Molecular, enzymatic responses and in vitro embryonic developmental competency of heat-shocked buffalo embryos co-cultured with granulosa cells monolayer.

The present study was designed to establish a suitable alternative approach to mitigate the adverse effect of high culture temperature on in vitro embryo development and the related molecular response in buffalo. Pre-cultured granulosa cells (GCs) were used as a monolayer during in vitro embryo culture until day 8 (day of fertilization = D0). Post fertilization, presumptive embryos were randomly assigned into two culture conditions: embryos cultured in the presence of GCs monolayer under normal culture temperature (N: 38.5 °C; GEN group) or heat shock (H: 40.5 °C; GEH group) and their counterpart groups of embryos cultured without GCs (EN and EH groups). Additionally, two groups of GCs monolayer were cultured without embryos up to day 8 under 38.5 °C (GN) or 40.5 °C (GH) for further spent culture media enzymatic analyses. Heat shock was administered for the first 2 h of culture then continued at 38.5 °C until day 8. The results indicated that under heat treatment, GCs enhanced (P ≤ 0.05) embryo cleavage and development (day 8) rates, which were comparable to the embryos cultured at 38.5 °C. On the molecular level, blastocysts of the GEH group showed similar expressions of metabolism-regulating genes (CPT2 and SlC2A1/GLUT1) and an antioxidant gene (SOD2) when compared to the blastocysts of the EN group. The relative expression of HSP90 was significantly up-regulated under heat shock and/or co-culture conditions. However, HSF1 expression was increased (P ≤ 0.05) in the GEH group. No statistical differences were observed among the study groups for the pluripotency gene NANOG, and stress resistance transcript NFE2L2. Regarding the enzymatic profile, the concentrations of SOD, total protein, and MDA were decreased (P ≤ 0.05) in the GEH group compared to the cultured GCs without embryos (GH group). In conclusion, GCs as a monolayer have a beneficial impact on alleviating heat stress at the zygote stage through the regulatory mechanisms of metabolic activity, defense system, and heat shock response genes.

Melatonin administration during the first half of pregnancy improves the reproductive performance of rabbits: Emphasis on ovarian and placental functions.

This study was designed to investigate the roles of melatonin administration during different sensitive windows of the first half of pregnancy in the function and gene expression of the ovary and placenta, hormone profile, and pregnancy outcomes in rabbits. Four equal experimental groups of 20 rabbits each were used. The first (FW), second (SW), and third (F + SW) groups comprised rabbits that orally received 0.7-mg melatonin/kg body weight during the first week, second weeks, and during both weeks of pregnancy; and the fourth group served as the control group (C). The total number of visible follicles significantly increased in all melatonin-treated groups compared with that in the C group. In all melatonin-treated groups, the number of absorbed fetuses was significantly reduced, whereas the weights of embryonic sacs and fetuses were higher than in the C group. The placenta efficiency was significantly increased in the F + SW group compared with that in the C group, followed by the SW group, whereas no significant difference in the placenta efficiency was found between the FW and C groups. Melatonin treatments significantly improved the expression of antioxidants, gonadotropin receptors, and cell cycle regulatory genes in the ovary, whereas only FW treatment upregulated steroidogenic acute regulatory gene. Compared with the C and FW groups, melatonin treatments during the SW and F + SW significantly upregulated the expression of most genes in the placenta. The concentrations of estradiol were significantly higher in the SW and F + SW groups than in the FW and C groups. The concentrations of progesterone were significantly increased in the FW group compared with those in the C and SW groups, whereas the F + SW group showed intermediate values. The litter size and weight at birth significantly increased in all melatonin-treated groups compared with those in the C group. The second week of pregnancy seems to be a sensitive window for melatonin actions during pregnancy. Thus, melatonin administration during the second week of pregnancy can be effective in improving pregnancy outcomes in rabbits.

Molecular and physiochemical evaluation of buck semen cryopreserved with antioxidants.

The current study evaluated the physiochemical quality and gene expression profile of post-thawed buck semen after supplementation with antioxidants [melatonin (M), L-carnitine (LC), cysteine (Cys), LC + M, M + Cys, LC + Cys, LC + Cys + M] in comparison with the non-treated control group. Physical and biochemical characteristics of semen were evaluated following freezing and thawing. Transcript abundance of six selected candidate genes was profile using quantitative real-time PCR. The data demonstrated significant enhancement of post-freezing total motility, progressive motility, percentage of live sperm, CASA parameters, plasma membrane and acrosome integrity in all groups supplemented with Cys, LC, M + Cys and LC + Cys compared with the control group. The biochemical analysis of semen indicated that semen groups supplemented with LC and LC + Cys recorded increased levels of GPX and SOD that were coupled with up-regulation of antioxidant genes (SOD1, GPX1 and NRF2) and mitochondrial transcripts (CPT2 and ATP5F1A). Moreover, H2O2 level and DNA fragmentation percentage were reduced compared with other groups. In conclusion, supplementation of Cys alone or in combination with LC positively improved the post-thaw physiochemical properties of rabbit semen through activation of bioenergetics-related mitochondrial genes and cellular antioxidant defence mechanism.

