Effects of Extender and Packaging Devices on Ultrastructural Changes and Reproductive Index of Cryopreserved Spermatozoa of Ossimi Rams.

Simple SummaryLitter size is one of the most important reproductive traits in sheep. Four single nucleotide polymorphisms (SNPs), g.70067210 T > C in SLC5A1, g.25350431 C > T and g.25360220 T > C in CCNA1, and g.14413132 C > T in ABCC1, were identified by mass spectrometry and may be associated with litter size in sheep. Four SNPs were genotyped in Small-Tail Han, Hu, Cele Black, Suffolk, Sunite, Prairie Tibetan, and Tan sheep, and the expression patterns of SLC5A1, CCNA1, and ABCC1 were determined in Small-Tail Han sheep with different fecundities. Furthermore, we also studied the FecB mutation’s association with litter size in Small-Tail Han sheep. The results indicated that all genes included in this study were differentially expressed in the ovary and uterus of polytocous and monotocous Small-Tail Han sheep. Furthermore, association analysis indicated that both g.70067210 T > C in SLC5A1 and the FecB mutation in BMPR-IB were significantly associated with litter size in Small-Tail Han sheep. Linear regression analysis of the association of multiple markers (FecB and g.70067210 T > C in SCL5A1) with litter size indicated that homozygous ewes carrying the BB/TT genotype had a larger litter size than any other genotype.SLC5A1, CCNA1, and ABCC1 have been extensively studied as candidate genes because of their great influence on the reproductive traits of animals. However, little is known about the association between polymorphisms of the SLC5A1, CCNA1, and ABCC1 genes and litter size in Small-Tail Han sheep. In this study, the expression levels of SLC5A1, CCNA1, and ABCC1 in HPG (hypothalamic–pituitary–gonadal) axis tissues of polytocous and monotocous Small-Tail Han sheep were analyzed by qPCR. To better understand the effects of four single nucleotide polymorphisms (SNPs) comprising of g.70067210 T > C in SLC5A1, g.25350431 C > T and g.25360220 T > C in CCNA1, and g.14413132 C > T in ABCC1, a population genetic analysis was conducted using data obtained from genotyping in 728 sheep from seven breeds. The results indicated that all genes included in this study were differentially expressed in the pituitary and uterus of polytocous and monotocous Small-Tail Han sheep (p < 0.05). The associations of these four SNPs and the FecB mutation with litter size in 384 Small-Tail Han sheep were analyzed, therefore, and it was found that both g.70067210T > C and the FecB mutation were significantly associated with litter size (p < 0.05). The linear regression analysis of the association of multiple markers (FecB and g.70067210 T > C in SCL5A1) with litter size indicated that homozygous ewes carrying the BB/TT genotype had larger litter size than any ewes with any other genotype. In conclusion, the SLC5A1 SNPs significantly affect litter size in sheep and are useful as genetic marker for litter size.

Litter size in sheep is a complex trait controlled by micro-effective polygenes. APAF1, CLSTN2, CTH, PLCB1, PLCB4, and CHST11 are all involved in mammalian reproduction. However, the effects of these genes on litter size in sheep are still unclear. Therefore, in this study, we used Sequenom MassARRAY® SNP assay technology to type the single nucleotide polymorphisms (SNPs) loci of six genes in five sheep breeds. The results showed that most sheep breeds contain three genotypes at each locus. Then, we conducted population genetic analysis on the SNPs of six genes and found that the polymorphic information content in all sheep breeds ranged from 0 to 0.37, and most sheep breeds were in Hardy-Weinberg equilibrium (p > 0.05). In addition, association analysis in Small Tail Han sheep indicated that the rs399534524 locus in CLSTN2 was highly associated with first parity litter size, and litter size in ewes with CT genotype was higher than that in ewes with CC genotype or TT genotype. Furthermore, the rs407142552 locus in CTH was highly associated with second parity litter size in Small Tail Han sheep, and litter size in ewes with CT genotype was higher than that in ewes with TT genotype. Finally, we predicted the CTH and CLSTN2 protein interaction network and found that HTR1E, NOM1, CCDC174 and ALPK3 interact with CLSTN2 and have been reported as candidate genes related to litter size in sheep. These results suggest that they may be useful genetic markers for increasing litter size in sheep.

