Effects of dietary Moringa oleifera leaf extract on semen characteristics, fertility, and hatchability in aged broiler breeder roosters.

Declining semen quality will have a negative impact on the fertility of aged roosters. Various factors influence this decrease in quality. This study was conducted to investigate the effects of different levels of Moringa plant extract on semen characteristics, fertility, and hatchability in aged broiler breeder roosters. A total of 24 roosters were fed 1 of 4 dietary supplements for 10 wk: Control, 100 µL/kg (Moringa oleifera leaf extract [MOLE]-100), 200 µL/kg (MOLE-200), or 400 µL/kg body weight (MOLE-400) of Moringa oleifera extract. Results showed supplementation with MOLE-200 significantly improved (P < 0.05) semen concentration, total motility, progressive motility, sperm membrane integrity compared to other treatments. However, semen volume and body weight were unaffected (P > 0.05). Sperm lipid peroxidation, as indicated by malondialdehyde concentration, was lowest in MOLE-200. There was a significant difference observed among the treatments in terms of total antioxidant capacity (TAC) results. The testosterone concentration in the MOLE-200 treatment was significantly higher than the other treatments (P < 0.05). However, no significant differences were observed in the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) hormones among the experimental treatments. Fertility and hatchability rates were measured at the end of the trial. Fertility, defined as the number of fertilized eggs, was greatest in the MOLE-200 treatment compared to the other treatments. Similarly, hatchability (hatched chicks/fertilized eggs %) was highest at 88.02% for MOLE-200. In conclusion, dietary supplementation with M. oleifera extract improved semen quality, fertility, and hatchability in aged broiler breeder roosters.

Effect of exogenous genistein on osteogenic differentiation of adipose-derived mesenchymal stem cells in laying hens.

Previous literature revealed that genistein might play a preventive role in osteoporosis. Therefore, we aimed to evaluate the effect of genistein on the osteogenic potency of laying hens' adipose-derived stem cells (LHASCs). The viability of LHASCs after isolation was investigated on tissue culture plastic (TCP) under exposure to genistein up to 50 µg/mL by MTT assay. Our preliminary result revealed that LHASCs cultured under genistein exposure up to 20 µg/mL are feasible. Then, we evaluated the osteogenic induction of LHASCs under exposure to 0, 10, and 20 µg/mL genistein. The Alizarin Red staining confirmed the calcium deposition. Our findings showed that osteogenic differentiation under exposure to 20 µg/mL genistein led to higher ALP activity and more calcium content. We then tried to see the probable additive effect of the genistein-plus Poly-L-lactic acid (PLLA) scaffold on the cell viability and osteogenic capacity of LHASCs. For this, cells were cultured on a PLLA scaffold and exposed to 20 µg/mL genistein. Cell growth rate, as indicated by the MTT assay, revealed no differences between the groups. LHASCs cultured on a genistein-plus PLLA scaffold showed higher ALP activity and more calcium content. The expressions of Osteocalcin, COL1A2, ALP, and Runx2 genes were increased in the genistein-plus PLLA group as compared with PLLA and TCP groups. Adequate proliferation rates and higher expression of osteogenic markers provide genistein as a suitable substrate to support the proliferation and differentiation of LHASCs. Genistein supports osteogenic induction as a further positive effect if such a PLLA scaffold is available.

Alpha-linolenic acid alleviates the detrimental effects of lipopolysaccharide during in vitro ovine oocyte development.

