Peripheral leucocyte molecular indicators of inflammation and oxidative stress are altered in dairy cows with embryonic loss.

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 < 0.05). In conclusion peripheral leucocyte molecular indicators of inflammation and plasma indicators of OS were altered in pregnant cows undergoing embryonic losses compared to cows with a sustained pregnancy.

Probiotics intake from proximal or distal gastrointestinal tract: The investigation on intestinal morphology and performance of Japanese quail.

The aim of the present study was to investigate the efficacy of early single-dose probiotics administered from proximal or distal gastrointestinal tract on quails' performance and small intestinal morphology. A total of 120 Japanese quail (Coturnix coturnix japonica) 1-d-old chicks were divided into the following experimental groups before being transferred to the raising room: (i) control (no probiotic administered); (ii) oral group (received probiotics via oesophageal gavage); and (iii) vent lip group (received probiotics directly into vent). Four replicates of 10 chicks per cage were considered for each treatment and birds were raised up to 35 days in the same conditions with ad libitum access to feed and water. A commercial probiotics (Protexin, Somerset, UK) used in this study contained 2 × 109  cfu/g of nine different micro-organisms. On day 35, all birds were weighed and then three birds in every pen were killed via cervical dislocation. Two cm of mid-point of duodenum and ileum were cut for histomorphology assays. Both probiotics-treated groups had higher body weight and body weight gain than control (p < .01). Ileum was longer in probiotics-treated groups than in the control (p < .01). Orally treated birds had the highest duodenum villus height and crypt depth (p < .01). In the cloacally treated quails, ileum villus length was higher than the both other groups, and crypts' depth was deeper than the control (p < .01). The numbers of both types of goblet cells (acidic mucin producer and neutral mucin producer) were higher in the birds receiving probiotics than control birds (p < .01). Although there were no statistical differences in performance values between the different ways of probiotics entrance into gut, some intestinal histomorphometric parameters were improved in ileum of the birds receiving probiotics through distal gut that is cloaca (p < .01).

Inflammatory status and its relationships with different patterns of postpartum luteal activity and reproductive performance in early lactating Holstein cows.

This study was undertaken to determine the effect of inflammatory status on different patterns of postpartum luteal activity and reproductive performance of Holstein cows during early lactation. The cows (n = 75) averaged 3.4 ±â€¯1.2 (mean ±â€¯SEM) in parity and 3.1 ±â€¯0.2 (mean ±â€¯SEM) in body condition score at calving. Transrectal ultrasonography was performed twice weekly from day 10-60 postpartum to consider ovarian dynamics. Plasma concentrations of progesterone (P4) and estradiol (E2) were measured twice weekly and plasma levels of tumor necrosis factor alpha (TNF-α) and lipopolysacchride binding protein (LBP) were measured weekly (week 3-6 postpartum). Based on plasma P4 and E2 results, 34 (45.3%) cows had normal luteal activity (NLA), whereas 19 (25.3%), 12 (16.0%), 7 (9.3%) and 3 (4.0%) cows had prolonged luteal phase (PLP), delayed first ovulation (DO), anovulation (AO) and short luteal phase (SLP), respectively. Plasma TNF-α and LBP concentrations were affected by postpartum luteal activity (NLA, SLP and PLP), ovulatory status (DO and AO) and number of weeks postpartum (P < .05). These concentrations were greater in cows with PLP (P < .05) as compared to NLA cows, and in cows that had delayed ovulation or anovulation compared to ovulated cows (P < .05). Cows with PLP had greater open days and lower conception rate as compared to NLA cows (P < .05). Healthy cows had a larger CL and greater plasma estradiol and progesterone concentrations at first and second cycle postpartum compared to inflamed cows and followed it with greater fertility (P < .05). In conclusion, inflammatory statuses were different in high-producing dairy cows showing PLP, AO and DO in comparison with the postpartum normal luteal activity (NLA) cows that influenced reproduction outcomes.

Evaluation of shortened timed-AI protocols for resynchronization of ovulation in multiparous Holstein dairy cows.

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.

Administration of prostaglandin F2α 14 d before initiating a G6G or a G7G timed artificial insemination protocol increased circulating progesterone prior to artificial insemination and reduced pregnancy loss in multiparous Holstein cows.

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.

Oral administration of supplementary biotin differentially influences the fertility rate and oviductal expression of avidin and avidin-related protein-2 in low- and high-fertility broiler line hens.

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<0.0001) expressions were influenced by a biotin×line interaction effect, where low-fertility line B hens receiving the high biotin level recorded respectively a 3.9 and 15.3% increase in avidin and AVR2 mRNA expression, although biotin did not affect these traits in line D hens. Control hens in line D had a dramatically higher AVR2 expression record (7.4-fold) compared with the control hens in line B. The correlation coefficients of fertility rate and avidin expression were 0.73 and 0.66 in lines B and D, respectively. However, the correlation of fertility and AVR2 (r=0.65) was significant for line D hens only. Overall, fertility rate and oviductal expression of avidin and AVR2 were dichotomously affected by oral biotin in low- and high-fertility line hens, where only low-fertility birds showed improvements in these attributes.