Integrative QTL mapping and selection signatures in Groningen White Headed cattle inferred from whole-genome sequences.

Here, we aimed to identify and characterize genomic regions that differ between Groningen White Headed (GWH) breed and other cattle, and in particular to identify candidate genes associated with coat color and/or eye-protective phenotypes. Firstly, whole genome sequences of 170 animals from eight breeds were used to evaluate the genetic structure of the GWH in relation to other cattle breeds by carrying out principal components and model-based clustering analyses. Secondly, the candidate genomic regions were identified by integrating the findings from: a) a genome-wide association study using GWH, other white headed breeds (Hereford and Simmental), and breeds with a non-white headed phenotype (Dutch Friesian, Deep Red, Meuse-Rhine-Yssel, Dutch Belted, and Holstein Friesian); b) scans for specific signatures of selection in GWH cattle by comparison with four other Dutch traditional breeds (Dutch Friesian, Deep Red, Meuse-Rhine-Yssel and Dutch Belted) and the commercial Holstein Friesian; and c) detection of candidate genes identified via these approaches. The alignment of the filtered reads to the reference genome (ARS-UCD1.2) resulted in a mean depth of coverage of 8.7X. After variant calling, the lowest number of breed-specific variants was detected in Holstein Friesian (148,213), and the largest in Deep Red (558,909). By integrating the results, we identified five genomic regions under selection on BTA4 (70.2-71.3 Mb), BTA5 (10.0-19.7 Mb), BTA20 (10.0-19.9 and 20.0-22.7 Mb), and BTA25 (0.5-9.2 Mb). These regions contain positional and functional candidate genes associated with retinal degeneration (e.g., CWC27 and CLUAP1), ultraviolet protection (e.g., ERCC8), and pigmentation (e.g. PDE4D) which are probably associated with the GWH specific pigmentation and/or eye-protective phenotypes, e.g. Ambilateral Circumocular Pigmentation (ACOP). Our results will assist in characterizing the molecular basis of GWH phenotypes and the biological implications of its adaptation.

Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum.

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

Co-expression of candidate genes regulating growth performance and carcass traits of Barki lambs in Egypt.

Sheep are considered one of the main sources of animal protein in Egypt and the producers of sheep mutton eagers to find biological criteria for selecting fast-growing lambs that reach market weight early. Therefore, the present study aimed to find a link between the expression profile of selected candidate genes with growth performance and carcass traits of Barki lambs. Thirty-eight Barki lambs were kept and fed individually after weaning till 12 months of age and were divided into 3 groups according to growth performance (fast, intermediate, and slow-growing). Three samples were taken from different body tissues (eye muscle, liver, and fat tail) of each group, directly during slaughtering and stored at - 80 °C until RNA isolation. Real-time PCR was used to profile selected candidate genes (RPL7, CTP1, FABP4, ADIPOQ, and CAPN3) and GAPDH was used as a housekeeping gene. The results indicated that the final body weight was significantly (P ≤ 0.05) greater in the fast (49.9 kg) and intermediate (40.7 kg) compared to slow-growing animals (30.8 kg). The hot carcass weight was heavier (P ≤ 0.05) in the fast and intermediate-growing (24.57 and 19.07 kg) than slow-growing lambs (15.10 kg). The blood profiles of T3 and T4 hormones in addition to other parameters such as total protein, total lipids, and calcium level showed no clear variations among different experimental groups. At the molecular level, our data demonstrated upregulation of genes involved in protein biosynthesis (RPL7), fatty acid oxidation (CPT1), and lipolysis (FABP4) in the fast and intermediate-growing lambs in all studied tissues which facilitate protein accretion, energy expenditure, and fatty acid partitioning required for muscle building up. Moreover, the expression profile of the gene involved in muscle development (CAPN3) was increased in fast and intermediate-growing compared to slow-growing lambs in order to support muscle proper development. On the other hand, a candidate gene involved in lipogenesis (ADIPOQ) was expressed similarly in fat and liver tissues; however, its expression was increased in muscles of fast and intermediate-growing lambs compared to slow-growing animals. In conclusion, the current study indicated that the expression profile of genes involved in metabolic activities of liver, muscle, and adipose tissue is linked with the growth performance of lambs although no variations were detected in blood parameters. This provides an evidence for the importance of co-expression of these genes in body tissues to determine the final body weight and carcass characteristics of Barki sheep.

