Eggshell translucency in late-phase laying hens and its effect on egg quality and physiological indicators.

Eggshell translucency severely affects external egg quality, and variations in the eggshell or eggshell membrane are considered the structural basis of the trait. Research has shown that 1.85% additional mixed fatty acids in the diet would greatly decrease the occurrence of eggshell translucency. Only a few studies have examined the phenotypic regularity of eggshell translucency with the increasing age of hens. Therefore, two strains, 1139 Rhode Island Red-White (RIR-White) and 836 Dwarf Layer-White (DWL-White), were used, and from each strain, 30 hens each that consecutively laid translucent or opaque eggs at 67 wks of age were selected. Subsequently, eggshell translucency, internal quality and external quality of eggs, and total cholesterol, albumin, calcium binding protein and other physiological indicators related to lipid, lipoprotein, and calcium metabolisms at the 75th, 79th, and 83rd wks of age in the late phase of the laying cycle were determined. Results: (1) In terms of flocks, for both strains, the translucency scores of the translucent groups were significantly higher than those of the opaque groups (P < 0.05); in terms of individuals, 81.1% RIR-White and 82.8% DWL-White hens consecutively laid eggs of the same or similar translucency, indicating the stability of the trait with increasing hen age; (2) In RIR-White, the eggshell strength of the translucent group at 75 weeks was significantly higher than that of the opaque group (P < 0.05); in DWL-White, the eggshell membrane thickness of the translucent group at the 75th and 83rd weeks was significantly lower than that of the opaque group (P < 0.05); (3) Compared to the opaque groups, the translucent groups had lower total cholesterol content in both RIR-White and DWL-White, lower albumin content in DWL-White at the 79th weeks (P < 0.05), and higher calcium-binding protein (CALB1) in RIR-White at the 83rd weeks (P < 0.05). In summary, this study illustrates the stability of eggshell translucency in late-phase laying hens and provides a reference of physiological indicators for exploring the formation of translucent eggs.

Restorative effects of Lactobacillus rhamnosus LR-32 on the gut microbiota, barrier integrity, and 5-HT metabolism in reducing feather-pecking behavior in laying hens with antibiotic-induced dysbiosis.

The development of abnormal feather-pecking (FP) behavior, where laying hens display harmful pecks in conspecifics, is multifactorial and has been linked to the microbiota-gut-brain axis. Antibiotics affect the gut microbial composition, leading to gut-brain axis imbalance and behavior and physiology changes in many species. However, it is not clear whether intestinal dysbacteriosis can induce the development of damaging behavior, such as FP. The restorative effects of Lactobacillus rhamnosus LR-32 against intestinal dysbacteriosis-induced alternations need to be determined either. The current investigation aimed to induce intestinal dysbacteriosis in laying hens by supplementing their diet with the antibiotic lincomycin hydrochloride. The study revealed that antibiotic exposure resulted in decreased egg production performance and an increased tendency toward severe feather-pecking (SFP) behavior in laying hens. Moreover, intestinal and blood-brain barrier functions were impaired, and 5-HT metabolism was inhibited. However, treatment with Lactobacillus rhamnosus LR-32 following antibiotic exposure significantly alleviated the decline in egg production performance and reduced SFP behavior. Lactobacillus rhamnosus LR-32 supplementation restored the profile of the gut microbial community, and showed a strong positive effect by increasing the expression of tight junction proteins in the ileum and hypothalamus and promoting the expression of genes related to central 5-HT metabolism. The correlation analysis revealed that probiotic-enhanced bacteria were positively correlated, and probiotic-reduced bacteria were negatively correlated with tight junction-related gene expression, and 5-HT metabolism, and butyric acid levels. Overall, our findings indicate that dietary supplementation with Lactobacillus rhamnosus LR-32 can reduce antibiotic-induced FP in laying hens and is a promising treatment to improve the welfare of domestic birds.

Effect of natural garlic essential oil on chickens with artificially infected Eimeria tenella.