During the transition period and early lactation of ruminants with higher production, the reproductive organs are exposed to various stressors, like inflammation stimulators such as lipopolysaccharides (LPS), as a consequence of high concentrate consumption. In this study, we aimed to determine the probable potential of α-linolenic acid (ALA) in alleviating LPS-induced effects in ovine oocytes in vitro as well as the underlying controlling mechanisms. Different concentrations of LPS (0, 0.01, 0.1, 1, and 10 µg/mL) were added to the oocyte maturation medium to evaluate its effect on oocyte developmental competence. Likewise, different concentrations of ALA (0, 10, 50, 100, and 200 µM/mL) were added to the maturation medium to define its effects on oocyte developmental competence. Accordingly, a combination of ALA and LPS in a dose-dependent manner was added to the maturation medium to elucidate their effect on oocyte developmental competence and uncover any possible potential of ALA to alleviate the detrimental effect induced by the presence of LPS. The expressions of candidate genes were measured in mature oocytes treated either with ALA, LPS, or ALA plus LPS. Adding LPS to the maturation medium decreased the cleavage rate of the treated oocytes, and those oocytes reached the blastocyst stage at a lower rate. Adding ALA to the maturation medium in the presence of LPS alleviated the detrimental effects of LPS in a dose-dependent manner, which ultimately led to higher cleavage and blastocyst formation. A higher expression of Trim26, GRHPR, NDUFA, PGC-1α, SOD, CS, SDH, p53, and CAT was observed in LPS-treated oocytes compared with the ALA and control groups. Additionally, CS and CAT transcripts were down-regulated in oocytes in LPS plus ALA-treated group compared to that of the LPS-treated group. These findings revealed that ALA has the potential to alleviate the detrimental effects induced by LPS on in ovine oocytes during maturation in vitro. Thus, LPS-detrimental effect and ALA-preventing mechanisms seem to be regulated through the expression of genes involved in mitochondrial biogenesis and function, oxidative stress, and antioxidant systems.

A soy lecithin nanoparticles-based extender effectively cryopreserves Holstein bull sperm.

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL.

A nutrigenomics approach to study the effects of ω-3 fatty acids in laying hens under physiological stress.

Supplement of ω-3 fatty acids can decrease the harmful effects of stress. However, the potential molecular mechanisms that are modulated by dietary ω-3 fatty acids in laying hens under stress remain unknown. Hence, RNA-sequencing (RNA-Seq) technology was used to gain new insights into different gene expression profiles and potential pathways involved in response to stress in the liver of 35-week-old Lohmann LSL-Lite laying hens supplemented with ω-3. Three groups including control (non-stress), stress, and stress_ω-3 fatty acids (three layers per each group) were applied. A total of 1,321 genes were detected as differentially expressed genes of which 701, 1,049, and 86 DEGs belonged to stress vs. control, stress_ω-3 vs. control, and stress vs. stress_ω-3 pairwise comparisons, respectively. Gene ontology and KEGG pathway analysis indicated that the DEGs were enriched in particular regulation of steroid and cholesterol biosynthetic process, fatty acid degradation, AMPK signaling pathway, fatty acid biosynthesis, and immune response. Our data represented a promising approach regarding the importance of ω-3 as anxiolytic and anti-stress. In this context, UNC13B and ADRA1B genes were downregulated in the stress_ω-3 group compared to the stress group, which are associated with decreased activity of glutamatergic stimulatory neurons and probably play important role in facilitating the response to stress. This study extends the current understanding of the liver transcriptome response to physiological stress, and provides new insights into the molecular responses to stress in laying hens fed a diet supplemented with ω-3 fatty acids.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences.

