Further screening of SNP loci of eggshell translucency related genes and evaluation of genetic effects.

Eggshell translucency is a widespread issue in the field of egg quality. Previous research has established that the heritability of eggshell translucency is relatively low or moderate. Scientists have also successfully identified SNP loci related to eggshell translucency on different chromosomes by using gene chips and single-variant GWAS. However, the specific impact of single or multiple genes on the trait of eggshell translucency remains unknown. In an effort to investigate this, we examined 170 SNPs associated with eggshell translucency obtained by our research group. We selected 966 half-sibling laying hens from 2 generations in 3 pure lines: Dwarf Layer-White, Rhode Island Red-White Strain, and Rhode Island Red. Eggs were collected from each hen over a period of 5 consecutive days, and eggshell translucency was measured using a grading method in which the hens were divided into 2 groups: an opaque group and a translucent group. We collected blood samples from the laying hens and extracted DNA. Time of flight mass spectrometry (TOF-MS) was used for genotyping to identify SNP loci that influence the trait of eggshell translucency. The results of our analysis revealed that using TOF-MS in 3 chicken strains, we were able to eliminate loci with low gene polymorphism, genetic effect contribution less than 1%, and deviation from Hardy-Weinberg equilibrium. Ultimately, 5 SNPs (Affx-50362599, rs15050262, rs312943734, rs316121113, and rs317389181) were identified on chromosomes 1, 5, and 19. Additionally, nine candidate genes (DCN, BTG1, ZFP92, POU2F1, NUCB2, FTL, GGNBP2, ACACA, and TADA2A) were found to be associated with these SNPs. No linkage disequilibrium relationship was observed between the 2 pairs of SNP loci on chromosomes 1 and 19. Based on previous studies on the formation mechanism of eggshell translucency, we hypothesize that NUCB2, FTL, and ACACA genes may be affecting the eggshell structure through different mechanisms, such as increase the water permeability or make thin of eggshell membrane, which promote moisture or part of other egg contents and ultimately lead to the formation of eggshell translucency. These findings validate and identify five SNP loci that regulate the translucency trait, and provide molecular markers for breeding non-translucent populations. Furthermore, this study serves as a reference for further investigation of the genetic regulatory mechanisms underlying eggshell translucency.

Comparative Analysis of the Ultrastructure, Bubble Pores, and Composition of Eggshells of Dwarf Layer-White and Guinea Fowl.

The decrease in eggshell quality seriously affects production efficiency. Guinea fowl (GF) eggs possess strong eggshells because of their unique crystal structure, and few systematic studies have compared laying hen and GF eggs. Sixty eggs were collected from both 40-week-old Dwarf Layer-White (DWL-White) laying hens and GF, and the eggshell quality, ultrastructure, bubble pores, and composition were measured. The results showed that the DWL-White eggs had a higher egg weight and a lower eggshell strength, strength per unit weight, thickness, and ratio than the GF eggs (p < 0.01). There were differences in the mammillary layer thickness ratio, the effective layer thickness ratio, the quantity of bubble pores (QBPs), the ratio of the sum of the area of bubble pores to the area of the eggshell in each image (ARBE), and the average area of bubble pores (AABPs) between the DWL-White and GF eggs (p < 0.01). The composition analysis demonstrated that there were differences in the organic matter, inorganic matter, calcium, and phosphorus between the DWL-White and GF eggs (p < 0.01). There were positive associations between the mammillary knob number in the image and the QBPs and ARBE and a negative correlation with the AABPs in the DWL-White eggs (p < 0.01). This study observed distinctions that offer new insights into enhancing eggshell quality.

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.

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.

The relationship between the mTOR signaling pathway and ovarian aging in peak-phase and late-phase laying hens.

