The effect of Tempo and MitoTEMPO on oocyte maturation and subsequent embryo development in bovine model.

Reactive oxygen species (ROS) are one of the factors which reduces oocyte quality and viability of the in vitro produced embryos. Oocyte mitochondria are the major source of ROS production, hence, and the addition of mitochondrion-specific antioxidants could be suggested to minimize the damage caused by ROS during culture. MitoTEMPO, a targeted mitochondrial antioxidant, is formed by conjugating TEMPO to triphenylphosphonium and has an activity like that of superoxide dismutase. It can pass through lipid bilayers easily and accumulate selectively in mitochondria. The goal of this study was to investigate the effects of MitoTEMPO and its non-targeted form, TEMPO, on the developmental competence of bovine oocytes. Accordingly, oocytes were cultured in maturation medium supplemented with either five mM TEMPO (T5) or one µM MitoTEMPO (M1), or T5 + M1 (MT15), or without the antioxidants (C). Nuclear maturation to metaphase II (MII) stage, intracellular glutathione (GSH) content and ROS levels in matured oocytes were analyzed. In addition, cleavage after in vitro fertilization, and blastocyst rates, total cell number in blastocysts as well as the relative abundance of apoptosis-related genes (BAX and BCL2) in blastocysts were determined. Results revealed that the proportion of oocytes at the MII stage, embryos at the blastocyst stage and total cell number in blastocysts increased significantly in the M1 group compared to the C and T5 groups. The levels of intracellular GSH and ROS in oocytes decreased in the M1 group than in the C group (P < 0.05). The expression level of the pro-apoptotic gene (BAX) reduced in blastocysts from the M1 group in comparison to the C and T5 groups (P < 0.05). On the other hand, the expression level of anti-apoptotic gene (BCL2) in obtained blastocysts was not affected by TEMPO and MitoTEMPO. However, the ratio of BAX/BCL2 in blastocysts from the M1 and MT15 groups decreased significantly compared to the C group. These findings suggest that MitoTEMPO can mitigate the adverse effects of oxidative stress on the developmental competence of bovine oocytes.

Effects of dietary curcumin supplementation on seminal quality indices and fertility rate in broiler breeder roosters.

1. Decreased semen quality is an underlying contributor to age-related subfertility in broiler breeder roosters. This study investigated the effects of dietary curcumin (derived from turmeric) supplementation as an antioxidant source on semen quality and fertility in broiler breeder roosters. 2. Twenty-eight Ross 308 roosters were randomly allotted to four groups with seven birds in each and were fed a standard diet supplemented with different levels of curcumin at 0 (C0), 10 (C10), 20 (C20) and 30 (C30) mg/bird per day from 48 through to 61 weeks of age. Body weight and semen quality traits were evaluated on a weekly basis and seminal concentrations of malondialdehyde (MDA) as a measure of antioxidation status were quantified at one-week intervals during the first 11 weeks of the trial (48-59 weeks of age). Semen samples from last 2 weeks (60 and 61 weeks of age) were used to artificially inseminate to assess the sperm-egg penetration (SP) in perivitelline membrane and fertility rates. 3. Except for body weight and ejaculate volume, other characteristics, including semen concentration, total sperm production, progressive motility and plasma membrane integrity were linearly improved by the increasing levels of curcumin supplementation (P < 0.01). However, dietary curcumin levels were linearly and quadratically associated with decreased seminal concentration of MDA (P < 0.01 and P < 0.03), percentage of abnormal sperm (P < 0.01 and P < 0.07) and increased plasma membrane functionality (P < 0.01 and P < 0.04), respectively. The SP holes in perivitelline membrane were increased in a linear and quadratic manner in response to increasing levels of curcumin (P < 0.01). Moreover, fertility rate was linearly improved (P < 0.01) as the dosage of curcumin increased, and resulted in 8, 12 and 14% improvements in the birds fed C10, C20 and C30, compared to C0, respectively. 4. In conclusion, the results showed that increasing levels of dietary supplementation of curcumin was associated with beneficial effects on semen quality indices and fertility rate in aged broiler breeder roosters.

TGF-ß4 and HSP70 responses in breeder hens treated with thyroxine.