Low temperatures during cryopreservation activate a cascade of changes, which may lead into irreversible damage and reduction of the fertilization potential, including the process of premature capacitation. The aim of our study was to evaluate the range of cell damage following the cryopreservation process and possible activation of cryocapacitation in bovine spermatozoa. For the experiments semen samples were obtained from 30 sexually mature Holstein bulls. Within the analysed parameters, we focused on the functional activity, structural integrity, capacitation status and oxidative profile. The samples were divided into three experimental groups, control (CTRL), in vitro capacitated (CAP) and cryopreserved (CRYO). Based on the collected data, there was a significant decrease in the sperm motility, mitochondrial membrane potential and concentration of cyclic adenosine monophosphate in the CRYO group when compared to CAP and CTRL (P<0.0001). A significant decrease (P<0.01; P<0.0001) in the membrane and acrosome integrity as well as DNA fragmentation index and a significant increase (P<0.0001) of necrotic cells were observed in the CRYO group. Following capacitation, a significant increase (P<0.01; P<0.0001) was recorded in the number of cells which underwent the acrosome reaction in the CRYO group against CAP and CTRL. Changes in the oxidative profile of the CRYO group indicates an increase (P<0.0001) in the reactive oxygen species generation, except for the superoxide radical, which was significantly higher (P<0.0001; P<0.001) in the CAP group in comparison with CRYO and CTRL. In summary, premature capacitation may be considered a consequence of cryopreservation and the assessed parameters could serve as physical markers of cryogenic damage to bovine spermatozoa in the future.

Aflatoxin B1 impairs in vitro early developmental competence of ovine oocytes.

Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 µM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 µM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 µM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 µM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 µM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.

Effects of different ultrastructures of lecithin on cryosurvival of goat spermatozoa.

This study was to evaluate the effects of two different ultrastructures of lecithin including nanoparticles (NPE mostly nanomicelles) and lecithin nanoliposome (NLE) with egg yolk extender (EYE) on goat sperm cryopreservation. Semen samples were collected from 6 goats, then pooled, diluted and then frozen. Motility and motion parameters, plasma membrane integrity and functionality, morphology, apoptosis status (Annexin V-PI), acrosome integrity, DNA fragmentation and in vitro fertilisation were assessed. Total motility and most motion parameters were higher in EYE (p < .05) compared with the two lecithin extenders, while there were no significant differences between NLE and NPE. NLE and NPE had higher values for viable spermatozoa (Annexin V-PI) (p < .05) compared with EYE. The highest value for dead spermatozoa was observed in EYE (p = .08). A higher percentage of DNA fragmentation (p < .05) was detected in EYE compared with NPE. Plasma membrane integrity and functionality, morphology, acrosome integrity and fertility of spermatozoa indicated no significant differences between extenders. Data suggested that ultrastructural changes of lecithin (micelles versus. liposome) could not improve the sperm cryosurvival of goat spermatozoa. Moreover, we cannot also claim that lecithin-based diluent supplies better protection compared with the egg yolk in goat.

Effects of dietary L-carnitine on puberty indices in the young breeder rooster.

The aim of current study was to investigate the effect of dietary L-Carnitine (LC) in immature roosters on reproductive hormones, lipid profile and testicular histology at the time of maturity. Eighteen 12-wk-old breeder roosters (Ross 308) of similar weights were randomly allocated into 3 dietary treatments (LC-0: basic diet, LC-250: basic diet + 250 mg LC/kg of diet, LC-500: basic diet + 500 mg of LC/kg of diet) in 6 replicates. The feeding program and photoperiod regimen were performed based on ROSS 308 management handbook. Dietary LC supplementation markedly improved testicle weight and testicle index (p < 0.05). Comb height was also affected by LC supplementation (p < 0.05). The testicle weight and index, comb height, and shank lengths improved linearly with increasing levels of dietary LC (p < 0.05). The LC-250 and LC-500 diets significantly improved the number of sertoli cells (NSC), height epithelium seminiferous tubules (HEST), seminiferous tubules diameter (STD), spermiogenesis index (SI) and tubular differentiation index (TDI) of rooster's testis tissue (p < 0.05). The number of seminiferous tubules (NST) was affected by of the amount of LC (p < 0.05). The roosters on the LC-250 mg/kg diet had longer HEST compared to roosters that received the LC-500 mg/kg diet (p < 0.05). Testicular histology parameters increased in a linear and quadratic manner in response to increasing levels of LC (p < 0.05). Dietary LC significantly increased (p < 0.05) plasma concentrations of testosterone, GnRH, LH, FSH and High-Density Lipoprotein (HDL), but reduced the plasma concentration of Low-Density Lipoprotein (LDL). However, no significant differences were observed between LC-250 and LC-500 groups in these parameters. Plasma testosterone, GnRH, LH, LDL and HDL were affected in a linear and quadratic manner in response to increasing levels of LC (p < 0.05). Similarly, FSH increased linearly with increasing dietary LC (p < 0.05). Thus, adding up to 250g of LC per kg of the rooster chicken can improve reproductive hormones, blood lipids and testicular histology parameters at the time of maturity.

