A 1.1 Mb duplication CNV on chromosome 17 contributes to skeletal muscle development in Boer goats.

The Boer goat is one of the top meat breeds in modern animal husbandry and has attracted widespread attention for its unique growth performance. However, the genetic basis of muscle development in the Boer goat remains obscure. In this study, we identified specific structural variants in the Boer goat based on genome-wide selection signals and analyzed the basis of the molecular heredity of related candidate genes in muscle development. A total of 9 959 autosomal copy number variations (CNVs) were identified through selection signal analysis in 127 goat genomes. Specifically, we confirmed that the highest signal CNV (HSV) was a chromosomal arrangement containing an approximately 1.11 Mb (CHIR17: 60062304-61171840 bp) duplicated fragment inserted in reverse orientation and a 5 362 bp deleted region (CHIR17:60145940-60151302 bp) with overlapping genes (e.g., ARHGAP10, NR3C2, EDNRA, PRMT9, and TMEM184C). The homozygous duplicated HSV genotype (+/+) was found in 96% of Boer goats but was not detected in Eurasian goats and was only detected in 4% of indigenous African goats. The expression network of three candidate genes ( ARHGAP10, NR3C2, and EDNRA) regulating dose transcription was constructed by RNA sequencing. Results indicated that these genes were involved in the proliferation and differentiation of skeletal muscle satellite cells (SMSCs) and their overexpression significantly increased the expression of SAA3. The HSV of the Boer goat contributed to superior skeletal muscle growth via the dose effects of overlapping genes.

Active kisspeptin DNA vaccines oral immunization disrupt mRNA hormone receptors expression in ram lambs.

To evaluate the efficacy of oral immunization with active kisspeptin DNA vaccine on the expression of hormone receptor mRNA. For this study, ten 56-day-old Hu breed ram lambs were randomly assigned to the treatment and control groups (n = 5). Treatment Experimental group received C500/pKS-asd and the control group received C500/pVAX-asd (aspartate-ß semialdehyde dehydrogenase orally on days 0, 28, and 56, and blood samples were taken at each immunization interval (14-day) and tissues samples were collected at the end of the experimental period (day 98). The collected samples were stored in the refrigerator at -20 °C and liquid nitrogen, respectively, for laboratory examination. Total RNA was extracted from samples using TRIzol reagent and quantitative real-time polymerase chain reaction (QPCR) was used to quantify the levels of KISS1, G protein-coupled receptor-54 (Kiss1r), and gonadotrophin-releasing hormone (GnRH) mRNA in the hypothalamus. Levels of luteinizing hormone receptor (LHR) and luteinizing hormone beta (LHß) mRNA, and follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone beta (FSHß) mRNA in the testes and pituitary were analyzed, respectively. Further, gonadotropin-releasing hormone receptor (GnRHR) mRNA expression level in the pituitary was measured. Moreover, the Kiss1r concentration level in the blood was measured using an indirect ELISA. The concentration of Kiss1r in the blood was lower in the treatment group than in the control group (p < 0.05). The levels of testicular FSHR and LHR mRNA were significantly lower in the treatment group (p < 0.05) when compared to the control group. Furthermore, the treatment group's levels of hypothalamic KISS1, Kiss1r, and GnRH mRNA were significantly lower (p < 0.05) than the controls. LH, FSH, and GnRHR mRNA expression in the pituitary were also significantly lower in the treatment group (p < 0.01 and p < 0.05, respectively). These findings imply that oral immunization with active kisspeptin DNA vaccine suppresses hormone receptor mRNA expression in the ram lambs.

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.

Kisspeptin recombinant oral vaccine: A master gene vaccine inhibiting the reproductive physiology and behavior of ram lambs.