Adaptive and Biological Responses of Buffalo Granulosa Cells Exposed to Heat Stress under In Vitro Condition.

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

Genetic Features of Reproductive Traits in Bovine and Buffalo: Lessons From Bovine to Buffalo.

Bovine and buffalo are important livestock species that have contributed to human lives for more than 1000 years. Improving fertility is very important to reduce the cost of production. In the current review, we classified reproductive traits into three categories: ovulation, breeding, and calving related traits. We systematically summarized the heritability estimates, molecular markers, and genomic selection (GS) for reproductive traits of bovine and buffalo. This review aimed to compile the heritability and genome-wide association studies (GWASs) related to reproductive traits in both bovine and buffalos and tried to highlight the possible disciplines which should benefit buffalo breeding. The estimates of heritability of reproductive traits ranged were from 0 to 0.57 and there were wide differences between the populations. For some specific traits, such as age of puberty (AOP) and calving difficulty (CD), the majority beef population presents relatively higher heritability than dairy cattle. Compared to bovine, genetic studies for buffalo reproductive traits are limited for age at first calving and calving interval traits. Several quantitative trait loci (QTLs), candidate genes, and SNPs associated with bovine reproductive traits were screened and identified by candidate gene methods and/or GWASs. The IGF1 and LEP pathways in addition to non-coding RNAs are highlighted due to their crucial relevance with reproductive traits. The distribution of QTLs related to various traits showed a great differences. Few GWAS have been performed so far on buffalo age at first calving, calving interval, and days open traits. In addition, we summarized the GS studies on bovine and buffalo reproductive traits and compared the accuracy between different reports. Taken together, GWAS and candidate gene approaches can help to understand the molecular genetic mechanisms of complex traits. Recently, GS has been used extensively and can be performed on multiple traits to improve the accuracy of prediction even for traits with low heritability, and can be combined with multi-omics for further analysis.

Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation.

This study was conducted to monitor the cellular and molecular changes of buffalo cumulus-oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB- and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB- group. BCB- oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB- under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB-) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB- oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.

In Vivo and In Vitro Evaluation of Bull Semen Processed with Zinc (Zn) Nanoparticles.

Defective sperms cause fertilization failure under both in vivo and in vitro conditions. Therefore, providing optimal conditions during semen storage is a prerequisite for maintaining viability. The current study investigated bull semen quality in vitro and in vivo when zinc (Zn) nanoparticles were used as antioxidant during semen processing and cryopreservation. In total, 32 ejaculates were collected from four Holstein bulls. All ejaculates were pooled and diluted with Bioxcell-extender containing 0 (control group), 10-6, 10-5, 10-4, 10-3, and 10-2 M of Zn nanoparticles. Several physical and biochemical sperm parameters were determined after freeze-thawing process. In vitro embryo development rate and pregnancy rate were monitored after in vitro fertilization or artificial insemination using semen treated with Zn nanoparticles. Plasma membrane integrity was improved (P < 0.05) in bull semen treated with 10-6 M (69.3%), and 10-2 (62.4%) of Zn nanoparticles compared to untreated group (51.3%). In addition, proportions of live spermatozoa with active mitochondria were increased (P < 0.05) in semen supplemented with Zn nanoparticles at concentration of 10-6 M (67.3%), and 10-2 (85.3%) compared to control group (49.8%). Moreover, the level of MDA was lower (P < 0.05) in semen with Zn nanoparticles at 10-6 M (2.97 mol/mL) and 10-2 (2.7 mol/mL) concentrations than control semen samples (3.77 mol/mL). However, sperm total and progressive motility, sperm viability, DNA fragmentation, and pregnancy rate were not affected by treatment of semen with Zn nanoparticles. On the other hand, supplementation of in vitro maturation media with 10-6 M Zn nanoparticles has increased blastocyst rate (P < 0.05) compared to other experimental groups, while addition of Zn nanoparticles-treated sperm during in vitro fertilization did not affect embryo development rate. In conclusion, supplementation of Zn nanoparticles to semen has improved its quality without affecting embryo development rate in vitro. However, in vitro embryo development rate was increased when Zn nanoparticles were supplemented to IVM media. This support the notion of Zn nanoparticles beneficial action on improving bovine gametes quality without affecting pregnancy rate.

