indicated a likely diagnosis of TCBZ resistance in all the flocks with a high fluke burden. In contrast, nitroxynil and closantel were found to be fully effective against TCBZ-resistant flukes in each of the flocks bearing a high chronic fluke burden. All of the flocks with a high fluke burden and TCBZ resistance were managed on lowland in the South and East of NI. Upland flocks, in the North and West, had low fluke burdens, or were clear of infection; and FECs were too low to allow valid resistance testing. The study highlights the high level of penetration of TCBZ resistance throughout F. hepatica populations in areas of intensively managed sheep production with a high level of fluke challenge. Further, it emphasises the importance of pre-emptive chemotherapeutic action against chronic fasciolosis, using flukicides effective against the egg-producing adult flukes to minimise pasture contamination for the next season's lamb crop. This study also exemplifies the use of several complementary methods (FECRT; CRT; fluke histology; comparative anthelmintic efficacy testing) for confirmation of a diagnosis of fluke drug resistance.

Toxicity to the hematopoietic and lymphoid organs of piglets treated with a therapeutic dose of florfenicol.

Florfenicol (FLO) is a broad-spectrum antibacterial agent for treatment of bacteriosis of piglets in veterinary practice. To study the toxicity to the hematopoietic and lymphoid organs of piglets treated with a therapeutic dose of FLO, 20 healthy weaned piglets were selected and randomly divided into two groups. Piglets in the FLO group were fed with fodder supplemented with 30mg/kg BW of FLO twice a day for 10 days. Blood samples were drawn at four time points: 1 day before FLO administration and 1, 7, and 14 days post-withdrawal. Three or four piglets were euthanized at each time point post-withdrawal and tissue samples (bone marrow, thymus and spleen) were collected for fixation and cryostorage. The levels of classical swine fever virus (CSFV) antibody against the vaccine, the concentrations of Hsp70 and IL-6 in serum and Hsp70 in tissues, and the mRNA expression levels of B-cell lymphoma 2 (bcl-2) and tumor suppressor p53 were detected, the hematology of the piglets were analyzed, and the histopathology and the status of apoptosis of the hematopoietic and lymphoid organs was examined. The results showed changes in several indicators in the FLO group 1 day post-withdrawal: the concentration of red blood cells (RBCs) was decreased, and that of platelets (PLTs) was significantly lower (p<0.05); the volumes of RBC and PLT were increased; the sum of blood lymphocytes was statistically decreased (p<0.05); the concentration of IL-6 was significantly increased (p<0.05); the concentrations of Hsp70 in serum and tissues were increased; obvious atrophy of the hematopoietic cell lines and partial replacement by fat cells were observed in bone marrow; thymus and spleen tissues showed lower concentrations and sparser arrangement of lymphocytes in the thymic medulla and white pulp of the spleen respectively; and the mRNA expression levels of bcl-2 in the three tissues were up-regulated, while that of p53 was down-regulated. With time after cessation of FLO administration, the indicators of the FLO group gradually returned to close to that of the control group and the histological lesions of the tissues gradually recovered, and the differences in the densities of lymphocytes and cell arrangements in the tissues between two groups gradually decreased. In conclusion, a therapeutic dose of FLO induces temporary toxicity in the hematopoietic and lymphoid organs of piglets to some extent, and influences hemopoiesis and immune function. These effects gradually decrease after cessation of FLO administration.

Protective roles of sodium selenite against aflatoxin B1-induced apoptosis of jejunum in broilers.