Reproductive performance and oviductal expression of avidin and avidin-related protein-2 in young and old broiler breeder hens orally exposed to supplementary biotin.

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 (T0), 0.30 (T1), or 0.45 (T2) 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.

Improvements in semen quality, sperm fatty acids, and reproductive performance in aged Cobb 500 breeder roosters fed diets containing dried ginger rhizomes (Zingiber officinale).

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.

Prestorage in ovo injection of biological buffers: an approach to improve hatchability in long-term stored eggs.

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 (P < 0.0001). 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.

Oral exposure of broiler breeder hens to extra thyroxine modulates early adaptive immune responses in progeny chicks.

Based on the findings of a recent study suggesting a decreased cold-induced ascites incidence in broiler progeny from hyperthyroid (HYPER) breeder hens, and a controversy on the effects of hyperthyroidism on immunocompetence, the present study was conducted to determine the probable adverse effect of induced maternal hyperthyroidism on immune function in progeny chicks. Breeder hens (n = 88) were randomly allotted to the control or HYPER groups and received common or thyroxine (T4)-added (1 mg/L) water, respectively. The hens were artificially inseminated, and hatching eggs (n = 924) were incubated. Thereafter, the male hatchlings (n = 288) were reared for 42 d, and several cellular and humoral immune responses were evaluated at standard or low ambient temperature. Prevaccination antibody titers to Newcastle disease, infectious bronchitis, and infectious bursal disease virus were higher in HYPER chicks during 1 wk of age, although not different in their dams. For primary response to SRBC administered at 7 d of age, HYPER chicks recorded higher total, IgM (d 14), and IgG (d 21) anti-SRBC antibody titers. Higher cutaneous basophilic hypersensitivity response in HYPER chicks (d 10) was not observed at 35 d of age. Carbon clearance assay showed no difference, but in vitro lymphoproliferative response to concanavalin A was higher in 19-d-old HYPER chicks, independent of temperature treatment. An increase in lymphocyte percentage coincided with a decreased heterophil percentage and heterophil to lymphocyte ratio (d 14) in the HYPER group. The weight of lymphoid organs in progeny was not influenced by the oral exposure of dams to extra T4. Independent of T4 treatment, cold exposure was generally associated with decreased immune functions at early stages. The data suggested that oral exposure of broiler breeder hens to 1 mg/L of T4 not only had no adverse effect on immune function, but also modulated early adaptive immune responses in progeny chicks for which the causal mechanisms remain to be unraveled.

Maternal hyperthyroidism is associated with a decreased incidence of cold-induced ascites in broiler chickens.

A hypothesis was tested that providing the breeder hens with exogenous thyroxine (T(4)) would help their offspring to better survive the ascites-inducing condition during the growing period. In total, 132 broiler breeder hens were randomly assigned to one of 3 treatments: control (CON), hypothyroid [HYPO; 6-N-propyl-2-thiouracil (PTU)-treated], and hyperthyroid (HYPER; T(4)-treated). The hens were artificially inseminated, and the hatching eggs (n = 1,320) were incubated. No eggs in the HYPO group hatched. The 1-d-old male chicks (n = 288) from other groups were reared for 42 d under standard or low ambient temperature to induce ascites. Blood samples were drawn from the hens, embryos, and broilers for determination of T(4) and triiodothyronine (T(3)). The hematocrit was also determined in broilers. The PTU-treated hens had an increased BW along with lower plasma T(3) and T(4) concentrations. Plasma T(4) was higher in the HYPER hens compared with CON hens, but T(3) concentration was not different between these groups. The fertility rate was not affected by either hypo- or hyperthyroidism. The embryos in the HYPO group had lower plasma T(3) and T(4) concentrations at d 18 of embryonic development and internal pipping. Higher plasma T(4) was recorded in the HYPER birds at internal pipping, although plasma T(3) concentration was not affected at this stage. Maternal hyperthyroidism decreased the overall incidence of ascites in the cold-exposed chickens (10.0 vs. 33.4% for HYPER and CON groups, respectively). Although the effect of maternal PTU or T(4) treatment on plasma thyroid hormones and on the right ventricle-to-total ventricular weight ratio in the broilers was not significant, the cold-exposed healthy CON chicks showed higher hematocrit values, compared with the HYPER birds. It was concluded that maternal hyperthyroidism could decrease the incidence of cold-induced ascites in broiler chickens; however, probable causal mechanisms remain to be elucidated.