Preparation of a high-immunoglobulin product from bovine colostrum

An effective colostrum management programme is the most important factor in determining the health and survival of the neonatal calf. Commercially available colostrum replacers (CR) and colostrum supplements (CS) are an alternative to colostrum on farms that do not have an adequate, high-quality colostrum supply, or those farms that want to prevent transmission of disease from cow to calf. The present study aimed to obtain a high immunoglobulin (Ig), dried bovine colostrum product that could be used as a CR or CS for dairy calves. Dried whey was made from 6 batches of colostrum and an additional 6 batches were used to produce dried whey and curds. Dried whey had higher IgG concentration (P<0.05) and lactose (P<0.05), and less fat (P<0.05) compared to curds or the original colostrum. There was a strong linear relationship between initial colostrum IgG concentration and whey (R² = 0.86) with approximately 0.45 of the initial colostral IgG residing in curd. The high IgG and the composition of colostrum whey powder suggests it could be an effective CS product for use with dairy calves. The high fat and IgG content of the curd by-product indicate that it might be a potential weaning supplement in piglets or even a product for human consumption.     

Hydroxyflutamide alters the characteristics of live boar spermatozoa

Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 μg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the “C-Ruch” computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P < 0.05) in sperm mitochondrial membrane potential and a concomitant increase (P < 0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 μg OH-Flu compared with the respective controls and other doses used (P < 0.05). The adverse effects of OH-Flu become strengthened over time (P < 0.05). Notably, 50 and 100 μg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P < 0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P < 0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P < 0.05) only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility.     

Relationship between intake of tannin-containing tropical tree forage,PEG supplementation,and salivary haze development in hair sheep and goats

The objective of this study was to estimate the relationship between tannin binding salivary protein (TBSP) and condensed tannins (CT) intake in hair sheep and creole goats. Foliage was obtained from trees with different levels of CT content; animals were offered foliage ad libitum, with or without polyethylene glycol (PEG). Saliva haze development (SHD) was evaluated as evidence for TBSP. PEG consumption did not affect dry matter intake (DMI) (P > 0.05). Lignin (R = −0.714, P < 0.001) and Crude Protein (CP) (R = 0.622, P < 0.001) contents had a stronger association with DMI than CT (R = 0.622, P < 0.011) in sheep; no significant association was found in goats. The positive relationship between tannin intake and SHD (P < 0.05) was not confirmed after PEG supplementation in sheep (P > 0.09), but remained significant for goats (P < 0.01), except for those fed Lysiloma latisiliquum (P = 0.07). Foliage lignin or CP contents are better predictors of foliage intake than CT. Sheep and goats fed with tropical tree forages containing different levels of tannins exhibited differences in intake behavior; moreover, individual variations in TBSP expression helps explaining foliage DMI.     

Effects of dietary Bacillus subtilis and inulin supplementation on performance,eggshell quality,intestinal morphology and microflora composition of laying hens in the late phase of production

Eighty Lohmann White laying hens were used to investigate the effect of dietary inclusion of Bacillus subtilis and inulin, individually or in combination, on egg production, eggshell quality, tibia traits, Ca retention, and small intestine morphology and microflora composition from 64 to 75 weeks of age. Hens were randomly distributed into 4 treatment groups, with 5 replicates per treatment and 4 hens per replicate. Treatment groups were fed basal diet (control), basal diet plus 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g), basal diet plus 1 g/kg inulin, or basal diet plus a synbiotic combination of 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g) and 1 g/kg inulin. Dietary supplementation of B. subtilis, inulin or synbiotic improved (P<0.05) feed conversion, egg performance, eggshell quality and calcium retention compared with the control. B. subtilis and synbiotic groups exhibited the highest (P<0.05) increase in egg production and egg weight. Inulin and synbiotic groups exhibited the highest (P<0.05) increase in eggshell thickness and eggshell calcium content, and the lowest (P<0.05) eggshell deformations. Unmarketable eggs were 8.4% (P<0.05) of the total eggs produced by the control group compared to 3.5%, 1.7%, and 1.5% for the B. subtilis, inulin and synbiotic groups, respectively. Tibia density, ash, and Ca content increased (P<0.05) by inulin and synbiotic inclusions, compared with the control. B. subtilis, inulin, and their synbiotic combination increased (P<0.05) villus height and crypt depth in all intestinal segments, compared with the control. B. subtilis and inulin modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of bifidobacteria and lactobacilli, compared with the control. Colonization of the beneficial microflora along with increasing the villi–crypts absorptive area were directly associated with the improvements in performance and eggshell quality. It can be concluded that egg production and eggshell quality of laying hens can be improved (P<0.05) in the late phase of production by dietary inclusion of B. subtilis and inulin.     