TGF‐β induced factor homeobox 1 (TGIF1) and splicing factor 1 (SF1) are important for mammalian reproduction; however, the effects of these genes on litter size in sheep remain unexplored. In this study, we genotyped 768 ewes from seven sheep breeds at two loci: g.37871539C>T, a synonymous mutation of TGIF1; and g.42314637T>C, a 3′UTR variant of SF1. Our analysis of polymorphism revealed only two genotypes at locus g.37871539C>T in TGIF1, with most sheep populations being moderately polymorphic (0.25 < PIC < 0.5) at this site. In contrast, most breeds exhibited low polymorphism (PIC ≤0.25) at the SF1 locus g.42314637T>C. The association analysis revealed that a synonymous mutation at g.37871539C>T in TGIF1 was highly associated with litter size in Small Tail Han sheep, in which it causes a significant decrease in litter size. Conversely, while the SF1 3′UTR variant g.42314637T>C was also highly associated with litter size in sheep, it causes a significant increase in the number of litter size. Combined, these data provide valuable information regarding candidate genetic markers for sheep breeding programs.

(1) Background: Litter size is one of the most important economic traits of sheep. The FecB locus has been extensively studied due to its significant impact on litter size in Hu sheep, and BMP15 and GDF9 have also been reported as major genes associated with litter size in sheep. This study aimed to identify variants of BMP15 and GDF9 and perform an association analysis of these variants with litter size in the Hu sheep breed. (2) Methods: In this study, exons of the BMP15 and GDF9 genes were fully sequenced to identify polymorphisms in Hu sheep. Population genetic parameters and haplotype frequencies were estimated, and an association analysis between these polymorphic loci and litter size was performed. Additionally, the protein structures of the wild-type and mutated BMP15 and GDF9 genes were predicted. (3) Results: The polymorphisms of the BMP15 and GDF9 genes were investigated within their exon regions, revealing mutations at four previously reported sites: BMP15 c.31_33CTTdel and GDF9 (G2, G3, and G4) in Hu sheep, with no novel variants were detected. Genetic analysis indicated that the GDF9-G3 and GDF9-G4 loci have low polymorphisms, whereas the BMP15 c.31_33CTTdel and the GDF9-G2 locus are moderately polymorphic. The mutation sites in the BMP15 and GDF9 genes were under Hardy-Weinberg equilibrium. Association analysis revealed that the BMP15 c.31_33CTTdel and GDF9 (G2, G3, and G4) mutations are not associated with litter size in Hu sheep. Protein structure prediction indicated that the mutations in BMP15 and GDF9 resulted in alterations to their tertiary structures. (4) Conclusions: In this study, four reported mutations in the BMP15 and GDF9 genes can also be detected in the Hu sheep breed. In these mutations, the G2 and G3 mutations of GDF9 did not alter the amino acid sequence, while the BMP15 c.31_33CTTdel mutation and the GDF9 G4 mutation resulted in protein structure alteration. Furthermore, the BMP15 c.31_33CTTdel mutation and the GDF9 mutations (G2, G3, G4) were associated with an increased tendency in litter size. However, no significant difference was observed (p > 0.05). This study provides valuable insights for improving the lambing performance of Hu sheep.

