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Objective of experiment was to determine whether oxidative stress (OS) and inflammation altered embryonic loss in dairy cows. Blood samples were collected at days 0, 16, 32 and 60 after timed (AI) from 200 Holstein cows to determine embryonic loss based on interferon-stimulated gene-15 (ISG15) mRNA expression (day 16) and ultrasound at day 32 and day 60. Leucocyte expressions of mRNA TLR2, TLR4, TNF-α, IL1B, IL10, STAT3 (inflammation), PTGS2, PTGES (prostaglandin synthesis), and PLA2G4A and ALOX5AP (eicosanoid metabolism) at days 0 and 16 were determined. Plasma redox status for antioxidant enzymatic activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), total antioxidant capacity (TAC), and concentrations of malondialdehyde (MDA) were determined at days 0, 16, 32 and 60. All antioxidant-redox responses were beneficially significant in pregnant cows diagnosed pregnant at day16 and sustained pregnancy to day 60 compared to non-pregnant cows at day16 or pregnant at day16 and lost embryos by days 32 or 60. The leucocyte mRNA expressions of TLR2, TLR4, STAT 3, IL1B, PTGS2, PLA2G4A and ALOX5AP were greater and PTGES was lower at day16 in pregnant cows that lost embryos early (P

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.

The objective was to compare three shortened protocols for timed-AI (TAI) on ovarian responses, pregnancy per AI (P/AI) and pregnancy loss after resynchronization of ovulation in multiparous Holstein cows. Cows (n = 370), at one location, were randomly assigned at non-pregnancy diagnosis (approximately 32 d after AI) to one of three ovulation resynchronization protocols. Cows in the OS group received a 5-d Ovsynch [100 μg GnRH on Day 1, 500 μg cloprostenol (PGF) on Days 6 and 7, GnRH on Day 8.5 and TAI on Day 9 (16 h after second GnRH)]. Cows in the OS + P4 (progesterone) group received a 5-d Ovsynch as described for OS group plus an intravaginal device (Cue-Mate), containing 1.56 g of progesterone (P4), between Days 1 and 6. Cows in the J-synch group received a Cue-Mate and 2.5 mg of estradiol benzoate (EB) on Day 0, PGF and Cue-Mate removal on Day 6, another PGF on Day 7, and 100 μg of GnRH on Day 8.5, with TAI on Day 9. Ovarian response and pregnancy status at 32 and 60 d after the resynchronization TAI were determined by transrectal ultrasonography. Blood samples were collected at first PGF treatment and at TAI from a subset of 40 cows per group to determine P4 concentrations. Percentage of cows with CL at initiation of the protocol did not differ (P > 0.05) among resynchronization groups. Plasma P4 concentrations at first PGF treatment were greater (P < 0.01) in cows that received a Cue-Mate (OS + P4 and J-synch) compared to OS cows. Luteal regression was greater (P < 0.01) for J-synch (88.6%) compared with OS (76.9%) and OS + P4 (78.8%). More (P < 0.01) cows in the OS + P4 and J-synch groups had their estrous cycle synchronized and were pregnant at 32 d after TAI (48.7 and 34.7%, 67.8 and 48.0%, and 72.4 and 50.0% for OS, OS + P4 and J-synch, respectively). However, more cows subjected to J-synch remained pregnant at 60 d after TAI and, hence, had fewer (P < 0.05) pregnancy losses (19.2, 18.8 and 5.0% for OS, OS + P4 and J-synch, respectively). In summary, cows resynchronized with either OS + P4 or J-synch had greater P4 concentrations at first PGF treatment and a greater response to treatments compared to cows subjected to OS. Although OS + P4 and J-synch resynchronization protocols resulted in increased P/AI at 32 d, pregnancy losses were significantly reduced in cows subjected to the J-synch protocol.

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.