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.     

Nutraceutical effects of bioactive peptides obtained from Pterophylla beltrani (Bolivar & Bolivar) protein isolates

Edible insects have been important sources of food proteins for human consumption and animal feed. In this study, a protein isolate from Pterophylla beltrani Bolivar & Bolivar, 1942 (Orthoptera: Tettigoniidae) was enzymatically processed and its nutraceutical properties were evaluated. Protein isolates were obtained from an insect flour and then was hydrolyzed for 5 h with a sequential process using pepsin and trypsin-chymotrypsin to simulate the gastric intestinal fluids. To evaluate the effect of peptide molecular size on nutraceutical properties, the peptides obtained from Total hydrolyzed (TH) were fractionated by ultrafiltration (UF) with 10 kDa and 3 kDa UF membranes giving fractions (F) with different molecular size (F < 3KDa, F < 10KDa and F > 10KDa). The inhibition assay of Angiotensin-Converting Enzyme (ACE) showed that the best treatment (P < 0.05) was the (TH) with an IC₅₀ value of 0.5 mg/mL while the F < 3KDa was the lowest (P < 0.05) with an IC50 of 1.44 mg/mL, and the peptide size had no effect. However, an α-amylase inhibition was observed with an increase of the IC50 value between TH, F > 10KDa and F < 10KDa although no significative difference (P > 0.05) was found between the TH and F < 3KDa with IC50 of 0.48 and 0.68 mg/mL, respectively. In antioxidant activity, significant differences (P < 0.05) were observed between TH and UF fractions where the best response was in the F < 3KDa. In conclusion, P. beltrani proteins isolate are a source of bioactive peptide, and these could be considered as potential edible insect and sustainable food with nutraceutical effects.     

The potential role of the gut microbiota in modulating renal function in experimental diabetic nephropathy murine models established in same environment

Recent studies have shown that laboratory murine autoimmunity models under the same environment display different outcomes. We established diabetic nephropathy model mice under the same environment using the classic streptozotocin method. Renal dysfunction was different among the mice. Proteinuria was more significant in the severe proteinuria group (SP) than in the mild proteinuria group (MP). We hypothesized a role for the gut microbiota in the outcome and reproducibility of induced DN models. 16S rDNA gene sequencing technology was used to analyze the differences in the gut microbiota between the two groups. Here, through fecal microbiota transplantation (FMT) and gas chromatography mass spectrometry (GC–MS), we verified the role of the gut microbiota and its short-chain fatty acid (SCFA) generation in DN mouse renal dysfunction. In the SP group, there was a reduced abundance of Firmicutes (P < 0.0001), and the dominant genus Allobaculum [linear discriminant analysis (LDA) >3, P < 0.05] was positively correlated with body weight (Rho = 0.767, P < 0.01) and blood glucose content (Rho = 0.648, P < 0.05), while the dominant genus Anaerosporobacter (LDA > 3, P < 0.05) was positively correlated with 24-hour urinary protein content (Rho = 0.773, P < 0.01). In the MP group, the dominant genus Blautia (LDA > 3, P < 0.05) was negatively correlated with 24-hour urinary protein content (Rho = −0.829, P < 0.05). The results indicated that Allobaculum and Anaerosporobacter may worsen renal function, while Blautia may be a protective factor in DN. These findings suggested that the gut microbiota may contribute to the heterogeneity of the induced response since we observed potential disease-associated microbial taxonomies and correlations with DN.     