The growth differentiation factor 9 (GDF9) gene has been regarded as having major impacts on ovulation rate and litter size in sheep. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) of the GDF9 gene and their association with litter size in Garut sheep. For this purpose, a total of 60 ewes of Garut sheep were included in this study. Based on the sheep GDF9 reference sequences (Genbank Acc. No. AF078545.2), one pair of primers (5’-CTGCTGTTTAACCTGGATCGTG-3 5’-GGAGAGCCATACCGATGTCC-3 as forward and reverse, respectively) was used for PCR amplification. The results revealed that four SNPs (g.54C&gt;T, g.60G&gt;A, g.304G&gt;A, and g.333G&gt;A) were found in Garut sheep by direct sequencing. For SNP g.54C&gt;T, the sheep exhibited the highest frequency of allele C and genotype CC. On the other hand, SNPs g.60G&gt;A, g.304G&gt;A, and g.333G&gt;A showed a higher frequency of allele G than allele A, and the GG genotype was predominant in the population. SNP g.333G&gt;A had a significant effect on litter size (p &lt; 0.05), and ewes with the GG genotype had a higher litter size than those with the GA genotype. Genotype distributions for all identified SNPs were in agreement with Hardy-Weinberg equilibrium. We highlight that SNP g.333G&gt;A may be useful as a genetic marker for litter size in Garut sheep.

Association between polymorphism of GPR54 gene and litter size in Small Tail Han sheep

In mammals, the melatonin receptor gene has been widely studied since it has a great influence on reproductive traits. However, little is known about the association between polymorphism of the coding region of the MTNR1B gene and year‐round oestrus or the litter size in Small Tail Han sheep. To better understand the effects of single nucleotide polymorphism (SNP) rs400827589 in MTNR1B, a population polymorphism analysis was conducted using genotyping data in 45 sheep breeds around the world. The results indicated that TT was the dominant genotype in all sheep breeds. The associations of this SNP with reproductive seasonality and litter size in Small Tail Han sheep showed rs400827589 was correlated with fecundity as assessed by reproductive seasonality and litter size (p < .05). Bioinformatics analysis indicated the change in amino acid from Ile to Leu may affect the function of the MTNR1B protein by impacting the secondary and tertiary protein structures. The present results demonstrate that rs400827589 could be used in the marker‐assisted selection of the litter size in Small Tail Han sheep.

The genetic polymorphisms of TGFβ superfamily genes are associated with litter size in a Chinese indigenous sheep breed (Hu sheep)

Increasing the litter size in sheep is a primary objective in breeding programs, driving sustained interest in identifying candidate functional genes and molecular markers associated with fecundity. The known FecD mutation in the LEPR gene has been shown to regulate reproductive traits by influencing the ovulation rate of Davisdale sheep. However, the relationship between single-nucleotide polymorphisms (SNPs) in the LEPR and litter size remains unknown in Gobi short tail sheep and Sonid sheep. In this research, we found one novel variant and thirteen known variants through direct sequencing in Sonid sheep and Gobi short tail sheep and performed an association study in a large-scale population. The association analysis identified two SNPs (c.240C>T (rs159694506) and c.279C>T (rs159694508)) with significant litter size associations in Gobi short tail sheep (p < 0.01 ). In Sonid sheep, the c.240C>T (rs159694506), c.279C>T (rs159694508), g.41249772C>T (rs412130067), g.41249873A>C (rs425490800), g.41250357T>C (rs424307284), and g.41250358T>C (rs404651806) SNPs were significantly associated with litter size (p < 0.05 ). In addition, in GB sheep, the frequency of the litter-size-associated C allele at the c.240C>T (rs159694506) and c.279C>T (rs159694508) variants were significantly lower than that in Sonid sheep (SN), Mongolia sheep (MG), Ujimqin sheep (UM), Tan sheep (Tan), Hu sheep (Hu), and Small-tailed Han sheep (STH) populations. In SN sheep, the frequency of the litter-size-associated C allele at the c.240C>T (rs159694506) and c.279C>T (rs159694508) variant was significantly lower than that in the DPU. These findings provided valuable molecular markers pertinent to the fecundity of sheep, offering scientific evidence for the genetic improvement of these breeds.

