Investigation of a multicomponent mycotoxin detoxifying agent for aflatoxin B1 and ochratoxin A-induced blood profile in broiler chickens.

Background and Aim: Mycotoxins such as aflatoxin B1 and ochratoxin A (OTA) are secondary metabolites in molds that grow in raw materials or commercial feed. This interaction has a synergistic effect on mortality, body weight, feed intake, embryo abnormalities, egg production, and lymphoid organ atrophy. This study was conducted to determine the effect of a mycotoxin detoxifier on the blood profile of broilers that were given feed contaminated with mycotoxin, such as the number of heterophils, lymphocytes, monocytes, mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC). Materials and Methods: A total of 20 day-old chicks (DOC) of Cobb broilers were given four treatments with five replicates. The number of chickens used in this research was determined using statistical calculations, and the data obtained was homogeneous so that the population was represented. Treatments included negative control with basal feed (C-), positive control with mycotoxins contamination (C+), treatment 1: Mycotoxins contamination and mycotoxin detoxification 1.1 g/kg (T1), and treatment 2: Mycotoxins contamination and mycotoxin detoxification 1.6 g/kg (T2). Mycotoxin contamination comprised 0.1 mg/kg aflatoxin B1 and 0.1 mg/kg OTA. The treatment period for chickens was 28 days, from 8 to 35 days. A battery cage was used in this study. Chickens were kept in a closed, ventilated room and the room temperature (27°C) was monitored during the treatment period. Results: Based on the results of statistical data processing, a significant difference (p < 0.05) was observed between chickens fed mycotoxin-contaminated feed (C+) and chickens not fed mycotoxin-contaminated feed (C-) and chickens given 1.6 g/kg mycotoxin detoxification (T2). Mycotoxin detoxification at a dose of 1.6 g/kg had a significant (p < 0.05) effect on the heterophil, lymphocyte, and heterophil lymphocyte ratio, leukocyte, erythrocyte, and hemoglobin levels of the blood broiler in this experiment. On other parameters such as monocytes, MCH, and MCHC, treatment 2 at dose 1.6 g/kg was the best treatment, although there was no significant effect with C- and T1. Conclusion: The administration of mycotoxin detoxifiers at a dose of 1.6 g/kg increased the number of heterophils and the ratio of heterophil lymphocytes, leukocytes, erythrocytes, and hemoglobin in broilers fed mycotoxin-contaminated feed.

Role of apoptotic inhibitors, viability, and differentiation in low oxygen tension of mesenchymal stem cells cultured in a rat model of ovarian failure.

Background: Stem cell therapy shows applications potential for malnutrition-induced ovarian failure in rat models. However, it is ineffective because of the lack of viability and differentiation of transplanted stem cells, resulting in low adaptation and survival rates. We aimed to determine whether stem cells cultured under low oxygen (O 2) tension improves the adaptability and viability of stem cells, as well as ovarian failure. Methods: After four days of culturing mesenchymal stem cells (MSCs) in 21% oxygen (normoxia) as the T2 group and 1% oxygen (low O 2 or hypoxia) as the T1 group, 200 million bone marrow-derived MSCs per rat were transplanted into female rats with ovarian failure (15 rats per treatment group). A total of 15 fertile and 15 infertile rats were categorized as the C+ and C- groups, respectively. Results: The slight increase in cells expressing HSP70 (C+, T2, T1, and C- groups were 0.5 a±0.53, 1.7 a±0.82, 6.2 b±1.5, and 9.6 c±1.3, respectively), decrease in cells expressing caspase-3 as an apoptotic inhibitor (C+, T2, T1, and C- groups were 0.2 a±0.42, 0.6 a±0.52, 4.8 b±1.03, and 7.3 c±1.42, respectively), and increase in cells expressing VEGF-1 (C+, T2, T1, and C- groups were 10.8 c±1.55, 8.7 b±0.48, 0.4 a±0.52, and 0.2 a±0.42, respectively) and GDF-9 (C+, T2, T1, and C- groups were 5.8 c±1.47, 4.6 b±0.97, 0.5 a±0.53, and 0.3 a±0.48, respectively) were used as markers for viability and differentiation in ovarian tissue, indicating that MSCs cultured under low O 2 tension were more effective than those cultured under normoxic conditions as a treatment for female rats with ovarian failure. Furthermore, infertile female rats treated with MSCs cultivated under low O 2 tension had an enhanced ovarian tissue shape, as indicated by the increasing Graafian follicle count (C+, T2, T1, and C- groups were 8.9 c±0.74, 4.5 b±0.71, 0.5 a±0.53, and 0.4 a±0.52, respectively). Conclusions: MSCs cultured under low O 2 tension are an effective treatment for malnourished rats with ovarian failure.

