Spirulina ameliorates arsenic induced reproductive toxicity in male rats.

Spirulina (Spirulina platensis), has numerous health benefits including antioxidant, immunomodulatory, and anti-inflammatory activities, works against heavy metal toxicity, and is often used as a food supplement in human, animals, birds and fishes. This study aimed to evaluate the protective ability of the dietary spirulina against the toxic effects of inorganic arsenic (iAs) on male reproductive parameters in rats. Seventy-two mature Long-Evans male rats, dividing into six groups (T0, T1, T2, T3, T4 and T5) (12 rats/group) were included in this study. The T3, T4 and T5 group rats were treated with three consecutive doses (1.0 g, 1.5 g and 2.0 g/kg feed) of spirulina in feed along with 3.0 mg NaAsO2/kg body weight (BW) in drinking water (DW) daily for 90 days. Each rat of group T1 received NaAsO2 (3.0 mg/kg BW) in DW, and those of T2 group were fed with spirulina (2.0 g/kg feed) daily for 90 days. The rats of group T0 served as the control with normal feed and water. Total arsenic (tAs) contents, reproductive parameters (testicular weight, sperm motility and morphology), and histological changes in the testicles were evaluated in these rats. Arsenic dosing significantly (p=0.003, Kruskal-Wallis test) increased the tAs contents in the testicles, decreased testes weight, sperm morphology and motility compared to the controls. The effect of arsenic dosing was also evidenced by the histological changes like decreased germinal layers in the seminiferous tubules of the treated rats. Moreover, dietary spirulina (2.0 g/kg feed) supplementation significantly (p=0.011, Kruskal-Wallis test) lowered tAs contents in testicles and increases testes weights, sperm motility and morphology. Therefore, spirulina can be used as an effective dietary supplement to ameliorate the adverse effects of arsenic induced reproductive toxicities. However, further study is required to elucidate the underlying molecular mechanisms of reduction of arsenic induced reproductive toxicity by spirulina.

The colon and terminal ileum in patients with ankylosing spondylitis and controls in Bangladesh: a macroscopic and microscopic study.

OBJECTIVE: Little is known about gut lesions in AS patients in a developing country, such as Bangladesh. METHODS: Full colonoscopy, including the terminal ileum, was performed in 60 AS patients and 20 controls, without diarrhoea, to study macroscopic and microscopic lesions. RESULTS: In the colon, in 60 AS patients 17 macroscopic lesions were found, of which 11 were in the rectum; only one lesion was found in 20 controls. The prevalence of microscopic lesions in the ascending colon, sigmoid colon and rectum was 51, 44 and 50 in patients, respectively, and 13, 9 and 8 in controls. In the terminal ileum, macroscopic and microscopic lesions were seen in 21/56 and 43/56 AS patients, respectively, and in 1/20 and 9/20 controls. In the AS group, macroscopic (38.5 vs 5%, P < 0.01) and microscopic (76.8 vs 45%, P = 0.009) lesions were more frequent than in controls; no IBD was diagnosed. Findings were comparable in the axial AS group (n = 25) and the mainly peripheral group (n = 35). In AS patients, marked eosinophilic infiltration was observed in the ascending colon and sigmoid colon but not in the rectum, and this infiltration was more than in controls. The colonic mucosa in controls was otherwise comparable with western studies. Anaemia was seen in 18/60 cases. No association was found between anaemia or HLA-B27 status and gut lesions. CONCLUSION: There was an equal percentage of microscopic lesions in the whole gut in AS cases and healthy controls. Previous helminth invasions might have played a role. Lesions differ significantly between AS and controls only in the ileum; therefore, the ileal lesions might be more disease related than the colonic ones.

Economic benefit in repeat breeder cows using intrauterine infusion of penicillin and estrus synchronization followed by timed artificial insemination.

OBJECTIVE: Until recently, management of repeat breeding in cattle remains a major problem to dairy farmers. This study was carried out to improve the fertility in cows, those did not conceive after three or more consecutive services. MATERIALS AND METHODS: Twenty-three repeat breeding cows were selected from the Central Cattle Breeding Station and Dairy Farm (CCBS & DF), Savar, Dhaka, Bangladesh. Data of another 23 repeat breeding cows that conceived after four or more services were collected from the same farm as controls. The repeat breeders were synchronized for estrus with two injections of PGF2α 11 days apart and timed AI (TAI) was performed. RESULTS: Seventeen of 23 cows had uterine infections. Intrauterine infusion of penicillin daily for three successive days from the following day of the first PGF2α injection resulted in 94.1% (16/17) recovery. Of 23 treated cows, four with estrus synchronization and TAI, and one with only AI after hormone treatment were detected pregnant by per rectal examination of the genital tract. By this pregnancy, the treated cows have advanced average of 131.6 days calving interval and days open, and thereby saved neat US $3,045.3 in comparison to control cows. Control group cows incurred losses of US $15,134.0 compared with average days open of the treated pregnant cows. CONCLUSION: Intrauterine infusion of penicillin can successfully recover the uterine infections, and estrus synchronization followed by TAI resulted in pregnancy in a proportion of repeat breeder cows with economic benefit that had a uterine infection.