The KISS1 gene product, kisspeptin, stimulates gonadotrophic steroid hormone (GNRH) neuronal signaling through the G-protein coupled receptor, kiss1r. Disturbance of this signaling pathway causes hypogonadotropic hypogonadism in mammals. As part of this cutting-edge research project, we analyzed the efficacy of an oral kisspeptin recombinant vaccine on the reproductive physiology and behavior of ram lambs. Ten 56-day old ram lambs were randomly divided into treatment and control groups to receive the experimental recombinant vaccines, C500/pKS-asd or C500/pVAX-asd (aspartate-ß semialdehyde dehydrogenase), respectively. The vaccines were orally administered at day 0, 28 and 56 and blood samples were taken and scrotal circumference data recorded at 14-day intervals (days 0, 14, 28, 42, 56, 70, and 84). At the end of the experimental period, day 98, sexual behaviors were assessed, scrotal circumferences were measured, and blood samples were collected. Testicular samples were also collected after the animals were sacrificed. Anti-kisspeptin antibody and testosterone serum levels were measured by indirect ELISA. Results demonstrated that the levels of anti-kisspeptin antibodies were significantly higher in the treatment group compared to controls (P<0.05, P<0.01 and P<0.001). However, serum testosterone levels were lower in the treatment group (P<0.01). Interestingly, vaccine administration contributed to a significant reduction (P<0.01) in sexual behavior propensity. These results suggest that the kisspeptin recombinant oral vaccine regulates and inhibits the reproductive physiology and behavior of ram lambs.

Reversibility and safety of KISS1 metastasis suppressor gene vaccine in immunocastration of ram lambs.

OBJECTIVE: The aim of this study was to investigate the reversibility and safety of KISS1 metastasis suppressor (KISS1) gene vaccine in immunocastration. METHODS: Six eight-week old ram lambs were randomly divided into vaccinated and control groups. The vaccine (1 mg/ram lamb) was injected at weeks 0, 3, and 6 of the study. Blood samples were collected from the jugular vein before primary immunization and at weeks 2, 4, 6, 10, 14, 22, and 30 after primary immunization. All ram lambs were slaughtered at 38 weeks of age, and samples were collected. RESULTS: The specific anti-KISS1 antibody titers in vaccinated animals were significantly higher and the serum testosterone level was significantly lower than those in the control groups from week 4 to 14 after primary immunization (p<0.05). No significant difference was observed at weeks 22 and 30 after the primary immunization. Similar results were also found for scrotal circumference, testicular weight, length, breadth, and spermatogenesis in seminiferous tubules in week 30 after primary immunization. KS (KISS1-hepatitis B surface antigen S) fusion fragment of KISS1 gene vaccine was not detected in host cell genomic DNA of 9 tissues of the vaccinated ram lambs by polymerase chain reaction. CONCLUSION: The effects of KISS1 gene vaccine in immunocastration were reversible and no integration events were recorded.

Investigation of individual heterozygosity correlated to growth traits in Tongshan Black-boned goat.

Ten single nucleotide polymorphisms were used for genotyping of 176 Tongshan Black-boned goats, which are Chinese indigenous goat colony for meat production. The average individual heterozygosity was 0.292. To assess the correlations between individual heterozygosity and growth in Tongshan Black-boned goat individuals, and the potential of using individual heterozygosity as an indicator of growth, the data of growth traits, including body weight, height at withers, body length, chest girth and cannon circumference, were collected. Significant correlations were observed between individual heterozygosity and body weight, height at withers, body length, heart girth, cannon circumference (P < 0.05). All the significant regression showed positive slope with R square values ranged from 0.0251 to 0.0368. These data suggests that individual heterozygosity is positively correlated with growth traits in Tongshan Black-boned goat individuals and associative overdominance may affect Tongshan Black-boned goat growth significantly. Therefore it is possible to use individual heterozygosity as an indicator of growth. Our results also provide a strong support to the overdominance hypothesis.

Positive selection drives lactoferrin evolution in mammals.

