Identification of lncRNAs Differentially Expressed during Natural and Induced Estrus in Sheep

Background: The manipulation of the estrous cycle or induction of estrus is a commonly used technique in sheep industry. The goal of this study was to identify and characterize differences of non-coding RNAs (lincRNAs) expression between induced estrus and natural estrus using the BGISEQ-500 plat form in 7 Mongolian sheep, which will provide insights into the regulation mechanisms of lncRNAs in different reproduction mode of sheep. Methods: During the late spring, ovarian, pituitary, hypothalamic, pineal and uterine tissue samples were collected from four artificially induced estrus and three naturally estrus Mongolian sheep. Total RNA was extracted from the five tissues using TRIzol reagent (Invitrogen) and treated with DNase I following the manufacturer’s instructions. A total of 35 sheep samples were sequenced using the BGISEQ-500 plat form. Bioinformatics methods were used to analysis expression difference analysis between groups, SNP and InDel, alternative splicing, lncRNA’s miRNA precursor prediction, lncRNA target gene and family prediction. Result: 211 novel lncRNAs were systematically identified using RNA-Seq technology. Meanwhile, we found that there are diversifications of lncRNAs in induced estrus vs. nature estrus of ewes. Therefore, we predict that, under the action of exogenous hormones, many physiological processes of ewes may be affected to varying degrees through the change of LncRNA to a variety of pathways.

