Royal jelly may improve sperm characteristics during preservation of rooster semen: Gene expression of antioxidant enzymes.

Sustainable production and the increasing number of embryonated hatching eggs are critical aspects of the poultry production industry. The present paper aims to appraise the effectiveness of royal jelly (RJ) on the semen characteristics of Native Mazandaran roosters in both liquid and frozen storage conditions. Semen collected from 10 sexually mature roosters and following dilution was supplemented with RJ at 0.0 (control), 5 (RJ5), 10 (RJ10), 20 (RJ20) and 40 (RJ 40) mg/ml. After cooling and freezing-thawing, the percentage of forward progressive motility, viability, abnormality, hypo-osmotic swelling test (HOST) and the mRNA abundance of antioxidant enzymes of spermatozoa were measured. Our results revealed that the addition of 5 mg/ml RJ to the semen extender significantly increased (p < .05) the percentages of forward progressive motility, viability and HOST during liquid and frozen storage. The abnormality of spermatozoa in the RJ5 group was significantly lower compared to the other groups. During liquid storage, a significant decrease in forward progressive motility was found after 48 hr in comparison with 24 hr at 4°C. High levels of RJ (from 10 to 40 mg/ml) were severely decreased the characteristics of rooster spermatozoa in comparison with RJ5 and the control group. The inclusion of RJ at 5 mg/ml to the semen extender enhanced the mRNA transcript of antioxidant enzymes of spermatozoa during liquid preservation. The mRNA abundance of antioxidant enzymes did not influence by cryostorage. Overall, these data suggest that supplementation of RJ at 5 mg/ml to the extender improved semen characteristics and redox status of rooster spermatozoa.

Oral administration of royal jelly may improve the preservation of rooster spermatozoa.

The aim of the present study was to investigate the effect of dietary supplementation of royal jelly (RJ) on liquid and frozen storage of rooster spermatozoa. Twenty-five 30-week-old of Mazandaran native breeder roosters were randomly divided into five treatments (n = 5 roosters/group). Experimental treatments are designed to include a control group and various levels (0.0 (RJ0), 100 (RJ100), 200 (RJ200), 300 (RJ300) mg kg-1  BW-1 ) of royal jelly (RJ) that were fed to the roosters using force-feed method. The percentage of forward progressive motility, abnormal spermatozoa, membrane integrity and viability of spermatozoa evaluated after 24 and 48 hr of cooling (at 4°C) and after the freeze-thawing process. Also, mitochondrial activity and DNA fragmentation in fresh (24 hr) and post-thawed spermatozoa were assessed. The result of this study showed that the spermatozoa forward progressive motility, abnormality, membrane integrity, and viability were improved by the RJ100 group compared to the other groups after 24 and 48 hr storage period at 4°C. The percentage of membrane integrity and forward progressive motility after freeze-thawing in the RJ100 group was significantly higher than the other groups, and the percentage of abnormal spermatozoa was lower. A significant decrease in semen quality parameters was seen after 24 and 48 hr of refrigeration, but there was no observed change between 2 and 24 hr in the RJ100. The viability percentage of spermatozoa in both RJ100 and RJ200 groups was not different. Moreover, after freeze-thawing, DNA integrity and mitochondrial activity in the RJ100 group were significantly higher than the other groups. According to our results, feeding of RJ at 100 mg kg-1  BW-1 to the roosters was improved spermatozoa characteristics during liquid and cryopreservation conditions.

Antioxidant levels, copper and zinc concentrations were associated with postpartum luteal activity, pregnancy loss and pregnancy status in Holstein dairy cows.

The role of oxidative stress in the control of female reproduction has not been fully elucidated in ruminants; however, it seems that antioxidants can make influence to the reproductive axis at different levels. Therefore, the aim of this study was to determine the relationship between antioxidant status and concentrations of trace minerals (chromium (Cr), copper (Cu), iron (Fe), and zinc (Zn)) with postpartum luteal activity and fertility in Holstein dairy cows. The cows (n = 100, a parity range of 2-5, and a body condition score (BCS) of 3.0 ±â€¯0.25, mean ±â€¯SEM) were assigned to the experiment at second week post calving. Ovarian follicular dynamics were monitored twice a week (from 3rd to 6th weeks post calving) by transrectal ultrasonography (US). Blood samples were collected twice weekly from the 3rd to the 6th weeks post calving at timed artificial insemination (TAI), and days 32 and 50 post AI to determine superoxide dismutase (SOD), glutathione peroxidase (GPX), total antioxidant capacity (TAC), and trace mineral concentrations. There were associations between plasma concentrations of SOD, GPX, and TAC with postpartum luteal activity (PLA, P = 0.01) and ovulation (P = 0.03). Mean plasma SOD and GPX activities and TAC levels (U/mL) were greater in cows with normal luteal activity (NLA) than prolonged luteal phase (PLP) and anovulation (AO) cows, as well as in ovulated compared to AO cows (P = 0.03). Pregnant cows had greater levels of SOD, GPX, and TAC (U/mL) at TAI than non-pregnant cows (P = 0.01). Plasma Cu and Zn concentrations increased in pregnant compared to non-pregnant cows at TAI. In conclusion, antioxidant levels and Cu and Zn concentrations were associated with PLA and fertility.