The molecular mechanisms underlying reproductive aging in avian species are poorly understood. Previous studies have shown the importance of mechanistic target of rapamycin (mTOR) signaling pathway in aging. In this study, we investigated the relationship between the mTOR signaling pathway and ovarian aging in the peak phase and late phase of egg production in laying hens. The egg production rate and egg quality were tracked for 5 consecutive weeks in 30-week-old and 70-week-old Dawu Jinfeng hens (N = 30/group). During the peak phase (week 35) and late phase (week 75), antioxidant and immune indicators were detected by enzyme-linked immunosorbent assay, and mTOR signaling-related genes (CLIP-170, GRB10, LIPIN-1, ATG1, 4E-BP1, S6K, PKC, RHO, and SGK1) were detected in the follicles by quantitative reverse transcription-PCR technology. The protein expression of key genes (mTOR, PKC, 4EBP1) was evaluated by Western blot analysis. The egg production rate and the antioxidant indexes superoxide dismutase and glutathione peroxidase and the levels of total antioxidant capacity and immunoglobulins (IgM and IgG) were significantly higher at week 35 than those at week 75 (P < 0.01), while malondialdehyde levels were significantly lower (P < 0.01). At week 75, there were fewer follicles in the different stages of development than were detected at week 35. The number of white follicles (large and small) and primary follicles were significantly higher at week 75 than those detected at week 35 (P < 0.01). The mRNA expression of avTOR, CLIP-170, GRB10, LIPIN-1, 4E-BP1, S6K, RHO, and SGK genes in small white follicles (SWF), large white follicles (LWF), F3, F1, and ovary at week 75 was lower than that in the hens at week 35 (P < 0.05). The mRNA expression in small yellow follicle (SYF) was significantly higher than that at week 35 (P < 0.05), while the mRNA expression of ULK1 in SWF, LWF, F3, F1, and ovary at week 75 was higher than that of hens at week 35 (P＜0.01), and SYF was lower (P < 0.05). Treatment of chicken granulosa cells with the mTOR agonist MHY1485 significantly enhanced granulocyte proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) and significantly increased avTOR, S6K, 4E-BP1, and PKC gene expression (P < 0.01). The protein expression levels of mTOR, S6K, p-mTOR, and p-S6K were consistent with mRNA expression levels. The mTOR activity is age-specific, and a compensatory enhancement of the mTOR signaling cascade can regulate ovarian follicular development in aged laying hens.

Melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptors.

Melatonin is a key regulator of follicle granular cell maturation and ovulation. The mammalian target of rapamycin (mTOR) pathway plays an important role in cell growth regulation. Therefore, our aim was to investigate whether the mTOR signaling pathway is involved in the regulation of melatonin-mediated proliferation and apoptotic mechanisms in granulosa cells. Chicken follicle granular cells were cultured with melatonin (0, 2, 20, or 200 µmol/L) for 48 h. The results showed that melatonin treatment enhanced proliferation and suppressed apoptosis in granular cells at 20 µmol/L and 200 µmol/L (P < 0.05) by upregulation of cyclin D1 (P < 0.01) and Bcl-2 (P < 0.01) and downregulation of P21, caspase-3, Beclin1, and LC3-II (P < 0.01). The effects resulted in the activation of the mTOR signaling pathway by increasing the expression of avTOR, PKC, 4E-BP1, S6K (P < 0.05), p-mTOR, and p-S6K. We added an mTOR activator and inhibitor to the cells and identified the optimal dose (10 µmol/L MHY1485 and 100 nmol/L rapamycin) for subsequent experiments. The combination of 20 µmol/L melatonin and 10 µmol/L MHY1485 significantly enhanced granulosa cell proliferation (P < 0.05), while 100 nmol/L rapamycin significantly inhibited proliferation and enhanced apoptosis (P < 0.05), but this action was reversed in the 20-µmol/L melatonin and 100-nmol/L rapamycin cotreatment groups (P < 0.05). This was confirmed by mRNA and protein expression that was associated with proliferation, apoptosis, and autophagy (P < 0.05). The combination of 20 µmol/L melatonin and 10 µmol/L MHY1485 also activated the mTOR pathway upstream genes PI3K, AKT1, and AKT2 and downstream genes PKC, 4E-BP1, and S6K (P < 0.05), as well as protein expression of p-mTOR and p-S6K. Rapamycin significantly inhibited the mTOR pathway-related genes mRNA levels (P < 0.05). In addition, activation of the mTOR pathway increased melatonin receptor mRNA levels (P < 0.05). In conclusion, these findings demonstrate that melatonin regulates chicken granulosa cell proliferation and apoptosis by activating the mTOR signaling pathway via its receptor.

Melatonin regulates the ovarian function and enhances follicle growth in aging laying hens via activating the mammalian target of rapamycin pathway.

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P < 0.01) was found in the M. The levels of mTOR and p-mTOR protein were increased in the M (P < 0.05). The mTORC1-dependent 4E-BP1 and p-4E-BP1 were increased in the M (P < 0.05). This study indicated that MT may enhance follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR and downstream components in aging laying hens.

Identification of a Goat Intersexuality-Associated Novel Variant Through Genome-Wide Resequencing and Hi-C.

