The functions and mechanisms of the genomes from the different gametes on the epigenetic reprogramming of embryos are still unclear. In this study, the expression of enzymes typically associated with changes in epigenetic markers was measured in parthenogenetically-activated and in-vitro fertilised embryos. General control of nucleotide synthesis 5 (GCN5), histone deacetylase 1 (HDAC1) and DNA methyltransferases 1 (DNMT1), were analysed in early diploid PA and control embryos using fluorescent immunocytochemistry. Levels of GCN5 expression of two-cell embryos were similar between the PA and IVF groups, but the distribution of GCN5 in PA embryos at the four-cell stage was significantly decreased. HDAC1 and DNMT1 expression was also significantly decreased in PA embryos. In addition, the observed localisation of HDAC1 expression within and surrounding the nucleus in IVF embryos was not present in PA embryos. Embryos with only the maternal genome have altered expression patterns of key enzymes required for embryonic epigenetic reprogramming.
Contamination and penetration of salmonellae into hatching eggs may comprise an important link in the transmission of these bacteria to growing birds, processed carcasses, and eventually to the consumer. In this study, a predictive model for Salmonella typhimurium as a function of initial cell number and storage or incubation time at a nearly constant temperature and humidity was developed and evaluated to compute the bacterial load after 1 d (holding), 10 d (candling), 17 d (incubation), and 21 d (chick processing). Experiments were conducted for S. typhimurium with both high initial bacterial load (HIBL) and low initial bacterial load (LIBL) of 6.0 and 3.5 log cfu/egg, respectively. Eggs with HIBL experienced 2.0 log reduction in the bacterial load after holding at 4 degrees C for 24 h and 3.0 log increase in the bacterial load during incubation and hatch at approximately 37 degrees C between 17 d and 21 d. Experimental data showed that bacterial load of S. typhimurium from holding to chick processing changed from 3.7 to 6.6 log cfu/egg and from 3.7 to 2.7 log cfu/egg in HIBL and LIBL eggs, respectively. The developed model was able to predict bacterial load of S. typhimurium from 3.6 to 6.6 log cfu/egg in HIBL eggs and from 3.4 to 2.7 log cfu/egg in LIBL eggs from holding to chick processing. Root mean square errors and plot of predicted compared with observed bacterial load of S. typhimurium in contaminated eggs yielded a good fit and prediction. The predicted and experimental results indicated that incubated broiler eggs have an increase in internal bacterial loads between incubation and hatch. This model can be used as a tool to predict bacterial load of S. typhimurium in contaminated eggs as well as help predict the behavior of S. typhimurium during hatch.
The influences of F-strain Mycoplasma gallisepticum (FMG) vaccine inoculation during the pullet period on the subsequent productive and reproductive performance of parent broiler chicken breeders on a multi-age farm were evaluated. Three thousand breeders were randomly divided into 2 treatment groups that were either vaccinated with FMG (FMG-vaccinated group) or not vaccinated with FMG (FMG-free group). Body weight and egg production were determined through approximately 50 wk of age. Egg weight and feed conversion was determined at 26, 32, 35, 38, and 43 wk of age. Egg quality parameters, including eggshell strength, egg-specific gravity, egg shape index, blood-meat spots, Haugh unit score, eggshell thickness, yolk:albumen ratio, percentage yolk, albumen and eggshell weights, and percentage fertility, hatchability, and second-quality chicks were determined at 26, 32, and 43 wk of age. Air sacs were examined and lesions were scored at 20, 32, and 50 wk of age. The number of mature ovarian follicles, histologies of ovary, and lengths, and histologies of the infundibulum, magnum, isthmus, uterus, and vagina were determined. In the present study, an increase in egg production of broiler breeder hens in the FMG-vaccinated group during peak of lay was compared with the FMG-free group. Feed conversion of hens in the FMG-vaccinated group was significantly less at 32, 35, 38, and 43 wk of age. Eggs from hens in the FMG-vaccinated group had a significantly higher Haugh units score at 26 wk of age and had a significantly higher eggshell thickness and lower incidence of blood-meat spots at 32 wk. Hatching eggs from hens in the FMG-vaccinated group had a significantly higher hatchability. The mean lesion score of air-sac lesion of birds in the FMG-vaccinated group was significantly less than FMG-vaccinated group. Uteruses of hens in the FMG-vaccinated group had a significantly longer length compared with the FMG-free group at 32 wk of age. The results indicate that inoculation of commercial parent broiler chicken breeders with the FMG vaccine before laying may prevent infection by field M. gallisepticum, and facilitate productive and reproductive performance.