Dietary Guanidinoacetic acid modulates testicular histology and expression of c-Kit and STRA8 genes in roosters.

Decline in semen quality is considered as a major contributing factor in age-related subfertility of broiler breeder flocks. This study was aimed to investigate the effect of dietary supplementation of Guanidinoacetic acid (GAA), as an alternative energy source along with antioxidant potential, on testicular histology and relative gene expression of some spermatogonial markers (c-Kit and STRA8) in aged roosters. Sixteen 24-week-old male broiler breeders were randomly allocated into four groups and fed a basal diet supplemented with increasing levels of GAA including 0 (GAA-0), 600 (GAA-600), 1200 (GAA-1200) or 1800 (GAA-1800) mg/kg diet/day for 26 successive weeks. At the end of the experiment, all the birds were killed and two ipsilateral testicle samples were taken to either quantify relative gene expression or do histology. Except for seminiferous tubules' diameter, testicular weight, and the number of blood vessels, dietary supplementation of GGA improved the epithelium thickness of seminiferous tubules, the number of spermatogonia and Leydig cells and the relative gene expression of c-Kit and STRA8 (P < 0.01). Increasing levels of GAA cubically affected (P < 0.01) the diameter of seminiferous tubules and their epithelium thickness as well as the number of spermatogonia. However, number of Leydig cells and relative expression of c-Kit were linearly, and relative expression of STRA8 was quadratically (P < 0.01) enhanced in response to graded levels of GAA supplementation. Taking all parameters into account, daily supplementation of 1300-1450 mg of GAA/kg diet was estimated as an optimum dosage maximizing the evaluated traits.

Oocyte maturation with royal jelly increases embryo development and reduces apoptosis in goats.

Royal jelly (RJ) was supplemented to goat oocyte in vitro maturation (IVM) medium at three different concentrations (2.5, 5, and 10 mg/ml). Maturation rate, embryo cleavage, and blastocyst rate were recorded. Gene expression of apoptosis-related transcripts was investigated in matured oocytes. Percentage of oocytes that reached MII-stage was increased in RJ-treated groups compared to the control group. Glutathione (GSH) content of mature oocytes was enhanced when RJ was added to IVM medium at any supplementation compared with control. Percentage of cleaved embryos and blastocysts was higher in the RJ-treated groups at a concentration of 5 than in the 2.5 mg/ml and control group. Total number of cells per blastocyst was not different in the control and RJ-treated group at 5 mg/ml. However, number of apoptotic cells per blastocyst was higher in the control group than in the RJ-treated group at 5 mg/ml. Expression profile of Bax, and p53 was down-regulated while Bcl-2 was up-regulated in oocytes treated with RJ at 5 and 10 mg/ml compared with the control group. Addition of RJ at concentrations of 5 mg/ml improved embryo production through increasing maturation rate. RJ seems to improve the IVM microenvironment by reducing expression of genes inducing apoptosis, enhancing GSH content, and reducing incidence of apoptosis in blastocysts.

D-Aspartate amends reproductive performance of aged roosters by changing gene expression and testicular histology.

