In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane.

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C - PKC; phospholipases A2 and Cζ - PLA2, PLCζ; soluble adenylyl cyclase 10 - sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 - Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

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.

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.

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.

OOgenesis_Pred: A sequence-based method for predicting oogenesis proteins by six different modes of Chou's pseudo amino acid composition.

Regarding to critical roles of oogenesis in formation of ova or unfertilized eggs from the oogonia by mitotic division and subsequent differentiation, the identification of oogenesis-related proteins is of great interest. However, the experimental determination of proteins involved in oogenesis is expensive, time consuming and labor-intensive. Therefore, a new powerful discriminating model is indispensable for classifying oogenesis/non-oogenesis-related proteins with high accuracy and precision. Hereby, for the first time we developed a support vector machine based oogenesis protein prediction method which differentiates oogenesis from non-oogenesis proteins. By means of informative protein physicochemical properties and in addition parameter optimization scheme, our method yields a robust and consistent performance. Our model achieved 87.68% and 84.82% prediction accuracy by five-fold cross validation test for datasets with 90% and 50% identity, respectively. The prediction model was also assessed using the independent dataset and yielded 91.62% and 85.38% prediction accuracy for datasets with 90% and 50% identity, respectively, which further demonstrates the effectiveness of our method. Moreover, by applying 10 different feature weighting methods, the more important protein features for oogenesis/non-oogenesis-related proteins discrimination, including serine and glycine frequency, quasi-sequence-order, pseudo-amino acid composition, distribution and conjoint triad, were determined. The success rates revealed that our model can be considered as a new encouraging and strong model for predicting proteins involved in oogenesis with appropriate performance. To enhance the value of the practical applications of the proposed method, we developed a standalone software for predicting oogenesis candidate proteins called OOgenesis_Pred. This software is the first predictor ever established for identifying oogenesis proteins. We also showed the capability of OOgenesis_Pred by making oogenesis-related proteins prediction for some of the oogenesis candidate proteins. It is anticipated that OOgenesis_Pred will become a powerful tool for future proteomic studies related to oogenesis.

Fat harvesting site is an important determinant of proliferation and pluripotency of adipose-derived stem cells.

To define the optimal fat harvest site and detect any potential differences in adipose-derived stem cells (ASCs) proliferation properties in camels, aspirates from the abdomen and hump sites were compared. Obtained results revealed that ASCs from both abdomen and hump exhibited spindle-shaped and fibroblast-like morphology with hump-derived ASCs being smaller in size and narrower in overall appearance than abdominal ASCs. Abdominal ASCs required a greater time for proliferation than the hump-derived cells. These results were further confirmed with a tetrazolium-based colorimetric assay (MTT) which showed a greater cell proliferation rate for hump ASCs than for the abdomen. Under inductive conditions, ASCs from both abdominal and hump fat deposits maintained their lineage differentiation potential into adipogenic, chondrogenic, and osteogenic lineages during subsequent passages without any qualitative difference. However, expression of alkaline phosphatase was higher in osteogenic differentiated cells from the hump compared with those of the abdomen. Moreover, the increase in calcium content in hump-derived stem cells was higher than that in abdominal-derived stem cells. In conclusion, our findings revealed that ASCs can be obtained from different anatomical locations, although ASCs from the hump fat region may be the ideal stem cell sources for use in cell-based therapies.

Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 µM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 µM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 µM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 µM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 µM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 µM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

Effect of ethanol induced mild stress on post-thawed bull sperm quality.

This study was performed to investigate the effect of sub-lethal exposure of bull semen to ethanol on the post-thaw spermatozoa quality. Semen samples (n=24, 6 ejaculates/bull) from 4 Holstein bulls were collected and pooled. Pooled samples were divided into 4 equal parts and each part was frozen after being diluted with Optidyl® extender containing 0 (O-E0), 0.03 (O-E3), 0.09 (O-E9) and 0.15 (O-E15) % (v/v) absolute ethanol. Sperm motility and velocity, plasma membrane integrity and functionality, mitochondrial activity, malondialdehyde concentration, and apoptosis status were evaluated after thawing. A higher percentage of total motility was observed in the O-E9 group as compared to the O-E0, O-E3 and O-E15 groups (p<0.05). Also, plasma membrane integrity was higher (p<0.05) in the O-E9 group compared to the O-E3, and O-E15 groups. However, the difference between the O-E9 and O-E0 groups was not significant (p>0.05). In terms of the proportion of sperm abnormality and plasma membrane functionality no differences (p>0.05) were observed between the groups. Our results revealed that malondialdehyde level was lower in ethanol treated (O-E3, O-E9 and O-E15) groups compared to the O-E0 group (p<0.05). Furthermore, the percentage of live spermatozoa with active mitochondria was higher in the O-E9 and O-E15 groups compared to the O-E0 and O-E3 groups (p<0.05). The O-E3 and O-E9 groups resulted in the highest and lowest percentage of apoptotic spermatozoa, respectively (p<0.05). The results of this study demonstrate that supplementation of semen extender with sub-lethal concentration of ethanol influences post-thawed bull sperm quality in a dose dependent manner.

Effect of Linolenic acid during in vitro maturation of ovine oocytes: embryonic developmental potential and mRNA abundances of genes involved in apoptosis.