Omega-3 fatty acids reduce the negative effects of dexamethasone-induced physiological stress in laying hens by acting through the nutrient digestibility and gut morphometry.

In this study, the effects of omega-3 fatty acids on egg production, nutrients digestibility, eggs yolk lipid peroxidation, and intestinal morphology in laying hens under physiological stress were investigated. Ninety-six 35-wk-old Lohmann LSL-Lite laying hens were used in 2 × 3 factorial arrangement with 2 levels of dexamethasone (DEX) (0 and 1.5 mg/kg of the diet) and 3 levels of omega-3 fatty acids (0, 0.24, or 0.48% of the diet) in a completely randomized design. At 41 wk of age, the stress groups were continuously fed with a DEX 1.5 mg/kg diet for 1 wk. Egg production, egg mass, feed intake, egg weight, and feed conversion ratio were recorded. In addition, the AME, digestibility of CP, crude fat (CF), and organic matter were measured during the stress induction period. At the end of 41 wk of age, malondialdehyde and cholesterol concentrations in the egg yolk and intestinal morphology were investigated. The results showed that egg production, egg mass (P < 0.0001), egg weight (P = 0.043), and BW (P = 0.0005) were lower in DEX layers. Feed intake was reduced by the interaction between DEX and omega-3 fatty acid (P = 0.042). Malondialdehyde value (P = 0.002) and cholesterol concentration (P = 0.001) in egg yolk increased by DEX administration. The combination of DEX administration and omega-3 fatty acids supplementation was found in the indices of intestinal morphology such as villus height and width and crypt depth (P < 0.05). Administration of DEX decreased the CP digestibility (P < 0.0001) and AME (P = 0.006). Digestibility of CF and AME in the group of 0.48% omega-3 fatty acids were higher (P < 0.05) than those of 0 and 0.24%. In conclusion, we found that dietary omega-3 fatty acids had beneficial effects on gut morphology and nutrient digestibility in laying hens under physiological stress. However, they could not alleviate the negative effects of physiological stress on performance.

In Vivo and In Vitro Evaluation of Bull Semen Processed with Zinc (Zn) Nanoparticles.

Defective sperms cause fertilization failure under both in vivo and in vitro conditions. Therefore, providing optimal conditions during semen storage is a prerequisite for maintaining viability. The current study investigated bull semen quality in vitro and in vivo when zinc (Zn) nanoparticles were used as antioxidant during semen processing and cryopreservation. In total, 32 ejaculates were collected from four Holstein bulls. All ejaculates were pooled and diluted with Bioxcell-extender containing 0 (control group), 10-6, 10-5, 10-4, 10-3, and 10-2 M of Zn nanoparticles. Several physical and biochemical sperm parameters were determined after freeze-thawing process. In vitro embryo development rate and pregnancy rate were monitored after in vitro fertilization or artificial insemination using semen treated with Zn nanoparticles. Plasma membrane integrity was improved (P < 0.05) in bull semen treated with 10-6 M (69.3%), and 10-2 (62.4%) of Zn nanoparticles compared to untreated group (51.3%). In addition, proportions of live spermatozoa with active mitochondria were increased (P < 0.05) in semen supplemented with Zn nanoparticles at concentration of 10-6 M (67.3%), and 10-2 (85.3%) compared to control group (49.8%). Moreover, the level of MDA was lower (P < 0.05) in semen with Zn nanoparticles at 10-6 M (2.97 mol/mL) and 10-2 (2.7 mol/mL) concentrations than control semen samples (3.77 mol/mL). However, sperm total and progressive motility, sperm viability, DNA fragmentation, and pregnancy rate were not affected by treatment of semen with Zn nanoparticles. On the other hand, supplementation of in vitro maturation media with 10-6 M Zn nanoparticles has increased blastocyst rate (P < 0.05) compared to other experimental groups, while addition of Zn nanoparticles-treated sperm during in vitro fertilization did not affect embryo development rate. In conclusion, supplementation of Zn nanoparticles to semen has improved its quality without affecting embryo development rate in vitro. However, in vitro embryo development rate was increased when Zn nanoparticles were supplemented to IVM media. This support the notion of Zn nanoparticles beneficial action on improving bovine gametes quality without affecting pregnancy rate.