Male broiler breeders (n=32) of 55 weeks of age were administered four different doses of capsulated d-aspartate (DA; 0, 100, 200 or 300mgkg-1day-1, p.o. (DA0, DA100, DA200 and DA300 respectively)) for 12 successive weeks to assess reproductive performance, blood testosterone, testicular histology and transcript levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), androgen receptor (AR), LH receptor (LHR), 3ß-hydroxysteroid dehydrogenase (3BHSD), proliferating cell nuclear antigen (PCNA), glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) and glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B). Blood samples and ejaculates were collected, and bodyweight was recorded weekly for 10 weeks. AI was performed weekly for the last 2 weeks to determine the number of sperm penetration holes in the perivitelline layer, fertility and hatchability. Testes histology and transcript levels were evaluated in the 12th week. Bodyweight, numbers of Leydig cells and blood vessels, testis index and levels of sperm abnormalities were not affected (P>0.05) by the treatment. However, sperm total and forward motility, plasma membrane integrity and functionality of sperm, ejaculate volume, testosterone concentration and fertility were higher (P<0.05) in both the DA200 and DA300 groups compared with the other groups. In the DA100 and DA200 groups, sperm concentration, number of spermatogonia, thickness of the seminiferous epithelium and the diameter of tubules were significantly higher (P<0.05) than the other DA-treated groups. The number of penetration holes, hatchability and malondialdehyde concentration were higher in the DA200, all DA-treated and DA300 groups respectively compared with the control and other treatment groups. Except for P450scc, AR, LHR and PCNA transcript levels in the DA300 groups, the relative expression of the genes evaluated improved significantly in the other DA-treated groups. Based on these experimental findings, it is concluded that DA improves reproductive performance of aged roosters.

Letrozole, an aromatase inhibitor, reduces post-peak age-related regression of rooster reproductive performance.

This study was designed to evaluate orally administrated Letrozole (Lz) on reproductive performance, plasma testosterone and estradiol concentrations and relative abundance of mRNA of GnRH, FSH and LH in roosters. Ross 308 roosters (n=32) that were 40-weeks of age were individually housed and received a basal standard diet supplemented different amounts of capsulated Lz [0 (Lz-0), 0.5 (Lz-0.5), 1 (Lz-1) or 1.5 (Lz-1.5), mg Lz/bird/day] for 12 weeks. Sperm quality variables and plasma testosterone and estradiol concentrations were assessed from the first to the tenth week of the treatment period. Semen samples from the 11th to 12th week were used for artificial insemination and eggs were collected and allotted to assess fertility and hatchability rates. Relative abundance of hypothalamic and pituitary GnRH, LH and FSH mRNA was evaluated at the end of 12th week. The results indicated that total and forward sperm motility as well as egg hatchability rate were greater in the Lz-0.5 group. Greater sperm concentrations, ejaculate volume, sperm plasma membrane integrity, testis index and fertility rates were recorded for both Lz-0.5 and Lz-1 groups compared with the Lz-0 group (P<0.05). Body weight, percentage of sperm abnormalities, and sperm plasma membrane functionality were not affected by treatment. Testosterone and estradiol concentrations were negatively related with greater testosterone concentrations in the Lz-1.5 group which had lesser estradiol concentrations. Relative mRNA transcript abundance for GnRH, LH and FSH was Lz dose responsive being greater in the treated groups; however, this trend plateaued for GnRH and for the relative abundance of both LH and FSH mRNA was less in the Lz-1.5 group than the other treatment groups. It is concluded that Lz may be an effective treatment to improve age related post-peak reproductive performance of roosters.

Royal jelly may improve the metabolism of glucose and redox state of ovine oocytes matured in vitro and embryonic development following in vitro fertilization.

The aim of this study was to investigate the effect of different concentrations of royal jelly (RJ) on in vitro maturation (IVM), fertilization, cleavage, blastocyst rates, glutathione (GSH) content in ovine oocyte, mRNA abundance of antioxidant enzymes in both oocyte and cumulus, and glucose metabolism-related genes in cumulus cells. In vitro maturation of oocyte was performed in the presence of control (RJ0), 2.5 (RJ2.5), 5 (RJ5), and 10 (RJ10) mg/mL of RJ. Nuclear status, intracellular GSH content in oocytes, and mRNA abundance of selected genes were evaluated following 24 hours of IVM. Following the IVM, fertilization and embryo culture were carried out in all the groups and embryonic development was examined. The addition of 10-mg/mL RJ to maturation media not only yielded a higher number of oocytes at MII stage but also showed an increased level of intracellular GSH content than did RJ2.5 and control groups. Fertilization, cleavage, and blastocyst rate were higher in the RJ10 treatment group in comparison to the control one. In cumulus cells, the expression of PFKM, PFKL, and G6PDH were increased following the addition of RJ to the maturation media. Supplementation of 10-mg/mL RJ to IVM medium increased the GPx mRNA abundance in both oocyte and cumulus cells and SOD expression in the cumulus cells. The CAT mRNA abundance was not influenced by the addition of RJ to the maturation media in either oocyte or cumulus cells. It seems that the improvement of oocyte maturation and its subsequent development in RJ10 group may be associated with amelioration of redox status in the oocytes and activation of glucose metabolic pathways in their surrounding cumulus cells.

