In vitro production of haploid germ cells from murine spermatogonial stem cells using a two-dimensional cell culture system.

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3, Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol.

Dietary curcumin supplementation effects on blood immunological profile and liver enzymatic activity of laying hens after exposure to high temperature conditions.

Various environmental factors affect livestock production but heat stress is a major challenge in the poultry farming. Poultry exposes to high temperature alters blood immunological parameters and liver enzymatic function which in turn, suppress the immunity and disease resistance of chickens. Thus, the purpose of present study was to explore the effect of dietary curcumin supplementation on blood immunological biomarker and liver enzymatic activity of laying hens under heat stress conditions. Experimental groups contained two control groups (normal temperature control (NC) and heat stress control (HC) and 3 heat stress curcumin treatment groups (HT100, HT200 and HT300). Hens in HC group with basal diet and heat stress curcumin treatment groups were exposed 6 h/day heat stress (32 ± 1 °C) from 10:00 a.m. to 16:00 p.m. for 9 week. The results of present study showed that heat stressed curcumin treatment group had improved liver weight, WBC values and immunoglobulin level as compared to untreated HC and NC groups. The available results also indicated that laying hens supplemented with curcumin under high temperature conditions had reduced H/L ratio, serum corticosterone levels, inflammatory cytokines response and liver enzymatic activity (ALT) which enhanced the immunity of laying hens under hot climatic conditions. Therefore, it is concluded that curcumin has ability to combat harsh environmental conditions which can be used as anti-inflammatory and immune booster feed additive in the poultry nutrition.

Effect of dietary curcumin on the antioxidant status of laying hens under high- temperature condition.

Heat stress induces oxidative stress, and reduces antioxidant defenses of birds, which may affect poultry-production performance. Dietary antioxidants may protect against heat stress. We evaluated the effect of increasing concentrations of dietary curcumin on antioxidant parameters of hens under high-temperature conditions for nine weeks. Roman laying hens (n = 336, 22 weeks old, 1420 g weight) were divided into three treatment groups. The first group served as a thermo-neutral control (kept at 25 ±â€¯1 °C). The second group was exposed to high temperatures (32 ±â€¯1 °C, 6 h/day), and fed a basal diet. The third group was further divided into five groups, and all were exposed to high temperatures (32 ±â€¯1 °C, 6 h/day) and provided a basal diet supplemented with 100, 150, 200, 250, 300 mg/kg curcumin (H1, H2, H3, H4, H5). All treatments included four replicates of 12 hens. Total superoxide dismutase (SOD) activity was significantly higher in H2 and H3 groups, and total antioxidant capacity (T-AOC) was higher in H2, H3, and H5 groups. Catalase (CAT) and glutathione peroxidase (GSH-Px) activities were significantly higher in the H3 group. Malondialdehyde concentrations were lower in curcumin supplemented hens compared to control groups hens. Hens in all curcumin treatment groups had slightly (but non-significantly) higher activities of CAT, SOD, GSH-Px, and T-AOC in liver, heart, and lung tissues, compared to heat stressed control group. It is concluded that dietary curcumin given to laying hens under heat stress may enhance their antioxidant status, and ameliorate stressful environmental conditions.

Chicken toll-like receptors and their significance in immune response and disease resistance.

Infectious diseases are a major challenge for the poultry industry that causes widespread production losses. Thus, management and control of poultry health and diseases are essential for the viability of the industry. Toll-like receptors are best characterized as membrane-bound receptors that perform a central role in immune homeostasis and disease resistance by recognition of pathogen-associated molecular patterns. In response to pathogen recognition, TLRs initiate both innate and adaptive immune responses which may help to develop immunomodulatory therapeutics for TLR associated diseases. Vaccination produces specific immunity in the animal's body towards pathogens. However, due to certain disadvantages of vaccines, (inactivation of attenuated pathogens into the virulent strains and weak immunogenicity of inactivated vaccines) there is a crucial need to develop the safe and effective therapeutic intervention. TLR ligands have been classified as a potential adjuvant against the infectious diseases in farm animals. TLR adjuvants induce both specific and nonspecific immune responses in chickens to combat several bacterial, viral and parasitic diseases. Therefore, the aim of this review was to explore the chicken TLR4 and their role in immune responses and disease resistance to develop disease resistance poultry breeds in future.