Candidate gene approach for parasite resistance in sheep--variation in immune pathway genes and association with fecal egg count.

Sheep chromosome 3 (Oar3) has the largest number of QTLs reported to be significantly associated with resistance to gastro-intestinal nematodes. This study aimed to identify single nucleotide polymorphisms (SNPs) within candidate genes located in sheep chromosome 3 as well as genes involved in major immune pathways. A total of 41 SNPs were identified across 38 candidate genes in a panel of unrelated sheep and genotyped in 713 animals belonging to 22 breeds across Asia, Europe and South America. The variations and evolution of immune pathway genes were assessed in sheep populations across these macro-environmental regions that significantly differ in the diversity and load of pathogens. The mean minor allele frequency (MAF) did not vary between Asian and European sheep reflecting the absence of ascertainment bias. Phylogenetic analysis revealed two major clusters with most of South Asian, South East Asian and South West Asian breeds clustering together while European and South American sheep breeds clustered together distinctly. Analysis of molecular variance revealed strong phylogeographic structure at loci located in immune pathway genes, unlike microsatellite and genome wide SNP markers. To understand the influence of natural selection processes, SNP loci located in chromosome 3 were utilized to reconstruct haplotypes, the diversity of which showed significant deviations from selective neutrality. Reduced Median network of reconstructed haplotypes showed balancing selection in force at these loci. Preliminary association of SNP genotypes with phenotypes recorded 42 days post challenge revealed significant differences (P<0.05) in fecal egg count, body weight change and packed cell volume at two, four and six SNP loci respectively. In conclusion, the present study reports strong phylogeographic structure and balancing selection operating at SNP loci located within immune pathway genes. Further, SNP loci identified in the study were found to have potential for future large scale association studies in naturally exposed sheep populations.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle.

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n=1239) were collected at three different time points (12, 18 and 24h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P>0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P<0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

Bovine spermatozoa react to in vitro heat stress by activating the mitogen-activated protein kinase 14 signalling pathway.

Heat stress has long been recognised as a cause of subfertility in farm animals. The objectives of the present study were to elucidate the effect of heat stress on sperm function and involvement of the mitogen-activated protein kinase (MAPK) 14 signalling pathway. Spermatozoa incubated for 4 h at a physiological temperature (38.5°C) exhibited significantly (P<0.05) reduced motility, plasma membrane integrity and mitochondrial potential compared with non-incubated spermatozoa; the reductions in these parameters were more severe following incubation at a hyperthermic (41°C) temperature (P<0.01). Percentages of fertilisation and embryo development were highly affected in spermatozoa incubated at 41°C compared with non-incubated spermatozoa (P<0.01). Similarly, embryo quality was adversely affected by sperm incubation at 41°C, as indicated by a higher apoptotic cell ratio in Day 7 blastocysts compared with that in the non-incubated control group (14.6% vs 6.7%, respectively; P<0.01). Using SB203580 (10 µgmL(-1)), a specific inhibitor of the p38 MAPK pathway, during sperm hyperthermia reduced MAPK14 activation (24.9% vs 35.6%), increased sperm motility (45.8% vs 26.5%) and reduced DNA fragmentation (16.9% vs 23.4%) compared with the untreated control group, but did not improve subsequent fertilisation and embryo development. In conclusion, heat stress significantly affects the potential of spermatozoa to penetrate oocytes, as well as subsequent embryo development and quality. Notably, the data show that the MAPK14 signalling pathway is largely involved in heat-induced sperm damage. However, further research is needed to elucidate other signalling pathways possibly involved in heat-induced sperm damage.

Oocyte quality determines bovine embryo development after fertilisation with hydrogen peroxide-stressed spermatozoa.

Exposure of gametes to specific stressors at sublethal levels can enhance the gametes' subsequent performance in processes such as cryopreservation. In the present study, bull spermatozoa were subjected to H2O2 for 4 h at 100-, 200- and 500-µM levels; computer-assisted sperm analysis (CASA) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used for evaluation of subsequent sperm motility and DNA integrity, respectively. Exposure of spermatozoa to H2O2 did not affect sperm motility but DNA integrity was negatively affected by 500 µM H2O2 compared with mock-exposed spermatozoa, whereas both motility and DNA integrity were affected compared with untreated spermatozoa. Nevertheless, insemination of oocytes with spermatozoa exposed to 200 µM H2O2 increased fertilisation, cleavage and blastocyst rates (P < 0.05). Furthermore, the higher blastocyst yield after fertilisation of oocytes with spermatozoa exposed to 200 µM H2O2 was related to oocyte diameter, with large-medium oocytes yielding higher blastocyst rates, while small-diameter oocytes consistently failed to develop into blastocysts. In conclusion, the results indicate that exposure of spermatozoa to 200 µM H2O2 before sperm-oocyte interaction may enhance in vitro embryo production in cattle. However, this increased embryo production is largely dependent on the intrinsic quality of the oocytes.

Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production.

BACKGROUND: Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers' inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA) to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes. METHODS: Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS) and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding), Days 10-12 and Days 22-24. Defatted milk was preserved at -80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70-90. RESULTS: Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate). Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17) and 83 to 135 (n = 13) days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (<1 ng/ml), high (≥ 1 ng/ml) and high (≥ 1 ng/ml) on Day 0, Days 10-12 and Days 22-24, respectively. Buffaloes cycling later in the postpartum period had fewer missed oestruses (P < 0.05). Buffaloes with a superior BCS had a shorter calving to oestrus interval and produced more milk (P < 0.05). CONCLUSIONS: Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.

Preovulatory follicular and subsequent luteal size influence pregnancy success in water buffaloes.

The diameter of the preovulatory follicle (POF) and its effects on subsequent corpus luteum (CL) size and conception were studied in 38 lactating indigenous cycling buffaloes in the Mymensingh district of Bangladesh. Body condition score (BCS) at estrus was estimated for the buffaloes. The buffaloes were synchronized with two injections of a synthetic analogue of PGF2α administered 11 days apart. Transrectal ultrasonography was carried out at estrus and on days 5, 9, 12 and 16 post ovulation to determine the POF and successive CL size. Pregnancy was confirmed by ultrasound examination on day 40-45 post ovulation. Twenty one (55.3%) buffaloes were diagnosed as pregnant. The conception rates of thin (BCS ≤2.0), good (BCS 2.5-3.5) and fat (BCS glt;3.5) buffaloes were 7.7, 88.2 and 62.5% (χ² = 19.54; P<0.05), respectively. The mean diameter of the POF at estrus was larger (P<0.01) in buffaloes that ultimately were diagnosed as pregnant compared with their nonpregnant counterparts (13.7 ± 0.3 vs. 11.2 ± 0.5 mm, respectively). The conception rates of buffaloes having small (9 to ≤ 12 mm), medium (>12 to ≤14 mm) and large (>14 to 16 mm) POFs at estrus were 9.1, 70.0 and 85.7% (χ² = 13.87, P<0.01), respectively. On day 5 post ovulation, CL size was positively correlated (CL: r=.74, P<0.01) with POF diameter. Retrospective analysis revealed that on day 5 post ovulation, the pregnant buffaloes had higher (P<0.01) post ovulation CL sizes than their nonpregnant counterparts (15.6 vs. 11.8 mm). Similarly, on day 9 post ovulation, the difference in CL size (14.3 vs. 13.6 mm) between pregnant and nonpregnant buffaloes was significant (P<0.05). In conclusion, the diameter of the POF in buffaloes has a positive impact on the size of the post ovulation CL and conception.

Scrotal insulation and its relationship to abnormal morphology, chromatin protamination and nuclear shape of spermatozoa in Holstein-Friesian and Belgian Blue bulls.

The objectives of this study were to identify the stages of spermatogenesis susceptible to elevated testicular temperature in terms of sperm motility, viability, morphology, chromatin protamination and nuclear shape. The latter two valuable parameters are not included in routine semen analysis. Scrotal insulation (SI) was applied for 48 h in 2 Holstein-Friesian (HF) and 2 Belgian Blue (BB) bulls and semen was collected at 7 d intervals along with semen collection of a non-insulated bull of each breed. Semen samples were frozen and assigned to 4 groups: period 1 (preinsulation) = -7 d and 0 d, where 0 d = initiation of SI after semen collection; period 2 = 7 d (sperm presumed in the epididymis during SI); period 3 = 14 d to 42 d (cells presumed at spermiogenesis and meiosis stages during SI); period 4 = 49 d to 63 d (cells presumed at spermatocytogenesis stage during SI). The percentages of progressively motile and viable spermatozoa as assessed by computer-assisted sperm analysis (CASA) and fluorescence microscopy, respectively were decreased whereas abnormal sperm heads, nuclear vacuoles and tail defects were increased at period 3 (P < 0.05) compared to period 1, 2 or 4 in SI bulls of both HF and BB breeds. Protamine deficient spermatozoa as observed by chromomycin A(3) (CMA(3)) staining were more present (P < 0.05) at period 2 and 3 in both breeds compared to period 1 or 4. Sperm nuclear shape as determined by Fourier harmonic amplitude (FHA) was most affected by heat stress during period 3 (P < 0.01) and a higher response was observed in BB bulls than HF bulls. In conclusion, sperm cells at the spermiogenic and meiotic stages of development are more susceptible to heat stress. The lack of chromatin protamination is the most pertinent result of heat stress, together with subtle changes in sperm head shape, which can be detected by FHA but not by conventional semen analysis.