Effects of type of ionophore and carrier on in vitro ruminal dry matter disappearance,gas production,and fermentation end products of a concentrate substrate

Effects of ionophore type and carrier on in vitro ruminal digestion and fermentation patterns of a concentrate substrate were evaluated at various incubation times. Treatments were: control (no ionophore); lasalocid sodium commercial premix (Bov); lasalocid sodium mycelium cake (LasBio); laidlomycin sodium salt (LaidNa); laidlomycin propionate commercial premix (LaidPro); monensin sodium salt (Mon); and monensin sodium commercial premix (Rum). The Bov, LasBio, Mon, and Rum treatments supplied 4 μg of ionophore/mL of culture volume, whereas the LaidNa and LaidPro treatments supplied 1.33 μg of ionophore/mL. Total gas and methane production did not differ among treatments at any of the incubation times (P>0.09). Similarly, in vitro dry matter disappearance (IVDMD) was not affected by treatment (P>0.28) at 6, 18, and 24 h of incubation; however, IVDMD (P=0.03) was greater for ionophores than for the control at 12 h of incubation. Molar proportions of acetate (P<0.01), acetate:propionate (P<0.01), and total volatile fatty acid (VFA) concentrations (P<0.01) were decreased and propionate was increased (P<0.001) for the average of all ionophore-containing substrates compared with the control. Total VFA were decreased by Bov, LaidNa, and Rum, contrasted with their specific counterparts (LasBio, LaidPro, and Mon, respectively; P<0.05). Differences were detected among ionophore types for acetate (lasalocid vs. laidlomycin; P<0.05), propionate (lasalocid vs. monensin; P<0.05), and butyrate (monensin vs. lasalocid or laidlomycin; P<0.05). Capture of metabolic hydrogen in end products of fermentation was greater for ionophore-containing treatments (P<0.01) than for the control. These data suggest limited unique effects of ionophore type or carrier on IVDMD, total gas production, and methane; however, VFA proportions varied among ionophore types and carriers, which deserves further study.     

Nutritional characterization of Mucuna pruriens: 3. Effect of replacing soybean meal with Mucuna on intake,digestibility, N balance and microbial protein synthesis in sheep

Mucuna pruriens seeds have relatively high crude protein (CP) concentrations, but little is known about their potential to replace commonly used CP supplements in ruminant rations. The aim of this experiment was to determine effects of replacing soybean meal (SB) with Mucuna on the performance of lambs. Forty Rambouillet lambs (33.2 ± 5.73 kg) fed a basal diet of maize grain, cottonseed hulls and urea were randomly assigned to one of four supplements formulated by substituting 0 (SB), 330 (Lo), 670 (Med) or 1000 g/kg (Hi) of soybean meal with rolled Mucuna seeds. Lambs were housed individually in metabolic crates and allowed ad libitum access to isocaloric (metabolizable energy=11.7 MJ/kg dry matter, DM) and isonitrogenous (CP = 146 g/kg, DM) diets for 14 d of adaptation and 7 d of total fecal collection. Fecal egg counts and coccidian oocyst scores were determined on d 14. Dry matter intake (1.7 kg/d versus 1.5 kg/d; P<0.05), CP digestibility (774 g/kg versus 714 g/kg DM; P<0.05) and N retention (28.0 g/d versus 20.4 g/d; P<0.01) were higher and amylase-pretreated neutral detergent fiber digestibility (617 g/kg versus 686 g/kg DM) was lower (P<0.05) in sheep fed SB versus Mucuna diets. However, supplementary protein source did not affect rumen pH, blood urea N or glucose concentration, or fecal egg counts. Increasing the level of Mucuna supplementation increased (P<0.05) level and efficiency of microbial protein synthesis, ruminal fluid acidity, total volatile fatty acid concentration, decreased (P<0.05) coccidian oocyst scores, and tended (P<0.10) to increase N retention. Therefore, SB is a better supplement than Mucuna to support performance of lambs. Nevertheless, Mucuna seeds are a promising CP supplement for situations where cost or availability precludes use of SB in ruminant rations.     