Chicken coccidiosis is a kind of parasitic protozoosis caused by Eimeria parasitizing in the chicken intestinal epithelial cells. Eimeria tenella is considered as a significantly virulent and harmful parasite. At present, drug resistance remains a major problem and a large number of drug residues have been found to be produced in the treatment of the disease. Hence, novel strategies are needed to avoid the harmful effects caused by the generation of various chemical drug residues to the human body and also reduce the economic loss caused by coccidiosis to the chicken industry. In this study, natural garlic essential oil was used to control Eimeria tenella infection. The anticoccidial index (ACI) was calculated according to the clinical symptoms, body weight gain, oocyst excretion and cecal lesions. The immune organ index and serum biochemical indexes were measured to verify the possible anticoccidial effects. The results showed that: compared with the infected group, continuous feeding of different doses of natural garlic essential oil could significantly reduce the clinical symptoms, cecal lesions, the number of oocysts, but increase the weight of sick chickens, and effectively improve the intestinal functions. Moreover, compared with diclazuril control group, 0.06 mL/L garlic essential oil exhibited similar anticoccidial index. The content of immune organ index, serum biochemical index IgM, IgG and IgA in 0.06 mL/L garlic essential oil group was the highest, which indicated that garlic essential oil had a significant tendency to improve the immune function of the chickens. This study also showed that the natural garlic essential oil exhibited the same beneficial effects as that of diclazuril on chicken coccidiosis, and the anti-coccidiosis index of 0.06 mL/L garlic essential oil was favorable. Thus based on the above evidences and its relatively low cost, garlic essential oil can be potentially be used as an efficient anti parasitic drug.

[A nonsurgical embryo transfer technique in mice].

Although efficient nonsurgical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of nonsurgical transfer has hampered its acceptance and use. Here, a plastic catheter was used to mimic embryo transfer process and then the transfer efficiency was evaluated by intrauterine trypan blue dye dispersion. Also 3.5-day blastocysts from natural pregnant mice were transferred through cervix into uterine horns. The results show that 70.9% of CD-1 mouse 3.5-day blastocysts transferred into unilateral uterine horns of pseudopregnant 2.5-day recipients can be developed to live newborns, and an efficient mouse nonsurgical embryo transfer technique was established. The technique was simple, rapid, inexpensive, unlikely to get contaminated, ethical and do not need specialized apparatus, and can completely replace surgical embryo transfer techniques. Moreover, the mouse nonsurgical embryo transfer technique provides a research model for human and other large animal embryo transfer.

Generation of fully pluripotent female murine-induced pluripotent stem cells.

The high quality of induced pluripotent stem cells (iPSCs) has been determined to be high-grade chimeras that are competent for germline transmission, and viable mice can be generated through tetraploid complementation. Most of the high-quality iPSCs described to date have been male. Female iPSCs, especially fully pluripotent female iPSCs, are also essential for clinical applications and scientific research. Here, we show, for the first time, that a gender-mixed induction strategy could lead to a skewed sex ratio of iPSCs. After reprogramming, 50%, 70%, and 90% female initiating mouse embryonic fibroblasts at different male ratios resulted in 14.1 ± 6.8% (P < 0.05), 31.8 ± 5.4% (P < 0.05), and 80.1 ± 2.8% (P < 0.05) female iPSCs, respectively. Furthermore, these female iPSCs had pluripotent properties typical of embryonic stem cells. Importantly, these fully pluripotent female iPSCs could generate viable mice by tetraploid complementation. These findings indicate that high-quality female iPSCs could be derived effectively, and suggest that clinical application of female iPSCs is feasible.

Transcervical embryo transfer in mice.

Although efficient transcervical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of transcervical transfer has hampered its acceptance and use. Here, we describe a novel device and protocol for transcervical embryo transfer in mice. Blastocysts from CD1 female mice were transferred into the uteri of 2.5-d pseudopregnant CD1 mice by using this method resulted in the successful development of 66.7% to 73.5% of the transferred blastocysts into live-born fetuses. Our method is as efficient as surgical embryo transfer yet is much simpler, easier, and markedly less traumatic to the recipient. In addition, our method provides hygienic and economic advantages and conforms to the principles of humane experimental technique. More importantly, our method provides a model for studying transcervical embryo transfer in cattle, other large animals, and humans.

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells.

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of "4N-ON" and the corresponding "4N-OFF" iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.

Melatonin improves the reprogramming efficiency of murine-induced pluripotent stem cells using a secondary inducible system.

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all-iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10(-6), 10(-7), 10(-8), 10(-9), or 10(-10 )m) or with vitamin C (50 µg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10(-8) (0.81 ± 0.04%), and 10(-9 )m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT2 was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT1 and MT2 receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis-related genes p53 and p21 was lower in the group treated with 10(-9) m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53-mediated apoptotic pathway.