Effects of longissimus muscle fatty acid composition in mongolian sheep breeds

This survey to comparative investigates on fat and fatty acid composition in muscles depends on season effects of pastoral and grain fed Mongolian breeds sheep (Mongolian grassland pastoral Mongolian sheep, Inner Mongolian grassland pastoral and grain fed Ujumqin sheep) muscle’s longissimus dorsi. Two breeds three kinds Mongolian sheep muscle’s longissimus dorsi IMF content and fatty acids were analyzed by gas chromatography after methyl esterification. The result reveals that: (1) The IMF content in pastoral of mongolian and Ujumqin sheep muscles was between winter and summer seasons observed extreme differences (p<0.01), but no extreme differences (p>0.01) in grain fed Ujumqin sheep. (2) The result showed that there were 29 identified fatty acids in the muscle’s longissimus dorsi means of winter season pastoral Mongolian, Ujumqin and grain fed Ujumqin sheep, where in the content of palmitic acid, stearic acid and oleic acid were 71.45%, 73.17%, 72.02% of the total content of fatty acids respectively. Also saturated fatty acids accounted for 44.49%, 46.27%, 45.37%; monounsaturated fatty acids accounted for 35.68%, 36.09%, 35.9; polyunsaturated fatty acids accounted for 8.18%, 8.33%, 9.32% respectively. In the summer muscle’s longissimus dorsi means of Mongolian, Ujumqin and grain fed Ujumqin sheep were 31, 31, 26 identified fatty acids, where in the content of palmitic acid, stearic acid and oleic acid were 75.96%, 77.17%, 72.79% of the total content of fatty acids respectively. Also saturated fatty acid accounted for 48.35%, 48.09%, 46.31; monounsaturated fatty acids accounted for 36.46%, 37.66%, 36.54; polyunsaturated fatty acids accounted for 7.07%, 7.43%, 8.05% respectively. Therefore, positive feature of pastoral Mongolian and Ujumqin sheep in muscle’s longissimus dorsi means that levels of the nutritionally important long chain PUFA EPA C20:5, DHA C22:6 was in winter 1.2-1.8%; in summer of the total content of fatty acids 1-1.2% respectively. But grain fed Ujumqin sheep muscle’s different parts means only identified long chain PUFA EPA C20:5 was in winter and summer 0.8% of the total content of fatty acids and lower than pastoral Mongolian and Ujumqin sheep 1%. Монгол хонины нурууны булчин махны тосны хүчлийн бүрдлийн судалгаа Энэхүү судалгаагаар өвөл болон зуны улирлын 2 үүлдэрийн 3 төрлийн Монгол хонины (Бэлчээрийн Монгол, Үзэмчин болон суурин маллагаатай Үзэмчин) нурууны булчин маханд (M. longissimus dorsi) тос болон тосны хүчлийн бүрдлийг харьцуулан судалсан болно. Дээрх 3 төрлийн хонины нурууны булчин эдийн тосыг ялган хэмжээг тогтоож, метил эфиржүүлсний дараа хийн хромотаграфаар тосны хүчлийн бүрэлдэхүүнийн тодорхойлсон. Булчин эдийн тосны агууламжийн үр дүн: Бэлчээрийн Монгол болон Үзэмчин хонины гуяны булчин эдийн тосны агууламж нь өвөл болон зуны улирлын хооронд хэт их утга (p<0.01) илэрсэн. Тосны хүчлийн бүрэлдэхүүний үр дүн: Өвлийн улирлын бэлчээрийн Монгол, Үзэмчин болон суурин маллагаатай Үзэмчин хонины longissimus dorsi булчин эдэд нийт тосны 89.35%, 91.19%, 90.6% -ийг 29 төрлийн тосны хүчил бүрдүүлж түүнээс пальмитины (C16:0), стеарины (C18:0), олеины хүчлүүд (C18:1) хүчлүүд 71.45%, 73.17%, 72.02%-ийг эзэлж байв. Зуны улиралд хонины нурууны булчин эдийн нийт тосны 91.88%, 93.18%, 90.9% -ийг 26-31 төрлийн тосны хүчил бүрдүүлж түүнээс пальмитины (C16:0), стеарины (C18:0), олеины хүчлүүд (C18:1) хүчлүүд 75.96%, 77.17%, 72.79%-ийг тус тус бүрдүүлж байна. Өвлийн улирлын бэлчээрийн Монгол, Үзэмчин болон суурин маллагаатай Үзэмчин хонины нурууны булчин эдийн дундаж ханасан тосны хүчил 44.49%, 46.27%, 45.37%’; мониен хүчил 35.68%, 36.09%, 35.9%; полиен хүчил 8.18%, 8.33%, 9.32%; Зуны улиралд ханасан хүчил 48.35%, 48.09%, 46.31%; мониен хүчил 36.46%, 37.66%, 36.54%; полиен хүчил 7.07%, 7.43%, 8.05% тус тус агуулагдаж байв. Эмчилгээний өндөр ач холбогдолтой гэгдээд байгаа EPA C20:5 болон DHA C22:6 олон холбоот тосны хүчлүүд бэлчээрийн Монгол болон Үзэмчин хонинд эдгээр хүчлийн нийлбэр өвлийн улирал 1.2-1.8%, зуны улиралд 1-1.2% агуулагдаж суурин маллагаатай Үзэмчин хонины нурууны булчин махнаас 1% орчим өндөр агуулагдаж байгаа бэлчээрийн хонины махны ач холбогдлыг илэрхийлэх үндэслэлтэй. Түлхүүр үг: Монгол хонь, Үзэмчин хонь, тосны хүчил, өвөл, зун

GLIS1, a potential candidate gene affect fat deposition in sheep tail

Fat deposition in sheep tails is as a result of a complicated mechanism. Mongolian sheep (MG) and Small Tail Han sheep (STH) are two fat-tailed Chinese indigenous sheep breeds while DairyMeade and East Friesian (DS) are two thin-tailed dairy sheep breeds recently introduced to China. In this study, population genomics analysis was applied to identify candidate genes associated with sheep tails based on an in-depth whole-genome sequencing of MG, STH and DS. The selective signature analysis demonstrated that GLIS1, LOC101117953, PDGFD and T were in the significant divergent regions between DS and STH–MG. A nonsynonymous point mutation (g.27807636G>T) was found within GLIS1 in STH–MG and resulted in a Pro to Thr substitution. As a pro-adipogenic factor, GLIS1 may play critical roles in the mesodermal cell differentiation during fetal development affecting fat deposition in sheep tails. This study gives a new insight into the genetic basis of species-specific traits of sheep tails.