Effect of estrogen on the expression of GnRH and kisspeptin in the hypothalamus of rats during puberty

The aim of this study was to assess whether changes in kisspeptin and GnRH levels could be attributed to sex steroids at puberty onset. We used the ovariectomy (OVX) model in rats treated with 17β-estradiol (E₂; OVX + E₂), or oil (OVX + oil), and in intact rats treated with E₂ (intact + E₂) or oil only (intact + oil) to determine gene expression changes of Kiss1 and Gnrh1 in the hypothalamus and protein expression of kisspeptin and GnRH in the different areas of the hypothalamus. In the intact + E₂ and OVX + E₂ rats on the day of the onset of puberty, GnRH-immunoreactive (ir) cell numbers decreased (P < 0.05) in the arcuate nucleus but were increased in the preoptic area; Kisspeptin-ir cells increased (P < 0.05) in the arcuate nucleus, periventricular nucleus, and preoptic area; no difference (P > 0.05) was found in the paraventricularis nucleus for GnRH-ir or kisspeptin-ir cells. Additionally, levels of Kiss1 and Gnrh1 messenger RNA in the hypothalamus were significantly higher (P < 0.05) in the OVX + E₂ or intact + E₂ rats than in the OVX + oil or intact + oil animals, respectively. In the OVX + oil rats, OVX significantly increased (P < 0.05) levels of Gnrh1 and Kiss1 messenger RNA and the expression of GnRH and kisspeptin in the hypothalamus compared to intact + oil animals. These results suggest that kisspeptin and GnRH play major roles in modulating the activity of estrogen circuits at the onset of puberty.     

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.     

Protective role of scutellarin on LPS induced – Acute lung injury and regulation of apoptosis,oxidative stress and reduction of mitochondrial dysfunction

Lung fluid accumulation was determined using wet/dry lung mass ratio. Rats subjected to LPS-induced acute lung injury (2.8 ± 0.33, P < 0.05) presented with a significantly higher wet to dry lung weight ration ratio than sham rats (1.6 ± 0.23, P < 0.05). These results demonstrate that acutely inured rats' lungs were oedematous. On the other hand, treatment with scutellarin alone and in combination with a JNK inhibitor, SP600125, both significantly attenuated pulmonary edema as shown via reduced wet/dry lung mass ratios (1.7 ± 0.09 and 1.8 ± 0.23; P < 0.05, respectively). These results showed that the interventions were effective against LPS-induced edema of the lungs. However, the difference between treatment groups' weight ratios was not statistically significant (P > 0.05). In the sham control rats, the levels of ROS and SOD production were maintained at a low and at a high concentration, respectively (P < 0.05). However, following LPS infusion, the ROS levels skyrocketed while that of SOD decreased significantly relative to the control rats (P < 0.05). Furthermore, we noted that pre-treatment with scutellarin reduced the ROS levels in LPS-injured rats while the SOD was increased to near control levels (P < 0.05). Moreover, the combined effect of scutellarin and JNK inhibitor SP600125 on the levels of ROS and the SOD activity followed a similar trend to that of scutellarin alone albeit with a lower magnitude of change. Our results also showed that the combinatorial treatment was not significantly different from scutellarin alone in terms of influence on the levels of ROS production and SOD activity (P > 0.05). The effect of Scutellarin on broncho-alveolar lavage fluid (BALF) cytokine secretion The expression of interleukins-1β, ?18 and ?6 in the broncho-alveolar lavage fluid were significantly upregulated by LPS infusion (P < 0.05). The rise was, however, attenuated via pre-treatment with scutellarin only or in conjunction with SP600125, a JNK inhibitor (all P < 0.05). On the contrary, we observed that LPS injection caused a reduction of interlekins ?4 and ?10 secreted in the BALF. Pre-treatment with scutellarin alone (P < 0.05) and not in combination with SP600125 or SP600125 was able to significantly reverse this noted down-regulation (all P > 0.05).     

