Maternal supplementation of organic selenium during gestation improves sows and offspring antioxidant capacity and inflammatory status and promotes embryo survival.

Selenium (Se) is an essential trace element in humans and sows, having a biological function mediated in part by its incorporation into selenoproteins. This study was conducted to investigate the effects of maternal 2-hydroxy-4-methylselenobutanoic acid (HMSeBA), an organic Se source, on reproductive performance, antioxidant capacity and inflammatory status of sows and their offspring. Forty-three Landrace × Yorkshire sows were randomly allocated to receive one of the following three diets during gestation: control diet (control, basal diet, n = 15), sodium selenite (Na2SeO3) supplemented diet (Na2SeO3, basal diet + Na2SeO3 at 0.3 mg Se per kg, n = 13), and HMSeBA supplemented diet (HMSeBA, basal diet + HMSeBA at 0.3 mg Se per kg, n = 15). Blood samples of sows and piglets, placentas and piglet liver samples were analyzed for selenium status, antioxidant capacity and inflammatory cytokines. Results showed that, as compared to the control group, HMSeBA supplementation increased the number of born alive piglets and plasma concentrations of total selenium and selenoprotein P in both sows and piglets. Besides, the activities of antioxidant enzymes in the blood of sows, umbilical cord and piglets, placentas and piglets' liver were increased by dietary HMSeBA supplementation as compared to the control group, while malondialdehyde concentration (p < 0.05) was decreased in the blood of sows, umbilical cord and newborn piglets. In addition, maternal HMSeBA intake during gestation up-regulated antioxidant-related selenoprotein gene expression in the placenta (GPx2, GPx3, p < 0.05) and in the liver of newborn piglets (GPx1, GPx2, GPx3, TXNRD2, p < 0.05). Moreover, as compared to the control group, sows and newborn piglets in the Na2SeO3 and HMSeBA groups had a lower serum interleukin-6 (p < 0.05) concentration, and placentas in the HMSeBA group had lower IL-1ß, IL-6 and IL-8 gene expression (p < 0.05). In conclusion, maternal supplementation of HMSeBA during pregnancy improved antioxidant capacities and reduced the inflammation level in mater, placenta, and fetus. This finding may highlight the important role of selenoproteins (especially GPXs) in preventing negative consequences of over-production of free radicals and inflammatory cytokines during gestation and at births.

Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA.

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

L-carnitine supplementation in culture media improves the pregnancy rate of in vitro produced embryos with sexed semen from Bos taurus indicus cows.

The in vitro embryo production industry in the actual world presents some difficulties related to low embryonic production rates, a problem that could be associated with in vitro culture conditions that differed from the in vivo (oviductal) conditions, mainly related to cytoplasmic lipid accumulation. L-carnitine is known as a modulator of ß-oxidation in the developing embryo, as it has been demonstrated that it improves embryo quality without affecting the in vitro embryo production rate. The aim of the present work was to evaluate the effect of L-carnitine supplemented during the in vitro maturation and culture processes on the implantation rate of in vitro produced embryos. Supplementation with 3.8 mM of L-carnitine was used during in vitro maturation, and later, during late in vitro culture, it was added at 1.5 mM. A control group contained no L-carnitine supplementation. Bovine oocytes obtained by ultrasound-guided follicle aspiration from healthy Bos taurus indicus cows were matured, fertilized and cultured in vitro. Multiparous F1 (Bos taurus taurus × Bos taurus indicus) cows were used as recipients. Overall, 460 oocytes were processed in three independent replicates from in vitro maturation until day 8 of the in vitro culture. No significant difference was found between treatments of in vitro embryo production. However, pregnancy rate at days 45 and 72 was significantly higher in blastocysts derived from L-carnitine treatment (31.55 ± 9.78%) compared to the control group (18.68 ± 6.31%). In conclusion, addition of L-carnitine at 3.8 mM and 1.5 mM in the maturation, and culture medium after day 3 of in vitro production process, significantly improved pregnancy rate after embryo transfer.

Media optimization to promote rat embryonic development to the blastocyst stage in vitro.

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.

Yy1 regulates Senp1 contributing to AMPA receptor GluR1 expression following neuronal depolarization.