Manipulation of fatty acid profiles in roosters' testes, alteration in sexual hormones, improvements in testicular histology characteristics and elevation sperm quality factor by L-carnitine.

The present study was designed to investigate the effects of different levels of l-carnitine (LC) on sperm quality factor (SQF), alterations in testis fatty acid profiles, testicular histology and reproductive hormones in young roosters. Eighteen broiler breeders (Ross 308) weighed at 3 months of age. They were randomly classified while each group had six birds. There were three experimental groups based on the LC concentrations (i.e. LC-0, LC-250, LC-500 mg per kg of diet). After two weeks of adaptation, semen samples were collected and evaluated for seminal attributes every two weeks (from week 24 to week 34). At the end of the experiments, four roosters from each treatment group were sacrificed in order to analyze testicular histology, testis fatty acid profiles and reproductive hormones. Supplementing the diet with two of the LC levels for 22 weeks caused significant rise in sperm concentration, viability and SQF compared to that of the control group (P < 0.05). Quadratic analysis in terms of number of seminiferous tubules and spermatogenesis index were significant (P<0.05). Tubular differentiation index improved linearly by the increasing levels of LC supplementation (P<0.01). The analysis of fatty acid profiles showed that LC significantly (P < 0.05) reduced the percentages of C14:0, C21:0, total saturated fatty acids, total odd-chain fatty acids and n-6/n-3 ratio. Moreover, LC significantly increased the percentage of C20:5n-3 (Eicosapentaenoic acid; EPA) (P < 0.05). Analysis of the correlation coefficient revealed that the SQF is in consistency with EPA (r = 0.98; P < 0.04). In contrast, SQF negatively and significantly correlates with odd-chain fatty acids (r = - 0.99; P < 0.001). The desaturation index for C16 fatty acids (16:1cis/C16:0) negligibly increased linearly as LC was added to the diet (P < 0.05). Furthermore, LC caused the roosters to have significant (P < 0.05) high levels of total testosterone and FSH concentrations. The concentration of LH in different treatment groups, however, turned out to be similar in response to the different levels of LC. In conclusion, long-term supplementation of rooster diet with LC can have beneficial effects on SQF and testis histology. The benefits include alterations in testicular histology, reproductive hormones and testicular fatty acid profiles.

Dietary L-carnitine affects the expression of genes involved in apoptosis and fatty acid metabolism in rooster testes.