PURPOSE: To study the effect of α-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes. METHODS: A preliminary experiment was conducted to analyze the concentration of ALA in "small" (≤2 mm) and "large" (≥6 mm) follicles using gas chromatography/mass spectrometry analysis. The concentration of ALA in small and large follicles was determined to be in a range of 75.4 to 125.7 µM, respectively. In vitro maturation (IVM) of oocyte was then performed in presence of 0 (control), 10 (ALA-10), 50 (ALA-50), 100 (ALA-100), and 200 (ALA-200) µM of ALA. Meiotic maturation and mRNA abundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryonic cleavage and blastocyst formation following parthenogenetic activation were also determined for each group. RESULTS: The highest concentration of ALA (ALA-200) decreased the oocyte maturation rate compared with the control group. Analysis of apoptosis-related genes in oocytes after IVM revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in ALA-treated oocytes as compared with the control oocytes. In term of cleavage rate (considered as 2-cell progression), we did not observe any differences among the groups. However, ALA-100 group promoted more blastocyst formation as compared with the control group. CONCLUSION: Our results suggested that ALA treatment during IVM had a beneficial effect on developmental competence of ovine oocytes by increasing the blastocyst formation and this might be due to the altered abundance of apoptosis-regulatory genes.

Dietary supplementation of Gracilariopsis persica is associated with some quality related sera and egg yolk parameters in laying quails.

BACKGROUND: Gracilariopsis persica (Gp) is one of the most abundant red algae distributed in the Persian Gulf, containing various bioactive components with hypolipedimic, hypoglycemic and antioxidant properties. Therefore using laying quails as a model we aimed to investigate the effect of dietary Gp on body weight, feed conversion, estradiol, progesterone, calcium and lipid levels in serum, as well as the high-density:low-density lipoprotein (HDL:LDL) ratio. Yolk cholesterol and yolk lipid oxidation were also evaluated. To accomplish this, diets containing 0, 10, 30 and 50 g kg(-1) Gp were fed to 5-week-old laying quails for 12 weeks. RESULTS: Our data revealed that Gp had no effect on body weight, feed conversion, triglycerides and estradiol levels of serum. Dietary Gp decreased the serum and yolk cholesterol in a dose-dependent manner. In addition, the sera progesterone and calcium levels and HDL:LDL ratios were increased by feeding diets containing 50 g kg(-1) Gp. Our results relating to yolk lipid oxidation showed that malondialdehyde content was decreased in Gp-fed laying quails. CONCLUSIONS: The results of the present study demonstrate that not only serum and egg yolk cholesterol levels, but also susceptibility of yolk lipids to oxidation, can be decreased by feeding Gp to laying quails.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells.

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression.

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze-thawing process of ram sperm.

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p < 0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p < 0.05) viable and lowest (p < 0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p > 0.05) by different levels of rosemary aqueous extract. Lower (p < 0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner.

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte.

PURPOSE: To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts. METHODS: Goat oocytes were exposed to brilliant cresyl blue (BCB) staining test and categorized into BCB(+) (blue-cytoplasm), and BCB(-) (colorless-cytoplasm) groups. A group of oocytes were not exposed to BCB test and was considered as a control group. After maturation in vitro, a group of oocytes were used for determination of nuclear status and intracellular GSH content while another group was subjected to parthenogenetic activation followed by in vitro embryo culture. RESULTS: We found that BCB(+) oocytes not only yielded higher rate of maturation, but also showed an increased level of intracellular GSH content than BCB(-) and control oocytes. Furthermore, BCB(+) oocytes produced more blastocysts than BCB(-) and control oocytes. Our data revealed that the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes were interacted with G6PDH-activity in mature oocyte, their surrounding cumulus cells, and blastocyst-stage embryos. CONCLUSIONS: The results of this study demonstrate that selection of goat oocytes based on G6PDH-activity through the BCB test improves their developmental competence, increases intracellular GSH content, and affects the expression of the apoptosis-related genes.

Association of glucose-6-phosphate dehydrogenase activity with oocyte cytoplasmic lipid content, developmental competence, and expression of candidate genes in a sheep model.

PURPOSE: To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers. METHODS: Before brilliant cresyl blue (BCB) staining test, oocytes were classified as high, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after the test as having low or high G6PDH-activity (BCB(+) and BCB(-), respectively). After maturation in vitro, a group of oocytes were subjected to IVF followed by in vitro embryo culture and another group was used for evaluation of expression of candidate genes. RESULTS: The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group, intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More (P < 0.05) oocytes in HCLC group were BCB(+), and higher (P < 0.05) maturation, cleavage, and blastocyst rates were seen for BCB(+) oocytes than the BCB(-) oocytes. Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3, BMP15, and GDF9 genes was similar between BCB oocytes groups. However, the expression of ATP1A1 was higher (P < 0.05) for BCB(+) oocytes compared to BCB(-) oocytes. In addition, BAX transcript abundance was similar (P > 0.05) among BCB(+), BCB(-), and control groups, before and after maturation in vitro. CONCLUSION: Activity of G6PDH in sheep oocytes is highly associated with lipid content, and compared with the morphological parameters might be a more precise and objective predictor for subsequent developmental competence in vitro.

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.

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.

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.

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.