α-Tocopherol restores semen quality in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Background and Aim: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant toxic to the human reproductive system. This study aimed to evaluate the effect of α-Tocopherol administration on the male fertility parameters of a rat model exposed to TCDD. Materials and Methods: Fifty healthy 12-week-old male rats were randomly divided into five groups. Rats in the control group were given corn oil twice daily in 4 h intervals. In the treatment groups, all rats were given TCDD at a dose of 700 ng/kg of body weight (BW)/day for 45 days. Four hours after receiving the TCDD, T0 rats were given corn oil, and T1, T2, and T3 rats were given α-Tocopherol at doses of 77, 140, and 259 mg/kg BW/day, respectively, for 45 days. On day 46, experimental animals were sacrificed to collect blood and testicular samples. Results: TCDD exposure decreased superoxide dismutase activity, plasma membrane integrity, Leydig cell count, sperm cell count, sperm viability and motility, and increased malondialdehyde levels, serum testosterone levels, and sperm morphological abnormalities. The administration of α-Tocopherol mitigated the effects of TCDD exposure, and the 140 and 259 mg/kg BW/day treatments returned those male fertility parameters to normal levels. Conclusion: The administration of 140 mg/kg BW/day α-Tocopherol restored male semen quality in rats exposed to TCDD. We found dynamics serum testosterone levels in rats exposed to TCDD that need to be further studied.

Effectiveness of forest honey ( Apis dorsata) as therapy for ovarian failure causing malnutrition.

Background: Malnutrition is the imbalance between intake and nutritional needs, resulting in a decrease in body weight, composition, and physical function. Malnutrition causes infertility due to intestinal and liver degeneration,which may progress to testicular and ovarian degeneration. Methods: An infertile female rat model with a degenerative ovary was induced with malnutrition through a 5-day food fasting but still had drinking water. The administration of (T1) 30% (v/v) and (T2) 50% (v/v) forest honey ( Apis dorsata) were performed for ten consecutive days, whereas the (T+) group was fasted and not administered forest honey and the (T-) group has not fasted and not administered forest honey. Superoxide dismutase, malondialdehyde, IL-13 and TNF-α cytokine expressions, and ovarian tissue regeneration were analyzed. Results: Superoxide dismutase was significantly different ( p<0.05) in T1 (65.24±7.53), T2 (74.16±12.3), and T- (65.09±6.56) compared with T+ (41.76±8.51). Malondialdehyde was significantly different ( p<0.05) in T1 (9.71±1.53), T2 (9.23±0.96), and T- (9.83±1.46) compared with T+ (15.28±1.61). Anti-inflammatory cytokine (IL-13) expression was significantly different ( p<0.05) in T1 (5.30±2.31), T2 (9.80±2.53), and T- (0.30±0.48) compared with T+ (2.70±1.57). Pro-inflammatory cytokine (TNF-α) expression was significantly different ( p<0.05) in T1 (4.40±3.02), T2 (2.50±1.65), and T- (0.30±0.48) compared with T+ (9.50±1.78). Ovarian tissue regeneration was significantly different ( p<0.05) in T- (8.6±0.69) and T2 (5.10±0.99) compared with T1 (0.7±0.95) and T+ (0.3±0.67). Conclusion: The 10-day administration of 50% (v/v) forest honey can be an effective therapy for ovarian failure that caused malnutrition in the female rat model.