Thirty-six 12-week-old breeder roosters (Ross 308) were randomly allocated into three groups to receive L-carnitine (LC): LC-0, LC-250 or LC-500 mg/kg of diet to evaluate the effects of dietary LC on the expression of apoptotic-related genes and desaturases and elongase mRNA transcript levels, in the cockerel testicles. Alteration of Bak (Bcl2 antagonist/killer), Bcl2, Cas3, Cas8, Cas9, Elovl2, Elovl4, Elovl5, Fads1, Fads2 and Scd expression at 24 and 34 weeks of age was compared by real-time quantitative PCR. The expression of Bcl2 and Elovl5 was significantly up-regulated (p < .05), while Cas8 expression (p < .05) and Bak/Bcl2 ratio were reduced (p < .02) in the cockerel testicles at 24 weeks of age. Although Bak mRNA abundance decreased by dietary LC, Bak/Bcl2 ratio was not affected by the treatments at 34 weeks of age. The expression of Cas3 was down-regulated, while Fads2 was up-regulated in the cockerel testicles by dietary LC at 34 weeks of age (p < .05). The results demonstrate the beneficial effects of LC supplementation in suppression of the Bak/Bcl2 ratio by altering Bak and Bcl2 mRNA abundance and, ultimately, prevention of apoptosis. Furthermore, LC increased the expression of Elovl5 and Fads2 genes which are involved in the metabolism of long chain fatty acids.

Transcriptional profile of ovine oocytes matured under lipopolysaccharide treatment in vitro.

Lipopolysaccharide (LPS) derived from gram negative bacteria cell wall is known to cause ruminal acidosis and/or infectious diseases such as metritis and mastitis which has a significant negative impact on the reproductive performance. This study aimed to investigate the effect of LPS on oocyte maturation and subsequent development in vitro. Ovine cumulus oocyte complexes (COCs) were matured in a medium supplemented with 0 (control), 0.01, 0.1, 1 and 10 µg/mL LPS. Nuclear maturation, cleavage and blastocyst rate, mitochondrial membrane potential (ΔΨm), intracellular reactive oxygen species (ROS) content and changes to the transcript abundance were evaluated. In case of the maturation rate, the percentage of oocytes reaching the MII stage was lower following exposure to 10 µg/mL LPS in comparison to the control group (P < 0.05). Moreover, the blastocyst rate decreased in case of 1 and 10 µg/mL LPS when compared to the control group (P < 0.05). ROS overproduction accompanied by a decreased ΔΨm were recorded in LPS treated oocytes in comparison to the control group (P < 0.05). The 3' tag digital gene expression profiling method revealed that 7887 genes were expressed while only seven genes exhibited changes in the transcript abundance following exposure to LPS. Tripartite motif containing 25 (TRIM25), Tripartite motif containing 26 (TRIM26), Zona Pellucida glycoprotein 3 (ZP3), Family with sequence similarity 50-member A (FAM50A), Glyoxalate and hydroxy pyruvate reductase (GRHPR), NADH ubiquinase oxireductase subunit A8 (NDUFA8) were down-regulated (P < 0.05), while only Centrin 3 (CETN3) was up-regulated (P < 0.05). Our findings show that LPS has undesirable effects on the maturation competence of ovine oocytes and subsequent embryo development. In addition, the transcriptomic profiling results may shed more light on the molecular mechanisms of LPS-induced infertility in ruminants.

Effects of increasing lipopolysaccharide concentrations on in vitro developmental competence of ovine oocytes.

Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewe's ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.

Effect of dietary supplementation of whole flaxseed on sperm traits and sperm fatty acid profile in aged broiler breeder roosters.