The candidate genes and pathways affecting litter size in sheep

Aim of study: To assess selection methods via introgression to improve litter size in native and synthetic sheep breeds.Area of study: Sanandaj, Kurdistan, Iran.Material and methods: Selection approaches were performed using classical, genomic, gene-assisted classical (GasClassical) and gene-assisted genomic (GasGenomic) selection. Litter size trait with heritability of 0.1 including two chromosomes was simulated. On chromosome 1, a single QTL as the major gene was created to explain 40% of the total additive genetic variance. After simulation of a historical population, the animals from the last historical population were split into two populations. For the next 7 generations, animals were selected for favorable or unfavorable alleles to create distinct breeds of A or B, respectively. Then from the last generation, both males and females from breed B were selected to create a native population. On the other hand, males from breed A and females from breed B were mated to simulate a synthetic population. Finally, intra-population selections were carried out based on high breeding values during the last five generations.Main results: The genetic gain in the synthetic breed was higher than that of the native breed under all selection methods. The frequencies of favorable alleles after five generations in the classical, genomic, GasClassical and GasGenoimc selection approaches in the synthetic breed were 0.623, 0.730, 0.850 and 0.848, respectively.Research highlights: Combining gene-assisted selection with classical or genomic selection has the potential to improve genetic gain and increase the frequencies of favorable allele for litter size in sheep.

Simple SummaryLitter size is one of the most important reproductive traits in sheep. Two single nucleotide polymorphisms (SNPs), g.71874104G>A and g.71833755T>C, in the Histone Cell Cycle Regulator (HIRA) gene, were identified by whole-genome sequencing (WGS) and may be correlated with litter size in sheep. The two SNPs were genotyped and expression patterns of HIRA was determined in sheep breeds with different fecundity and in groups of Small Tail Han sheep producing large or small litters. Association analysis indicated that both SNPs were significantly correlated with litter size in Small Tail Han sheep. Furthermore, high levels of HIRA expression may have a negative effect on litter size in Small Tail Han sheep.Maintenance of appropriate levels of fecundity is critical for efficient sheep production. Opportunities to increase sheep litter size include identifying single gene mutations with major effects on ovulation rate and litter size. Whole-genome sequencing (WGS) data of 89 Chinese domestic sheep from nine different geographical locations and ten Australian sheep were analyzed to detect new polymorphisms affecting litter size. Comparative genomic analysis of sheep with contrasting litter size detected a novel set of candidate genes. Two SNPs, g.71874104G>A and g.71833755T>C, were genotyped in 760 Small Tail Han sheep and analyzed for association with litter size. The two SNPs were significantly associated with litter size, being in strong linkage disequilibrium in the region 71.80–71.87 Mb. This haplotype block contains one gene that may affect litter size, Histone Cell Cycle Regulator (HIRA). HIRA mRNA levels in sheep with different lambing ability were significantly higher in ovaries of Small Tail Han sheep (high fecundity) than in Sunite sheep (low fecundity). Moreover, the expression levels of HIRA in eight tissues of uniparous Small Tail Han sheep were significantly higher than in multiparous Small Tail Han sheep (p < 0.05). HIRA SNPs significantly affect litter size in sheep and are useful as genetic markers for litter size.

Litter size is one of the important economic traits of livestock. Seasonal oestrus, ovulation and lambing of sheep have severely restricted the development of sheep farming in Xinjiang, China. The purpose of this study was to investigate the polymorphisms and genetic correlation between GRM1, GNAQ and HCRTR1 genes and the seasonal reproduction and litter size in three sheep breeds. The DNA mixed pool sequencing and PCR-SSCP methods were used to detect single nucleotide polymorphisms (SNPs) of GRM1, GNAQ and HCRTR1 genes in seasonal oestrous (Kazakh and Chinese Merino [Xinjiang Junken type]) and perennial oestrous (Hu) sheep breeds. The association between genetic polymorphism and litter size was also analysed. The results showed that T945C in exon 2 of GRM1 gene, C589T in exon 2 of HCRTR1 gene and A191G in exon 2 of GNAQ gene were identified by Sanger sequencing, and three genotypes were existed in each SNP site, which all belonged to the synonymous mutation. GRM1 (CC), GNAQ (GA) and HCRTR1 (TC) were the dominant genotypes of seasonal reproduction and litter size in Kazakh sheep and Chinese Merino sheep, respectively, while, in perennial oestrous Hu sheep populations, the dominant genotypes were GRM1 (TC), GNAQ (GA) and HCRTR1 (TC), respectively, and association analysis also confirmed the results. The above results implied that GRM1, GNAQ and HCRTR1 genes are significantly associated with lambing traits in Kazakh, Chinese Merino and Hu sheep. Among them, the locus of GRM1 (T945C), GNAQ (A191G) and HCRTR1 (C589T) might be considered as a potential molecular marker, which controls seasonal reproduction and litter size in sheep.