Kaliandra honey improves testosterone levels, diameter and epithelial thickness of seminiferous tubule of white rat (Rattus norvegicus) due to malnutrition through stimulation of HSP70.

Background: Malnutrition can cause an increase in oxidative stress as it triggers the expression of heat shock protein70 (HSP70), a chaperon molecule that is needed to repair damaged cells within optimal levels. Honey is a source of feed that can stimulate HSP70 expression, which can be given to the malnourished in the animal trial. Aim: The purpose of this study was to prove that Kaliandra honey can improve testosterone levels, diameter, and epithelial thickness of the seminiferous tubule of rat testes (Rattus norvegicus) due to malnutrition through stimulation of HSP70, which is expressed immunohistochemically. Methods: This study used 40 male rats, which were divided into four treatment groups: T0 (negative control): normal rats and not given honey; T1 (positive control): malnourished rats and not given honey; T2 (treatment 2): malnourished rats and given 30% Kaliandra honey (v/v) for 10 days; T3 (treatment 3), malnourished rats and given 50% Kaliandra honey (v/v) for 10 days. The condition of malnutrition is carried out by fasting the feed for five consecutive days resulting in damage to the male reproductive organs, especially the testes. Results: The results showed that Kaliandra honey at a dose of 50% (v/v) had a significant effect in improving testosterone levels, diameter, and epithelial thickness of seminiferous tubule of malnourished male rats through stimulation of HSP70 expression. The HSP70 expression scores by IHC at T0, T1, T2, and T3 were 0.15a ± 0.5, 3.15c ± 0.4, 2.95c ± 0.35, and 1.75b ± 0.15, sequentially. enzyme-linked immunosorbent assay indirect testosterone levels at T0, T1, T2, and T3 (in µg/dl) were 36.39c ± 0.35, 6.12a ± 0.51, 7.45a ± 0.15, 25.27b ± 0.63, sequentially. The diameter and epithelial thickness of the seminiferous tubule of the testes (in µm) in the four treatments T0, T1, T2, and T3 were 362.40c ± 4.71, 248.46a ± 3.90, 255.22a ± 2.34, 318.37b ± 4.23 and 117.60d ± 11.30, 3.86a ± 1.57, 9.72b ± 3.96, 29.84c ± 4.02 sequentially. Conclusion: The conclusion of the study showed that Kaliandra honey at a dose of 50% (v/v) had a significant effect in improving testosterone levels, diameter, and epithelial thickness of the seminiferous tubule of malnourished rats through stimulation of HSP70, although not significantly the same as negative control (T0).

Effectiveness of mesenchymal stem cells cultured under hypoxia to increase the fertility rate in rats (Rattus norvegicus).

BACKGROUND AND AIM: Mesenchymal stem cells (MSCs) transplanted into the testes of rats with testicular failure can help rescue fertility. However, the low viability of transplanted MSCs limits the success of this treatment. This study aimed to determine the effectiveness of MSCs cultured under hypoxia to increase the fertility rate in rats (Rattus norvegicus). MATERIALS AND METHODS: Bone marrow-derived MSCs (200 million cells/rat) were transplanted into male rat models with induced infertility (10 rats/treatment group) after 4 days of culture in 21% O2 (normoxia) and 1% O2 (hypoxia). Ten fertile and 10 untreated infertile rats served as controls. In the infertile male rats that had been fasted from food for 5 days, the fasting condition induced malnutrition and then resulted in testicular failure. RESULTS: The results indicated that the MSCs cultured under hypoxic conditions were more effective than those cultured in normoxic conditions as a treatment for testicular failure in infertile male rats based on the increased number of cells expressing p63 as a quiescent cell marker and ETV5 as a transcription factor expressed in Sertoli and germ cells. Furthermore, the structure of the seminiferous tubules, which contain spermatogonia, primary and secondary spermatocytes, and spermatid, Sertoli, and Leydig cells, was improved in infertile male rats treated with the MSCs cultured under hypoxic conditions. CONCLUSION: The testicular transplantation of MSCs cultured under hypoxic conditions was an effective treatment for testicular failure in rats.