Effects of graded dose dietary supplementation of Piper betle leaf meal and Persicaria odorata leaf meal on growth performance,apparent ileal digestibility,and gut morphology in broilers

Due to antimicrobial resistance and the public health hazard of antibiotic growth promoters, there is a grave need to find potential alternatives for sustainable poultry production. Piper betle (PB) and Persicaria odorata (PO) are herbs, which have been reported for antimicrobial, antioxidant, and anti-inflammatory properties. The present study aimed to estimate the influence of different dose supplementation of Piper betle leaf meal (PBLM) and Persicaria odorata leaf meal (POLM) on growth performance, ileal digestibility and gut morphology of broilers chickens. A total of 210 one day-old broiler chicks were randomly grouped into 7 treatments, and each treatment group has 3 replicates (n = 10) with a total number of 30 chicks. The treatments included T₁ control (basal diet (BD) with no supplementation), T₂ (BD + 2 g/kg PBLM); T₃ (BD + 4 g/kg PBLM), T₄ (BD + 8 g/kg PBLM), T₅ (BD + 2 g/kg POLM), T₆ (BD + 4 g/kg POLM), T₇ (BD + 8 g/kg POLM). Growth performance, gut morphology and ileal digestibility were measured. Except for T₄ (8 g/kg PBLM), graded dose inclusion of PBLM and POLM increased (P < 0.05) the body weight gain (BWG), positively modulated the gut architecture and enhanced nutrient digestibility in both stater and finisher growth phases of broiler chickens. Birds fed on PBLM 4 g/kg (T₃), and POLM 8 g/kg (T₇) had significantly higher (P < 0.05) BWG with superior (P < 0.05) feed efficiency in the overall growth period. Chickens fed on diets T₃ and T₇ had longer (P < 0.05) villi for duodenum as well as for jejunum. Furthermore, the birds fed on supplementations T₃ and T₇ showed improved (P < 0.05) digestibility of ether extract (EE), and dry matter (DM) compared to the control group. However, least (P < 0.05) crude protein (CP) digestibility was recorded for T_4. In conclusion, dietary supplementations of PBLM 4 g/kg and POLM 8 g/kg were positively modulated the intestinal microarchitecture with enhanced nutrient digestibility, resulted in maximum body weight gain, thus improved the growth performance of broiler chickens.     

Seasonal modulation of immunity by melatonin and gonadal steroids in a short day breeder goat Capra hircus

Role of melatonin in regulation of immunity and reproduction has never been studied in detail in goats. The aim of the present study was to explore hormonal regulation of immunity in goats with special reference to melatonin. Plasma of male and female goats (n = 18 per sex per season) was processed for hormonal (estrogen, testostrone, and melatonin) and cytokine (interleukin [IL-2], IL-6, and tumor necrosis factor α) measurements during three seasons, i.e., summer, monsoon, and winter. To assess cell-mediated immune response, percent stimulation ratio of thymocytes was recorded during three seasons. To support and establish the modulation by hormones, Western blot analysis for expressions of melatonin receptors (MT1, MT2), androgen receptor, and estrogen receptor α and estimations of marker enzymes, arylalkylamine N-acetyltransferase for melatonin and 3β-hydroxysteroid dehydrogenase activities for steroidogenesis were performed in thymus. All the hormones and cytokines were estimated by commercial kits. Biochemical, immunologic, and Western blot analyses were done by standardized protocols. We noted a significant increase in estrogen and testosterone levels (P < 0.05) in circulation during monsoon along with melatonin (P < 0.05) presenting a parallel relationship. Expressions of melatonin receptors (MT1 and MT2) in thymus of both the sexes were significantly high (P < 0.01) during winter. Estrogen receptor α expression in female thymus was significantly high during monsoon (P < 0.05). However, androgen receptor showed almost static expression pattern in male thymus during three seasons. Further, both arylalkylamineN-acetyltransferase and 3β-hydroxysteroid dehydrogenase enzyme activities were significantly high (P < 0.05; P < 0.01, respectively) during monsoon. These results suggest that there may be a functional parallelism between gonadal steroids and melatonin as melatonin is progonadotrophic in goats. Cell-mediated immune parameters (percent stimulation ratio of thymocytes) and circulatory levels of cytokines (IL-2, IL-6, and tumor necrosis factor α) were significantly high (P < 0.01) during monsoon. In vitro supplementation of gonadal steroids to T-cell culture suppressed immunity but cosupplementation with melatonin restored it. Further, we may also suggest that reproductive and immune seasonality are maintained by variations in circulatory hormones and local synthesis of melatonin and gonadal steroids. These functional interactions between melatonin and gonadal steroid might be of great importance in regulating the goat immunity by developing some hormonal microcircuit (gonadal steroid and melatonin) in lymphatic organs.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection,helminthiasis, thrombosis and neuropharmacological disorders