In this study, the effects of hydroalcoholic extract of red algae (Laurencia caspica) at different levels were evaluated on antioxidant defense, innate immune responses, and expression of immune genes in rainbow trout (Oncorhynchus mykiss). A total of 750 rainbow trout (39.43 ± 0.44 g) were randomly assigned to five treatments with three replicates (50 fish per replicate) in 15 ponds and reared for 8 weeks. Fish samples were fed a diet containing different levels of algal extract (0, 5, 10, 15, and 20 g extract per kg of basal diet). At the end of feeding period, the fish were challenged with Aeromonas hydrophila and fish mortality was recorded over a 10-day period. To evaluate the antioxidant and immune parameters, fish were sampled at day 0, end of weeks 4 and 8, and after bacterial challenge. Expression of immune genes (IL-1β, lysozyme II, Complement C3, and TNFα) was also examined after bacterial challenge. Results indicated that application of algal powder in fish diet improved lysozyme activity, complement system, and immunoglobulin levels of extract fed-fish compared to control group (p

The aim of this study was to investigate the effects of different dietary levels of L-arginine on the growth performance, blood parameters, and lipogenic gene expression of Arian broiler chickens. For this purpose, 168 Arian broiler chicks (40.33±1.7 g) were assigned to four treatments with three replicates of14 birds each, according to completely randomized design. The experimental treatments consisted of 100, 124, 139, and 154% dietary arginine levels relative to the published requirements of Arian broilers. On 42 d of the experiment, blood samples were collected from two birds (six birds per treatment) for blood metabolite measurements. These birds were then euthanized for carcass evaluation and collection of tissue samples. Increasing dietary arginine levels reduced (p

Exposure to high levels of polyunsaturated fatty acid predisposes spermatozoa to lipid peroxidation, resulting in their decreased fertility. Ginger powder (GP), which is high in antioxidative compounds, was fed to aged breeder roosters to improve their reproductive performance. Seventy-five 52-wk-old Cobb 500 breeder roosters randomly received either 0 (GP0), 15 (GP15), or 30 (GP30) g of GP/kg of diet for 14 consecutive wk, during which time their seminal characteristics were evaluated every 2 wk. At the end of the trial, semen samples were tested for determination of sperm fatty acid (FA) concentration and seminal plasma total antioxidant capacity. Furthermore, sperm penetration was assayed, and using 225 artificially inseminated hens, fertility and hatchability rates were determined. Dietary GP improved sperm forward motility, live sperm percentage, and sperm plasma membrane integrity. These were associated with a decrease in the percentage of abnormal sperm. The seminal TBA reactive species concentration was lower in birds belonging to the GP30 treatment in comparison with those in the GP15 and GP0 treatments. The feeding of GP resulted in overall decreases and increases in sperm saturated and unsaturated FA, respectively. The n-6:n-3 FA ratio of sperm was decreased in the GP30 group in comparison with controls. The highest levels of sperm C20:4(n-6) and C22:6(n-3) FA were recorded in the GP15 and GP30 treatments, respectively. A higher percentage of sperm C22:4(n-6) FA was found in GP-fed roosters. Seminal plasma total antioxidant capacity was considerably improved by the GP15 and GP30 treatments. Further, a higher number of perivitelline membrane sperm penetration holes was recorded for the GP30 treatment in comparison with the GP15 and GP0 treatments. Interestingly, although hatchability, chick quality, and embryonic mortality were not affected by dietary treatment, fertility rate was improved by the feeding of GP. In conclusion, dietary GP improved most of the seminal characteristics evaluated in aged roosters of this study, suggesting that it has potential for use in attenuating age-related subfertility in senescent male commercial broiler breeders.