A hypothesis was tested that long-term administration of thyroxine (T4) in broiler breeder hens would affect fertility, sperm penetration rate, and the duration of fertility. Relative abundance of oviductal TGF-ß4 and HSP70 mRNA was determined to ascertain whether T4 treatment affected these genes, and modulated the sustained storage of spermatozoa within the uterovaginal sperm storage tubules of hens. A total of 70, 47-week-old Cobb 500 breeder hens was randomly allotted to two treatment groups (T4 treatment (ET) and control). The T4 was orally administered to the ET group (0.3 mg T4/bird/day) for 100 consecutive days; whereas the control group was not administered T4 during the experimental period. Breeder hens were artificially inseminated to evaluate specific reproductive variables. On the last day of the treatment period two hens /replicate were randomly killed to estimate oviductal gene expression. The T4 treatment resulted in an increase in plasma concentration of T4; however, the T3 concentration was not affected. The long term administration of T4 had no effect on fertility; however, it resulted in a decreased sperm penetration rate and decreased the duration of fertility compared with the control group. The relative abundance of TGF-ß4 and HSP70 mRNA in the SST was not influenced by T4 supplementation. The correlation coefficients between fertility and sperm penetration rate with relative abundance of TGF-ß4 and HSP70 were not significant. Overall, among the diverse reproductive variable assessed in the current study, the sperm penetration rate and the duration of fertility were most responsive to long-term treatment with T4.

Histomorphometrical changes of oviduct during the long-term exposure of breeder hens to extra thyroxine.

In this research a 100 day long treatment period was considered to unmask the probable adverse effects of long-term induced hyperthyroidism on histomorphometrical attributes of the oviduct in broiler breeder hens. A total of seventy 47-week-old Cobb 500 breeder hens were randomly allotted to two treatment groups (5 replicates of 7 hens each). Thyroxine (T4) was orally administered to the hyperthyroid group (0.3 mg/bird/d) for 100 consecutive days; whereas the control group received drinking water only throughout the trial. At 64 weeks of age, 2 birds per replicate were killed by cervical dislocation and their oviducts were removed. For histomorphometrical observations, segments were taken from five different regions. After tissue preparation and staining with haematoxylin and eosin, histological layers were evaluated using light microscopy. The assessment of histomorphometrical characteristics of oviduct showed the height of mucosal folds in the magnum, thickness of mucosal folds of the magnum and uterus, thickness of tunica muscularis in the magnum and vagina, epithelial thickness of the isthmus and vagina, and uterine tubular glands percentage were decreased in the hyperthyroid birds compared with the control counterparts. The results showed long-term induced hyperthyroidism was associated with a decrease in a number of histomorphometrical traits in different regions of the oviduct. Some studies should be done to clarify to what extent the long-term maternal hyperthyroidism might affect the egg production, fertility rate, duration of fertility, and sperm penetration rate to make a final decision on exploitation of this preventative treatment to diminish the ascites incidence in progeny chicks.

Orally administered Chrysin improves post-thawed sperm quality and fertility of rooster.

Chrysin is a bioflavonoid compound found in passion flower, chamomile, propolis and honey at high levels. Post-thawed sperm quality and fertility of Chrysin-fed roosters were assessed in this study. Twenty 40-week-old male broiler breeders were randomly divided into four groups and fed basal diet supplemented with different levels of Chrysin including 0 (Ch-0), 25 (Ch-25), 50 (Ch-50) or 75 (Ch-75) mg/day for 12 consecutive weeks. Semen samples were weekly collected from 6th to 9th week of experiment to evaluate some sperm quality parameters including total and progressive motility, plasma membrane integrity and functionality (in fresh and post-thawed samples) and mitochondrial activity (only in post-thawed samples). Also, collected semen samples from 10th, 11th and 12th week of experiment were frozen and then artificially inseminated to test fertility rate. According to the results, an improvement in both fresh and post-thawed sperm quality including total [fresh: 88.00 ± 0.58 and 87.25 ± 0.67 (p < .01); post-thawed: 51.07 ± 2.05 and 52.72 ± 1.96 (p < .01)] and progressive motility [fresh: 76.00 ± 0.58 and 78.25 ± 0.65 (p < .01); post-thawed: 40.61 ± 2.01 and 39.88 ± 2.01 (p < .01)], plasma membrane integrity [fresh: 91.60 ± 0.58 and 89.85 ± 0.59 (p < .01); post-thawed: 56.99 ± 1.86 and 54.39 ± 1.86 (p < .01)] and functionality [fresh: 75.40 ± 0.42 and 77.90 ± 0.96 (p < .01); post-thawed: 45.69 ± 1.71 and 46.35 ± 1.71 (p < .01)] was noted for both Ch-50 and Ch-75, respectively, groups compared to control group. Despite no significant change in mitochondrial activity, fertility rate of post-thawed spermatozoa was significantly improved in all Chrysin-fed groups compared to Ch-0 group. In conclusion, oral Chrysin administration to roosters could ameliorate cryopreservation-induced impairment of sperm quality and fertility rate.