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes.

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle's salt with L-glutamine and sodium bicarbonate) for 27 h at 39°C under 5% CO(2) in humidified air. The oocytes retrieval and efficiency (mean ± SD) per prepubertal and pubertal goats were 5.2 ± 0.6 and 6.8 ± 0.6, and 77.3 ± 0.1% and 80.5 ± 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 ± 0.8 vs. 2.6 ± 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode's albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 ± 0.9% vs. 32.7 ± 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 ± 1.7 vs. 32.2 ± 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.

Comparison of metabolic rates and feed nutrient digestibility in conventional, genetically improved (GIFT) and genetically male (GMNT) Nile tilapia, Oreochromis niloticus (L.).

Various aspects of energy metabolism and feed digestibility were evaluated in two reportedly improved strains of Nile tilapia (Oreochromis niloticus) namely GIFT (genetically improved farmed tilapia) and GMNT (genetically male Nile tilapia) and compared with those of CNT (conventional Nile tilapia). Fish were stocked individually in a computer-controlled respirometer system at 27+/-0.1 degrees C for 10 weeks. Metabolic rates were measured at three different feeding levels: starved, maintenance (3.0 g kg(-0.8) day(-1)) and growth (7.5 g kg(-0.8) day(-1)) using a fishmeal based feed containing TiO2 marker (41% crude protein, 9% crude lipid and 19 kJ (g DM)(-1) gross energy). The standard metabolic rate (SMR), measured at the beginning of the experiment (45.4+/-4.6, 52.4+/-7.7 and 46.8+/-4.6 mg O2 kg(-0.8) h(-1) respectively for GIFT, GMNT and CNT), did not differ significantly between the groups (p<0.05). Similarly, non-significant differences were also observed in the routine metabolic rates under starved, maintenance and growth conditions but the variability was higher in the case of GMNT and CNT than in GIFT. The latter group showed a significantly lower active metabolic rate (145 mg O2 kg(-0.8) h(-1)) compared to GMNT and CNT (232 and 253 mg O2 kg(-0.8) h(-1), respectively) at maintenance feeding level. The specific dynamic action (% offered feed energy) showed no significant differences among the groups. Digestibility coefficients of feed dry matter, protein, lipid and energy for the three tilapia groups also did not differ significantly. Therefore, we concluded that the genetic improvement or modification in the GIFT or GMNT might not upgrade the inherent physiological potential compared to CNT as far as energy metabolism and digestion efficiencies are concerned.

A randomized, placebo-controlled trial of thymosin-alpha1 and lymphoblastoid interferon for HBeAg-positive chronic hepatitis B.

Combination therapy between two immunomodulators used for treatment of chronic hepatitis B was explored based on reported therapeutic efficacy of interferon-alpha, and thymosin-alpha1 as monotherapeutic agents to determine if combination therapy was superior to interferon alone. This double-blinded, randomized, placebo-controlled trial compares the addition of thymosin-alpha1, 1.6 microg taken three times per week (combination therapy) or thymosin placebo (monotherapy) to lymphoblastoid interferon (Wellferon), 5 million international units (MIU) taken three times per week, for 24 weeks. Entry criteria included positive hepatitis B e antigen (HBeAg); alanine aminotransferease (ALT) > or = 1.5 x upper normal limit, but < or = 10 x upper normal limit; positive HBV DNA; absence of cirrhosis; treatment naivety and no co-morbid factors. A total of 98 HBeAg-positive patients were recruited, of which 48 were randomized to combination therapy and 50 to monotherapy. The primary endpoint was the loss of HBeAg at 72 weeks. The secondary endpoints were HBeAg seroconversion, normalization of ALT, loss of HBV DNA and improvement in histology. The HBeAg loss was 45.8% and 28.0% for combination therapy and monotherapy, respectively (difference, 17.8%; 95% CI -1.2%-35.3%, P = 0.067). There was a trend towards HBeAg loss when using combination therapy. There were also no statistically significant differences between the different therapies with respect to the secondary endpoints of HBeAg seroconversion, changes in histology, normalization of ALT or loss of HBV DNA. In conclusion, this trial showed a 17.8% improvement in HBeAg loss rates using combination therapy over interferon monotherapy. This could clinically indicate a potential important difference that would need confirmation in subsequent trials.