The objectives of this study were to determine the effect of PGF2α treatment 14 d before the initiation of a G6G or G7G (PGF2α, 2 d, GnRH, 6 or 7 d, Ovsynch) protocol on ovarian response, synchronization protocol on ovarian response, progesterone (P4) concentration, pregnancy per AI (P/AI), and pregnancy loss in multiparous Holstein cows. Cows (3.6±1.1 lactations and yielding >30kg/d of milk) were randomly assigned to 1 of 4 timed artificial insemination (TAI) protocols: 1) G6G (n=240), one injection of PGF2α, GnRH 2 d later and a 7-d Ovsynch protocol (GnRH, 7 d, PGF2α, 56 h, GnRH 16 h TAI) was initiated 6 d later; 2) PG6G (n=250), PGF2α 14 d before the initiation of the G6G protocol; 3) G7G (n=200), one injection of PGF2α, GnRH 2 d later, and a 7-d Ovsynch protocol initiated 7 d later; and (4) PG7G (n=200), a PGF2α injection 14 d before the initiation of the G7G protocol. Blood samples from a subset of 269 cows were collected at the times of first and second GnRH, and PGF2α of the Ovsynch protocol to measure P4. Ultrasound examinations were performed to evaluate ovarian response to GnRH and PGF2α of Ovsynch, and to determine pregnancy status at 32 and 60 d after TAI. The proportion of cows with high (≥1ng/mL) P4 at first GnRH of Ovsynch was greater for PG6G and PG7G compared with G6G and G7G groups (combined 79.7 vs. 59.3%). In addition, mean (±SEM) plasma P4 concentration (ng/mL) at PGF2α of Ovsynch was also greater in PG6G (6.5±0.2) and PG7G (6.7±0.3) compared with G6G (5.1±0.2) and G7G (5.0±0.2). Cows given PGF2α 14 d before initiating a G6G or a G7G TAI (PG6G and PG7G) tended to have a greater P/AI at 32 d compared with those cows not receiving PGF2α (G6G and G7G). However, P/AI at 60 d was greater in cows subjected to PG6G and PG7G protocols (31.1 vs. 39.2%), with a lower pregnancy loss between 32 and 60 d (11.65 vs. 19.7%). In summary, administration of PGF2α 14 d before initiating a G6G or a G7G TAI protocol increased P4 concentrations before artificial insemination and late embryonic/early fetal survival in multiparous Holstein cows.

Probable involvement of avidin and avidin-related protein-2 (AVR2) in sperm viability in the sperm storage tubules of turkeys has been suggested. The high affinity of biotin to avidin and its analogs is also well documented. The present study aimed to determine the effect of oral biotin on reproductive performance and oviductal mRNA expression of avidin and AVR2 in 2 broiler hen lines with different fertility rates. Low-fertility (line B) and high-fertility (line D) hens (n=144) were randomly allotted to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg/L biotin in drinking water from 30 through 33 wk of age. The reproductive performance of the hens was evaluated using artificial insemination. At the end of the treatment period, 24 hens per line were killed to assay the expression of avidin and AVR2 in the uterovaginal junction. Supplementary biotin increased egg production from 73.5% for T0 to 87.8% for T2. Hens administered with biotin in line B, but not in line D, showed an increase (8.4%) in fertility rate. Hatchability, chick quality, and overall embryonic mortality were not different among the experimental groups. Real-time PCR data showed that both avidin (P=0.0013) and AVR2 (P

To study the effect of alpha-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes., Purpose To study the effect of α-linolenic acid (ALA) on meiotic maturation, mRNAabundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation inovine oocytes.Methods A preliminary experiment was conducted to analyze the concentration of ALAin "small" (≤2 mm) and "large" (≥6 mm) follicles using gas chromatography/massspectrometry analysis. The concentration of ALA in small and large follicles wasdetermined 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 mRNAabundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryoniccleavage and blastocyst formation following parthenogenetic activation were alsodetermined for each group.Results The highest concentration of ALA (ALA-200) decreased the oocyte maturationrate compared with the control group. Analysis of apoptosis-related genes in oocytesafter IVM revealed lesser transcript abundances for Bax gene, and higher transcriptabundances for Bcl-2 gene in ALA-treated oocytes as compared with the controloocytes. In term of cleavage rate (considered as 2-cell progression), we did notobserve any differences among the groups. However, ALA-100 group promoted moreblastocyst 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 blastocystformation and this might be due to the altered abundance of apoptosis-regulatorygenes.