Effect of curcumin supplementation on TLR4 mediated non-specific immune responses in liver of laying hens under high-temperature conditions.

The liver performs a significant role in innate and adaptive immunity. Heat stress causes oxidative stress in liver tissues and reduces the immune responses of laying hens which can cause several diseases affecting poultry-production performance. Hepatic inflammation is a common trigger of liver disease, which is reflected by hepatic tissue damage leading to fibrogenesis and hepatocellular carcinoma. Dietary manipulation of curcumin has been proposed to ameliorate the immune status of chickens under heat stress. Thus, this study aimed to investigate the effect of curcumin supplementation on TLR4 mediated non-specific immune response in liver of laying hens under high-temperature conditions. Experimental groups contained two controls groups (high temperature and thermo-neutral control (HC and NC) fed basal diet) and three high-temperature curcumin treatments groups (HT100, HT200 and HT300). Laying hens in HC and HT groups exposed 6 h/day heat stress (32 ±â€¯1 °C). The results of present study showed that heat stress curcumin treatment group had reduced inflammatory responses (IL-6, IL-1ß, TNF-α) as compared to HC and NC group. Pathological lesions and DNA damage of immune tissues were decreased in heat stress curcumin supplementation as compared to HC and NC group. Furthermore, PCNA, TLR4 and its downstream gene expression as well as protein expression (TLR4, NF-κB and PCNA) were significantly down regulated in heat stress curcumin supplemented group as compared to HC and NC group. Therefore, it is concluded that heat stressed hens supplemented with dietary curcumin enhance the immunity of laying hens and combat stressful environmental conditions.

Pancreatic ß-cell replacement: advances in protocols used for differentiation of pancreatic progenitors to ß-like cells.

Insulin-producing cells derived from in vitro differentiation of stem cells and non-stem cells by using different factors can spare the need for genetic manipulation and provide a cure for diabetes. In this context, pancreatic progenitors differentiating to ß-like cells garner increasing attention as ß-cell replacement source. This kind of cell therapy has the potential to cure diabetes, but is still on its way of being clinically useful. The primary restriction for in vitro production of mature and functional ß-cells is developing a physiologically relevant in vitro culture system which can mimic in vivo pathways of islet development. In order to achieve this target, different approaches have been attempted for the differentiation of pancreatic stem/progenitor cells to ß-like cells. Here, we will review some of the state-of-the-art protocols for the differentiation of pancreatic progenitors and differentiated pancreatic cells into ß-like cells with a focus on pancreatic duct cells.

Cryptic footprints of rare earth elements on natural resources and living organisms.

BACKGROUND: Rare earth elements (REEs) are gaining attention due to rapid rise of modern industries and technological developments in their usage and residual fingerprinting. Cryptic entry of REEs in the natural resources and environment is significant; therefore, life on earth is prone to their nasty effects. Scientific sectors have expressed concerns over the entry of REEs into food chains, which ultimately influences their intake and metabolism in the living organisms. OBJECTIVES: Extensive scientific collections and intensive look in to the latest explorations agglomerated in this document aim to depict the distribution of REEs in soil, sediments, surface waters and groundwater possibly around the globe. Furthermore, it draws attention towards potential risks of intensive industrialization and modern agriculture to the exposure of REEs, and their effects on living organisms. It also draws links of REEs usage and their footprints in natural resources with the major food chains involving plants, animals and humans. METHODS: Scientific literature preferably spanning over the last five years was obtained online from the MEDLINE and other sources publishing the latest studies on REEs distribution, properties, usage, cycling and intrusion in the environment and food-chains. Distribution of REEs in agricultural soils, sediments, surface and ground water was drawn on the global map, together with transport pathways of REEs and their cycling in the natural resources. RESULTS: Fourteen REEs (Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sm, Tb, Th and Yb) were plighted in this study. Wide range of their concentrations has been detected in agricultural soils (<15.9-249.1 µg g-1) and in groundwater (<3.1-146.2 µg L-1) at various sites worldwide. They have strong tendency to accumulate in the human body, and thus associated with kidney stones. The REEs could also perturb the animal physiology, especially affecting the reproductive development in both terrestrial and aquatic animals. In plants, REEs might affect the germination, root and shoot development and flowering at concentration ranging from 0.4 to 150 mg kg-1. CONCLUSIONS: This review article precisely narrates the current status, sources, and potential effects of REEs on plants, animals, humans health. There are also a few examples where REEs have been used to benefit human health. However, still there is scarce information about threshold levels of REEs in the soil, aquatic, and terrestrial resources as well as living entities. Therefore, an aggressive effort is required for global action to generate more data on REEs. This implies we prescribe an urgent need for inter-disciplinary studies about REEs in order to identify their toxic effects on both ecosystems and organisms.