Impact of supplementary royal jelly on in vitro maturation of sheep oocytes: genes involved in apoptosis and embryonic development.

Optimizing culture conditions lead to the improvement of oocyte developmental competence and additives with anti-oxidative activity in culture media improved embryonic development. Royal jelly (RJ) is a product from the cephalic glands of nurse bees that has considerable health effects. The aim of this study was to investigate the effect of different concentrations of RJ on the maturation, cleavage, and blastocyst rates and gene expression in the oocyte and cumulus cells during in vitro maturation (IVM) of sheep oocyte. IVM of oocyte was performed in the presence of control (RJ0), 2.5 (RJ2.5), 5 (RJ5), 10 (RJ10), 20 (RJ20), and 40 (RJ40) mg/mL of RJ. Following the maturation period, parthenogenetic activation was carried out in two treatment groups (RJ0 and RJ10) and embryonic development was examined three and eight days thereafter. Moreover, the relative expression of BCL2 and BAX in oocyte as well as BCL2, BAX, HAS2, PTGS2, and STAR in cumulus cells were assessed. The results indicated that the addition of 10 mg/mL of RJ (90 ± 4.51%) to the maturation medium linearly increased the oocyte maturation rate compared to the control group (57 ± 2.42%), then it remained constant to the RJ40 (93 ± 3.10%) group. The higher RJ concentrations were associated with increased (p < 0.01) cleavage (53.3 ± 1.55% to 82.3 ± 2.82%) and blastocyst rate (15.5 ± 1.16% to 33.8 ± 3.09%) from the RJ0 to the RJ10 group. The relative mRNA expression of BCL2 and BAX in the oocyte was higher at RJ10. In cumulus cells, the expression of BCL2 was not affected, but that of BAX decreased, and expression of HAS2, PTGS2, and STAR were increased following the addition of RJ to the maturation media. In conclusion, the addition of 10 mg/mL of RJ to maturation medium improved blastocyst formation and decreased the apoptotic incidence in sheep cumulus cells and the oocyte during the in vitro development.

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.

Post-ovulatory ageing of mouse oocytes affects the distribution of specific spindle-associated proteins and Akt expression levels.

The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 post-translational modifications and on v-akt murine thymoma viral oncogene homolog 1 (Akt) expression levels. To this end, oocytes were retrieved 13, 29 and 33h after human chorionic gonadotrophin (hCG) treatment. The presence and distribution at the meiotic spindle of acetylated tubulin, γ-tubulin, polo kinase-1 and Ser473/Thr308 phosphorylated Akt (pAkt) as well as histone H3 and H4 acetylation and phosphorylation levels were assayed via immunofluorescence. Akt expression levels were determined via reverse transcription-polymerase chain reaction and western blotting analyses. Spindles from oocytes recovered 13h and 29h after hCG treatment showed similar levels of acetylated tubulin but ageing induced: (1) translocation of γ-tubulin from spindle poles to microtubules, (2) absence of Thr308- and Ser473-pAkt in 76% and 30% of oocytes, respectively, and (3) a significant reduction in phosphorylation levels of serine 10 on histone 3. At 29h, a significant decrease in Akt mRNA, but not in pAkt or Akt protein levels, was recorded. By contrast, protein content significantly decreased 33h after hCG. We conclude that POA impairs oocyte viability and fertilisability by altering the expression levels and spindle distribution of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.

Prediction of oocyte developmental competence in ovine using glucose-6-phosphate dehydrogenase (G6PDH) activity determined at retrieval time.

PURPOSE: To determine whether G6PDH-activity measured by Brilliant Cresyl Blue known as BCB dye, predicts developmental competence within cohorts of ovine oocytes. METHODS: Ovine oocytes were exposed to BCB staining and categorized into two groups: BCB+ (blue cytoplasm, low G6PDH-activity) and BCB- (colorless cytoplasm, high G6PDH-activity). After maturation in vitro, oocytes were subjected to fertilization followed by in vitro embryo culture. RESULTS: We observed a significant difference in oocyte diameter considering BCB+ and BCB- oocytes. BCB+ and Control groups showed significantly higher maturation rates compared to BCB- group. There were significantly more cleaved embryos in BCB+ and control groups than in BCB- group. Blastocyst rate was significantly higher for BCB+ group compared to control and BCB- groups with control group being significantly higher than BCB- group. CONCLUSION: G6PDH-activity is a strong predictive marker of oocyte competence and may be useful in identifying oocytes with a good prognosis for further develop.