Orexin A promotes progesterone secretion in luteinized granulose cells of Mongolian Ovis aries ovary by PRRT2 and ABCG1 genes

Summary To study the role of orexin A in the reproductive regulation of Mongolian sheep, ovine ovarian granulosa cells were cultured in vitro. The cells were divided into groups after luteinization, the experimental group was given orexin A and the transcriptome was sequenced together with that of the control group. The different genes related to reproduction were screened out. qRT-PCR, western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the selected genes and detect the effect on progesterone secretion. In total, 123 differentially expressed genes were obtained by sequencing. Six genes with high expression related to reproduction (PRRT2, ABCG1, SOX4, TBX3, ID1 and ATP8) were screened. The results of qRT-PCR were consistent with those of sequencing; western blot and ELISA were used to verify the protein levels of steroidogenic acute regulatory protein (StAR) and its related PRRT2 and ABCG1, and to detect their effect on progesterone secretion. Validation results were consistent with those of qRT-PCR and sequencing. The experimental group was given orexin A and compared with the control group. Expression of PRRT2 protein was significantly increased (P < 0.05), ABCG1 protein expression was significantly decreased (P < 0.05), StAR expression was significantly increased (P < 0.05), and progesterone secretion was significantly increased (P < 0.05). The results showed that orexin A promoted the expression of StAR by upregulating PRRT2 and downregulating ABCG1, therefore affecting secretion of progesterone. Gene expression characteristics of orexin A affecting progesterone secretion were preliminarily explored; this study provides a theoretical basis for further study on signalling pathways and reproductive regulation in Mongolian sheep.

Whole Genome Sequencing Reveals the Effects of Recent Artificial Selection on Litter Size of Bamei Mutton Sheep

Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on detecting candidate genes associated with the increasing of the litter size in this breeds under recent artificial selection to improve the efficiency of mutton production. We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH (gonadotropin-releasing hormone), oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 (forkhead box A1) and ESR1 (estrogen receptor 1). Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions that are responsible for litter size can in turn be used for further selection.

Phylogenetic analysis of Mongolian sheeppox and goatpox viruses

Sheeppox and goatpox are caused by sheep pox virus (SPPV) and goat pox virus (GTPV), members of Capripoxvirus genus, Poxviridae family. SPPV and GTPV damage host animal’s wool and skin and reduce production of mutton and milk. Because of morbidity and mortality of the diseases, they bring huge economic burden to the country. Main goal was to compare Mongolian sheep pox, goat pox sequences with other strains that were registered in Genebank. In this study, two SPPV and two GTPV field strains from Mongolia and Perego M strain (Biocombinat SOI, Mongolia), Russian and Chinese alive vaccine strains were used. The common DNA extraction method was used and samples were amplified on a nested polymerase chain reaction (nested-PCR) which amplify the full p32 gene of Capripoxvirus. The primers were designed based on the conserved sequences just outside of the p32 gene of SPPV or GTPV. By applying this method to the sheep and goat samples, suspected with SPPV and GTPV infection in Mongolia, the nested-PCR products were obtained from all samples on the predicted size, and the presence of SPPV and GTPV were confirmed via full length sequence analysis of P32 gene. Sequence comparison was performed using the online BLAST program. Sequence identities of nucleotides were analyzed using MUSCLE algorithm. A phylogenetic tree derived from nucleotide sequences was constructed for the Capripoxvirus using the neighbor joining method of MEGA (version X) software. Based on the phylogenetic tree, the Mongolian sheep pox virus, 2017 clustered together with Zabaikalsk strain and Perego strain (Biocombinat SOI, Mongolia). The Mongolian sheep pox virus, 2015 was closer to Tunisian and Chinese Gansu, Shanxi province strains. Chinese vaccine strain AV41, sequenced in this study was clustered with EF522181.1 Chinese Goat pox vaccine strain but Russian sheep pox vaccine strain, sequenced in this study was close to Mongolian goat pox viruses, 2009. The present data provides theoretical references to improve the preventive and control strategy. Based on the phylogenetic tree that we made, we conclude that SPPV and GTPV sequences in Mongolia were closer to Chinese SPPV, GTPV sequences therefore they were most likely imported from China.

Whole-Genome Sequencing of Bamei Mutton Sheep for Screening the Genes and SNPs Associated with Litter Size under Selection

Background: Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on increasing the litter size of this breed to improve the efficiency of mutton production.Results: We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH, oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 and ESR1. Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B.Conclusion: These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions of KDM4B that are responsible for litter size can in turn be used for further selection.