BACKGROUND: Neuronal activity-induced changes in gene expression patterns are important mediators of neuronal plasticity. Many neuronal genes can be activated or inactivated in response to neuronal depolarization. Mechanisms that activate gene transcription are well established, but activity-dependent mechanisms that silence transcription are less understood. It is also not clear what is the significance of inhibiting these genes during neuronal activity. METHODS: Quantitative Real Time-PCR, western blot and immunofluorescence staining were performed to examine the expression of Senp1 and GluR1 in mouse cortical neurons. The alterations of Yy1 phosphorylation upon neuronal depolarization and the interaction of Yy1 with Brd4 were studied by protein co-immunoprecipitation. The regulators of Yy1 phosphorylation were identified by phosphatase inhibitors. Chromatin immunoprecipitation, in vitro DNA binding assay, luciferase assay and gene knockdown experiments were used to validate the roles of Yy1 and its phosphorylation as well as Brd4 in regulating Senp1 expression. RESULTS: We report that neuronal depolarization deactivates the transcription of the SUMO protease Senp1, an important component regulating synaptic transmission, scaling, and plasticity, through Yy1. In un-stimulated neurons, Senp1 transcription is activated by a Yy1-Brd4 transcription factor protein complex assembled on the Senp1 promoter. Upon membrane depolarization, however, Yy1 is dephosphorylated and the Yy1-Brd4 complex is evicted from the Senp1 promoter, reducing Senp1 transcription levels. Both Yy1 and Senp1 promote the expression of AMPA receptor subunit GluR1, a pivotal component in learning and memory. CONCLUSIONS: These results reveal an axis of Yy1/Brd4-Senp1 which regulates the expression of GluR1 during neuronal depolarization. This implicates a regulation mechanism in silencing gene expression upon neuronal activity.

The biochemistry surrounding bovine conceptus elongation†.

Conceptus elongation is a fundamental developmental event coinciding with a period of significant pregnancy loss in cattle. The process has yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by systemic progesterone. To better understand the environment facilitating this critical reproductive phenomenon, we interrogated the biochemical composition of uterine luminal fluid from heifers with high vs physiological circulating progesterone on days 12-14 of the estrous cycle-the window of conceptus elongation-initiation-by high-throughput untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. A total of 233 biochemicals were identified, clustering within 8 superpathways [amino acids (33.9%), lipids (32.2%), carbohydrates (8.6%), nucleotides (8.2%), xenobiotics (6.4%), cofactors and vitamins (5.2%), energy substrates (4.7%), and peptides (0.9%)] and spanning 66 metabolic subpathways. Lipids dominated total progesterone (39.1%) and day (57.1%) effects; however, amino acids (48.5%) and nucleotides (14.8%) accounted for most day by progesterone interactions. Corresponding pathways over-represented in response to day and progesterone include (i) methionine, cysteine, s-adenosylmethionine, and taurine (9.3%); (ii) phospholipid (7.4%); and (iii) (hypo)xanthine and inosine purine metabolism (5.6%). Moreover, under physiological conditions, the uterine lumen undergoes a metabolic shift after day 12, and progesterone supplementation increases total uterine luminal biochemical abundance at a linear rate of 0.41-fold day-1-resulting in a difference (P ≤ 0.0001) by day 14. This global metabolic analysis of uterine fluid during the initiation of conceptus elongation offers new insights into the biochemistry of maternal-embryo communication, with implications for improving ruminant fertility.

Flaxseed improves embryo production in Boer goats.

Flaxseed is a source of polyunsaturated fatty acids and could be used as a dietary ingredient to enhance reproductive performance of ruminants. The objectives of this study were to determine the effect of feeding diets with different levels of flaxseed on the nutrient intake, and quantity and quality of embryos in Boer goats. A total of 24 multiparous Boer goats were fed with a diet containing either 0, 4, 8 or 12% of flaxseed (n = 6 per group) and subjected to superovulation to determine the quantity and quality of embryos collected on 7 d after natural service. The nutrient intake was linearly associated with levels of flaxseed in the diet and, whereas while the fat (measured as ether extract) intake was positively associated, the non-fiber carbohydrate intake had a negative association with increasing levels of flaxseed in the diet. The quantity, quality and stage of embryonic development on 7 d after natural service were significantly different between levels of flaxseed in the diet. The number of viable embryos was greater in goats fed with a diet containing 4, 8, and 12% flaxseed (94, 84, and 87%, respectively) than those fed with a diet containing 0% flaxseed (65%). On the other hand, the number of degenerated embryos was greater for goats fed with a diet containing 0% flaxseed (35%) than those fed with a diet containing 4, 8, and 12% flaxseed (6, 16, and 13%, respectively). The proportion of grade 1 embryo collected was greater for goats fed with a diet containing 4 and 8% flaxseed (74 and 83%, respectively) than those fed with a diet containing 0 and 12% flaxseed (40 and 46%, respectively). In summary, our study demonstrated that feeding a diet with moderate levels of flaxseed could produce a greater number of better-quality embryos in Boer goats.