Physiological and molecular aspects of heat-treated cultured granulosa cells of Egyptian buffalo (Bubalus bubalis).

The physiological and molecular responses of granulosa cells (GCs) from buffalo follicles were investigated when there were in vitro heat stress conditions imposed. The cultured GCs were heat-treated at 40.5 °C for 24, 48 or 72 h while GCs of the control group were not heat-treated (37 °C). There were no differences in viability between control and heat-treated groups. There was an upward trend in increase in E2 secretion as the duration of heat stress advanced, being greater (P ≤ 0.05) for the GCs on which heat stress was imposed for 72 as compared with 24 h. In contrast, P4 release was less (P ≤ 0.05) from GCs heat-treated for 48 h than those cultured for 24 h and GCs of the control group. The relative abundance of ATP5F1A and SOD2 mRNA transcripts was consistent throughout the period when there was imposing of heat stress to sustain mitochondrial function. The relative abundance of CPT2 transcript was less in heat-treated GCs than in GCs of the control group. There was a greater relative abundance of SREBP1 and TNF-α mRNA transcripts after 48 h of heat-treatment of GCs than GCs of the control group. In conclusion, the results from the current study indicate buffalo GCs cultured when there was imposing of heat stress maintained normal viability, steroidogenesis and transcriptional profile. The stability of antioxidant status and increased transcription of genes regulating cholesterol biosynthesis and stress resistance may be defense mechanisms of buffalo GCs against heat stress.

Effect of flunixin meglumine and aspirin administration on conception rate and estrous cycle characteristics of Egyptian Baladi cows during hot season.

The current investigation aims to evaluate the effects of flunixin meglumine (FM) and aspirin as non-steroid anti-inflammatory drug (NSAID) administration on estrous cycles characteristics and conception rate of Egyptian Baladi cows during hot season. In the first phase, 30 cows were divided into 3 groups, 10 cows for each treatment. The first group was treated with FM at the rate of 1.1 mg/kg body weight (BW) intramuscular, while the second group was administrated aspirin solution orally at the rate of 50 mg/kg BW. The third group was assigned as control (CG) that has no treatment. The FM group was administrated on day 14 after mating, while aspirin was given on day 14 and day 15 post-mating. All cows were mated naturally after showing estrus signs. Pregnancy diagnosis was carried 60 days after mating by rectal palpation. In the second phase, cows were monitored for estrus behavior by visual observation twice a day. The length of normal estrous cycles was 20, 23, and 22 days in cows treated with FM, aspirin, and control cows, respectively. There was no significant effect of treatment on the length of normal estrous cycles in Egyptian cows (P < 0.05). Proportions of long cycles in Egyptian cows that treated with FM or aspirin and control were 75, 67.7, and 57.1%, respectively. Short cycles were completely absent in cows that treated with FM or aspirin, but it was 29% in CG. Mounting behavior and tail rising were not detected in CG compared to 0 and 33% in FM or 25 and 33% in aspirin treated cows, respectively. Conception or pregnancy rate were 60, 40, and 30%, respectively, in FM, aspirin treated, and CG. Treatment cows whether FM or aspirin group did not influence (P < 0.05) progesterone concentration during the 14 days and 21 days from estrous cycle in pregnant and non-pregnant Egyptian Baladi cows than CG. In conclusion, the results of this study clearly indicated beneficial effect of FM and aspirin administration on intense of displayed estrous behavior and conception rate of Egyptian Baladi cows during the hot season.

Transcriptome profile and association study revealed STAT3 gene as a potential quality marker of bovine gametes.

The present study was aimed to investigate differences in molecular signatures in oocytes derived from Holstein-Friesian heifers with different genetic merit for fertility, euthanized during day 0 or day 12 of the estrous cycle. Moreover, association between single nucleotide polymorphisms (SNPs) of ODC1 and STAT3 genes and bull fertility traits was investigated. The gene expression patterns were analyzed using cDNA array and validated with quantitative real-time polymerase chain reaction (PCR). The result revealed that several genes have shown not only to be regulated by fertility merit but also by the day of oocyte recovery during the estrous cycle. The STAT3 gene was found to be upregulated in oocytes recovered from animals with high fertility merit at both day 0 and day 12. Some other genes like PTTG1, ODC1 and TUBA1C were downregulated at day 0 and upregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In contrast, the transcript abundance of TPM3 was upregulated at day 0 and downregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In addition, ODC1 and STAT3 were found to be associated (P < 0.05) with sperm quality traits as well as flow cytometry parameters. Therefore, the expression of several candidate genes including ODC1 and STAT3 was related to the genetic merit of the cow. In addition polymorphisms in these two genes were found to be associated with bull semen quality.