[Experiment study on ultrashort wave for treating vascular crisis after rat tail replantation].

OBJECTIVE: To explore the effect and mechanism of ultrashort wave (USW) for prevention and treatment of vascular crisis after rat tail replantation. METHODS: Eighty 3-month old female Sprague Dawley rats (weighing 232.8-289.6 g) were randomly divided into 5 groups. In each group, based on the caudal vein and the coccyx was retained, the tail was cut off. The tail artery was ligated in group A; the tail artery was anastomosed in groups B, C, D, and E to establish the tail replantation model. After surgery, the rats of group B were given normal management; the rats of group C were immediately given intraperitoneal injection (3.125 mL/kg) of diluted papaverine hydrochloride injection (1 mg/mL); the rats of groups D and E were immediately given the local USW treatment (once a day) at anastomotic site for 5 days at the dosage of 3 files and 50 mA for 20 minutes (group D) and 2 files and 28 mA for 20 minutes (group E). The survival rate of the rat tails was observed for 10 days after the tail replantation. The tail skin temperature difference between proximal and distal anastomosis was measured at pre- and post-operation; the change between postoperative and preoperative temperature difference was calculated. The blood plasma specimens were collected from the inner canthus before operation and from the tip of the tail at 8 hours after operation to measure the content of nitric oxide (NO). RESULTS: The survival rates of the rat tails were 0 (0/14), 36.4% (8/22), 57.1% (8/14), 22.2% (4/18), and 75.0% (9/12) in groups A, B, C, D, and E, respectively, showing significant overall differences among 5 groups (chi2 = 19.935, P = 0.001); the survival rate of group E was significantly higher than that of group B at 7 days (P < 0.05), but no significant difference was found between the other groups by pairwise comparison (P > 0.05). At preoperation, there was no significant difference in tail skin temperature difference among 5 groups (P > 0.05); at 8 hours, 5 days, 6 days, and 7 days after operation, significant overall difference was found in the change of the skin temperature difference among groups (P < 0.05); pairwise comparison showed significant differences after operation (P < 0.05): group B > group D at 8 hours, group C > group D at 5 days, groups A, B, and C > group D at 6 days, groups B and C > groups A and E, and group B > group D at 7 days; but no significant difference was found between the other groups at the other time points (P > 0.05). Preoperative plasma NO content between each group had no significant difference (P > 0.05). The overall differences had significance in the NO content at postopoerative 8 hours and in the change of the NO content at pre- and post-operation among groups (P < 0.05). Significant differences were found by pairwise comparison (P < 0.05): group D > groups A, B, and C in the plasma NO content, group D > groups A and B in the change of the NO content at pre- and post-operation; but no significant difference was found between the other groups by pairwise comparison (P > 0.05). CONCLUSION: Rat tail replantation model in this experiment is feasible. USW therapy can increase the survival rate of replanted rat tails, reduce skin temperature at 7 days, improve blood supply, increase the content of nitric oxide at the early period and prevent vascular crisis.

Decreased surfactant protein B expression in mice derived completely from embryonic stem cells.

ES mice that are derived completely from embryonic stem (ES) cells can be obtained by tetraploid embryo complementation. Many neonate ES mice die because of respiratory distress, but it is not clear what contributes to the phenomenon. Using five microsatellite DNA markers, we confirmed that our ES mice were completely derived from ES cells and contained no tetraploid component. The neonatal ES mice that exhibited respiratory distress were tested for surfactant protein B (SP-B) expression by Western blotting. These mice had no SP-B expression, and even apparently healthy adult ES mice had decreased SP-B levels and aberrant SP-B phenotypes. These data suggest that the expression of SP-B protein is an important factor in the survival of ES mice to term and adulthood.

Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation.

The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.

The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation.

The aim of this paper was to determine whether the genetic background of tetraploid embryos contributed to the survival of mice derived from embryonic stem (ES) cells by tetraploid embryo complementation. Twenty-five newborns were produced by aggregation of hybrid ES cells and tetraploid embryos with different genetic backgrounds. These newborns were entirely derived from ES cells judged by microsatellite DNA (A specific sequence of DNA bases or nucleotides that contains mono, di, tri or tetra repeats) and coat colour phenotype and germline transmission. Fifteen survived to adulthood while seven died of respiratory failure. All newborns were derived from outbred or hybrid tetraploid aggregates and no newborns were from the inbreds. Our results demonstrate that the genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation. In addition, this method represents a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.