The effects of aflatoxin B1 (AFB1) exposure and sodium selenite supplementation on cell apoptosis of jejunum in broilers were studied. A total of 240 one-day-old male AA broilers were randomly assigned four dietary treatments containing 0 mg/kg of AFB1 (control), 0.3 mg/kg AFB1 (AFB1), 0.4 mg/kg supplement Se (+ Se) and 0.3 mg/kg AFB1 + 0.4 mg/kg supplement Se (AFB1 + Se), respectively. Compared with the control broilers, the number of apoptotic cells, the expression of Bax and Caspase-3 mRNA were significantly increased, while the expression of Bcl-2 mRNA and the Bcl-2/Bax ratio were significantly decreased in AFB1 broilers. The number of apoptotic cells and the expression of Caspase-3 mRNA in AFB1 + Se broilers were significantly higher than those in the control broilers, but significantly lower than those in AFB1 broilers. There were no significant changes in the expression of Bax mRNA between AFB1 + Se and control broilers; the expression of Bcl-2 mRNA and the Bcl-2/Bax ratio in AFB1 + Se broilers were significantly lower than those in the control broilers, but significantly higher than those in AFB1 broilers. In conclusion, 0.3 mg/kg AFB1 in the diet can increase cell apoptosis, decrease Bcl-2 mRNA expression, and increase of Bax and Caspase-3 mRNA expression in broiler's jejunum. However, supplementation of dietary sodium selenite at the concentration of 0.4 mg/kg Se may ameliorate AFB1-induced apoptosis by increasing Bcl-2 mRNA expression, and decreasing Bax and Caspase-3 mRNA expression.

Immunohistochemical localization of inflammatory cells and cell cycle proteins in the gills of Loma salmonae infected rainbow trout (Oncorhynchus mykiss).

Microsporidial gill diseases particularly those caused by Loma salmonae incur significant economic losses to the salmonid aquaculture industry. The gill responses to infection include the formation of xenomas and the acute hyperplastic inflammatory responses once the xenomas rupture releasing infective spores. The aim of this work was to characterize the inflammatory responses of the gill to both the presence of the xenomas as well as the hyperplasia associated with L. salmonae infection in the rainbow trout gill following an experimental infection using immunohistochemistry. Hyperplastic lesions demonstrated numerous cells expressing PCNA as well as an apparent increased expression of caspase-3 and number of apoptotic cells (TUNEL positive cells). There was an expression of TNFα in individual cells within the gill and increased expression of a myeloid cell line antigen indicating the presence of granulocyte infiltration of both the hyperplastic lesions as well as the xenomas. Similar immune-reactivity was seen in gill EGCs. Hyperplastic gill lesions showed a marked infiltration of CD8+ cells and expression of MHC class I antigens. These findings suggest that L. salmonae xenomas may be subject to infiltration by the host immune cells as well as the mounting or a marked cellular cytotoxic immunoreaction in the resultant hyperplasia following xenoma rupture and spore release.

The low expression of Dmrt7 is associated with spermatogenic arrest in cattle-yak.

Dmrt7 is a member of the DM domain family of genes. Dmrt7 deficiency is also a strong candidate as a cause for male cattle-yak infertility, as it is regarded as essential for male spermatogenesis, between the pachynema and diplonema stages. In our study, the coding region sequence of yak and cattle-yak Dmrt7 was cloned by molecular cloning techniques, and the sequence, conserved domains, functional sites, and secondary and tertiary structures of the Dmrt7-encoded protein were predicted and analyzed using bioinformatics methods. The coding region sequences of the Dmrt7 gene, encoding 370 amino acids, were consistent in yak and cattle-yak. The protein encoded by yak and cattle-yak Dmrt7 contains a DM domain. We detected Dmrt7 mRNA expression in testis, but not in any other tissue. Dmrt7 mRNA and protein expression was significantly higher in testis of cattle and yak than that in cattle-yak (p < 0.01). Histological analysis indicated that seminiferous tubules in male cattle-yak were highly vacuolated and contained primarily Sertoli cells and spermatogonia, while those of cattle and yak contained abundant primary spermatocytes. Male cattle-yak testis contained a significantly larger number of apoptotic cells than those in cattle and yak assessed by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) analysis. The accumulation of SCP3-positive spermatocytes indicated the arrest of spermatogenesis at the pachynema stage in the cattle-yak. These results suggest low levels of Dmrt7 expression lead to male sterility in cattle-yak. The molecular function of Dmrt7 and the regulation of its expression warrant need to be examined in future studies.