The effect of ginger powder and Chinese herbal medicine on production performance, serum metabolites and antioxidant status of laying hens under heat-stress condition.

This study was done to evaluate the effects of Chinese herbal medicine (CHM) and ginger powder on layers-production performance, serum metabolites and antioxidant status under heat stress condition. Two hundred and fifty Lohmann layers were randomly divided into 5 different, including two controls and three experimental groups (H1, H2, and H3). Control groups were fed the basic diet without supplementation, while, the feed of three experimental groups was supplemented with 3.32 g CHM, 10 g ginger powder, and 10 g ginger powder+ 3.32 g CHM per kg of diet, respectively. Results showed that feed consumption and production rate were decreased in the HC group, while, feed intake and production significantly improved when birds were given supplemented diet. The production rate and feed intake of the H3 group were even significantly higher than the NC group. The birds that received supplemented diet had higher glucose level compared to HC. Triglycerides and serum cholesterol had significantly decreased level in supplemented groups compared to HC. Fungal catalase (CAT) level was significantly improved in H2 and H3 groups. Nitric oxide (NO), glutathione peroxidase (GSH-PX) and Total Protein (T-AOC) level were significantly improved in supplemented groups. Findings showed that ginger powder and CHM could be a viable alternative to the synthetic antibiotic in poultry feed.

Curcumin inhibits heat-induced oxidative stress by activating the MAPK-Nrf2 / ARE signaling pathway in chicken fibroblasts cells.

Curcumin is a natural phenolic component of yellow curry spice, exhibits antioxidant and anti-inflammatory properties. In this study we investigated whether curcumin suppresses heat-induced apoptosis in chicken embryonic fibroblast cells (CEF) and the underlying mechanism. CEF cells line was divided into 6 groups (4 repetitions per group) including normal temperature group (NC), high temperature control group (H) and 4 experimental groups (H1(5 µmol/L), H2(10 µmol/L), H3(20 µmol/L) and H4(40 µmol/L)). Control groups were cultured in basic medium without Curcumin, while, the experimental groups were supplemented with 5, 10, 20 and 40 µmol/L, respectively. The experimental groups and H control group were cultured at 43 ℃ (95% air/5% CO2), whereas NC group cells were cultured at 37 °C. After 6, 12 and 24 h of culture, cells were collected for viability, proliferation, apoptosis, antioxidant status and gene expression analysis. Results showed that heat stress trigged the ROS production and induced the apoptosis, leading to decrease the cell viability and proliferation. The enzymatic activities of antioxidants (SOD, CAT, and GPX) were down-regulated. The expression of antioxidant enzyme (CAT, SOD1, SOD2, GSTO1, GSTT1 and GSTA3) and MAPK-Nrf2 pathway genes (Nrf2, Jnk, Erk and P38) were down-regulated under heats stress condition. While, the Curcumin treated groups had decreased ROS and MDA content. Down-regulation of the activity and expression of antioxidant enzyme induced by heat were also reversed by curcumin. Furthermore the up-regulation in expression of Nrf2, Jnk, Erk and P38 in supplemented groups revealed the involvement of MAPK-Nrf2 signaling pathway to alleviate oxidative stress induced by heat stress. This study demonstrates that curcumin has the ability to ease the oxidative damage through activating the MAPK-Nrf2 signaling pathway in CEF cells.