Antioxidative effect of BHA in soya bean lecithin-based extender containing Glycerol or DMSO on freezing capacity of goat semen.

The aim of this study was to evaluate the effects of butylated hydroxyanisole (0 or 4 mM) along with different concentrations (5 or 7%) of glycerol (G) and dimethyl sulphoxide (DMSO) as cryoprotectant (CPAs) on freezability of goat semen. Semen was collected from four bucks (3-4 years) twice a week for five weeks. The pooled ejaculates were diluted with extender containing two different concentrations of G or DMSO in combination with BHA. Afterwards, the diluted samples were loaded into 0.25 ml straws and frozen using a standard protocol. After thawing motility parameters, viability, membrane integrity and total abnormality were assessed. The Results showed that the presence of BHA in extender, type and level of CPAs as main factors had significant effects on goat sperm viability, total and progressive motility after freezing-thawing processes (p < .05). Also, the interaction of BHA (0 and 4 mM) and levels of G or DMSO (5 or 7%) had a significant effects (p < .05) on total motility, viability and some characteristic. In this case, the addition of 5% G or DMSO with BHA resulted in highest motility and viability than the other groups (p < .05). The addition of G5 (with and without BHA) increased VSL and reduced abnormality than the other groups (p < .05). The results showed that the main effects of CPAs and CPAs level on membrane functionality were significant (p < .05). Also there were no significance differences in the interactive effects of MDA, VCL, VAP, ALH, LIN and STR among the groups (p > .05). Finally, it can be concluded that the use of 5% CPAs with or without BHA may result in better post-thaw sperm quality of goat.

Effect of dietary fish oil supplementation on ram semen freeze ability and fertility using soybean lecithin- and egg yolk-based extenders.

Ram semen cryopreservation is not efficient for artificial insemination in commercial herds. Beneficial effects of dietary fish oil have been evaluated for cryopreservation of ram semen in soybean lecithin (SL) and egg yolk (EY)-based extenders. A factorial study (two diets × two extenders) was used to analyze the effects of two diets supplemented with fish oil (n-3 fatty acid) or palm oil (saturated fatty acids; [SFAs]) to freeze ram semen in two extenders containing SL or EY. Motility characteristics, membrane integrity, abnormal morphology, mitochondria activity, acrosome integrity, apoptotic status, and fertilizing ability were assessed after freeze-thawing. Although diet had significant (P ≤ 0.05) effects on the quality parameters of frozen-thawed sperm, effects of extenders on these traits were not significant (P > 0.05). The higher significant (P ≤ 0.05) percentage of total motility and progressive motility were observed in n-3/SL (44.83 ± 1.56 and 28.33 ± 1.4) and n-3/EY (43.33 ± 1.56 and 28.50 ± 1.4) than SFA/SL (32.16 ± 1.56 and 14.00 ± 1.4) and SFA/EY (31.66 ± 1.56 and 12.66 ± 1.4) groups. Moreover, n-3/SL and n-3/EY produced the higher significant (P ≤ 0.05) percentage of membrane integrity of sperm (39.83 ± 1.4 and 37.33 ± 1.4) than SFA/SL and SFA/EY (29.83 ± 1.4 and 28.5 ± 1.4). For viability results, the higher significant percentage of live sperm was observed in n-3/SL and n-3/EY (43.16 ± 1.38 and 45.66 ± 1.38) than SFA/SL and SFA/EY (28.66 ± 1.38 and 27.5 ± 1.38). For fertility trials, n-3-based diets (n-3/SL and n-3/EY) improved significantly (P ≤ 0.05) pregnancy rate (44% and 46%), parturition rate (42% and 42%), and lambing rate (46% and 44%) compared with the SFA-based diets (SFA/SL and SFA/EY). No interaction effects have been found between diets and extenders (P > 0.05). It seems that dietary fish oil can improve the semen performance after freezing-thawing process and artificial insemination aside from type of extenders.