Effect of low oxygen tension on transcriptional factor OCT4 and SOX2 expression in New Zealand rabbit bone marrow-derived mesenchymal stem cells.

BACKGROUND AND AIM: Octamer-binding transcription factor 4 (OCT4) and sex-determining region Y-box 2 (SOX2) are transcription factors whose functions are essential to maintain the pluripotency of embryonic stem cells. The purpose of this study was to derive stem cells for in vitro culture and to maintain their viability and pluripotency, with the goal to obtain a cell line for transplantation in patients with degenerative diseases or injuries. This research focused on examining the effect of low oxygen tension on the ability of bone marrow-derived mesenchymal stem cells (BM-MSCs) to express OCT4 and SOX2 in vitro. MATERIALS AND METHODS: BM-MSCs were obtained from femurs of 2000 to 3000 g New Zealand male rabbits. BM-MSCs were divided into three groups to test different culture conditions: A control group under hyperoxia condition (21% O2) and two treatment groups with low oxygen tension (1% and 3% O2). We characterized the BM-MSCs using flow cytometric measurement of cluster differentiation 44 (CD44) and cluster differentiation 90 (CD90) expression. The expression of OCT4 and SOX2 was measured by immunofluorescence staining after 48 h of incubation in chambers with normal or low oxygen tension with controlled internal atmosphere consisting of 95% N2, 5% CO2, and 1% O2 (T1) and 3% O2 (T2). We considered OCT4 and SOX2 as two markers of pluripotency induction. All immunofluorescence data were subjected to a post hoc normality Tukey's honestly significant difference test; all differences with p<5% were considered significant. RESULTS: BM-MSCs were positive for CD44 and CD90 expression after isolation. Oxygen tension culture conditions of 1% and 3% O2 led to OCT4 and SOX2 expression on culture days 2 and 4 (p<0.05), respectively, as compared to the hyperoxia condition (21% O2). CONCLUSION: Based on the OCT4 and SOX2 immunofluorescence data, we conclude that the stem cells were pluripotent at low O2 tension (at 1% O2 on day 2 and at 3% O2 on day 4), whereas under 21% O2 the OCT4 and SOX2 were not expressed.

The effect of crude guava leaf tannins on motility, viability, and intact plasma membrane of stored spermatozoa of Etawa crossbred goats.

AIM: The aim of this study was to know crude guava leaf tannins effect on motility, viability, and intact plasma membrane of stored spermatozoa of Etawa crossbred goats. MATERIALS AND METHODS: Macroscopic assessment of normal Etawa crossbred semen was followed by dilution with a glucose solution at a 1:10 ratio to increase volume. The diluted semen was treated by adding crude guava leaf tannins into 1 ml of the semen glucose diluent, and five treatments were obtained, namely, control group (C), with no added tannins; treatment Group 1 (T1), with 3%; treatment Group 2 (T2), with 6% tannins; treatment Group 3 (T3), with 12% tannins; and treatment Group 4 (T4), with 24% tannins. Each treatment used five replications. Then, microscopic analysis of the treated and control semen was carried out after 15 days of storage at 4-5°C temperature. The parameters observed were motility, pH, viability, abnormality, and intact spermatozoa plasma membrane. RESULTS: The spermatozoa motility in Group C was the highest (76.60±1.47). The motility in Group T1 did not differ from that in Group C, but was different and higher than that in Groups T2, T3, and T4. The pH of Group C tended to be acidic after 15 days of storage (4.78±0.01) as compared to the initial pH of fresh semen (6.76±0.12). The pH in Group C did not differ from that in the Groups T1 and T2, but differed from that in the T3 and T4 groups; the pH in the T3 and T4 groups was similar. The viability of spermatozoa in the T1 group was higher than that in all treatments (64.60±2.76); the lowest values were found in Group C (28.94±1.02). Group C had the lowest number of normal spermatozoa, with a mean of 72.58±3.48. The total number of abnormalities in the T2 group did not differ from those in the T3 group, and abnormalities in the T4 group did not differ from those in Group C, which exhibited the highest abnormalities in the head, neck, and tail. The most significant decrease was observed in the intact plasma membrane of spermatozoa on addition of 12% and 24% crude guava leaf tannin in glucose diluents. CONCLUSION: The addition of 3% crude guava leaf tannin to crossbred Etawa goat semen diluted with glucose diluent and stored for 15 days at 4-5°C resulted in a significant effect on spermatozoa motility, viability, and intact plasma membrane, whereas the administration of 24% crude guava leaf tannin resulted in low live percentage of spermatozoa.