The objective of this study was to evaluate the effect of dietary supplementation of whole flaxseed on sperm traits and sperm fatty acid profile in aged broiler breeder roosters. Twelve Ross 308 broiler breeder roosters (age: 52 weeks; weight: 4,900 ± 210 g) haphazardly allotted to three dietary treatments (each treatment contained four replicates and one bird in each replicate) for six weeks. Treatments were different levels of flaxseed (0% flaxseed [GFL0], 2% flaxseed [GFL2] and 4% flaxseed [GFL4]). The feed intake quadratically decreased (p < .05) with increasing whole flaxseed levels for the period (58 to 60 weeks). Sperm traits (semen volume and sperm concentration, sperm total and forward motility, sperm viability and morphology, sperm plasma membrane functionality) were evaluated every two weeks (four times), and sperm fatty acid profile was assessed at the end of the experiment. Semen volume, sperm concentration and sperm morphology were not affected by treatments. On week 60, GFL2 group showed a significantly lower percentage of total and progressive sperm motility and sperm membrane functionality in comparison with the control and GFL4 groups. Also, sperm viability was lower in GFL2 group compared with other groups on week 58 (p < .05). In terms of sperm fatty acid profile, GFL2 group significantly reduced the percentage of linoleic acid (C18:2 [n-6]) in comparison with other groups. However, any of the other fatty acids were not affected by dietary flaxseed. In conclusion, dietary supplementation of whole flaxseed could not improve the quality of aged broiler breeder roosters' sperm in this study, nor it could alter the sperm fatty acid profile; thus, it seems necessary to use some antioxidants such as vitamin E in the diet of aged broiler breeder roosters, when supplementing the diets with oils or oilseeds such as flaxseed.

Maternal supplementation with fish oil modulates inflammation-related MicroRNAs and genes in suckling lambs.

Dietary n-3 long-chain fatty acids (n-3 LCFA) have been shown to modify lipid metabolism and immune function. The objective of this study was to evaluate the effect of periparturient fish oil (FO) supplementation on the inflammation and metabolic health of ewes and their lambs at a molecular level. Prepartum ewes were fed control diet (CON, n = 12) or CON supplemented with 2% DM of calcium soap of FO (n = 12) from 28 days before until 21 days after parturition. The ewes were evaluated for plasma metabolites and milk composition. The experiment was followed by analyzing the relative transcript abundance of circulating microRNAs (miRNAs) in plasma and targeted miRNA/mRNA expression in peripheral blood mononuclear cells (PBMCs) in both ewes and lambs. FO treatment decreased prepartum feed intake (1812 ± 35 vs 1674 ± 33 g/day, P < 0.01), whereas the influence on plasma metabolites was negligible. Dietary FO supplementation decreased milk fat percentage (8.82 ± 0.49 vs 7.03 ± 0.45, P = 0.02) and reduced milk n-6/n-3 (P < 0.05). Also, it altered the expression of plasma-circulating miRNAs in both ewe and lamb (P < 0.05). Furthermore, maternal nutrition of FO downregulated the relative expression of miR-33a and miR-146b and transcript abundance of genes IL-1ß (0.41-fold) and NF-κB (0.25-fold) in lambs' PBMC. In conclusion, results showed that FO supplementation starting antepartum affects milk composition and circulating miRNA in dams and the inflammatory markers in lambs delivered by the supplemented ewes. These may provide a strategy to maintain immune balance during gestation and develop the immune system in lambs.

Comparison of two different antioxidants in a nano lecithin-based extender for bull sperm cryopreservation.

The objective of the present study was to assess the effect of two different antioxidants, enzymatic compared with non-enzymatic, in a nano lecithin-based extender on post-thaw bull sperm quality. Semen samples (n = 36) were collected from six bulls. In the first experiment, 11 different extenders were prepared by adding five quantities of vitamin E (α-tocopherol) as a non-enzymatic antioxidant (VE: 0.1, 0.2, 0.4, 0.6 and 1.0 mM), or four quantities of glutathione peroxidase (GPx) as an enzymatic antioxidant (GPx: 0.5, 1, 2 and 3 mM) to the extender. Other extenders were a Control 1 (C1: Extender with ethanol) and Control 2 (C2: Extender without ethanol). Sperm motility (CASA), plasma membrane functionality test (HOST) and lipid peroxidation (MDA) were assessed to determine the optimal treatment in the first experiment. In the second experiment, the optimally supplemented group from the first experiment (GPx-1) was compared to C2 group. Apoptotic-like changes (Annexin staining), mitochondrial activity (Rhodamine-123 staining), acrosome integrity (PSA staining), DNA fragmentation (SCSA test) and in vitro embryo production capacity were evaluated. In the first experiment, there were the greatest percentages of plasma membrane functionality and least MDA (P ≤ 0.05) in sperm diluted GPx-1 group. In the second experiment, percentage of live sperm, blastocyst formation and hatching rate were greater (P ≤ 0.05) in the GPx-1 group compared with C2 group. In conclusion, data indicate adding 1.0 mM GPx as an enzymatic antioxidant to the nano lecithin-based extender can improve post-thaw quality and in vitro fertility of bull sperm.