Oxidative stress differentially impacts male and female bovine embryos depending on the culture medium and the stress condition.

Male and female embryos are known to differ for their metabolism and response to environmental factors very early in development. The present study aimed to evaluate the response to oxidative stress of male and female bovine embryos at the morula-blastocyst stages in terms of developmental rates, total cell number and apoptotic rates in two culture conditions. Embryos where cultured in a medium supplemented with either 5% fetal calf serum (FCS) or 4 mg/mL bovine serum albumin and a mixture of insulin, transferrin and selenium (BSA-ITS). Oxidative stress was applied at Day-5 post insemination (pi) by adding either AAPH or menadione to the culture medium, and blastocysts were analyzed at Day-7pi. The impact on development and blastocyst quality was dependent on the culture medium and the stress inducer but differed between male and female embryos. Male embryos resisted better to oxidative stress in FCS supplemented medium, no matter the stress inducer. Accordingly, the impact on blastocyst cell number tended to be higher in female blastocysts after stress induction with AAPH in FCS supplemented medium. On the other hand, in BSA-ITS supplemented medium, female embryos were more resistant to AAPH induced stress, while menadione had no impact on sex ratio. The weaker resistance of males to AAPH in this medium is in accordance with their trend to show a higher increase in apoptotic rates than females in this condition. In conclusion, this study shows that oxidative stress has differential impact on male and female bovine blastocysts depending on the culture condition and on the way oxidative stress is induced.

l-carnitine supplementation during vitrification or warming of in vivo-produced ovine embryos does not affect embryonic survival rates, but alters CrAT and PRDX1 expression.

l-carnitine is an antioxidant and ß-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies.

Cobalamin supplementation during in vitro maturation improves developmental competence of sheep oocytes.

Pregnancies obtained by Assisted Reproductive Technologies are at higher risk of miscarriage than those obtained naturally. Previously, we reported impaired placental vascular development of in vitro produced (IVP) sheep embryos and defective DNA methylation in the placentae of those embryos. One reason behind these observed defects may be an impaired One Carbon Metabolism (OCM) The present study was performed to test the hypothesis that Cobalamin (Vitamin B12, an important OCM co-factor) supplementation during IVM corrects DNA methylation of IVP embryos and, consequently, ameliorates placental vasculogenesis. To this aim, embryos derived from oocytes matured with Cobalamin (B12 group) or without (negative control group, -CTR) were transferred to synchronized recipient sheep. At day 20 of pregnancy, collected embryos were morphologically evaluated while placentae were subjected to qPCR and histological analysis. The positive control group (+CTR) consisted of conceptuses obtained from naturally mated sheep. Results showed an increased fertilization rate in the B12 group vs -CTR (69.56% vs 57.91% respectively, P = 0.006) not associated with quantitative improvement in blastocyst and/or implantation rate (44.32% vs 36.67% respectively, P > 0.05). Moreover, Cobalamin supplementation during oocyte IVM ameliorated resulting conceptuses quality, in terms of placental vascularization (vessels' maturity and vasculogenetic factors' expression). The expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was also improved in placentae from the B12 group. In conclusion, Cobalamin supplementation during oocyte IVM improves IVP embryo quality. These results suggest that Cobalamin should be included in standard IVM media.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Activation of the neurokinin 3 receptor promotes filopodia growth and sprouting in rat embryonic hypothalamic cells.

Members of the tachykinin family have trophic effects on developing neurons. The tachykinin neurokinin 3 receptor (NK3R) appears early in embryonic development; during the peak birthdates of hypothalamic neurons, but its involvement in neural development has not been examined. To address its possible role, immortalized embryonic hypothalamic neurons (CLU209) were treated with CellMask, a plasma membrane stain, or the membranes were imaged in CLU209 cells that were transfected with a pEGFP-NK3R expression vector. Nontransfected cells and transfected cells were then treated with senktide, a NK3R agonist, or Dulbecco's Modified Eagle's Medium (DMEM) and time-lapse confocal images were captured for the following 30 min. Compared to DMEM, senktide treatment led to filopodia initiation from the soma of both nontransfected and transfected CLU209 cells. These filopodia had diameters and lengths of approximately 200 nm and 3 µm, respectively. Pretreatment with an IP3 receptor blocker, 2-aminoethoxydiphenyl borate (2-APB), prevented the senktide-induced growth in filopodia; demonstrating that NK3R-induced outgrowth of filopodia likely involves the release of intracellular calcium. Exposure of transfected CLU209 cells to senktide for 24 h led to further growth of filopodia and processes that extended 10-20 µm. A mathematical model, composed of a linear and population model was developed to account for the dynamics of filopodia growth during a timescale of minutes. The results suggest that the ligand-induced activation of NK3R affects early developmental processes by initiating filopodia formation that are a prerequisite for neuritogenesis.