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds’ phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 μg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2′-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.     

Evaluation of analgesic,antiamnesic and antidiarrheal potentials of Medicago denticulata extract using animal model

The analgesic, antidiarrheal, and neuro-pharmacological potentials of Medicago denticulata leaves extract were screened in animal models. Potential analgesic response was noted (*P < 0.05, ^**P < 0.01, ^***P < 0.001) in formalin, acetic acid and heat-induced pain models in a dose-dependent manner. Maximum activity by means of writhing inhibition was documented for Medicago denticulata at 300 mg/kg that was found to be 71.79% (17.43 ± 1.31). In first phase, the Medicago denticulata at a dose of 150 and 300 mg/kg showed analgesic activity and reduced the pain by 54.18% (18.39 ± 1.67) and 62.90% (14.89 ± 1.56), respectively. In second phase, the Medicago denticulata at a dose of 150 and 300 mg/kg showed analgesic activity and reduced the pain by 69.48% (19.78 ± 1.44) and 70.89% (18.86 ± 1.58), respectively. In hot plate method, the Medicago denticulata at a dose of 150 and 300 mg/kg showed the maximum response of 61.16% (8.47 ± 1.23) and 67.39% (10.09 ± 1.04), respectively at 60 min. Scopolamine significantly reduces spontaneous alteration in Y-maze model for antiamnesic activity. Medicago denticulata significantly increased the discrimination index in a dose-dependent manner using novel object recognition test (NORT) model. Exploration time in sec for the novel object was increased significantly (P < 0.001) by donepezil decreased for familiar one with a discrimination index (DI) of 62.18%. Medicago denticulata significantly increased the discrimination index by 60.86% and 57.24% at 300 and 150 mg/kg b.w, respectively. The lowest DI of 53.80% at 75 mg/kg was observed in comparison to the amnesic group. The Medicago denticulata significant decreased the elevated levels of acetylcholinesterase (AChE) and malondialdehyde (MDA and enhancing level of acetylcholine (ACh), superoxide dismutase (SOD) and catalase (CAT) acting as an antioxidant agent. Medicago denticulata reduced the total number of diarrheal feces to lesser extent at dose-dependent manner. From the study results, it is suggested that the Medicago denticulata extract possess good analgesic and antiamnesic activity however the antidiarrheal effects of plant were negligible. In the current study, the traditional use of the plant as a source of medicine has been validated.     

Effect of inoculum and pepsin–pancreatin hydrolysis on fibre fermentation measured by the gas production technique in pigs

Two experiments were undertaken to adapt the in vitro gas production technique in syringes, used for ruminants, to fibre fermentation studies in the large intestine of pigs.In a first experiment, two inocula (faeces and large intestine content) were compared at four dilution levels in a buffer solution (0.025, 0.05, 0.1 and 0.2 g ml⁻¹) with two substrates: wheat bran and sugar–beet pulp. The accumulated gas produced over 72 h was modelled and the kinetics parameters compared. The time to half asymptote was lower for the intestinal inoculum (5.5 versus 8.0 h, P<0.02), but the 2 inocula yielded similar fractional rates of degradation (0.16 h⁻¹) and gave equal final gas production (252 ml g⁻¹ substrate). No interaction (P>0.05) was observed between inocula and substrates. The dilution of the samples in the buffer solution increased (P<0.001) the lag time (from 0.9 to 2.1 h for dilution rates ranging from 0.2 to 0.025 g ml⁻¹, respectively) and decreased (P<0.001) the rates of substrate degradation (from 0.18 to 0.13 h⁻¹).A second experiment aimed to study the effect of an in vitro pepsin–pancreatin hydrolysis of the sample prior to the gas test. Six substrates were tested: maize, wheat bran, sugar–beet pulp, lupins, peas and soybean meal. The enzymatic hydrolysis affected (P<0.001) the kinetics parameters and the ranking order of the fermented substrates. The lag times also increased for all ingredients. The rate of degradation decreased when peas, lupins, maize and wheat bran were hydrolysed (P<0.001) but it increased with soybean meal (P=0.014) and sugar–beet pulp (P<0.001). Final gas production increased with peas and soybean meal (P<0.001), remained unchanged for lupins and decreased for the other substrates (P<0.001).In conclusion, the method using faeces as a source of microbial inoculum is reliable to characterise the fermentation kinetics of ingredients in the large intestine of pigs. However, it is important to hydrolyse the substrates with pepsin and pancreatin before the gas tests.     