Lactoferrin (LF) is a member of the transferrin family that is abundantly expressed and secreted by glandular epithelial cells. The biological functions of LF involve in iron homeostasis regulation of the body and antibacterial activity. Previous studies demonstrated that it had a high cationic N-terminal domain that could interact with glycosaminoglycans, lipopolysaccharides and the bacterial virulence protein. Two anti-microbial peptides, lactoferricin (LFcin) and lactoferrampin (LFampin), were also isolated and identified in N-terminal of LF. Although the antibacterial mechanism was carefully studied, little was known about the molecular evolution of LF. In this study, we estimated the nonsynonymous-to-synonymous substitution ratios ( ) per site using maximum likelihood method to analyze the LF evolution. The results of omega > 1 and five identified positive selection sites of amino acid suggested that the evolution of LF gene was characterized by positive selection. Further study found that the positive selection sites were either located in the LF-bacteria binding region or the peptides of LFcin and LFampin, indicating that the selection pressure was related to LF-bacteria interaction. The identification of these sites may contribute to the mechanism of bacteria-LF interaction.

[Effects of FUT1 gene on meat quality and carcass traits in swine].

A total of 139 hybrid finishing pigs from Large White x Meishan were slaughtered at about 88kg body weight. Fourteen meat quality traits and 8 carcass traits were assayed for each pig. FUT1 gene was scored by PCR-RFLP. The values of meat pH and meat color for AA genotype pig were higher than those of AG genotype pig (P<0.05). Water holding capacity (WHC) of AA genotype pig was higher than that of AG genotype pig(91.02% VS 86.70%, P<0.05). Backfat at 6-7 vertebra (BV) and backfat at vertebra-lumbar (BVL) of AA genotype pig were lower than those of AG genotype pig 4.26 mm and 3.96 mm respectively (P<0.05). Meat factor of AA genotype pig was higher than that of AG genotype pig 3.31% (53.46% VS 50.15%, P<0.05). These results indicated that FUT1 gene had good genetic effects on pig meat quality and carcass traits.

[Effects of individual gene heterozygosity on growth traits in swine].

Genotypes of 24 microsatellite loci were assayed in total of 139 F2 finishing pigs from resource population(Large White x Meishan) in the present study. There were not significant correlation in sex, family and microsatellite loci effects on birth weight (BW), average daily gain (ADG), adjusted weight of 180 d (LWT180), relative growth rate (RGR) and kleiber ratio (KR). Individual gene heterozygosity (Hi) was estimated by using 24 microsatellites. The 139 individuals were classified into five clusters based on Hi by stepwise 0.05 and the traits mentioned above were compared between Hi levels. Birth weight decreased significantly from gene heterozygosity level 1(0.5101 Hi and 1.5185 kg) to level 2(0.5796 Hi and 1.3063 kg, P < 0.05), the difference was 0.2123 kg equal to 14.66% of population average birth weight (1.4479 kg). And then the birth weight increased significantly with the gene heterozygosity increasing (P < 0.05). There were nonlinear relationships between LWT180, ADG, RGR, KR and individual gene heterozygosity, and mostly like the pattern of sinusoidal curve. The peaks were located at about heterozygosity level 2(averagely Hi 0.5796). ADG, RGR and KR at heterozygosity level 2 were significantly higher than level 4(28.7 g/d, 0.0375 and 0.0003 respectively, P < 0.05). The differences were equal 7.38%, 3.99% and 2.20% to respective average population values.

Detection of quantitative trait loci for protein percentage in muscle in resource pig population.

An intercross between Meishan and Large White pig population was used to map quantitative trait loci(QTL) for protein percentage in muscle. In the present study, all 135 F2 animals, their parents and their grandparents were genotyped for 24 microsatellites, which were in 1, 2 and 6 chromosome. The marker genotypes were used to calculate the additive and dominant coefficients at fixed position in the genome of each F2 animal, and the protein percentage were regressed on to the coefficients in intervals of 1 cM. Three significant QTLs effects were found for protein percentage in muscle. They were located between markers Sw1332 and Sw2130 at chromosome 1, between Sw2516 and Sw1201 at chromosome 2 and between Sw322 and Sw607 at chromosome 6(P < 0.05), and the explained residual variance were 0.91%, 12.76% and 4.60%, respectively. The results show that QTLs, which affect protein percentage, may be located in different chromosomes.