Background: Polled intersex syndrome (PIS) leads to reproductive disorders in goats and exerts a heavy influence on goat breeding. Since 2001, the core variant of an 11.7 kb deletion at ~129 Mb on chromosome 1 (CHI1) has been widely used as a genetic diagnostic criterion. In 2020, a ~0.48 Mb insertion within the PIS deletion was identified by sequencing in XX intersex goats. However, the suitability of this variation for the diagnosis of intersex goats worldwide and its further molecular genetic mechanism need to be clarified. Results: The whole-genome selective sweep of intersex goats from China was performed with whole-genome next-generation sequencing technology for large sample populations and a case-control study on interbreeds. A series of candidate genes related to the goat intersexuality phenotype were found. We further confirmed that a ~0.48 Mb duplicated fragment (including ERG and KCNJ15) downstream of the ~20 Mb PIS region was reversely inserted into the PIS locus in intersex Chinese goats and was consistent with that in European Saanen and Valais black-necked goats. High-throughput chromosome conformation capture (Hi-C) technology was then used to compare the 3D structures of the PIS variant neighborhood in CHI1 between intersex and non-intersex goats. A newly found structure was validated as an intrachromosomal rearrangement. This inserted duplication changed the original spatial structure of goat CHI1 and caused the appearance of several specific loop structures in the adjacent ~20 kb downstream region of FOXL2. Conclusions: Results suggested that the novel complex PIS variant genome was sufficient as a broad-spectrum clinical diagnostic marker of XX intersexuality in goats from Europe and China. A series of private dense loop structures caused by segment insertion into the PIS deletion might affect the expression of FOXL2 or other neighboring novel candidate genes. However, these structures require further in-depth molecular biological experimental verification. In general, this study provided new insights for future research on the molecular genetic mechanism underlying female-to-male sex reversal in goats.

Study of measurement methods on phenotype of translucent eggs.

Scoring is a common method to evaluate eggshell translucency, and it mainly depends on the area and the density of translucent spots in eggshells. However, the lack of common scoring criteria and the difficulty of quantitatively measuring spots in eggshells impede effective comparisons between research papers and greatly hinder the progress of research on translucent eggshell. To make measurement of translucent eggshells more objective, we optimized the scoring method and compared it with 2 new methods: grayscale recognition and the colorimeter method. Briefly, a total of 354 eggs from 600, 395-day-old dwarf brown laying hens were collected and classified into 4 score groups according to their degree of translucency. This subjective process was repeated 5 times. Then, we captured the profile side of each egg using a camera and measured spot characteristics using grayscale recognition, which involved measuring the quantity of spots (QS), diameter of each spot (DS), average area of each spot (AAES), sum of spot areas (SUSA), sum of shell area (SUSHA), and ratio of SUSA to SUSHA (RSS) on the eggshell. Furthermore, the L, A, and B values of each egg at the sharp, middle, and blunt ends were separately measured using a colorimeter. As a result, average values of 31.31, 29.78, 29.81, and 9.08% of all eggs were divided into score levels 1, 2, 3, and 4 (from opaque to translucent), which correspond with RSS values of 1.34, 3.23, 6.21, and 11.89%, respectively. By grayscale recognition, QS, DS, AAES, SUSA, SUSHA, and RSS all increased along with increased translucency scores (P < 0.05). Using scoring, an egg with a specific RSS value was more easily assigned to a specific score level (50%) or adjacent score levels (50%). The L, A, and B values of eggshells in score level 1 were significantly different from those of eggshells in levels 3 or 4; however, there were no significant differences between any adjacent score levels. In summary, the present study explored objective reference metrics to measure eggshell translucency.

Genetic variation of major histocompatibility complex BLB2 gene exon 2 in Hebei domestic chicken.

Genetic variation of MHC BLB2 gene exon 2 in Hebei domestic chicken was investigated, after PCR and sequencing of a 374bp fragment (containing entire exon 2 (270bp) of BLB2 gene) in 76 individuals. The results showed that along this fragment, there were 69 variable sites, of which 18 were novel variations, and 82 estimated haplotypes with the diversity of 0.960. In Hebei domestic chicken, the nucleotide diversity (π), the average number of nucleotide differences (k), the average number of nucleotide diversity of synonymous substitution (π(s)) and non-synonymous substitution (π(a)) in BLB2 gene exon 2 were 0.098, 24.688, 0.075, and 0.106, respectively; nine non-synonymous substitutions was exclusively found in the peptide-binding sites (PBS) region of BLB2 gene exon 2, inferring that these unique substitutions might be helpful to resist some special bacteria and pathogens. The higher genetic diversity of MHC BLB2 gene exon 2 in Hebei domestic chicken might be consistent with its more robust disease resistance.