Comparison of biotechnological culture of hypoxia-conditioned rat mesenchymal stem cells with conventional in vitroculture of normoxia-conditioned rat mesenchymal stem cells for testicular failure therapy with low libido in rats.

AIM: Biotechnological culture of hypoxia-conditioned (CH) rat mesenchymal stem cells (rMSC-CH) for testicular failure therapy with low libido improves the functional outcome of the testicle for producing spermatogenic cells and repairs Leydig cells in rats (Rattus norvegicus). MATERIALS AND METHODS: In the first group (T1), rats with testicular failure and low libido were injected with normoxia-conditioned (CN) rMSCs (21% oxygen); in the second group (T2), rats with testicular failure and low libido were injected with rMSC-CH (1% oxygen); in the negative control group (T-), rats with normal testis were injected with 0.1 mL phosphate-buffered saline (PBS); and in the sham group (TS), rats with testicular failure and low libido were injected with 0.1 mL of PBS. RESULTS: Vascular endothelial growth factor expression, as the homing signal, in the groups T2, T-, T1, and TS was 2.00±0.5%, 2.95±0.4%, 0.33±0.48%, and 0±0%, respectively. The number of cluster of differentiation (CD)34+ and CD45+ cells in the groups T- and TS was <20%, whereas that in T1 and T2 groups was >30% and >80%, respectively, showing the mobilization of hematopoietic stem cells (HSCs). The number of spermatogenic cells (spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatid) decreased significantly (p<0.05) in TS compared with that in T-, T1, and T2, whereas that in T2 did not show a significant (p>0.05) decrease compared to that in T-. The improvement in libido, based on the number of Leydig cells producing the hormone testosterone for libido expression, did not increase in T1, whereas T2 was able to maintain the number of Leydig cells significantly compared to that between TS and T1. CONCLUSION: rMSC-CH culture for testicular failure with low libido showed improvement in the functional outcome of the testicle and in repairing Leydig cells.

The improvement of eggs quality of Mojosari duck (Anas javanica) with soybean husk fermentation using cellulolytic bacteria of Spodoptera litura.

AIM: This study was aimed to improve the quality of the eggs of Mojosari duck (Anas javanica) through complete feeding containing soybean husk was fermented using cellulolytic bacteria of Spodoptera litura. MATERIALS AND METHODS: This study consisted of three stages: The first stages, isolation and identification of cellulolytic bacteria from S. litura; the second stage, the fermentation of soybean husk through the application of bacterial cellulolytic isolate from the first stage; and the third stage, the application of the best complete feed formulation from the second stage to Mojosari duck. RESULTS: There are four dominant bacteria: Bacillus sp., Cellulomonas sp., Pseudomonas sp., and Cytophaga sp. Furthermore, the best reduction of the crude fiber of soybean husks is the use of Cellulomonas sp. bacteria. The final of the study, the quality of the eggs of Anas javanica, was improved, as indicated by cholesterol decrease from the yolk without the decrease of egg weight and eggshell thickness, although the decrease in egg yolk color was inevitable. CONCLUSION: Soy husk fermentation using cellulolytic bacteria of S. litura was added to complete feeding can be performed to improve the quality of the eggs of Mojosari duck.