Simple SummaryBMP15 is a critical gene in sheep reproduction. Most of its variations have been reported in European sheep. In this study, the entire open reading frame (ORF) region of BMP15 was sequenced in 154 Luzhong mutton sheep. Among 13 identified variations, six were novel. Four SNPs (ENSOART00000010201.1:c.352+342C>A, c.352+1232T>C, c.352+1165A>G and c.353-2036T>A) were significantly associated with litter size, and could be used as candidate genetic markers for improving litter size. The results also suggested possible interaction between BMP15 and FecB/GDF9.Litter size is an important economic trait in the mutton sheep industry. BMP15 is one of the key candidate genes for litter size in sheep. In this study, the entire ORF region of BMP15 was sequenced in 154 Luzhong mutton ewes, and the novel variations were determined. The association between polymorphism in BMP15 and litter size was analyzed using a general linear model. Six out of a total of thirteen variations were identified to be novel. Association analysis indicated that four (SNPs ENSOART00000010201.1:c.352+342C>A, c.352+1232T>C, c.352+1165A>G and c.353-2036T>A) were significantly associated with litter size. The joint analysis among three major genes (BMP15, BMPR1B and GDF9) exhibited significant interaction effects in three combinations (FecB and c.352+1232T>C of BMP15; FecB and c.352+1165A>G of BMP15; c.352+342C>A of BMP15 and ENSOART00000014382.1:c.994G>A of GDF9). For the SNPs c.352+1232T>C and c.352+342C>A, the global distribution of allele frequencies showed that the highest variation frequency occurs in Western Europe. In conclusion, the results demonstrated that BMP15 is a major gene for litter size in Luzhong mutton sheep and candidate SNPs associated with litter size were identified.

Simple SummaryThe NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on litter size in sheep are unknown. In this study, we conducted population genetic and association analyses on five NLRP5 and NLRP9 loci of sheep. Our results suggested that a mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as the growth differentiation factor 9 (GDF9), whereas a mutation in g. g.59030623T > C may enhance the NLRP9-combining capacity with these proteins. Consequently, these mutations may lead to differences in ovulation rate and even litter size. Overall, this study provided useful genetic markers that can be used to improve sheep breeding.Previous studies showed that the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on sheep litter size are unknown. Therefore, in this study, we first genotyped seven sheep breeds via the MassARRAY® SNP system at the loci g.60495375A > G, g.60495363G > A, and g.60499690C > A in NLRP5, and g.59030623T > C and g.59043397A > C in NLRP9. Our results revealed that each locus in most sheep breeds contained three genotypes. Then, we conducted population genetic analysis of single nucleotide polymorphisms in NLRP5 and NLRP9, and we found that the polymorphism information content value in all sheep breeds ranged from 0 to 0.36, and most sheep breeds were under Hardy–Weinberg equilibrium (p > 0.05). Furthermore, association analysis in Small Tail Han sheep indicated that two loci, g.60495363G > A in NLRP5 and g.59030623T > C in NLRP9, were highly associated with litter size. The mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as GDF9, whereas the mutation in g.59030623T > C may enhance the combining capacity of NLRP9 with these proteins; consequently, these mutations may influence the ovulation rate and even litter size. The findings of our study provide valuable genetic markers that can be used to improve the breeding of sheep and even other mammals.