The effect of feeding excess arginine on lipogenic gene expression and growth performance in broilers.

1. Reducing excess fat accretion is important for both human health and animal production. The present study was conducted to investigate the effects of arginine (Arg) on the regulation of lipogenic gene expression and on growth performance. 2. One-d-old female broiler chicks (Ross, n = 192) were used in a completely randomised design with 4 dietary treatments in which diets included 100% (CTL), 153% (LArg), 168% (MArg) and 183% (HArg) of the recommended concentration of digestible Arg. 3. Results showed that high concentrations of Arg improved body weight gain, feed efficiency, meat production, fat and crude protein content of breast muscle and plasma thyroid hormones. Conversely, abdominal fat, cholesterol, triglyceride and urea were lower with higher concentrations of Arg. Dietary arginine increased lipogenic gene expression in muscles, while decreasing those in adipose tissue and liver. 4. It was concluded that increasing Arg in the diet reduced abdominal fat content, enhanced intramuscular fat and increased muscle and protein gain. Furthermore, Arg supplementation at the MArg concentration improved growth performance, and at HArg had the greatest effect on fat reduction.

Optimization of ram semen cryopreservation using a chemically defined soybean lecithin-based extender.

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin-based semen extender as a substitute for egg yolk-based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris-based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post-thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early-apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post-thawed necrotic and late-apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.

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.

A twice-a-day feeding regimen optimizes performance in broiler breeder hens.

To evaluate the effects of different methods of feed allocation on performance, plasma hormone concentration, and ovarian morphology, an experiment was conducted using 32 Cobb 500 broiler breeder hens. The experiment was started at 27 wk and lasted to 39 wk of age. Feeding regimens included: i) hens with an ad libitum feeding program, ii) hens that received their restricted feed once a day, iii) hens that received their restricted feed twice a day, and iv) hens that received their restricted feed 3 times a day. Each hen was assumed as an experimental unit and treatments were replicated 8 times. Daily egg production, BW, and egg and yolk weights were measured. Two blood samples were taken 3 and 6 h after the first feed allocation every 2 wk. Plasma samples were assayed for glucose, triacylglycerol (TAG), cholesterol, as well as leptin-like concentration, glucagon, triiodothyronine, thyroxine, progesterone, estradiol, and testosterone. Liver, abdominal fat pad, and ovary were collected at necropsy. Ovaries were weighed and follicles were characterized as large yellow follicles, small yellow follicles, and large white follicles. Results showed inferior egg production in ad libitum-fed birds along with high levels of plasma glucose, TAG, cholesterol, leptin-like concentration, and testosterone. Twice-a-day-fed birds produced more egg in the entire production period than once-a-day-fed birds. Better performance of twice-a-day-fed hens was associated with lower plasma glucose, TAG, and leptin-like concentration, whereas their estradiol and glucagon were higher than once-a-day-fed hens. Results obtained in our study suggest that allocation of restricted feed 2 times a day may alleviate or delay lipotoxicity development and improve reproductive performance in broiler breeder hens.

The relationship of plasma leptin concentration and puberty in Holstein bull calves (Bos taurus).

The objective of this experiment was to study the changes of plasma leptin concentration during puberty and its relationship with testosterone level and testis dimensions in Holstein bull calves. Six Iranian Holstein bull calves with approximately 6 months of age were used. Semen evaluation was conducted at 1-month interval to determine the puberty state. To detect the plasma leptin and testosterone changes, blood samples were collected from the jugular vein during pre-puberty (6-7 months of age), puberty (8-9 months of age) and post-puberty (10-11 months of age). In addition, body weight (BW), body condition score (BCS) and testicular width and length were measured at 3-week intervals. The effects of time (age) on total sperm number and percentage of progressive motility of sperm, plasma concentration of leptin and testosterone, amplitude and frequencies of testosterone, BW, BCS, testicular dimensions were significant. Sperm number and progressive motility during post-puberty were higher than those during puberty and pre-puberty. Plasma concentration of leptin during the pre-puberty was higher than those during puberty and post-puberty (p < 0.01). Mean plasma testosterone concentrations during puberty were higher than those during pre-puberty (p < 0.05). BW, BCS and testicular dimensions consistently increased throughout the trial. Results indicated that in growing bull calves, plasma concentrations of leptin decreased during puberty, while circulating testosterone increased.