The optimized condition for the isolation and in vitro propagation of mouse spermatogonial stem cells.

INTRODUCTION: The ability for isolation and in vitro propagation of spermatogonial stem cells (SSCs) offer a base for studies on spermatogenesis, and also contribute to the development of new methods for the preservation of livestock and animal genetic modification. The aim of this study was to find the optimal isolation and culture condition for efficient propagation of SSCs. METHODS: Three different isolation methods (mechanical, one-, and two-step enzymatic digestion) were compared to find the optimal isolation method. To find the best culture conditions for in vitro propagation, isolated SSCs were cultured for 7 days in three different culture conditions supplemented with 10% FBS, 0.25% BSA, and 10% KSR, respectively. RESULTS: The result showed that two-step enzymatic digestion produced a significant high fraction of live cells compared the other two. Non-adhering cells collected after 48 hr and cultured in BSA- and KSR-supplemented medium had a significantly high number of SSCs clump formation compared to FBS-supplemented group. The expression of CD9 confirmed that cell clumps were SSCs clumps. Spermatogonial stem cells cultured in BSA-supplemented medium were positive for NGN3 and PLZF expressions, whereas negative for Stra8 (a meiotic-specific gene) expression, suggesting that most of the cells were undifferentiated SSCs in BSA culture system. In contrast, in FBS- and KSR-supplemented groups, the SSCs were positive forNGN3, PLZF, and Stra8. CONCLUSION: These data revealed that two-step enzymatic digestion is the best method for the isolation, and 0.25% BSA-supplemented culture condition is effective for optimal in vitro propagation of SSCs.

Heat stress in poultry production: Mitigation strategies to overcome the future challenges facing the global poultry industry.

Worldwide, the effect of climatic variations has become a great challenge in poultry production. As global climate is changing, it alters the environmental temperatures, precipitation patterns and atmospheric carbon dioxide. Poultry farming mainly depends on climatic conditions such as temperature and humidity. Several factors can be involved but heat stress is one of most important environmental factor influencing a wide range of chickens performances including reduced feed intake which, in turn, affects growth rate, body weight, meat quality, egg quality, egg production, semen quality and fertility; these negative influences result in great economic losses. Heat stress associated food safety issues have gained special importance due to public awareness and an abundance of available scientific information. Environmental modifications (early heat conditioning, open sheds and cooling systems) and nutritional strategies (early feed restriction, electrolyte, vitamin and mineral balance) cannot satisfy the special needs of stressed poultry. Therefore, there exists a crucial need to explore effective strategies including genetic markers to enhance thermo-tolerance and productivity of poultry birds in hot regions of the world.

Progress and future prospect of in vitro spermatogenesis.

Infertility has become a major health issue in the world. It affects the social life of couples and of all infertility cases; approximately 40-50% is due to "male factor" infertility. Male infertility could be due to genetic factors, environment or due to gonadotoxic treatment. Developments in reproductive biotechnology have made it possible to rescue fertility and uphold biological fatherhood. In vitro production of haploid male germ cell is a powerful tool, not only for the treatment of infertility including oligozoospermic or azoospermic patient, but also for the fertility preservation in pre-pubertal boys whose gonadal function is threatened by gonadotoxic therapies. Genomic editing of in-vitro cultured germ cells could also potentially cure flaws in spermatogenesis due to genomic mutation. Furthermore, this ex-vivo maturation technique with genomic editing may be used to prevent paternal transmission of genomic diseases. Here, we summarize the historical progress of in vitro spermatogenesis research by using organ and cell culture techniques and the future clinical application of in vitro spermatogenesis.