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries).

BACKGROUND: The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. RESULTS: Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. CONCLUSIONS: The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation.

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

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

Identification and characterization of miRNAs in the ovaries of a highly prolific sheep breed.

Until recently, there have been few studies concerning miRNAs or miRNA-mediated biological processes in sheep (Ovis aries). In the present study, we used a deep-sequencing approach to examine ovarian miRNAs and the mRNA transcriptomes in two ewes of a highly prolific breed, Finnsheep. We identified 113 known sheep miRNAs, 131 miRNAs conserved in other mammals and 60 novel miRNAs, the expression levels of which accounted for 78.22%, 21.73% and 0.05% of the total respectively. Furthermore, the 10 most abundantly expressed miRNAs in the two libraries were characterized in detail, and the putative target genes of these miRNAs were annotated using GO annotation and KEGG pathway enrichment analyses. Among the target genes, intracellular transducers (SMAD1, SMAD4, SMAD5 and SMAD9) and bone morphogenetic protein (BMP) receptors (BMPR1B and BMPR2) were involved in the transforming growth factor ß (TGFß) signaling pathway in the reproductive axis, and the most significant GO terms were intracellular part (GO:0044424), binding (GO:0005488) and biological_process (GO:0008150) for cellular component, molecular function and biological process respectively. Thus, these results expanded the sheep miRNA database and provided additional information on the prolificacy trait regulated through specific miRNAs in sheep and other mammals.

Effect of peroxiredoxin II on the quality and mitochondrial activity of pre-implantation bovine embryos.

Endogenous peroxiredoxin II (PRDX II) protein plays a vital role in early embryonic development. This study assessed the beneficial effects of exogenous PRDX II on bovine embryo development at the cellular and molecular levels. To this end, in vitro maturation (IVM) medium was supplemented with various concentrations of PRDX II (0, 6.25, 12.5, 25, 50, and 100µg/mL). Of these, 12.5µg/mL PRDX II was the most effective and significantly promoted embryonic development. Therefore, this concentration of PRDX II was used in subsequent experiments. The percentage of embryos that developed into Day 8 blastocysts and the total number of cells per blastocyst (38.2% and 150.6±5.1) was higher in the PRDX II-treated group than in the control (26.4% and 128.9±3.9, respectively). Moreover, the percent of TUNEL positive cells was higher (P<0.05) in the control than in the PRDX II-treated. Furthermore, PRDX II added to the IVM media increased mitochondria content in blastocysts and decreased the intracellular ROS levels in oocytes and blastocysts compared with the control (P<0.05). The expression of genes associated with blastocyst quality (CDX2 and IFNτ), antioxidant activity (SOD2), and mitochondrial activity (TFAM) was higher, whereas the expression of a gene involved in the apoptotic pathway (c-FOS) was lower, in the PRDX II-treated than in the control group. In conclusion, supplementation of IVM medium with PRDX II promotes development to the blastocyst stage and improves blastocyst quality through reducing ROS, enhancing embryonic mitochondrial activity, and modulating development- related target genes expression.

Differential expression of selected candidate genes in bovine embryos produced in vitro and cultured with chemicals modulating lipid metabolism.

Lipid accumulated in embryos produced in vitro has been linked to reductions in both quality and postcryopreservation viability. Therefore, the objective of the present study was to investigate the influence of lipid-reducing chemicals on embryo development, quality, and postcryopreservation viability, in addition to expression profiles of selected lipid metabolism-regulating genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro; eight-cell stage embryos were cultured in IVC medium supplemented with phenazine ethosulfate (PES), L-carnitine (LC), PES + LC, or no supplementation (control). Culturing embryos in medium with LC increased (P < 0.05) blastocyst rate (38.8%) compared with the other groups (control = 28.1%, PES = 27.1%, PES + LC = 26.3%). Embryos cultured with supplements had greater total cell number and fewer apoptotic cells than the control. Cytoplasmic lipid content was reduced, whereas mitochondria density was increased in embryos treated with culture supplements; this was linked to altered expression profiles of selected genes regulating lipid metabolism. For example, transcript abundance of transmembrane lipid gene (SGPP1) was greater in LC- and PES-treated embryos, and they had increased postcryopreservation hatching ability (indicative of embryo cryotolerance). In conclusion, the two lipid metabolism regulators added to the culture media had improved embryo quality and cryotolerance, but embryo development rate and downstream lipid metabolism-regulating genes were more influenced with LC supplementation.

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression.

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.