Developmental competence of bovine early embryos depends on the coupled response between oxidative and endoplasmic reticulum stress.

The stress produced by the coupling of reactive oxygen species (ROS) and endoplasmic reticulum (ER) has been explored extensively, but little is known regarding their roles in the early development of mammalian embryos. Here, we demonstrated that the early development of in vitro-produced (IVP) bovine embryos was governed by the cooperative action between ROS and ER stress. Compared with the tension produced by 5% O2, 20% O2 significantly decreased the blastocyst formation rate and cell survival, which was accompanied by increases in ROS and in levels of sXBP-1 transcript, which is an ER stress indicator. In addition, treatment with glutathione (GSH), a ROS scavenger, decreased ROS levels, which resulted in increased blastocyst formation and cell survival rates. Importantly, levels of sXBP-1 and ER stress-associated transcripts were reduced by GSH treatment in developing bovine embryos. Consistent with this observation, tauroursodeoxycholate (TUDCA), an ER stress inhibitor, improved blastocyst developmental rate, trophectoderm proportion, and cell survival. Moreover, ROS and sXBP-1 transcript levels were markedly decreased by supplementation with TUDCA, suggesting a possible mechanism governing the mutual regulation between ROS and ER stress. Interestingly, knockdown of XBP-1 transcripts resulted in both elevation of ROS and decrease of antioxidant transcripts, which ultimately reduced in vitro developmental competence of bovine embryos. Based on these results, in vitro developmental competence of IVP bovine embryos was highly dependent on the coupled response between oxidative and ER stresses. These results increase our understanding of the mechanism(s) governing early embryonic development and may improve strategies for the generation of IVP embryos with high developmental competence.

Role of melatonin on production and preservation of gametes and embryos: a brief review.

The aim of this brief review is to clarify the role of melatonin in the production and preservation of mammalian gametes and embryos. Melatonin is an indoleamine synthesized from tryptophan in the pineal gland and other organs that operates as a hypothalamic-pituitary-gonadal axis modulator and regulates the waxing and waning of seasonal reproductive competence in photoperiodic mammals. A major function of the melatonin rhythm is to transmit information about the length of the daily photoperiod to the circadian and circannual systems in order to provide time-of-day and time-of-year information, respectively, to the organism. Melatonin is also a powerful antioxidant and anti-apoptotic agent, which is due to its direct scavenging of toxic oxygen derivatives and its ability to reduce the formation of reactive species. Mammalian gametes and embryos are highly vulnerable to oxidative stress due to the presence of high lipid levels; during artificial breeding procedures, these structures are exposed to dramatic changes in the microenvironment, which have a direct bearing on their function and viability. Free radicals influence the balance between oxidation-reduction reactions, disturb the transbilayer-phospholipid asymmetry of the plasma membrane and enhance lipid peroxidation. Melatonin, due to its amphiphilic nature, is undoubtedly useful in tissues by protecting them from free radical-mediated oxidative damage and cellular death. The supplementation of melatonin to semen extender or culture medium significantly improves sperm viability, oocyte competence and blastocyst development in vitro.

Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro.

High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

Green tea polyphenols added to IVM and IVC media affect transcript abundance, apoptosis, and pregnancy rates in bovine embryos.

Three experiments were conducted to examine the effects of green tea polyphenols (GTP) during IVM and IVC on apoptosis and relative transcript abundance (RA) of three genes controlling antioxidant enzymes, as well as subsequent pregnancy rates. In experiment 1, oocytes were matured in the presence of 0, 10, 15, or 25 µM GTP for 24 hours. The GTP dose applied to IVM medium was followed by the same dose supplemented to IVC medium, so oocytes and embryos of a given group were cultured in similar conditions. This resulted in a total of four groups (three experimental groups and the control). After IVF, presumptive zygotes were cultured in medium containing 0 to 25 µM GTP for 8 days. The addition of 15 µM GTP during IVM and IVC increased RA of SOD1, CAT, and GPX genes in blastocysts compared with the control (P < 0.05). Increase in GTP doses from 15 to 25 µM did not further increase the transcript level. In experiment 2, effects of GTP doses on apoptosis were investigated in bovine blastocysts. Two of the applied GTP doses (10 and 15 µM) decreased the apoptotic index (AI) in blastocysts (7.4% and 6.2% respectively) compared with the control (9.3%; P < 0.05). However, the highest GTP dose used (25 µM) caused an increase in AI compared with a dose of 15 µM (P < 0.05). Considering the results of experiment 1 and 2, the effects of 15 µM GTP treatment during IVM and IVC on pregnancy rate was evaluated after embryo transfer in experiment 3. Cows receiving embryos treated with 15 µM GTP had higher pregnancy rates on Day 30 (34.8% vs. 28.6%) and Day 60 (34.8% vs. 23.9%) than those receiving control embryos (P < 0.05). In conclusion, addition of 15 µM GTP during IVM and IVC improved pregnancy rates; this improvement seemed to be associated with the increase of RA of antioxidant enzyme genes and the decrease in AI in bovine blastocysts.