Accumulating evidence suggests that ghrelin plays an important role in female reproduction. The objective of the present study was to investigate the effect of ghrelin on pre-implantation development of porcine in vitro-fertilized (IVF) and parthenogenetic embryos. Cumulus–oocyte complexes were matured for 44 h in BSA-free NCSU23 supplemented with 10 ng mL-1 epidermal growth factor, 10 ng mL-1 leptin, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, and 10 IU mL-1 hCG. After removal of the cumulus cells, some oocytes were fertilized with fresh boar semen (1 � 105 sperm mL-1) in modified Tween medium B with milk powder (Abeydeera and Day 1997 Theriogenology 48, 537–544) and some oocytes were activated by a single, 100-�s, direct current pulse of 1.4 kV cm-1. Presumptive zygotes (Experiment 1) and parthenogenetic oocytes (Experiment 2) were subsequently cultured in PZM3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) supplemented with ghrelin at 0 (control), 0.5, 5, 50, and 500 ng mL-1 (ghrelin 0.5, 5, 50, 500 groups, respectively) under 5% CO2, 5% O2, 90% N2 and 100% humidity at 39.0�C. Cleavage and blastocyst rates were assessed on Days 2 and 6 (Day 0: the day IVF and activation were conducted). The total cell number in blastocysts was determined by Hoechst 33342 staining on Day 6. All data were analyzed by using SPSS (13.0) with one-way ANOVA. All experiments were done at least 4 times. In Experiment 1, the rate of blasotcyst formation in IVF embryos was significantly (P < 0.05) increased in the ghrelin 500 group compared with that in the control group (26.1 � 1.8 vs. 12.4 � 6.0%, mean � SEM). Furthermore, increased total cell numbers (P < 0.05) were observed in the ghrelin 50 and 500 groups compared with that in the control group (63 � 6.6 and 64 � 5.5 vs. 42 � 6.6). In Experiment 2, we found that the blastocyst rate of parthenogenetic embryos was significantly (P < 0.05) higher in the ghrelin 5 and 500 groups than in the others (24.6 � 4.7 and 25.0 � 3.3 vs. 13.3 � 2.7, 14.9 � 2.4, 18.1 � 2.3% in the control and ghrelin 0.5 and 50 groups, respectively; P < 0.05). The total cell number per blastocyst was significantly increased in the ghrelin 50 group compared with that of the control group (85 � 10.2 vs. 56 � 8.0, P < 0.05). The maximum total cell number in the ghrelin treatment groups of parthenogenetic embryos was higher than in the control group (82, 93, 102, 100 in the ghrelin 0.5, 5, 50, 500 groups, respectively, vs. 69; P < 0.05). We also found that more embryos were developed to the morula stage and fewer embryos died early at the 2- to 4-cell stage in the ghrelin treatment groups than in the control group (data not shown) in both Experiments 1 and 2. The results suggest that supplementation with ghrelin in the embryo culture medium could enhance the pre-implantation development of porcine IVF and parthenogenetic embryos. This study was funded by the Natural Scientific Foundation of Beijing (5030001).
The Mof gene (males absent on the first) is crucial to X-chromosome dosage compensation in the fly. It acts specifically to catalyse acetylation of histone H4 lysine 16 (H4K16ac) as one histone acetyltransferase of the MYST family, playing essential roles during mammalian development. However, little is known about Mof gene in pigs. The present study was designed to explore effects of Mof on pre-implantation development of pig parthenogenetic embryos obtained as reported by Cao et al. (2012 Zygote 20, 229–236). Immunofluorescent staining was performed to examine protein expression level of porcine Mof (pMof) and H4K16ac, and fluorescent intensity was measured by Image J software (NIH, Bethesda, MD, USA). Data are presented as mean ± standard error, and statistical analyses of the fluorescent intensity value, embryo development rate, and quality were performed using ANOVA with SPSS software (version 15.0, SPSS Inc., Chicago, IL, USA), and P-value <0.05 was considered significant. First, the coding sequence (CDS) of the pMof gene was cloned and the spatio-temporal expression patterns of the CDS were determined in pig oocytes, early embryos, and other tissues. A 1471-bp-long cDNA of pMof was obtained with 99.34% and 98.25% amino acid sequence homology and 92.88% and 88.96% nucleotide sequence homology to the human and mouse MOF homologues. We observed that pMof is expressed predominantly in oocytes and early embryos but at low levels in sperm and other organs. We found that pMof decreased from pronuclear to 8-cell stages and remained low until the blastocyst stage based on RT-qPCR results (n = 10 for each stage embryos). In contrast, pMof protein expression as examined by immunofluorescent staining (n = 15 for each stage embryos) remained high throughout the pre-implantation development period. After porcine embryonic genome activation, pMOF remained detectable in 4-cell (n = 10) and 8-cell (n = 10) embryos despite amanitin treatment for 24 h. Thus, the mRNA level of MOF was not decreased after transcription inhibition suggesting that MOF is a maternal gene. To assess functional significance, we examined the expression of H4K16ac, a target of pMof, and found that the level of H4K16ac was constantly low from pronuclear to morula stage, but increased dramatically in blastocysts. When we knocked down pMOF by cytoplasmic injection of siRNA into porcine MII oocytes, rate and cell number of blastocysts declined significantly [blastocyst rate: uninjected (n = 228) v. negative-siRNA (n = 220) v. MOF-siRNA (n = 230) = 65.8 ± 3.75% v. 57.5 ± 4.30% v. 46.3 ± 5.72%; cell numbers: uninjected v. negative-siRNA v. MOF-siRNA = 84.73 ± 5.25 v. 77 ± 5.50 v. 55.08 ± 6.56]. A marker for DNA double-strand breaks and repair, γ-H2AX, increased in parallel to more apoptotic cells. Knockdown of MOF reduced H4K16ac. Overall, pMof is highly conserved among human, mouse, and pig; pMof is essential to pre-implantational development of pig parthenogenetic embryos involved in regulating H4K16ac.Research was supported by NSFC (31272442).
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