Effects of Epichloë gansuensis on root-associated fungal communities of Achnatherum inebrians under different growth conditions

This study was conducted to investigate how Epichloë gansuensis endophyte and soil disturbance affect root-associated fungi (RAF) of Achnatherum inebrians (drunken horse grass, DHG), using Illumina sequencing techniques. The rhizosphere soil of wild endophyte-infected (W-EI) DHG had significantly (P < 0.05) higher available phosphorous and potassium, total organic matter, ammonium and nitrate nitrogen than cultivated soil. In addition, the rhizosphere soil of endophyte-infected DHG had significantly (P < 0.05) lower pH and nitrate nitrogen, and higher available phosphorous, than endophyte-free DHG under cultivated conditions. The sequencing provided a total of 54,413 sequences and these were assigned into 190 operational taxonomic units (OTUs) with 97% similarity. Ascomycota was the most dominant phylum in roots of three DHG populations. W-EI DHG had significantly (P < 0.05) higher RAF diversity than cultivated endophyte-infected (C-EI) DHG. The presence of endophyte significantly (P < 0.05) decreased RAF diversity under cultivation. The principal component analysis (PCA) and sample similarity analysis results indicated that both endophyte and soil disturbance could bring changes to RAF community composition. The RDA results demonstrated the RAF of W-EI DHG were positively correlated with soil properties, and the RAF of cultivated DHG roots were negatively correlated with soil properties. This study demonstrated that both endophyte and soil disturbance resulted in changes to the RAF communities.     

Effects of Acrylamide on the Nervous Tissue Antioxidant System and Sciatic Nerve Electrophysiology in the Rat

To investigate the time-dependent effects of acrylamide (ACR) on the antioxidative status in rat nerve tissues, adult male Wistar rats were given ACR (40 mg/kg, i.p., 3 times/week) for 2, 4, 6 and 10 weeks, respectively. The time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues were investigated. The electrophysiology indices (nerve conduction velocity, NCV; compound action potential duration, CAPD; compound action potential amplitude, CAPA; compound action potential latency, CAPL) in the sciatic nerve were determined using BL-420E Biologic Function Determining System. The results showed that MDA levels increased significantly (P < 0.05) in nerve tissues, while GSH levels markedly decreased (P < 0.05) in a time-dependent manner. SOD activity (in the spinal cord and sciatic nerve) and GR activity (in the sciatic nerve) increased significantly after 4 weeks ACR treatment (P < 0.01), but then decreased (P < 0.05). The anti-ROS activity in the sciatic nerve was markedly decreased at the end of week 6 and 10 (P < 0.01). The above indices changed most in the sciatic nerve. The levels of GSH, MDA and anti-ROS in rat sciatic nerve were in high correlation (P < 0.05, |r| > 0.80) with the electrophysiology indices according to the exposure time. Thus, ACR-induced neurotoxicity may be associated with the enhancement of lipid peroxidation and reduction of the antioxidative capacity. Depletion of neural GSH level might be one of the primary events in ACR-induced neuropathy. Ying-Jian Zhu and Tao Zeng––These authors contributed equally to this work.