Genotoxic and cytotoxic study of Tecoma stans Bignoniaceae.

Tecoma stans (Bignoniaceae) is a central and south American tree used for the control of diabetes. This plant is cultivated in Iraq. The dried leaves were soaked in ethanol and water separately for 3 days then filtered and dried. The genotoxic potential of Tecoma stans was studied by in vivo and in vitro system. This study examined the genotoxic activity of aqueous and ethanolic extracts on bone marrow cells from BALB/c mice through evaluation of mitotic index and chromosomal aberrations and cytotoxic effect of the two extracts on Mouse Embryo Fibroblast (MEF) cell line. No alteration in the total number of chromosomal aberrations or the number of cells with chromosomal aberrations observed and percentage of mitotic index at the concentrations tested remained unchanged. The higher concentrations used of the plant extracts had a cytotoxic effect on the MEF cell line. Both extracts had no significant clastogenic effect in vivo but showed cytotoxic effects on mouse embryo in vitro, caution should be exercised in the use of this substance as a medicine.

Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success.

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b)-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

Oocyte and embryo production and quality after OPU-IVF in dairy heifers given diets varying in their n-6/n-3 fatty acid ratio.

Dietary fat supplementation can improve oocyte quality in ruminants. The influence of the type of dietary fat on the number and quality of oocytes collected by ovum pick-up and on the production of embryos in vitro was investigated in Holstein heifers. Heifers were given hay plus one of two dietary supplements for 42 days. The supplements were linseed (L, rich in linolenic acid, C18:3n-3, n = 9) or soya bean (S, rich in linoleic acid, C18:2n-6, n = 9). Oocytes were collected by ovum pick-up (OPU) for 6 wks (2 sessions/wk) and morphologic quality assessed. Half the oocytes were frozen and the other half was used to produce embryos. Blood samples were analyzed for: insulin, insulin-like growth factor-1, glucose, non-esterified fatty acids, ß-hydroxy butyrate and urea and the proportions of fatty acids. Neither growth rate nor plasma hormone and metabolite concentrations were affected by dietary supplement. However, L significantly increased the proportion of plasma C18:3n-3 while S significantly increased the proportion of C18:2n-6(P < 0.001). Neither oocyte characteristics (number, their quality and number fertilized and cleaved per heifer per session) nor embryo characteristics (number and quality per heifer per session) and embryo development stages were affected by dietary treatment. Real-time RT-PCR was performed on immature and mature cumulus-oocyte complexes (COC). Prostaglandin E synthase-1 expression increased in L compared to S heifers. In conclusion, the type of fatty acid did not modify the numbers of oocytes and embryos produced by OPU-IVF and their quality in dairy heifers. Upregulation of prostaglandin E synthase-1 may ensure sufficient PGE(2) production for oocyte maturation even when its precursor is low.

Valproic acid improves the in vitro development competence of bovine somatic cell nuclear transfer embryos.

The present study was carried out to examine the effect of valproic acid (VPA), an important histone deacetylase inhibitor, on the in vitro development and expression of the epigenetic marker histone H3 lysine 9 (H3K9ac) in bovine somatic cell nuclear transfer (SCNT) embryos. We found that treatment with 4 mM VPA for 24 h could significantly improve the development of bovine SCNT embryos. Compared with the no-treatment group, the cleavage rate was higher (69.79 ± 0.99% vs. 65.11 ± 1.02%, p<0.05), as was the blastocyst rate (39.99 ± 1.29% vs. 34.87 ± 1.74%, p<0.05). Moreover, the rate of apoptosis (1.91 ± 0.48% vs. 5.67 ± 0.40%, p<0.05) in blastocysts was greatly reduced after VPA treatment. Valproic acid treatment also increased the immunofluorescent signal for H3K9ac in SCNT embryos in a pattern similar to that of in vitro fertilized (IVF) embryos. In conclusion, we demonstrated that VPA can significantly improve the in vitro developmental competence and enhance the nuclear reprogramming of bovine SCNT embryos.