The effect of l-arginine and flaxseed on plasma testosterone concentration, semen quality and some testicular histology parameters in old broiler breeder roosters.

The objective of this study was to investigate the effect of dietary supplementation of l-arginine and flaxseed on testosterone and lipid parameters of serum and semen quality, as well as histological and morphometric parameters of testes in old broiler breeder rooster. Thirty six 52-week-old Ross 308 broiler breeder roosters with similar weights (4900 g ±â€¯210) were used for a 8-weeks trial period in a 3 × 3 factorial arrangement of dietary treatments that three levels of l-arginine (0.52, 0.68 and 0.83%) and flaxseed (0, 2 and 4%) with four replications in each. The ratio of left testicle weight/total testicular weight was significantly higher in A68F0 group. The maximum and minimum seminiferous tubule diameters were recorded for roosters fed no flaxseed and F2 flaxseed, respectively. The roosters fed A68 showed the highest serum testosterone. Seminal volume and sperm concentration, were not significantly affected by l-arginine × flaxseed interaction; however, sperm forward motility were significantly affected by arginine × flaxseed interaction (P < 0.05). A68F0 had significantly greatest tubular differentiation indices (TDI) in all of the treatments. Also, repopulation indices (RI) and spermiogenesis indices (SI) in A68F0 were significantly higher than other groups, but A52F0 and A52F4. However, testis index, testis density, serum lipids (cholesterol, LDL, HDL and HDL: LDL ratio), epithelium height of seminiferous tubules and number of Sertoli cells were not affected by the treatments. It is concluded that dietary supplementation of arginine (0.68%) has positive effects on blood testosterone, semen quality and spermatogenesis index of aged roosters.

Zinc silicate mineral-coated scaffold improved in vitro osteogenic differentiation of equine adipose-derived mesenchymal stem cells.

In current study we aimed to coat the PLLA scaffold with zinc (Zn) silicate mineral nanoparticles. Then, using equine adipose-derived stem cells (ASCs) we intended to compare the osteogenic induction potency of Zn silicate mineral-coated PLLA scaffold with uncoated PLLA scaffold and tissue culture plastic (TCPS). Adipose tissues were collected from 3 horses, and isolation of ASCs was achieved by enzymatic digestion. PLLA scaffold was successfully prepared using a phase separation method and coated with Zn silicate mineral nanoparticles. The coating efficiency was then characterized by scanning electron microscopy and further evaluated with the application of fourier transform infrared microscopic imaging. Viability and growth characteristics of ASCs on TCPS, uncoated and coated PLAA scaffolds were investigated by MTT assay. Alizarin Red staining was performed for determination of calcium deposition following the osteogenic induction. Furthermore, other common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content, as well as osteogenic (Runx2, ALP, osteonectin, and collagen I) marker genes were also evaluated. Our data showed that Zn silicate mineral nanoparticles was coated successfully on PLLA scaffold and such scaffold had no detrimental effect on cell growth rate as indicated by MTT assay. Moreover, ASCs that differentiated on Zn silicate mineral-coated PLLA scaffold indicated higher ALP activity, more calcium content, and higher expression of bone-related genes than that on uncoated PLLA scaffold and TCPS. Adequate proliferation rate and higher expression of osteogenic markers of stem cells, provides this scaffold as a suitable substrate to support proliferation and differentiation of ASCs in equine.