Published data on the probable involvement of avidin and avidin-related protein-2 (AVR2) in sustaining sperm viability in sperm storage tubules in 38-wk-old turkeys, and the high affinity of avidin or its analogs to biotin suggest that supplementary biotin may increase oviductal avidin and AVR2 expression, thereby attenuating the adverse effect of aging on hen reproductive performance. Broiler breeder hens (n = 120) were randomly assigned to receive 0 (T-0), 0.30 (T-1), or 0.45 (T-2) mg of biotin/L of drinking water from 30 to 33 (young) and 53 to 56 (old) wk of age, and artificially inseminated to determine their reproductive performance. At the end of each period of biotin administration, 8 hens from each treatment group were killed for RNA extraction from the uterovaginal junction. Egg production was lower in the old hens (44%) compared with the young ones (82%), and biotin supplementation increased egg production only in the latter. Administering supplementary biotin to young hens increased their oviductal expression of AVR2, which was much higher in the old hens (1.0 and 4.6 for young and old groups, respectively). Fertility rate was not different between young and old hens, and was increased (4.4%) at the higher level of biotin supplementation. Hatchability and hatchling quality were not affected by biotin supplementation. Embryonic mortality between 17 to 21 d of incubation was higher in young (5.2%) compared with old (1.4%) birds. Egg fertility rate showed a moderate correlation (P < 0.05) with avidin (r = 0.59) and AVR2 (r = 0.55) expression in the young-age group, and very low correlations in old-age group (0.04 and 0.17). Regardless of the hen's age, the correlation coefficient of hatchability with avidin or AVR2 expression was very low (-0.16 and 0.18). Overall, the effect of biotin supplementation on AVR2 expression, and the relationship between biotin administration and oviductal expression of avidin and AVR2 was dependent on the hen's age, being higher in the young hens.

A hypothesis was tested that the in ovo injection of biological buffers may reinforce the buffering capacity of albumen, thereby withstanding the increase in albumen pH during storage and improving hatchability and chick quality in long-term stored eggs. Hatching eggs (n = 2,420) were randomly assigned to 11 treatment groups (4 replicates of 55 eggs each) and injected (d 1) with distilled water, 25 or 50 mM HEPES (H25 and H50), Bicine (B25 and B50), Tris (T25 and T50), and Bis-Tris-propane (BTP25 and BTP50) solutions or were not injected (intact: control; or pricked with a needle: N). The eggs were then stored for 14 d during which the egg internal characteristics were evaluated at 1, 2, 3, 4, 5, 8, and 13 d of storage (n = 924 in total) and the remaining eggs (n = 1,496) were incubated. A decrease in albumen pH was found for H25, H50, B50, and BTP25 groups from 2 through 5 d postinjection. Eggs receiving H25, H50, and B50 recorded a higher albumen index (at 13 d of storage) and Haugh unit (between 8 and 13 d of storage) compared with the control. Interestingly, the hatchability of fertile eggs was influenced by the treatment effect (P = 0.0001) where the eggs receiving H25 (88.3%), H50 (88.9%), B50 (88.4%), and BTP25 (87.6%) recorded higher values than that of control (82.1%), associated with a decreased early embryonic mortality rate (P0.0001). In ovo injection of Tris buffer, however, profoundly decreased the hatchability (47.2 and 29.0% for T25 and T50, respectively) and percentage of first-grade chicks (67.5 and 63.6% for T25 and T50, respectively) compared with the control (90.1%). In conclusion, prestorage in ovo injection of H25, H50, B50, and BTP25 improved hatchability in long-term stored eggs in which a decreased albumen pH during the d 2 through 5 of storage period might be involved.

To determine whether G6PDH-activity measured by Brilliant Cresyl Blue known as BCB dye, predicts developmental competence within cohorts of ovine oocytes.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.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.G6PDH-activity is a strong predictive marker of oocyte competence and may be useful in identifying oocytes with a good prognosis for further develop.