Mycotoxin binders potential on histological of ovary mice exposed by zearalenone.

AIM: This study was conducted to examine the potential of mycotoxin binder in ceasing zearalenone (ZEN) effect on mice reproduction. ZEN mycotoxin can induce reactive oxygen species that may cause damage and cell death. ZEN is estrogenic so that it may affect the reproductive organs failure. MATERIALS AND METHODS: Mycotoxin binder administration to female mice exposed to ZEN was aimed to count the number of primary follicles, secondary follicles, tertiary follicles, de Graaf's follicles, and the corpus luteum (CL). Negative control group (C) was not exposed to ZEN and without the administration of mycotoxin binders, while positive control group (C+) was exposed to 0.1 mg/mouse/day ZEN and without the provision of mycotoxin binders. Treatment groups (T1, T2, T3) were exposed to 0.1 mg/mouse/day ZEN and mycotoxin binders 0.5; 1; 2 mg/BW/day. RESULTS: ZEN and mycotoxin binders administration was conducted for 10 days. The number of primary follicles, secondary, tertiary, de Graaf's follicles and CL in negative control (C-) was 14.2±1.36, 11.2±0.28, 6.5±0.53, 7.5±0.74, and 2.3±0.35. The number in positive control (C+) group was as follows 7.1±0.12, 3.7±1.17, 3.8±1.21, 1.5±0.62, and 2.3±0.34. Results in treatment 1 (T1) were as follows 6.2±0.16, 5.2±0.16, 3.6±0.16, 2.6±0.19, and 2.6±0.10; in treatment 2 (T2) 7.8±0.28, 5.8±0.53, 3.7±0.26, 2.7±0.26, and 2.5±0.10; and in treatment 3 (T3) 8.4±0.34, 8.4±0.34, 4.6±0.34, 4.5±1.01, and 3.4±0.23. CONCLUSION: The number of follicles and CL more in line with increasing doses of mycotoxin binders. Required more than 2 mg/mouse/day mycotoxin binders to inhibit the effects of ZEN so that its can maintain the number of primary follicle, secondary follicle, tertiary follicle, the de Graaf's follicle, and the number of CL in the ovary of ZEN-exposed female mice (Mus musculus).

Honeybee product therapeutic as stem cells homing for ovary failure.

AIM: Complexity of the method of isolation, cultivation in vitro and the expensive cost of transplantation process of stem cells, it would require an innovation to homing and differentiation of stem cells and increase folliculogenesis. The stem cells homing was achieved through the provision of food or beverages derived from natural materials like honeybee product. Through honeybee product, there will be homing of stem cells and accompany with the sources from the body itself will take place in regeneration of the ovary. MATERIALS AND METHODS: Female rats model of degenerative ovary was obtained through food fasting but still have drinking water for 5 days. It caused malnutrition and damage of the ovarian tissue. The administration of 50% honeybee product (T1) was performed for 10 consecutive days, while the positive control group (T0+) was fasted and not given honeybee product and the negative control (T0-) not fasted and without honeybee product. Observations were taken for homing of stem cells, raised of folliculogenesis, differentiation of stem cells, and regeneration of the ovarian tissue using routine H&E staining. RESULTS: Homing of stem cells shown the vascular endothelial growth factor and granulocyte colony-stimulating factor expression; enhancement of folliculogenesis was indicated by an increase of follicle dee Graaf count; enhancement of differentiation of stem cells was indicated by growth differentiation factor-9 expression; and regeneration of ovarian tissue indicated by intact ovarian tissue with growing follicles. CONCLUSION: Honeybee product can be induced endogenous stem cells in regeneration of ovary failure due to malnutrition.